Validation of internal controls for gene expression analysis in the intestine of rats infected with Hymenolepis diminuta

The non-invasive parasitic cestode Hymenolepis diminuta induces hypertrophy, hyperplasia and other changes in cell activity in the intestine of rats which are indicated in the expression of mRNA. We have investigated various house-keeping genes (GAPDH, β-actin, 18S and HPRT) and other internal controls (total RNA/unit biomass, total RNA/unit length of intestine) to validate gene expression in the rat intestine after cestode infection and drug-induced neuromodulation. Variation in GAPDH, β-actin, 18S and HPRT expression was observed in rat jejunal tissue according to treatment. Total RNA/unit length of intestine was found to be the most suitable internal control for normalizing target gene mRNA expression in both infected and/or drug-induced rat intestine. This normalization method may be applied to studies of gene expression levels in intestinal tissue where hypertrophy, hyperplasia, rapid growth and cell differentiation generally occur.     

A novel approach for predicting the evolutionary time of acquisition of foreign genes by bacteria: Application of codon usage analyses

Lysogenic bacteriophages are considered as a major player for the introduction of foreign genes into bacterial strains. At the time of introduction foreign genes do not fit well into the translation system of the recipient host bacterium as they tend to retain the characteristics of the donor bacterium from which they have been transferred. Consequently foreign genes are poorly transcribed at the early phase of their evolution within the host bacterium. This is largely due to the difference in the codon usage pattern between the horizontally transferred genes and the host bacterium. In this study we present detailed analyses of various parameters of the codon usages such as codon adaptation index (CAI), mean difference (MD) of the relative adaptiveness, synonymous substitution rate (SSR) of six different phage encoded toxin genes (cholera toxin, shiga toxin, diphtheria toxin, neurotoxin C1, enterotoxin type A and cytotoxin), and proposed conceptual relationship between the evolutionary time of acquisition of the foreign genes and the selected set of parameters of the codon usage. On the basis of the observed data we hypothesize that CAI, MD and SSR of the phage encoded toxin genes are correlated with the evolutionary time of their acquisition, and have developed a novel approach based on the analyses of these parameters, which can be used to predict the evolutionary time of their acquisition by the corresponding host bacterium.     

Effect of exogenous insulin administration on ovarian function, embryo/fetal development during pregnancy in goats

The effect of insulin was investigated on ovarian follicle population, ovulation rate, hormonal profiles and embryo/fetal development during pregnancy using transrectal ultrasonography in goats. Twelve goats synchronized in estrus were selected for the experiment. They were divided into two groups, viz. (untreated control, n=6) and (insulin treated, n=6). In treated group long acting bovine insulin was administered @ 0.2IU/kg body weight subcutaneously for three consecutive days, i.e. days 7-9 of estrous cycle. Thereafter, weekly single injection of insulin was continued for rest of the experiment. However, in control group only normal saline was injected as placebo. Breeding was allowed by natural service in both the groups. The does were subjected to B-mode transrectal ultrasound scanning of ovary and uterus weekly up to 120 and 98 days of gestation, respectively. Blood samples were collected weekly up to 135 days of gestation for the estimation of estradiol 17beta and progesterone (P4). The result revealed no difference in mean number of total follicles between the control and insulin treated groups. The diameter of medium follicle did not differ where as diameter of large follicle was comparatively higher in treated than control goats. The average number of corpus luteum (CL) was higher in insulin treated group as compared to control (1.66 vs. 1.16). However, the number as well as mean diameter of CL did not differ significantly between treated and control group. Serum concentrations of estradiol 17beta and progesterone were significantly (P<0.01) higher in treated than control goats. Embryonic vesicle was detected by day 21 in both the groups, however, its diameter did not differ significantly (0.73 and 0.72cm) between the groups. The twinning percentage was higher (50 vs. 16%) in insulin treated than the control goats. Placentome diameter was also higher (P>0.05) in treated animals. The results demonstrated beneficial effect of exogenous administration of insulin on ovarian function and twinning percentage in goats.     

Effect of exogenous administration of buffalo follicular fluid on follicular development, estrus response and luteal function in anoestrous goats (Capra hircus)

The effect of buffalo follicular fluid (buFF) on follicular development, estrus response and luteal function was investigated in anoestrous does. Treatment with buFF (18 ml/doe) had no significant effect on the number of antral follicles of all class categories during the period of administration. However, after cessation of buFF treatment, the number of total antral follicles increased significantly with time (P < 0.003) as well as due to the treatment × time interaction (P < 0.02), without any influence on follicle size. Injection of buFF also caused a marked increase (P < 0.049) with time in the number of medium-sized follicles at cessation. Approximately 60 and 20% of buFF-treated anoestrous does showed behavioural and silent estrus, respectively, compared to none in the control. The mean interval between cessation of buFF treatment to onset of oestrus and oestrus duration was 67.0 ± 18.5 and 17.0 ± 3.6 h, respectively. Corpus lutea size varied between 4.6 and 5.8 mm with an average diameter of 5.2 ± 0.3 mm. Only 33.3% of does showed serum progesterone levels above 1 ng/ml, while the remainder (66.7%) had below 0.5 ng/ml. Our results indicate that exogenous administration of buFF causes enhanced follicular activity following cessation of treatment, which results in behavioural oestrus and corpus luteum (CL) development in anoestrous does. CL development and its function is, however, inadequate in buFF-treated anoestrous does.     

