[Prevalence, awareness, treatment and control of hypertension in elderly residents in Hebei province].

Objective: To understand the prevalence, awareness, treatment and control of hypertension in elderly residents in Hebei province. Methods: Elderly residents aged ≥60 were selected though multistage clustering sampling during August to December, 2015. Design based methods were adopted to analyze the prevalence, awareness, treatment and control of hypertension in local residents of Hebei. Results: A total of 2 501 elderly adults were included in the study. The overall prevalence rate of hypertension was 63.7% (58.3% in males, 69.0% in females), the awareness rate of hypertension was 42.4% (35.7% in males, 48.0% in females), the treatment rate was 38.2% (32.0% in males, 43.3% in females), and the control rate was 9.0% (8.1% in males, 9.7% in females). The results of multivariate analysis indicated that age, sex, degree of education, BMI and central obesity were the factors influencing the prevalence, awareness, treatment and control of hypertension in elderly population in Hebei. Conclusions: The prevalence of hypertension was high, but the rates of awareness, treatment and control of hypertension were low in elderly residents in Hebei. The influences of overweight, obesity and central obesity on hypertension were significant in the elderly. It is necessary to standard the management of hypertension and reduce the risk factors for hypertension in elderly population to improve the control of hypertension.

Evaluating the diagnostic and prognostic value of long non-coding RNA SNHG15 in pancreatic ductal adenocarcinoma.

OBJECTIVE: Long non-coding RNA SNHG15 (SNHG15) has been reported to play very important roles in the malignancy behaviors of various tumors, including pancreatic ductal adenocarcinoma (PDAC). However, its clinical significance in PDAC remains largely unclear. The aim of this study was to investigate whether the aberrant expression of SNHG15 can be used as potential prognostic and diagnostic markers of human PDAC. MATERIALS AND METHODS: TaqMan Real Time-PCR was performed to investigate the expression of SNHG15 in PDAC tissues and serum samples. Receiver operator characteristic (ROC) analysis was applied to obtain the diagnostic utility of SNHG15. Association between SNHG15 levels and clinicopathological factors was analyzed. Kaplan-Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. RESULTS: SNHG15 levels were significantly up-regulated in both sera and tumors tissues from PDAC patients. ROC curve analysis revealed that SNHG15 may be a potential biomarker for differentiating PDAC tissues from normal pancreatic tissues, and the plasma levels of SNHG15 may be a potential biomarker for differentiating PDAC patients from healthy controls. Clinicopathologic analysis revealed that high SNHG15 expression was associated with tumor differentiation (p = 0.000), lymph node metastasis (p = 0.001) and tumor stage (p = 0.005). Furthermore, patients with high SNHG15 expression had a shorter overall survival compared with the low SNHG15 expression group (p = 0.003). Also, Cox multivariate analyses confirmed that SNHG15 expression was an independent prognostic factor in PDAC (p < 0.004). CONCLUSIONS: Our study firstly indicated the potential value of SNHG15 as an important biomarker for the diagnosis and prognosis prediction of PDAC.

Interleukin-27 augments the inhibitory effects of sorafenib on bladder cancer cells.

Both sorafenib and interleukin-27 (IL-27) are antineoplastic drugs. This study aimed to investigate the synergistic effect of these two drugs on bladder cancer cells. HTB-9 and T24 cells were stimulated with IL-27 (50 ng/mL), sorafenib (2 µM) or the synergistic action of these two drugs. The cells without treatment acted as control. Cell proliferation, apoptosis and invasion were measured by bromodeoxyuridine assay, flow cytometry and modified Boyden chamber, respectively. Simultaneously, both modified Boyden chamber and scratch assay were used to assess cell migration. Finally, the phosphorylation levels of key kinases in the Akt/mechanistic target of rapamycin (mTOR)/mitogen-activated protein kinase (MAPK) pathway, and expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were detected by western blot analysis. Stimulation with IL-27 or sorafenib repressed proliferation, migration and invasion but promoted apoptosis, and the effects were all enhanced by the combination of these two drugs in HTB-9 cells. The effect of the combined treatment on bladder cancer cells was verified in T24 cells. Additionally, the phosphorylation levels of AKT, mTOR and MAPK as well as the expression levels of MMP-2 and MMP-9 were all decreased by a single treatment of IL-27 or sorafenib, and further decreased by the combined treatment of these two drugs. The combination of IL-27 and sorafenib inhibited proliferation, migration and invasion and promoted apoptosis of bladder cancer cells compared with mono-drug treatment. Additionally, the AKT/mTOR/MAPK pathway might be implicated in the functional effects by down-regulations of MMP-2 and MMP-9.

