In vitro and in vivo synthesis of dolichol and other main mevalonate products in various organs of the rat

The relative rate of biosynthesis of dolichol from [3H]mevalonate in nine rat organs was studied in slices and in the whole animal. This biosynthesis was also compared to that of cholesterol and ubiquinone. All tissues examined are able to synthesize dolichol, as well as ubiquinone and cholesterol. Comparison of the data from slices in vitro with the in vivo studies demonstrated relatively good agreement for dolichol and ubiquinone synthesis. Although dolichol of high specific radioactivity was recovered in the blood, redistribution between organs, such as occurs with cholesterol, appears to be insignificant. The highest rates of dolichol biosynthesis were found in kidney, spleen and liver. On the other hand, muscle makes the largest contribution to total body dolichol synthesis. Newly synthesized dolichol also appears in the bile, but excretion by this route is far from sufficient to account for dolichol turnover. Incorporation of mevalonate into the final products is mainly dependent on biosynthetic activity. For comparison of the biosynthetic rates in different organs, possible sources of errors (such as variations in the size of the precursor pool, limitation by the rate of precursor uptake or non-linear incorporation) were investigated the size of the mevalonate pool in various organs. Equilibration of this pool with exogenous mevalonate is a rapid and passive process. The size of the mevalonate pool does not determine the rates of cholesterol and dolichol biosynthesis, indicating the presence of regulatory steps in the terminal portion of these biosynthetic pathways.     

Factors affecting malate dehydrogenase activity in freezing-thawing processes

Malate dehydrogenases from several sources show different behaviour when frozen-thawed in 100 mM sodium phosphate buffer, pH 7.4, containing chaotropic ions. The effects produced by the addition of various metabolites, protein concentration and buffer medium used on the loss of activity induced by the freezing-thawing process are reported. The major part of the loss of activity is caused by the formation of "wrong" aggregates of high mol. wt.     

Potential secondary structure at translation-initiation sites.

Since translational start codons also occur internally, more-complex features within mRNA must determine initiation. We compare the potential secondary structure of 123 prokaryotic mRNA start regions to that of regions coding for internal methionines. The latter display an unexpectedly-uniform, almost-periodic pattern of pairing potential. In contrast, sequences 5' to start codons have little self-pairing, and do not pair extensively with the proximal coding region. Pairing potential surrounding start codons was found to be less than half of that found near internal AUGs. In groups of random sequences where the distribution of nucleotides at each position, or of trinucleotides at each in-frame codon position, matched the observed natural distribution, there was no periodicity in the pairing potential of the internal sequences. Randomized internal sequences had less pairing: the ratio of pairing intensity between internals and starts was reduced from 2.0 to 1.6 by randomization. We propose that the transition from the relatively-unstructured start domains to the highly-structured internal sequences may be an important determinant of translational start-site recognition.     

An acridine-linked oligodeoxynucleotide targeted to the common 5' end of trypanosome mRNAs kills cultured parasites

Anti-messenger oligodeoxynucleotides covalently linked to an intercalating agent were tested for their ability to inhibit translation of Trypanosoma brucei mRNAs in a cell-free system. The sequence of these oligodeoxynucleotides was complementary to part of the 35-nucleotide (nt) sequence which is present at the 5' end of all trypanosome mRNAs (the so-called mini-exon sequence). In a rabbit reticulocyte lysate, a nonadeoxynucleotide linked to an acridine derivative, specifically inhibited protein synthesis from T. brucei mRNAs much more efficiently than unmodified oligodeoxynucleotides of similar length. These oligodeoxynucleotides were tested on cultured trypanosomes. The acridine-linked nonadeoxynucleotide had a lethal effect on the parasites. No effect was observed with the homologous unmodified 9-mer nor with those 9-mers linked to the acridine derivative which were not complementary to the mini-exon sequence. These effects are probably a result of hybrid formation between the anti-messenger and mini-exon sequence. Trypanocidal activity of the acridine-modified nonadeoxynucleotide is most likely due to (i) increased affinity for its target, (ii) improved resistance to 3' exonucleases, and (iii) promoted membrane penetration of living parasites.     

Unexpected influence of ionic strength on branched-pathway interactions between beta-lactamases and beta-halogenopenicillanates.

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.     

The Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region.

Fragments of the lipophosphoglycan of Leishmania donovani were generated by phospholipase C digestion and mild acid hydrolysis. The fragments were purified and examined for inhibitory activity on protein kinase C isolated from rat brains. On a molar basis, the 1-O-alkylglycerol portion of LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not as effective. In addition, several glycolipid antigens from L. major, which contain short carbohydrate chains attached to phosphatidylinositol, were also efficient inhibitors of the enzyme. These results are consistent with the hypothesis that protein kinase C may be a key target for the parasites to overcome within host macrophages.     

Plasma norepinephrine and heart rate dynamics during recovery from submaximal exercise in man

The time course of heart rate (HR) and venous blood norepinephrine concentration [NE], as an expression of the sympathetic nervous activity (SNA), was studied in six sedentary young men during recovery from three periods of cycle ergometer exercise at 21% +/- 2.8%, 43% +/- 2.1% and 65% +/- 2.3% of VO2max respectively (mean +/- SE). The HR decreased mono-exponentially with tau values of 13.6 +/- 1.6 s, 32.7 +/- 5.6 s and 55.8 +/- 8.1 s respectively in the three periods of exercise. At the low exercise level no change in [NE] was found. At medium and high exercise intensity: (a) [NE] increased significantly at the 5th min of exercise (delta [NE] = 207.7 +/- 22.5 pg.ml-1 and 521.3 +/- 58.3 pg.ml-1 respectively); (b) after a time lag of 1 min [NE] decreased exponentially (tau = 87 s and 101 s respectively); (c) in the 1st min HR decreased about 35 beats.min-1; (d) from the 2nd to 5th min of recovery HR and [NE] were linearly related (100 pg.ml-1 delta [NE] congruent to 5 beats.min-1). In the 1st min of recovery, independent of the exercise intensity, the adjustment of HR appears to have been due mainly to the prompt restoration of vagal tone. The further decrease in HR toward the resting value could then be attributed to the return of SNA to the pre-exercise level.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase of Actinomadura R39.

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).