Dual effects of acupuncture on gastric motility in conscious rats

The effects of manual acupuncture on gastric motility were investigated in 35 conscious rats implanted with a strain gauge transducer. Twenty (57.1%) rats showed no cyclic groupings of strong contractions (type A), whereas 15 (42.9%) rats showed the phase III-like contractions of the migrating motor complex (type B) in the fasting gastric motility. Acupuncture at the stomach (ST)-36 (Zusanli), but not on the back [Weishu, bladder (BL)-21], increased the peak amplitude of contractions to 172.4 +/- 25.6% of basal in the type A rats (n = 20, P < 0.05). On the other hand, the motility index for 60 min after the acupuncture was not affected by the acupuncture in this group. On the contrary, acupuncture decreased the peak amplitude and motility index to 72.9 +/- 14.0% and 73.6 +/- 16.2% in the type B rats (n = 15, P < 0.05), respectively. The stimulatory and inhibitory effects of acupuncture observed in each type were reproducible on the separate days. In 70% of type A rats, acupuncture induced strong phase III-like contractions lasting for over 3 h that were abolished by atropine, hexamethonium, atropine methyl bromide, and vagotomy. Naloxone significantly shortened the duration of the stimulatory effects from 3.52 +/- 0.21 to 1.02 +/- 0.15 h (n = 3, P < 0.05). These results suggest that acupuncture at ST-36 induces dual effects, either stimulatory or inhibitory, on gastric motility. The stimulatory effects are mediated in part via vagal efferent and opioid pathways.     

Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.     

Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina

One of the major roles of brain-derived neurotrophic factor (BDNF) is to promote the differentiation and support the survival of neurons in the central nervous system. The objective of the present study was to evaluate the effect of BDNF on the fate of adult rat hippocampus-derived neural stem cells (AHPCs) transplanted into the developing rat retina. Immunohistochemical analysis showed a significant increase in the ratio of grafted AHPCs stained for MAP2ab (P<0.05) and a marked decrease in the ratio of nestin-positive grafted cells in the slow-releasing BDNF group compared with the control group. The respective changes in the ratios of MAP5 and GFAP-positive grafted cells were comparable for the two groups. The results reported here suggest a potentially beneficial role for extended delivery of BDNF in the differentiation of grafted neural stem cells, which may lead to a novel modification of stem cell transplantation.     

Crystal structure of chartreusin derivative A132

The crystal structure of chartreusin derivative A132 (benzilidene chartreusin) has been determined by single-crystal X-ray diffraction. The space group is C2 with unit cell dimensions, a=18.482(4), b=8.749(3), c=43.906(2) A, beta=94.87(2) degrees, and the structure was refined to R-factors of 0.2365 (6585 all unique reflections) and 0.087 (2914 reflections with F(o)>4 sigma(F(o))) by a full-matrix least-squares method. There are two molecules in an asymmetric unit. Both molecules have similar structures, which are favorable to bind with DNA in the minor groove. A modeling study of the A132-DNA complex based on the X-ray structures suggests that the sugar moiety of A132 may play an important role in recognizing the sequence of DNA base pairs.     

High-cut characteristics of the baroreflex neural arc preserve baroreflex gain against pulsatile pressure

A transfer function from baroreceptor pressure input to sympathetic nerve activity (SNA) shows derivative characteristics in the frequency range below 0.8 Hz in rabbits. These derivative characteristics contribute to a quick and stable arterial pressure (AP) regulation. However, if the derivative characteristics hold up to heart rate frequency, the pulsatile pressure input will yield a markedly augmented SNA signal. Such a signal would saturate the baroreflex signal transduction, thereby disabling the baroreflex regulation of AP. We hypothesized that the transfer gain at heart rate frequency would be much smaller than that predicted from extrapolating the derivative characteristics. In anesthetized rabbits (n = 6), we estimated the neural arc transfer function in the frequency range up to 10 Hz. The transfer gain was lost at a rate of -20 dB/decade when the input frequency exceeded 0.8 Hz. A numerical simulation indicated that the high-cut characteristics above 0.8 Hz were effective to attenuate the pulsatile signal and preserve the open-loop gain when the baroreflex dynamic range was finite.     

Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.     

Mitochondrial permeability transition mediates apoptosis induced by N-methyl(R)salsolinol,an endogenous neurotoxin,and is inhibited by Bcl-2 and rasagiline,N-propargyl-1(R)-aminoindan

The role of mitochondrial permeability transition (PT) in apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], was studied by use of dopaminergic neuroblastoma SH-SY5Y cells. NM(R)Sal reduced mitochondrial membrane potential, DeltaPsim, in the early phase of apoptosis, which was not suppressed by a pan-caspase inhibitor, but was antagonized by Bcl-2 and cyclosporin A, suggesting the involvement of the PT in NM(R)Sal-induced loss of DeltaPsim. NM(R)Sal-induced apoptosis was completely inhibited not only by Bcl-2 and a pan-caspase inhibitor, but also by cyclosporin A, suggesting the essential role of the PT in NM(R)Sal-induced apoptosis. In mitochondria isolated from rat liver, NM(R)Sal induced swelling and reduced DeltaPsim, which was inhibited by cyclosporin A and Bcl-2 overexpression. These results indicate that NM(R)Sal induced the PT by direct action on the mitochondria. Rasagiline, N-propargyl-1(R)-aminoindan, which is a now under a clinical trial for Parkinson's disease, suppressed the DeltaPsim reduction, release of cytochrome c, and apoptosis induced by NM(R)Sal in SH-SY5Y cells. Rasagiline also inhibited the NM(R)Sal-induced loss of DeltaPsim and swelling in the isolated mitochondria, proving that rasagiline directly targets the mitochondria also. Altogether, mitochondrial PT plays a key role both in NM(R)Sal-induced cell death and the neuroprotective effect of rasagiline.     

Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase,in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.     

Conformation of beta 2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond

Beta2-microglobulin (beta2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of beta2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mm dithiothreitol. Because beta2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.