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The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors (GEFs). Here, we found the N-terminal amphipathic motifs of the Golgi-localized Arf family protein, Arfrp1, and the endosome- and plasma membrane-localized Arf family protein, Arl14, play critical roles in spatial determination. Exchanging the amphipathic helix motifs between these two Arf proteins causes the switch of their localizations. Moreover, the amphipathic helices of Arfrp1 and Arl14 are sufficient for cytosolic proteins to be localized into a specific cellular compartment. The spatial determination mediated by the Arfrp1 helix requires its binding partner Sys1. In addition, the acetylation of the Arfrp1 helix and the myristoylation of the Arl14 helix are important for the specific subcellular localization. Interestingly, Arfrp1 and Arl14 are recruited to their specific cellular compartments independent of GTP binding. In sum, our results demonstrate that the GTP binding and membrane association are of Arfrp1 and Arl14 are uncoupled. The amphipathic motifs of Arfrp1 and Arl14 are sufficient for them to be located to specific cellular compartments in a GTP-independent manner. The insulin-like growth factor 2 (IGF2) plays critical roles in cell proliferation, growth, migration, differentiation and survival. Despite its importance, the molecular mechanisms mediating secretion of IGF2 remain unclear. Here we utilized a Retention Using Selective Hook (RUSH) system to analyze molecular mechanisms that regulate secretion of IGF2. We found that a type I transmembrane protein, TMED10, is essential for secretion of IGF2. Further analyses indicate that the residues from position 112 to position 140 in IGF2 is important for the secretion of IGF2 and these residues directly interact with the GOLD domain of TMED10. We then reconstituted release of IGF2 into COPII vesicles and this assay suggests that TMED10 regulates the pac

Circulating insulin-like growth factor-1 (IGF-1) is consistently associated with prostate cancer risk. IGF-1 binds to IGF-1 receptor (IGF1R) and insulin receptor (IR), activating cancer hallmark pathways. Experimental evidence suggests that TMPRSS2:ERG may interact with IGF/insulin signaling to influence progression. We investigated IGF1R and IR expression and its association with lethal prostate cancer among 769 men. Protein expression of IGF1R, IR and ERG (i.e. a surrogate of ERG fusion genes) were assayed by immunohistochemistry. Cox models estimated hazard ratios (HR) and 95% confidence intervals (CI) adjusted for clinical characteristics. Among patients, 29% had strong tumor IGF1R expression and 10% had strong IR expression. During a mean follow-up of 13.2 years through 2012, 80 men (11%) developed lethal disease. Tumors with strong IGF1R or IR expression showed increased cell proliferation, decreased apoptosis and a higher prevalence of ERG. In multivariable models, strong IGF1R was associated with a borderline increased risk of lethal prostate cancer (HR 1.7; 95% CI 0.9-3.1). The association appeared greater in ERG-positive tumors (HR 2.8; 95% CI 0.9-8.4) than in ERG-negative tumors (HR 1.3; 95% CI 0.6-3.0, p-heterogeneity 0.08). There was no association between IR and lethal prostate cancer (HR 0.8; 95% CI 0.4-1.9). These results suggest that tumor IGF1R expression may play a role in prostate cancer progression to a lethal phenotype and that ERG-positive tumors may be more sensitive to IGF signaling. These data may improve our understanding of IGF signaling in prostate cancer and suggest therapeutic options for disease subtypes.

We report a six-year-old boy who presented with short stature, microcephaly, dysmorphic features, and developmental delay and who was identified with a terminal deletion of 15q26.2q26.3 containing the insulin-like growth factor receptor (IGF1R) gene in addition to a terminal duplication of the 4q35.1q35.2 region. We compare our case with other reports of deletions and mutations affecting the IGF1R gene associated with pre-and postnatal growth restriction. We report the dramatic response to growth hormone therapy in this patient which highlights the importance of identifying patients with IGF1R deletion and treating them early.

Exportin-1 mediates nuclear export of multiple tumor suppressor and growth regulatory proteins. Aberrant expression of exportin-1 is noted in human malignancies, resulting in cytoplasmic mislocalization of its target proteins. We investigated the efficacy of selinexor against liposarcoma cells both in vitro and in vivo. Exportin-1 was highly expressed in liposarcoma samples and cell lines as determined by immunohistochemistry, western blot, and immunofluorescence assay. Knockdown of endogenous exportin-1 inhibited proliferation of liposarcoma cells. Selinexor also significantly decreased cell proliferation as well as induced cell cycle arrest and apoptosis of liposarcoma cells. The drug also significantly decreased tumor volumes and weights of liposarcoma xenografts. Importantly, selinexor inhibited insulin-like growth factor 1 (IGF1) activation of IGF-1R/AKT pathway through upregulation of insulin-like growth factor binding protein 5 (IGFBP5). Further, overexpression and knockdown experiments showed that IGFBP5 acts as a tumor suppressor and its expression was restored upon selinexor treatment of liposarcoma cells. Selinexor decreased aurora kinase A and B levels in these cells and inhibitors of these kinases suppressed the growth of the liposarcoma cells. Overall, our study showed that selinexor treatment restored tumor suppressive function of IGFBP5 and inhibited aurora kinase A and B in liposarcoma cells supporting the usefulness of selinexor as a potential therapeutic strategy for the treatment of this cancer.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder in which the MECP2 (methyl CpG-binding protein 2) gene is mutated. Recent studies showed that RTT-derived neurons have many cellular deficits when compared to control, such as: less synapses, lower dendritic arborization and reduced spine density. Interestingly, treatment of RTT-derived neurons with Insulin-like Growth Factor 1 (IGF1) could rescue some of these cellular phenotypes. Given the critical role of IGF1 during neurodevelopment, the present study used human induced pluripotent stem cells (iPSCs) from RTT and control individuals to investigate the gene expression profile of IGF1 and IGF1R on different developmental stages of differentiation. We found that the thyroid hormone receptor (TRalpha 3) has a differential expression profile. Thyroid hormone is critical for normal brain development. Our results showed that there is a possible link between IGF1/IGF1R and the TRalpha 3 and that over expression of IGF1R in RTT cells may be the cause of neurites improvement in neural RTT-derived neurons.

It has been generally accepted that integrin cell adhesion receptors are involved in growth factor signaling (integrin-growth factor crosstalk), since antagonists to integrins often suppress growth factor signaling. Partly because integrins have been originally identified as cell adhesion receptors to extracellular matrix (ECM) proteins, current models of the crosstalk between IGF1 and integrins propose that ECM ligands (e.g., vitronectin) bind to integrins and IGF1 binds to IGF receptor type 1 (IGF1R), and two separate signals merge inside the cells. Our research proves otherwise. We discovered that IGF1 interacts directly with integrins, and induces integrin-IGF-IGF1R complex formation on the cell surface. IGF1 signaling can be detected in the absence of ECM (anchorage-independent conditions). Integrin antagonists block both ECM-integrin interaction and IGF-integrin interaction, and do not distinguish the two. This is one possible reason why integrin-IGF1 interaction has not been detected. With these new discoveries, we believe that the direct IGF-integrin interaction should be incorporated into models of IGF1 signaling. The integrin-binding defective mutant of IGF1 is defective in inducing IGF signaling, although the mutant still binds to IGF1R. Notably, the IGF1 mutant is dominant-negative and suppresses cell proliferation induced by wt IGF1, and suppresses tumorigenesis in vivo, and thus the IGF1 mutant has potential as a therapeutic.

To investigate the role of IGF-I signaling in osterix (OSX)-expressing cells in the skeleton, we generated IGF-I receptor (IGF-IR) knockout mice ((OSX)IGF-IRKO) (floxed-IGF-IR mice × OSX promoter-driven GFP-labeled cre-recombinase [(OSX)GFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), (OSX)GFP-cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, (OSX)GFP-cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the (OSX)IGF-IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of (OSX)IGF-IRKOs. Quantitative real-time PCR revealed that the mRNA levels of the matrix degradation markers, MMP-9, 13 and 14, were decreased in the (OSX)IGF-IRKOs compared with the controls. The (OSX)IGF-IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF-I signaling in OSX-expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF-I signaling in the OSX-expressing PHC is required for growth plate maturation and osteoblast differentiation in the de

Computational models using metabolic reconstructions for in silico simulation of metabolic disorders such as type 2 diabetes mellitus (T2DM) can provide a better understanding of disease pathophysiology and avoid high experimentation costs. There is a limited amount of computational work, using metabolic reconstructions, performed in this field for the better understanding of T2DM. In this study, a new algorithm for generating tissue-specific metabolic models is presented, along with the resulting multi-confidence level (MCL) multi-tissue model. The effect of T2DM on liver, muscle, and fat in MKR mice was first studied by microarray analysis and subsequently the changes in gene expression of frank T2DM MKR mice versus healthy mice were applied to the multi-tissue model to test the effect. Using the first multi-tissue genome-scale model of all metabolic pathways in T2DM, we found out that branched-chain amino acids' degradation and fatty acids oxidation pathway is downregulated in T2DM MKR mice. Microarray data showed low expression of genes in MKR mice versus healthy mice in the degradation of branched-chain amino acids and fatty-acid oxidation pathways. In addition, the flux balance analysis using the MCL multi-tissue model showed that the degradation pathways of branched-chain amino acid and fatty acid oxidation were significantly downregulated in MKR mice versus healthy mice. Validation of the model was performed using data derived from the literature regarding T2DM. Microarray data was used in conjunction with the model to predict fluxes of various other metabolic pathways in the T2DM mouse model and alterations in a number of pathways were detected. The Type 2 Diabetes MCL multi-tissue model may explain the high level of branched-chain amino acids and free fatty acids in plasma of Type 2 Diabetic subjects from a metabolic fluxes perspective.

Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adult hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. These data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer.

