Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding.
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To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans. (+info)
Diversity and cell type specificity of local excitatory connections to neurons in layer 3B of monkey primary visual cortex.
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In the primary visual cortex of macaque monkeys, laminar and columnar axonal specificity are correlated with functional differences between locations. We describe evidence that embedded within this anatomical framework is finer specificity of functional connections. Photostimulation-based mapping of functional input to 31 layer 3B neurons revealed that input sources to individual cells were highly diverse. Although some input differences were correlated with neuronal anatomy, no 2 neurons received excitatory input from the same cortical layers. Thus, input diversity reveals far more cell types than does anatomical diversity. This implies relatively little functional redundancy; despite trends related to laminar or columnar position, pools of neurons contributing uniquely to visual processing are likely relatively small. These results also imply that similarities in the anatomy of circuits in different cortical areas or species may not indicate similar functional connectivity. (+info)
Cloning and characterization of bonnet monkey GnRH receptor.
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Gonadotrophin releasing hormone (GnRH) plays an important role in the reproductive processes of both males and females. It is synthesized by the hypothalamus and binds to a specific receptor on the pituitary to bring about the release of the gonadotrophins, lutineizing hormone and follicle stimulating hormone, which in turn bring about the release of the gonadal steroids. Although the structure of the GnRH receptor (GnRHR) has been elucidated from a number of sources, no information is available about the receptor from the non-human primate species. Here we report the cloning and characterization of the receptor from the pituitary of the bonnet monkey. Antiserum to a bacterially expressed recombinant fragment was used in Western blot analysis and fluorescence microscopy to demonstrate the presence of GnRHR in both human and monkey placentae and pituitary. (+info)
Thalamic and cortical contributions to neural plasticity after limb amputation.
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Little is known about the substrates for the large-scale shifts in the cortical representation produced by limb amputation. Subcortical changes likely contribute to the cortical remodeling, yet there is little data regarding the extent and pattern of reorganization in thalamus after such a massive deafferentation. Moreover, the relationship between changes in thalamus and in cortex after injuries of this nature is virtually unexplored. Multiunit microelectrode maps were made in the somatosensory thalamus and cortex of two monkeys that had long-standing, accidental forelimb amputations. In the deprived portion of the ventroposterior nucleus of the thalamus (VP), where stimulation to the hand would normally activate neurons, new receptive fields had emerged. At some recording sites within the deprived zone of VP, neurons responded to stimulation of the remaining stump of the arm and at other sites neurons responded to stimulation of both the stump and the face. This same overall pattern of reorganization was present in the deprived hand representation of cortical area 3b. Thus thalamic changes produced by limb amputation appear to be an important substrate of cortical reorganization. However, a decrease in the frequency of abnormal stump/face fields in area 3b compared with VP and a reduction in the size of the fields suggests that cortical mechanisms of plasticity may refine the information relayed from thalamus. (+info)
Effect of dichloracetate on infarct size in a primate model of focal cerebral ischaemia.
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Acidosis is a major contributing factor towards spread of the ischaemic focus in the brain. Drugs that increase pyruvate dehydrogenase activity could decrease the formation of lactic acidosis. The sodium salt of dichloracetic acid (DCA) has been found to be effective in reducing lactate. This study was undertaken to study the efficacy of DCA in reducing infarct size in experimental focal ischaemia in monkeys. Macaca radiata monkeys in the treatment group were given 35 mg per kilogram of dichloracetate intravenously immediately before occluding and interrupting the middle cerebral artery, and the control group was given saline as placebo under similar conditions. Mean infarct size expressed as a percentage of the size of the hemisphere in all the three brain slices was 35.38 in the control group as against l2.06 in the treated group (p=0. 0008). (+info)
Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli.
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An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 M) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both alpha-helical and beta-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization. (+info)
Ethane dimethylsulphonate selectively destroys Leydig cells in the adult bonnet monkeys (Macaca radiata).
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AIM: To study the effect of intratesticular administration of ethane-1,2-dimethylsulphonate (EDS) which has been extensively used to selectively destroy Leydig cells in rats and study the role of gonadotropin in regulation of differentiation of Leydig cells (LC) in the adult male bonnet monkey. METHODS AND RESULTS: In vitro studies with cultured interstitial cells isolated from monkey testis revealed an inhibitory effect of EDS on LC as assessed by decrease in testosterone production. Intratesticular administration of EDS (5, 10, 20, 50 mg/testis) resulted in a dose-dependent rapid decrease in serum testosterone levels, with a 65% decrease with 5 mg of EDS by the 3rd day, which returned to control levels by the 45th day. EDS treatment resulted in a significant decrease in testicular testosterone. In addition a significant decrease in [125I]hCG binding and phenylesterase activity in the interstitial cells was noticed. Histological analysis of the testes on the 5th day after administration of EDS revealed an interstitium devoid of LC indicating the destructive action of EDS. CONCLUSION: The monkey LC are sensitive to destructive action of EDS. (+info)
Purified and refolded recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa.
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Bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (bmZPB), excluding the N:-terminal signal sequence and the C:-terminus transmembrane-like domain, has been expressed in Escherichia coli as polyhistidine fusion protein. A requirement of 4 M urea to maintain the purified protein in soluble state rendered it unsuitable for biological studies. Purification of refolded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate methodology that involved purification of inclusion bodies to homogeneity and solubilization in the presence of a low concentration of chaotropic agent (2 M urea) and high pH (pH 12). The solubilized protein was refolded in the presence of oxidized and reduced glutathione. The circular dichroism spectra revealed the presence of both alpha helical and beta sheet components in the secondary structure of the refolded r-bmZPB. The binding of the refolded r-bmZPB to the spermatozoa was evaluated by an indirect immunofluorescence assay and also by direct binding of the biotinylated r-bmZPB. The binding was restricted to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. In the acrosome-reacted spermatozoa a shift in the binding pattern of r-bmZPB was observed and it bound to the equatorial segment, postacrosomal domain, and midpiece region. Binding of biotinylated r-bmZPB was inhibited by cold r-bmZPB as well as by monoclonal and polyclonal antibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acrosome-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization. (+info)