Does beta-adrenoceptor activation stimulate Ca2+ mobilization and inositol trisphosphate formation in parotid acinar cells?

The effects of the beta-adrenoceptor agonist, isoprenaline, on Ca2+ mobilization and inositol phosphate formation in parotid acinar cells were examined. Isoprenaline (2 microM) failed to increase cytosolic [Ca2+] in acinar cells, as measured by Fura-2 fluorescence, even in the presence of a phosphodiesterase inhibitor. Likewise, neither the 8-bromo nor the dibutyryl derivatives of cAMP (both at 2 mM concentration) increased [Ca2+]i. However, in confirmation of results previously published, a higher concentration of isoprenaline (200 microM) increased cytosolic [Ca2+]i of rat parotid acinar cells, from 104 +/- 4 nM to 151 +/- 18 nM. The increase in [Ca2+]i in response to isoprenaline, while transient in the absence of extracellular Ca2+, was sustained in Ca2(+)-containing medium. This isoprenaline-stimulated Ca2+ signal was more potently antagonized by phentolamine than by propranolol, suggesting that the higher concentration of isoprenaline activated alpha-adrenoceptors. Furthermore, the Ca2+ signal generated in response to the alpha-adrenoceptor agonist, phenylephrine, also was blocked by the same concentrations of propranolol necessary to block the effects of isoprenaline, suggesting that propranolol may block alpha-adrenoceptors under certain experimental conditions. The high concentration of (-)isoprenaline (200 microM) also increased inositol (1,4,5) trisphosphate and inositol (1,3,4) trisphosphate formation 45% within 30 s. Analogous to the increase in intracellular Ca2+, the formation of inositol phosphates stimulated by isoprenaline was more potently antagonized by the alpha-adrenoceptor antagonist, phentolamine, than by the beta-adrenoceptor antagonist, propranolol, again suggesting that isoprenaline interacts with alpha-adrenoceptors on parotid cells. Thus, the effects of isoprenaline on [Ca2+]i do not appear to be mediated by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium.

Bacterial DNA ligases use NAD as an energy source. In this study we addressed two questions about these enzymes. First, what is the physiological consequence of completely removing the NAD-dependent enzyme and replacing it with an ATP-dependent DNA ligase? We constructed Salmonella typhimurium strains in which the endogenous NAD-dependent DNA ligase activity was inactivated by an insertion mutation and the ATP-dependent enzyme from bacteriophage T4 was provided by a cloned phage gene. Such strains were physiologically indistinguishable from the wild type, even under conditions of UV irradiation or treatment with alkylating agents. These results suggest that specific functional interactions between DNA ligase and other replication and repair enzymes may be unimportant under the conditions tested. Second, the importance of DNA ligation as the initiating event of the bacterial pyridine nucleotide cycle was critically assessed in these mutant strains. Surprisingly, our results indicate that DNA ligation makes a minimal contribution to the pyridine nucleotide cycle; the Salmonella strains with only an ATP-dependent ligase had the same NAD turnover rates as the wild-type strain with an NAD-dependent ligase. However, we found that NAD turnover was significantly decreased under anaerobic conditions. We suggest that most intracellular pyridine nucleotide breakdown occurs in a process that protects the cell against oxygen damage but involves a biochemical mechanism other than DNA ligation.     

The roles of phospholipase D and a GTP-binding protein in guanosine 5''-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-[alpha beta-methylene]triphosphate, but not adenosine 5'-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5'-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.     

The effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold

The purpose of this investigation was to determine the effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold (PWCFT). Ten adult males (mean age 23 years, SD 3) volunteered as subjects for this study. During the first laboratory visit the subjects performed a maximal bicycle ergometer test for the determination of maximum oxygen consumption (VO2max). Between 48 and 72 h later, the subjects pedaled to exhaustion at a power output which corresponded to a mean of 76% of VO2max (range, 72-80%) for the purpose of glycogen depletion. For the next 3 days, the subjects were fed a 10.5 MJ.day-1 low carbohydrate diet which consisted of 7.5% carbohydrates, 22.0% protein and 70.5% fat. The subjects then performed an incremental cycle ergometer test to the onset of fatigue or PWCFT, which was estimated from integrated electromyographic voltages of the vastus lateralis muscle. For the next 3 days the subjects were fed a 10.5 MJ high carbohydrate diet which consisted of 72.2% carbohydrates, 12.4% protein and 15.4% fats for the purpose of glycogen supercompensation. The subjects then performed a second PWCFT test. A paired t-test indicated that there was no significant (p greater than 0.05) difference between the means of the PWCFT values (depletion 246 W, SD 30; supercompensation 265 W, SD 28) and they were highly correlated at r = 0.884. The results of this investigation suggested that the methods commonly used to affect glycogen depletion or supercompensation had no effect on PWCFT.     