Diabetes mellitus is a chronic metabolic disease characterized by elevated blood glucose that results from defects in insulin secretion and/or action. Until now, the cellular mechanisms and molecular components responsible for the biogenesis of insulin granules are still not fully understood. PICK1 (Protein Interacting with C Kinase 1) is a peripheral membrane protein which forms a tight heteromeric complex with ICA69 (Islet Cell Autoantigen 69kD). This complex is able to tether membrane proteins to vesicles and hence regulate their trafficking. Previously, our lab found that PICK1-ICA69 was associated with insulin granules and deficiency of PICK1-ICA69 in mice led to glucose intolerance and decreased insulin level. However, the detailed molecular mechanism underlying the defects is unclear. In my study, I further investigated the PICK1-ICA69’s role in insulin granule biogenesis. I first examined the glucose metabolism in Ica69-/- mice and found that they mimicked the phenotype of Pick1-/- mice, which was manifested by impaired glucose tolerance and insufficient insulin secretion. Molecular studies showed that PICK1 and ICA69 specifically affected the dense-core insulin granules in the regulated secretory pathway but not other types of granules in β cells. In the absence of PICK1-ICA69, ~50% of the insulin granule contents were lost due to the missorting for lysosomal degradation. Upon glucose stimulation, the budding and maturation of insulin granules were also compromised. Moreover, the important insulin granule metal ion Zn2+ was significantly reduced in Pick1-/- and Ica69-/- islets. PICK1 interacted with β cell specific zinc transporter, ZnT8, regulated its expression and subcellular localization, hence Zn2+ homeostasis in β cells. Taken together, these results provide evidence that PICK1-ICA69 complex plays important roles in cargo sorting, vesicle budding and Zn2+ homeostasis in pancreatic β cells.

Calorie restriction (CR) inhibits prostate cancer progression, partially through modulation of the IGF axis. IGF-1 receptor (IGF-1R) blockade reduces prostate cancer xenograft growth. We hypothesized that combining calorie restriction with IGF-1R blockade would have an additive effect on prostate cancer growth. Severe combined immunodeficient mice were subcutaneously injected with 22Rv1 cells and randomized to: (1) Ad libitum feeding/intraperitoneal saline (Ad-lib); (2) Ad-lib/20 mg/kg twice weekly, intraperitoneal ganitumab [anti-IGF-1R antibody (Ad-lib/Ab)]; (3) 40% calorie restriction/intraperitoneal saline (CR); (4) CR/ intraperitoneal ganitumab, (CR/Ab). CR and ganitumab treatment were initiated one week after tumor injection. Euthanasia occurred 19 days post treatment. Results showed that CR alone decreased final tumor weight, plasma insulin and IGF-1 levels, and increased apoptosis. Ganitumab therapy alone reduced tumor growth but had no effect on final tumor weight. The combination therapy (CR/Ab) further decreased final tumor weight and proliferation, increased apoptosis in comparison to the Ad-lib group, and lowered plasma insulin levels relative to the Ad-lib and Ad-lib/Ab groups. Tumor AKT activation directly correlated with plasma IGF-1 levels. In conclusion, whereas ganitumab therapy modestly affected 22Rv1 tumor growth, combining IGF-1R blockade with calorie restriction resulted in a significant decrease in final tumor weight and improved metabolic profile.

Biomaterials that are similar to skeletal muscle extracellular matrix have been shown to augment regeneration in ischemic muscle. In this study, treatment with a collagen-based matrix stimulated molecular myogenesis in an mdx murine model of necrosis. Matrix-treated animals ran ≥ 40% further, demonstrating functional regeneration, and expressed increased levels of myogenic transcripts. By contrast, matrix treatment was unable to induce transcriptional or functional changes in an MLC/SOD1G93A atrophic mouse model. In vitro, satellite cells were cultured under standard conditions, on matrix, in the presence of myocyte debris (to simulate a necrotic-like environment) or with both matrix and necrotic stimuli. Exposure to both matrix and necrotic stimuli induced the greatest increases in mef2c, myf5, myoD and myogenin transcripts. Furthermore, conditioned medium collected from satellite cells cultured with both stimuli contained elevated levels of factors that modulate satellite cell activation and proliferation, such as FGF-2, HGF and SDF-1. Application of the conditioned medium to C2C12 myoblasts accelerated maturation, as demonstrated by increased mef2c, myf5 and myogenin transcripts and fusion indexes. In summary, the collagen matrix required a necrotic stimulus to enhance the maturation of satellite cells and their secretion of a myogenic cocktail. Considering that matrix treatment supports myogenesis only in in vivo models that exhibit necrosis, this study demonstrates that a necrotic environment is required to maximize matrix-mediated myogenesis.

Background: Mexican Americans are the fastest aging segment of the U.S. population, yet little scientific literature exists regarding the Alzheimer's disease (AD) among this segment of the population. The extant literature suggests that biomarkers of AD will vary according to race/ethnicity though no prior work has explicitly studied this possibility. The aim of this study was to create a serum-based biomarker profile of AD among Mexican American. Methods: Data were analyzed from 363 Mexican American participants (49 AD and 314 normal controls) enrolled in the Texas Alzheimer's Research & Care Consortium (TARCC). Non-fasting serum samples were analyzed using a luminex-based multi-plex platform. A biomarker profile was generated using random forest analyses. Results: The biomarker profile of AD among Mexican Americans was different from prior work from non-Hispanic populations with regards to the variable importance plots. In fact, many of the top markers were related to metabolic factors (e.g., FABP, GLP-1, CD40, pancreatic polypeptide, insulin-like-growth factor, and insulin). The biomarker profile was a significant classifier of AD status yielding an area under the receiver operating characteristic curve, sensitivity, and specificity of 0.77, 0.92, and 0.64, respectively. Combining biomarkers with clinical variables yielded a better balance of sensitivity and specificity. Conclusion: The biomarker profile for AD among Mexican American cases is significantly different from that previously identified among non-Hispanic cases from many large-scale studies. This is the first study to explicitly examine and provide support for blood-based biomarkers of AD among Mexican Americans. Areas for future research are highlighted.

An antiserum (R8) raised against a purified specific low-M(r) (16,000-18,000) insulin-like-growth-factor (IGF)-inhibitor/binding protein, which is immunologically related to the native growth hormone (GH)-dependent M(r)-150,000 IGF-binding protein in serum, has been used to probe a possible additional relationship to the predominant non-GH-dependent IGF-binding protein (M(r) ~ 30,000) of human amniotic fluid. Amniotic-fluid fractions and an IGF-inhibitory fraction of serum were analysed by covalent corss-linking, ligand-blotting and immunoblotting techniques. Western blotting of the serum fraction using the R8 antiserum gave five immunoreactive (ir) bands, of which at least three (M(r) 38,000, 34,000 and 23,000) possessed IGF-binding activity, as indicated by ligand (125I-IGG-I) blotting. Western blotting of two differently prepared amniotic-fluid fractions, which both showed potent IGF-inhibitory bioactivity, gave several (M(r) range 14,500-73,000) ir bands, of which two (M(r) 28,000 and 17,000) were most prominent. Ligand-blotting analysis gave a single intensely labelled band at M(r) 28,000, consistent with the major presence of the M(r) 28,000-30,000 amniotic-fluid IGF-binding protein. Covalent cross-linking of the amniotic-fluid fractions to 125I-IGF-I gave three specifically cross-linked complexes (M(r) 36,000, 32,000 and 23,000), which, assuminmg a 1:1 binding stoichiometry with IGF (M(r) 7500), represent binding proteins of M(r) 28,500, 24,500 and 15,500 respectively. The M(r)-15,000 binding protein, very similar to the M(r)-17,000 ir band, in all likelihood represents the IGF-inhibitor protein. Our results indicate that inhibitor-sized binding proteins and IGF-inhibitor bioactivity are present in human amniotic fluid, and that the IGF-inhibitor protein (M(r) 16,000) and amniotic-fluid IGF-binding protein (M(r) 28,000) are immunologically related. Since the IGF-inhibitor protein is also immunologically and structurally related to the native, GH-dependent, M(

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.