1,4‐butanediyl‐bismethanethiosulfonate (BMTS) induces apoptosis through reactive oxygen species‐mediated mechanism

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4‐butanediyl‐bismethanethiosulfonate (BMTS)‐induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase‐9 and caspase‐3, and downregulation of Bcl‐2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti‐oxidant N‐acetyl cysteine (NAC) inhibited BMTS‐mediated caspase‐9 and ‐3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox‐linked apoptosis induced by sulfhydryl‐reactive chemicals. J. Cell. Biochem. 108: 1059–1065, 2009. © 2009 Wiley‐Liss, Inc.     

The effects of methylglyoxal on glutathione S-transferase from Nicotiana tabacum

Methylglyoxal (MG) is one of the aldehydes that accumulate in plants under environmental stress. Glutathione S-transferases (GSTs) play important roles, including detoxification, in the stress tolerance systems of plants. To determine the effects of MG, we characterized recombinant GST. MG decreased GST activity and thiol contents with increasing K(m). GST can serve as a target of MG modification, which is suppressed by application of reduced glutathione.     

Evaluation of Pneumonia Severity and Acute Physiology Scores to Predict ICU Admission and Mortality in Patients Hospitalized for Influenza

Background

The demand for inpatient medical services increases during influenza season. A scoring system capable of identifying influenza patients at low risk death or ICU admission could help clinicians make hospital admission decisions.

Methods

Hospitalized patients with laboratory confirmed influenza were identified over 3 influenza seasons at 25 Ontario hospitals. Each patient was assigned a score for 6 pneumonia severity and 2 sepsis scores using the first data available following their registration in the emergency room. In-hospital mortality and ICU admission were the outcomes. Score performance was assessed using the area under the receiver operating characteristic curve (AUC) and the sensitivity and specificity for identifying low risk patients (risk of outcome <5%).

Results

The cohort consisted of 607 adult patients. Mean age was 76 years, 12% of patients died (71/607) and 9% required ICU care (55/607). None of the scores examined demonstrated good discriminatory ability (AUC≥0.80). The Pneumonia Severity Index (AUC 0.78, 95% CI 0.72–0.83) and the Mortality in Emergency Department Sepsis score (AUC 0.77, 95% 0.71–0.83) demonstrated fair predictive ability (AUC≥0.70) for in-hospital mortality. The best predictor of ICU admission was SMART-COP (AUC 0.73, 95% CI 0.67–0.79). All other scores were poor predictors (AUC <0.70) of either outcome. If patients classified as low risk for in-hospital mortality using the PSI were discharged, 35% of admissions would have been avoided.

Conclusions

None of the scores studied were good predictors of in-hospital mortality or ICU admission. The PSI and MEDS score were fair predictors of death and if these results are validated, their use could reduce influenza admission rates significantly.     

Both spike and background genes contribute to murine coronavirus neurovirulence

Various strains of mouse hepatitis virus (MHV) exhibit different pathogenic phenotypes. Infection with the A59 strain of MHV induces both encephalitis and hepatitis, while the highly neurovirulent JHM strain induces a fatal encephalitis with little, if any, hepatitis. The pathogenic phenotype for each strain is determined by the genetic composition of the viral genome, as well as the host immune response. Using isogenic recombinant viruses with A59 background genes differing only in the spike gene, we have previously shown that high neurovirulence is associated with the JHM spike protein, the protein responsible for attachment to the host cell receptor (J. J. Phillips, M. M. Chua, G. F. Rall, and S. R. Weiss, Virology 301:109-120, 2002). Using another set of isogenic recombinant viruses with JHM background genes expressing either the JHM or A59 spike, we have further investigated the roles of viral genes in pathogenesis. Here, we demonstrate that the high neurovirulence of JHM is associated with accelerated spread through the brain and a heightened innate immune response that is characterized by high numbers of infiltrating neutrophils and macrophages, suggesting an immunopathogenic component to neurovirulence. While expression of the JHM spike is sufficient to confer a neurovirulent phenotype, as well as increased macrophage infiltration, background genes contribute to virulence as well, at least in part, by dictating the extent of the T-cell immune response.     

Purification, properties and amino acid sequence of a low-Mr abundant seed protein from pea (Pisum sativum L.).

The seeds of pea (Pisum sativum L.) contain several proteins in the albumin solubility fraction that are significant components of total cotyledonary protein (5-10%) and are accumulated in developing seeds concurrently with storage-protein synthesis. One of these proteins, of low Mr and designated 'Psa LA', has been purified, characterized and sequenced. Psa LA has an Mr of 11000 and contains polypeptides of Mr 6000, suggesting that the protein molecules are dimeric. The amino acid sequence contains 54 residues, with a high content (10/54) of asparagine/aspartate. It has no inhibitory action towards trypsin or chymotrypsin, and is distinct from the inhibitors of those enzymes found in pea seeds, nor does it inhibit hog pancreatic alpha-amylase. The protein contains no methionine, but significant amounts of cysteine (four residues per polypeptide), suggesting a possible role as a sulphur storage protein. However, its sequence is not homologous with low-Mr (2S) storage proteins from castor bean (Ricinus communis) or rape (Brassica napus). Psa LA therefore represents a new type of low-Mr seed protein.