催产素对大鼠背根神经节分离细胞GABA激活电流的调制作用

在急性分离的大鼠背根神经节（ｄｏｒｓａｌ ｒｏｏｔ ｇａｎｇｌｉｏｎ,ＤＲＧ）细胞上,应用全细胞膜片箝技术观察了预知催产素（ｏｘｙｔｏｃｉｎ,ＯＴ）对ＧＡＢＡ激活电流的调制作用。结果如下：（１）大多数细胞（４８／５２,９０．５％）对ＧＡＢＡ敏感。（２）ＯＴ可引起５１．３％（２０／３９）的受检细胞出现外向膜电流;４３．６％（１７／３９）无明显膜反应;５．１％（２／３９）出现内向膜电流。（３）预加ＯＴ     

脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用

在乌拉坦麻醉的成年ＳＤ大鼠上,用玻璃微电极细胞外记录的方法,观察了脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用。结果发现：脑室内注射一氧化氮供体硝普钠对下丘脑室旁核中的加压素神经元产生剂量依赖性抑制作用;脑室内注射一氧化氮合酶抑制剂对加压素神经元也产生抑制作用。上述两种药物对催产素神经元均无作用。这些结果提示：一氧化氮可能在调节加压素和催产素神经元活动中起着不同的作用。     

In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid

Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10(-4) M retinoic acid for 3 h differentiated into pancreas-like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus-like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas-specific molecular markers, XIHbox8 and insulin, were induced in retinoic acid-treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas-like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.     

Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early development

In the eggs and embryos of sea urchins, the activity of protein phosphatase type 2A (PP2A) increased during the developmental period between fertilization and the morula stage, decreased after the prehatching blastula stage and increased again after hatching. The PP2A activity changed keeping pace with alteration to the activities of cAMP-dependent protein kinase (A kinase), Ca2+/calmodulin-dependent protein kinase (CaM kinase) and casein kinase. Probably, PP2A contributes to the quick turning off of cellular signals because of protein phosphorylation. The activity of protein phosphatase type 1 (PP1) was not detectable up to the morula stage and appreciably increased thereafter. In the isolated nucleus fraction, specific activities of PP1 and PP2A were higher than in whole embryos at all stages in early development. Exponential increase in the number of nuclei because of egg cleavage probably makes PP1 activity detectable in whole embryos after the morula stage. In isolated nuclei, the activities of PP1 and PP2A appreciably decreased after hatching, whereas the activities of A kinase, Ca2+/phospholipid-dependent protein kinase (C kinase) and CaM kinase, as well as casein kinase, became higher. In nuclei, cellular signals caused by protein phosphorylation after hatching do not seem to be turned off by these protein kinases so quickly as before hatching. The PP1 and PP2A in nuclei also seem to contribute to the elimination of signal noise.     

Ectopic adenoviral vector-directed expression of Sema3A in organotypic spinal cord explants inhibits growth of primary sensory afferents

Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF-dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc-DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949-959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord.     

The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells

The spinal muscular atrophy protein, SMN, is a cytoplasmic protein that is also found in distinct nuclear structures called "gems." Gems are closely associated with nuclear coiled bodies and both may have a direct role in snRNP maturation and pre-RNA splicing. There has been some controversy over whether gems and coiled bodies colocalize or form adjacent/independent structures in HeLa and other cultured cells. Using a new panel of antibodies against SMN and antibodies against coilin-p80, a systematic and quantitative study of adult differentiated tissues has shown that gems always colocalize with coiled bodies. In some tissues, a small proportion of coiled bodies (<10%) had no SMN, but independent or adjacent gems were not found. The most striking observation, however, was that many cell types appear to have neither gems nor coiled bodies (e.g., cardiac and smooth muscle, blood vessels, stomach, and spleen) and this expression pattern is conserved across human, rabbit, and pig species. This shows that assembly of distinct nuclear bodies is not essential for RNA splicing and supports the view that they may be storage sites for reserves of essential proteins and snRNPs. Overexpression of SMN in COS-7 cells produced supernumerary nuclear bodies, most of which also contained coilin-p80, confirming the close relationship between gems and coiled bodies. However, when SMN is reduced to very low levels in type I SMA fibroblasts, coiled bodies are still formed. Overall, the data suggest that gem/coiled body formation is not determined by high cytoplasmic SMN concentrations or high metabolic activity alone and that a differentiation-specific factor may control their formation.     

Stability and nuclear distribution of mammalian replication protein A heterotrimeric complex

Replication protein A (RPA), a stable complex of three polypeptides, is the single-stranded DNA-binding protein essential for DNA replication in eukaryotic cells. Previous studies of the subcellular distribution and stability of the RPA heterotrimer during the mammalian cell cycle have produced conflicting results. Here, we present evidence that these inconsistencies can be accounted for by the presence of an extractable pool of soluble RPA within the nucleus. Indirect immunofluorescence experiments in both CHO and HeLa cells showed that all three RPA subunits associated specifically with sites of ongoing DNA synthesis, similar to the replication fork protein proliferating cell nuclear antigen. Furthermore, we found no evidence for disassembly of the chromatin-bound heterotrimeric RPA complex in vivo. Our results are consistent with a role for RPA in the initiation and elongation steps of replication, as previously defined in the viral in vitro replication systems.     

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.     

The human circadian clock and aging

The suprachiasmatic nucleus (SCN) of the hypothalamus is implicated in the timing of a wide variety of circadian processes. Since the environmental light-dark cycle is the main zeitgeber for many of the rhythms, photic information may have a synchronizing effect on the endogenous clock of the SCN by inducing periodic changes in the biological activity of certain groups of neurons. By studying the brains obtained at autopsy of human subjects, marked diurnal oscillations were observed in the neuropeptide content of the SCN. Vasopressin, for example, one of the most abundant peptides in the human SCN, exhibited a diurnal rhythm, with low values at night and peak values during the early morning. However, with advancing age, these diurnal fluctuations deteriorated, leading to a disrupted cycle with a reduced amplitude in elderly people. These findings suggest that the synthesis of some peptides in the human SCN exhibits an endogenous circadian rhythmicity, and that the temporal organization of these rhythms becomes progressively disturbed in senescence. (Chronobiology International, 17(3), 245-259, 2000)     

Primary Vagal Projection to the Contralateral Non-NTS Region in the Embryonic Chick Brainstem Revealed by Optical Recording

Using multiple-site optical recording with the voltage-sensitive dye, NK2761, we found that vagus nerve stimulation in the embryonic chick brainstem elicits postsynaptic responses in an undefined region on the contralateral side. The characteristics of the contralateral optical signals suggested that they correspond to the monosynaptic response that is related to the vagal afferent fibers. The location of the contralateral response was different from the vagal motor nucleus (the dorsal motor nucleus of the vagus nerve) and sensory nucleus (the nucleus of the tractus solitarius), and other brainstem nuclei that receive primary vagal projection. These results show that the vagus nerve innervates and makes functional synaptic connections in a previously unreported region of the brainstem, and suggest that sensory information processing mediated by the vagus nerve is more complex than expected.