研究目的：脊髓損傷是中樞神經系統的嚴重創傷，致殘率高。脊髓損傷後的再生修復一直是當前醫學的難題。迄今為止，脊髓損傷依然缺乏一種有效地治療方法。既往研究證明，胰島素樣生長因子-1對鼠和兔脊髓損傷有保護作用，為了進一步把這些發現應用到臨床方面，我們採用與人類生理更相近的豬只作為實驗動物，構建與臨床相似的脊髓損傷動物模型，并以此為基礎，系統性研究胰島素樣生長因子-1的脊髓保護作用，評估該治療的功效。, 研究方法：以運動誘發電位為指導，通過直接壓迫和牽拉造成脊髓損傷。18頭猪只隨機分為3組：胰島素樣生長因子-1治療組、生長激素治療組及生理鹽水對照組。脊髓損傷后1小時、24小時及48小時經鞘內注射給藥。于術後第1天、第3天及第21天收集腦脊液檢測胰島素樣生長因子-1和生長激素濃度。連續21天使用修正的 Tarlov 評分標準對動物的運動功能進行評估。第21天處死動物並取材，檢測脊髓中NeuN, GFAP, caspase-3 的活性，并通過TUNEL染色觀察細胞凋亡情況，比較各組之間有無差別。, 研究結果：通過這種方法建立的脊髓損傷動物模型穩定可靠，各組之間無明顯差異。鞘內給藥24小時及48小時后，腦脊液中胰島素樣生長因子-1和生長激素濃度明顯升高，術後21天檢測，其濃度恢復至基礎值。胰島素樣生長因子-1治療組的運動功能的恢復優於其它各組。與生理鹽水對照組比較，胰島素樣生長因子-1治療組可以明顯提高脊髓損傷后神經元的存活數量，抑制星形膠質細胞增生，減少細胞凋亡。而生長激素治療組僅抑制星形膠質細胞增生，其它方面與生理鹽水對照組無明顯差別。, 結論：胰島素樣生長因子-1通過提高神經元存活數量，抑制星形膠質細胞增生，以及減少細胞凋亡促進脊髓損傷的恢復。, Objective: Spinal cord injury is a devastating condition that leads to long-term disabilities. Currently, there is no effective treatment that minimizes spinal cord damage or enhances neurological recovery. Recent studies in rats or rabbits suggested that neurologic recovery after spinal cord injury could be improved with the administration of neurotropic hormones, such as insulin-like growth factor-1 (IGF-1). In order to apply such bench-side discovery to clinical practice, we conducted a study in a higher animal model, akin to human physiology, to evaluate the effectiveness of intrathecal injections of IGF-1to improve neurological recovery in a porcine model of acute traumatic spinal cord injury., Methods: Traumatic spinal cord injury model was produced by controlled compression and distraction of the exposed T12 segment of the spinal cord. Eighteen pigs were randomly assigned to receive intrathecal injections of either IGF-1, growth hormone or saline at 1, 24 and 48 hours after spinal cord injury. Locomotor function was assessed daily using the validated modified Tarlov’s scale for 21 days. Spinal cord segments were then harvested and the survival of neurons, reactive astrogliosis and apoptosis were determined using neuronal-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays., Results: Intrathecal injections of IGF-1 and growth hormone significantly increase the concentrations of the neurotropic hormones in the cerebrospinal fluid after injury (p < 0.01). These concentrations returned to baseline by 21 days after drug delivery. Motor deficits on the first day after injury were comparable between animals in the treatment and control groups. By the end of the third week, neurologic recovery was better in animals receiving IGF-1 treatment (p < 0.05). Immunohistological and western blot studies of the injured segments of spinal cord showed that treatment with both IGF-1 and growth hormone prevented reactive astrogliosis (p < 0.05) while only IGF-1 improved the survival of mature neurons (p < 0.05). IGF-1 also inhibited apoptosis after spinal cord injury (p < 0.05)., Conclusions: In our clinically relevant model of traumatic spinal cord injury in pigs, intrathecal injection of IGF-1 demonstrated beneficial effects on neurological and histological recovery., Detailed summary in vernacular field only., Wang, Qinzhou., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 105-122)., also in Chinese., Declaration of origination --- p.I, p.II, Acknowledgements --- p.VI, Table of Contents --- p.VIII, List of Tables --- p.XII, List of Figures --- p.XIII, Abbreviations --- p.XVIII, Chapter Part 1 --- Spinal Cord Injury: A Review --- p.1, Chapter Chapter 1-1 --- Acute Spinal Cord Injury: Epidemiology, Socioeconomic Impact --- p.2, Chapter 1.1.1 --- Epidemiology of Spinal Cord Injury --- p.2, Chapter 1.1.2 --- Socioeconomic Impact of Acute Spinal Cord Injury --- p.5, Chapter Chapter 1-2 --- Mechanisms of Spinal Cord Injury --- p.6, Chapter Chapter 1-3 --- Putative Treatments for Spinal Cord Injury --- p.8, Chapter 1.3.1 --- Methylprednisolone --- p.8, Chapter 1.3.2 --- Stem Cell Therapy --- p.11, Chapter 1.3.3 --- Riluzole --- p.11, Chapter 1.3.4 --- Other Pharmacological Therapies for Spinal Cord Injury --- p.12, Chapter Chapter 1-4 --- Insulin-like Growth Factor-1 for the Treatment of Spinal Cord Injury --- p.13, Chapter Chapter 1-5 --- Summary --- p.17, Chapter Part 2 --- Insulin-like Growth Factor-1 and Growth Hormone for Spinal Cord Injury --- p.18, Chapter Chapter 2-1 --- Hypothesis and Objectives --- p.19, Chapter Chapter 2-2 --- Establishment of Animal Models for Acute Spinal Cord Injury --- p.22, Chapter 2.2.1 --- Introduction --- p.22, Chapter 2.2.2 --- Experimental Animals --- p.22, Chapter 2.2.3 --- Anesthesia --- p.23, Chapter 2.2.4 --- Transcranial Electrical Motor Evoked Potential --- p.26, Chapter 2.2.5 --- Surgery --- p.28, Chapter 2.2.6 --- Statistics --- p.34, Chapter 2.2.7 --- Results --- p.34, Chapter 2.2.8 --- Discussion --- p.38, Chapter Chapter 2-3 --- Optimal Stimulation Protocols for Transcranial Electrical Motor Evoked Potential. --- p.42, Chapter 2.3.1 --- Introduction --- p.42, Chapter 2.3.2 --- Methods --- p.42, Chapter 2.3.2.1 --- Experimental Animals and Anesthesia --- p.42, Chapter 2.3.2.2 --- Transcranial Electrical Motor Evoked Potential Recording --- p.44, Chapter 2.3.2.3 --- Stimulation Protocol --- p.44, Chapter 2.3.3 --- Analyses --- p.44, Chapter 2.3.4 --- Results --- p.45, Chapter 2.3.5 --- Discussion --- p.52, Chapter Chapter 2-4 --- Evaluation of the Efficacy of Insulin-like Growth Factor-1 and Growth Hormone in a Porcine Model --- p.54, Chapter 2.4.1 --- Introduction --- p.54, Chapter 2.4.2 --- Materials and Methods --- p.54, Chapter 2.4.2.1 --- Study Design --- p.54, Chapter 2.4.2.2 --- Intrathecal Injection and Collection of Cerebrospinal Fluid --- p.58, Chapter 2.4.2.3 --- Measurements --- p.58, Chapter 2.4.2.3.1 --- Clinical Evaluation --- p.58, Chapter 2.4.2.3.2 --- Biochemical Assessments --- p.58, Chapter 2.4.2.3.3 --- Spinal Cord Section, Histological and Immunochemical Staining --- p.63, Chapter 2.4.2.3.4 --- Western Blot --- p.69, Chapter 2.4.3 --- Statistical Analysis and Sample Size Calculation --- p.72, Chapter 2.4.3.1 --- General Analysis --- p.72, Chapter 2.4.3.2 --- Sample Size --- p.72, Chapter 2.4.4 --- Results --- p.73, Chapter 2.4.4.1 --- Changes of TceMEP --- p.73, Chapter 2.4.4.2 --- Motor Deficit after Spinal Cord Injury at Baseline --- p.75, Chapter 2.4.4.3 --- Insulin-like Growth Factor-1 and Growth Hormone in Cerebrospinal Fluid --- p.77, Chapter 2.4.4.4 --- Clinical Assessment --- p.80, Chapter 2.4.4.5 --- Demyelination, Neuron Survival and Astrocyte Reaction --- p.85, Chapter 2.4.4.6 --- Apoptosis --- p.89, Chapter 2.4.5 --- Discussion --- p.93, Chapter 2.4.5.1 --- Principal Findings --- p.93, Chapter 2.4.5.2 --- Insulin-like Growth Factor-1 and Neuroprotection after Spinal Cord Injury --- p.93, Chapter 2.4.5.3 --- Growth Hormone and Neuroprotection after Spinal Cord Injury --- p.95, Chapter 2.4.5.4 --- Strengths and Limitations of Our Study --- p.96, Chapter 2.4.5.5 --- Summary --- p.97, Chapter Part 3 --- Summary and Future Directions --- p.99, Chapter Chapter 3-1 --- Summary --- p.100, Chapter Chapter 3-2 --- Future Directions --- p.103, Chapter Part 4 --- References and appendixes --- p.104, References --- p.105, Appendixes --- p.123, http://library.cuhk.edu.hk/record=b5549520, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

As a functional unit involved in both maintaining endocrine homeostasis and also producing mature eggs, the ovary plays a central role in female reproduction. The development and function of the ovarian follicles are controlled by gonadotropins released from the pituitary. It is widely accepted that the action of gonadotropins on ovarian follicles is mediated by paracrine/autocrine factors produced by the somatic cells surrounding the oocyte. Increasing evidence indicates that the Igf system is involved in mediating the action of gonadotropins in the ovary. Previously, we identified a gonad-specific Igf subtype (Igf3) distinct from Igf1 and Igf2. This fmding further highlights the importance of the Igf system in the fish ovary. In this thesis, efforts were made to understand the role of the Igf system in ovary using zebrafish as the model organism, and attention was focused on Igf3., Because the expression of Igf3 is correlated with the LH receptor in zebrafish follicles, the regulation of igf3 by gonadotropins was subsequently studied in the ovary. The expression of igf3 was significantly up-regulated in both ovarian fragments and isolated follicles upon treatment with hCG in dose- and time-dependent manners. Treatment with 8-Br-cAMP or IBMX mimicked the effects of hCG on the expression of igf3 in follicles of different stages., Four Igfs are present in zebrafish, and our results show that all four igfs are expressed in the ovary of zebrafish and exhibit the differential expression profiles during folliculogenesis. Using a primary culture of zebrafish follicle cells, we demonstrated that hCG stimulated igf2b and igf3 expression but suppressed igf2a expression. Moreover, the effect of gonadotropin could be mimicked by IBMX, which increased the intracellular levels of cAMP, suggesting the possible involvement of cAMP in the gonadotropin-based regulation and differential expression of igf2a, igf2b and igf3. These results also show that the Igf3 is the Igf subtype most sensitive to gonadatropin and cAMP., In addition, the expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were also studied during zebrafish embryogenesis. The unique temporal and spatial expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were revealed by both real-time PCR and whole mount in situ hybridization, the results suggest divergent functions for these Igfs in early zebrafish development., Taken together, the present studies provide substantial information about the Igf system, especially that of Igf3 in the zebrafish ovary. Data were gathered regarding Igf3 expression, regulation and functions, which is not only helpful for the understanding of the role of the Igf system in fish reproduction, but also contributes toward uncovering the ovarian signaling network involved in oocyte maturation across vertebrates. This study of igfs gene expression provides direct information to the study of Igf signaling in zebrafish., To study the function of Igf3, bioactive recombinant Igf3 proteins were prepared using a bacterial expression system. Incubation of follicles with recombinant zebrafish Igf3 significantly enhanced oocyte maturation in time-, dose- and stage-dependent manners. The potential mechanisms of Igf3-induced oocyte maturation were then investigated. Igf3 stimulated oocyte maturation via a steroid-independent manner. Igf3 induced oocyte maturation through Igf1rs and the PI3 kinase, PDE3 and MAP kinase were necessary for Igf3-mediated oocyte maturation in zebrafish., We first examined the gene expression patterns of Igf3 in the ovary. The igf3 gene in zebrafish was found to be alternatively spliced into two transcripts, with transcript variant 1 exclusively expressed in the gonads and transcript variant 2 only expressed during early development. Using specific antibodies developed for zebrafish Igf3, both the prepropeptide and the mature peptide forms of Igf3 were found to be predominantly expressed in the zebrafish ovary. Real-time PCR and in situ hybridization revealed that igf3 mRNA levels were relatively low in the early follicles but significantly increased after the mid vitellogenic stage (midstage III) and were high in the full grown follicles. In the full grown follicles, igf3 mRNA was detected primarily in the somatic follicular cells, with a low level of expression in the oocytes. Igf3 immunoreactivity was confined to the follicular cells alone., Li, Jianzhen., Advisers: Hui Zhao; Hon Ki Christopher Cheng., Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: ., Thesis (Ph.D.)--Chinese University of Hong Kong, 2011., Includes bibliographical references (leaves 122-150)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., isbn: 9781267214201, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

