Insertional inactivation of the methionine s-methyltransferase gene eliminates the s-methylmethionine cycle and increases the methylation ratio

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.     

Does terbutaline damage the developing heart?

BACKGROUND: β₂‐Adrenoceptor (βAR) agonists, such as terbutaline, are widely used to arrest preterm labor. They also cross the placenta where they stimulate receptors in fetal tissues, which in turn use βAR input for trophic control of cell replication and differentiation. METHODS: As rats are altricial, we administered terbutaline in two different postnatal exposure periods (10 mg/kg given daily on Days 2–5 or 11–14). RESULTS: Hearts were examined twenty‐four hours after the last dose and on postnatal day 30 for cardiac damage. Neither treatment paradigm caused an increase in cardiac abnormalities compared to controls but quantitative analysis of the number of nuclei indicated reductions in females. CONCLUSIONS: These findings do not support earlier case reports of outright myocardial necrosis after terbutaline tocolysis in human infants. Nevertheless, the significant statistical association between terbutaline and cardiac anomalies in epidemiological studies suggest that terbutaline may sensitize the developing heart to other insults that affect development. Birth Defects Res B 68:449–455, 2003. © 2003 Wiley‐Liss, Inc.     

A genetic analysis of the London strain of rainbow trout

The London strain of rainbow trout (Oncorhynchus mykiss) was created by interbreeding three other strains of rainbow trout and therefore was expected to have higher levels of genetic variation than other strains of rainbow trout. We examined 129 London strain rainbow trout from Indiana by allozyme electrophoresis to assess levels of genetic variation and to examine the relationship between the London strain and other hatchery strains. When using the same loci to compare with other hatchery strains the London strain showed levels of genetic variation within the range of other hatchery strains: mean heterozygosity of 0.053 (0.031-0.099), 1.27 (1.20-1.60) alleles per locus and 20.0% (20.0-40.0%) of the loci were polymorphic. The London strain is somewhat distinct from other hatchery strains (D=0.009-0.072), in part because of the high frequency of the sIDHP*40 allele.     

Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia

Human obesity and high fat feeding in rats are associated with the development of insulin resistance and perturbed carbohydrate and lipid metabolism. It has been proposed that these metabolic abnormalities may be reversible by interventions that increase plasma leptin. Up to now, studies in nongenetic animal models of obesity and in human obesity have concentrated on multiple injection therapy with mixed results. Our study sought to determine whether a sustained, moderate increase in plasma leptin, achieved by administration of a recombinant adenovirus containing the leptin cDNA (AdCMV-leptin) would be effective in reversing the metabolic abnormalities of the obese phenotype. Wistar rats fed a high-fat diet (HF) were heavier (P < 0.05), had increased fat mass and intramuscular triglycerides (mTG), and had elevated plasma glucose, insulin, triglyceride, and free fatty acids compared with standard chow-fed (SC) control animals (all P < 0.01). HF rats also had impaired glucose tolerance and were markedly insulin resistant, as demonstrated by a 40% reduction in insulin-stimulated muscle glucose uptake (P < 0.001). Increasing plasma leptin levels to 29.0 +/- 1.5 ng/ml (from 7.0 +/- 1.4 ng/ml, P < 0.001) for a period of 6 days decreased adipose mass by 40% and normalized plasma glucose and insulin levels. In addition, insulin-stimulated skeletal muscle glucose uptake was normalized in hyperleptinemic rats, an effect that correlated closely with a 60% (P < 0.001) decrease in mTG. Importantly, HF rats that received a control adenovirus containing the beta-galactosidase cDNA and were calorically matched to AdCMV-leptin-treated animals remained hyperglycemic, hyperinsulinemic, insulin resistant, and maintained elevated mTG. We conclude that a gene-therapeutic intervention that elevates plasma leptin moderately for a sustained period reverses diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance, and that these improvements are tightly linked to leptin-induced reductions in mTG.     

