Effects of mechanical stretching on caspase and IGF-1 expression during the proliferation process of myoblasts

It has been reported that the synthesis, degradation, and metabolism of muscle proteins in myoblasts, as well as the proliferation and differentiation of cells, are influenced by various related to extracellular signaling molecules, such as neural transmitters, growth factors, and hormones, when muscle tissue has been exposed to mechanical stimulation. However, reports regarding the expression of growth factors during mechanical stimulation of myoblasts are few, and many questions remain unanswered. We examined the mRNA expression of insulin-like growth factor 1 (IGF-1) in myoblasts subjected to mechanical stretching in vitro. In addition, apoptosis caused by intracellular stress has been reported to occur during muscle development at the embryonic stage. To clarify the expression of intracellular stress factors, we here investigated related gene expression. Expression of IGF-1 increased in the early stage of cell stretching, followed by a decrease in the late stage. This suggests that mechanical stimulation resulted in an immediate increase in IGF-1 expression, followed by a decrease as cells acclimated to the inducing environment. Caspase was significantly expressed in a stretch group at 12 hours after the beginning of mechanical stimulation, compared with a control group. This suggests that cellular proliferation is also regulated by intracellular stress factors involving the endoplasmic reticulum, mitochondria, and other organelles during the process of muscle proliferation and differentiation.     

Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus

Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 μM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.     

Generation of cloned mice by direct nuclear transfer from natural killer T cells

Cloning mammals by nuclear transfer (NT) remains inefficient. One fundamental question is whether clones have really been derived from differentiated cells rather than from rare stem cells present in donor-cell samples. To date, cells, such as mature lymphocytes, with genetic differentiation markers have been cloned to generate mice only via a two-step NT involving embryonic stem (ES) cell generation and tetraploid complementation [1, 2 and 3]. Here, we show that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT. The pups and their placentas possessed the rearranged TCR loci specific for NKT cells. The NKT-cell-cloned embryos had a high developmental potential in vitro: Most (71%) developed to the morula/blastocyst stage, in marked contrast to embryos from peripheral blood T cells (12%; p < 1 x 10(-25)). Furthermore, ES cell lines were efficiently established from these NKT-cell blastocysts. These findings clearly indicate a high level of plasticity in the NKT-cell genome. Thus, differentiation of the genome is not always a barrier to NT cloning for either reproductive or therapeutic purposes, so we can now postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.     

活体冷冻固定及冷冻置换的小鼠睾丸组织内血清白蛋白和IgG的免疫组织化学定位

目的了解小鼠睾丸组织内血清白蛋白和IgG渗透性的时相性变化,揭示支持细胞间紧密连接的屏障功能。方法运用活体内冷冻固定技术对小鼠睾丸进行快速冷冻固定和冷冻置换,并作石蜡包埋。5μm厚连续切片用于抗鼠血清白蛋白和IgG的免疫组化染色和H．E染色。结果H．E染色切片显示距冷冻组织表面300-400μm深度范围内的精曲小管结构保存完好,无明显冰晶形成所致的人工损伤。血清白蛋白和IgG免疫反应产物主要定位于精曲小管管周肌样细胞周围,以及间质细胞之间和毛细血管内,部分免疫反应产物呈拱型出现在精曲小管上皮内,并严格限定在基底室,沿精原细胞表面分布。拱型免疫反应产物的数量与精曲小管上皮周期的发育时相密切相关。结论活体内快速冷冻固定和冷冻置换结合血清白蛋白和IgG的免疫组化方法,可很好地保存精曲小管组织结构并清楚地揭示支持细胞间紧密连接结构的时相变化。     

Novel small RNA-encoding genes in the intergenic regions of Bacillus subtilis

Small, non-coding RNAs (ncRNAs) perform diverse functions in a variety of organisms, but few ncRNAs have been identified in Bacillus subtilis. To search the B. subtilis genome for genes encoding ncRNAs, we focused on 123 intergenic regions (IGRs) over 500 bp in length and analyzed expression from these regions. Seven IGRs termed bsrC, bsrD, bsrE, bsrF, bsrG, bsrH and bsrI expressed RNAs smaller than 380 nt. All small RNAs except BsrD RNA were expressed in transformed Escherichia coli cells harboring a plasmid with PCR-amplified IGRs of B. subtilis, indicating that their own promoters independently express small RNAs. Under the non-stressed condition, depletion of the genes for the small RNAs did not affect growth. Although their functions are unknown, gene expression profiles at several time points showed that most of the genes except for bsrD were expressed during the vegetative phase (4-6 h), but undetectable during the stationary phase (8 h). Mapping the 5' ends of the 6 small RNAs revealed that the genes for BsrE, BsrF, BsrG, BsrH, and BsrI RNAs are preceded by a recognition site for RNA polymerase sigma factor sigma(A). These small RNAs might lack an SD sequence and exert their actions as ncRNAs.     

Involvement of Fas/Fas ligand in the induction of apoptosis in chronic sialadenitis of minor salivary glands including Sjögren's syndrome

The role of apoptosis and contribution of Fas/FasL systems in the pathogenesis of Sjogren's syndrome (SS) are still controversial. With serial sections, we explored apoptosis assessed by the dUTP nick end labeling (TUNEL) method and expression of Fas and FasL by immunohistochemistry, and compared their distribution in minor salivary gland (MSG) of SS and sialolithiasis (SIL) patient tissues. Fas and FasL were co-localized in ductular and acinar cells of SS and SIL TUNEL+ cells co-distributed with the Fas and FasL expressing cells in ductular and acinar cells of SS in the vicinity of lymphocytic infiltration, while not in those of SIL Moreover, to morphologically confirm apoptosis, we identified TUNEL-positive(+) cells in the MSGs of SS at the ultra structural level by applying an inversion method to paraffin-embedded sections stained by TUNEL method. Surprisingly, these cells did not show characteristic apoptotic figures although TUNEL products were deposited on the hyperchromatin of acinar and ductular cells. On the other hand, acinar and ductular cells of SIL included clusters of TUNEL+ apoptotic bodies as did those cells by phagocytosis or having fallen into the ductular lumen. These findings suggest that Fas and FasL expressed in ducts and acini of chronic sialadenitis in SS patients induce apoptosis, possibily in an autocrine and/or paracrine manner.