Positive regulation of amidase synthesis in Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa were isolated that were acetamide-negative in growth phenotype at 41 degrees C and constitutive for amidase synthesis at 28 degrees C. Two mutants were derived from the magno-constitutive amidase mutant PAC111 (C11), and a third from a mutant that had enhanced inducibility by formamide, PAC153 (F6). The three temperature-sensitive mutants produced amidases with the same thermal stabilities as the wild-type enzyme. Cultures growing exponentially at 28 degrees C, synthesizing amidase constitutively, ceased amidase synthesis almost immediately on transfer to 41 degrees C. Cultures growing at 41 degrees C were transferred to 28 degrees C and had a lag of about 0.5 of a generation before amidase synthesis became detectable. Pulse-heating for 10 min at 45 degrees C of a culture growing exponentially at 28 degrees C resulted in a lag of about 0.5 of a generation before amidase synthesis recommenced after returning to 28 degrees C. Acetamide-negative mutants that were unable to synthesize amidase at any growth temperature were isolated from an inducible strain producing the mutant B amidase PAC398 (IB10). Two mutants were examined that gave revertants producing B amidase but with novel regulatory phenotypes. It is suggested that amidase synthesis is regulated by positive control exerted by gene amiR.     

Verbal Autopsy: Reliability and Validity Estimates for Causes of Death in the Golestan Cohort Study in Iran

Background

Verbal autopsy (VA) is one method to obtain valid estimates of causes of death in the absence of valid medical records. We tested the reliability and validity of a VA questionnaire developed for a cohort study in Golestan Province in northeastern Iran.

Method

A modified version of the WHO adult verbal autopsy was used to assess the cause of death in the first 219 Golestan Cohort Study (GCS) subjects who died. The GCS cause of death was determined by two internists who independently reviewed all available medical records. Two other internists (“reviewers”) independently reviewed only the VA answers and classified the cause of death into one of nine general categories; they repeated this evaluation one month later. The reliability of the VA was measured by calculating intra-reviewer and inter-reviewer kappa statistics. The validity of the VA was measured using the GCS cause of death as the gold standard.

Results

VA showed both good validity (sensitivity, specificity, PPV, and NPV all above 0.81) and reliability (kappa>0.75) in determining the general cause of death independent of sex and place of residence. The overall multi-rater agreement across four reviews was 0.84 (95%CI: 0.78–0.89). The results for identifying specific cancer deaths were also promising, especially for upper GI cancers (kappa = 0.95). The multi-rater agreement in cancer subgroup was 0.93 (95%CI: 0.85–0.99).

Conclusions

VA seems to have good reliability and validity for determining the cause of death in a large-scale adult follow up study in a predominantly rural area of a middle-income country.     

Estimates of pregnancy outcomes based on selection of bovine embryos produced in vivo or in vitro

The objective of this study was to estimate the degree of variation among experienced evaluators selecting in vivo- or in vitro-produced embryos for transfer and to determine how this affects both the proportion of recipients becoming pregnant after transfer, and the number of embryo transfers required per pregnancy. Data from 6 experienced evaluators who graded Day 7 embryos produced either in vivo (n = 15) or in vitro (n = 15) were used to estimate these effects. The evaluators viewed video recorded images of the embryos and classified each embryo for stage of development and quality grade (1 = excellent, 2 = good, 3 = fair, 4 = degenerated and nontransferable). The statistical model considered transfer of embryos of the following individual or combined grades: Grade 1 only, Grade 2 only, Grade 3 only, Grades 1 and 2, Grades 2 and 3, and Grades 1, 2 and 3. Probabilities of pregnancy after embryo transfer were based on pregnancy rates of recipients at the facility of 1 of the 6 evaluators where the percentages of heifers pregnant after the transfer of Grade 1, 2 and 3 embryos, by embryo source, were 76, 65 and 54% (in vivo), and 59, 45 and 30% (in vitro). Within most grades, the proportion of embryos selected for transfer differed (P < 0.05) among the 6 evaluators. Although no significant differences (P > 0.10) were found among evaluators in the proportion of recipients pregnant after transfer within any embryo grade, there was substantial variation among evaluators in the proportion of recipients becoming pregnant, especially for embryos produced in vitro. Estimated percentages of heifers becoming pregnant for embryos classified as Grade 1, 2 or 3 were 66 to 76, 62 to 69, and 54 to 60%, respectively, for in vivo-produced embryos; and, 39 to 59, 15 to 45, and 24 to 32%, respectively, for in vitro-produced embryos. Approximately twice as many transfers were needed per pregnancy for embryos produced in vitro as for those produced in vivo regardless of the grade.     

Standardizing global gene expression analysis between laboratories and across platforms

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.     

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.     

In vitro production of embryos alters levels of insulin-like growth factor-II messenger ribonucleic acid in bovine fetuses 63 days after transfer

