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Objective: Diabetes results from a deficiency of functional beta-cells due to both an increase in beta-cell death and an inhibition of beta-cell replication. The molecular mechanisms responsible for these effects in susceptible individuals are mostly unknown. The objective of this study was to determine whether a gene critical for cell division and cell survival in cancer cells, survivin, might also be important for beta-cells.Research Design and Methods: We generated mice harboring a conditional deletion of survivin in pancreatic endocrine cells using mice with a Pax-6-Cre transgene promoter construct driving tissue-specific expression of Cre-recombinase in these cells. We performed metabolic studies and immunohistochemical analyses to determine the effects of a mono- and biallelic deletion of survivin.Results: Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in beta-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age. Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones. Exogenous expression of survivin in mature beta-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in beta-cell mass, confirming the specificity of the survivin effect in these cells.Conclusions: Our findings implicate survivin in the maintenance of beta-cell mass through both replication and antiapoptotic mechanisms. Given the widespread involvement of survivin in cancer, a novel role for survivin may well be exploited in beta-cell regulation in diseased states, such as diabetes. [ABSTRACT FROM AUTHOR]

Background: Because of the increasing prevalence of dementia in Japan, the government introduced financial incentives for specialized care for dementia at acute care hospitals in 2016. Our hospital then introduced a multidisciplinary collaborative specialized team, referred to as dementia-specialized care team. The aim of this study is to examine the influence of dementia-specialized care team on clinical outcomes for elderly inpatients., Methods: In this retrospective observational study at a general hospital with 650 beds in Japan, we compared clinical outcomes such as incidence of falls, prescription of hypnotics, length of hospital stay, in-hospital mortality, and discharge destinations in inpatients aged 65 years and older between a two-year pre-intervention period (2014-2015) and a two-year post-intervention period (2017-2018)., Results: During the observation period, a total of 34,097 patients were admitted, with 16,237 patients in the pre-intervention period and 17,860 patients in the post-intervention period. The proportion of patients receiving any hypnotics decreased from 21.2 % to 19.2 %, notably with benzodiazepine from 19.8 % to 13.2 %. The incidence of falls from a seated or lying position, particularly at night, was significantly lower (from 0.5 % to 0.2 %) as was the length of hospital stay (from 13.7 days to 13.2 days) during the post-intervention period., Conclusion: After the implementation of dementia-specialized care team, favorable outcomes such as a reduction in the use of hypnotics, the incidence of falls, and the length of hospitalization were observed. Introduction of the team and associated incentives may be effective in improving clinical outcomes in elderly inpatients., Competing Interests: Declaration of Competing Interest Drs. Shimomura, Nishimura, Murayama, and Yoshimura have no conflicts of interest. Dr. Mimura has received grants from Daiichi Sankyo, Eisai, Fronteo, Mitsubishi Tanabe Pharma, Shionogi, Takeda, and Tsumura; and speaker’s fees from Biogen, Byer, Daiichi Sankyo, Demant, Eisai, Eli Lilly, Fujifilm RI Pharma, Hisamitsu, H.U. Frontier, Janssen, Mochida, MSD, Mylan EPD, Nippon Chemipher, Novartis Pharma, Ono, Otsuka, Pfizer, Shionogi, Sumitomo Pharma, Takeda, Teijin Pharma, and Viatris. Dr. Uchida has received grants from Daiichi Sankyo, Eisai, Mochida, Otsuka, and Sumitomo Pharma; speaker’s fees from Boehringer Ingelheim, Eisai, Lundbeck, Meiji Seika Pharma, Otsuka, MSD, and Sumitomo Pharma; and consulting fees from Boehringer Ingelheim, Lundbeck, Sumitomo Pharma, and Takeda. Dr. Takeuchi has received grants from Daiichi Sankyo, Novartis Pharma, and Otsuka; speaker fees from EA Pharma, Eisai, Janssen, Kyowa, Lundbeck, Meiji Seika Pharma, Mitsubishi Tanabe Pharma, MSD, Otsuka, Sumitomo Pharma, Takeda, and Yoshitomiyakuhin; and consulting fees from Boehringer Ingelheim, Janssen, Mitsubishi Tanabe Pharma, Ono, and Sumitomo Pharma. Dr. Shishida has received speaker’s fee from MSD and Sumitomo Pharma., (Copyright © 2024. Published by Elsevier B.V.)

A lack of social relationships is increasingly recognized as a type 2 diabetes (T2D) risk. To investigate the underlying mechanism, we used male KK mice, an inbred strain with spontaneous diabetes. Given the association between living alone and T2D risk in humans, we divided the non-diabetic mice into singly housed (KK-SH) and group-housed control mice. Around the onset of diabetes in KK-SH mice, we compared H3K27ac ChIP-Seq with RNA-Seq using pancreatic islets derived from each experimental group, revealing a positive correlation between single-housing-induced changes in H3K27ac and gene expression levels. In particular, single-housing-induced H3K27ac decreases revealed a significant association with islet cell functions and GWAS loci for T2D and related diseases, with significant enrichment of binding motifs for transcription factors representative of human diabetes. Although these H3K27ac regions were preferentially localized to a polymorphic genomic background, SNVs and indels did not cause sequence disruption of enriched transcription factor motifs in most of these elements. These results suggest alternative roles of genetic variants in environment-dependent epigenomic changes and provide insights into the complex mode of disease inheritance., (© 2024 Nammo et al.)

The mechanisms of impaired glucose-induced insulin secretion from the pancreatic β-cells in obesity have not yet been completely elucidated. Here, we aimed to assess the effects of adipocyte-derived factors on the functioning of pancreatic β-cells. We prepared a conditioned medium using 3T3-L1 cell culture supernatant collected at day eight (D8CM) and then exposed the rat pancreatic β-cell line, INS-1D. We found that D8CM suppressed insulin secretion in INS-1D cells due to reduced intracellular calcium levels. This was mediated by the induction of a negative regulator of insulin secretion-NECAB1. LC-MS/MS analysis results revealed that D8CM possessed steroid hormones (cortisol, corticosterone, and cortisone). INS-1D cell exposure to cortisol or corticosterone increased Necab1 mRNA expression and significantly reduced insulin secretion. The increased expression of Necab1 and reduced insulin secretion effects from exposure to these hormones were completely abolished by inhibition of the glucocorticoid receptor (GR). NECAB1 expression was also increased in the pancreatic islets of db/db mice. We demonstrated that the upregulation of NECAB1 was dependent on GR activation, and that binding of the GR to the upstream regions of Necab1 was essential for this effect. NECAB1 may play a novel role in the adipoinsular axis and could be potentially involved in the pathophysiology of obesity-related diabetes mellitus., (© 2023. Springer Nature Limited.)

