Application of Most-Probable-Number Statistics to Direct Enumeration of Microorganisms

A novel method for rapid determination of total microbial cell numbers was investigated. The method involves the application of most-probable-number estimation statistics to direct microscopic counting of microbial cells by using a particle sizing graticule. Its accuracy and reliability were tested with computer simulations of bacterial cell distributions encountered in ecological studies. Good estimates of cell numbers were obtained when the cell density varied from 3 to 6,000 cells per field, i.e., over 3 orders of magnitude. Low levels of contagion did not markedly influence cell estimates, although high levels, corresponding to discrete scattered microcolonies, did. However, these could be recognized visually. Estimates of cell numbers in Breed smears confirmed its speed and good correlation with the standard quadrat counting technique under real experimental conditions.     

Oxidative metabolism of cholesterol precursors: sensitivity to ketoconazole, an inhibitor of cytochrome P-450

The effects of ketoconazole, an inhibitor of cytochrome P-450, on the metabolism of the cholesterol precursors lanosterol, dihydrolanosterol, lanost-8-en-3 beta,32-diol, and 3 beta-hydroxylanost-8-en-32-al were investigated in subcellular fractions of rat liver and in rat hepatocytes in culture. At low (1-2 microM) concentrations of the drug, the oxidative demethylation of lanosterol was inhibited by about 70% in the subcellular fractions but there was no effect on the metabolism of the 3 beta, 32-diol or the 32-aldehyde. Higher drug concentrations (10-20 microM) were required to inhibit the oxidative metabolism of these cholesterol precursors. Similar results were obtained during longer-term incubations using hepatocytes in culture medium, but higher concentrations of ketoconazole were required to effect the same degree of inhibition of each precursor. In the subcellular fractions, dihydrolanosterol, the 3 beta,32-diol and the 32-aldehyde were each metabolized to more polar sterols, in addition to cholesterol. Ketoconazole also inhibited the formation of these polar substances.     

Urinary hormone analysis as a diagnostic tool to evaluate the ovarian function of female gorillas (Gorilla gorilla)

Daily urine samples were collected from 4 adult female gorillas over 7 menstrual cycles. Urinary oestrone conjugate and pregnanediol-3-glucuronide (PDG) were measured by radioimmunoassay; LH was measured by enzyme immunoassay and each hormone was indexed by creatinine. The quantity of urinary LH during the ovulatory surge was positively correlated with the quantity of PDG excreted during the luteal phase (r = 0.87, P = 0.0013). The observations indicate a relationship between the quality of the LH surge and the levels of PDG in the luteal phase and suggest that both the LH surge and subsequent luteal phase function may be predictable from the oestrogen excretion profile during the follicular phase.     

Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases

ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803. The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (A_0.5 = 0.81 millimolar; n_H = 2.0). Activation by 3-phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (A_0.5 = 0.12 millimolar; n_H = 1.0), having a maximal stimulation of 17-fold. I_0.5 values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (A_0.5 = 10 micromolar; n_H = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia coli) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoresis and probed with Western blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase.     

Effect of long-term n-butanol ingestion on rat brain polypeptide synthesis directed by endogenous messengers.

Long-term ingestion of sublethal n-butanol doses by rats led to a noteworthy increase in the resistance of in vitro brain ribosomal function to the acute inhibitory action of ethanol and isopropanol. Withdrawal of n-butanol did not change this adaptation process immediately. The step affected seems to be the elongation of polypeptide chains. The dependence of in vitro translation on incubation temperature was affected by the adaptation process, the translation system of chronic animals being less stimulatable than that of control animals at low temperature.     

Evidence for a differential physiological modulation of brown fat iodothyronine 5'-deiodinase activity in the perinatal period

Brown adipose tissue iodothyronine 5'-deiodinase increases progressively in fetuses from the day 17 of pregnancy on, it reaches peak values on the 20th day of gestation and declines in the last days of fetal life as well as during the first day of life. Birth of premature fetuses causes a sudden drop in the enzyme activity. Postmaturity is associated to a decrease in brown fat 5'-deiodinase similar to that found after birth in fetuses born at term. In the first hours of life brown fat iodothyronine 5'-deiodinase is essentially insensitive to the cold-stimulus. Present data indicates that, differently from adult rats, brown fat iodothyronine 5'-deiodinase activity during the perinatal period is dissociated from the thermogenic activity of the tissue. It is suggested that factors different from the action of the sympathetic nervous system may play a main role in brown fat iodothyronine 5'-deiodinase activity modulation in the fetal and neonatal life.     

Protein kinase C from small intestine epithelial cells

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.