Fate of intravenously administered rat lymphokine-activated killer cells labeled with different markers

Summary Rat lymphokine-activated killer (LAK) cells, generated by adhering rat splenocytes isolated from the 52% Percoll density fraction to plastic flasks, demonstrate restricted in vivo tissue distribution, localizing in the lungs and liver after 2 h, but redistributing into the liver and spleen 24 h after i.v. administration. However, a different pattern of distribution was observed when this population of LAK cells was labeled with one of four commonly used radioisotopes. For example, LAK cells showed a high distribution into the lungs 30 min after administration when labeled with⁵¹Cr,¹²⁵I-dUrd or¹¹¹In-oxine, whereas¹¹¹InCl-labeled LAK cells showed an equal distribution into the blood, lungs and liver at this time. Two hours after administration, cells labeled with¹¹¹In-oxine showed an equivalent distribution into the lungs and liver, those labeled with¹²⁵I-dUrd or⁵¹Cr showed a high accumulation in the lungs, whereas those labeled with¹¹¹In-Cl entered more into the liver and blood. The pattern of distribution of¹¹¹In-Cl- or¹¹¹In-oxine-labeled cells was confirmed using gamma camera imaging analysis. By 24 h, LAK cells labeled with¹¹¹InCl,¹¹¹In-oxine or⁵¹Cr distributed in the liver and spleen in variable concentrations. In contrast, cells labeled with¹²⁵I-dUrd were not detected in any organ tested.This study was paralleled by monitoring the distribution of LAK cells labeled with Hoechst 33342 (H33342) and analyzed for the presence of fluoresceinated cells in different organs either by flow cytometry analysis, or in frozen section. The data indicate that the distribution pattern of LAK cells labeled with¹¹¹In-oxine is the closest to the distribution of H33342-labeled cells. Of all the radioisotopes used,¹²⁵I-dUrd has the most disadvantages and is not recommended for monitoring the in vivo distribution of leukocytes.     

The effect of salinity on the composition of fatty acid double-bond isomers andsn-1/sn-2 positional distribution in membrane phospholipids of a moderately halophilic eubacterium

We report the first study of the effect of NaCl on the double-bond isomeric composition of fatty acids and theirsn-1/sn-2 positional distribution in the membrane phospholipids of a moderately halophilic eubacterium. The major phospholipids, phosphatidylethanolamine and phosphatidylglycerol, ofVibrio costicola grown in 1M or 3M NaCl both have ansn-1 saturated,sn-2 unsaturated distribution of fatty acids. There is a greater effect of salinity on the fatty acid composition of phosphatidylglycerol compared with phosphatidylethanolamine. The fatty acids in phosphatidylethanolamine of cultures grown in 1M compared with 3M NaCl have the same unsaturation index and average chain length, but different double-bond isomeric compositions. In comparison, the fatty acid composition of phosphatidylglycerol is more unsaturated, with a different double-bond isomeric distribution, and has a shorter average chain length in cultures grown in 3M compared with 1M NaCl. The pattern of fatty acid isomers of 16:1 and 18:1 shows thatV. costicola uses the anaerobic pathway of fatty acid biosynthesis. The presence of the isomers 16:1c11 and 18:1c13 in the phospholipids of cultures grown in 3M but not in 1M NaCl indicates that external salinity affects the specificity of fatty acid synthetase in this moderately halophilic bacterium.     

The flocculation of bacteria using cationic synthetic flocculants and chitosan

Summary Bacteria are flocculated with high molecular weight cationic synthetic flocculants and chitosan. High charge density polymers are the most effective of the synthetic flocculants. Only chitosan is effective in flocculating the E. coli and B. subtilis cultures in complex broth. The difference in effectiveness between the synthetic flocculants and chitosan for flocculating E. coli, B. subtilis and Z. mobilis may be attributed to hydrogen bonding between the polysaccharide flocculant and cell surface polymers in addition to electrostatic interactions, and, in complex media, complexation of synthetic polymers with anionic polyelectrolytes.