Effect of propionate and pyruvate on citrulline synthesis and ATP content in rat liver mitochondria.

Propionate inhibits citrullinogenesis when succinate (plus rotenone) or glutamate are the oxidizable substrates used. Propionate decreases the intramitochondrial concentration of carbamylphosphate by decreasing the ATP content. When the energy supply for citrullinogenesis is provided by an influx of exogenous ATP, propionate is no longer an inhibitor. Pyruvate inhibits citrullinogenesis with glutamate but not with succinate (plus rotenone) as oxidizable substrates. Propionate and pyruvate deplete mitochondrial ATP but probably by different mechanisms.     

Characterization of cDNA of lycopene beta-cyclase responsible for a high level of beta-carotene accumulation in Dunaliella salina.

Lycopene beta-cyclase (Lyc-B) is the key enzyme in the catalysis of linear lycopene to form cyclic beta-carotene, an indispensable part of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Studies showing that the microalga Dunaliella salina can accumulate a high level of beta-carotene are lacking. We hypothesize that D. salina is closely involved with the catalytic mechanism of Lyc-B and the molecular regulation of its gene. In this study, we used RT-PCR and RACE-PCR to isolate a 2475 bp cDNA with a 1824 bp open reading frame, encoding a putative Lyc-B, from D. salina. Homology studies showed that the deduced amino acid sequence had a significant overall similarity with sequences of other green algae and higher plants, and that it shared the highest sequence identity, up to 64%, with Lyc-B of Chlamydomonas reinhardtii. Codon analysis showed that synonymous codon usage in the enzyme has a strong bias towards codons ending with adenosine. Two motifs were found in the Lyc-B sequence, one at the N terminus, for binding the hypothetical cofactor FAD, and the other was a substrate carrier motif in oxygenic organisms shared by an earlier carotenogenesis enzyme, phytoene desaturase, and Lyc-B. A tertiary structure prediction suggested that the catalytic or binding site structure within LycB from D. salina is superior to that of both H. pluvialis and C. reinhardtii. The LycB protein from D. salina was quite removed from that of H. pluvialis and C. reinhardtii in the phylogenetic tree. Taken as a whole, this information provides insight into the regulatatory mechanism of Lyc-B at the molecular level and the high level of beta-carotene accumulation in the microalga D. salina.     

Active compounds in Populus nigra L. wilted leaves responsible for attracting Helicoverpa armigera (Hübner) (Lep., Noctuidae) and new agaropectin formulation

Abstract:  The leaf extracts of Populus nigra were collected and identified by steam distillation, air entrainment and gas chromatographic–mass spectrometric analysis. Electroantennograms were recorded from Helicoverpa armigera adults in response to the chemicals identified. Both aromatic compounds and green-leaf volatiles elicited strong responses. Field experiments revealed that the active compounds responsible for attracting H. armigera moths are mainly short-side-chain aromatic alcohols and aldehydes. We, for the first time, used agaropectin as the controlled-release matrix of insect attractants. A five-component lure containing all the aromatics without phenolics, mixed in the proportions as found in the steam distillate of the leaves collected in August, produced the best trap catch. The results showed that the volatiles of wilted leaves of P. nigra can attract H. armigera adults by feeding attraction.     

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.