Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli

The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown. Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity. These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O. Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction.     

G1 arrest and the division/conjugation decision in Tetrahymena.

The transformation from the asexual proliferative stage of Tetrahymena to the sexual stage, during which cells of complementary mating types pair and nuclear fertilization occurs, provides an opportunity to study the relationship between the division cycle and differentiation. Conjugation is induced in cells starved for at least 2 hr by mixing complementary mating types. To determine the effect of starvation on the cell cycle, dividing cells were selected from a log growth culture and stepped down to non-nutrient conditions. The G₁ stage is operationally divisible into two sectors, A and B. In the A stage, cells arrest in nutrient-free medium. In the B stage, they proceed through the division cycle. Arrested G₁A cells may conjugate directly when challenged with similar cells of a complementary mating type. It is thereby demonstrated that Tetrahymena cells in G₁A can be directed to divide (nutrient conditions) or can be directed to differentiate (non-nutrient conditions plus complementary mating type) without an intervening division cycle. This rules out a requirement for reprogramming via chromosomal replication or cell division and suggests that G₁A is a stage during which the division/differentiation decision is made in direct response to ambient conditions.     

Biosynthesis of Prostacyclin in Rat Cerebral Microvessels and the Choroid Plexus

Abstract: Microvessels, predominantly capillaries, were isolated from rat cerebrum by a modification of published procedures. The morphology and purity of the preparations were monitored by light and electron microscopy and by enrichment in alkaline phosphatase, γ-glutamyl transpeptidase, and prostacyclin synthetase. A reversed-phase high-pressure liquid chromatographic method was used in the purification of prostaglandins after extraction from aqueous incubation solutions. Prostacyclin synthesis in brain is localized in cerebral blood vessels and capillaries. The endogenous biosynthetic capacity of the isolated cerebral capillary fractions for prostacyclin, measured as its chemically stable breakdown product, 6-keto-prostaglandin F_1α, was 11 ng/mg protein/10 min. Choroid plexus and intact surface vessels synthesized 6-keto-prostaglandin F_1α at 37 and 35 ng/mg protein/10 min, respectively. The prostacyclin-synthesizing enzyme of the cerebral capillaries also converted the exogenously added prostaglandin endoperoxides to 6-keto-prostaglandin F_1α. Comparison of the synthesis of prostaglandins 6-keto-F_1α, E₂, and F_2α showed that 6-keto-prostaglandin F_1α was the major prostaglandin formed in the microvessels, in the larger surface vessels, and in the choroid plexus. Prostaglandin D₂ was not detected. Prostacyclin synthesis by the cerebral vasculature is similar to that in other blood vessels and cultured human endothelial cells. Possible physiological roles of prostacyclin in the cerebral microvasculature are discussed with special regard to the autoregulation of cerebral blood flow.     

Substrate recognition properties of oligopeptidase B from Salmonella enterica serovar Typhimurium

Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P(1). While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu(576) and Glu(578), that define P(1) specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp(460) and Asp(462), that may be involved in defining P(2) specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.     

Antituberculous drug resistance in Manitoba from 1980 to 1989.

OBJECTIVES: To estimate the magnitude of antituberculous drug resistance and identify the risk factors for its development in tuberculosis patients in Manitoba over a 10-year period. As well, to examine the clinical course of the patients whose initial or subsequent isolates of Mycobacterium tuberculosis were resistant to one or more drugs. DESIGN: Comparison of drug-resistant and non-drug-resistant cases of tuberculosis. SETTING: Manitoba. PATIENTS: All people with tuberculosis reported to the Central Tuberculosis Registry of Manitoba between Jan. 1, 1980, and Dec. 31, 1989. MAIN OUTCOME MEASURES: Of 1478 cases of active tuberculosis 1086 were culture positive, and drug susceptibility testing was performed in these cases. The clinical course, including outcome of treatment, of all drug-resistant cases was described. RESULTS: Of 1086 culture-positive cases of tuberculosis 77 (7.1%) were drug resistant. Odds ratios suggested that the risk of drug resistance was significantly higher among the immigrants than among the other Canadians. Compared with the other Canadians the risk of drug resistance was 9.9 times greater among the immigrants in whom tuberculosis developed within the first year after arrival in Canada and 5.4 times greater among the immigrants in whom it developed 2 to 5 years after arrival in Canada. Of the 71 patients with drug-resistant disease whose type of resistance was known 62% had never taken antituberculous drugs before and 38% had. Most (91%) of the 77 cases of drug-resistant disease were resistant to first-line drugs, especially isoniazid and streptomycin. Thirty-two (42%) of the 77 cases were resistant to two or more first-line drugs. Of patients with drug-resistant disease a subgroup of 10 had disease that became resistant to several drugs over the 10-year period. The outcome of treatment in these individuals was poor, and they presented a particular public health problem. CONCLUSION: Resistance to one or more first-line antituberculous drugs continues to complicate the treatment of tuberculosis and may facilitate the spread of the disease.     

