DNA of some anaerobic rumen fungi: G + C content determination

The nuclear DNAs from five species of anaerobic rumen fungi have been isolated and purified by means of two extraction methods (with and without 8 M urea). Their G + C contents have been characterized by the thermal denaturation procedure of Marmur and Doty. As has already been shown in Neocallimastix frontalis, the results obtained by the two techniques demonstrated a very low G + C content (less than 20%) and the constant presence of satellite DNA.     

Platelet-activating factor induces tyrosine phosphorylation in human neutrophils

The addition of platelet-activating factor (PAF) to human neutrophils increases the levels of the tyrosine phosphorylation in several proteins. These proteins have molecular weights of 41 (pp41), 54 (pp54), 66 (pp66), 104 (pp104), and 116 (pp116) kDa. The effect of PAF was dose-dependent and could be seen at concentrations as low as 1 nM. The nonmetabolizable bioactive PAF analog, C-PAF, caused an increase in the level of phosphorylation of the same proteins in a time- and dose-dependent manner. On the contrary, lyso-PAF, enantio-PAF, and L-beta,gamma-dihexadecyl-alpha-lecithin failed to stimulate the phosphorylation of any of the aforementioned proteins. The response to PAF was prevented by the PAF antagonist BN-52021. The PAF-induced increases in tyrosine phosphorylation in pp66, pp116, and pp104 were selectively inhibited by pertussis toxin. In contrast, the level of pp41 phosphorylation remained unchanged after the pertussis toxin treatment. The calcium chelator EGTA significantly inhibited the PAF-produced phosphorylation of the pp41 protein. The intracellular calcium chelator 1,2-bis-(O-aminophenoxil)ethane-N,N,N',N'-tetraacetic acid (BAPTA) potentiated the PAF-enhanced levels of tyrosine phosphorylation on the pp41 protein. On the other hand, the PAF-induced phosphorylations of pp66, pp104, and pp116 were inhibited in BAPTA-treated cells. The calcium ionophore A23187 selectively potentiated the phosphorylation of the pp41 protein and reduced the phosphorylation in the pp54 protein. This phosphorylation was dependent on the extracellular calcium and was inhibited in toxin-treated cells. The results suggest that PAF is able to affect either directly or indirectly tyrosine kinase and/or phosphotyrosine phosphatase activities. The phosphorylation of the high and low molecular weight proteins are mediated by two different sets of kinases and/or phosphatases.     

Subunit IV (Mr = 14,384) of the cytochrome b-c1 complex from Rhodobacter sphaeroides. Cloning, DNA sequencing, and ubiquinone binding domain.

The Rhodobacter sphaeroides gene encoding subunit IV of the cytochrome b-c1 complex (fbcQ) was cloned and sequenced. The fbcQ cistron is 372 base pairs long and encodes 124 amino acid residues. The molecular mass of subunit IV, deduced from the nucleotide sequence, is 14,384 Da. A hydropathy plot of the predicted amino acid sequence revealed only one transmembrane helix; it is near the C-terminal end. The 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone ([3H]azido-Q)-labeled subunit IV was isolated from the [3H]-azido-Q-treated cytochrome b-c1 complex. A ubiquinone-binding peptide was obtained by digesting the labeled subunit IV with V8 protease followed by high performance liquid chromatography separation. Amino acid analysis and partial N-terminal sequencing of this ubiquinone-binding peptide revealed that it corresponded to residues 77-124 of subunit IV. Based on the hydropathy profile and predicted tendency to form alpha-helices and beta-sheets, we propose a structural model for subunit IV. In this model the ubiquinone-binding domain is located near the surface of the membrane.     

Receptor and G protein-mediated responses to thrombin in HEL cells.

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.     

