Mumol Min-1 Research Articles

Gene Cloning, Expression, and Characterization of a Family 51 α-l-Arabinofuranosidase from Streptomyces sp. S9

An alpha-L-arabinofuranosidase gene, abf51S9, was cloned from Streptomyces sp. S9 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene consisted of 1,506 bp and encoded 501 amino acids with a calculated mass of 55.2 kDa. The deduced amino acid sequence was highly homologous with the alpha-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by Ni-NTA affinity chromatography and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.0 and 60 approximately 65 degrees C, respectively. The enzyme showed a broad pH range of stability, retaining over 75% of the maximum activity at pH 5.0 to 11.0. The specific activity, K (m), and V (max) with p-nitrophenyl-alpha-L-arabinofuranoside as substrate were 60.0 U mg(-1), 1.45 mM, and 221 mumol min(-1) mg(-1), respectively. Abf51S9 showed a mild but significant synergistic effect in combination with xylanase on the degradation of oat-spelt xylan and soluble wheat arabinoxylan substrates with a 1.19- and 1.21-fold increase in the amount of reducing sugar released, respectively. These favorable properties make Abf51S9 a good candidate in various industrial applications.

Purification and Characterization of a (R)-1-Phenyl-1,3-propanediol-producing Enzyme fromTrichosporon fermentansAJ-5152 and Enzymatic (R)-1-Phenyl-1,3-propanediol Production

An (R)-1-phenyl-1,3-propanediol-producing enzyme was purified from Trichosporon fermentans AJ-5152. It was NADPH-dependent and converted 3-hydroxy-1-phenylpropane-1-one (HPPO) to (R)-1-phenyl-1,3-propanediol [(R)-PPD] with anti-Prelog's specificity. It showed maximum activity at pH 7.0 and 40 degrees C. Its K(m) and V(max) values toward HPPO were 20.1 mM and 3.4 mumol min(-1) mg protein(-1) respectively. The relative molecular weight of the enzyme was estimated to be 68,000 on gel filtration and 32,000 on SDS-polyacrylamide gel electrophoresis. An (R)-PPD-producing reaction using the (R)-PPD-producing enzyme and an NADPH recycling system was carried out by successive feeding of HPPO. A total (R)-PPD yield of 8.9 g/l was produced in 16 h. The molar yield was 76%, and the optical purity of the (R)-PPD produced was over 99% e.e.

Physiological role and redox properties of a small cytochrome c5, cytochrome c-552, from alkaliphile, Pseudomonas alcaliphila AL15-21T

Cytochrome c-552 from Pseudomonas alcaliphila AL15-21(T), which is a small cytochrome c(5) from Pseudomonas spp., was first purified and characterized in our previous study. Although it has been found that cytochrome c-552 is induced at a high pH under air-limited condition, the physiological role of this cytochrome c has not been clarified yet. Therefore, to understand its physiological role, further characterization of this cytochrome expressed in Escherichia coli was performed. The yield of the recombinant protein reached 2.8 mg/l of culture, which was 76.4-fold larger than that of native cells. Analytical data of the recombinant protein exactly agreed with that of native cytochrome c-552. The recombinant cytochrome c-552 was oxidized by partially purified cb-type cytochrome c oxidase from P. alcaliphila AL15-21(T) at a rate of 9.6 mu mol min(-1) mg oxidase(-1). Unlike reported cytochromes c from other Pseudomonas spp., the E degrees ' values between pHs 5.0 and 10.0 were nearly unchanged. Cytochrome c-552 oxidized very slowly at pHs 8.0 (6.1 x 10(-4) h(-1)), 9.0 (1.4 x 10(-3) h(-1)) and 10.0 (1.6 x 10(-3) h(-1)), whereas it oxidized more rapidly at pH 7.0 (2.5 x 10(-3) h(-1)). On the other hand, horse heart cytochrome c showed higher oxidation rates at pHs 6.0-10.0 than cytochrome c-552. It is considered that the high electron-retaining ability of cytochrome c-552 at high pHs is important for its physiological function in the environmental adaptation of this bacteria for superior growth at high pHs under air-limited conditions.

A beta-galactosidase from pea seeds (PsBGAL): purification, stabilization, catalytic energetics, conformational heterogeneity, and its significance.