The mechanisms of neuronal death produced by mitochondrial toxin 3-nitropropionic acid: the roles of N-methyl-D-aspartate glutamate receptors and mitochondrial calcium overload.

Previous studies showed that 3-nitropropionic acid, an irreversible inhibitor of succinate dehydrogenase, produced neuronal death secondary to perturbed intracellular calcium homeostasis. However, the response of intramitochondrial calcium ([Ca(2+)](m)) to 3-nitropropionic acid remains unknown. In this study, we investigated the roles of and relationships among [Ca(2+)](m) overload, mitochondrial reactive oxygen species, and mitochondrial membrane depolarization in 3-nitropropionic acid-induced neuronal death. Following 1 mM 3-nitropropionic acid treatment on primary rat neuronal cultures, there was a gradual increase of [Ca(2+)](m) beginning at 2-4 h post 3-nitropropionic acid application, and a twofold increase of mitochondrial reactive oxygen species at 4 h. These were followed by mitochondrial membrane depolarization at 6-8 h post-treatment. By inhibiting [Ca(2+)](m) uptake, Ruthenium Red attenuated the production of reactive oxygen species, and prevented the 3-nitropropionic acid-induced mitochondrial membrane depolarization and 70% of apoptotic neuronal death (P<0.001). Inhibition of caspase activation attenuated the elevation of [Ca(2+)](m) (P<0.001), indicating that caspase activation plays a role in the elevation of [Ca(2+)](m). MK-801, an antagonist of N-methyl-D-aspartate (NMDA) glutamate receptors, prevented 3-nitropropionic acid-induced [Ca(2+)](m) elevation, caspase-3 activation, mitochondrial depolarization, and neuronal death. We conclude that the activation of NMDA glutamate receptor contributes to mitochondrial alterations induced by 3-nitropropionic acid. Inhibition of its activation and [Ca(2+)](m) overload with subsequent mitochondrial membrane depolarization can therefore attenuate the neuronal death induced by 3-nitropropionic acid.

Amphetamine induces differential changes in the gene expression of metabotropic glutamate receptor 5 in cultured cortical and hippocampal neurons.

Mechanisms underlying the short-term effects of amphetamine (AMPH) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) in cultured rat neurons. The cortical and hippocampal neurons were incubated with 0.1-100 microM of AMPH for 1 h or 1 microM of AMPH for 10 min to 3 h. Immunocytochemical and in situ hybridization (ISH) analyses revealed that the levels of mGluR5 immunoreactivity and mRNA in the cortical neurons were initially increased with the treatment time and dosage, to reach maximal elevations of 34 and 53% from control values following 1 h incubation of 1 microM, and then returned toward the controls. When the cortical neurons were preincubated with the antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG) to mGluRs, before treated with 1 M of AMPH for 1 h, the levels of mGluR5 protein and mRNA became 120 and 116% of control values. In hippocampal neurons, the AMPH treatment persistently upregulated the mGluR5 protein by 50-62%; however, the mRNA responded with the bell-shaped pattern to the treatment times and doses, with 20-43% increases from controls. These modifications of the receptor were reversible, since removal of AMPH resulted in regular levels of the receptor. Notably, the AMPH-generated increases in mGluR5 protein and mRNA were completely blocked by the pretreatment with cycloheximide and actinomycin D, respectively. The data indicate differential responsive patterns of mGluR5 in the cortical and hippocampal neurons to the drug perturbation. The action of AMPH may involve regulation to transcriptional and translational events in the neurons, and the activation of the MCPG-sensitive receptors.