Background: Chronic rhinosinusitis (CRS) can be divided into endotypes by functional or pathophysiologic findings., Objective: The aim of this study was to analyze the expression of cytokines, prostaglandin (PG) synthases, and their receptors related to the pathogenesis of CRS, especially those contributing to nasal polyp (NP) formation., Methods: NPs and uncinate tissue (UT) samples were collected from 90 patients who underwent endoscopic sinus surgery. They included 75 patients with CRS (including 45 with eosinophilic CRS [eCRS] and 30 with non-eCRS) and 15 patients without CRS. A total of 30 genes were selected for our original DNA array plate to analyze the levels of expression of 10 cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, IL-25, IL-33, and TSLP), 4 prostaglandin synthases (prostaglandin D 2 [PGD 2 ] synthase, prostaglandin E 2 synthase, COX-1, and COX-2), and their 16 receptors. Clustering analysis was performed according to the expression results, and clinical findings of patients from each cluster were investigated., Results: The samples could be divided into 3 clusters. Cluster 1 showed elevated levels of expression of IL4, IL5, IL13, TSLP, IL1RL1 (ST2 [an IL-33 receptor]), HPGDS , and GPR44 (CRTH2, a PGD 2 receptor); cluster 2 showed elevated levels of expression of IL17A and PTGES ; and cluster 3 showed an elevated level of expression of IL25 . Regarding clinical features, the main characteristics of each cluster were as follows: NPs from patients with eCRS for cluster 1, NPs and/or UT samples from patients with non-eCRS for cluster 2, and UTs from patients with non-CRS for cluster 3., Conclusion: The results suggest that there are associations between type 2 inflammation/PGD 2 and eCRS and also between type 3 inflammation/prostaglandin E 2 and non-eCRS., (© 2023 The Author(s).)

Pancreatic β-cells are specialized to properly regulate blood glucose. Maintenance of the mature β-cell phenotype is critical for glucose metabolism, and β-cell failure results in diabetes mellitus. Recent studies provide strong evidence that the mature phenotype of β-cells is maintained by several transcription factors. These factors are also required for β-cell differentiation from endocrine precursors or maturation from immature β-cells during pancreatic development. Because the reduction or loss of these factors leads to β-cell failure and diabetes, inducing the upregulation or inhibiting downregulation of these transcription factors would be beneficial for studies in both diabetes and stem cell biology. Here, we discuss one such factor, i.e., the transcription factor MAFA. MAFA is a basic leucine zipper family transcription factor that can activate the expression of insulin in β-cells with PDX1 and NEUROD1. MAFA is indeed indispensable for the maintenance of not only insulin expression but also function of adult β-cells. With loss of MAFA in type 2 diabetes, β-cells cannot maintain their mature phenotype and are dedifferentiated. In this review, we first briefly summarize the functional roles of MAFA in β-cells and then mainly focus on the molecular mechanism of cell fate conversion regulated by MAFA.

Energetic ion beam experiments with major space radiation elements, 1 H, 4 He, 16 O, 28 Si and 56 Fe, have been conducted to investigate the radiation shielding properties of composite materials. These materials are expected to be used for parts and fixtures of space vehicles due to both their mechanical strength and their space radiation shielding capabilities. Low Z materials containing hydrogen are effective for shielding protons and heavy ions due to their high stopping power and large fragmentation cross section per unit mass. The stopping power of the composite materials used in this work is intermediate between that of aluminum and polyethylene, which are typical structural and shielding materials used in space. The total charge-changing cross sections per unit mass, σ UM , of the composite materials are 1.3-1.8 times larger than that of aluminum. By replacing conventional aluminum used for spacecraft with commercially available composite (carbon fiber / polyether ether ketone), it is expected that the shielding effect is increased by ∼17%. The utilization of composite materials will help mitigate the space radiation hazard on future deep space missions., (Copyright © 2021. Published by Elsevier B.V.)

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-β-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion., (© 2021 John Wiley & Sons Ltd.)

Long-term and mild confinement or isolation in an enclosed environment can occur in situations such as disasters, specific political, economic or social events, nuclear shelters, seabed exploration, polar expeditions, and space travel. To investigate the effects of stress caused by long-term confinement in an enclosed environment in mammals, we divided 8-week-old C57BL/6J mice into four groups that were housed in a closed environment with a narrow metabolic cage (stress group), normal metabolic cage (control group), conventional cage (conventional group) or conventional cage with wire mesh floor (wire mesh group). The phenotypes of the mice were examined for four weeks, followed by behavioral tests. Weight gain suppression was observed in the stress group. Continuous analysis of these mice every two minutes for four weeks using an implanted measuring device showed a significantly decreased amount of spontaneous activity and subcutaneous temperature in the stress group. After housing in each environment for four weeks, the behavioral tests of mice in the stress group also revealed a shorter latency to fall off in the rotarod test and shorter stride length and interstep distance in the footprint test. Interestingly, the lower spontaneous activity of mice in the stress group was rescued by housing in conventional cages. These results suggest a temporary effect of long-term confinement in an enclosed environment as a chronic and mild stress on homeostasis in mammals., (Copyright © 2021. Published by Elsevier Inc.)

The bicaudal D homolog 2 ( BICD2 ) gene encodes a protein required for the stable complex of dynein and dynactin, which functions as a motor protein working along the microtubule cytoskeleton. Both inherited and de novo variants of BICD2 are reported with autosomal dominant spinal muscular atrophy with lower extremity predominance (SMALED2). Here, we report a male patient with a novel mutation in the BICD2 gene caused by a heterozygous substitution of arginine with cysteine at residue 162 (Arg162Cys); inherited from his asymptomatic mother. The patient showed typical clinical symptoms of SMALED2, which was genetically confirmed by sequencing. The Arg162Cys mutant clusters with four previously reported variants (c.361C>G, p.Leu121Val; c.581A>G, p.Gln194Arg; c.320C>T, p.Ser107Leu; c.565A>T, p.Ile189Phe) in a region that binds to the dynein-dynactin complex (DDC). The BICD2 domain structures were predicted and the Arg162Cys mutation was localized in the N-terminus coiled-coil segment 1 (CC1) domain. Protein modeling of BICD2's CC1 domain predicted that the Arg162Cys missense variant disrupted interactions with dynein cytoplasmic 1 heavy chain 1 within the DDC. The mutant did this by either changing the electrostatic surface potential or making a broader hydrophobic unit with the neighboring residues. This hereditary case supports the complex and broad genotype-phenotype correlation of BICD2 mutations, which could be explained by incomplete penetrance or variable expressivity in the next generation., Competing Interests: The authors have no conflicts of interest to disclose., (2021, International Research and Cooperation Association for Bio & Socio - Sciences Advancement.)

