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PDF file - 757KB, Effect of the cell growth and the intracellular signals by BGT226. (A) Growth inhibitory effect of BGT226, rapamycin and LY294002. FaDu and OECM1 cells were treated with each compound in the identical concentration-dependent manner. The result was plotted (X axis, logarithm scale of concentration; y axis, ratio). (B) Effect of BGT226 and the other PI3K/mTOR targeting agents in OECM1. In the left panel, cells were treated with BGT226 120 nM or LY294002 2μM in a time-dependent manner. In the right panel, cells were treated with various PI3K/mTOR targeting agents for 30 min before whole cell lysate collection (G, BGT226 120 nM; K, BKM120 2μM; Rad, RAD001, 0.5 or 2 nM; L, LY294002 2μM; Rapa, rapamycin 100 or 200 nM; C, control).

PDF file - 468KB, (A) The inhibitory effect of BGT226 in cetuximab-resistant Hep3B cell line. Growth inhibition assay was used in this study. After subculture, cells were treated with cetuximab or BGT226 at least three generation time. The results of cetuximab were represented as curve plot in the left panel, and the IC50 with BGT226 were shown in the right table. (B) Basal expression of PTEN in all tested cell lines. All cell lysate was extracted at the time in steady growth. (C) Combination treatment of BGT226 with radiation in FaDu cells. Clonogenic assay was applied for the cell survival study, with 400 cells implanted into each of 6-well plate. After attachment, BGT226 was administrated 2 h prior to the radiation, and then replaced with drug-free medium after 24 h. The result was then assessed after incubation for 10 days.

PDF file - 515KB, Body weight change of the mice model. The body weight was recorded before treatment for the baseline, and throughout the period of experiment. The plots represent the curve of (A) the dose-dependent BGT226 study, and (B) the comparison study of individual PI3K and mTOR inhibitors.

PDF file - 1659KB, Apoptosis and autophagy-associated reaction by BGT226. (A) Apoptotic effect of BGT226. HSC3 cells incubated with either BGT226 or cisplatin for 24 h were analyzed for apoptosis-associated protein expression. Arrows indicated the cleavage-form of protein. (B) Effect of AVOs formation in FaDu cells treated with BGT226. Cells were stained with acridine orange. The extent of staining intensity in tested cells was analyzed and plotted by flow cytometry. The left rows represented cells incubated in DMSO or 3MA. The right row showed cells that were treated with BGT226 120 nM with or without the co-treatment of 3MA. The percentage of detected cells in each quadrant is marked on the panel. (C) Evaluation of cathepsin B/D/L (Cat B/D/L) activity in FaDu cells treated with BGT226. The activity of each factor was assessed by its individual activity kit (BioVisiion, CA). For the preparation, we followed manifacturer's protocol after BGT226 treatment of the cells. The samples were then transferred to 96-well plate, with the result read using fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. The data was then compared to the control, with the ratio ploted and shown in the bar chart.

Summary Enterohaemorrhagic Escherichia coli (EHEC) causes life-threatening infections in humans as a consequence of the production of Shiga-like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga-like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen-activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1-dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.

Group B streptococcus (GBS) is a common asymptomatic colonizer in acidic vagina of pregnant women and can transmit to newborns, causing neonatal pneumonia and meningitis. Biofilm formation is often associated with bacterial colonization and pathogenesis. Little is known about GBS biofilm and the effect of environmental stimuli on their growth along with biofilm formation. The objective of this study was to investigate the survival and biofilm formation of GBS, isolated from pregnant women, in nutrient-limited medium under various pH conditions. Growth and survival experiments were determined by optical density and viable counts. Crystal violet staining, scanning electron microscopy, and atomic force microscopy (AFM) were used to analyze the capacity of biofilm production. Our results showed that GBS isolates proliferated with increasing pH with highest maximum specific growth rate (μmax) at pH 6.5, but survived at pH 4.5 for longer than 48 h. Biofilm formation of the 80 GBS isolates at pH 4.5 was significantly higher than at pH 7.0. This difference was confirmed by two other methods. The low elastic modulus obtained from samples at pH 4.5 by AFM revealed the softness of biofilm; in contrast, little or no biofilm was measured at pH 7.0. Under acidic pH, the capability of biofilm formation of serotypes III and V showed statistically significant difference from serotypes Ia and Ib. Our finding suggested that survival and enhanced biofilm formation at vaginal pH are potentially advantageous for GBS in colonizing vagina and increase the risk of vaginosis and neonatal infection.