[Truncated abstract] Meat yield and product quality are important to the Australian sheep meat production industry as the country faces more and more competition from overseas imports. The Callipyge genotype in sheep is a natural mutation in chromosome 18, the mode of inheritance has been termed polar over-dominance which leads to significantly greater muscle yield. Although this genetic polymorphism has been well studied, the biochemical mechanisms responsible for the muscle hypertrophy are still not fully understood. It has been shown in many systems, that insulin-like growth factor-1 (IGF-1) is one of the effectors of skeletal muscle hypertrophy. IGF-1 is involved in both the mitogen activated protein kinase (MAPK: RAS-RAP-MEK-ERK) pathway related to cell proliferation, and the phosphatidylinositol 3-kinase PI3K/Akt1/p70S6K pathway related to cell differentiation. IGF-1 causes changes in muscle protein accretion. Recent reviews describe how IGF-1 induces hypertrophy by stimulating, PI3K/Akt1/p70S6K signalling pathway, resulting in the downstream activation of protein synthesis and inhibition of protein degradation in muscle cells. Many studies have demonstrated that IGF-1 is necessary and sufficient to induce hypertrophic growth in human and mouse skeletal muscle. Thus the role of IGF-1 signalling was investigated in primary muscle cultures of Callipyge sheep. Meat yield and product quality are important to the Australian sheep meat production industry as the country faces more and more competition from overseas imports. The Callipyge genotype in sheep is a natural mutation in chromosome 18, the mode of inheritance has been termed polar over-dominance which leads to significantly greater muscle yield. Although this genetic polymorphism has been well studied, the biochemical mechanisms responsible for the muscle hypertrophy are still not fully understood. It has been shown in many systems, that insulin-like growth factor-1 (IGF-1) is one of the effectors of, skeletal muscle hypertrophy. IGF-1 is involved in both the mitogen activated protein kinase (MAPK: RAS-RAP-MEK-ERK) pathway related to cell proliferation, and the phosphatidylinositol 3-kinase PI3K/Akt1/p70S6K pathway related to cell differentiation. IGF-1 causes changes in muscle protein accretion. Recent reviews describe how IGF-1 induces hypertrophy by stimulating, PI3K/Akt1/p70S6K signalling pathway, resulting in the downstream activation of protein synthesis and inhibition of protein degradation in muscle cells. Many studies have demonstrated that IGF-1 is necessary and sufficient to induce hypertrophic growth in human and mouse skeletal muscle. Thus the role of IGF-1 signalling was investigated in primary muscle cultures of Callipyge sheep. I initially verified the procedures using the C2C12 immortalized mouse muscle myogenic cell line. The data clearly showed that IGF-1 treatment results in hypertrophy of myotubes in C2C12 and sheep (ovine) primary myoblast cell culture. Hypertrophy of myotubes in both cultures was apparent after desmin staining but was also reflected as an increase in the myotube size and increase in the number of myonuclei per myotube in both the C2C12 and ovine cells after IGF-1 treatment..., Thesis (Ph.D.)--University of Western Australia, 2010

Mong, Lok Yee., Thesis submitted in: December 2007., Thesis (M.Phil.)--Chinese University of Hong Kong, 2008., Includes bibliographical references (leaves 124-145)., s in English and Chinese; some text in appendix also in Chinese., Acknowledgements --- p.i, (English version) --- p.iii, (Chinese version) --- p.v, Table of Contents --- p.vii, List of Tables --- p.ix, List of Figures --- p.xi, List of Abbreviations --- p.xiii, Chapter Chapter 1 - --- Introduction, Chapter 1.1 --- Childhood obesity: a worldwide epidemic --- p.1, Chapter 1.2 --- Health consequences of childhood obesity --- p.3, Chapter 1.3 --- Determinants of childhood obesity --- p.5, Chapter 1.4 --- Hormonal dysregulation and obesity --- p.9, Chapter 1.5 --- Project objectives and long term significance --- p.14, Chapter Chapter 2 - --- Research Plan and Methodology, Chapter 2.1 --- Study cohort, Chapter 2.1.1 --- Subject recruitment --- p.15, Chapter 2.1.2 --- Ethics --- p.16, Chapter 2.1.3 --- Measurements and blood sample collections --- p.16, Chapter 2.1.4 --- Subgroup for dietary assessment --- p.18, Chapter 2.1.5 --- Cohort re-visits in 2006 --- p.19, Chapter 2.2 --- Genetic study, Chapter 2.2.1 --- Sample size estimation and research subjects --- p.21, Chapter 2.2.2 --- DNA samples --- p.22, Chapter 2.2.3 --- Candidate genes --- p.24, Chapter 2.2.4 --- SNP tagging and prioritizing --- p.25, Chapter 2.2.5 --- Genotyping methods & quality control --- p.28, Chapter 2.2.6 --- Statistical analysis --- p.31, Chapter 2.3 --- Dietary assessment, Chapter 2.3.1 --- Three-day 24-hour dietary recalls --- p.36, Chapter 2.3.2 --- Lifestyle questionnaire --- p.37, Chapter 2.3.3 --- Data management --- p.38, Chapter 2.3.4 --- Statistical methods --- p.39, Chapter Chapter 3 - --- Results Page, Chapter 3.1 --- Study cohort --- p.41, Chapter 3.2 --- Genetic study, Chapter 3.2.1 --- Subjects --- p.41, Chapter 3.2.2 --- SNPs selection --- p.41, Chapter 3.2.3 --- Factor analysis of adiposity in the study population --- p.44, Chapter 3.2.4 --- Genotyping and association testing in stage1 --- p.50, Chapter 3.2.5 --- Genotyping and association testing in stage2 --- p.52, Chapter 3.2.6 --- Association of the CART gene with adiposity --- p.55, Chapter 3.2.7 --- Association of the GHR gene with adiposity --- p.60, Chapter 3.2.8 --- Association of the GHRHR gene with adiposity --- p.69, Chapter 3.2.9 --- Association of the IGFBP3 gene with adiposity --- p.75, Chapter 3.2.10 --- Association of the POMC gene with adiposity --- p.83, Chapter 3.2 --- Dietary assessment, Chapter 3.3.1 --- Nutrient intakes of the subgroup in2004 --- p.87, Chapter 3.3.2 --- Nutrient intakes of the subgroup in2006 --- p.92, Chapter 3.3.3 --- Lifestyle pattern of the cohort in2006 --- p.97, Chapter Chapter 4 - --- Discussion, Chapter 4.1 --- The role of GH-related genes with adolescent adiposity --- p.102, Chapter 4.2 --- Nutrient intakes and lifestyle pattern of the adolescents --- p.120, Chapter 4.3 --- Conclusion of this study --- p.123, References --- p.124, Appendices, Chapter A --- Information of the SNPs selected --- p.146, Chapter B --- Comparison of SNPs minor allele frequency (MAF) among two genotyping stages and HapMap data --- p.154, Chapter C --- Hardy-Weinberg Equilibrium (HWE) of SNPs in two genotyping stages --- p.162, Chapter D --- Factor score coefficient matrix --- p.170, Chapter E --- Association of SNPs with factors scores --- p.172, Chapter F1 --- Consent form (English version) --- p.207, Chapter F2 --- Consent form (Chinese version) --- p.209, Chapter G1 --- 24-hour dietary recall forms (English version) --- p.211, Chapter G2 --- 24-hour dietary recall forms (Chinese version) --- p.218, Chapter H --- Food photo booklet --- p.225, Chapter I1 --- Lifestyle questionnaire (English version) --- p.236, Chapter I2 --- Lifestyle questionnaire (Chinese version) --- p.238, http://library.cuhk.edu.hk/record=b5893425, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Insulin-like growth factor-I (IGF-I) and its principle binding protein (IGFBP-3) are believed to play a role in mediating the anabolic effects of exercise. The purpose of this study was to assess the effect of 4 months of training on IGF-I and IGFBP-3, and to determine if changes in IGF-I or IGFBP-3 were related to changes in training status. Twelve varsity swimmers (5 males, 7 females) were tested pre-season, and again after 8 and 16 weeks of training. Measures included: VO2 max, nutritional status, athletic performance, subjective symptoms of overtraining, and serum levels of IGF-I and IGFBP-3. There was no significant change across time in VO2 max, athletic performance, IGF-I or IGFBP-3. Resting IGFBP-3 was positively correlated to symptoms of overtraining at week 0 (p=0.017), however, this relationship did not persist at week 8 or 16. These findings can not confirm that resting levels of IGF-I and IGFBP-3 are sensitive markers of training status.