Regulation of the subcellular distribution and gene expression of GABA(A) receptor by microtubules and microfilaments in cultured brain neurons.

Mechanisms underlying the intracellular transport of gamma-aminobutyric acid(A) receptor (GABA(A)R) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and (3)H-flunitrazepam (FNZ) binding assay were employed to determine the cell surface and intracellular distribution of the receptor. A 3-h treatment of the cells with 1 microM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABA(A)R density by about 36% and decreased that of the cell surface receptors by 18% from respective control values, whereas the 3-h incubation with 2 microM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the (3)H-FNZ binding sites of the neurons and the affinity of the ligand. Moreover, the exposure to colchicine seemed to produce a stronger cytoplasmic immunostaining of the GABA(A)R alpha subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular (3)H-FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of alpha(1)+51kDa proteins. In addition, the colchicine or cytochalasin D treatment inhibited approximately 21 or 18% of the rate of general protein synthesis in the culture. Notably, in situ hybridization assay showed that the GABA(A)R alpha(1) or alpha(2) mRNA was present in 92 +/- 2% or 94 +/- 2% of the cytochalasin D-treated neurons, both of which were higher than 71 +/- 2-74 +/- 3% of the control and colchicine-treated cells. The data suggest that by regulating the intracellular transport, the microtubular system participates in the maintenance of normal subcellular distribution of GABA(A)R in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABA(A)R subunits.

Changes in the gene expression of GABA(A) receptor alpha1 and alpha2 subunits and metabotropic glutamate receptor 5 in the basal ganglia of the rats with unilateral 6-hydroxydopamine lesion and embryonic mesencephalic grafts.

By using an animal model of parkinsonism, we examined the expression of GABA(A) receptor (R) and metabotropic glutamate receptor (mGluR) 5 in the basal ganglia after transplantation with dopamine-rich tissue. The adult rats were unilaterally lesioned by the injection of 6-hydroxydopamine to their left medial forebrain bundles. At 5-10 weeks following the dopaminergic denervation, the levels of GABA(A)R in the left caudate-putamen and globus pallidus were about 20 and 16% lower than that of the right intact (control) sides, as shown by [3H]flunitrazepam binding autoradiography on the brain sections. However, the receptor density increased to around 132 and 130% of control levels in the entopeduncular nucleus and substantia nigra pars reticulata of the lesioned sides. Furthermore, in situ hybridization analysis exhibited parallel trends of changes in the levels of the GABA(A)R alpha1 and alpha2 subunit and mGluR5 mRNAs in the neurons of the brain regions with that of the proteins detected by the binding assay. A number of the rats 5 weeks postlesion were transplanted with the ventral mesencephalon of the embryonic rat into their left striata. Five weeks later, the changes in the [3H]flunitrazepam binding seemed to be recovered by approximately 50-63% on the grafted sides of the areas. Moreover, the transplantation appeared to produce a nearly complete reversal of the lesion-induced alterations in the levels of the mRNAs. Thus, the data indicate the mechanism of gene regulation for the modified expression of the receptors and could implicate the participation of the receptors in the pathogenesis of Parkinson's disease.

An enzyme-linked immunosorbent assay for the detection of antibody to avian reovirus by using protein sigma B as the coating antigen.

An enzyme-linked immunosorbent assay using the expressed protein sigma B as the coating antigen (sigma B-ELISA) for detecting antibody to avian reovirus (ARV) in chickens was developed and compared with a conventional ELISA. Both ELISA s and a serum neutralisation (SN) test were used to test the sera from experimentally vaccinated and farm chickens. The sigma B-ELISA could clearly distinguish the SN-positive and -negative sera in 38-week-old chickens. The correlation rate between SN and a sigma B-ELISA was 100 per cent (65/65), and that between SN and conventional ELISA was 84 per cent (55/65). With the sigma B-ELISA, all SN-negative sera had low absorbance values (below 0.06), and the absorbance values correlated closely with the SN titres. However, the sera which were antibody-negative by SN had various absorbance values, ranging from 0.07 to 0.39 in the conventional ELISA. Hence, the sigma B-ELISA had lower non-specific binding reactions than the conventional ELISA against sera from ARV -negative birds. Antibody against ARV could be detected by sigma B-ELISA after vaccination. Absorbance values peaked 4 weeks after vaccination at 2 weeks of age and were maintained until the birds were 27 weeks old. The results suggest that the presence of antibody against viral protein sigma B in birds may be used as a good indicator by the sigma B - ELISA for detecting immune status of a chicken flock or to detect chickens infected with ARV.

