Liver size and lipid content differences between BALB/c and BALB/cJ mice on a high-fat diet are due,in part,to Zhx2

Mammalian Genome - BALB/cJ mice exhibit considerable phenotypic differences with other BALB/c substrains. Some of these traits involve the liver, including persistent postnatal expression of genes...     

Biology of Francisella tularensis subspecies holarctica live vaccine strain in the tick vector Dermacentor variabilis

Background

The γ-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis.

Methodology/Principal Findings

Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.8±0.8×10¹ and 1.1±0.03×10³ CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42% of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50% of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then to the oocytes, but the pathogen was not recovered from the subsequently-hatched larvae.

Conclusions/Significance

This study demonstrates that D. variabilis can be efficiently colonized with F. tularensis using artificial methods. The persistence of F. tularensis in D. variabilis suggests that this tick species may be involved in the maintenance of enzootic foci of tularemia in the central United States.     

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.     

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.     

Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Role of intracellular calcium in Pasteurella haemolytica leukotoxin-induced bovine neutrophil leukotriene B4 production and plasma membrane damage

Isolated neutrophils were used to study the intracellular calcium ([Ca2+]i) dependency of Pasteurella haemolytica leukotoxin-induced production of leukotriene B4 and plasma membrane damage. Exposure of neutrophils to leukotoxin caused a rapid and concentration-dependent increase in [Ca2+]i, followed by simultaneous plasma membrane damage and production of leukotriene B4. Removal of extracellular Ca2+, replacement of Ca2+ with other divalent cations, or exposure to high concentration of verapamil, an inhibitor of voltage-dependent calcium channels, inhibited leukotoxin-induced increases in [Ca2+]i, leukotriene B4 production, and membrane damage, thus indicating that influx of extracellular Ca2+ is necessary to produce these leukotoxin-induced neutrophil responses.     

High-throughput screening of a diversity collection using biodefense category a and B priority pathogens

One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute-based high-throughput screening (HTS) 96-well-based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 μg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.