Evaluation of automated analysis of 15N and total N in plant material and soil

Simultaneous determination of ¹⁵N and total N using an automated nitrogen analyser interfaced to a continuous-flow isotope ratio mass spectrometer (ANA-MS method) was evaluated. The coefficient of variation (CV) of repeated analyses of homogeneous standards and samples at natural abundance was lower than 0.1%. The CV of repeated analyses of ¹⁵N-labelled plant material and soil samples varied between 0.3% and 1.1%. The reproductibility of repeated total N analyses using the automated method was comparable to results obtained with a semi-micro Kjeldahl procedure. However, the automated method gave results which were 3% to 5% higher than those obtained with the Kjeldahl procedure. Since only small samples can be analysed, careful sample homogenization and fine grinding are very important. Evaluation of a diffusion method for preparing nitrate and ammonium in solution for automated ¹⁵N analysis showed that the recovery of inorganic N in the NH₃ trap was lower when the N was diffused from water than from 2 M KCl. The results also indicated that different proportions of the NO₃ ^- and the NH₄ ⁺ in aqueous solution were recovered in the trap after combined diffusion. The method is most suited for diffusing either NO₃ ^- or NH₄ ⁺ alone, but can be used for combined diffusion of the two ions.     

A comparative histological and histochemical study of the post-gastric alimentary canal from three species of pleuronectid, the Atlantic halibut, the yellowtail flounder and the winter flounder

The histology and mucus histochemistry of the pleuronectid post-gastric alimentary canal was examined using light and electron microscopy. Distinct differences in goblet cell mucus histochemistry were observed between species, with the two closest taxonomic species, the winter flounder and the yellowtail flounder showing the most diversity and the halibut showing regional variation. Numbers of goblet cells within post-gastric regions did not differ significantly between species, but were significantly different between regions within species increasing toward the rectum. The post-gastric region was divisible into two areas based upon the ultrastructural features of lipid digestion and absorption in the intestine and pyloric caeca, and of exogenous protein in the rectum. The combination of species-specific histochemical differences in mucus and general histological and ultrastructural differences within the post-gastric regions between these species suggest a correlation between lumenal environmental conditions/histology and natural prey preference.     

RESTRICTION ENZYME ANALYSIS OF DNA FROM SPECIES OF BULINUS (BASOMMATOPHORA: PLANORBIDAE) USING A CLONED RIBOSOMAL RNA GENE PROBE

A ribosomal RNA gene probe (pSM889) has been used to study restrictionenzyme digests of various species of Bulinus. In order to minimiseproblems of DNA shearing associated with snail tissues a methodof extracting nucleic acids from material embedded in agaroseblocks has been used. Restriction enzyme digests with Bgl IIand Bam HI hybridised to pSM889 showed clear differences betweenB. truncatus, B. wrighti, B. africanus and B. forskalii, representingthe four species groups of Bulinus. No differences were observedbetween samples of B. tropicus and B. truncatus digested withBam HI, Bgl II and Pst I. Intra-specific variation was observedbetween samples of B. forskalii from Säo Tomé andAngola digested with Bgl II and Hind III although restrictionprofiles for Bam HI, Pst I and Bst EII digests were similar.Intra-specific variation was also observed between two differentpopulation samples of B. wrighti from South Yemen using BamHI and Bgl II digested genomic DNA hybridised to pSM889. (Received 5 December 1989; accepted 19 April 1990)     

Dietary fluoride,unlike bromide or iodide,counteracts phosphorus-induced nephrocalcinosis in female rats

Supplemental dietary F has been shown to counteract P-induced nephrocalcinosis in female rats. In order to obtain information as to the specificity of this F effect, the effect of other halogens, namely Br and I, on P-induced nephrocalcinosis was studied in weanling female rats. Supplemental dietary Br (5.24 mmol/kg of diet) and I (1.43 mmol/kg of diet) did not influence P-induced nephrocalcinosis, whereas F at equimolar dietary concentrations had marked antinephrocalcinogenic activity. The halogens were added to the diets in the form of KBr, KI, and NaF; the diets were balanced for the kations with Cl salts. The addition of KI to the diet to a concentration of 5.24 mmol/kg caused pronounced growth retardation, decreased feed intake, hepatomegaly, and signs of lethargy. It is concluded that the protective effect of dietary F against P-induced nephrocalcinosis does not extend to other halogens.