Particulate carbonic anhydrase in homogenates of human kidney.

About 2% of human kidney carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been found in particulate fractions. Its distribution in the particulate fractions obtained by differential centrifugation suggests that it may be concentrated in the brush border. The particulate enzyme is like red cell carbonic anhydrace C in its susceptibility to inhibition by anions. Particulate carbonic anhydrase is firmly bound to the membrane and is not released by incubation at pH 10.6 and 37 degrees C or by addition of Triton X-100 or deoxycholate. In 10% Triton X-100 at pH 11.3 and 37 degrees C, the particulate enzyme is inactivated with a half time of about 20 min, and this is at least an order of magnitude slower than the inactivation of soluble enzymes in the presence or absence of membranes. The soluble enzymes are inactivated within a few minutes at 25 degrees C in 3-4% sodium dodecyl sulfate, but the particulate enzyme is relatively stable under those conditions, and its half-time of inactivation at 14 degrees C with a detergent-protein ratio of 25 was about 24 h. Gel filtration with Ultragel AcA-44 in sodium dodecyl sulfate indicates that the membrane carbonic anhydrase has a molecular weight of less than 66 000, so its stability is not due to association with large membrane fragments or vesicles. These results suggest that the membrane enzyme may be a different isozyme than the soluble carbonic anhydrases. Although present in relatively small amounts, its localization on the membrane could give it functional significance.     

Effects of Petroleum Hydrocarbons on Plant Litter Microbiota in an Arctic Lake

The effects of petroleum hydrocarbons on the microbial community associated with decomposing Carex leaf litter colonized in Toolik Lake, Alaska, were examined. Microbial metabolic activity, measured as the rate of acetate incorporation into lipid, did not vary significantly from controls over a 12-h period after exposure of colonized Carex litter to 3.0 ml of Prudhoe Bay crude oil, diesel fuel, or toluene per liter. ATP levels of the microbiota became elevated within 2 h after the exposure of the litter to diesel fuel or toluene, but returned to control levels within 4 to 8 h. ATP levels of samples exposed to Prudhoe Bay crude oil did not vary from control levels. Mineralization of specifically labeled ¹⁴C-[lignin]-lignocellulose and ¹⁴C-[cellulose]-lignocellulose by Toolik Lake sediments, after the addition of 2% (vol/vol) Prudhoe Bay crude oil, motor oil, diesel fuel, gasoline, n-hexane, or toluene, was examined after 21 days of incubation at 10°C. Diesel fuel, motor oil, gasoline, and toluene inhibited ¹⁴C-[lignin]-lignocellulose mineralization by 58, 67, 67, and 86%, respectively. Hexane-treated samples displayed an increase in the rate of ¹⁴C-[lignin]-lignocellulose mineralization of 33%. ¹⁴C-[cellulose]-lignocellulose mineralization was inhibited by the addition of motor oil or toluene by 27 and 64%, respectively, whereas diesel fuel-treated samples showed a 17% increase in mineralization rate. Mineralization of the labeled lignin component of lignocellulose appeared to be more sensitive to hydrocarbon perturbations than was the labeled cellulose component.     

Renal nerve inhibition by central NaCl and ANG II is abolished by lesions of the lamina terminalis

The lamina terminalis is situated in the anterior wall of the third ventricle and plays a major role in fluid and electrolyte homeostasis and cardiovascular regulation. The present study examined whether the effects of intracerebroventricular infusion of hypertonic saline and ANG II on renal sympathetic nerve activity (RSNA) were mediated by the lamina terminalis. In control, conscious sheep (n = 5), intracerebroventricular infusions of 0.6 M NaCl (1 ml/h for 20 min) and ANG II (10 nmol/h for 30 min) increased mean arterial pressure (MAP) by 6 +/- 1 (P < 0.001) and 14 +/- 3 mmHg (P < 0.001) and inhibited RSNA by 80 +/- 6 (P < 0.001) and 89 +/- 7% (P < 0.001), respectively. Both treatments reduced plasma renin concentration (PRC). Intracerebroventricular infusion of artificial cerebrospinal fluid (1 ml/h for 30 min) had no effect. In conscious sheep with lesions of the lamina terminalis (n = 6), all of the responses to intracerebroventricular hypertonic saline and ANG II were abolished. In conclusion, the effects of intracerebroventricular hypertonic saline and ANG II on RSNA, PRC, and MAP depend on the integrity of the lamina terminalis, indicating that this site plays an essential role in coordinating the homeostatic responses to changes in brain Na(+) concentration.     

