Efficacy and safety of Touchi extract, an alpha-glucosidase inhibitor derived from fermented soybeans, in non-insulin-dependent diabetic mellitus

The water-extracted Touchi, a traditional Chinese food, exerted a strong inhibitory activity against rat intestinal alpha-glucosidase in foodstuffs. In borderline and developed diabetic subjects, 0.3 g of Touchi-extract (TE) significantly inhibited postprandial blood glucose levels. For confirmation of safety, 9 healthy subjects were given 1 g of TE before every meal (3 g/day) for 12 weeks. None indicated changes in hematological and relevant biochemical parameters, body weight or BMI. In a non-comparative study, 18 type-2 diabetic patients ingested 0.3 g of TE before every meal (0.9 g/day) for 6 months (mo). Blood glucose (mean; 9.31 +/- 0.71 mmol/L) and HbA(1c) (mean: 10.24 +/- 0.58%) levels gradually decreased, and significant effects were elicited on the blood glucose levels (8.61 +/- 0.66 mmol/L; p < 0.01) after 6 mo and HbA(1c) after 3 (9.13 +/- 0.43%; p < 0.05) and 6 mo (8.96 +/- 0.30%; p < 0.05) post-ingestion of TE. Indexes for serum lipids and total cholesterol level revealed moderate decreases with a slight increase in the high-density lipoprotein (HDL) level after TE ingestion. However, triglyceride (TG) levels significantly decreased at 3 (p < 0.05) and 6 mo (p < 0.01) post-ingestion of TE. In this study, other biochemical parameters were not affected in any of the patients, and no one complained of any side-effects or abdominal distension. TE, exhibiting alpha-glucosidase inhibitory activity, demonstrated an anti-hyperglycemic effect and may prove useful for improving glycemic control in patients suffering from non-insulin-dependent diabetic mellitus.     

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.     

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.     

A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 X g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P-450.     

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer na?ve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (na?ve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (na?ve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their na?ve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.     

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.     

Wax ester production from n-alkanes by Acinetobacter sp. strain M-1: ultrastructure of cellular inclusions and role of acyl coenzyme A reductase

Acinetobacter sp. strain M-1 accumulated a large amount of wax esters from an n-alkane under nitrogen-limiting conditions. Under the optimized conditions with n-hexadecane as the substrate, the amount of hexadecyl hexadecanoate in the cells reached 0.17 g/g of cells (dry weight). Electron microscopic analysis revealed that multilayered disk-shaped intracellular inclusions were formed concomitant with wax ester formation. The contribution of acyl-CoA reductase to wax ester synthesis was evaluated by gene disruption analysis.     

Diabetes is not a potent inducer of neuronal cell death in mouse sensory ganglia,but it enhances neurite regeneration in vitro

We examined the effects of diabetes on the morphological features and regenerative capabilities of adult mouse nodose ganglia (NG) and dorsal root ganglia (DRG). By light and electron microscopy, no apoptotic cell death was detected in the ganglia obtained from either streptozotocin (STZ)-induced diabetic or normal C57BL/6J mice in vivo. Neurite regeneration from transected nerve terminals of NG and DRG explants in culture at normal (10 mM) and high (30 mM) glucose concentrations was significantly enhanced in the diabetic mice. Chromatolytic changes (i.e. swelling and migration of the nucleus to an eccentric position in the neurons, and a loss of Nissl substance in the neuronal perikarya) and apoptotic cell death (less than one-fifth of the neurons) in the cultured ganglia were present, but neither hyperglycemia in vivo nor high glucose conditions in vitro altered the morphological features of the ganglia or the ratios of apoptotic cells at 3 days in culture. By semiquantitative RT-PCR analysis, the mRNA expressions of ciliary neurotrophic factor (CNTF) in DRG from both mice were down-regulated at 1 day in culture. The expression in diabetic DRG, but not in control DRG, was significantly up-regulated at later stages (3 and 7 days) in culture. In summary, hyperglycemia is unlikely to induce cell death in the sensory ganglia, but enhances the regenerative capability of vagal and spinal sensory nerves in vitro. The up-regulation of CNTF mRNA expression during the culture of diabetic DRG may play a role in the enhanced neurite regeneration.     

pH-dependent aggregate forms and conformation of Alzheimer amyloid beta-peptide (12-24)

The conformational transition to a beta-structure and the aggregation process of Alzheimer amyloid beta-peptide (12-24) [abbreviated as A beta(12-24)] were studied. The influence of sample dissolution methods for the aggregate structure was examined by electron microscopy (EM). The difference in the width of the aggregate of A beta(12-24) depended on the pH immediately after sample dissolution. Two types of sample dissolution methods, F and R, were employed. For dissolution method F, the peptide sample was immediately dissolved in water and then adjusted to pH 2.2 by adding buffer, while for dissolution method R, the peptide was directly dissolved in the buffer solution. In the latter case, the starting pH was 3.0. Slight fibrils (10-12 nm in diameter) were observed with method F, and wider ribbon-like aggregates (17-20 nm in diameter) with method R, despite the same pH range. A difference between methods F and R was also detected in the CD spectra, especially at pHs near 5.0. The CD intensity of the 214 nm band with method R changed with pH, with the highest value at pH 3.7, whereas that with method F was unchanged at pHs below 5.0. The temperature-dependent CD results showed that a thermostable aggregate of A beta(12-24) occurs at higher pHs than 3.0. NMR analysis showed that deprotonation of the C-terminal carboxylate group in A beta(12-24) triggered the aggregate formation, and the transition from a random coil to a beta-conformation in the C-terminal region of V18-V24 was detected on analysis of the (3)Ja(N) coupling constant in the pH range of 2.2 to 3.0.