The objective of this study was to determine the effect of embryo production systems on the expression of insulin-like growth factor (IGF)-II mRNA in fetal bovine tissues at Day 70 of gestation (63 days after transfer). Oocytes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. Zygotes were cultured in either tissue culture medium (TCM)-199 + 10% estrous cow serum (ECS; in vitro-produced with serum [IVPS]) or TCM-199 + 1% BSA (in vitro-produced with serum restriction [IVPSR]). At 72 h postinsemination, IVPSR embryos were transferred into fresh TCM-199 + 10% ECS whereas IVPS embryos had fresh medium replaced. All embryos were cultured for an additional 96 h. In vivo-produced embryos were harvested from superovulated Holstein cows (multiple ovulations [MO]). Grade 1 blastocysts from all groups were transferred singly into Angus heifers. At Day 70 of gestation, fetuses (n = 14, 13, and 11 for MO, IVPS, and IVPSR, respectively) were collected; liver and skeletal muscle samples were snap frozen, and whole-cell RNA (wcRNA) was extracted. Levels of IGF-II mRNA were determined by RNase protection assay and quantified relative to 18S rRNA (mean arbitrary units +/- SEM). WcRNA from adult and Day 90 fetal bovine liver were used as controls. Adult liver contained 9-fold less IGF-II mRNA than liver from Day 90 fetuses (P < 0.05). Fetal livers of males originating from IVPS and IVPSR groups possessed approximately 2-fold greater levels of mRNA for IGF-II than those from MO males (0.25 +/- 0.07, 0.33 +/- 0.04, and 0.14 +/- 0.03, respectively; P < 0.05). Levels of mRNA for IGF-II tended to be lower (P = 0.07) in skeletal muscle of fetuses originating from the IVPSR group (0.043 +/- 0.005) compared to MO controls (0.070 +/- 0.008). In conclusion, at Day 70 of gestation, fetuses originating from in vitro production systems possessed altered levels of IGF-II mRNA in both liver and skeletal muscle.     

The Effect of CYP19 and COMT Polymorphisms on Exercise‐Induced Fat Loss in Postmenopausal Women

Objective:To examine whether genetic polymorphisms in CYP19 [intron 4 (TTTA)_n; n = 7 to 13 and a 3‐base pair deletion, which is in strong linkage disequilibrium with the seven repeat] and COMT (Val^108/158Met) modified the change in BMI, total and percentage body fat, or subcutaneous and intra‐abdominal fat during a year‐long exercise intervention trial. These genes metabolize estrogens and androgens, which are important in body fat regulation. Research Methods and Procedures: A randomized intervention trial was used, with an intervention goal of 225 min/wk of moderate‐intensity exercise for one year. Participants (n = 173) were postmenopausal, 50 to 75 years old, sedentary, overweight or obese, and not taking hormone therapy at baseline. Results: Exercisers with two vs. no CYP19 11‐repeat alleles had a larger decrease in total fat (?3.1 kg vs. ?0.5 kg, respectively, p = 0.01) and percentage body fat (?2.4% vs. ?0.6%, respectively, p = 0.001). Exercisers with the COMT Met/Met vs. Val/Val genotype had a smaller decrease in percentage fat (?0.7% vs. ?1.9%, respectively, p = 0.05). Among exercisers, women with the COMT Val/Val genotype and at least one copy of the CYP19 11‐repeat allele vs. those with neither genotype/allele had a significantly larger decrease in BMI (?1.0 vs. +0.1 kg/m², respectively, p = 0.009), total fat (?2.9 vs. ?0.5 kg, respectively, p = 0.004), and percentage body fat (?2.6% vs. ?0.4%, respectively, p < 0.001). Discussion: Genetic polymorphisms in CYP19 and COMT may be important for body fat regulation and possibly modify the effect of exercise on fat loss in postmenopausal women.     

Differential gene expression in primary and recurrent carotid stenosis

Apoptosis of the cellular components of complex atherosclerotic plaque may lead to plaque instability and rupture. In this study, five primary plaques and one recurrent fibrointimal lesion obtained from patients undergoing carotid endarterectomy for symptomatic carotid stenosis > or = 70% were analyzed by immunohistochemistry and cDNA microarray to identify gene expression patterns that may determine plaque susceptibility or resistance to apoptosis. Immunohistochemistry showed expression of active caspase 3, an effector of apoptosis, in macrophages and lymphocytes surrounding the lipid core, in smooth muscle cells in the fibrous cap, and media of primary plaques as well as in occasional smooth muscle cells in the recurrent lesion. Among the genes demonstrating increased expression in primary plaques were IGFR2, DR4, DAPK1, Bak, and ERK 1 and 2 and those showing decreased expression included the TNF receptors 1 and 2, akt1, and IGFBP3. When comparing the recurrent lesion to the normal tissue, the expression of 13 genes was decreased by 3-fold, including IGFBP2 and IGFBP3, and none were increased by more than 1.5-fold. The analysis of gene expression patterns in primary and recurrent stenotic lesions provides a powerful approach to identify the signaling pathways that alter cellular apoptotic patterns in such lesions.     

Birth of a Holstein freemartin calf co-twinned to a schistosomus reflexus fetus

An unusual case of a live Holstein freemartin calf co-twinned with schistosomus reflexus fetus is presented here. Delivery of the schistosomus reflexus was achieved by fetotomy 24 h after manual delivery of a live heifer calf. The dam subsequently experienced concurrent metritis and left displacement of the abomasum; however, she conceived following insemination 85 d post partum. Cytogenetic examination of the live heifer using lymphocyte culture and cutaneous fibroblast cell culture failed to demonstrate chromosomal chimerism, whereas polymerase chain reaction (PCR) detected the presence of the bovine Y-chromosome marker BRY-1. Low concentrations of testosterone, estradiol and progesterone at 3, 6, 24 and 48 h after administration of hCG were detected in the serum of the freemartin heifer. Genetic, hormonal, histological and clinical findings established the live female co-twin calf was a freemartin, an abnormality of phenotypic sex. Failure to detect any significant peripheral concentrations of androgen supports the hypothesis that masculinization of the freemartin reproductive tract arises from diffusion of androgen and possibly other substances from the male co-twin rather than from endogenous production of androgen within the freemartin. This report documents that the freemartin condition can be induced by a male fetus with severe developmental abnormalities.