Cheung Chun Kai., Thesis submitted in: December 2003., Thesis (M.Phil.)--Chinese University of Hong Kong, 2004., Includes bibliographical references (leaves 133-146)., s in English and Chinese., Acknowledgements --- p.ii, p.iv, 摘要 --- p.vii, Table of Contents --- p.ix, List of Tables --- p.xv, List of Figures --- p.xvi, List of Abbreviations --- p.xxi, Chapter Chapter 1 --- Overview --- p.1, Chapter Chapter 2 --- Literature Review --- p.3, Chapter 2.1 --- Historical background --- p.3, Chapter 2.2 --- Insulin-like growth factor --- p.5, Chapter 2.2.1 --- Structure and synthesis --- p.5, Chapter 2.2.2 --- Physiologic role and biological actions --- p.6, Chapter 2.3 --- Insulin-like growth factor binding protein-3 --- p.8, Chapter 2.3.1 --- Structure and synthesis --- p.8, Chapter 2.3.2 --- Physiologic role and biological actions --- p.8, Chapter 2.4 --- Clinical aspects --- p.10, Chapter 2.4.1 --- Metabolic effects of IGF-1 --- p.10, Chapter 2.4.1.1 --- Similarities between IGF-I and insulin --- p.11, Chapter 2.4.1.2 --- Differences between IGF-I and insulin --- p.13, Chapter 2.4.2 --- Glucose and protein metabolism --- p.14, Chapter 2.4.3 --- Therapeutic use of IGF-I --- p.15, Chapter 2.4.3.1 --- Type 1 diabetes mellitus --- p.16, Chapter 2.4.3.2 --- Type 2 diabetes mellitus --- p.17, Chapter 2.4.4 --- Side effects --- p.19, Chapter 2.5 --- World demands --- p.21, Chapter 2.5.1 --- Significance of large-scale production --- p.21, Chapter 2.5.2 --- IGF-I production --- p.21, Chapter 2.6 --- Plants as bioreactors --- p.24, Chapter 2.6.1 --- Medical molecular farming --- p.24, Chapter 2.6.2 --- Advantages of plant bioreactor --- p.24, Chapter 2.6.3 --- Commercial biopharmaceutical protein --- p.25, Chapter 2.7 --- Tobacco expression system --- p.26, Chapter 2.7.1 --- Tobacco model plant --- p.26, Chapter 2.7.2 --- Transformation methods --- p.26, Chapter 2.8 --- Hypotheses and aims of study --- p.28, Chapter Chapter 3 --- Expression of Human IGF-I and IGFBP-3 in Transgenic Tobacco --- p.30, Chapter 3.1 --- Introduction --- p.30, Chapter 3.2 --- Materials and methods --- p.31, Chapter 3.2.1 --- Chemicals --- p.31, Chapter 3.2.2 --- Plant materials --- p.31, Chapter 3.2.3 --- Bacterial strains --- p.32, Chapter 3.2.4 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.32, Chapter 3.2.5 --- Transient assay to study IGF-I or IGFBP-3 translatability --- p.39, Chapter 3.2.5.1 --- Construction of chimeric genes for particle bombardment --- p.39, Chapter 3.2.5.2 --- Particle bombardment of GUS fusion constructs --- p.42, Chapter 3.2.6 --- Construction of chimeric genes for tobacco transformation --- p.44, Chapter 3.2.6.1 --- Construction of chimeric genes with different promoters --- p.44, Chapter 3.2.6.1.1 --- Construction of chimeric gene with CaMV 35S promoter --- p.44, Chapter 3.2.6.1.2 --- Construction of chimeric genes with phaseolin promoter --- p.46, Chapter 3.2.6.2 --- Construction of fusion constructs --- p.48, Chapter 3.2.6.2.1 --- Construction of GUS fusion constructs --- p.48, Chapter 3.2.6.2.2 --- Construction of LRP fusion constructs --- p.51, Chapter 3.2.6.3 --- Construction of phaseolin targeting constructs --- p.56, Chapter 3.2.6.3.1 --- Construction of phaseolin targeting constructs without AFVY --- p.56, Chapter 3.2.6.3.2 --- Construction of phaseolin targeting constructs with AFVY --- p.60, Chapter 3.2.6.4 --- Cloning of chimeric genes into Agrobacterium binary vector pBI 121 --- p.64, Chapter 3.2.7 --- Confirmation of sequencing fidelity of chimeric genes --- p.66, Chapter 3.2.8 --- Transformation of Agrobacterium by electroporation --- p.66, Chapter 3.2.9 --- Transformation of tobacco --- p.67, Chapter 3.2.10 --- Selection and regeneration of transgenic tobacco --- p.67, Chapter 3.2.11 --- GUS assay --- p.68, Chapter 3.2.12 --- Extraction of leaf genomic DNA --- p.68, Chapter 3.2.13 --- PCR of genomic DNA --- p.69, Chapter 3.2.14 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.69, Chapter 3.2.15 --- Southern blot analysis --- p.70, Chapter 3.2.16 --- Extraction of total RNA from leaves or developing seeds --- p.70, Chapter 3.2.17 --- Northern blot analysis --- p.71, Chapter 3.2.18 --- Extraction of total protein --- p.71, Chapter 3.2.19 --- Tricine SDS-PAGE --- p.72, Chapter 3.2.20 --- Western blot analysis --- p.72, Chapter 3.2.21 --- Enterokinase digestion of fusion protein --- p.73, Chapter Chapter 4 --- Results --- p.74, Chapter 4.1 --- Particle bombardment for transient assay --- p.74, Chapter 4.1.1 --- Construction of GUS fusion genes for particle bombardment --- p.74, Chapter 4.1.2 --- Transient expression of GUS fusion genes in soybean cotyledons and tobacco leaves --- p.76, Chapter 4.2 --- Construction of chimeric genes for tobacco transformation --- p.78, Chapter 4.3 --- "Tobacco transformation, selection and regeneration" --- p.81, Chapter 4.4 --- Detection of GUS activity --- p.83, Chapter 4.5 --- Detection of transgene integration --- p.84, Chapter 4.5.1 --- Extraction of genomic DNA and PCR --- p.84, Chapter 4.5.2 --- Southern blot analysis --- p.88, Chapter 4.6 --- Detection of transgene transcription --- p.92, Chapter 4.6.1 --- Extraction of total RNA --- p.92, Chapter 4.6.2 --- Northern blot analysis --- p.92, Chapter 4.7 --- Detection of transgene translation --- p.99, Chapter 4.7.1 --- Extraction of total protein and Tricine SDS-PAGE --- p.99, Chapter 4.7.2 --- Western blot analysis --- p.102, Chapter 4.7.3 --- Enterokinase digestion of fusion protein --- p.109, Chapter Chapter 5 --- Discussion --- p.111, Chapter 5.1 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.114, Chapter 5.2 --- Transient expression of IGF-I and IGFBP-3 cDNAs --- p.116, Chapter 5.3 --- Fusion of IGF-I and IGFBP-3 cDNA with LRP gene --- p.118, Chapter 5.4 --- Enterokinase digestion --- p.120, Chapter 5.5 --- Phaseolin targeting signal --- p.122, Chapter 5.6 --- Gene silencing --- p.124, Chapter 5.7 --- Future perspectives --- p.128, Chapter Chapter 6 --- Conclusion --- p.131, References --- p.133, http://library.cuhk.edu.hk/record=b5892009, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Tse Chui-ling., "July 2004.", Thesis (Ph.D.)--Chinese University of Hong Kong, 2004., Includes bibliographical references (p. 172-185)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., Mode of access: World Wide Web., http://library.cuhk.edu.hk/record=b6073681, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

by Vong Puinga Queenie Maria., "August 2002.", Thesis (Ph.D.)--Chinese University of Hong Kong, 2002., Includes bibliographical references (p. 176-194)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., Mode of access: World Wide Web., s in English and Chinese., http://library.cuhk.edu.hk/record=b6073497, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Most of the insulin-like growth factors (IGFs) in adult rat or human plasma circulate in 150-kDa heterotrimeric complexes with IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). These 150-kDa complexes are not present, however, in rat serum at birth. As ALS is the critical determinant in the formation of the 150-kDa complexes in adult rat serum, the present study asks whether the absence of 150-kDa complexes in fetal rat serum results from a low abundance of ALS. We report that ALS mRNA is expressed in term fetal rat liver at 30% of the levels in adult liver, that radioiodinated rat ALS is not proteolyzed by incubation with fetal rat serum, and that sufficient functional ALS is present in fetal rat serum to form 150- kDa complexes with recombinant human IGFBP-3. These results indicate that the low levels of 150-kDa complexes in perinatal rat serum are not due to low circulating levels of ALS.

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the p

Insulin-like growth factor (IGF) -I and -II play a significant role in determining a cell's fate, being involved in cellular growth and differentiation, tumourigenesis and as survival factors against apoptosis. These diverse biological actions of IGFs are mediated by their interaction with the type I IGF receptor (IGF-IR), with their availability to this receptor mediated by IGF binding proteins (IGFBPs) present within the extracellular compartments. The IGF-I and -II genes synthesise multiple mRNAs by alternate promoter usage and alternative splicing. This potentially contributes to the diverse biological actions of IGF-I and -II. The aim of this doctoral study was to establish whether the differential expression of the IGF-I mRNAs, and to a lesser extent IGF-Il mRNAs, is a mechanism which determines their particular biological function. Using the IGF-I expressing human neuroblastoma cell line SK-N-MC and the non-expressing SK-N-SH cell line, I investigated the pattern of mRNA expression of IGF-I, IGF-II, the IGF-IR and the six IGFBPs, during growth and differentiation in vitro ( +1- foetal+ bovine serum). This study has identified that the four major IGF-I mRNAs, the IGF-IR and IGFBP-2, -4, -5 and -6 are expressed by the SK-N-MC cells, while IGF-II, its Ser-29 variant and the IGF-IR mRNAs were expressed by the SK-N-SH cells. During cell growth, the expression of all four IGF-I mRNAs and the IGF-IR increased, with no change in the pattern of expression over time, suggesting that these mRNAs each have a consistent and coordinate role during cell growth. Differentiation of the SK-N-MC cells was stimulated by the removal of exogenous growth factors, marked by a change in morphology and a drop in plasminogen activator activity. Of the IGF-I mRNAs, only class I Eb was expressed at detectable levels and increased with proliferation. IGF-IR mRNA was also detected in these cells, as well as an mRNA coding for a potential novel IGF-IR mRNA lacking the cysteine binding domai

The baculovirus p35 protein is a potent inhibitor of programmed cell death induced by a variety of stimuli in insects, nematodes, and mammalian cell lines. The broad ability of p35 in preventing apoptosis has led us to investigate its effect on mouse embryo fibroblasts in vitro and in vivo. For this purpose, we have used R- cells (3T3-like fibroblasts derived from mouse embryos with a targeted disruption of the insulin-like growth factor I receptor (IGF-IR) genes) and R508 cells (derived from R- and with 15 x 10(3) IGF-IRs per cell). Both cell lines grow normally in monolayer, but they do not form colonies in soft agar, and they are non-tumorigenic in nude mice. We show here that, in addition to its anti-apoptotic effect, p35 causes transformation of R508 cells, as evidenced by the following: 1) decreased growth factor requirements, 2) ability to form foci in monolayer and colonies in soft agar, and 3) ability to form tumors in nude mice. Since R- cells stably transfected with p35 do not transform, our observations suggest that in addition to its effect as an inhibitor of apoptosis, the baculovirus p35 protein has transforming potential that requires the presence of the IGF-IR. The possibility that these two properties could be separated was confirmed by demonstrating that R508 cells expressing another anti-apoptotic protein, Bcl-2, could not form tumors in nude mice.