Dedifferentiation has been implicated in β cell dysfunction and loss in rodent diabetes. However, the pathophysiological significance in humans remains unclear. To elucidate this, we analyzed surgically resected pancreatic tissues of 26 Japanese subjects with diabetes and 11 nondiabetic subjects, who had been overweight during adulthood but had no family history of diabetes. The diabetic subjects were subclassified into 3 disease stage categories, early, advanced, and intermediate. Despite no numerical changes in endocrine cells immunoreactive for chromogranin A (ChgA), diabetic islets showed profound β cell loss, with an increase in α cells without an increase in insulin and glucagon double-positive cells. The proportion of dedifferentiated cells that retain ChgA immunoreactivity without 4 major islet hormones was strikingly increased in diabetic islets and rose substantially during disease progression. The increased dedifferentiated cell ratio was inversely correlated with declining C-peptide index. Moreover, a subset of islet cells converted into exocrine-like cells during disease progression. These results indicate that islet remodeling with dedifferentiation is the underlying cause of β cell failure during the course of diabetes progression in humans.

Aims/introduction: Taste receptors, T1rs and T2rs, and the taste-selective G-protein, α-gustducin, are expressed outside the taste-sensing system, such as enteroendocrine L cells. Here, we examined whether α-gustducin also affects nutrition sensing and insulin secretion by pancreatic β-cells., Materials and Methods: The expression of α-gustducin and taste receptors was evaluated in β-cell lines, and in rat and mouse islets either by quantitative polymerase chain reaction or fluorescence immunostaining. The effects of α-gustducin knockdown on insulin secretion and on cyclic adenosine monophosphate and intracellular Ca 2+ levels in rat INS-1 cells were estimated. Sucralose (taste receptor agonist)-induced insulin secretion was investigated in INS-1 cells with α-gustducin suppression and in islets from mouse disease models., Results: The expression of Tas1r3 and α-gustducin was confirmed in β-cell lines and pancreatic islets. Basal levels of cyclic adenosine monophosphate, intracellular calcium and insulin secretion were significantly enhanced with α-gustducin knockdown in INS-1 cells. The expression of α-gustducin was decreased in high-fat diet-fed mice and in diabetic db/db mice. Sucralose-induced insulin secretion was not attenuated in INS-1 cells with α-gustducin knockdown or in mouse islets with decreased expression of α-gustducin., Conclusions: α-Gustducin is involved in the regulation of cyclic adenosine monophosphate, intracellular calcium levels and insulin secretion in pancreatic β-cells in a manner independent of taste receptor signaling. α-Gustducin might play a novel role in β-cell physiology and the development of type 2 diabetes., (© 2020 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Aims/hypothesis: Epigenetic regulation of gene expression has been implicated in the pathogenesis of obesity and type 2 diabetes. However, detailed information, such as key transcription factors in pancreatic beta cells that mediate environmental effects, is not yet available., Methods: To analyse genome-wide cis-regulatory profiles and transcriptome of pancreatic islets derived from a diet-induced obesity (DIO) mouse model, we conducted chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) of histone H3 lysine 27 acetylation (histone H3K27ac) and high-throughput RNA sequencing. Transcription factor-binding motifs enriched in differential H3K27ac regions were examined by de novo motif analysis. For the predicted transcription factors, loss of function experiments were performed by transfecting specific siRNA in INS-1, a rat beta cell line, with and without palmitate treatment. Epigenomic and transcriptional changes of possible target genes were evaluated by ChIP and quantitative RT-PCR., Results: After long-term feeding with a high-fat diet, C57BL/6J mice were obese and mildly glucose intolerant. Among 39,350 islet cis-regulatory regions, 13,369 and 4610 elements showed increase and decrease in ChIP-Seq signals, respectively, significantly associated with global change in gene expression. Remarkably, increased H3K27ac showed a distinctive genomic localisation, mainly in the proximal-promoter regions, revealing enriched elements for nuclear respiratory factor 1 (NRF1), GA repeat binding protein α (GABPA) and myocyte enhancer factor 2A (MEF2A) by de novo motif analysis, whereas decreased H3K27ac was enriched for v-maf musculoaponeurotic fibrosarcoma oncogene family protein K (MAFK), a known negative regulator of beta cells. By siRNA-mediated knockdown of NRF1, GABPA or MEF2A we found that INS-1 cells exhibited downregulation of fatty acid β-oxidation genes in parallel with decrease in the associated H3K27ac. Furthermore, in line with the epigenome in DIO mice, palmitate treatment caused increase in H3K27ac and induction of β-oxidation genes; these responses were blunted when NRF1, GABPA or MEF2A were suppressed., Conclusions/interpretation: These results suggest novel roles for DNA-binding proteins and fatty acid signalling in obesity-induced epigenomic regulation of beta cell function., Data Availability: The next-generation sequencing data in the present study were deposited at ArrayExpress. RNA-Seq: Dataset name: ERR2538129 (Control), ERR2538130 (Diet-induced obesity) Repository name and number: E-MTAB-6718 - RNA-Seq of pancreatic islets derived from mice fed a long-term high-fat diet against chow-fed controls. ChIP-Seq: Dataset name: ERR2538131 (Control), ERR2538132 (Diet-induced obesity) Repository name and number: E-MTAB-6719 - H3K27ac ChIP-Seq of pancreatic islets derived from mice fed a long-term high-fat diet (HFD) against chow-fed controls.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved lipid binding proteins found in organisms ranging from yeast to mammals. Recent findings have indicated that these proteins mainly localize to contact sites of 2 different membranous organelles. ORP6, a member of the ORP subfamily III, is one of the least studied ORPs. Using approaches in molecular cell biology, we attempted to study the characteristics of ORP6 and found that ORP6 is abundantly expressed in mouse cultured neurons. Deconvolution microscopy of cultured cerebellar granular cells revealed that ORP6 is localized to the endoplasmic reticulum (ER) and ER-plasma membrane (PM) contact sites, where it co-localized with extended synaptotagmin2 (E-Syt2), a well-known ER-PM contact site marker. E-Syt2 also co-localized with ORP3, another subfamily III member, and ORP5, a subfamily IV member. However, ORP5 does not distribute to the same ER-PM contact sites as subfamily III members. Also, the co-expression of ORP3 but not ORP5 altered the distribution of ORP6 into the processes of cerebellar neurons. Immunoprecipitation demonstrated binding between the intermediate region of ORP6 and ORP3 or ORP6 itself. Additionally, the localization of ORP6 in the PM decreased when co-expressed with the intermediate region of ORP6, in which the pleckstrin homology (PH) domain and OSBP-related ligand binding domain (ORD) are deleted. Over-expression of this intermediate region shifted the location of a phophtidylinositol-4-phosphate (PI4P) marker from the Golgi to the PM. Knockdown of ORP6 resulted in the same shift of the PI4P marker. Collectively, our data suggests that the recruitment of ORP6 to ER-PM contact sites is involved in the turnover of PI4P in cerebellar granular neurons., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glucose metabolism is regulated by insulin, which is produced from β-cells in the pancreas. Because insulin is secreted into vessels in response to blood glucose, vascular structures of the pancreas, especially the relationship between vessels and β-cells, are important for physiological and pathological glucose metabolism. Here, we developed a system to visualize vessels surrounding mature β-cells expressing transcription factor MafA in a three-dimensional manner. Optical clearing of the pancreas prevented light scattering of fluorescence driven by the bacterial artificial chromosome (BAC)-mafA promoter in β-cells. Reconstruction of confocal images demonstrated mature β-cells and the glomerular-like structures of β-cell vasculatures labeled with DyLight 488-conjugated lectin in normal mice as well as in low-dose streptozotocin-injected diabetes model mice with reduced β-cell mass. This technological innovation of organ imaging can be used to investigate morphological changes in vascular structures during transplantation, regeneration and diabetes development.

