Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na/Ca exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca²⁺ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca²⁺ concentration [Ca²⁺]_i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca²⁺-mediated [Ca²⁺]_i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca²⁺ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca²⁺ entry. KB-R7943, a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and/or replacement of Na⁺ with NMDG⁺ inhibited thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca²⁺ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca²⁺ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca²⁺ influx pathways requiring Ca²⁺ signals for its effect on fibroblast proliferation.     

Osteocyte network; a negative regulatory system for bone mass augmented by the induction of Rankl in osteoblasts and Sost in osteocytes at unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.     

Structural changes in the digestive glands of larval Antarctic krill (<Emphasis Type="Italic">Euphausia superba</Emphasis>) during starvation

The effects of starvation on ultrastructure of digestive gland cells were studied in furcilia larvae of Antarctic krill (Euphausia superba: hereafter krill). Under laboratory conditions, larvae were starved for 0, 5, 10, 15, 20 and 25 days, and their R-cells were investigated by transmission electron microscope. R-cells are thought to play a role in the storage and absorption of nutrients. In fed larvae, numerous mitochondria scattered homogenously, and densely packed microvilli were observed on the apical surface of R-cells. After 5 days of starvation, mitochondria were swollen and were found concentrated in the apical region in R-cells. A decrease in cell volume and an increase in thickness of the basal lamina with many irregular infoldings were observed after 10–15 days of starvation. Lipid droplets were rarely found in the R-cells regardless of whether larvae had been fed or starved suggesting an inability to store lipid. Without the ability to store energy in the form of lipid, survival would be dependant on sourcing continuous food until maturation.     

Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.     

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes

Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles’ medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, α-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was α-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. γ-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.     

Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia

Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3 ^?/?) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3 ^?/? mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3 ^?/? mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3 ^?/? mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3 ^?/? mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm–egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm–egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.