by Yu Wai Fu, Jason., Thesis (M.Phil.)--Chinese University of Hong Kong, 1997., Includes bibliographical references (leaves 112-120)., ACKNOWLEDGMENTS --- p.i, p.ii, TABLE OF CONTENTS --- p.iii, Chapter CHAPTER 1 --- INTORDUCTION --- p.1, Chapter 1.1 --- General Introduction --- p.1, Chapter 1.2 --- Historical Overview --- p.3, Chapter 1.2.1 --- Insulin-Like Growth Factors I and II --- p.3, Chapter 1.2.2 --- IGF Receptors --- p.5, Chapter 1.2.3 --- IGF Binding Proteins --- p.7, Chapter 1.3 --- Origin and Production of IGFs --- p.7, Chapter 1.3.1 --- The Hypothalamo-Pituitary-GH-IGF-I Axis --- p.7, Chapter 1.3.2 --- Factors Regulating IGF production --- p.9, Chapter 1.3.3 --- Expression of IGFs in the Central Nervous System --- p.11, Chapter 1.4 --- Actions of IGFs --- p.12, Chapter 1.4.1 --- Insulin-like Metabolic Effects --- p.12, Chapter 1.4.2 --- Mitogenic Effects --- p.13, Chapter 1.4.3 --- Effects on Differentiation --- p.13, Chapter 1.4.4 --- IGFs in Reproductive System --- p.14, Chapter 1.4.5 --- IGF Actions in the Central Nervous System --- p.14, Chapter 1.5 --- Transgenic and Knockout Animal Models for IGFs --- p.15, Chapter 1.6 --- Molecular Biology of IGFs --- p.17, Chapter 1.6.1 --- Structure of the IGF Genes --- p.17, Chapter 1.6.2 --- Expression of IGF Genes --- p.21, Chapter 1.6.3 --- Regulation of IGF Gene Expression --- p.23, Chapter 1.7 --- IGF Receptors --- p.24, Chapter 1.7.1 --- IGF-I Receptor --- p.24, Chapter 1.7.2 --- IGF-II Receptor --- p.26, Chapter 1.8 --- IGFBPs --- p.26, Chapter 1.9 --- Teleost IGFs --- p.28, Chapter 1.9.1 --- The GH-IGF-Axis in Teleost --- p.28, Chapter 1.9.2 --- Osmoregulation and Other Biological Actions of IGFin Teleost --- p.29, Chapter 1.9.3 --- Molecular Biology of IGFs in Teleost --- p.30, Chapter 1.9.4 --- IGFBPs and IGF Receptors in Teleost --- p.31, Chapter 1.10 --- Rationale and Aim of the Present Study --- p.32, Chapter CHAPTER 2 --- SEARCH OF IGF-I PROMOTER BY GENOMIC DNA POLYMERASE CHAIN REACTION --- p.34, Chapter 2.1 --- Introduction --- p.34, Chapter 2.2 --- Materials --- p.35, Chapter 2.3 --- Methods --- p.39, Chapter 2.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.39, Chapter 2.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.40, Chapter 2.3.3 --- Polymerase Chain Reaction --- p.40, Chapter 2.3.3.1 --- Ligation of the Cassette to Digested Genomic DNA --- p.40, Chapter 2.3.3.2 --- Amplification by PCR --- p.40, Chapter 2.3.4 --- Agarose Gel Electrophoresis --- p.42, Chapter 2.3.5 --- Gene Clean Using Sephaglas´ёØ BandPrep Kit (Pharamica) --- p.42, Chapter 2.3.6 --- Cloning of PCR Products --- p.43, Chapter 2.3.7 --- Transformation of Competent Cell (Heat Shock Method) --- p.43, Chapter 2.3.8 --- Small Scale Alkaline Preparation of Plasmid DNA --- p.44, Chapter 2.3.9 --- Restriction Enzyme Digestion to Release the Insert --- p.45, Chapter 2.3.10 --- Large Scale Plasmid Preparation of the Positive Clone --- p.45, Chapter 2.3.11 --- DNA Sequencing of the Positive Clone Using the T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.46, Chapter 2.4 --- Results and Discussion --- p.49, Chapter CHAPTER 3 --- ISOLATION OF GENOMIC CLONES CARRYING THE IGF-I GENE --- p.55, Chapter 3.1 --- Introduction --- p.55, Chapter 3.2 --- Materials --- p.56, Chapter 3.3 --- Methods --- p.58, Chapter 3.3.1 --- Preparation of the Plating Host Cells --- p.58, Chapter 3.3.2 --- Phage Titering --- p.58, Chapter 3.3.3 --- Primary Screening of Common Carp Genomic Library --- p.59, Chapter 3.3.4 --- Preparation of Radioactive Nucleic Acid Probes --- p.60, Chapter 3.3.5 --- Purification of the Positive Clones --- p.60, Chapter 3.3.6 --- Purification of DNA from Lambda Phage Using Sephaglas´ёØ PhagePrep Kit (Pharmacia) --- p.61, Chapter 3.3.7 --- Restriction Enzyme Digestion Release of Inserts --- p.62, Chapter 3.3.8 --- Capillary Transfer of DNA to Nylon Membrane Under Alkaline Condition --- p.62, Chapter 3.3.9 --- Southern Analysis of the 10 Positive Clones --- p.63, Chapter 3.3.10 --- Restriction Mapping of the Clone P1 --- p.64, Chapter 3.3.11 --- Subcloning of the Fragments of the Clone PI into Plasmid Vector --- p.64, Chapter 3.3.12 --- IGF-I Specific PCR --- p.64, Chapter 3.3.13 --- Amplification of Introns from the Clone P1 Using PCR --- p.67, Chapter 3.4 --- Results and Discussion --- p.70, Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION --- p.83, Chapter 4.1 --- Introduction --- p.83, Chapter 4.2 --- Materials --- p.85, Chapter 4.3 --- Methods --- p.86, Chapter 4.3.1 --- Administration of Hormones --- p.86, Chapter 4.3.1.1 --- Injection Time Course1 --- p.86, Chapter 4.3.1.2 --- Injection Time Course2 --- p.86, Chapter 4.3.2 --- Total RNA Extraction --- p.87, Chapter 4.3.2.1 --- Rapid RNA Isolation --- p.87, Chapter 4.3.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.88, Chapter 4.3.4 --- Rapid Isolation of PolyA+ mRNA from Total RNA --- p.89, Chapter 4.3.5 --- IGF-I Specific RT-PCR --- p.90, Chapter 4.4 --- Results and Discussion --- p.92, Chapter CHAPTER 5 --- SEARCH FOR IGF-II GENE USING GENOMIC SOUTHERN BLOT ANALYSIS --- p.101, Chapter 5.1 --- Introduction --- p.101, Chapter 5.2 --- Materials --- p.103, Chapter 5.3 --- Methods --- p.104, Chapter 5.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.104, Chapter 5.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.104, Chapter 5.3.3 --- Southern Blotting of the Digested Genomic DNA --- p.104, Chapter 5.3.4 --- Preparation of the Trout IGF-II Specific Probe --- p.104, Chapter 5.3.5 --- Genomic Southern Hybridization --- p.105, Chapter 5.4 --- Results and Discussion --- p.106, Chapter CHAPTER 6 --- GENERAL DISCUSSION AND CONCLUSION --- p.109, REFERENCES --- p.112, http://library.cuhk.edu.hk/record=b5895734, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

by Liang Yiu-hon., Thesis (M.Phil.)--Chinese University of Hong Kong, 1995., Includes bibliographical references (leaves 104-117)., ACKNOWLEDGMENTS --- p.i, p.ii, ABBREVIATIONS --- p.iii, AMINO ACIDS SHORTHAND --- p.v, TABLE OF CONTENTS --- p.vi-x, Chapter CHAPTER 1 --- INTRODUCTION, Chapter 1.1 --- General Introduction --- p.1, Chapter 1.2 --- The Discovery of IGFs --- p.1, Chapter 1.3 --- The Growth Promoting Actions of IGFs --- p.3, Chapter 1.4 --- Molecular Biology of IGFs in Mammals --- p.6, Chapter 1.4.1 --- IGF Genes and Transcripts --- p.6, Chapter 1.4.2 --- Regulation of IGF Gene Expression --- p.8, Chapter 1.5 --- IGF Binding Proteins --- p.11, Chapter 1.5.1 --- Regulation of IGF Action by IGF Binding Proteins --- p.11, Chapter 1.6 --- The Insulin and IGF Receptors --- p.13, Chapter 1.6.1 --- IGF-I Receptor --- p.13, Chapter 1.6.2 --- IGF-II Receptor --- p.13, Chapter 1.6.3 --- Insulin/IGF-I Hybrid Receptor --- p.15, Chapter 1.7 --- IGF in Mammalian Fetal Growth --- p.17, Chapter 1.8 --- The Role of IGFs in Fish --- p.19, Chapter 1.9 --- Aims of the Present Study --- p.26, Chapter CHAPTER 2 --- GENERAL METHODOLOGY, Chapter 2.1 --- Materials --- p.27, Chapter 2.2 --- Methods --- p.32, Chapter 2.2.1 --- Gene Clean --- p.32, Chapter 2.2.1A --- Gene Clean by Glassmilk Method --- p.32, Chapter 2.2.1B --- Gene Clean by Sephaglas´ёØ BandPrep Kit --- p.32, Chapter 2.2.2 --- Preparation of Radioactive Nucleic Acid Probes --- p.33, Chapter 2.2.3 --- Sephadex G-50 Spun-column Chromatography --- p.33, Chapter 2.2.4 --- Small Scale Alkali Preparation of Plasmid DNA --- p.34, Chapter 2.2.5 --- Large Scale Preparation of Plasmid DNA 36 - using Wizard Maxiprep Kit (Promega), Chapter 2.2.6 --- DNA Sequencing using T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.37, Chapter 2.2.7 --- Restriction Enzyme Digestion --- p.38, Chapter 2.2.8 --- Agarose Gel Electrophoresis --- p.39, Chapter 2.2.9 --- Dephosphorylation of Linearized Plasmid DNA --- p.39, Chapter 2.2.10 --- Ligation of Foreign DNA with Linearized Plasmid --- p.40, Chapter 2.2.11 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.40, Chapter 2.2.12 --- Blotting : Transfer of DNA to Nylon Membrane --- p.41, Chapter 2.2.12A --- Capillary Transfer of DNA to Nylon Membrane in 10X SSC --- p.41, Chapter 2.2.12B --- Capillary Transfer of DNA to Nylon Membrane under Alkaline Condition --- p.42, Chapter CHAPTER 3 --- SCREENING OF COMMON CARP LIVER cDNA LIBRARY, Chapter 3.1 --- Introduction --- p.44, Chapter 3.2 --- Materials and Methods --- p.45, Chapter 3.2.1 --- Materials --- p.45, Chapter 3.2.2 --- Methods --- p.48, Chapter 3.2.2.1 --- Preparation of the Plating Host --- p.48, Chapter 3.2.2.2 --- Phage Titering --- p.48, Chapter 3.2.2.3 --- Primary Screening of Common Carp Liver cDNA Library --- p.48, Chapter 3.2.2.4 --- Purification of the Positive Clone --- p.49, Chapter 3.2.2.5 --- Checking the Insert Size of the Positive Clone --- p.50, Chapter 2.2.2.6 --- In vivo Excision to Release Phagemid from the Phage vector --- p.51, Chapter 3.2.2.7 --- Plasmid Minipreparation of the Positive Clone --- p.52, Chapter 3.2.2.8 --- Restriction Enzyme Digestion to Release the Insert --- p.52, Chapter 3.2.2.9 --- Large Scale Plasmid Preparation of the Positive Clone --- p.53, Chapter 3.2.2.10 --- DNA Sequencing of the Positive Clone --- p.53, Chapter 3.2.2.11 --- Restriction Mapping of the Positive Clone --- p.53, Chapter 3.2.2.12 --- Subcloning of the Positive Clone into Plasmid Vectors --- p.53, Chapter 3.2.2.13 --- DNA Sequencing of the Subclones --- p.54, Chapter 3.3 --- Results and Discussion --- p.55, Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION, Chapter 4.1 --- Introduction --- p.70, Chapter 4.2 --- Materials and Methods --- p.71, Chapter 4.2.1 --- Materials --- p.71, Chapter 4.2.2 --- Methods --- p.72, Chapter 4.2.2.1 --- Tissue Preparation --- p.72, Chapter 4.2.2.2 --- Total RNA Extraction --- p.72, Chapter 4.2.2.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.73, Chapter 4.2.2.4 --- First Strand cDNA Synthesis --- p.74, Chapter 4.2.2.5 --- IGF-I Specific PCR --- p.75, Chapter 4.2.2.6 --- Preparation of Carp IGF Conserve Region --- p.75, Chapter 4.2.2.7 --- Southern Hybridization of PCR Products --- p.76, Chapter 4.3 --- Results and Discussion --- p.76, Chapter CHAPTER 5 --- GENOMIC SOUTHERN ANALYSIS, Chapter 5.1 --- Introduction --- p.80, Chapter 5.2 --- Materials and Methods --- p.81, Chapter 5.2.1 --- Materials --- p.81, Chapter 5.2.2 --- Methods --- p.82, Chapter 5.2.2.1 --- Preparation of Genomic DNA from Carp Testis --- p.82, Chapter 5.2.2.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.82, Chapter 5.2.2.3 --- Southern Blotting of the Digested Genomic DNA --- p.83, Chapter 5.2.2.4 --- Preparation of the Carp IGF-I Specific Probe --- p.83, Chapter 5.2.2.5 --- Genomic Southern Hybridization --- p.83, Chapter 5.3 --- Results and Discussion --- p.84, Chapter CHAPTER 6 --- THE SEARCH OF OTHER IGF cDNA SUBTYPES IN COMMON CARP, Chapter 6.1 --- Introduction --- p.88, Chapter 6.2 --- Materials and Methods --- p.89, Chapter 6.2.1 --- Materials --- p.89, Chapter 6.2.2 --- Methods --- p.91, Chapter 6.2.2.1 --- Screening using a Conserve Region cDNA Probe of Carp IGF-I --- p.91, Chapter 6.2.2.2 --- PCR using IGF-I Specific Primers --- p.92, Chapter 6.2.2.3 --- PCR Using T3 and T7 Primers --- p.92, Chapter 6.2.2.4 --- Southern Blot Analysis of T3 and T7 PCR Products of cDNA Insert --- p.93, Chapter 6.2.2.5 --- DNA Sequencing of Positive Clones --- p.94, Chapter 6.3 --- Results and Discussion --- p.94, Chapter CHAPTER 7 --- GENERAL DISCUSSION AND CONCLUSION --- p.99, REFERENCES --- p.104-117, http://library.cuhk.edu.hk/record=b5895591, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

