Investigations of the pathogenicity of Salmonella enteritidis serotype Amsterdam following a naturally occurring infection in rats

A naturally occurring infection of an unusual Salmonella sp. was detected in research rats during routine health monitoring procedures. The organism, Salmonella enteritidis serotype Amsterdam appeared to have a low pathogenicity and did not cause clinical disease in adult rats. Experimental mouse and guinea pig inoculations suggest that the organism is more pathogenic in mice.     

A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression.

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.     

High-performance liquid chromatography assay for N-acetylcysteine in biological samples following derivatization with N-(1-pyrenyl)maleimide

N-Acetylcysteine is a thiol antioxidant with expanding clinical importance. A sensitive, rapid method for determining reduced N-acetylcysteine (NAC) concentration in biological samples has been developed which uses a modified reversed-phase high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl)maleimide (NPM). The NAC-NPM adduct was analyzed by HPLC with fluorescence detection. The calibration curve for NAC was linear over the range 8–2500 nM and the coefficient of variation obtained for the within-run precision and the between-run precision for 0.5 mM NAC was 1.5% and 2.7%, respectively. Relative recovery of NAC from biological materials ranged between 86% and 96% and the limit of quantitation from biological samples was 32 nM. These results suggest practical advantages relative to other widely-accepted methods of NAC measurement.     

Expression of carbonic anhydrase II mRNA and protein in oligodendrocytes during toxic demyelination in the young adult mouse

The aim of this study was to identify events that might take place in oligodendrocytes early in the process of demyelination, i.e., before the occurrence of massive loss of myelin. It was considered important to focus on demyelination and remyelination in young adults, in whose brains there would be relatively few juvenile glial precursor cells. CAII mRNA and protein were used to monitor changes in oligodendrocytes during cuprizone intoxication in the mice. After four or eight weeks of cuprizone feeding CAII message became less plentiful in oligodendrocyte processes. Two days after removal of cuprizone CAII message had appeared in those cell processes. Four or eight weeks after beginning cuprizone feeding CAII protein had decreased∼25% in forebrain homogenates. The loss of CAII protein was reversible after four weeks on cuprizone, but not after eight weeks. After four weeks of cuprizone feeding the numbers of CAII mRNA-prositive oligodendrocytes had decreased by ∼50%m and after eight weeks, by ∼80%. By 12 weeks, however, the number of oligodendrocytes expressing CAII mRNA had spontaneously returned to normal levels. Before eight weeks of cuprizone feeding, loss of myelinated tracts in the corpus striatum was reversible. Demyelination appreared to become irreversible after nine weeks of intoxication, although expression of CAII mRNA remained reversible. The results suggest that in the brain of the young adult, oligodendrocytes expressing message for CAII can be generated spontaneously shortly before demyelination becomes irreversible, and can survive and continue to express CAII mRNA but not CAII protein. Special issue dedicated to Dr. Marion E. Smith.     

Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication

Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later, as cyclin A accumulates and cells enter S phase, hOrc1p is ubiquitinated on chromatin and then degraded. hOrc1p destruction occurs through the proteasome and is signaled in part by the SCF(Skp2) ubiquitin-ligase complex. Other hORC subunits are stable throughout the cell cycle. The regulation of hOrc1p may be an important mechanism in maintaining the ploidy in human cells.     

Structure of an atypical epoxide hydrolase from Mycobacterium tuberculosis gives insights into its function

Epoxide hydrolases are vital to many organisms by virtue of their roles in detoxification, metabolism and processing of signaling molecules. The Mycobacterium tuberculosis genome encodes an unusually large number of epoxide hydrolases, suggesting that they might be of particular importance to these bacteria. We report here the first structure of an epoxide hydrolase from M.tuberculosis, solved to a resolution of 2.5 A using single-wavelength anomalous dispersion (SAD) from a selenomethionine-substituted protein. The enzyme features a deep active-site pocket created by the packing of three helices onto a curved six-stranded beta-sheet. This structure is similar to a previously described limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis and unlike the alpha/beta-hydrolase fold typical of mammalian epoxide hydrolases (EH). A number of changes in the mycobacterial enzyme create a wider and deeper substrate-binding pocket than is found in its Rhodococcus homologue. Interestingly, each structure contains a different type of endogenous ligand of unknown origin bound in its active site. As a consequence of its wider substrate-binding pocket, the mycobacterial EH is capable of hydrolyzing long or bulky lipophilic epoxides such as 10,11-epoxystearic acid and cholesterol 5,6-oxide at appreciable rates, suggesting that similar compound(s) will serve as its physiological substrate(s).