Proteinaceous factor(s) in culture supernatant fluids of bifidobacteria which prevents the binding of enterotoxigenic Escherichia coli to gangliotetraosylceramide.

We have examined the competitive binding of several species of Bifidobacterium and Escherichia coli Pb176, an enterotoxigenic E. coli (ETEC) strain, to gangliotetraosylceramide (asialo GM1 or GA1), a common bacterium-binding structure, and identified a factor(s) in the Bifidobacterium culture supernatant fluid that inhibits the binding of E. coli Pb176 to GA1. The ETEC strain we used expresses colonization factor antigen (CFA) II, which consists of coli surface-associated antigens CS1 and CS3. Competitive exclusion of ETEC from GA1 molecules by Bifidobacterium cells was found by an in vitro thin-layer chromatography overlay binding suppression assay. However, the ETEC cells were less effective in blocking the adherence of Bifidobacterium cells to GA1. These findings suggest that the two bacterial species recognize different binding sites on the GA1 molecule and that the mechanism of competitive exclusion is not due to specific blockage of a common binding site on the molecule. The neutralized culture supernatant fluids of Bifidobacterium species, including that of Bifidobacterium longum SBT 2928 (BL2928), showed remarkable inhibition of the ETEC binding to GA1. Our results suggest that the binding inhibitor produced by BL2928 is a proteinaceous molecule(s) with a molecular weight around or over 100,000 and a neutral isoelectric point. The binding inhibitor produced by BL2928 and other Bifidobacterium species is estimated to contribute to their normal anti-infectious activities by preventing the binding of pathogenic strains of E. coli to GA1 on the surface of the human intestinal mucosa.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.     

Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54^−/−/KU70^−/− DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54^−/−/LIG4^−/− Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

Impact of overweight on left ventricular function in type 2 diabetes mellitus

Background

Coexistence of left ventricular (LV) longitudinal myocardial systolic dysfunction with LV diastolic dysfunction could lead to heart failure with preserved ejection fraction (HFpEF). Diabetes mellitus (DM) is known as a significant factor associated with HFpEF. Although the mechanisms of DM-related LV myocardial injury are complex, it has been postulated that overweight contributes to the development of LV myocardial injury in type 2 diabetes mellitus (T2DM) patients. However, the precise impact of overweight on LV longitudinal myocardial systolic function in T2DM patients remains unclear.

Methods

We studied 145 asymptomatic T2DM patients with preserved LV ejection fraction (LVEF) without coronary artery disease. LV longitudinal myocardial systolic function was assessed by global longitudinal strain (GLS), which was defined as the average peak strain of 18-segments obtained from standard apical views. Overweight was defined as body mass index (BMI) ≥ 25 kg/m². Ninety age-, gender- and LVEF-matched healthy volunteers served as controls.

Results

GLS of overweight T2DM patients was significantly lower than that of non-overweight patients (17.9 ± 2.4% vs. 18.9 ± 2.6%, p < 0.05), whereas GLS of both overweight and non-overweight controls was similar (19.8 ± 1.3% vs. 20.4 ± 2.1%, p = 0.38). Furthermore, multiple regression analysis revealed that for T2DM patients, BMI was the independent determinant parameters for GLS as well as LV mass index.

Conclusions

Overweight has a greater effect on LV longitudinal myocardial systolic function in T2DM patients than on that in non-DM healthy subjects. Our finding further suggests that the strict control of overweight in T2DM patients may be associated with prevention of the development of HFpEF.

     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.