Adhesive interactions between cells are needed to maintain tissue architecture during development, tissue renewal and wound healing. Thrombomodulin (TM) is an integral membrane protein that participates in cell-cell adhesion through its extracellular lectin-like domain. However, the molecular basis of TM-mediated cell-cell adhesion is poorly understood. Here, we demonstrate that TM is linked to the actin cytoskeleton via ezrin. In vitro binding assays showed that the TM cytoplasmic domain bound directly to the N-terminal domain of ezrin. Mutational analysis of the TM cytoplasmic domain identified (522)RKK(524) as important ezrin-binding residues. In epidermal epithelial A431 cells, TM colocalized with ezrin and actin filaments at cell-cell contacts. Knockdown of endogenous TM expression by RNA interference induced morphological changes and accelerated cell migration in A431 cells. Moreover, epidermal growth factor, upstream of ezrin activation, stimulated the interaction between ezrin and TM. In skin wound healing of mice, TM and ezrin were highly expressed in neoepidermis, implying that both proteins are key molecules in reepithelialization that requires collective cell migration of epithelial cells. Finally, exogenous expression of TM in TM-deficient melanoma A2058 cells promoted collective cell migration. In summary, TM, which associates with ezrin and actin filaments, maintains epithelial morphology and promotes collective cell migration.

Cathepsin S is a cellular cysteine protease, which is frequently over-expressed in human cancer cells and plays important role in tumor metastasis. However, the role of cathepsin S in regulating cancer cell survival and death remains undefined. The aim of this study was to determine whether targeting cathepsin S could induce autophagy/apoptosis in cancer cells. In this study, we demonstrated that targeting cathepsin S by either specific small molecular inhibitors or cathepsin S siRNA induced autophagy and subsequent apoptosis in human cancer cells, and the induction of autophagy was dependent on the phosphorylation of EGFR and activation of the EGFR-related ERK/MAPK-signaling pathway. In conclusion, the current study reveals that cathepsin S plays an important role in the regulation of cell autophagy through interference with the EGFR-ERK/MAPK-signaling pathway.

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine-5'-triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12(D197N)) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12(D197N) presented with oligoasthenozoospermia, whereas the SEPT12(T89M) patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12(D197N) patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation.

Intragastric inoculation of mice with Klebsiella pneumoniae can cause liver abscesses, necrosis of liver tissues, and bacteremia. A newly isolated phage (φNK5) with lytic activity for K. pneumoniae was used to treat K. pneumoniae infection in an intragastric model. Both intraperitoneal and intragastric administration of a single dose of φNK5 lower than 2 × 10 8 PFU at 30 min after K. pneumoniae infection was able to protect mice from death in a dose-dependent manner, but the efficacy achieved with a low dose of φNK5 by intragastric treatment provided the more significant protection. Phage φNK5 administered as late as 24 h after K. pneumoniae inoculation was still protective, while intraperitoneal treatment with phage was more efficient than intragastric treatment as a result of the dissemination of bacteria into the circulation at 24 h postinfection. Surveys of bacterial counts for mice treated with φNK5 by the intraperitoneal route revealed that the bacteria were eliminated effectively from both blood and liver tissue. K. pneumoniae -induced liver injury, such as liver necrosis, as well as blood levels of aspartate aminotransferase and alanine aminotransferase and inflammatory cytokine production, was significantly inhibited by φNK5 treatment. These data suggest that a low dose of φNK5 is a potential therapeutic agent for K. pneumoniae -induced liver infection.

