Improvement of cardio-ankle vascular index by glimepiride in type 2 diabetic patients.

AIMS: Glimepiride, a third generation sulfonylurea (SU), is known to have extrapancreatic effects, but its vascular effect is unclear. We investigated the efficacy of glimepiride in improving arterial stiffness assessed by cardio-ankle vascular index (CAVI) in type 2 diabetic patients, compared with glibenclamide, a conventional SU. METHODS: Forty type 2 diabetic patients were randomly assigned to two groups. One group was administered glimepiride 1.5 mg/day, and the other group was administered glibenclamide 1.25 mg/day for 6 months. RESULTS: No significant difference in hypoglycaemic effect was observed between two groups. CAVI significantly decreased only in glimepiride group (9.4 ± 1.4→8.9 ± 0.8, p < 0.05). Decrease in CAVI was greater in glimepiride group than in glibenclamide group (-0.50 ± 0.98 vs. -0.04 ± 0.57, p = 0.048). Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) decreased in glimepiride group and increased in glibenclamide group, and the changes were significantly different between groups (-1.5 ± 3.5 vs. + 1.8 ± 3.6, p = 0.009); whereas serum lipoprotein lipase mass increased in glibenclamide group and decreased in glibenclamide group, and the changes tended to be different between groups (+ 2.1 ± 19.1 vs. -7.4 ± 19.2, p = 0.096). Change in urinary 8-OHdG was a significant independent predictor for change in CAVI in all subjects. CONCLUSIONS: These results suggest that glimepiride improves CAVI compared with glibenclamide. Reduced oxidative stress and improved insulin resistance may contribute to the improvement of CAVI by glimerpiride.

Hepatocyte growth factor secreted by cultured adipocytes promotes tube formation of vascular endothelial cells in vitro.

OBJECTIVE: Adipose tissue is closely associated with angiogenesis, but the mechanisms are not fully understood. Some of the adipocyte-derived cytokines are hypothesized to play an important role in angiogenesis. We evaluated tube formation of human umbilical vascular endothelial cells (HUVECs) cultured in type I collagen gel when overlaid with the supernatant of 3T3-L1 cell culture, and expression of tube-forming factor(s) in 3T3-L1 cells with or without pioglitazone. We also studied plasma growth factor levels in patients with type 2 diabetes mellitus treated with pioglitazone. RESULTS AND METHODS: The supernatant of 3T3-L1 cells increased tube formation of HUVECs by 9.03-fold of control. Reverse transcription-polymerase chain reaction showed that hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) mRNA were expressed in 3T3-L1 cells. Western blot analysis also demonstrated HGF and VEGF protein expression. When 3T3-L1 cells were treated with 100 nM small interfering RNAs (siRNAs) for HGF, the HGF mRNA and protein were suppressed. The VEGF mRNA and protein in the cells were also suppressed by siRNA for VEGF. The supernatant of 3T3-L1 cells treated with HGF siRNA suppressed tube formation of HUVECs by 61% compared with the supernatant of cells treated with control siRNA. Addition of VEGF siRNA resulted in no significant changes. The supernatant conditioned with pioglitazone further promoted the tube formation. Pioglitazone enhanced HGF mRNA expression in 3T3-L1 cells. After 12 weeks of pioglitazone treatment, the changes of plasma HGF levels in patients treated with pioglitazone were significantly higher than those in control. CONCLUSION: These results suggest that HGF secreted from 3T3-L1 cells may be the major factor regulating the tube formation, and agents that enhance the differentiation of adipocytes may promote tube formation of HUVECs mediated by HGF secreted by adipocytes.

Effect of weight loss using formula diet on renal function in obese patients with diabetic nephropathy.

OBJECTIVE: To evaluate the effect and safety of treatment with low-calorie formula diet on renal function and proteinuria in obese patients with diabetic nephropathy. DESIGN: Prospective study on safety and efficacy of a 4-week low-calorie (11-19 kcal/kg/day) normal-protein (0.9-1.2 g/kg/day) diet partly supplemented with formula diet. SUBJECTS: In all, 22 obese patients with diabetic nephropathy (BMI: 30.4+/-5.3 kg/m(2), HbA1c: 7.1+/-1.4%, serum creatinine: 172.4+/-57.5 micromol/l, urinary protein: 3.3+/-2.6 g/day). RESULTS: The mean body weight decreased by 6.2+/-3.0 kg. The mean systolic blood pressure, creatinine, blood urea nitrogen, urinary protein, and 8-hydroxydeoxyguanosine decreased significantly by 7.5+/-12.7 mmHg, 41.6+/-23.9 micromol/l, 1.50+/-1.61 mmol/l, 1.8+/-1.7 g/day, and 3.1+/-3.6 ng/mg creatinine, respectively. No patient had increased serum creatinine and urinary protein. Mean creatinine clearance (40.6+/-17.9 to 46.1+/-14.6 ml/s/1.73 m(2)) and serum albumin showed no significant changes. Delta serum creatinine and Delta urinary protein correlated with Delta body weight (r=0.62 and 0.49, respectively) and Delta visceral fat area (r=0.58 and 0.58, respectively), but did not correlate with Delta systolic blood pressure, Delta fasting blood glucose and Delta subcutaneous fat area. CONCLUSION: These results suggested that weight reduction using formula diet might improve renal function and proteinuria safely for a short term in obese patients with diabetic nephropathy.