Pathological complete response (pCR) is considered to be a useful prognostic marker for neoadjuvant chemotherapy to improve the survival rate of patients with operable breast cancer. In the present study, we identified differentially expressed microRNAs (miRNAs) between pCR and non-pCR groups of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer who received neoadjuvant chemotherapy with trastuzumab. Expression profiles were examined by miRNA microarrays using total RNA extracted from formalin-fixed, paraffin-embedded tissues from pretreatment biopsy specimens. Significant differences were observed in miRNAs associated with pCR between the luminal B-like (HER2-positive) and HER2-positive (nonluminal) subtypes, which were further classified according to their estrogen receptor (ER) status. Prediction models constructed with differentially expressed miRNAs performed well. In conclusion, the combination of miRNA profiles and ER status may improve the accuracy of pCR prediction in patients with HER2-positive breast cancer and enable the development of personalized treatment regimens.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor critical for synaptic plasticity, neuronal development and neurite extension. BDNF mRNA is transported to dendrites and axons, where it is expressed locally. We previously reported that dendritic targeting elements in the BDNF 3' UTR are necessary for dendritic transport and interact with cytoplasmic polyadenylation element binding protein 1. Here, we demonstrated that the short 3' UTR directs local translation of BDNF and that locally synthesized BDNF exists in a novel compartment that does not co-localize with markers of endosomes, endoplasmic reticulum, Golgi or the trans-Golgi network. Further, locally synthesized BDNF vesicles co-localized with Bicaudal-D2 (BicD2), a member of dynein motor complex proteins. Silencing BicD2 significantly reduced BDNF local synthesis in dendrites. These new findings may underlie the mechanism of local neuronal response to environmental stimuli.

We report a patient with multiple myeloma who developed severe hemostatic dysfunction during spinal fusion surgery. A 74-year-old man presented with lower limb paralysis due to thoracic supine pathological fracture and was scheduled for spinal fusion surgery. He suffered from multiple myeloma for decades but did not present with any significant abnormalities in a preoperative blood exam. Prothrombin time international normalized ratio (PT-INR, 0.97) and activated partial thromboplastin time (APTT, 31 sec) were normal. Surgeons confronted hemostatic difficulty during surgery. At the point of 1,000 ml blood loss, PT-INR was 9.99, APTT was 300 sec, and platelet count was 116,000 x μl(-1). The patient was administered 1,400 ml of frozen plasma concentrate; PT-INR and APTT recovered to 1.04 and 39.6 sec, respectively. Hemostatic dysfunction in this patient may have resulted from an inherent coagulation deficiency associated with multiple myeloma.

Early in pancreatic development, epithelial cells of pancreatic buds function as primary multipotent progenitor cells (1°MPC) that specify all three pancreatic cell lineages, i.e., endocrine, acinar and duct. Bipotent "Trunk" progenitors derived from 1°MPC are implicated in directly regulating the specification of endocrine progenitors. It is unclear if this specification process is initiated in the 1°MPC where some 1°MPC become competent for later specification of endocrine progenitors. Previously we reported that in Pdx1tTA/+;tetOMafA (bigenic) mice inducing expression of transcription factor MafA in Pdx1-expressing (Pdx1+) cells throughout embryonic development inhibited the proliferation and differentiation of 1°MPC cells, resulting in reduced pancreatic mass and endocrine cells by embryonic day (E) 17.5. Induction of the transgene only until E12.5 in Pdx1+ 1°MPC was sufficient for this inhibition of endocrine cells and pancreatic mass at E17.5. However, by birth (P0), as we now report, such bigenic pups had significantly increased pancreatic and endocrine volumes with endocrine clusters containing all pancreatic endocrine cell types. The increase in endocrine cells resulted from a higher proliferation of tubular epithelial cells expressing the progenitor marker Glut2 in E17.5 bigenic embryos and increased number of Neurog3-expressing cells at E19.5. A BrdU-labeling study demonstrated that inhibiting proliferation of 1°MPC by forced MafA-expression did not lead to retention of those progenitors in E17.5 tubular epithelium. Our data suggest that the forced MafA expression in the 1°MPC inhibits their competency to specify endocrine progenitors only until E17.5, and after that compensatory proliferation of tubular epithelium gives rise to a distinct pool of endocrine progenitors. Thus, these bigenic mice provide a novel way to characterize the competency of 1°MPC for their ability to specify endocrine progenitors, a critical limitation in our understanding of endocrine differentiation.

