Activation of α-1 adrenergic receptors modulates the control of left/right sidedness in rat embryos

Presomite stage rat embryos were cultured for 45–49 hr with medium containing various adrenergic agonists and antagonists. -Norepinephrine but not -norepinephrine (several orders of magnitude less potent than the -isomer at α-1 adrenergic receptors) resulted in a dose-dependent increase of situs inversus similar to that found for phenylephrine, an α-1 adrenergic agonist. Prazosin, an α-1 adrenergic antagonist, inhibited phenylephrine-induced situs inversus in a dose-dependent manner. Neither dexmedetomidine, an α-2 adrenergic agonist, nor isoproterenol, a β adrenergic agonist, caused situs inversus. These results provide pharmacological evidence that stimulation of α-1 but not of α-2 and β adrenergic receptors modulates the control of left/right sidedness in rat embryos.     

A mechanism for the development of Clara cell lesions in the mouse lung after exposure to trichloroethylene.

Female CD-1 mice exposed to trichloroethylene (6 h/day) at concentrations from 20-2000 ppm developed a highly specific lung lesion after a single exposure, characterised by vacuolation of the Clara cells, the number of cells affected increasing with increasing dose level. At the highest dose levels pyknosis of the Clara cells was apparent. After 5 days of repeated exposures the lesion had resolved but exposure of mice following a 2-day break resulted in recurrence of the lesion. The changes in mouse lung Clara cells were accompanied by a marked loss of cytochrome P-450 activities. No morphological changes were seen in the lungs of rats exposed to either 500 or 1000 ppm trichloroethylene. Isolated mouse lung Clara cells were shown to metabolize trichloroethylene to chloral, trichloroethanol and trichloroacetic acid. Chloral was the major metabolite. Trichloroethanol glucuronide was not detected. In comparative experiments using mouse hepatocytes the major metabolites were trichloroethanol and its glucuronide conjugate. The activity of UDP-glucuronosyltransferase was compared in mouse lung Clara cells and hepatocytes using two phenolic substrates and trichloroethanol. Hepatocytes readily formed glucuronides from all three substrates whereas Clara cells were only active with the two phenolic substrates. The three major metabolites of trichloroethylene, chloral, trichloroethanol and trichloroacetic acid were each dosed to mice and of these metabolites, only chloral had an effect on mouse lung causing a lesion (Clara cell) identical to that seen with trichloroethylene. It is proposed that the failure of Clara cells to conjugate trichloroethanol leads to an accumulation of chloral which results in cytotoxicity. The known genotoxicity of chloral suggests that this lesion may be related to the development of lung tumours in mice exposed to trichloroethylene by inhalation.     

Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils.

The subcellular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotaurine and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50:1. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiring cells by exposure to opsonized zymosan. These results prompted in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypotaurine competes with 5,5'-dimethyl-1-pyrroline N-oxide) (DMPO) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.     

Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: Recovery and renaturation from bacterial inclusion bodies

A synthetic gene encoding the Group 11 phospholipase A₂ (PLA₂) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA₂'s were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA₂. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA₂ demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.     

Two Types of Genetic Interaction Implicate the Whirligig Gene of Drosophila Melanogaster in Microtubule Organization in the Flagellar Axoneme

The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an alpha-tubulin locus, alpha 1t, mutations in a beta-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known alpha- or beta-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrlnc4 and B2tn are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2tn, for a deficiency of alpha 1t, or for the haync2 allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrlnc4 or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules.     

The involucrin gene of the galago. Existence of a correction process acting on its segment of repeats

The involucrin gene of the galago, a prosimian, has been cloned and sequenced. The coding region contains a segment of repeats homologous to the segment of repeats in the gene of another prosimian, the lemur, and different from the segments of repeats in the genes of higher primates. The repeats lengths in the two prosimians are similar; and except for a single duplication of a block of repeats in the lemur alone, the number of repeats is the same. However, the nucleotide consensus sequences of the repeats differ between the two species at 3 out of 39 nucleotide positions. The repeats therefore appear to have been modified by a correction process that led toward homogeneity in the repeats of each species while permitting divergence between the two species. The correction process, an example of concerted evolution, has taken place preferentially between adjacent repeats. The numerous differences between the segments of repeats of higher primates and the segments of repeats of lower animals reveal a discontinuity in the evolutionary processes acting on the gene.     

Specificity and pH dependence for acylproline cleavage by prolidase

Catalytic pH dependence for the hydrolytic activity of the enzyme prolidase with a series of dipeptide substrates is found to be generally bell-shaped (kcat/Km) or simple sigmoidal (kcat). An enzymic residue with a pKa value of 6.6 is found to be critically involved in the catalytic mechanism, as is the substrate amino group. Significant catalysis at a pH of 6.6 is also observed for prolidase with (alkylthio)acetylprolines and with haloacetylprolines. A reverse-protonation state mechanism for substrate binding and activation is postulated, involving a chelative interaction of the aminoacylamide portion of substrate with a strongly Lewis-acidic active site metal ion.     

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera)

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.     

Occurrence,localization, and possible significance of an ornithine-containing lipid in Paracoccus denitrificans

A ninhydrin-positive, phosphorus-negative lipid from Paracoccus denitrificans ATCC 13543 has been isolated and purified by mild alkaline methanolysis followed by silicic acid column chromatography and preparative thin-layer chromatography. The lipid was identified as an ornithine-containing lipid. The major ester-linked fatty acid was cis vaccenic acid. Major amide-linked fatty acids were 3-OH-20:1 and 3-OH-18:0. Ornithine-containing lipid was a major lipid component of P. denitrificans. Phospholipids made up about 57% and ornithine-containing lipid about 14% of the weight of the total lipid of the organism. The ratios of lipid ornithine: lipid phosphorus were 0.23, 0.65 and 0.58 in cytoplasmic membrane, outer membrane, and an NaCl extract, which is thought to represent chiefly outer membrane, respectively. Thus ornithine-containing lipid appears to be present in larger amounts in outer membrane than cytoplasmic membrane. No substantial variations in lipid ornithine levels were noted in stationary phase versus exposnential phase organisms, organisms grown in complex medium versus organisms grown in minimal medium with and without amino acid supplements, or in organisms grown in low phosphate-containing medium.Non standard abbreviations TLC thin-layer chromatography - Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride - TMS trimethylsilyl - TFA triluoroacetyl - NPPN ninhydrin-positive, phosphorus-negative - ECL equivalent chain length