Protection against experimental encephalomyelitis. Idiotypic autoregulation induced by a nonencephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene.

The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti-idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope.     

Determinants of human myelin basic protein that induce encephalitogenic T cells in Lewis rats

Due to critical amino acid changes in the 72-89 sequence, the determinant of human (Hu) basic protein (BP) that induces experimental autoimmune encephalomyelitis (EAE) in Lewis rats most likely differs from rat and guinea pig BP. To discern encephalitogenic sequence(s), the immunodominant epitopes recognized by Hu-BP-specific T cell lines were identified using synthetic peptides that corresponded to the Hu-BP sequence. The Hu-BP-reactive T cell line contained two distinct specificities, one directed at the 87-99 (Hu) sequence restricted by I-E, and the second directed at the 55-74 (Hu) sequence restricted by I-A. T cells specific for the 87-99 determinant recognized both Hu- and Rt-BP, were highly encephalitogenic, and accounted for the experimental autoimmune encephalomyelitis-inducing activity of the Hu-BP line. T cells directed at the S55-74 (Hu) sequence did not recognize Rt-BP and were not encephalitogenic. The same TCR V genes (homologous to the mouse V alpha 2 and V beta 8 families) that we showed previously were utilized preferentially in response to the I-A restricted 72-89 encephalitogenic sequence were also present in T cell lines specific for both the S55-74 and S87-99 epitopes. These data indicate that encephalitogenic activity of BP in Lewis rats is related to discrete T cell epitopes that are present on or cross-react with rat-BP. Furthermore it would appear that genes in the TCR V alpha 2 and V beta 8 families are widely used in response to different BP epitopes restricted by either I-A or I-E molecules.     

Porphyromonas gingivalis regulates the RANKL-OPG system in bone marrow stromal cells

Porphyromonas gingivalis is a Gram-negative anaerobe implicated in chronic periodontitis, a bacterial-induced inflammatory condition that causes destruction of the periodontal connective tissues and underlying alveolar bone. The receptor activator of nuclear factor-kappaB ligand (RANKL) is a cytokine that directly stimulates osteoclastogenesis and bone resorption, whereas its decoy receptor osteoprotegerin (OPG) blocks this action. This study aimed to investigate the effects of P. gingivalis culture supernatants on RANKL and OPG expression in W20-17 bone marrow stromal cells, and evaluate the involvement of its virulence factors, particularly gingipains and lipopolysaccharide. P. gingivalis up-regulated RANKL and down-regulated OPG mRNA expression and protein production. These effects were blocked by indomethacin, suggesting mediation by prostaglandins. Furthermore, P gingivalis induced the production of prostaglandin E(2). Heat-inactivation, or chemical inhibition of P. gingivalis gingipains did not affect RANKL and OPG regulation. However, lipopolysaccharide depletion by polymyxin B abolished RANKL induction, and partly rescued the suppression of OPG. In conclusion, P. gingivalis regulates the RANKL-OPG system via prostaglandin E(2) in bone marrow stromal cells, in a manner that favours osteoclastogenesis. A non-proteolytic and non-proteinaceous P. gingivalis component is involved in these events, most probably its lipopolysaccharide. This activity may contribute to the bone loss characteristic of periodontitis.     

Venom induces alarm behaviour in the social wasp Polybioides raphigastra (Hymenoptera: Vespidae): an investigation of alarm behaviour, venom volatiles and sting autotomy

Chemicals from the venom gland elicited alarm behaviour and attack in the Asian polistine wasp Polybioides raphigastra. When presented with crushed venom glands workers of this wasp respond with a mass stinging attack. Gas chromatography–mass spectrometry analyses show that the major volatiles in the venom gland are alkanes, monounsaturated alkenes and 2-alcohols. Several pyrazines, a spiroacetal and aromatics were also identified as trace compounds. The anatomy and morphology of the sting apparatus are reported, and we describe sting autotomy in this wasp. This is the first such report for the Ropalidiinae. The structures responsible for autotomy are likely to be large barbs present on the sting lancets, and a conspicuous tooth present on the medial side of the left lancet. Sting autotomy in P. raphigastra probably plays an important role in the localization of sites of attack by wasps defending the nest.     

Methylation of Chromosomal Proteins in Neuronal and Glial Nuclei Purified from Cerebral Hemispheres of Rat During Postnatal Development

The process of methylation of chromosomal proteins [histones and nonhistone proteins (NHP)] in neuronal and glial cell nuclei obtained from cerebral hemispheres of rats at 1, 10, and 30 days of age was investigated. Purified neuronal and glial nuclei were incubated in the presence of S-adenosyl[methyl-3H]methionine. Histone and NHPs were extracted and fractionated by polyacrylamide gel electrophoresis. The results obtained indicate remarkable differences in the process of methylation of histones and NHPs between neuronal and glial nuclei, especially during the first period of postnatal development. In both nuclear populations the histone fraction H3 was labeled to a greater degree than the other fractions and showed the major changes during postnatal development. The densitometric and radioactive patterns of NHPs show considerable changes in the two nuclear populations at the various ages examined. The main difference between neuronal and glial nuclei consists in the intense methylation of proteins with a molecular weight of approximately 100,000, which are present in neuronal nuclei and virtually absent in glial ones. The results obtained may be correlated with the different chromatin structures of neuronal and glial nuclei and with the patterns of maturation and differentiation of neuronal and glial cells during postnatal development.     