Background: Anesthesiologists often encounter LMA-ProSeal(®) (ProSeal) insertion difficulty due to its large cuff size. We performed a randomized clinical trial to examine how insertion efficacy and sealing pressure of ProSeal are affected by muscle relaxant administration in anesthetized patients., Methods: Our adult patients were either administered rocuronium (0.9 mg kg(-1)) as a muscle relaxant (R group; 40 patients) or not (C group; 40 patients). Anesthesia was induced with propofol and fentanyl. We compared the two groups with regard to the number of attempts required for successful insertion, sealing pressure, and subjective difficulty for insertion., Results: Total insertion attempts required for successful ventilation in the two groups were one (R group, 38 patients; C group, 28 patients), two (R group, one patient; C group, seven patients), and three (R group, one patient; C group, five patients), revealing a significant difference between groups (p < 0.001). Sealing pressure was significantly higher in the R group than in the C group (R group, 27.4 ± 5.4 cmH2O; C group, 21.2 ± 5.2 cmH2O; p < 0.001). Leakage volume by mechanical ventilation was significantly smaller in the R group than in the C group (R group, 17.4 ± 29.1 ml; C group, 46.8 ± 45.5 ml; p < 0.001). Subjective difficulty of insertion was significantly lower in the R group than in the C group (R group, 12.3 ± 23.1 mm; C group, 39.4 ± 31.9 mm; p < 0.001)., Conclusions: Muscle relaxation appears to facilitate ProSeal insertion efficacy by enabling higher successful insertion rates, higher sealing pressure, lower leakage volume, and lower subjective difficulty of insertion in anesthetized patients.

Recent studies suggest that dedifferentiation of pancreatic β-cells is involved in compromised β-cell function in diabetes mellitus. We have previously shown that the promoter activity of MafB, which is expressed in α-cells of adult islets and immature β-cells in embryonic pancreas but not in mature β-cells in mice, is increased in compromised β-cells of diabetic model mice. Here, we investigated a rat β-cell line of INS1 cells with late-passage numbers, which showed extremely low expression of MafA and insulin, as an in vitro model of compromised β-cells. In these INS1 cells, the mRNA expression and the promoter activity of MafB were upregulated compared with the early-passage ('conventional') INS1 cells. Analysis of the MafB promoter in these late-passage INS1 cells revealed that specific CpG sites in the MafB promoter were partially demethylated. The reporter assay revealed that the unmethylated promoter activity of the 373 bp region containing these CpG sites was higher than the in vitro methylated promoter activity. These results suggest that the chronic culture of the rat β-cell line resulted in partial DNA demethylation of the MafB promoter, which may have a role in MafB promoter activation and possible dedifferentiation in our compromised β-cell model., (© 2015 Society for Endocrinology.)

Genetic factors are important determinants of the onset and progression of diabetes mellitus. Numerous susceptibility genes for type 2 diabetes, including potassium voltage-gated channel, KQT-like subfamily Q, member1 (KCNQ1), have been identified in humans by genome-wide analyses and other studies. Experiments with genetically modified mice have also implicated various genes in the pathogenesis of diabetes. However, the possible effects of the parent of origin for diabetes susceptibility alleles on disease onset have remained unclear. Here, we show that a mutation at the Kcnq1 locus reduces pancreatic β-cell mass in mice by epigenetic modulation only when it is inherited from the father. The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the Kcnq1 locus and regulates the expression of neighboring genes on the paternal allele. We found that disruption of Kcnq1 results in reduced Kcnq1ot1 expression as well as the increased expression of cyclin-dependent kinase inhibitor 1C (Cdkn1c), an imprinted gene that encodes a cell cycle inhibitor, only when the mutation is on the paternal allele. Furthermore, histone modification at the Cdkn1c promoter region in pancreatic islets was found to contribute to this phenomenon. Our observations suggest that the Kcnq1 genomic region directly regulates pancreatic β-cell mass and that genomic imprinting may be a determinant of the onset of diabetes mellitus.

Although recent genome-wide association studies (GWAS) have been extremely successful, it remains a big challenge to functionally annotate disease‑associated single nucleotide polymorphisms (SNPs), as the majority of these SNPs are located in non‑coding regions of the genome. In this study, we described a novel strategy for identifying the proteins that bind to the SNP‑containing locus in an allele‑specific manner and successfully applied this method to SNPs in the type 2 diabetes mellitus susceptibility gene, potassium voltage‑gated channel, KQT‑like subfamily Q, member 1 (KCNQ1). DNA fragments encompassing SNPs, and risk or non‑risk alleles were immobilized onto the novel nanobeads and DNA‑binding proteins were purified from the nuclear extracts of pancreatic β cells using these DNA‑immobilized nanobeads. Comparative analysis of the allele-specific DNA-binding proteins indicated that the affinities of several proteins for the examined SNPs differed between the alleles. Nuclear transcription factor Y (NF‑Y) specifically bound the non‑risk allele of the SNP rs2074196 region and stimulated the transcriptional activity of an artificial promoter containing SNP rs2074196 in an allele‑specific manner. These results suggest that SNP rs2074196 modulates the affinity of the locus for NF‑Y and possibly induces subsequent changes in gene expression. The findings of this study indicate that our comparative method using novel nanobeads is effective for the identification of allele‑specific DNA‑binding proteins, which may provide important clues for the functional impact of disease‑associated non‑coding SNPs.