Characterization of avian reovirus non structural protein sigmaNS synthesized in Escherichia coli.

The coding region of avian reovirus S1133 genomic segment S4, encoding the non structural protein sigmaNS, was inserted into expression vector pET28a and the protein was expressed in Escherichia coli BL21(DE3) as a fusion protein containing a C-terminal peptide with six tandem histidines (His-tag). The expressed protein (esigmaNS) consistent with the expected molecular size of the avian reovirus protein sigmaNS synthesized in infected cells was readily purified by His-Bind Resin. The esigmaNS was further confirmed to be indistinguishable from viral sigmaNS by immunoblot analysis. The esigmaNS binds 32P-labeled ssRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding activity is blocked by heterologous yeast rRNA, but not by homologous avian reovirus dsRNA and heterologous infectious bursal disease virus dsRNA and salmon sperm dsDNA. Therefore, the ssRNA-binding activity of the expressed protein sigmaNS is non sequence-specific, similar to that previously described for viral sigmaNS purified from avian reovirus infected cell extracts. In addition, the recent data also show that the optimal salt (NaCl) concentration and pH for its binding are 100-150 mM and 7.0, respectively, in terms of the UV cross-linking and RNase A treatment of the reaction mixtures prior to the denaturing gel analysis.

Synthesis in Escherichia coli of avian reovirus core protein varsigmaA and its dsRNA-binding activity.

The genome segment S2 of p6ian reovirus (ARV) S1133 was cloned and sequenced. The entire S2 nucleotide sequence is 1325 bp long with one long open reading frame that encodes a protein of 415 amino acids, corresponding to varsigmaA, a major core protein of ARV. S2 possesses a pentanucleotide, TCATC, at the 3'-terminus of its plus strand, common to other known genome segments of ARV and to 10 genome segments of mammalian reovirus. Amino acid sequence analysis revealed that varsigmaA contains a carboxy-terminal region (one-fourth of the protein) that is formed from alpha-helices and beta-turns, and the remainder (three-fourths of the protein) is formed predominantly from beta-strands and beta-turns. Analysis of binding activity to poly(rI)-poly(rC)-agarose suggested that ARV protein A present in total virus-infected chicken embryo fibroblasts (CEF) had dsRNA-binding activity. To further characterize the binding activity, protein varsigmaA was subsequently expressed in Escherichia coli BL21(DE3) cells as a fusion protein and isolated by metal chelate affinity chromatography. The expressed protein evarsigmaA was further purified through a Superdex 75 HR 10/30 column after digestion of the purified fusion peptide with enterokinase. The expressed protein evarsigmaA has the same molecular weight as virion protein varsigmaA purified from ARV-infected CEF and is indistinguishable from virion protein varsigmaA by immunoblot analysis. The evarsigmaA binds cooperatively alpha (32)P-labeled dsRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding reaction is blocked by homologous ARV dsRNA or heterologous infectious bursal disease virus dsRNA and poly(rI)-poly(rC), but not by salmon sperm DNA. The results indicate that the expressed protein evarsigmaA has dsRNA-binding activity similar to that of varsigmaA obtained from infected cells, and its binding is sequence-independent.

Effect of amphetamine on the expression of the metabotropic glutamate receptor 5 mRNA in developing rat brain.

