Urea metabolism in the cyanobacterium Anabaena cycadeae: regulation of urea uptake and urease by ammonia

A total of 160 Escherichia coli positive for F165 fimbrial antigen and isolated from diarrheic and septicemic animals, were examined for the presence of the pap, afa, and sfa/foc operons or related nucleotide sequences using colony hybridization. Most isolates shared DNA sequences with the pap operon sequences alone or in association with afa or sfa. Thus, our results indicate that F165-positive E. coli from diseased animals share DNA sequences with operons coding for adhesins important in human extra-intestinal disease and that multiple adhesin systems are often found in single isolates. However, 20% of the F165-positive isolates did not show any homology with the probes representing the three adhesin systems, suggesting that one of the operons responsible for F165 production could be different from the pap, sfa/foc, and afa operons.     

Assay of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.     

Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.     

Differing amounts of genetic polymorphism in testes and male accessory glands ofDrosophila melanogaster andDrosophila simulans

We surveyed genetic polymorphism by two-dimensional gel electrophoresis of male reproductive tract proteins in 20 isofemale lines each ofDrosophila melanogaster andDrosophila simulans. After classifying 244 such proteins ofDrosophila melanogaster and 271 ofDrosophila simulans by their distribution between testes and accessory glands within the reproductive tract, significant correlations were found between genetic polymorphism and tissue distribution. In both species, gland-specific proteins were significantly more polymorphic than testis-specific proteins, as well as those found in both testes and glands. Simultaneously, inDrosophila simulans, proteins found in roughly equivalent relative abundance in both testes and glands were significantly less variable than gland-specific and testis-specific proteins, as well as those with a quantitative difference in relative abundance between testes and glands. These correlations may reflect general differences in variability between extracellular and intracellular proteins and between proteins with broad as opposed to tissue-specific distributions.We thank the Natural Sciences and Engineering Research Council of Canada for financial support (Grant A0235 to R.S.S.).     

Plant water relations as selection criteria for drought tolerance in mustard

Genotypes of mustard (B. juncea) were evaluated for concurrent changes in leaf water potential (Ψ), leaf osmotic potential (π), leaf turgor potential (P) and leaf relative water content (RWC) during moisture stress at reproductive stage of growth. The slope ‘b’ in the regression between Ψ and π varied from 0.43 to 0.97 and was positively correlated with P and RWC. The genotypes with ‘b’ around 0.7 were able to maintain P of about 0.5 MPa at Ψ of − 2.5 MPa and thus such value of ‘b’ seems to provide enough degree of tolerance against drought.     

Cell cycle dependent variation of calmodulin in Tetrahymena

The level of calmodulin (CaM), a ubiquitous calcium-binding protein of eukaryotic cells was determined at different phases of the cell cycle in a synchronized Tetrahymena population. It was found that the concentration of CaM at G1 was approximately half of the concentration of S and this 2 x G1 level of CaM was maintained through the G2 and M stages of the cell cycle. To ascertain the role of CaM in the initiation of DNA synthesis, the cells were treated with trifluoperazine (TFP), a CaM antagonist, and EGTA (Ca2+-chelator) at the G1/S boundary. It was found that DNA synthesis was inhibited in these drug-treated cells. The uptake of the nucleotide precursor was not affected in TFP and EGTA treated cells, thus excluding the possibility of alteration in the membrane transport properties. Treatment with TFP failed to inhibit the synchronous mitotic division in Tetrahymena. The existence of a variable content of CaM through the cell cycle of Tetrahymena was demonstrated, suggesting the possible involvement of this Ca2+-binding protein in the nuclear DNA replication process.     

The inhibitory effects of some adenosine analogues on transmitter release at the mammalian neuromuscular junction

An electrophysiological study was made of the effects of four adenosine analogues, 2-chloroadenosine (2-CIA), 5'-N-ethylcarboxamidoadenosine (NECA), L-N6-phenylisopropyladenosine (L-PIA), and 2-(p-methoxyphenyl)-adenosine (CV-1674) on neurotransmitter release in the mouse phrenic nerve - hemidiaphragm preparation. All four drugs decreased miniature end-plate potential frequency in a dose-dependent manner. Evoked transmitter release in the cut diaphragm preparation was depressed by 2-CIA and CV-1674 to a similar extent. The ability of theophylline to antagonize the inhibitory effect of CV-1674 on spontaneous transmitter release was also established. On the basis of these results, the rank order of potencies was: L-PIA greater than NECA greater than 2-CIA greater than CV-1674. A clear classification of receptor type could not be made, since the ratio of potencies of L-PIA and NECA was narrow. Different slopes of the concentration-effect curves for 2-CIA and CV-1674 compared with L-PIA and NECA suggest an additional component to simple agonist action in their overall effects.     

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.