The generation and characterization of antagonist RNA aptamers to human oncostatin M

Oncostatin M (OSM) is a multifunctional member of the interleukin-6 cytokine family. OSM has been implicated as a powerful proinflammatory mediator and may represent a potentially important, novel therapeutic opportunity for treatment of established rheumatoid arthritis. To further investigate the role of OSM in inflammatory disorders, we have isolated a series of RNA aptamers that bind specifically to human OSM. The highest affinity aptamer, designated ADR58, has been characterized in a series of in vitro and cell based assays. ADR58 has an affinity of 7 nm for human OSM, and it can antagonize OSM binding to the gp130 receptor and specifically antagonize OSM mediated signaling. The aptamer has been truncated in length to 33 bases, all pyrimidine positions are substituted with 2' fluorine, and 14 of 18 purine positions have been substituted with 2' O-methyl to increase stability toward nucleases. This truncated, modified form of ADR58 retains complete affinity and functional activity for OSM. This aptamer may be used as a tool to further investigate the role of OSM in inflammatory disorders and may also have role as a therapeutic agent.     

Characterization of oxytetracycline-resistant heterotrophic bacteria originating from hospital and freshwater fishfarm environments in England and Ireland

This ecotaxonomic study compared the antibiotic tolerance among culturable oxytetracyline-resistant (Ot(r)) heterotrophic strains isolated from two aquatic environments representing human activities in health care and aquaculture, namely hospital effluents and freshwater fishfarms. Using a standardized methodology, samples taken in England and Ireland were analyzed to determine the antibiotic tolerance profiles of two groups of culturable Ot(r) bacterial isolates at the intergeneric and intrageneric level comprising heterotrophs (189 strains) and mesophilic Aeromonas spp. (153 strains), respectively. Antibiogram data of heterotrophic isolates revealed that Irish hospital strains comprised higher frequencies of multi-tolerance than those originating from fishfarm environments whereas a reverse correlation was found among the English heterotrophs. Polyphasic identification of the isolates using fatty acid analysis and API 20E profiling showed that this difference arose from the unique taxonomic diversity within each heterotrophic strain set. Acinetobacter (27%) and Brevundimonas (22%) were predominant among the Irish Ot(r) fishfarm isolates, whereas isolates originating from the English aquaculture site almost entirely consisted of Stenotrophomonas maltophilia (86%) exhibiting high frequencies of tolerance to ampicillin and streptomycin. Within both the English and the Irish Ot(r) Aeromonas strain sets, on the other hand, the hospital strain sets displayed higher numbers of multi-tolerant isolates than to fishfarm isolates although country-specific differences were observed for individual antimicrobial agents. The typical occurrence of kanamycin-tolerant aeromonads in the Irish hospital site could to some extent be linked to the typical presence of A. hydrophila DNA hybridization group (HG) 3 strains as determined by fatty acid analysis and fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting. Essentially, these data indicate that tolerance profiles in a specific environment of one country do not necessarily reflect the corresponding tolerance profiles of the same type of environment in another country, and this mainly as a result of the unique taxonomic composition of each site. Ot(r) representatives of Acinetobacter, S. maltophilia, and A. veronii biovar sobria HG8 were common to most if not all of the four sites under study, indicating that these three taxa may serve as potential indicator organisms for monitoring antibiotic tolerance among indigenous bacterial populations in various aquatic environments.     

Taxonomic anxieties: Axis I and Axis II in prison

This essay describes the use of the Diagnostic and Statistical Manual of Psychiatry in the prison setting. The distinction between the Axis I disorders (major mental illness) and those designated Axis II (character disorders) is explored in terms of the professional division of labor in prison and the problems posed for prison discipline by "behaviorally disturbed" inmates. Conflicts over diagnosis are placed in ethnographic and historical perspective and form the basis for a discussion of the problematic relationship between disciplinary space and issues of subjection and agency.     

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.