Macromolecular solvation energies derived from small molecule crystal morphology.

The morphology of small molecule crystals provides a model for evaluating surface solvation energies in a system with similar packing density to that observed for amino acid residues in proteins. The solvation energies associated with the transfer of methylene and carboxyl groups between vacuum and aqueous phases are estimated to be approx. $40 and -260 cal/A2, respectively, from an analysis of the morphology of succinic acid crystals. These solvation energies predict values for contact angles in reasonable agreement with measurements determined from macroscopic monolayer surfaces. Transfer free energies between vapor and water phases for a series of carboxylic acids are also predicted reasonably well by these solvation energies, provided the surface exposure of different groups is quantitated with the molecular surface area rather than the more traditional accessible surface area. In general, molecular surfaces and molecular surface areas are seen to have important advantages for characterizing the structure and energetics of macromolecular surfaces. Crystal faces of succinic acid with the lowest surface energies in aqueous solution are characteristically smooth. Increasing surface roughness and apolarity are associated with higher surface energies, which suggests an approach for modifying the surface properties of proteins and other macromolecules.     

Cholecystokinin binding and degradation by isolated rat liver cells

The present studies were directed to examine and quantify binding and degradation of radiolabelled cholecystokinin (CCK) peptides by isolated rat liver cells. After incubation with liver cells (4.5 x 10(6) cells/ml) at 14 degrees C, minimal binding (less than 5%) of labelled CCK33 was detected. When labelled nonsulfated (nsCCK8) and sulfated CCK8 (sCCK8) were incubated, 16.2 +/- 1.8% (mean +/- S.E.) and 7.2 +/- 0.1% of 125I-nsCCK8 and 125I-sCCK8, respectively, were bound to the cell fraction. However, no inhibition of binding of either labelled nsCCK8 or sCCK8 was observed when incubated in the presence of excess unlabelled peptide (10 ng-10 micrograms). Preferential binding of labelled sCCK8, the biologically active form of the octapeptide, appeared to be to the nonparenchymal liver cell, rather than the hepatocyte, fraction; when corrected for cell size and protein content, binding of sCCK8 was approximately 15-times greater by the nonparenchymal cell population. When incubated with hepatocytes at 37 degrees C for 60 min, no degradation of labelled sCCK8 was detected by high pressure liquid chromatography. In contrast, progressive degradation of sCCK8 was observed when the peptide was incubated with the nonparenchymal cells. The results of these studies confirm previous observations that CCK33 is not bound by the liver. They further demonstrate that to some degree CCK8 is preferentially bound and degraded by hepatic nonparenchymal cells; however, this binding appears to be noncompetitive and, therefore, probably not receptor-mediated.     

Regional and Ontogenic Expression of the NMDA Receptor Subunit NR2D Protein in Rat Brain Using a Subunit-Specific Antibody

Abstract: A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an ∼160-kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross-reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N -glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.     

In vivo kinematic behavior of the radio-capitate joint during wrist flexion-extension and radio-ulnar deviation.

The capitate is often considered the "keystone" of the carpus, not simply because of its central and prominent position in the wrist, but also because of its mechanical interactions with neighboring bones. The purpose of this study was to determine in vivo three-dimensional capitate kinematics. Twenty uninjured wrists were investigated using a recently developed, non-invasive markerless bone registration (MBR) technique. Surface contours of the capitate, third metacarpal and radius were extracted from computed tomography images of seven wrist positions and the three-dimensional motions of the capitate and third metacarpal were calculated with respect to the radius in wrist flexion-extension and radio-ulnar deviation. We found that in vivo capitate motion does not simply occur about a single pivot point like a universal joint, as demonstrated by non-intersecting rotation axes for different capitate motions. The distance between flexion and ulnar deviation axes was 3.9+/-2.0 mm, and the distance between extension and ulnar deviation axes was 3.9+/-1.4 mm. Furthermore, capitate axes for males tended to be located more distally than axes for females. However, we believe that this result is related to subject size and not to gender. We also found that there is minimal relative motion between the capitate and third metacarpal during these in vivo wrist motions. These findings demonstrate the complexity of capitate kinematics, as well as the different mechanisms through which wrist flexion, extension, radial deviation and ulnar deviation occur.