CXC chemokines modulate IgE secretion and pulmonary inflammation in a model of allergic asthma

The pathophysiology of asthma is influenced by exposure to allergens and endotoxin. Although the role of allergen-induced eosinophilia has been widely studied, neutrophil-mediated responses remain elusive. A role for neutrophils in the asthmatic responses is likely since human neutrophils have been shown to express IgE receptors, as well as receptors for many cytokines and chemokines implicated in the pathogenesis of asthma. In this study we investigated neutrophil involvement in a novel, house dust extract (HDE) induced model of asthma-like pulmonary inflammation. Mice were immunized and challenged with HDE containing high levels of cockroach allergens, 377 U/ml Bla g1 and 6249 ng/ml Bla g2. The biological activity of the murine chemokines KC and MIP-2 was inhibited with specific rabbit antisera. Differential counting of cells recovered from the bronchoalveolar lavage (BAL) fluid showed that neutralization of KC and MIP-2 significantly decreased pulmonary recruitment of neutrophils (reduced 86%) and lymphocytes (reduced 76%). Neutralization of these chemokines also exerted a systemic effect with a significant decrease in plasma IgE levels, 547 ng/ml+/-65 compared to 1314 ng/ml+/-247 for control sera treated animals. This study shows that CXC chemokines play an important role in allergy and asthma both at the level of pulmonary cell recruitment and systemic immune responses.     

Characterization of the Phd repressor-antitoxin boundary

The P1 plasmid addiction operon (a classic toxin-antitoxin system) encodes Phd, an unstable 73-amino-acid repressor-antitoxin protein, and Doc, a stable toxin. It was previously shown by deletion analysis that the N terminus of Phd was required for repressor activity and that the C terminus was required for antitoxin activity. Since only a quarter of the protein or less was required for both activities, it was hypothesized that Phd might have a modular organization. To further test the modular hypothesis, we constructed and characterized a set of 30 point mutations in the third and fourth quarters of Phd. Four mutations (PhdA36H, V37A, I38A, and F44A) had major defects in repressor activity. Five mutations (PhdD53A, D53R, E55A, F56A, and F60A) had major defects in antitoxin activity. As predicted by the modular hypothesis, point mutations affecting each activity belonged to disjoint, rather than overlapping, sets and were separated rather than interspersed within the linear sequence. A final deletion experiment demonstrated that the C-terminal 24 amino acid residues of Phd (preceded by a methionine) retained full antitoxin activity.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.     

Preclinical TSPO Ligand PET to Visualize Human Glioma Xenotransplants: A Preliminary Study

Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [¹⁸F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [¹⁸F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [¹⁸F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [¹⁸F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.     

Breast Tissue Composition and Immunophenotype and Its Relationship with Mammographic Density in Women at High Risk of Breast Cancer

Aim

To investigate the cellular and immunophenotypic basis of mammographic density in women at high risk of breast cancer.

Methods

Mammograms and targeted breast biopsies were accrued from 24 women at high risk of breast cancer. Mammographic density was classified into Wolfe categories and ranked by increasing density. The histological composition and immunophenotypic profile were quantified from digitized haematoxylin and eosin-stained and immunohistochemically-stained (ERα, ERβ, PgR, HER2, Ki-67, and CD31) slides and correlated to mammographic density.

Results

Increasing mammographic density was significantly correlated with increased fibrous stroma proportion (rs (22) = 0.5226, p = 0.0088) and significantly inversely associated with adipose tissue proportion (rs (22) = -0.5409, p = 0.0064). Contrary to previous reports, stromal expression of ERα was common (19/20 cases, 95%). There was significantly higher stromal PgR expression in mammographically-dense breasts (p=0.026).

Conclusions

The proportion of stroma and fat underlies mammographic density in women at high risk of breast cancer. Increased expression of PgR in the stroma of mammographically dense breasts and frequent and unexpected presence of stromal ERα expression raises the possibility that hormone receptor expression in breast stroma may have a role in mediating the effects of exogenous hormonal therapy on mammographic density.