Aims/hypothesis: The plasticity of adult somatic cells allows for their dedifferentiation or conversion to different cell types, although the relevance of this to disease remains elusive. Perturbation of beta cell identity leading to dedifferentiation may be implicated in the compromised functions of beta cells in diabetes, which is a current topic of islet research. This study aims to investigate whether or not v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker, is involved in maintaining mature beta cell phenotypes., Methods: The fate and gene expression of beta cells were analysed in Mafa knockout (KO) mice and mouse models of diabetes in which the expression of MafA was reduced in the majority of beta cells., Results: Loss of MafA reduced the beta to alpha cell ratio in pancreatic islets without elevating blood glucose to diabetic levels. Lineage tracing analyses showed reduced/lost expression of insulin in most beta cells, with a minority of the former beta cells converted to glucagon-expressing cells in Mafa KO mice. The upregulation of genes that are normally repressed in mature beta cells or transcription factors that are transiently expressed in endocrine progenitors was identified in Mafa KO islets as a hallmark of dedifferentiation. The compromised beta cells in db/db and multiple low-dose streptozotocin mice underwent similar dedifferentiation with expression of Mafb, which is expressed in immature beta cells., Conclusions/interpretation: The maturation factor MafA is critical for the homeostasis of mature beta cells and regulates cell plasticity. The loss of MafA in beta cells leads to a deeper loss of cell identity, which is implicated in diabetes pathology.

Chemotherapy-induced nausea and vomiting is a serious adverse side-effect of anthracycline-based chemotherapy regimens, in patients with breast cancer. A combination of three drugs, 5-hydroxytryptamine (5-HT 3 ) receptor antagonist, aprepitant and dexamethasone, is recommended for antiemetic therapy. Palonosetron (PALO), a novel 5-HT 3 receptor antagonist has been identified to be effective against delayed nausea and vomiting. In this study, the results of PALO for patients who received anthracycline-based chemotherapy were compared with that of granisetron (GRA) using a crossover study design. This study evaluated the efficacy of antiemetics in the first cycle of chemotherapy, as well as the second and third cycles. A total of 21 patients and 19 patients were assigned to PALO and GRA treatment groups during the first cycle of chemotherapy, respectively. The patients switched to the other antiemetic drug for the second chemotherapy cycle (PALO followed by GRA or GRA followed by PALO). The patients could select PALO or GRA antiemetics for the third cycle, according to their preference. A total of 21 patients selected PALO and 18 patients selected GRA in the third cycle, and one patient was withdrawn from the study as their third cycle questionnaire was not obtained. No significant differences between PALO and GRA were identified in first and second cycles. However, during the third cycle, a significant difference was observed in acute-phase complete control of emetic events between the PALO and GRA groups, which was defined as no emetic episode, no additional antiemetic treatment and no more than mild nausea, between PALO and GRA. These results demonstrated that changing antiemetics may affect the efficacy of antiemetics. This study indicates that alteration of antiemetic regimens, including drug combination and order, may improve the efficacy of antiemetic treatment.

MafA and MafB are basic leucine zipper transcription factors expressed in mature pancreatic β- and α-cells, respectively. MafA is not only an insulin gene transcription factor but is also critical for the maturation and maintenance of β-cell function, whereas MafB is expressed in immature β-cells during development and in compromised β-cells in diabetes. In this study, we developed a mouse model to easily trace the promoter activity of MafA in β-cells as a tool for studying β-cell differentiation, maturation, regeneration and function using the expression of the fluorescent protein Kusabira Orange (KOr) driven by the BAC-mafA promoter. The expression of KOr was highly restricted to β-cells in the transgenic pancreas. By crossing MafA-KOr mice with MafB(GFP/+) reporter mice, simultaneous monitoring of MafA and MafB expressions in the isolated islets was successfully performed. This system can be a useful tool for examining dynamic changes in the differentiation and function of pancreatic islets by visualizing the expressions of MafA and MafB.

The postnatal proliferation and maturation of insulin-secreting pancreatic β-cells are critical for glucose metabolism and disease development in adults. Elucidation of the molecular mechanisms underlying these events will be beneficial to direct the differentiation of stem cells into functional β-cells. Maturation of β-cells is accompanied by increased expression of MafA, an insulin gene transcription factor. Transcriptome analysis of MafA knockout islets revealed MafA is required for the expression of several molecules critical for β-cell function, including Glut2, ZnT8, Granuphilin, Vdr, Pcsk1 and Urocortin 3, as well as Prolactin receptor (Prlr) and its downstream target Cyclin D2 (Ccnd2). Inhibition of MafA expression in mouse islets or β-cell lines resulted in reduced expression of Prlr and Ccnd2, and MafA transactivated the Prlr promoter. Stimulation of β-cells by prolactin resulted in the phosphorylation and translocation of Stat5B and an increased nuclear pool of Ccnd2 via Prlr and Jak2. Consistent with these results, the loss of MafA resulted in impaired proliferation of β-cells at 4 weeks of age. These results suggest that MafA regulates the postnatal proliferation of β-cells via prolactin signaling.

The luciferase reporter system is useful for the assessment of various biological processes in vivo. The transcription factor pancreatic and duodenal homeobox 1 (Pdx1) is critical for the formation and the function of pancreatic β-cells. A novel reporter system using secreted Gaussia princeps luciferase (GLuc) under the control of a Pdx1 promoter was generated and activated in rat and mouse β-cell lines. This Pdx1-GLuc construct was used as a transgene for the generation of reporter mice to monitor Pdx1 promoter activity in vivo via the measurement of secreted GLuc activity in a small aliquot of blood. Significantly increased plasma GLuc activity was observed in Pdx1-GLuc mice. Analysis of Pdx1-GLuc mice by bioluminescence imaging, GLuc reporter assays using homogenates of various organs, and immunohistochemistry revealed that GLuc expression and activity were exponentially higher in pancreatic β-cells than in pancreatic non-β-cells, the duodenum, and other organs. In addition, GLuc activity secreted into the culture medium from islets isolated from Pdx1-GLuc mice correlated with the number of islets. The transplantation of Pdx1-GLuc islets into severe combined immunodeficiency mice elevated their plasma GLuc activity. Conversely, a partial pancreatectomy in Pdx1-GLuc mice reduced plasma GLuc activity. These results suggest that a secreted luciferase reporter system in vivo enables not only the monitoring of promoter activity but also a quantitative and minimally invasive assessment of physiological and pathological changes in small cell masses, such as pancreatic β-cells.