Hook2 localizes to the centrosome, binds directly to centriolin/CEP110 and contributes to centrosomal function

Centrosomes serve as microtubule-organizing centers. However, centrosome function depends on microtubule organization and protein transport because the formation, positioning and maintenance of centrosomes require microtubule-dependent retrograde transport. Linker proteins that associate with the motor protein dynein, organelles and microtubules facilitate loading of cargos for retrograde transport and thus contribute to the composition and placement of the centrosome and other juxtanuclear protein complexes. Members of the hook family of proteins may function as adaptors to link various organelle cargos to dynein for transport and have also been implicated directly in centrosome positioning. Here, we show that mammalian hook2, a previously uncharacterized member of the hook family, localizes to the centrosome through all phases of the cell cycle, the C-terminal domain of hook2 directly binds to centriolin/CEP110, the expression of the C-terminal domain of centriolin/CEP110 alters the distribution of endogenous hook2 and mislocalized wild-type or mutant hook2 proteins perturb endogenous centrosomal and pericentrosomal proteins in cultured mammalian cells. In addition, interference with hook2 function results in the loss of the radial organization of microtubules and a defect in regrowth of microtubules following their nocodazole-induced depolymerization. Thus, we propose that hook2 contributes to the establishment and maintenance of centrosomal structure and function.     

Expression and activity of paraoxonase 1 in human cataractous lens tissue

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that is believed to be involved in the protection against oxidative stress. There is evidence that paraoxonase activity is reduced in patients with diabetes and cataract. In the current study, we analyzed mRNA expression of PON1 as well as other members of the paraoxonase family, PON2 and PON3, in human cataractous lens samples. Our results indicate that only PON1 is expressed at the gene and protein levels in human lens tissues. We quantified MDA levels and measured PON1 (paraoxonase/arylesterase) enzymatic activities in subjects suffering from cataract due to aging and diabetes. Decreased PON1 activity was more pronounced in diabetic patients (p  <  0.001) compared to senile subjects, which may be due to glycation and increased oxidative insult. To examine the structural alterations that occur in response to glycation, we constructed a three-dimensional model of PON1 and its glycated variant. Glycation at Lys70 and Lys75 is predicted to cause hindrance in binding of substrate to the active site of the enzyme.     

Hidden Structural Modules in a Cooperative RNA Folding Transition

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Nicotine-Induced Changes in Neurotransmitter Levels in Brain Areas Associated with Cognitive Function

Nicotine, one of the most widespread drugs of abuse, has long been shown to impact areas of the brain involved in addiction and reward. Recent research, however, has begun to explore the positive effects that nicotine may have on learning and memory. The mechanisms by which nicotine interacts with areas of cognitive function are relatively unknown. Therefore, this paper is part of an ongoing study to evaluate regional effects of nicotine enhancement of cognitive function. Nicotine-induced changes in the levels of three neurotransmitters, dopamine (DA), serotonin (5-HT), norepinepherine (NE), their metabolites, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), and their precursor, L-DOPA, were evaluated in the ventral and dorsal hippocampus (VH and DH), prefrontal and medial temporal cortex (PFC and MTC), and the ventral tegmental area (VTA) using in vivo microdialysis in awake, freely moving, male Sprague-Dawley rats. The animals were treated with acute nicotine (0.5 mg/kg, s.c.) halfway through the 300-min experimental period. The reuptake blockers, desipramine (100 microM) and fluoxetine (30 microM), were given to increase the levels of NE and 5-HT so that they could be detected. Overall, a nicotine-induced DA increase was found in some areas, and this increase was potentiated by desipramine and fluoxetine. The two DA metabolites, HVA and DOPAC, increased in all the areas throughout the experiments, both with and without the inhibitors, indicating a rapid metabolism of the released DA. The increase in these metabolites was greater than the increase in DA. 5-HT was increased in the DH, MTC, and VTA in the presence of fluoxetine; its metabolite, 5-HIAA, was increased in the presence and absence of fluoxetine. Except in the VTA, NE levels increased to a similar extent with desipramine and fluoxetine. Overall, nicotine appeared to increase the release and turnover of these three neurotransmitters, which was indicated by significant increases in their metabolites. Furthermore, DA, and especially HVA and DOPAC, increased for the 150 min following nicotine administration; 5-HT and NE changes were shorter in duration. As gas chromatography experiments showed that nicotine levels in the brain decreased by 75% after 150 min, this may indicate that DA is more susceptible to lower levels of nicotine than 5-HT or NE. In conclusion, acute nicotine administration caused alterations in the levels of DA, 5-HT, and NE, and in the metabolism of DA and 5-HT, in brain areas that are involved in cognitive processes.     

Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein which binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to identify, isolate, sequence, and synthesize the region of SHBG that interacts with its membrane receptor. To accomplish this, highly purified human SHBG was digested with trypsin. The SHBG-derived tryptic peptides were separated by high performance liquid chromatography. They were evaluated for their ability to compete with 125I-SHBG for binding to the SHBG receptor solubilized from human prostatic membranes. Only a single peptide, corresponding to residues 48-57 of the known sequence of human SHBG, inhibited receptor binding. A synthetic decapeptide with this amino acid sequence also competitively inhibited SHBG binding.