Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central gray in the rat.

The central nucleus of the amygdala, bed nucleus of the stria terminalis, and central gray are important components of the neural circuitry responsible for autonomic and behavioral responses to threatening or stressful stimuli. Neurons of the amygdala and bed nucleus of the stria terminalis that project to the midbrain central gray were tested for the presence of peptide immunoreactivity. To accomplish this aim, a combined immunohistochemical and retrograde tracing technique was used. Maximal retrograde labeling was observed in the amygdala and bed nucleus of the stria terminalis after injections of retrograde tracer into the caudal ventrolateral midbrain central gray. The majority of the retrogradely labeled neurons in the amygdala were located in the medial central nucleus, although many neurons were also observed in the lateral subdivision of the central nucleus. Most of the retrogradely labeled neurons in the BST were located in the ventral and posterior lateral subdivisions, although cells were also observed in most other subdivisions. Retrogradely labeled neurotensin, corticotropin releasing factor (CRF), and somatostatin neurons were mainly observed in the lateral central nucleus and the dorsal lateral BST. Retrogradely labeled substance P-immunoreactive cells were found in the medial central nucleus and the posterior and ventral lateral BST. Enkephalin-immunoreactive retrogradely labeled cells were not observed in the amygdala or bed nucleus of the stria terminalis. A few cells in the hypothalamus (paraventricular and lateral hypothalamic nuclei) that project to the central gray also contained CRF and neurotensin immunoreactivity. The results suggest the amygdala and the bed nucleus of the stria terminalis are a major forebrain source of CRF, neurotensin, somatostatin, and substance P terminals in the midbrain central gray.     

Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR

The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.     

Surviving winter hypoxia: behavioral adaptations of fishes in a northern Wisconsin winterkill lake

Synopsis Winterkill lakes often have a characteristic fish community, presumably composed of species able to survive winter hypoxia. Our research on a small winterkill lake in northern Wisconsin indicates that fishes common in winterkill lakes have behavioral adaptations for tolerating or avoiding winter hypoxia. We examined the distribution of fishes within the lake during one winter (December through May), and fish migrations into and out of the lake for two consecutive years. As DO within the lake declined in late fall, adult-sized fishes of four species, brook stickleback, finescale dace, redbelly dace, and fathead minnow, moved to the ice-water interface where DO levels were highest. Stickleback, and to a lesser extent, fathead minnows, also moved toward the more highly oxygenated water near the inlet. During the first year, young-of-the-year fishes of blacknose shiner, Iowa darter, redbelly dace, and fathead minnow, avoided hypoxic conditions by emigrating from the lake via the outlet stream in late fall and early winter while DO within the lake was still relatively high. Blacknose shiner, redbelly dace, and fathead minnow returned to the lake in spring. Almost no fishes were trapped leaving the lake in the second fall-winter season. Central mudminnows neither moved to the ice-water interface nor emigrated from the lake as DO dropped. Mudminnows survive winter hypoxia by breathing oxygen-containing bubbles trapped beneath the ice. These relatively simple behavioral adaptations allow fishes to survive or avoid hypoxic conditions lethal to other species and may help explain the consistency in fish communities of winterkill lakes.     

Butyrate-induced reversal of dexamethasone resistance in autonomous rat Nb2 lymphoma cells