Chronic intractable pain caused by postherpetic neuralgia (PHN) can be alleviated by intrathecal (i.t.) steroid therapy. We investigated the possibility that interleukin-6 (IL-6) release in an in vitro system could be a potential marker for evaluating the effectiveness of i.t. steroid therapy in PHN patients. We studied 32 patients who received a course of i.t. injection of water-soluble dexamethasone. Their therapeutic index was calculated as such: ((Pain score before treatment - Pain score after treatment)÷Pain score before treatment)×100%, and they were divided into two groups, therapy effective (index>50%) and ineffective (index<50%). Cerebrospinal fluid (CSF) from the patients was used to stimulate cultures of T98G glioblastoma cells, and the subsequent IL-6 release was measured by enzyme-linked immunosorbent assay (ELISA). Our results showed that the CSF triggered IL-6 release from T98G cells in a volume-dependent manner. IL-6 release was significantly lower when using CSF from the therapy effective patient group (p<0.001) compared to the therapy ineffective group. In particular, therapy effective patients had less IL-6 release even before treatment as compared to therapy ineffective patients. In the therapy effective group, in vitro steroid treatment suppressed the CSF's IL-6 releasing effect almost completely, whereas in the therapy ineffective group, the IL-6 release was significantly reduced but remained detectable. These in vitro tests may provide an objective evaluation on the efficacy of i.t. steroid therapy administered to PHN patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR). It is widely accepted that the progression of DR is mainly due to a local imbalance of pro- versus anti-angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithelial (RPE) cells produced pro-angiogenic factors under high glucose (HG) conditions in vitro., Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium from retinal pigment epithelium cells (ARPE-19) grown in HG medium and assessed for tube formation. Based on the expression profiles of ARPE-19, we investigated whether ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions using cultured human RE cells as the test system for experiments with recombinant protein, conditioned medium from ARPE-19 and RNA interference (RNAi)., Results: The conditioned medium from ARPE-19 cultured under HG conditions promoted tube formation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all of the elements of angiogenesis in human RE cells in vitro. The results of experiments using conditioned medium from ARPE-19 combined with RNAi demonstrated that ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions., Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angiogenic activity on RE cells. Our results are unique and may potentially add a new candidate to the long list of molecules involved in diabetic retinopathy., (© 2013 The Authors. Acta Ophthalmologica © 2013 Acta Ophthalmologica Scandinavica Foundation.)

Postherpetic neuralgia (PHN) is the most frequent complication of herpes zoster, and the risk of it increases with age. By comparing proteomes of the cerebrospinal fluid (CSF) before and after the treatment, it may be possible to identify proteins that play a role in PHN and to predict responses to various treatments. To address this issue, we enrolled eight outpatients with PHN over 55 years of age and treated them with intrathecal methylprednisolone and lidocaine four times every week, collecting CSF samples before the treatment at each visit. We used 2D DIGE to investigate differentially expressed proteins in the CSF before and after repetitive treatments individually. Of 145 differentially expressed spots, the levels of nine proteins were decreased by the treatment including lipocalin-type prostaglandin D synthase (L-PGDS), and five were increased by it. The time course of alterations in the L-PGDS concentration in the CSF of each patient, detected by a pairwise and sandwich ELISA by SPR constructed here was well correlated with that by 1DE Western blots with anti-L-PGDS antibody, but was not related with that of the pain relief. The present study demonstrates that the real-time ELISA was precise and sensitive enough to measure L-PGDS in the CSF and that the steroid treatment decreased the L-PGDS concentration in CSF., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The adult pancreas has considerable capacity to regenerate in response to injury. We hypothesized that after partial pancreatectomy (Px) in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a reproducible dedifferentiation and redifferentiation. We support this hypothesis by the detection of an early loss of the ductal differentiation marker Hnf6 in the mature ducts, followed by the transient appearance of areas composed of proliferating ductules, called foci of regeneration, which subsequently form new pancreatic lobes. In young foci, ductules express markers of the embryonic pancreatic epithelium - Pdx1, Tcf2 and Sox9 - suggesting that these cells act as progenitors of the regenerating pancreas. The endocrine-lineage-specific transcription factor Neurogenin3, which is found in the developing embryonic pancreas, was transiently detected in the foci. Islets in foci initially resemble embryonic islets in their lack of MafA expression and lower percentage of beta-cells, but with increasing maturation have increasing numbers of MafA(+) insulin(+) cells. Taken together, we provide a mechanism by which adult pancreatic duct cells recapitulate aspects of embryonic pancreas differentiation in response to injury, and contribute to regeneration of the pancreas. This mechanism of regeneration relies mainly on the plasticity of the differentiated cells within the pancreas.

The transcription factor MafA regulates glucose-responsive expression of insulin. MafA-deficient mice have a normal proportion of insulin+ cells at birth but develop diabetes gradually with age, suggesting that MafA is required for maturation and not specification of pancreatic beta-cells. However, several studies show that ectopic expression of MafA may have a role in specification as it induces insulin+ cells in chicken gut epithelium, reprograms adult murine acinar cells into insulin+ cells in combination with Ngn3 and Pdx1, and triggers the lens differentiation. Hence, we examined whether MafA can induce specification of beta-cells during pancreatic development. When the MafA transgene is expressed in Pdx1+ pancreatic progenitors, both pancreatic mass and proliferation of progenitors are reduced, at least partially due to induction of cyclin kinase inhibitors p27 and p57. Expression of MafA in Pdx1+ cells until E12.5 was sufficient to cause these effects and to disproportionately inhibit the formation of endocrine cells in the remnant pancreas. Thus, in mice, MafA expression in Pdx1+ pancreatic progenitors is not sufficient to specify insulin+ cells but in fact deters pancreatic development and the differentiation of endocrine cells. These findings imply that MafA should be used to enhance maturation, rather than specification, of beta-cells from stem/progenitor cells.

Mammalian MafA/RIPE3b1 is an important glucose-responsive transcription factor that regulates function, maturation, and survival of beta-cells. Increased expression of MafA results in improved glucose-stimulated insulin secretion and beta-cell function. Because MafA is a highly phosphorylated protein, we examined whether regulating activity of protein kinases can increase MafA expression by enhancing its stability. We demonstrate that MafA protein stability in MIN6 cells and isolated mouse islets is regulated by both p38 MAPK and glycogen synthase kinase 3. Inhibiting p38 MAPK enhanced MafA stability in cells grown under both low and high concentrations of glucose. We also show that the N-terminal domain of MafA plays a major role in p38 MAPK-mediated degradation; simultaneous mutation of both threonines 57 and 134 into alanines in MafA was sufficient to prevent this degradation. Under oxidative stress, a condition detrimental to beta-cell function, a decrease in MafA stability was associated with a concomitant increase in active p38 MAPK. Interestingly, inhibiting p38 MAPK but not glycogen synthase kinase 3 prevented oxidative stress-dependent degradation of MafA. These results suggest that the p38 MAPK pathway may represent a common mechanism for regulating MafA levels under oxidative stress and basal and stimulatory glucose concentrations. Therefore, preventing p38 MAPK-mediated degradation of MafA represents a novel approach to improve beta-cell function.