not available

By Pong Tsang Wai Hai., Acknowledgements --- p.i, p.ii, Abbreviations --- p.v, Chapter Chapter 1 --- Introduction, Chapter 1.1 --- Biological functions and structure of GH --- p.1, Chapter 1.2 --- Application of recombinant GH --- p.2, Chapter 1.3 --- Expression of eukaryotic gene in E.coli --- p.4, Chapter 1.4 --- Methods for increasing expression of a cloned gene --- p.6, Chapter 1.4.1 --- Changing the 5' end codons of the cDNA to E.coli preferred codons --- p.6, Chapter 1.4.2 --- Optimization of distance between SD sequence and the initiation codons --- p.6, Chapter 1.4.3 --- "Construction of a short ""dummy"" cistron at the 5' end of the cloned gene to improve attachment of ribosome"--Page 7, Chapter 1.4.4 --- Increasing the copy number of recombinant expression plasmid --- p.8, Chapter 1.4.5 --- Optimizing high density cell expression --- p.9, Chapter 1.5 --- Quantitating the expression of cloned gene --- p.10, Chapter 1.6 --- Inclusion bodies formation --- p.11, Chapter 1.7 --- The purification of eukaryotic polypeptides synthesized as inclusion bodies --- p.12, Chapter 1.7.1 --- Solubilization of the inclusion bodies --- p.13, Chapter 1.7.2 --- Refolding the polypetides and disulfide bond formation --- p.13, Chapter 1.8 --- Expression of secreted recombinant protein --- p.14, Chapter 1.9 --- Purpose of present study --- p.15, Chapter Chapter 2 --- Materials and Methods, Chapter 2.1 --- General techniques --- p.16, Chapter 2.1.1 --- Chemical Synthesis of DNA linkers and primers --- p.16, Chapter 2.1.2 --- Manipulation of DNA --- p.16, Chapter 2.1.3 --- Electro-elution of DNA from Agarose Gel --- p.17, Chapter 2.1.4 --- Preparation of Competent Cells and Transformation --- p.18, Chapter 2.1.5 --- Screening of the Expressed Clones --- p.19, Chapter 2.1.6 --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.21, Chapter 2.1.7 --- Western blot analysis --- p.21, Chapter 2.2 --- Purification procedures --- p.23, Chapter 2.2.1 --- Growing up the cells in large scale --- p.23, Chapter 2.2.2 --- Harvesting of cells from large scale culture --- p.23, Chapter 2.2.3 --- Sonication of the cells --- p.24, Chapter 2.2.4 --- Washing of the inclusion body --- p.24, Chapter 2.2.5 --- Solubilization of the inclusion bodies --- p.25, Chapter 2.2.6 --- Renaturation of r-gcGH --- p.26, Chapter 2.2.6.1 --- Step down dilution mehtod --- p.26, Chapter 2.2.6.2 --- Rapid dilution method --- p.26, Chapter 2.2.7 --- Separation by reverse phase chromatography --- p.27, Chapter 2.2.7.1 --- Octadodecylsilica (ODS) column separation --- p.27, Chapter 2.2.7.2 --- Fast performance Liquid Chromatography(FPLC) --- p.28, Chapter 2.3 --- Characterization methods --- p.29, Chapter 2.3.1 --- Radioimmunoassay --- p.29, Chapter 2.3.1.1 --- Iodination of r-gcGH --- p.29, Chapter 2.3.1.2 --- Binding assay --- p.31, Chapter 2.3.2 --- Preparation of anti-r-gcGH serum --- p.32, Chapter 2.3.3 --- Determination of amino acid composition and N-terminal sequence of r-gcGH --- p.32, Chapter Chapter 3 --- Results, Chapter 3.1 --- Recombinant plasmids construction --- p.34, Chapter 3.1.1 --- Basic construction of plasmid producing gcGH in E.coli --- p.34, Chapter 3.1.2 --- N-terminal modification of gcGH cDNA --- p.38, Chapter 3.1.3 --- Construction of a short 'dummy' cistron at the 5'end of gcGH cDNA --- p.40, Chapter 3.1.4 --- Optimization of distance between ribosomal binding site and initiation codon --- p.42, Chapter 3.1.5 --- Increasing expression level by increasing plasmid copy number --- p.44, Chapter 3.1.6 --- Optimizing the high density expression by changing the promoter --- p.48, Chapter 3.1.7 --- Construction of excretion plasmid for gcGH production from E. coli --- p.48, Chapter 3.2 --- Quantitation and qualitation of the expressed protein --- p.51, Chapter 3.3 --- Effect of IPTG on induction of r-gcGH in pp5 --- p.57, Chapter 3.4 --- Stability of overproducing strain pp5 during continuous culture --- p.59, Chapter 3.5 --- Stability of overproducing strain ppADH4 during continuous culture --- p.61, Chapter 3.6 --- "Optimization of culture condition for high level expression strains, pp5 and ppADH4"--Page 64, Chapter 3.7 --- Purification of r-gcGH --- p.67, Chapter 3.7.1 --- Distribution of r-gcGH as Soluble and insoluble protein in E. coli --- p.67, Chapter 3.7.2 --- Isolation and cleaning of the inclusion bodies --- p.69, Chapter 3.7.3 --- Solubilization and renaturation of r-gcGH --- p.71, Chapter 3.7.4 --- Purification of r-gcGH by chromatography --- p.73, Chapter 3.8 --- Characterization of r-gcGH --- p.78, Chapter 3.8.1 --- Amino acid composition and N-terminal sequence determination --- p.78, Chapter 3.8.2 --- Immunological property of r-gcGH --- p.81, Chapter 3.8.3 --- Physical Property of r-gcGH --- p.84, Chapter 3.8.4 --- Stability of r-gcGH --- p.84, Chapter 3.9 --- Expression and purification of r-gcGH in excretion vector ppSP14 --- p.86, Chapter Chapter 4 --- Discussion, Chapter 4.1 --- Evaluation of expression strains --- p.88, Chapter 4.1.1 --- Strain pKgcGH2 --- p.88, Chapter 4.1.2 --- Strain pKgcGH2-17 --- p.88, Chapter 4.1.3 --- Strain pSD78 --- p.89, Chapter 4.1.4 --- "Strains pLl, pL2 and pL4"--Page 90, Chapter 4.1.5 --- "Strains pp5,pplA, pp2I and pp4Q"--Page 90, Chapter 4.1.6 --- Strain ppADH4 --- p.91, Chapter 4.1.7 --- Strain ppSP14 --- p.91, Chapter 4.2 --- Disulfide bond formation during refolding process --- p.92, Chapter 4.2.1 --- Renaturaion in the presence of L-arginine and thiol reagent in oxidized form --- p.93, Chapter 4.2.2 --- Renaturation in the presence of thiol reagent and 3M guanidine hydrochloride --- p.94, Chapter 4.3 --- Stability of the r-gcGH --- p.94, Chapter 4.4 --- Further studies --- p.96, References --- p.98, Http://library.cuhk.edu.hk/record=b5887816, Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

The concentration of insulin-like growth factor I (IGF-I) in tissue taken from human non-small cell lung carcinoma (NSCLC) is 1.4- to 7-fold higher than the concentration of IGF-I in the surrounding normal lung tissue and therefore IGF-I may be involved in the growth of NSCLC. In this study it was determined that NSCLC cell lines (A549, A427, SK-LU-1) expressed the type I IGF-I receptor protein and IGF-I stimulated the proliferation of low density plated (2000 cells/cm² growth area) carcinoma cells by 1.6- to 3- fold above control after a four day inCUbation period under serum-free conditions (A549, A427) or in the presence of 0.25% serum (SK-LU-1). In addition, when added to detergent-solubilized type I IGF receptors from A549 cells, IGF-I stimulated [1] a dose-dependent increase in the autophosphorylation of the type I IGF receptor, and [2] a dose-dependent increase (1.5- to 4-fold) in the phosphorylation of a tyrosine kinase-specific substrate. These results suggest that the growth promoting activity of IGF-I for the lung carcinoma cells was mediated through the activation of the type I IGF receptor.