BackgroundLeptin is secreted from adipose tissue and plays an important role in obesity. Recent studies have shown that the relationship between leptin and body fat mass may have ethnic differences. The purpose of our study was to investigate the relationship between venous umbilical cord plasma leptin and anthropometric markers in term healthy Taiwanese newborns.MethodsUmbilical venous plasma samples were obtained from 98 term neonates (48 males and 50 females) and leptin levels were analyzed by enzyme-linked immunosorbent assay.ResultsUmbilical cord plasma levels of leptin were significantly higher in the female neonates than in males (p < 0.001). The large-for-gestational age and appropriate- for-gestational age newborns had significantly higher leptin cord plasma levels than the small-for-gestational age newborns (p < 0.01 and p < 0.05, respectively). In both male and female neonates, umbilical leptin levels showed significant positive correlations with birth weight and birth length. Multiple linear regression analysis revealed that birth weight was the only significant predictor of umbilical cord plasma leptin levels in both male and female neonates. However, the slopes of the regressions between leptin and birth weight in male and female neonates were not different.ConclusionIn Taiwanese healthy term neonates, leptin umbilical cord plasma levels are associated with sex and birth weight of the neonate. The relationship between leptin and birth weight may differ among different ethnic groups. These findings imply that the relationship between leptin and body fat mass may develop early in life.

Gold nanoparticles have been developed for the photoacoustic imaging, delivery of genes and laser induced photothermal therapy. In this study, we have developed oligonucleotide conjugated gold nanoparticles as the carrier for simultaneous DNA and anti-cancer nucleoside delivery. The polynucleotide-nanoparticle complex presented higher capacity in carrying 5-FU anti-cancer compounds than the original gold particles. The hydrodynamic size of the gold nanoparticles increased from 25 to 35 nm with an increase in the negative surface charge from -9.58 to 21.66 mV after polynucleotide conjugation and drug loading. A positive association between environmental pH and drug release was observed in PBS, which implied their potential use in the controlled localized drug release in the lower GI tract. The MTT assay revealed dose dependent cytotoxicity to colon cancer cell line than free compounds. These results suggest the potential use of this new polynucleotide-gold nanoparticles complex as the environmental controlled anti-cancer nanocapsule, especially suitable for per oral colon cancer chemotherapy.

The objective of this study was to search for brain-specific binding proteins that participated in Abeta aggregation. Immunoprecipitation of Abeta in Alzheimer's brain homogenate revealed a major co-precipitating 16-kDa protein band, which was identified through mass spectrometry as hemoglobin (Hb) alpha and beta chains. Hemoglobin was distributed in Alzheimer's disease (AD) patients in a brain region-dependent manner, with the highest levels in the hippocampus and parietal gray (PG) matter, followed by parietal white matter (PW), and the lowest in cerebellum (Cb). AD parietal gray and white matters exhibited higher Hb levels than those in the nondemented (ND) group. Likewise, RT-PCR revealed that the Hb mRNA levels in AD inferior temporal gyri were higher than those of ND subjects. Furthermore, Hb was shown to promote Abeta oligomer formation. Immunohistochemical studies indicated that Hb was localized within the cytosol of pyramidal neurons in the hippocampus, suggesting a potential source of intracerebral Hb. Finally, double immunofluorescent assay confirmed the co-localization of Hb with senile plaques (SP) and cerebral amyloid angiopathy (CAA). We propose that an elevation in brain Hb via circulation leakage or perturbations of Hb gene regulation may participate in AD pathogenesis.

