Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.     

Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Age-related decline in carriage of ampicillin-resistant Escherichia coli in young calves

The presence of ampicillin-resistant Escherichia coli (Amp(r) E. coli) in the fecal flora of calves was monitored on a monthly basis in seven cohorts of calves. Calves were rapidly colonized by Amp(r) E. coli, with peak prevalence in cohort calves observed in the 4 months after the calves were born. The prevalence of calves yielding Amp(r) E. coli in cohorts consistently declined to low levels with increasing age of the calves (P < 0.001).     

Ethanol production from H2 and CO2 by a newly isolated thermophilic bacterium, Moorella sp. HUC22-1

The thermophilic bacterium, Moorella sp. HUC22-1, newly isolated from a mud sample, produced ethanol from H(2) and CO(2) during growth at 55 degrees C. In batch cultures in serum bottles, 1.5 mM ethanol was produced from 270 mM H(2) and 130 mM CO(2) after 156 h, whereas less than 1 mM ethanol was produced from 23 mM fructose after 33 h. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were higher in cells grown with H(2) and CO(2) than those grown with fructose. The NADH/NAD(+) and NADPH/NADP(+) ratios in cells grown with H(2) and CO(2) were also higher than those in cells grown with fructose. When the culture pH was controlled at 5 with H(2) and CO(2) in a fermenter, ethanol production was 3.7-fold higher than that in a pH-uncontrolled culture after 220 h.     

Multiple resonances among time structures, chronomes, around and in US. Is an about 1.3-year periodicity in solar wind built into the human cardiovascular chronome?

AIMS: Velocity changes in the solar wind, recorded by satellite (IMP8 and Wind) are characterized by a solar cycle dependent approximately 1.3-year component. The presence of any approximately 1.3-year component in human blood pressure (BP) and heart rate (HR) and in mortality from myocardial infarction (MI) is tested and its relative prominence compared to the 1.0-year variation. MATERIALS AND METHODS: Around the clock manual or automatic BP and HR measurements from four subjects recorded over 5 to 35 years and a 29-year record of mortality from MI in Minnesota were analyzed by linear-nonlinear rhythmometry. Point and 95% confidence interval (CI) estimates were obtained for the approximately 1.3-year period and amplitude. The latter is compared with the 1.0-year amplitude for BP and HR records concurrent to the solar data provided by one of us (JDR). RESULTS: An approximately 1.3-year component is resolved nonlinearly for MI, with a period of 1.23 (95% CI: 1.21; 1.26) year. This component was invariably validated with statistical significance for BP and HR by linear rhythmometry. Nonlinearly, the 95% CI for the 1.3-year amplitude did not overlap zero in 11 of the 12 BP and HR series. Given the usually strong synchronizing role of light and temperature, it is surprising that 5 of the 12 cardiovascular series had a numerically larger amplitude of the 1.3-year versus the precise 1.0-year component. The beating of the approximately 1.3-year and 1.0-year components was shown by gliding spectra on actual and simulated data. DISCUSSION AND CONCLUSION: The shortest 5-year record (1998-2003) revealed an approximately 1.3-year component closer to the solar wind speed period characterizing the entire available record (1994-2003) than that for the concurrent 5-year span. Physiological variables may resonate with non-photic environmental cycles that may have entered the genetic code during evolution.     

Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor

The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, d-Phe7]-alphaMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 degrees C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.     

Effect of pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) on tissue oxygen content--treatment in central nervous system of mice

It has been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in preventing neuronal cell death and is also a potent vasodilator. Cerebral hypotension and hypoperfusion during cerebral ischemia and neurodegenerative diseases are well known as some of the negative factors which aggravate neuronal cell death. Nevertheless, the effect of PACAP on the cerebral circulation was not understood well. Therefore, in the present study, we determined the mean arterial blood pressure (MBP), regional cerebral blood flow (rCBF) and cerebral oxygen content (pO2) in mice, and estimated the therapeutically useful doses of PACAP. Under barbiturate anesthesia, polyethylene tubes were inserted into mice to monitor MBP and to administer PACAP (5 x 10(-13)-5 x 10(-8) mol/kg) or vasoactive intestinal peptide (VIP; 5 x 10(-12) and 5 x 10(-9) mol/kg). Then, MBP, rCBF and cerebral pO2 were simultaneously measured in the mice. PACAP (5 x 10(-10)-5 x 10(-9) mol/kg) injections transiently decreased MBP, and cerebral pO2. PACAP (5 x 10(-8) mol/kg) injections produced a long-lasting potent decline of MBP, rCBF and cerebral pO2. Therefore, PACAP should be applied at low doses which do not influence the MBP and cerebral circulation to determine the therapeutically useful doses of PACAP for neuroprotection.     

Pressure activates rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a central role in development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of the normal pancreas. We here evaluate the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure-loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. MAPK protein levels and alpha-smooth muscle actin (alpha-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-beta1 (TGF-beta1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative PCR and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and alpha-SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAPK. Treatment of PSCs with an MEK inhibitor and p38 MAPK inhibitor suppressed pressure-induced cell proliferation and alpha-SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-beta1 secretion, collagen type I mRNA expression, and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis.     

Monocyte chemotactic protein-1 regulates leukocyte recruitment during gastric ulcer recurrence induced by tumor necrosis factor-alpha

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.     

Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei

Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca²⁺-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.