Mechanisms underlying the acute effects of amphetamine (AMP) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) and specific 3H-glutamate binding in the developing rat brain. Each of the postnatal day (P) 4, P21 and P60 rats received one intraperitoneal injection of AMP, 5 mg/kg or saline and were sacrificed one hour later. In situ hybridization analysis revealed that the AMP treatment raised the levels of the mGluR5 mRNA by 9-28% in the neurons of the layer 5 of motor and somatosensory cortices, whereas reduced the levels by 12-28% in the layer 5 of perirhinal cortex and the ventromedial part of caudate-putamen of the 3 ages. In the layer 2/3 neurons of cingular cortex, an 18% higher and 14% and 22% lower than control levels of the mRNA were detected in the P4 and in the P21 and P60 rats injected with AMP. Moreover, the levels of mGluR5 mRNA in the hippocampi and dentate gyri were elevated by AMP to 110-151% of controls in the rats of 3 ages. Reversible 3H-glutamate binding assay showed an increase of 25% and a 12% decrease in the binding levels in the cortices of AMP-treated P4 and P21 rats. The AMP administration also produced a 27% reduction and 62% elevation in the binding of the hippocampi of P4 and P60 rats. The results reveal age- and region-dependent changes in the expression of the glutamate receptors induced by AMP and may indicate differential plastic capability of the neurons to the drug perturbation.

Toxicity of tunicamycin to cultured brain neurons: ultrastructure of the degenerating neurons.

Our previous study has shown that tunicamycin irreversibly downregulates the expression of GABA(A)R and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mechanisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brains were incubated with 5 microg/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons treated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), nuclear envelope and components of Golgi apparatus, in addition to the degranulation of rER and disaggregation of ribosomal rosettes. In neurons subjected to the prolonged incubation, some ribosomes reattached to the membranes of rER; the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle-like structure appeared in the perikarya. This structure is implicated to involve the neuronal degeneration. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons resemble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA-elicited current in the neurons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM treatment to the neurons and suggest differential mechanisms for the neuronal death among various populations of cells. It is evident that the N-glycosylation plays a critical role for neuronal survival.

Effects of pentobarbital on the expression of GABAA receptor beta 1 mRNA in the hippocampus: differential responses of CA1 and CA3.

Effects of barbiturates have been linked to the inhibitory GABAA receptor in the brain. The present study examines changes in the expression of GABAA receptor in the hippocampus of pentobarbital treated rat. Intraperitoneal pentobarbital injections were administered once daily for 9 days at an increasing dose schedule, 30 mg/kg at day 1-3, 60 mg/kg day 4-6, and 90 mg/kg day 7-9. Within each of the three dosage periods, the duration of sleep and extent of reduction in body temperature of the rats decreased with time. Two hours after the 9th injection, 3H-muscimol binding of the hippocampal homogenates of the animals showed that the maximal number of binding sites (Bmax), 10.2 +/- 1.6 pmol/mg protein, was not significantly greater than 9.5 +/- 1.2 of saline control, but strikingly about 7-fold control level of beta 1 mRNA was seen in the pyramidal cells of CA1 and CA2, as revealed by in situ hybridization analysis with digoxigenin-cRNA probes. However, when the rats were withdrawn from pentobarbital injection for 24 hours and 7 days, the Bmax of the hippocampi was lowered to 7.3 +/- 1.0 and 5.1 +/- 0.7, respectively, and the expression of beta 1 mRNA in CA1-2 returned toward control. The pentobarbital treatment did not significantly alter the affinity of the radioligand to the receptor in the hippocampus and the expression of beta 1 mRNA in CA3 and CA4. The results suggest the plasticity of the beta 1 mRNA in CA1-2 as well as differential involvement of CA1-2 and CA3-4 in response to the pentobarbital perturbation.

Downregulation and subcellular redistribution of the gamma-aminobutyric acidA receptor induced by tunicamycin in cultured brain neurons.

The significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of gamma-aminobutyric acidA receptor (GABA(A)R) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 microg/ml of tunicamycin for 24 h blocked the binding of 3H-flunitrazepam and 3H-muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%, respectively. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABA(A)R immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased to 24% from 34% of control in the presence of the agent, as revealed by trypsinization of cells in situ followed by 3H-flunitrazepam binding. The molecular weight of the receptor subunit protein was lowered around 0.5 kDa after tunicamycin treatment, in accordance with that following N-glycosidase F digestion, indicating the blockade of N-linked glycosylation of GABA(A)R by tunicamycin. Moreover, intense inhibitions of 91% and 44%, respectively, were detected to the general galactosylation and mannosylation in the tunicamycin-treated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of 3H-sugars and 3H-leucine. Nevertheless, treatment with castanospermine or swainsonine (10 microg/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABA(A)Ralpha1-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABA(A)R by producing irregularly glycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N-linked glycosylation may be important for the functional expression and intracellular transport of GABA(A)R.

Identification and characterization of RNA-binding activities of avian reovirus non-structural protein sigmaNS.

Cytoplasmic extracts prepared from avian reovirus (ARV) strain S1133-infected chicken embryo fibroblasts were examined for the presence of RNA-binding proteins in order to identify and characterize ARV RNA-binding proteins. Analysis of binding activity to poly(A)-Sepharose indicated that infected cells contained significant amounts of a protein that co-migrated with ARV protein sigmaNS present in total virus-infected cell extracts. Determination of the N-terminal amino acid sequence of several peptide fragments generated by V8 protease digestion of the poly(A)-Sepharose-purified protein confirmed that this viral protein was sigmaNS. Competition assays showed that single-stranded RNA from the unrelated avian pathogen infectious bursal disease virus was able to compete for binding of sigmaNS to poly(A)-Sepharose. These data suggest that ARV sigmaNS binds to single-stranded RNA in a nucleotide sequence non-specific manner and is functionally similar to its counterpart specified by mammalian reovirus.

Development and characterization of a nucleic acid probe for avian reoviruses.

A radiolabelled probe (S4-18), corresponding to 542 base pairs of the S4 genome segment of avian reovirus (ARV), was used in dot blot hybridization assays. The lowest limit of purified viral RNA detected was similar among three ARV strains and was 0.2 ng. The probe also detected nine heterologous strains of ARV RNA prepared directly from cell cultures, indicating that it had a broad specificity. In addition, the probe detected ARV RNA in tendon tissues of birds from commercial broiler raising farms following dot blot hybridization.

Characterization of the double-stranded RNA genome segment S3 of avian reovirus.

The double-stranded RNA genome segment S3 of avian reovirus (ARV) S1133 was cloned following polyadenylation of both strands and cDNA synthesis of S3 RNA. The complete segment S3 nucleotide sequence was determined. S3 is 1196 base pairs long with one long open reading frame (ORF). The ORF possesses the AUG initiation codon in an optimum context for translation and starts at the first initiation codon (residue 24) and extends for 367 codons, sufficient to encode a protein of the same size as the known S3 gene product, protein sigmaB, one of the major outer capsid proteins of avian reovirus (Mr 41471). Protein sigmaB was subsequently expressed in Escherichia coli. The expressed protein sigmaB was indistinguishable from virion protein sigmaB as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot assay, and N-terminal amino acid sequencing of several peptides generated by Staphyloccus aureus V8 protease digestion. ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3'-terminus of its plus strand. The pentanucleotide sequence is common to the other genome segment S1 of ARV and to ten genome segments of mammalian reovirus at the 3'-terminus of their plus strands. Amino acid sequence analysis revealed that ARV sigmaB does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus. The results of amino acid sequencing suggest that the most susceptible cleavage sites of sigmaB to V8 protease are located in a hydrophilic area between amino acids 95 and 140.

Differential expression of the GABAA receptor alpha 1 subunit in developing chicken brain.

