BDNF and trkB mRNA Expression in Neurons of the Neonatal Mouse Barrel Field Cortex: Normal Development and Plasticity after Cauterizing Facial Vibrissae

Development of the central somatosensory system is profoundly modulated by the sensory periphery. Cauterization of facial whiskers alters the segregation pattern of barrels in rodents only during a few days just after birth (critical period). Although a molecular basis of the segregation of barrel neurons and the critical period for the anatomical plasticity observed in layer IV barrel neuron is not clear yet, the accumulating evidence suggests that neurotrophins modulate synaptic connections including central nervous system. In this study, we showed by in situ hybridization that mouse barrel side neurons express brain-derived neurotrophic factor (BDNF) mRNA and both catalytic and non-catalytic forms of trkB mRNA. Cautery of row C vibrissae on the right side of the face within 24 h after birth (post natal day 0, PND0) reduced the expression of BDNF and trkB mRNA from the division region between the contralateral row C barrels at PND7. The vibrissae in row A, C, and E were cauterized at PND0 followed by quantitative RT-PCR for BDNF and trkB mRNA with total RNA isolated from the barrel region at PND7. The result showed that BDNF, but not trkB, mRNA was increased several-fold in the contralateral barrel region. These data suggest that the expression of BDNF mRNA is differentially regulated between injured barrels and actively innervated barrels. The differential expression of the mRNA encoding neurotrophins and their receptors may be important in regulating the injury-dependent re-segregation of barrels.     

Direct detection of circulating free radicals in the rat using electron spin resonance spectrometry.

We developed a new technique for directly observing in vivo free radical formation in the circulating blood of living rats using electron spin resonance (ESR) spectrometry without any labeling or trapping agents. It was found that a doublet peak spectrum was obtained following ferric citrate and ascorbic acid injection. The signals were confirmed in different ways to be due to ascorbic acid radicals. These results provide evidence to support the involvement of free radical intermediates in iron-ascorbic acid reactions, and further confirm the suggested mechanisms of both the adverse and protective effects of ascorbic acid in biological systems. Furthermore, this method of direct observation is a new application of ESR spectrometry to living animals.     

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.     

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.     

Royal jelly inhibits the production of proinflammatory cytokines by activated macrophages

In this study, we have examined the anti-inflammatory actions of royal jelly (RJ) at a cytokine level. When supernatants of RJ suspensions were added to a culture of mouse peritoneal macrophages stimulated with lipopolysaccharide and IFN-gamma, the production of proinflammatory cytokines, such as TNF-alpha, IL-6, and IL-1, was efficiently inhibited in a dose-dependent manner without having cytotoxic effects on macrophages. This suggests that RJ contains factor(s) responsible for the suppression of proinflammatory cytokine secretion. We named the factor for honeybees RJ-derived anti-inflammatory factor (HBRJ-AIF), and further investigated the molecular aspects of it. Size fractionation study showed that HBRJ-AIF is composed of substances of low (< 5 kDa) and high (> 30 kDa) molecular weights, with the former being a major component. Chromatographic analysis showed that MRJP3 is one candidate for the HBRJ-AIF with high molecular weights. Thus, our results suggest that RJ has anti-inflammatory actions through inhibiting proinflammatory cytokine production by activated macrophages.     

Highly potent PDE4 inhibitors with therapeutic potential

Based on the hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system (CNS), design and synthesis of a hydrophilic analogue is considered to be one approach to improving the side-effect profile of Ariflo 1. Water-soluble piperidine derivatives were found to possess therapeutic potential.