Though numerous animal thrombosis models have been introduced, an easy, reliable, and reproducible arterial thrombosis model remains a continuing challenge prior to a thrombolytic study. In an effort to evaluate the efficiency of various recombinant thrombolytic agents with specific affinity to activated platelets in vivo, we developed a novel double-opposing inverted-sutures model to create a platelet-rich thrombus in the femoral artery of rabbits. The arteriotomy was done semicircumferentially, and variously sized microsurgical sutures were introduced intraluminally in a double-opposing inverted manner. The animals were divided into three groups according to the double-opposing inverted-sutures used: Group 1 with 10-0 nylon ( n =6), Group 2 with 9-0 nylon ( n =6), and Group 3 with 8-0 nylon ( n =22). The superficial epigastric branch was cannulated with a thin polyethylene (PE) tube for intraarterial administration of the studied thrombolytic agent. The blood flow was continuously measured with a real-time ultrasonic flow meter. Within 2 h of installation of the sutures, there was no thrombus formation in either Group 1 or 2. In Group 3, the thrombosis rate was 91% (20 of 22) under a steady baseline flow (with an average of 12.23±2.40 ml/min). It was highly statistically significant with a P -value of .0000743 using Fisher's Exact Test. The averaged time to thrombosis was 21.8±9.8 min. The ultrasonic flow meter to record the dynamic real-time measurement of blood flow was a guideline for thrombus formation or dissolution, which was correlated with the morphological findings of stenotic status of the vessel detected by the Doppler sonography. The components of the thrombus were proven to be platelet-rich predominant by histological examination via hematoxylin and eosin (H&E) stain and transmission electron microscopy (TEM). To confirm that the double-opposing inverted-sutures model would be useful for a study of thrombolytic agents, we evaluated the effects of recombinant tissue-type plasminogen activator (rt-PA) and streptokinase-human plasminogen (SK-HPlg). The average time to thrombolysis post rt-PA infusion was 16.0±8.2 min and that of SK-HPlg was 79.6±23.1, which were similar to the previous reports. In conclusion, the novel double-opposing inverted-sutures (8-0 nylon) model provides a simple, reliable, and reproducible platelet-rich arterial thrombosis model with noninvasive and dynamic real-time measurement. It may be applied in assessing the efficiency of the recombinant thrombolytic agents and offers many advantages of an arterial platelet-rich in vivo thrombosis model.

The effect of a water-soluble trimalonic acid derivative of fullerene, carboxyfullerene (C63(COOH)6), on the opening of blood-brain barrier (BBB) in Escherichia coli (E. coli)-induced meningitis was tested. In E. coli-induced meningitis model, the increase of BBB permeability was manifested in two distinct phases based on cytokine expression pattern and neutrophilic infiltration in B6 mice. An early increase in BBB permeability was occurred at 1–5 h post injection that could be blocked by either anti-TNF-αor anti-IL-1β antibodies. A late one (neutrophil-associated BBB opening) was manifested at 6–12 h that was inhibited by vinblastine pretreatment. Inhibition of the early E. coli-induced BBB opening blocked the development of the late one. Furthermore, the neutrophil-associated BBB opening (the late phase), but not cytokine- induced one (the early phase), was inhibited by a water-soluble carboxyfullerene.

We have previously demonstrated that corticotropin-releasing hormone (CRH) treatment of MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of progesterone production. In view of this observation we wished to determine the effects of CRH on the synthesis of the steroidogenic acute regulatory (StAR) protein in these cells. StAR is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage enzyme. Treatment of MA-10 cells with the cAMP analogue dibutyryl cAMP (dbcAMP) resulted in a dose- and time-dependent increase in the levels of StAR protein that reached a maximum at 800 microM dbcAMP and within a time period of 6 h. Further, treatment of MA-10 cells with CRH also resulted in a dose-dependent increase in the synthesis of the StAR protein with a maximal response observed at 1 microM. Slightly different from that observed with dbcAMP, the maximal response to 1 microM CRH was seen at 4 h following stimulation. These results indicate that the observed increase in steroid production in response to CRH in MA-10 Leydig tumor cells is similar to that previously seen with trophic hormone stimulation acting through the cAMP second messenger pathway, and that it occurs as a result of an increase in the synthesis of the StAR protein.