During pancreatic development insulin(+) cells co-express the transcription factors MafB and Pax6, and transition from a MafA(-) to MafA(+) state. To examine the role of Pax6 and MafB in the development of beta-cells, we analyzed embryonic pancreata from Pax6- and MafB-deficient mice. Pax6 deficiency, as manifest in the Pax6(Sey-Neu) allele, reduced not only the number of cells expressing insulin or glucagon, but also the number of MafB, PDX-1 and MafA expressing cells. We show that MafB can directly activate expression of insulin and glucagon, and a MafB protein engineered to contain N248S mutation in the MafB (kr(ENU)) results in significantly reduced activation. Furthermore, pancreata from MafB deficient (kr(ENU)/kr(ENU)) mice exhibited reduced number of cells expressing insulin, glucagon, PDX-1 and MafA, with only a minor reduction in MafB expressing cells. MafB deficiency does not affect endocrine specification but does affect the lineage commitment of the endocrine cells and their maturation. Similar to Pax6 deficient mice, MafB deficient mice showed reductions both in insulin and glucagon expressing cells and in the ability of MafB and PDX-1 expressing cells to activate expression of these hormones. However, MafB deficient mice exhibited no effect on Pax6 expression. These results suggest that MafB may function as a downstream mediator of Pax6 in regulating the specification of insulin and glucagon expressing cells. Interestingly, the remaining insulin(+) cells in these knockouts preferentially express Hb9, suggesting the existence of an alternate pathway for the generation of insulin expressing cells, even in the absence of Pax6 and MafB function. Thus, Pax6 acts upstream of MafB, which in turn may trigger the expression of insulin and regulate the PDX-1 and MafA expression required for beta-cell maturation.

Background: D-Serine is a coagonist for the glycine-binding site of the N-methyl-D-aspartate receptors and has been implicated in various neuropsychiatric functions such as learning, memory, and nociception, as well as schizophrenia and Alzheimer disease. We developed an HPLC method for D- and L-serine in cerebrospinal fluid (CSF)., Methods: The dabsylated racemic serine peak, automatically collected using a previously reported HPLC separation process for CSF amino acids, was desalted and subjected to a chiral resolution HPLC step with a Sumichiral column using an ultraviolet-visible detector., Results: The limits of quantification (signal-to-noise ratio = 10) for D- and L-serine were 0.8 and 1.3 micromol/L, respectively. The mean imprecision values (CVs) for within-day measurements of D- and L-serine were 2.1% and 1.8%, respectively, and for between-day were 6.2% and 6.6%. Mean recovery of CSF serine (sum of D-serine + L-serine) applied to the Sumichiral column was 87%. The mean (SD) d-serine concentrations in 45 CSF samples obtained from 16 patients with chronic pain due to degenerative osteoarthritis of the knees, 16 with postherpetic neuralgia, and 13 with no pain were, respectively, 3.97 (0.44), 1.85 (0.21), and 2.72 (0.32) micromol/L., Conclusion: D- and L-serine can be quantified with ultraviolet-visible detection of dabsyl derivatives. The dabsyl derivatives are stable and allow duplicate analysis of CSF samples in multisample runs.

We established a simultaneous bioluminescent assay utilizing aequorin (Aq) and biotinylated firefly luciferase (b-Luc); furthermore, we developed a highly sensitive and rapid tandem bioluminescent immunoassay (BLIA) involving the Aq-labeled Fab fragment and b-Luc-streptavidin complex. Minimum detection limits of Aq and b-Luc were 9.4x10(-21) mol assay(-1) (blank + 3 S.D.) and 3.6x10(-19) mol assay(-1) (blank + 3 S.D.), respectively. Measurements of two luminescent proteins were completed in 4 s with a single assay medium. In this study, prostatic acid phosphatase (PAP) and prostate specific antigen (PSA), which served as analytes, were measured in the tandem BLIA. PAP and PSA were detected by the Aq-labeled anti-Dig Fab fragment and b-Luc-streptavidin complex, respectively. The measurable ranges of PAP and PSA were 0.04-100 and 0.2-200 ng mL(-1), respectively. This technique was also applied to the simultaneous measurement of PSA and alpha-fetoprotein (AFP). Measurable ranges of PSA and AFP were 0.2-200 and 1.95-1000 ng mL(-1), respectively. Levels of PAP and PSA or PSA and AFP in human serum could be accurately determined with the proposed BLIA. Satisfactory correlations were observed between results obtained from the proposed BLIA and those derived from commercial kits.

Major insulin gene transcription factors, such as PDX-1 or NeuroD1, have equally important roles in pancreatic development and the differentiation of pancreatic endocrine cells. Previously, we identified and cloned another critical insulin gene transcription factor MafA (RIPE3b1) and reported that other Maf factors were expressed in pancreatic endocrine cells. Maf factors are important regulators of cellular differentiation; to understand their role in differentiation of pancreatic endocrine cells, we analyzed the expression pattern of large-Maf factors in the pancreas of embryonic and adult mice. Ectopically expressed large-Maf factors, MafA, MafB, or cMaf, induced expression from insulin and glucagon reporter constructs, demonstrating a redundancy in their function. Yet in adult pancreas, cMaf was expressed in both alpha- and beta-cells, and MafA and MafB showed selective expression in the beta- and alpha-cells, respectively. Interestingly, during embryonic development, a significant proportion of MafB-expressing cells also expressed insulin. In embryos, MafB is expressed before MafA, and our results suggest that the differentiation of beta-cells proceeds through a MafB+ MafA- Ins+ intermediate cell to MafB- MafA+ Ins+ cells. Furthermore, the MafB to MafA transition follows induction of PDX-1 expression (Pdx-1(high)) in MafB+ Ins+ cells. We suggest that MafB may have a dual role in regulating embryonic differentiation of both beta- and alpha-cells while MafA may regulate replication/survival and function of beta-cells after birth. Thus, this redundancy in the function and expression of the large-Maf factors may explain the normal islet morphology observed in the MafA knockout mice at birth.