Involvement of a single periplasmic hydrogenase for both hydrogen uptake and production in some Desulfovibrio species

Various sulphate-reducing bacteria differing in the number of genes encoding hydrogenase were shown to ferment lactate in coculture with Methanospirillum hungatei, in the absence of sulphate. The efficiency of interspecies H2 transfer carried out by these species of sulphate-reducing bacteria does not appear to correlate with the distribution of genes coding for hydrogenase. Desulfovibrio vulgaris Groningen, which possesses only the gene for [NiFe] hydrogenase, oxidizes hydrogen in the presence of sulphate and produces some hydrogen during fermentation of pyruvate without electron acceptor. The hydrogenase of D. vulgaris was purified and characterized. It exhibits a molecular mass of 87 kDa and is composed of two different subunits (60 and 28 kDa). D. vulgaris hydrogenase contains 10.6 iron atoms, 0.9 nickel atom and 12 acid-labile sulphur atoms/molecule, and the absorption spectrum of the enzyme is characteristic of an iron-sulphur protein. Maximal H2 uptake and H2 evolution activities were 332 and 230 units/mg protein, respectively. D. vulgaris cells contain exclusively the [NiFe] hydrogenase, whatever the growth conditions, as shown by biochemical and immunological studies. Immunocytolocalization in ultrathin frozen sections of cells grown on lactate and sulphate, on H2 and sulphate and on pyruvate showed that the [NiFe] hydrogenase was located in the periplasmic space. Labelling was enhanced in cells grown on H2 and sulphate and on pyruvate. The results enable us to conclude that D. vulgaris Groningen contains a single hydrogenase of the [NiFe] type, located in the periplasmic space like that described for D. gigas. This enzyme appears to be involved in both H2 uptake and H2 production, depending on the growth conditions.     

How can and do Australian doctors promote physical activity?

We describe a modified technique for the salvage of a total knee arthroplasty after disruption of the extensor mechanism. Between January and December 1992, seven patients had reconstruction of the extensor mechanism with use of a medial or an extended medial gastrocnemius flap. Six of the seven patients were followed for a mean of thirty-three months (range, twenty-six to forty-one months) and were evaluated both preoperatively and postoperatively with regard to the knee and functional scores of The Knee Society as well as the range of motion, extensor lag, walking status, and patellar height. The seventh patient was lost to follow-up six months postoperatively and was excluded from the analysis of the results. Preoperatively, the knee and functional scores were 16 +/- 12.3 points and 12 +/- 12.1 points (mean and standard deviation), respectively; the mean range of motion was 70 +/- 44.0 degrees; and the mean extensor lag was 53 +/- 33.4 degrees. Postoperatively, the mean knee and functional scores improved to 82 +/- 12.4 points and 51 +/- 23.0 points, respectively; the mean range of motion improved to 100 +/- 21.8 degrees; and the mean extensor lag decreased to 24 +/- 18.8 degrees. After the procedure, all patients who previously had been dependent on a walker were able to walk about the community with or without a cane, and those who had been dependent on a wheelchair were able to walk with the assistance of a walker. Patellar height was measured according to the method of Insall and Salvati for the four patients who had a patella. Preoperatively, the patellar heights were grossly abnormal; postoperatively, they more closely approached accepted normal values for three of the four patients. Reconstruction of a complicated rupture of the extensor mechanism with use of a medial gastrocnemius transposition flap after total knee arthroplasty is a reliable option for treatment.     

Chromosome breakpoint distribution in nonmelanoma skin cancers

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330(233) primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood.     

Validity of screening for individuals at risk for type I diabetes by combined analysis of antibodies to recombinant proteins

OBJECTIVE: To determine whether screening for the presence of multiple antibody markers for IDDM is effective at identifying individuals with high risk for disease development. RESEARCH DESIGN AND METHODS: Antibodies to GAD and the tyrosine phosphatase-like protein 1A-2 were determined in sequential serum samples from 44 first-degree relatives of IDDM patients, identified as possessing islet cell antibody (ICA) and/or insulin autoantibody (IAA), who were followed prospectively for IDDM development, ICA, IAA, and antibodies to GAD and 1A-2 were also determined in 93 cases of new-onset nonfamilial IDDM. RESULTS: The presence of two or more antibodies in addition to ICA or IAA conferred high risk (61%) for development of IDDM within 5 years of entry into the study and identified 89% of those who have developed IDDM on current follow-up. None of the relatives positive for ICA or IAA alone, in the absence of other antibody markers, have developed IDDM. Antibodies to islet antigens could both appear and disappear in follow-up samples obtained after entry into the study. The majority (60%) of young (< 16 years), sporadic cases of IDDM had multiple antibodies to islet antigens, but this proportion was lower in older patients (37%). CONCLUSIONS: A screening strategy based on the analysis of antibodies to multiple islet antigens can predict IDDM at high sensitivity and specificity in families, and such a strategy may also be applicable to identify young individuals in the general population with high disease risk. Since appearance of antibodies to different antigens occurs sequentially rather than simultaneously, accurate assessment of diabetes risk based on the presence of multiple antibodies will require follow-up over a number of years after the first evidence of islet autoimmunity.     

