Hyperglycemia, hypoxia and their combination exert oxidative stress and changes in antioxidant gene expression: studies on cultured rat embryos

INTRODUCTION: Hyperglycemia and hypoxia are well‐known teratogens that may affect many animal species, including man. We hypothesize that a combination of hypoxia and hyperglycemia will increase embryonic damage produced by either factor individually. We investigated the interrelationship between hyperglycemia and hypoxia and their effects on genes involved in the balance of embryonic redox status. METHODS: Rat embryos (10.5‐day‐old) were cultured for 28 hr in culture medium with about 6 mg/ml of glucose and 20% oxygen (hyperglycemia), with 10% oxygen (hypoxia) and 2.4 g/ml glucose (normal) or a combination of both 6 mg/ml glucose and 10% oxygen. Antioxidant capacity was determined by activity and gene expression of antioxidant enzymes: Cu/Zn SOD, Mn‐SOD, CAT, and GSH‐px using real time PCR. RESULTS: Hyperglycemia, hypoxia, or their combination, decreased embryonic growth and induced a high rate (62–78%) of anomalies mainly of the nervous system, heart, and limbs. CAT mRNA and GSH‐px mRNA were decreased in the malformed embryos exposed to hyperglycemia, to hypoxia or their combination. CAT mRNA was also decreased in the nonmalformed embryos subjected to hyperglycemia and hypoxia. Cu/Zn SOD mRNA was increased in all experimental embryos whether malformed or not, whereas Mn‐SOD was drastically decreased. Total SOD and CAT like activity were changed very little in the experimental embryos compared to controls. CONCLUSIONS: Both hyperglycemia, hypoxia, and their combination reduce embryonic growth and development, induce embryonic anomalies, and modify the expression of the principle antioxidant genes. However, hypoxia does not seem to enhance the damaging effects of hyperglycemia except its effects of embryonic growth. Birth Defects Res (Part B) 92:231–239, 2011. © 2011 Wiley‐Liss, Inc.     

An inactivation gate in the selectivity filter of KCNQ1 potassium channels

Inactivation is an inherent property of most voltage-gated K⁺ channels. While fast N-type inactivation has been analyzed in biophysical and structural details, the mechanisms underlying slow inactivation are yet poorly understood. Here, we characterized a slow inactivation mechanism in various KCNQ1 pore mutants, including L273F, which hinders entry of external Ba²⁺ to its deep site in the pore and traps it by slowing its egress. Kinetic studies, molecular modeling, and dynamics simulations suggest that this slow inactivation involves conformational changes that converge to the outer carbonyl ring of the selectivity filter, where the backbone becomes less flexible. This mechanism involves acceleration of inactivation kinetics and enhancement of Ba²⁺ trapping at elevated external K⁺ concentrations. Hence, KCNQ1 slow inactivation considerably differs from C-type inactivation where vacation of K⁺ from the filter was invoked. We suggest that trapping of K⁺ at s₁ due to filter rigidity and hindrance of the dehydration-resolvation transition underlie the slow inactivation of KCNQ1 pore mutants.     

Effects of feeding cows in early lactation with soy hulls as partial forage replacement on heat production,retained energy and performance

This study measured the effects of replacing corn silage and vetch hay by soy hulls in total mixed rations (TMRs) fed to 25 pairs of cows through 90 d in milk, on dry matter (DM) intake, in vivo digestibility, milk yield and composition, onset of normal estrous activity, body condition score (BCS), health and the energy balance of lactating cows. The partitioning of metabolizable energy (ME) intake between heat production (HP) and retained energy (RE) in milk and body change of each cow was measured. The two TMRs differed in the content of forage and forage aNDFom [235 g/kg versus 350 g/kg; and 128 g/kg versus 187 g/kg DM, in the experimental (EXP) and control (CON) diets, respectively]. This was reflected by an increase in voluntary DM intake by 7.2% (P=0.02) in the EXP group as compared with the CON. In vivo DM and aNDFom digestibility were 4.9% (P=0.03) and 22.7% higher (P=0.01), respectively, in the EXP group than in the CON. The higher DM intake and digestibility of the EXP TMR were reflected by a concomitant increase of 7.4% in milk yield and 10.8% in RE (P=0.01) of the EXP cows as compared with the CON. The two dietary groups expressed similar somatic cell counts, and metabolic disorders (i.e., ketosis and/or lameness), as well as pedometer activity (steps/h) suggesting similar udder health, behavior and animal welfare. A trend to an earlier return to normal ovarian activity occurred in the EXP cows as reflected by fewer days to 1st ovulation and advanced outset of estrous cycle. Despite the higher RE of the EXP cows, the HP of both groups was maintained at an upper level of 141–136 MJ/cow/d during the 90 d of the experiment.     

The Treatment Cascade for Chronic Hepatitis C Virus Infection in the United States: A Systematic Review and Meta-Analysis

Background

Identifying gaps in care for people with chronic hepatitis C virus (HCV) infection is important to clinicians, public health officials, and federal agencies. The objective of this study was to systematically review the literature to provide estimates of the proportion of chronic HCV-infected persons in the United States (U.S.) completing each step along a proposed HCV treatment cascade: (1) infected with chronic HCV; (2) diagnosed and aware of their infection; (3) with access to outpatient care; (4) HCV RNA confirmed; (5) liver fibrosis staged by biopsy; (6) prescribed HCV treatment; and (7) achieved sustained virologic response (SVR).