A unique segment of chicken GABAA receptor alpha 1 subunit was expressed in E. coli and used to generate an antiserum 2A specific for the subunit. The DNA fragment encoding the segment of alpha 1 was obtained by selective amplification by polymerase chain reaction (PCR) from a chicken brain cDNA library. The antiserum is characterized by its capacity to immunoprecipitate a [3H]flunitrazepam binding protein of 50 kDa, the chicken GABAA receptor alpha 1 subunit. Subsequent immunoblotting and immunocytochemistry analyses reveal that alpha 1 is expressed in the optic tectum and cerebellum as early as embryonic day 15 (E15), in various areas of telencephalon as early as E20 and distributed heterogeneously among different cell types. The early expression of alpha 1 may imply its functional significance in neurotransmission.

Extrinsic inhibitory innervation to rubral neurons in rat brain-stem slices.

Synaptic connections between the neurons in the red nucleus (RN) and its extrinsic neurons were studied using rat brain-stem slices. Intracellular records were obtained from the RN neurons. Ipsilateral stimuli to areas in the dorsolateral mesencephalic reticular formation (DLMRF) or substantia nigra (SN) elicited monosynaptic hyperpolarizing postsynaptic potentials (PSPs) in about 95% of RN neurons recorded. The hyperpolarizing PSPs could be reversibly blocked by bicuculline, indicating that they were GABAA receptor-mediated-Cl(-)-inhibitory PSPs. The sites of most inhibitory synapses arising from DLMRF and SN are possibly located on the proximal half of the soma-dendritic membrane of RN neurons, according to the analysis of the IPSPs with Rall's model. In addition, tracing dyes were employed to examine the morphological pathways. After rhodamine B, a retrograde tracer, was applied to the RN in brain slices, the cell bodies of a number of neurons in DLMRF and SN were labeled. These labeled neurons were also immunopositive for glutamic acid decarboxylase (GAD) as revealed from double labeling with an anti-GAD antiserum. The anterograde tracer, tetramethylrhodamine dextran, was applied to the DLMRF or SN and taken up by many neurons in the areas. A portion of these cells extended their processes toward and terminated within the RN. Moreover, electron microscopic examination confirmed that the tetramethylrhodamine dextran-decorated synaptic terminals were present in the RN. The results indicate that the rubral neurons receive direct GABAA receptor-mediated inhibitory inputs from neurons in the DLMRF and SN, which may participate in modulation of rubral outputs.

Changes in the microtubule proteins in the developing and transected spinal cords of the bullfrog tadpole: induction of microtubule-associated protein 2c and enhanced levels of Tau and tubulin in regenerating central axons.

The distribution of tubulin, microtubule-associated protein 2 and Tau in the spinal cords of bullfrog tadpoles during development and after transection was studied. alpha-Tubulin or beta-tubulin immunoreactivity was present in the axons, neuronal perikarya and dendrites, as revealed by immunocytochemistry. The axonal staining intensity of the tubulins in the tadpoles was significantly stronger than that in the adult bullfrog. Microtubule-associated protein 2 immunoreactivity was localized largely to dendrites and expanded from distal to proximal dendrites with time; a high-molecular-weight microtubule-associated protein 2 was seen on the immunoblots of cord homogenates throughout development Tau1 stained mainly the axons. Two-dimensional gel immunoblotting disclosed that the tadpole contained a greater number of isoforms of Tau than the frog. Complete transection of the spinal cords of stage IV tadpoles was followed by regeneration of the damaged cord region. The levels of tubulin and Tau immunoreactivity in the regenerating axons of the ventral fasciculi were generally increased. Strikingly, microtubule-associated protein 2 immunoreactivity appeared in the regenerating axons and the chromatolytic cell bodies of axotomized motor neurons, paralleling the induction of microtubule-associated protein 2c in the regenerating cord segment shown by immunoblotting. The chromatolytic cell bodies were also markedly labeled by Tau1, whereas the high-molecular-weight microtubule-associated protein 2 diminished on the immunoblots, in accordance with the reduced level of staining for the dendrites. It is apparent that the changes in the cytoskeletal proteins in the regenerating axons mostly recapitulated their developmental patterns. Moreover, the data indicate a close relationship between tubulin and microtubule-associated proteins in axonal growth as well as providing evidence for similar molecular mechanisms underlying successful regeneration for central and peripheral axons.