We have identified an early-appearing intermediate filament-associated protein (IFAP-70/280 kDa) in radial glia and their immediate derivatives. This IFAP is absent in the adult CNS. In this study, we examined the reexpression of this early glial differentiation trait in rat reactive astrocytes induced by stab injury of the cerebrum. Double-label immunofluorescence microscopy demonstrated that by 36 h postlesion, IFAP-70/280 kDa was present in a few GFAP-positive astrocytes in the area adjacent to the wound. As the gliotic reaction progressed, the number of IFAP-positive reactive astrocytes increased and by 5–6 days postlesion, IFAP-70/280 kDa was present in most of the hypertrophied astrocytes in tissue immediately adjacent to the wound. By 8 days postlesion, while the number of IFAP-negative reactive astrocytes away from the wound diminished, the IFAP-containing reactive astrocytes close to the wound persisted. Concurrently, they began to change from a stellate form to an elongated shape, with their longitudinal axes radiating from the wound. The immunoreactivity of this IFAP started to diminish at 20 days postlesion, and by 30 days postlesion, it was not observed in the remaining gliotic cells. These results demonstrate that reactive astrocytes induced by stab-wound injury can be divided into two subtypes: persistent IFAP-70/280 kDa-containing cells which are close to the wound in the area of the glial scar and transient IFAP-70/280 kDa-negative cells which are farther from the wound. The reappearance of IFAP-70/280 kDa also suggests that some reactive astrocytes have the capacity to recapitulate early developmental stages.

It is estimated that 10-15% of couples are infertile and male factors account for about half of these cases. With the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring. However, teratozoospermia still remains a big challenge to tackle. Septins belong to a family of cytoskeletal proteins with GTPase activity and are involved in various biological processes e.g. morphogenesis, compartmentalization, apoptosis and cytokinesis. SEPTIN12, identified by c-DNA microarray analysis of infertile men, is exclusively expressed in the post meiotic male germ cells. Septin12(+/+)/Septin12(+/-) chimeric mice have multiple reproductive defects including the presence of immature sperm in the semen, and sperm with bent neck (defect of the annulus) and nuclear DNA damage. These facts make SEPTIN12 a potential sterile gene in humans. In this study, we sequenced the entire coding region of SEPTIN12 in infertile men (n = 160) and fertile controls (n = 200) and identified ten variants. Among them is the c.474 G>A variant within exon 5 that encodes part of the GTP binding domain. The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12. Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm

Purpose: Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway frequently accounts for the tumorigenesis in head and neck cancer. To develop a new treatment, we investigated the effect of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in head and neck cancer cells. Experimental Design: The in vitro antitumor effect of BGT226 was determined in various cancer cell lines. Animal models were also applied to examine drug potency. The inhibitory ability of BGT226 on the PI3K/AKT/mTOR signaling pathway was analyzed. Results: The growth inhibition assay revealed that BGT226 was active against all tested cancer cell lines. Cross-resistance was not observed in the cisplatin-resistant cell line. The activation of the AKT/mTOR signal cascade was suppressed by BGT226 in a concentration- and time-dependent manner. Flow cytometric analysis revealed an accumulation of cells in the G0–G1 phase with concomitant loss in the S-phase. Results of the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay and the analysis of caspase 3/7 and PARP indicated that BGT226 induced cancer cell death through an apoptosis-independent pathway. BGT226 induced autophagy as indicated by the aggregation and upregulation of the microtubule-associated protein light chain 3B-II, and p62 degradation. Gene silencing of Beclin1 or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited the BGT226-induced autophagy and led to the retrieval of colony survival. In a xenografted animal model, BGT226 significantly delayed tumor growth in a dose-dependent manner, along with suppressed cytoplasmic expression of p-p70 S6 kinase and the presence of autophagosome formation. Conclusions: These data indicate that BGT226 is a potential drug in the treatment of head and neck cancer. Clin Cancer Res; 17(22); 7116–26. ©2011 AACR.

Arsenic trioxide (As(2)O(3)) is an effective treatment for relapsed or refractory acute promyelocytic leukemia (APL). After the discovery of As(2)O(3) as a promising treatment for APL, several studies investigated the use of As(2)O(3) as a single agent in the treatment of solid tumors; however, its therapeutic efficacy is limited. Thus, the systematic study of the combination of As(2)O(3) with other clinically used chemotherapeutic drugs to improve its therapeutic efficacy in treating human solid tumors is merited. In this study, we demonstrate for the first time, using isobologram analysis, that As(2)O(3) exhibits a synergistic interaction with N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). The synergistic augmentation of the cytotoxicity of As(2)O(3) with BCNU is in part through the autophagic cell death machinery in human solid tumor cells. As(2)O(3) and BCNU in combination produce enhanced cytotoxicity via the depletion of reduced glutathione (GSH) and augmentation of reaction oxygen species (ROS) production. Further analysis indicated that the extension of GSH depletion by this combined regimen occurs through the inhibition of the catalytic activity of glutathione reductase. Blocking ROS production with antioxidants or ROS scavengers effectively inhibits cell death and autophagy formation, indicating that redox-mediated autophagic cell death involves the synergism of As(2)O(3) with BCNU. Taken together, this is the first evidence that BCNU could help to extend the therapeutic spectrum of As(2)O(3). These findings will be useful in designing future clinical trials of combination chemotherapy with As(2)O(3) and BCNU, with the potential for broad use against a variety of solid tumors.

