IMPROVED Candida methylica FORMATE DEHYDROGENASE FERMENTATION THROUGH STATISTICAL OPTIMIZATION OF LOW-COST CULTURE MEDIA

NAD⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is of use in the regeneration of NAD(P)H coenzymes, and therefore has strong potential for practical application in chemical and medical industries. A low-cost production of recombinant Escherichia coli (E. coli) containing FDH from Candida methylica (cmFDH) was optimized in molasses-based medium by using response surface methodology (RSM) based on central composite design (CCD). The beet molasses as a sole carbon source, (NH₄)₂HPO₄ as a nitrogen and phosphorus source, KH₂PO₄ as a buffer agent, and Mg₂SO₄ · 7H₂O as a magnesium and sulfur source were used as variables in the medium. The optimum medium composition was found to be 34.694 g L^?1 of reducing sugar (equivalent to molasses solution), 8.536 g L^?1 of (NH₄)₂HPO₄, 3.073 g L^?1 of KH₂PO₄, and 1.707 g L^?1 of Mg₂SO₄ · 7H₂O. Molasses-based culture medium increased the yield of cmFDH about three times compared to LB medium. The currently developed media has the potential to be used in industrial bioprocesses with low-cost production.     

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates)

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species‐level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near‐East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.     

Markers of Bone Metabolism Are Affected by Renal Function and Growth Hormone Therapy in Children with Chronic Kidney Disease

ObjectivesThe extent and relevance of altered bone metabolism for statural growth in children with chronic kidney disease is controversial. We analyzed the impact of renal dysfunction and recombinant growth hormone therapy on a panel of serum markers of bone metabolism in a large pediatric chronic kidney disease cohort.MethodsBone alkaline phosphatase (BAP), tartrate-resistant acid phosphatase 5b (TRAP5b), sclerostin and C-terminal FGF-23 (cFGF23) normalized for age and sex were analyzed in 556 children aged 6–18 years with an estimated glomerular filtration rate (eGFR) of 10–60 ml/min/1.73m². 41 children receiving recombinant growth hormone therapy were compared to an untreated matched control group.ResultsStandardized levels of BAP, TRAP5b and cFGF-23 were increased whereas sclerostin was reduced. BAP was correlated positively and cFGF-23 inversely with eGFR. Intact serum parathormone was an independent positive predictor of BAP and TRAP5b and negatively associated with sclerostin. BAP and TRAP5B were negatively affected by increased C-reactive protein levels. In children receiving recombinant growth hormone, BAP was higher and TRAP5b lower than in untreated controls. Sclerostin levels were in the normal range and higher than in untreated controls. Serum sclerostin and cFGF-23 independently predicted height standard deviation score, and BAP and TRAP5b the prospective change in height standard deviation score.ConclusionMarkers of bone metabolism indicate a high-bone turnover state in children with chronic kidney disease. Growth hormone induces an osteoanabolic pattern and normalizes osteocyte activity. The osteocyte markers cFGF23 and sclerostin are associated with standardized height, and the markers of bone turnover predict height velocity.     

Calcium, magnesium, and zinc status in experimental hyperthyroidism

In this study, experimental hyperthyroidism was established and used to investigate possible alterations in the calcium (Ca), magnesium (Mg), and zinc (Zn) homeostasis by assessing their concentrations in plasma and erythrocytes. In the L-thyroxine-induced hyperthyroidism condition, the experimental animals show a significant decrease in erythrocyte Ca, Mg, and Zn concentrations, and a significant decrease in plasma Mg concentration. Significant positive correlations were found for Mg and Zn both in plasma and in erythrocytes. The results suggest that the homeostasis of Ca, Mg, and Zn is altered during experimental hyperthyroidism.     

