Prevention of gut inflammation by Bifidobacterium in dextran sulfate-treated gnotobiotic mice associated with Bacteroides strains isolated from ulcerative colitis patients

Indigenous Bacteroides strains are closely associated with the occurrence and exacerbation of ulcerative colitis (UC). In this study, we aimed to clarify the effect of Bifidobacterium strains, another major member of colonic bacteria, on the development of gut inflammation using gnotobiotic mouse models associated with Bacteroides strains isolated from UC patients. Dextran sulfate (DSS) administration induced inflammation in the large intestine, in particular of the cecum, in the gnotobiotic mice associated with three strains of Bacteroides vulgatus, judging from the myeloperoxidase activity, occult blood score, and IgG leakage into the intestinal contents. However, the severity of the inflammation was greatly reduced in the gnotobiotic mice associated with both B. vulgatus and Bifidobacterium strains. The severity of the cecal inflammation was well correlated with the concentration of succinic acid in the cecum. Bacteriologically, the density of B. vulgatus strain A (BV-A) greatly decreased and the predominant strain changed from BV-A to BV-B on additional association with Bifidobacterium strains. Among gnotobiotic mice associated with a single B. vulgatus strain, the severity of cecal inflammation in BV-A-associated mice was greater than that in BV-B-associated mice. Each Bifidobacterium strain produced compound(s) more effectively inhibiting the growth of BV-A than BV-B in in vitro culture. Taken together, these results suggest that the severity of DSS-induced gut inflammation is closely associated with a particular B. vulgatus strain, and that Bifidobacterium strains may repress exacerbation of intestinal inflammation through growth inhibition of the B. vulgatus strain.     

Polar organic solvent added to an aqueous solution changes hydrolytic property of lipase

For developing further uses of lipase as a biocatalyst, its hydrolytic activity toward some esters was investigated in a miscible solution composed of a buffer and a polar organic solvent. Twenty percent dimethylformamide, 35% dimethylsulfoxide, 15% 1,4-dioxane, 15% dimethoxyethane, and 2% diethoxyethane promoted the hydrolysis by a lipase from Rhizomucor miehei toward some hydrophobic substrates, 4-methylumbelliferyl oleate, 4-methylumbelliferyl palmitate, and monoolein. While hydrolysis by this lipase toward the substrates with a relatively weak hydrophobicity (4-metylumbelliferyl heptanoate and 4-methylumbelliferyl nanoate) was suppressed by these solvents. A fluorometric analysis showed that the polar organic solvent in the buffer induced some conformational change around a tryptophan residue of R. miehei lipase. In addition to the influence of the miscible solvent on the solubility of the substrates, the conformational change of the protein induced by the miscible solvent would also affect the reactive properties of the lipase. Adding a polar organic solvent to an aqueous solution will be an efficient method for changing hydrolytic performance of lipases.     

Design of the linkers which effectively separate domains of a bifunctional fusion protein

With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.     

A kaiC-interacting sensory histidine kinase, SasA, necessary to sustain robust circadian oscillation in cyanobacteria

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.     

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.     

Calcium Antagonist Isradipine Reduces Metabolic Alterations in Acute Cerebral Ischemia in Spontaneously Hypertensive Rats

The present study was designed to examine the effect of a calcium antagonist isradipine (PN200-110: PN) on local cerebral blood flow and brain tissue metabolism after 1-hour supratentorial ischemia induced by bilateral carotid artery ligation (BCL) in spontaneously hypertensive rats (SHR). PN, dissolved in ethanol plus polyethylene glycol 400, diluted with saline to make the final concentration of 0.25mg/ml and 2.5mg/ml, was administered subcutaneously either 30 min prior to BCL or just after the induction of incomplete cerebral ischemia (n = 7 in each group). Vehicle injection was served as a control group (n = 7). Cerebral blood flow in the parietal cortex (CBF) and the cerebellar cortex (CeBF) was measured by hydrogen clearance technique, and the supra- and infratentorial metabolites of the brain frozen in situ were determined by the enzymatic method. Blood pressure was lowered, but CBF was increased by PN administration in pre-BCL treatment study. After 1 hour of BCL, CBF decreased to around 10% or less of the resting value, being insignificant among the groups. Brain adenosine triphosphate was better preserved in PN-administered groups. The increase in lactate level tended to reduce dose dependently by PN treatment. PN also reduced the metabolic alterations in brain tissue with significance, even when administered just after the induction of forebrain ischemia. It is considered that pre- as well as post-BCL administration of PN is beneficial to attenuate the metabolic alterations in incomplete forebrain ischemia in SHR.     