Glass ionomers have been used for perforation repair and retrograde filling where biointegration with periodontal tissue is required. Collagen has been demonstrated to promote cellular adhesion and enhance mineral tissue compressive strength. It was hypothesized that an appropriate concentration of collagen integrated into glass ionomer may improve both bio-compatibility and the mechanical properties of the material. By SEM and AFM, we discovered 70-nm granules appearing on the surfaces of glass-ionomer/collagen hybrids. Acid-etching revealed irregularly shaped particles interlinked by membrane-like sheets on the surface of the material with the typical 70-nm granules. WST-1 assay showed that acid-etching significantly enhanced the viability of attached gingival fibroblasts. However, the glass-ionomer/collagen hybrids’ combined surface-etching outperformed other groups. The glass-ionomer/collagen hybrids presented enhanced compressive strength when integrated with 0.01% collagen, while higher concentrations of collagen compromised their mechanical property. In summary, collagen improved both the mechanical and biocompatible properties of glass ionomers. Further in vivo study is warranted.

Nestin is an intermediate filament protein mainly expressed in muscle and neural progenitors. Recently, we reported that nestin is expressed in rat vascular smooth muscle cells (VSMCs), disappears after serum-deprivation and then is re-expressed again following EGF stimulation. As the function of nestin in VSMCs remains unknown, its anti-apoptotic function was investigated in this study. We first showed that cell viability of nestin-depleted cells following H(2)O(2) treatments decreased by nestin RNAi. Further DNA laddering analysis and flow cytometry results demonstrated that this loss of cell viability was mediated through apoptosis. In addition, caspase-9, caspase-3 and PARP were activated in nestin-depleted VSMCs following H(2)O(2) treatments, indicating that nestin has an upstream inhibitory effect on caspase activation. It is well known that EGF serves as a survival factor in rat VSMCs. Here, we show that the cytoprotective effect of EGF was prevented by nestin RNAi. In addition, the inhibition of Cdk5 prevented Bcl-2 phosphorylation and enhanced H(2)O(2)-induced caspase-3 activation as well as subsequent DNA fragmentation. Taken together, these results provide evidence for another cytoprotective role of EGF in that it is mediated through its stimulation of nestin expression which leads to the prevention of caspase activation by Cdk-5-induced Bcl-2 phosphorylation in rat VSMCs.

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.

The design and implementation of a high-performance controller for a field-oriented induction motor drive is presented. Dynamic modeling based on the stochastic technique is performed. Based on the estimated drive model, a two-degree-of-freedom controller is proposed so good dynamic responses in both the speed tracking and regulation characteristics can be achieved. The parameters of the controller are found using a proposed systematic design procedure according to the prescribed specifications. Having designed and tested the performance of the controller by simulation, the hardware implementation is successfully made, and some experimental results are given to demonstrate the effectiveness of the proposed controller. >

Photothermal therapy by the strategy of adding Au nanoparticles (NPs), UV-absorbed around near IR range of 800-1000 nm, had been studied and used as a novel tool to eliminate and kill cancer cells. However, we had successfully developed the bacteria of E. Coli. as a new template for growing and self-assembling Au NPs even nano-shell on E. Coli. surface and treated cells for photothermal therapy studies. And we not only found the E. Coli. coated with Au NPs were curative effect to cancer cells using photothermal therapy but also the those particles were non-toxicity to cells. Here, we had succeeded to develope the bacteria of E. Coli. as a new template for growing and self-assembling Au NPs on E. Coli. surface. We not only found the E. Coli. coated with Au NPs were curative effect to cancer cells using photothermal therapy but also the those particles were non-toxicity to cells.