I405V and TaqIB polymorphisms of the cholesteryl ester transfer protein and their relation to serum lipid and lipoprotein levels in a Turkish population

Cholesteryl ester transfer protein (CETP) plays a central role in high‐density lipoprotein (HDL) metabolism. Genetic polymorphisms of the CETP gene can influence levels of serum lipoproteins. It has been reported that mean HDL‐cholesterol (HDL‐C) concentrations are low in Turkish population. Thus, we investigated the frequencies of the common I405V and TaqIB polymorphisms of the CETP gene and their relation to serum lipid and lipoprotein levels in a Turkish population. The variant allele frequencies of I405V and TaqIB polymorphisms of the CETP gene were found to be 0.38 and 0.46, respectively and similar to some of the European populations. Subjects for the VV genotype of I405V polymorphism had higher HDL‐C levels than did II subjects. The covariance analysis showed that gender and triglyceride (TG) levels have an effect on the association of HDL‐C and I405V polymorphism. In conclusion, our results indicate that I405V polymorphism may affect the HDL‐C levels in Turkish population. The association of this polymorphism and HDL‐C levels could be modified by other factors, such as gender and TG levels. Copyright © 2009 John Wiley & Sons, Ltd.     

Biochemical effects of chlorpyrifos and deltamethrin on altered antioxidative defense mechanisms and lipid peroxidation in rat liver

Pesticides such as organophosphorus and organochlorine compounds commonly used in agriculture for achieving better quality products are toxic substances and lead to generation of reactive oxygen species (ROS) which have harmful effects on human health. While pyrethroid pesticides are used in preference to organophosphates and organochlorines due to their high effectiveness, low toxicity to non-target organisms and easy biodegradability, they may also produce oxidative stress. Thus, we investigated the effects of chlorpyrifos (CP, an organophosphate) and deltamethrin (DM, a pyrethroid pesticide) treatments at low and high doses on lipid peroxidation (LPO) and antioxidant enzyme activities such as SOD, GSH-Px and CAT in rat liver following 16 weeks exposure. Antioxidative defence mechanisms and lipid peroxidation in rat liver tissues display different responses depending on different pesticide treatments and doses. Biochemical analysis showed that administrations of the chlorpyrifos and deltamethrin cause liver damage. In the present study, we observed that lipid peroxidation levels are higher at high doses than at low doses, but DM caused more pronounced increase than CP. Experimentally, we have also observed that oxidant-antioxidant balance is more affected by deltamethrin treatment than by chlorpyrifos.     

Detection of Entamoeba histolytica antigen in stool samples in Mersin, Turkey

Entameoba histolytica, 1 of the 2 Entamoeba species with similar morphology that infect humans, causes invasive intestinal and extraintestinal diseases, whereas Entamoeba dispar is found commensally and is noninvasive. Because of their morphologic similarity, E. histolytica and E. dispar cannot be differentiated microscopically. The antigens of E. histolytica and E. dispar, however, may be detected by the ELISA method. Previous studies have found that the detection of antigens in the stool is as sensitive and specific as cultures and isoenzyme analyses. Stool samples from 272 patients with diarrhea in the province of Mersin, Turkey, were examined for the presence of Entamoeba species microscopically and for Entamoeba (E. histolytica/E. dispar) antigens using the ELISA method. An E. histolytica-specific ELISA test was used to examine 29 E. histolytica/E. disparpositive samples. Twenty-four (8.82%) of the samples tested positive for E. histolytica/E. dispar by trichrome staining, and 29 (10.6%) of the samples tested positive for E. histolytica/E. dispar by the Entamoeba screening test. Entamoeba histolytica was positive in 21 (7.72%) and E. dispar positive in 8 (2.94%) samples. The detection of true E. histolytica infection is possible with the use of E. histolytica-specific antigen ELISA tests. Thus, real cases of amoebiasis can be detected and treated, and overtreatment of the patients with E. dispar, which is the nonpathogenic species, will be prevented.     

Guanylin, uroguanylin, and heat-stable euterotoxin activate guanylate cyclase C and/or a pertussis toxin-sensitive G protein in human proximal tubule cells

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.