Dietary variety via sweetening and voluntary feed intake of lactating dairy cows

The effects of choice of diets on feed intake were studied using 12 lactating Holstein cows. A switchback design was used that had three periods and two sequential blocks. Diets were 1) a control total mixed ration (TMR), which consisted of alfalfa haylage, corn silage, and a concentrate mixture based on ground corn and soybean meal (25:25:50 on a dry matter basis) and 2) a sweetened TMR in which a brown sugar food product constituted 1.5% of the dietary dry matter. Treatments consisted of the control TMR fed on both sides of divided feed bunks, the sweetened TMR fed on both sides of divided feed bunks, or both TMR fed on alternating (daily) sides of divided feed bunks in tie stalls. Periods were 2 wk in duration, and cows averaged 67 and 53 d of lactation at the start of blocks 1 and 2, respectively. The dry matter intake, body weight, milk yield, and percentages of milk fat, protein, and solids not fat were similar when either TMR was fed alone. A choice of control TMR or sweetened TMR did not affect any of these variables. The dry matter intake, body weight, milk yield, and milk protein percentage were affected by block; however, these effects were probably caused by differences between the blocks in environment and stage of lactation. The results of this experiment might have been affected by the composition of the control TMR, its similarity to the sweetened TMR, availability of both diets simultaneously when a choice was offered, and use of a TMR instead of separate feeds or simpler mixtures.     

Isogeometric finite element data structures based on Bézier extraction of T‐splines

We develop finite element data structures for T‐splines based on Bézier extraction generalizing our previous work for NURBS. As in traditional finite element analysis, the extracted Bézier elements are defined in terms of a fixed set of polynomial basis functions, the so‐called Bernstein basis. The Bézier elements may be processed in the same way as in a standard finite element computer program, utilizing exactly the same data processing arrays. In fact, only the shape function subroutine needs to be modified while all other aspects of a finite element program remain the same. A byproduct of the extraction process is the element extraction operator. This operator localizes the topological and global smoothness information to the element level, and represents a canonical treatment of T‐junctions, referred to as ‘hanging nodes’ in finite element analysis and a fundamental feature of T‐splines. A detailed example is presented to illustrate the ideas. Copyright © 2011 John Wiley & Sons, Ltd.     

Glycated hemoglobin reference limits obtained by high performance liquid chromatography in adults and pregnant women

Pentachlorophenol (PCP) and molybdate have been shown to inhibit the sulfoconjugation of various chemicals in rats and therefore are useful to examine the role of sulfoconjugation on the toxicity of a chemical. PCP inhibits sulfation by competing with substrates for phenol-sulfotransferases, but not hydroxysteroid-sulfotransferases. In contrast, molybdate decreases sulfation by limiting sulfate availability and thereby decreasing the synthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which is the obligate cosubstrate for sulfation. Therefore, it was of interest to determine whether PCP or molybdate is effective in decreasing the in vivo sulfation of dehydroepiandrosterone (DHEA), which is a substrate for hydroxysteroid-sulfotransferases. PCP (40 micromol/kg ip) or molybdate (7.5 mmol/kg po) was given 45 min and 4 h, respectively, prior to the start of DHEA infusion. The effects of these two sulfation inhibitors on DHEA sulfation were dependent on the rate of DHEA infusion in rats. PCP had different effects on the sulfation of various infusion rates of DHEA in rats. PCP had little effect on the sulfation after the two lowest infusion rates of DHEA (12.5 and 25 mg/kg) and actually increased (233%) DHEA-sulfate serum concentrations with the highest DHEA infusion rate (50 mg/kg). Although molybdate had little affect on the sulfation of the lowest DHEA infusion rate, it significantly decreased (50-85%) DHEA-sulfate serum concentrations with the two higher DHEA infusion rates. These data indicate that molybdate, unlike PCP, decreases the sulfation of DHEA and may be a useful tool to decrease the sulfation of other substrates of hydroxysteroid-sulfotransferases.     

Long-term activation of capacitative Ca2+ entry in mouse microglial cells

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.