A basic glycosylated beta-galactosidase (PsBGAL) has been purified from pea seeds by 910-fold with a specific activity of 77.33 mumoL min(-1) mg(-1) protein. The purified enzyme is an electrophoretically homogeneous protein consisting of a single protein band with an apparent M(r) of 55 kDa, while the deglycosylated enzyme has a M(r) of 54.2 kDa on SDS-PAGE under reducing conditions. According to MALDI-TOF measurements of the 55 kDa band, the enzyme showed a homology with BGAL from other sources present in the SWISS-PROT database, while it showed no resemblance to any lectin. The N-terminal sequence of PsBGAL was determined as TIECK and showed a resemblance to BGAL from Arabidopsis thaliana (Q93Z24). The enzyme showed an unique property of multiple banding patterns on SDS-PAGE at 20 mA current, with tryptic digests of all bands having similar m/z values (using MALDI-TOF) while it showed only a single band at 10 mA current. PsBGAL is effectively compartmentalized during seed maturation inside vacuoles (pH approximately 5). The enzyme is capable of hydrolyzing pea seed xyloglucan, and it may be involved in modifying the cell wall architecture during seedling growth and development. The enzyme has a protonated carboxyl group at its active site as observed by ionization constant, thermodynamics, and chemical modification studies.

A novel alkalo- and thermostable phospholipase D from Streptomyces olivochromogenes

A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75 degrees C and the enzyme was stable from pH 7 to 13 and from 55 to 75 degrees C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca(2+)-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 mumol min(-1) mg(-1), respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50 degrees C. The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry.

Exercise training in late middle‐aged male Fischer 344 × Brown Norway F1‐hybrid rats improves skeletal muscle aerobic function

The Fischer 344 x Brown Norway F1-hybrid (F344BN) rat has become an increasingly popular and useful strain for studying age-related declines in skeletal muscle function because this strain lives long enough to experience significant declines in muscle mass. Since exercise is often considered a mechanism to combat age-related declines in muscle function, determining the utility of this strain of rat for studying the effects of exercise on the ageing process is necessary. The purpose of this study was to evaluate the plasticity of skeletal muscle aerobic function in late middle-aged male rats following 7 weeks of treadmill exercise training. Training consisted of 60 min per day, 5 days per week with velocity gradually increasing over the training period according to the capabilities of individual rats. The final 3 weeks involved 2 min high-intensity intervals to increase the training stimulus. We used in situ skeletal muscle aerobic metabolic responses and in vitro assessment of muscle mitochondrial oxidative capacity to describe the adaptations of aerobic function from the training. Training increased running endurance from 11.3 +/- 0.6 to 15.5 +/- 0.8 min, an improvement of approximately 60%. Similarly, distal hindlimb muscles from trained rats exhibited a higher maximal oxygen consumption in situ (23.2 +/- 1.3 versus 19.7 +/- 0.8 mumol min(-1) for trained versus sedentary rats, respectively) and greater citrate synthase and complex IV enzyme activities in gastrocnemius (29 and 19%, respectively) and plantaris muscles (24 and 28%, respectively) compared with age-matched sedentary control animals. Our results demonstrate that skeletal muscles from late middle-aged rats adapt to treadmill exercise by improving skeletal muscle aerobic function and mitochondrial enzyme activities. This rat strain seems suitable for further investigations using exercise as an intervention to combat ageing-related declines of skeletal muscle aerobic function.

Regional sympathetic denervation affects the relation between canine local myocardial blood flow and oxygen consumption

Myocardial blood flow and oxygen consumption are heterogeneously distributed. Perfusion and myocardial oxygen consumption are closely correlated in the normal heart. It is unknown how this metabolism-perfusion relation is influenced by sympathetic denervation. We investigated this question in seven chloralose-anaesthetized dogs, 3-4 weeks after regional sympathetic denervation of the left circumflex coronary artery area of supply of the left ventricle. Measurements were made of local myocardial blood flow (MBF, in ml min(-1) (g dry wt)(-1)), measured with microspheres, and myocardial oxygen consumption ( , in mumol min(-1) (g dry wt)(-1)) in the same location, calculated from the (13)C spectrum of tissue extracts after intracoronary infusion of 3-(13)C-lactate. Since both innervated and denervated regions are subject to the same arterial pressure, lower blood flow indicates higher resistance. Mean MBF was 5.56 ml min(-1) (g dry wt)(-1) (heterogeneity of 3.47 ml min(-1) (g dry wt)(-1)) innervated, 7.48 ml min(-1) (g dry wt)(-1) (heterogeneity of 3.62 ml min(-1) (g dry wt)(-1)) denervated (n.s.). Significant linear relations were found between MBF and M Vo2 of individual samples within the innervated and denervated regions. The slopes of these relations were not significantly different, but the adjusted mean was significantly higher in the denervated regions (+1.92 ml min(-1) (g dry wt)(-1), an increase of 38% of the mean MBF at the pooled mean M Vo2, P = 0.028, ANCOVA). The ratio MBF/M Vo2(in ml micromol(-1)) was significantly higher, being 0.296 +/- 0.167 ml micromol(-1) in the denervated region compared with the innervated region, 0.216 +/- 0.126 ml micromol(-1), P = 0.0182, Mann-Whitney U test. These results indicate that sympathetic tone under chloralose anaesthesia imposes a moderate vasoconstrictive effect in the myocardium that is not detected by comparison of the mean blood flow or resistance.

Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity.

A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40 degrees C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent K(m), K(i), and V(max) of 2.0 +/- 0.2 mM, 268.1 +/- 18.1 mM, and 6.6 +/- 0.2 mumol min(-1) mg(-1) of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides.

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacteriumAphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.

Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium

Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite and produce N(2). They reside in many natural ecosystems and contribute significantly to the cycling of marine nitrogen. Anammox bacteria generally live under ammonium limitation, and it was assumed that in nature anammox bacteria depend on other biochemical processes for ammonium. In this study we investigated the possibility of dissimilatory nitrate reduction to ammonium by anammox bacteria. Physically purified Kuenenia stuttgartiensis cells reduced (15)NO(3) (-) to (15)NH(4) (+) via (15)NO(2) (-) as the intermediate. This was followed by the anaerobic oxidation of the produced ammonium and nitrite. The overall end-product of this metabolism of anammox bacteria was (15)N(15)N dinitrogen gas. The nitrate reduction to nitrite proceeds at a rate of 0.3 +/- 0.02 fmol cell(-1) day(-1) (10% of the 'normal' anammox rate). A calcium-dependent cytochrome c protein with a high (305 mumol min(-1) mg protein(-1)) rate of nitrite reduction to ammonium was partially purified. We present evidence that dissimilatory nitrate reduction to ammonium occurs in Benguela upwelling system at the same site where anammox bacteria were previously detected. This indicates that anammox bacteria could be mediating dissimilatory nitrate reduction to ammonium in natural ecosystems.

Modeling of Oxygen Transport and Cellular Energetics Explains Observations on In Vivo Cardiac Energy Metabolism

Observations on the relationship between cardiac work rate and the levels of energy metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (CrP) have not been satisfactorily explained by theoretical models of cardiac energy metabolism. Specifically, the in vivo stability of ATP, ADP, and CrP levels in response to changes in work and respiratory rate has eluded explanation. Here a previously developed model of mitochondrial oxidative phosphorylation, which was developed based on data obtained from isolated cardiac mitochondria, is integrated with a spatially distributed model of oxygen transport in the myocardium to analyze data obtained from several laboratories over the past two decades. The model includes the components of the respiratory chain, the F0F1-ATPase, adenine nucleotide translocase, and the mitochondrial phosphate transporter at the mitochondrial level; adenylate kinase, creatine kinase, and ATP consumption in the cytoplasm; and oxygen transport between capillaries, interstitial fluid, and cardiomyocytes. The integrated model is able to reproduce experimental observations on ATP, ADP, CrP, and inorganic phosphate levels in canine hearts over a range of workload and during coronary hypoperfusion and predicts that cytoplasmic inorganic phosphate level is a key regulator of the rate of mitochondrial respiration at workloads for which the rate of cardiac oxygen consumption is less than or equal to approximately 12 μmol per minute per gram of tissue. At work rates corresponding to oxygen consumption higher than 12 μmol min−1 g−1, model predictions deviate from the experimental data, indicating that at high work rates, additional regulatory mechanisms that are not currently incorporated into the model may be important. Nevertheless, the integrated model explains metabolite levels observed at low to moderate workloads and the changes in metabolite levels and tissue oxygenation observed during graded hypoperfusion. These findings suggest that the observed stability of energy metabolites emerges as a property of a properly constructed model of cardiac substrate transport and mitochondrial metabolism. In addition, the validated model provides quantitative predictions of changes in phosphate metabolites during cardiac ischemia.

Partial purification and characterization of xylanase produced by Penicillium expansum

An extracellular xylanase was found to be the major protein in the filtrate culture of Penicillium expansum when grown on 0.3 % wheat bran, which showed no xylanase multiplicity. The enzime was partial purified by.ammonium sulfate fractioning, molecular exclusion chromatography, ultrafiltration and anion exchange chromatography. The protein eluation profile showed only one form of xylanase that was partially characterized. The activity of purified xylanase was optimal at pH 5.5 and 40 ºC. The enzyme was stable at pH between 5.5 and 6.5 and temperatures between 20-40 ºC. The enzyme showed a Km of 3.03 mM and Vmax of 0.027 mumol min-1 mug -1 of protein. The enzymatic activity was increased 31 % by Mg2+ and 28 % by Al3+.

Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39.

L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction. The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5).

The in vitro secretion of acetylcholinesterase by adult stages of Heligmosomoides polygyrus: the effects of broad-spectrum anthelmintics.

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 +/- 0.07 mumol min-1 l-1 mg-1) than for female (0.56 +/- 0.04 mumol min-1 mg-1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug-free medium (RPMI medium supplemented with 0.5% BSA) for 24 h or continuously exposed to the drugs in supplement-free medium (24 h), the concentration- and time-dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50% (IC50) relative to drug-free controls were estimated. The IC50 values ranged from 0.012 microM (IVM) to 2.96 microM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad-spectrum anti-nematodal activity is discussed.

Different effects of dopamine and dopexamine on the isolated perfused rat kidney

We have investigated the effect of dopamine and dopexamine on the isolated perfused rat kidney. After an equilibration period of 20 min and two control periods of 10 min, dopexamine 1.0, 2.5 or 4.0 micrograms kg-1 min-1 or dopamine 2.0 micrograms kg-1 min-1 were perfused for a further 40 min in random order. Renal blood flow, urine volume, glomerular filtration rate, absolute sodium excretion and fractional sodium reabsorption of the isolated perfused kidney were measured every 10 min during the experiment. Dopamine increased significantly urine production from mean 61.54 (SEM 4.7) to 117.2 (9.7) microliters min-1 g-1 and absolute sodium excretion from 0.4 (0.1) to 1.2 (0.1) mumol min-1 g-1, and decreased significantly fractional sodium reabsorption from 97.3 (0.5) to 90.7 (0.7)%. Renal blood flow and glomerular filtration rate were not altered. In contrast, dopexamine had no effect on the isolated kidney. These data suggest that the diuretic and natriuretic effects of dopexamine in humans may not result from a direct action on the kidney.

Quality of herbal remedies from Allium sativum: differences between alliinase from garlic powder and fresh garlic.

Alliinase (EC 4.4.1.4) has been isolated from commercially available garlic (Allium sativum L., Alliaceae) powder and was investigated with respect to its use as ingredient of herbal remedies. The enzyme was purified to apparent homogeneity and results were compared with those obtained from a sample of fresh A. sativum var. pekinense. The purification of the enzyme involved a gel filtration step as well as affinity chromatography on concanavalin-A agarose. Vmax using L-(+)-alliin as substrate (252 mumol min-1 mg-1) was at the lower range of data given in the literature (214-390 mumol min-1 mg-1). L-(-)-Alliin was also accepted as substrate (54 mumol min-1 mg-1). Vmax for alliinase from A. sativum var. pekinense was at 332 mumol min-1 mg-1 and 90 mumol min-1 mg-1 for L-(+)- and L-(-)-alliin, respectively. The Km values for alliinase from garlic powder were estimated to be 1.6 mM for L-(+)-alliin and 2.8 mM for L-(-)-alliin. In contrast to literature values, both temperature and pH optima were somewhat higher (36 degrees C and pH 7.0 versus 33 degrees C and pH 6.5, respectively). The enzyme was found to be active in a range from pH 5 to pH 10. Gel electrophoresis gave evidence that the alliinase obtained from garlic powder consisted of two slightly different subunits with molecular weights of 53 and 54 kDa whereas alliinase obtained from fresh garlic consists of two identical subunits. It is assumed that the alliinase gets significantly altered during the drying process of garlic powder but is still capable to convert alliin to allicin.

Characterization of the elevated esterase-associated insecticide resistance mechanism in Nilaparvata lugens (Stal) and other planthopper species

Insecticide-resistant strains of the brown planthopper, Nilaparvata lugens, from four locations all had a single diffuse elevated esterase band on native polyacrylamide gel electrophoresis. The white backed planthopper Sogatella furcifera had two elevated esterases with lower relative mobilities than the N. lugens esterase, while the insecticide-susceptible N. bakeri and the grass-associated sibling species of N. lugens sensu lato all had a single low intensity staining esterase with a lower relative mobility than the resistance associated N. lugens esterase. All the esterases were inhibited by pre-incubation with 0.1 mM paraoxon, but were not affected by permethrin up to its solubility limit, indicating their possible role in organophosphorus, but not pyrethroid insecticide, resistance. Partial purification of the elevated esterase from insecticide resistant Sri Lankan N. lugens showed that despite its diffuse nature on gels it purified as a single isoform, with a specific activity of 5.85 mumol min- 1 mg- 1 and an estimated molecular weight of approximately 60 kDa. Insecticide resistance was conferred by this elevated esterase through rapid binding and slow turnover of the carbamate or the insecticidally active oxon analogues of the phosphorothioates, i.e. sequestration rather than metabolism is the primary resistance mechanism. In contrast to earlier studies on the elevated esterases of N. lugens from Japan and the Philippines, we were unable to detect any malathion metabolism by this esterase, nor did malathion inhibit the esterase, although it was sensitive to inhibition by malaoxon. The elevated N. lugens esterase did not cross-react with antisera raised to the elevated mosquito or aphid esterases. The efficacy of the partially purified N. lugens esterase in insecticide sequestration and turnover is compared with other well characterized amplified esterases from mosquitoes and aphids.