Methods

We searched MEDLINE, EMBASE, and the Cochrane Database of Systematic Reviews for articles published between January 2003 and July 2013. Two reviewers independently identified articles addressing each step in the cascade. Studies were excluded if they focused on specific populations, did not present original data, involved only a single site, were conducted outside of the U.S., or only included data collected prior to 2000.

Results

9,581 articles were identified, 117 were retrieved for full text review, and 10 were included. Overall, 3.5 million people were estimated to have chronic HCV in the U.S. Fifty percent (95% CI 43–57%) were diagnosed and aware of their infection, 43% (CI 40–47%) had access to outpatient care, 27% (CI 27–28%) had HCV RNA confirmed, 17% (CI 16–17%) underwent liver fibrosis staging, 16% (CI 15–16%) were prescribed treatment, and 9% (CI 9–10%) achieved SVR.

Conclusions

Continued efforts are needed to improve HCV care in the U.S. The proposed HCV treatment cascade provides a framework for evaluating the delivery of HCV care over time and within subgroups, and will be useful in monitoring the impact of new screening efforts and advances in antiviral therapy.     

New developments reproductive technologies in deer

In vitro embryo production is the platform for advanced reproductive technologies, such as cloning. The in vitro embryo production system developed for farmed red deer (Cervus elaphus) evolved along similar lines to that pioneered by other domestic species researchers. However, applying existing in vitro embryo production methods from these other species resulted in limited success and has necessitated developing a species-specific methodology for red deer based on the their physiology. Analysis of oviduct fluid led to the development of a semi-defined fertilization and culture media system, Deer Synthetic Oviduct Fluid (DSOF), which resulted in successful culture of red deer embryos to the blastocyst stage. Transvaginal ultrasound-guided ovarian examination and ovum pickup has enabled the study of seasonality constraint and propagation from selected female genetics, respectively. During the 4-month breeding season (April-July), 15% of cleaved oocytes developed to blastocysts, whereas no blastocysts developed from oocytes collected after July. The process of developing an in vitro embryo production system for farmed red deer may serve as a beneficial model for the propagation of endangered cervine species.     

Intracellular domains interactions and gated motions of IKS potassium channel subunits

Voltage‐gated K⁺ channels co‐assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore‐forming subunits with KCNE1 β subunits generates the repolarizing K⁺ current I_KS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life‐threatening long or short QT syndromes. Here, we studied the interactions and voltage‐dependent motions of I_KS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage‐clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C‐terminus interacts with the coiled‐coil helix C of the Kv7.1 tetramerization domain. This association is important for I_KS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant‐negative C‐terminal domain. On channel opening, the C‐termini of Kv7.1 and KCNE1 come close together. Co‐expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K⁺ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C‐termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.     

Glycogen metabolism in rat heart muscle cultures after hypoxia

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (~ 180% of control) and the protein level of the glut-1 transporter (~ 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (~ 170% of control) and fractional activity of glycogen synthase (control, ~ 15%; hypoxia-treated, ~ 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (~ 50% of control) and fractional activity of glycogen phosphorylase (control, ~ 50%; hypoxia-treated, - 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.     

Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease

Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation (delta F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cystic fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57 (60%) are of Ashkenazi origin. Together with the delta F508 (23% in this group), G542X, N1303K, and 1717-1G----A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Patients homozygous for the W1282X mutation (n = 16) and patients heterozygous for the delta F508 and W1282X mutations (n = 22) had similarly severe disease, reflected by pancreatic insufficiency, high incidence of meconium ileus (37% and 27%, respectively), early age at diagnosis, poor nutritional status, and variable pulmonary function. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.     

Nitrogen nutrition of ginger (Zingiber officinale)

Summary Two fertilizer experiments were conducted in the field at Beerwah, South-East Queensland. In the first experiment leaf nitrogen concentrations, and the yield of ginger shoots and rhizomes at early and late harvests increased both with the total amount of nitrogen applied up to the highest level studied (336 kg N/ha as ammonium nitrate) and with the number of applications making up the total. At all levels of nitrogen application the apparent recovery of fertilizer nitrogen increased in the order 1 application <2 applications <4 applications. At 33.6 kg N/ha there appeared to be no advantage in dividing the total N applied into more than 4 applications but the data suggested higher recoveries of nitrogen with 8 applications at 112 kg N/ha and 336 kg N/ha. In the second experiment, ammonium nitrate, urea, and ammonium sulphate were found to be equally effective as nitrogen fertilizers for ginger when applied at equal rates of nitrogen per hectare. However, in terms of cost effectiveness they rated in the order urea > ammonium nitrate > ammonium sulphate.All three nitrogen sources acidified the soil, the decrease in soil pH during the growing season increasing with increasing rate of application. In Experiment 1 split applications, which increased the recovery of applied nitrogen in the crop, also increased the extent of acidification. In Experiment 2 ammonium sulphate tended to be more strongly acidifying than the other fertilizers but the difference was statistically significant only at the highest rate of nitrogen application. Because of the strong effects of nitrogen supply on both yield and soil pH, the highest yields were associated with end-of-season pH values below 5.0.