by Agnes Pui-Yee Chan., Thesis (M.Phil.)--Chinese University of Hong Kong, 1992., Includes bibliographical references (leaves 162-177)., ACKNOWLEDGEMENTS --- p.i, p.ii, TABLE OF CONTENTS --- p.iv, ABBREVIATIONS --- p.vii, Chapter CHAPTER 1 --- INTRODUCTION, Chapter 1.1 --- Physiology of growth --- p.3, Chapter 1.2 --- The anterior pituitary --- p.4, Chapter 1.3 --- Chemistry of GH and the GH gene family --- p.7, Chapter 1.4 --- Biochemical effects and mode of action of GH --- p.8, Chapter 1.5 --- Control of GH at cellular level --- p.10, Chapter 1.6 --- Control of GH gene expression at molecular level, Chapter 1.6.1 --- Introduction --- p.11, Chapter 1.6.2 --- Tissue-specific expression of GH gene, Chapter 1.6.2.1 --- Tissue-specific transcription factors of pituitary cells --- p.20, Chapter 1.6.2.2 --- Non-tissue specific transcription factors of pituitary cells --- p.27, Chapter 1.6.2.3 --- Negatively-acting transcription factors of non-pituitary cells --- p.34, Chapter 1.6.2.4 --- Theory for tissue-specific GH gene activation --- p.39, Chapter 1.7 --- Characteristic of growth in fish --- p.40, Chapter 1.8 --- Objectives of the present study --- p.42, Chapter CHAPTER 2 --- MATERIALS AND METHODS, Chapter 2.1 --- General techniques, Chapter 2.1.1 --- Preparation of DNA, Chapter 2.1.1.1 --- Minipreparation of DNA --- p.46, Chapter 2.1.1.2 --- Preparation of DNA using Qiagen column --- p.47, Chapter 2.1.1.3 --- Preparation of phage DNA --- p.48, Chapter 2.1.2 --- Elution of DNA from agarose gel --- p.51, Chapter 2.1.3 --- Preparation of competence cells and transformation --- p.52, Chapter 2.1.4 --- Ligation of DNA fragments --- p.53, Chapter 2.1.5 --- Cell feeding and subculturing --- p.54, Chapter 2.2 --- Special techniques, Chapter 2.2.1 --- DNA sequencing --- p.56, Chapter 2.2.2 --- Polymerase chain reaction (PCR) --- p.67, Chapter 2.2.3 --- Direct sequencing of PCR products --- p.72, Chapter 2.2.4 --- Nested-deletion --- p.75, Chapter 2.2.5 --- DNA transfection --- p.81, Chapter 2.2.6 --- CAT assay --- p.86, Chapter CHAPTER 3 --- RESULTS, Chapter 3.1 --- Sequencing of the grass carp GH gene, Chapter 3.1.1 --- Introduction --- p.93, Chapter 3.1.2 --- Sequencing strategy --- p.94, Chapter 3.2 --- Sequence analysis of the grass carp GH gene --- p.108, Chapter 3.3 --- Functional analysis of the grass carp GH gene --- p.115, Chapter CHAPTER 4 --- DISCUSSIONS, Chapter 4.1 --- DNA sequence comparison between grass carp GH gene and other organisms --- p.137, Chapter 4.2 --- Amino acid comparisons between grass carp GH and other organisms --- p.143, Chapter 4.3 --- Tissue-specific expression of GH gene, Activation of transcription --- p.154, Repression of transcription --- p.155, Chapter 4.4 --- Electroporation of zebrafish eggs --- p.157, Chapter 4.5 --- Further studies --- p.160, REFERENCES --- p.162, APPENDIX --- p.178, http://library.cuhk.edu.hk/record=b5886992, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Exogenous corticoids are known to be potent inhibitors of linear growth in children. We investigated the mechanisms underlying growth failure by evaluating growth hormone (GH) release during short-term high-dose prednisone treatment (40 mg/m2/day given orally in 3 divided doses) and 7 days after steroid withdrawal in 7 prepubertal children (4 males, 3 females, age range 3-12 years), affected by acute lymphoblastic leukemia. Patients also received weekly administrations of vincristine (1.5 mg/m2 i.v.), daunomycin (20 mg/m2 i.v.) and L-asparaginase (6,000 IU/m2 i.m.). Corticoid therapy suppressed GH secretion during deep sleep as well as in response to arginine, insulin and GH-releasing hormone (GHRH) administration. A significant recovery of GH responsiveness after drug discontinuation was observed during deep sleep (14.03 +/- 3.47 vs. 1.49 +/- 0.43 ng/ml, p < 0.025) as well as in response to arginine (13.63 +/- 2.73 vs. 4.95 +/- 1.54 ng/ml, p < 0.025) and GHRH (32.62 +/- 4.59 vs. 7.27 +/- 3.52 ng/ml, p < 0.005) but not to insulin (7.12 +/- 0.88 vs. 4.47 +/- 0.96 ng/ml, p = NS). Insulin-like growth factor 1 levels during deep sleep (0.61 +/- 0.13 IU/ml/min) were found to be low in the course of steroid therapy and did not increase after drug withdrawal (0.41 +/- 0.07 IU/ml/min). Our preliminary data suggest that recovery of adrenergic response to insulin does not immediately follow corticosteroid discontinuation.

by Wong Mee-wa., Thesis (M.Phil.)--Chinese University of Hong Kong, 1990., Bibliography: leaves 100-104., Contents, Abbreviations, Chapter Chapter 1 --- General Introduction --- p.1, Chapter 1.1 --- Introduction --- p.1, Chapter 1.2 --- Biological Functions and Structure of GH --- p.1, Chapter 1.3 --- Molecular Cloning of GH cDNA and its Expression --- p.3, Chapter 1.4 --- Molecular Cloning of the Genomic Sequence of Growth Hormone --- p.5, Chapter 1.4.1 --- The Isolation of Mammalian GH gene --- p.5, Chapter 1.4.2 --- Molecular Cloning of Rainbow Trout Growth Hormone Gene --- p.6, Chapter 1.4.3 --- Molecular Cloning of Atlantic Salmon Growth Hormone Gene --- p.9, Chapter 1.4.4 --- Molecular Evolution of GH gene --- p.9, Chapter 1.4.5 --- Control Elements on GH gene --- p.12, Chapter 1.4.6 --- Production of Transgenic Fish --- p.14, Chapter 1.5 --- Purpose of present Study --- p.15, Chapter Chapter 2 --- Construction of a Genomic Library --- p.17, Chapter 2.1 --- Introduction --- p.17, Chapter 2.2 --- Strategy --- p.19, Chapter 2.3 --- Materials and Methods --- p.20, Chapter 2.3.1 --- Materials --- p.20, Chapter 2.3.2 --- Procedure --- p.24, Chapter 2.3.2.1 --- Extraction of Total Genomic DMA --- p.24, Chapter 2.3.2.2 --- Southern Blotting and Hybridization --- p.25, Chapter 2.3.2.3 --- Extraction of the GH Enriched DNA Fraction --- p.28, Chapter 2.3.2.4 --- Cloning into Phage Vector Lambda GT11 --- p.29, Chapter 2.3.2.5 --- Studies on the Enriched Genomic Library --- p.31, Chapter 2.4 --- Result --- p.33, Chapter 2.5 --- Discussion --- p.43, Chapter Chapter 3 --- Screening --- p.46, Chapter 3.1 --- Strategy --- p.46, Chapter 3.2 --- Materials and Methods --- p.47, Chapter 3.2.1 --- Materials --- p.47, Chapter 3.2.2 --- Procedure --- p.48, Chapter 3.2.2.1 --- Primary Screening --- p.48, Chapter 3.2.2.2 --- Purificaiton of Positive Signal --- p.49, Chapter 3.2.2.3 --- DNA Extraction from Positive Clone --- p.49, Chapter 3.3 --- Result --- p.50, Chapter 3.4 --- Discussion --- p.58, Chapter Chapter 4 --- Studies on the Positive Clone --- p.59, Chapter 4.1 --- Introduction --- p.59, Chapter 4.2 --- Materials and Methods --- p.60, Chapter 4.2.1 --- Materials --- p.60, Chapter 4.2.2 --- Procedure --- p.62, Chapter 4.2.2.1 --- Insert DNA Preparation --- p.62, Chapter 4.2.2.2 --- Single Enzyme Digestion --- p.62, Chapter 4.2.2.3 --- Double Enzyme Digestion --- p.62, Chapter 4.2.2.4 --- Preparation of Position Specific Probes --- p.62, Chapter 4.2.2.5 --- Hybridization with Position Specific Probes --- p.64, Chapter 4.2.2.6 --- Preparation of Competent Cells --- p.65, Chapter 4.2.2.7 --- Subcloning into pUC18 --- p.65, Chapter 4.2.2.8 --- Plasmid Preparation --- p.66, Chapter 4.2.2.9 --- Sequencing --- p.67, Chapter 4.3 --- Result --- p.69, Chapter 4.4 --- Discussion --- p.78, Chapter Chapter 5 --- General Discussion --- p.80, Appendix A --- p.83, Appendix B --- p.93, References --- p.100, http://library.cuhk.edu.hk/record=b5886625, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

THIS SYMPOSIUM CONSISTS OF EIGHT ARTICLES, A PANEL DISCUSSION, 26 TABLES, AND 13 FIGURES (FOR QUICK REFERENCE). It includes articles on Cushing's disease diagnosis and treatment of acromegaly; diagnosis and followup of secretory pituitary adenomata; vitamin D; somatomedin and other growth factors; use of the computer in the management and study of diabetes mellitus; studies of the trophic properties of gastrin on the gut; and gastrin in the human small bowel. It also includes a pituitary panel discussion. (Author)

This thesis study was undertaken in order to investigate the nature and biological behavior of a somatornedin binding protein, identified in preterrn amniotic fluid (AF). Somatomedin (SM) is the generic designation applied to a family of serum peptide growth factors which are growth hormone dependent, stimulate incorporation of sulphate into cartilage, have insulin-like actions on nonskeletal tissues and increase mitosis in a wide variety of cultured cells. Thusfar two peptides have been fully characterized: insulin-like growth factor I (IGF-I), shown to be identical to sornatomedin-C, and insulin-like growth factor II (IGF-II). IGF-I and IGF-II have a 62% aminoacid sequence identity and are 38-48% homologous with the A and B domains of human pro-insulin.

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