The parental rat Nb2 lymphoma is a prolactin (PRL)-dependent T cell line. Exposure of a PRL-independent subline, Nb2-SFJCD1, to sodium butyrate (NaBT) causes transient reversal of their growth factor-independent proliferation in association with constitutive expression of protooncogenes pim-1and c-myc. In the present study, we investigated the effect of NaBT treatment on the sensitivity of Nb2-SFJCD1 cells to dexamethasone (DEX)-induced apoptosis. Pretreatment with NaBT (2 mM, 72 h) partially reversed resistance to apoptosis in Nb2-SFJCD1 cells exposed to DEX (100 nM) for 12 h, assessed by flow cytometric analyses of DNA fragmentation. However, the cytolytic effect of DEX was abrogated by PRL i n a time- and concentration-dependent manner. Eval uati on of apoptosis-associated gene expression in NaBT-pre-treated cultures incubated with DEX or DEX+PRL indicated that the apoptosis resistance did not stem from altered bcl-2 or bax expression. However, there was a strong correlation between the resistance to DEX-activated apoptosis and their enhanced expression of pim-1 mRNA and protein. The results show that it is possible to reverse DEX-induced apoptosis of Nb2 pre-T cells and suggest the pim-1 gene product has an important role as a suppressor of this process, perhaps functioning as a mediator of PRL action. This revised version was published online in June 2006 with corrections to the Cover Date.     

Defective thymocyte maturation in horses with severe combined immunodeficiency

Six monoclonal antibodies, designated EqT2, EqT3, EqT6, EqT7, EqT12, and EqT13, which identify T lymphocyte antigens present at different stages of T cell maturation were used to examine T lymphocyte development in foals with severe combined immunodeficiency (SCID). Flow microfluorimetry demonstrated the presence of EqT12+ and EqT13+ prothymocytes and a few phenotypically mature EqT2+ and EqT3+ thymocytes within the thymic remnants of SCID foals. However, very few EqT6+ and EqT7+ resident cortical thymocytes were detected. The near absence of EqT6+ and EqT7+ cortical thymocytes was confirmed by immunofluorescence analysis of thymic tissue from SCID foals. Those cells present were larger than normal cortical thymocytes. Furthermore, their activities of adenosine deaminase, adenosine monophosphate-deaminase, and 5' nucleotidase differed from those of normal cortical thymocytes. The combined evidence of monoclonal antibody analysis, size parameters, and purine enzyme activities demonstrate the near absence of cortical thymocytes in horses with this genetically defined immunodeficiency disorder.     

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.     

Metal ion binding to concanavalin A

Metal ion activation of saccharide binding has been studied for concana-valin A near pH 7.0. Although two metal ions, a transition metal ion and a Ca²⁺ ion, can bind, both are not required. Ca²⁺ alone, Mn²⁺ alone, or Ca²⁺ with other transition metal ions can activate this lectin. Only one Ca²⁺ ion per subunit or only one Mn²⁺ per subunit is sufficient. Metal ion binding was studied by magnetic resonance techniques and direct binding assays. Saccharide binding activity was monitored by following the fluorescence of 4-methylumbelliferyl a-D-mannopyranoside. When Ca²⁺ binds to demetalized concanavalin A, the transition metal ion site is hindered. When Mn²⁺ alone binds to demetalized concanavalin A, saccharide binding activity is induced. A subsequent conformational change, not necessary for carbohydrate binding activity, covers the Mn²⁺.     

Trisomy 15 with loss of the paternal 15 as a cause of Prader-Willi syndrome due to maternal disomy.

Uniparental disomy has recently been recognized to cause human disorders, including Prader-Willi syndrome (PWS). We describe a particularly instructive case which raises important issues concerning the mechanisms producing uniparental disomy and whose evaluation provides evidence that trisomy may precede uniparental disomy in a fetus. Chorionic villus sampling performed for advanced maternal age revealed trisomy 15 in all direct and cultured cells, though the fetus appeared normal. Chromosome analysis of amniocytes obtained at 15 wk was normal in over 100 cells studied. The child was hypotonic at birth, and high-resolution banding failed to reveal the deletion of 15q11-13, a deletion which is found in 50%-70% of patients with PWS. Over time, typical features of PWS developed. Molecular genetic analysis using probes for chromosome 15 revealed maternal disomy. Maternal nondisjunction with fertilization of a disomic egg by a normal sperm, followed by loss of the paternal 15, is a likely cause of confined placental mosaicism and uniparental disomy in this case of PWS, and advanced maternal age may be a predisposing factor.     

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)