DNA cleavage activities of Staphylococcus aureus gyrase and topoisomerase IV stimulated by quinolones and 2-pyridones.

We have cloned Staphylococcus aureus DNA gyrase and topoisomerase IV and expressed them in Escherichia coli as polyhistidine-tagged proteins to facilitate purification and eliminate contamination by host enzymes. The enzyme preparations had specific activities similar to previously reported values. Potassium glutamate (K-Glu) stimulated the drug-induced DNA cleavage activity and was optimal between 100 and 200 mM for gyrase and peaked at 100 mM for topoisomerase IV. Higher concentrations of K-Glu inhibited the cleavage activities of both enzymes. Using a common buffer system containing 100 mM K-Glu, we tested the enzyme-mediated DNA cleavage activities of both gyrase and topoisomerase IV with oxolinic acid, norfloxacin, ciprofloxacin, trovafloxacin, clinafloxacin, and the 2-pyridone ABT-719. As expected, all drugs tested demonstrated greater potency against topoisomerase IV than against gyrase. In addition, cleavage activity was found to correlate well with antibacterial activity.

Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes.

Spermatocytes of the teleost, Oryzias latipes, at meiotic prophase were cultured without contact with somatic cells. They began to divide, progressing through the meiotic divisions and differentiating into round spermatids within 48 h. The chromosome number in both the primary and secondary spermatocytes at metaphase was n = 24. In spermatids, a single flagellum was formed and the release of residual bodies was observed in vitro. The size and shape of the flagellum were the same as those seen in vivo. The expression of protamine mRNA was detected in round spermatids. This result suggests that gene expression, as well as morphological change, is regulated by the progression of spermatogenesis in cell culture. Furthermore, when the eggs of O. latipes were inseminated with germ cells cultured for 10 days, normal embryos developed and hatched out. These results suggest that the spermatocytes of O. latipes develop into fertile sperm in cell culture.

Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

Effect of left ventricular contractile performance on passive left atrial filling--clinical study using radionuclide angiography.

Many invasive and noninvasive methods have been used to study the cardiac atria; however, few allow quantitative measurement of atrial function. To determine the interaction between left ventricular (LV) contraction and left atrial (LA) filling, gated radionuclide angiography was conducted in 30 normal subjects (24 men and 6 women, mean age 58 +/- 10 years, range 26-68). LV and LA time-activity curves and their first-derivative curves were obtained simultaneously by using the method of Bough et al. The LV ejection fraction (64 +/- 18%) and LV peak ejection rate (LVPER; 3.42 +/- 0.27 EDV/s) were computed from these curves. As indices of LA filling, LA fractional emptying (38 +/- 12%) and LA peak filling rate (LAPFR; 2.86 +/- 0.17 LAVmax/s)--the latter being defined as the peak rate of LA filling during the LA filling phase--were also computed from these curves. In all subjects, the timing of the LVPER coincided with the occurrence of LAPFR, and there was a significant positive correlation between the LVPER and LAPFR (r = 0.81, p < 0.001), indicating that the LAPFR was strongly affected by the degree of LVPER. Thus, these results indicate that LV contractile performance plays an important role in determining LA passive filling during ventricular systole.

OmpF channel permeability of quinolones and their comparison with beta-lactams.

The diffusion rates of nalidixic acid, ofloxacin and ofloxacin's two optically active isomers through OmpF channels were measured in proteoliposomes and compared with the rates of beta-lactams. The four quinolones showed high diffusion rates, exceeding that of cephaloridine and being comparable to imipenem. There was no significant difference in diffusion rate between nalidixic acid and ofloxacin, or between the two optically active isomers. The diffusion rates of enoxacin and norfloxacin were also estimated to be higher than many beta-lactams.

Effect of physical fitness and training on physiological responses to hypogravity.

The studies on the orthostatic tolerance during the hypodynamics exposure seem to be significant in connection with the selection, training and health maintenance of astronauts. Using male human subjects of various physical fitness levels, fluctuations of their physical fitness through 2 weeks of vigorous athletic training were measured in many parameters. For some of the subjects, the effects of 6 hr thermal neutral water immersion exposure in head out supine position on the physical fitness parameters and orthostatic tolerability were compared before training with after training. The results obtained were as follows: (1) Before training, orthostatic tolerability before hypodynamics exposure increased, following the physical fitness levels; the value after the hypodynamics exposure decreased in all the cases, but no differences were observed between the physical fitness levels. (2) As a result of training an increase of the physical fitness capacity was observed. The increase of orthostatic tolerability before hypodynamics exposure was noticed except for athletes. (3) Before hypodynamics exposure the urinary excretion of noradrenaline on non-athlete subjects increased as the physicsl fitness level increased. The values were decreased by physical training, the more so the better the physical fitness. After hypodynamics exposure the same relation was observed. But for athletes the values remain more stable and the decrease by hypodynamics exposure was not so distinctive. Such decreased reaction to hypodynamic conditions seems to reveal the neuro hormonal mechanism for the detrimental adaptation of athletes to hypodynamics. These results suggest that stable athletes do not always have low orthostatic tolerability, but do not respond well to hypodynamic conditions, at least from the orthostatic point of view. The mechanism seems related to sympathetic nerve activity.