Effect of thyroparathyroidectomy on urinary acidification in diabetic rats.

In previous studies we have shown stimulation of renal acid excretion in the proximal tubules of rats with diabetes of short duration, with no important alterations in glomerular hemodynamics; on the other hand, in thyroparathyroidectomized rats (TPTX model), a significant decrease in renal acid excretion, glomerular filtration rate (GFR) and renal plasma flow (RPF) was detected. Since important changes in the parathyroid hormone-vitamin D-Ca axis are observed in the diabetic state, the present study was undertaken to investigate the renal repercussions of thyroparathyroidectomy in rats previously made diabetic by streptozotocin (45 mg/kg). Four to 6 days after the induction of diabetes (DM), a group of rats were thyroparathyroidectomized (DM + TPTX). Renal functional parameters were evaluated by measuring the inulin and sodium para-aminohippurate clearance on the tenth day. The decrease in the GFR and RPF observed in TPTX was not reversed by diabetes since the same alterations were observed in DM + TPTX. Net acid (NA) excretion was unchanged in DM (6.19 +/- 0.54), decreased in TPTX (3.76 +/- 0.25) and returned to normal levels in DM + TPTX (5.54 +/- 0.72) when compared to the control group (6.34 +/- 0.14 mumol min-1 kg-1). The results suggest that PTH plays an important vasodilator role regarding glomerular hemodynamics, since in its absence the impairment in GFR and RPF was not reversed by the diabetic state. However, with respect to acid excretion, the presence of diabetes was able to overcome the negative stimulus represented by TPTX.

A thiophosphate analog of dimyristoylphosphatidyl-inositol-4-phosphate is a substrate for mammalian phosphoinositide-specific phospholipase C.

1,2-Dimyristoyloxypropane-3-thiophosphate(rac-1-myo-inositol-4- phosphate), a thiophosphate analog of dimyristoyl phosphatidylinositol-4-phosphate was synthesized as a substrate for mammalian phosphoinositide-specific phospholipase C. Its activity with delta(1-132)-PI-PLC-delta 1 (a deletion mutant with the N-terminal pleckstrin homology domain removed) was studied in sonicated dispersions, with and without added Triton X-100. It had an initial activity of about 30 mumol min-1 mg-1, which rapidly decreased due to substrate depletion in the vesicle or micelle. The slower rate of hydrolysis appeared limited by enzyme hopping or exchange of substrate between vesicles or micelles, which was more rapid in the presence of detergent.

Desulfobulbus rhabdoformis sp. nov., a sulfate reducer from a water-oil separation system.

A mesophilic, Gram-negative, rod-shaped, marine, propionate-oxidizing sulfate reducer (strain M16T) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 31 degrees C, pH 6.8-7.2 and 1.5-2.0% (w/v) NaCl and 0.1-0.3% (w/v) MgCl2.6H2O in the medium. The growth yield with sulfate was 4.6 g cell biomass (mol propionate oxidized)-1. Strain M16T is nutritionally related to members of the genus Desulfobulbus, but differs in that it has no vitamin requirement and is able to utilize fumarate and malate as carbon and energy sources. Hydrogenase activity measured as hydrogen uptake was mainly membrane-bound and varied with the growth substrate. Highest activity [28 mumol min-1 (mg protein)-1] was found in cells grown with hydrogen and lowest [50 nmol min-1 (mg protein)-1] in cells grown with propionate as electron donors for sulfate reduction. Desulforubidin, menaquinone-5(H2) and cytochromes of the c- and b-type were present. The fatty acid pattern was similar to that found for Desulfobulbus propionicus. The DNA base composition was 50.6 mol% G + C. Strain M16T is equidistantly related to D. propionicus and Desulfobulbus elongatus with 96.1% 16S rDNA similarity. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain M16T (= DSM 8777T) is proposed as the type strain of a new species, Desulfobulbus rhabdoformis.