Adenocarcinomas in rats and humans frequently contain perivascular, degranulating mast cells that release heparin. Protamine is a low-molecular weight, cationic polypeptide that binds to heparin and neutralizes its anticoagulant properties. A novel magnetic resonance imaging (MRI) contrast agent containing protamine was synthesized. TTDASQ, the derivative of TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid), was also synthesized and the kinetic stability of [Gd(TTDASQ)]- chelate containing phosphate buffer and ZnCl2 to measure the relaxation rate (R1) at 20 MHz was studied by transmetallation with Zn(II). The water-exchange rate (k(ex)298) of [Gd(TTDASQ)]- is 6.4 x 10(6) s(-1) at 25.0 +/- 0.1 degrees C which was obtained from the reduced 17O relaxation rates (1/T(1r) and 1/T(2r)) and chemical shift (omega(r)) of H(2)17O, and it is compared with that previously reported for the other gadolinium(III) complex, [Gd(DO3ASQ)]. The binding affinity assay showed that the (TTDASQ)3-pro19 has higher activity toward heparin. On the other hand, the effect of heparin on the relaxivity of the [Gd(TTDASQ)3-pro19] conjugate shows the binding strength (K(A)) is 7669 dm3 mol(-1) at pH 7.4 and the relaxivity (r(b)1) of the [Gd(TTDASQ)3-pro19]-heparin adduct is 30.9 dm3 mmol(-1) s(-1).

Blood-brain barrier (BBB) permeability during sepsis with Escherichia coli or Streptococcus pneumoniae was examined in a mouse model and measured by a circulating beta-galactosidase tracer. The leakage of brain microvascular vessels during sepsis was confirmed by transmission electron microscopic examination of brain tissues stained with horseradish peroxidase. The increase of BBB permeability induced by E. coli and S. pneumoniae, which was maximal at 3 h and 12 h after injection, respectively, was transient because of rapid clearance of the bacteria from the blood. Tumour necrosis factor-alpha (TNF-alpha) was stained on microvascular vessels of the brain during sepsis and intravenous injection of recombinant TNF-alpha also increased the BBB permeability. The increase in BBB permeability induced by either E. coli or S. pneumoniae could be inhibited by anti-TNF-alpha antibody. It was concluded that circulating TNF-alpha generated during sepsis induced the increase in BBB permeability.

We have successfully transformed the infectious E. coli bacteria into biocompatible bacteria@Au composites for photothermal therapy.

Objective To understand the mechanisms of postpartum uterine involution, we investigated the uterine myometrial changes during pregnancy and the postpartum period. Materials and methods Nine groups of uterine myometrial samples from mice ( n = 4) were collected on gestational Day 0 (nonpregnant), Day 1, Day 2, Day 7, Day 14, and Day 21 and on postpartum Day 1, Day 2, and Day 7. Human samples of uterine myometrium on term ( n = 1) and postpartum Day 1 ( n = 2) were also collected. Ki-67 immunostaining was used to determine myometrial proliferation. For cell hypertrophy analysis, organelle proteins, β-actin, prohibin, calnexin, and golgin-97 were analyzed by Western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and evaluation of activated caspase-3 expression by Western blot analysis assay were used to detect apoptosis. Autophagy was assayed via the evaluation of LC3 expression by Western blotting, immunohistochemistry, and autophagosomes by electron microscopy. Results Uterine myocytes proliferated during the early stage of gestation with a peak at Day 2, whereas myocyte hypertrophy with increased cellular organelle production occurred gradually in later stages of pregnancy. Postpartum autophagy developed abruptly in uterine myocytes without obvious apoptosis. Conclusion Autophagy of myocytes may play an important role in uterine involution. These results have implications for our understanding of myometrial functional adaptations during pregnancy and the physiological role of autophagy in the uterine remodeling events in the postpartum period.