Sex Differences in the Default Mode Network with Regard to Autism Spectrum Traits: A Resting State fMRI Study

Autism spectrum traits exist on a continuum and are more common in males than in females, but the basis for this sex difference is unclear. To this end, the present study draws on the extreme male brain theory, investigating the relationship between sex difference and the default mode network (DMN), both known to be associated with autism spectrum traits. Resting-state functional magnetic resonance imaging (MRI) was carried out in 42 females (mean age ± standard deviation, 22.4 ± 4.2 years) and 43 males (mean age ± standard deviation, 23.8 ± 3.9 years) with typical development. Using a combination of different analyses (viz., independent component analysis (ICA), fractional amplitude of low-frequency fluctuation (fALFF), regional homogeneity (ReHo), and seed-based analyses), we examined sex differences in the DMN and the relationship to autism spectrum traits as measured by autism-spectrum quotient (AQ) scores. We found significant differences between female and male subjects in DMN brain regions, with seed-based analysis revealing a significant negative correlation between default-mode resting state functional connectivity of the anterior medial prefrontal cortex seed (aMPFC) and AQ scores in males. However, there were no relationships between DMN sex differences and autism spectrum traits in females. Our findings may provide important insight into the skewed balance of functional connectivity in males compared to females that could serve as a potential biomarker of the degree of autism spectrum traits in line with the extreme male brain theory.     

Mechanism of radiosensitizing effect of chloride ion on E. coli

Cells of E. coli capable of repairing DNA damage are sensitized to radiation in the presence of NaCl. However, the enhanced radiolethality was suppressed by the addition of compounds such as an amino acid to the irradiation buffer. The protective efficiencies of these compounds depend on their reactivities with Cl-.2 or OH.. ATP synthesis in the cells irradiated in the presence of NaCl was severely inhibited depending on the dose of irradiation. This reduced rate of ATP synthesis can account for the inhibition of protein, RNA and DNA synthesis in the irradiated cells with NaCl.     

Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification

We developed a technique for detecting the heat-labile I (LTI) and heat-stable I (STI) genes of enterotoxigenic Escherichia coli (ETEC) using a novel DNA amplification procedure designated Loop-Mediated Isothermal Amplification (LAMP). The detection limit of accelerated LAMP utilizing loop primers was 4 CFU/test for LTI and was 40 CFU/test for STI, which are 10-fold higher than those of conventional PCR assay (detection limit, 40 CFU/test and 400 CFU/test, respectively). No DNA amplification was observed in LT and ST non-producing E. coli or other bacterial strains; thus, high specificity was verified. The specificity of LAMP assay was also confirmed by digestion of LAMP products using restriction enzymes and DNA sequence analysis. In the accelerated LAMP assay, DNA amplification was detected within 35 min, and thus LAMP is superior to conventional PCR in terms of rapidity. It was confirmed that increased concentrations of primers and Bst DNA polymerase could further facilitate the reaction. Furthermore, with the high amplification efficiency of the LAMP assay, amplification can be visually observed by the turbidity caused by magnesium pyrophosphate, a byproduct of the reaction. Detection of LTI and STI in ETEC by LAMP is thus an extremely rapid procedure with high sensitivity and specificity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid diagnosis in ETEC infection.