Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Identification of the autophosphorylation sites in rhodopsin kinase.

Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.     

The sequence of the nucleoprotein gene of human influenza A virus, strain A/NT/60/68.

The nucleotide sequence of the nucleoprotein gene of influenza A/NT/60/68 was established after using improved cloning methods to obtain full length cDNA clones in pBr322. The gene is 1565 residues long and codes for a basic protein of 498 amino acids. There are only 30 amino acid differences between it and the homologous sequence in A/PR/8/35, all occurring as point mutations. Assuming a common lineage, the evolutionary rate of divergence of the two strains is 0.18% amino acid per year. This confirms there is a slow but significant rate of evolution.     

The complete sequence of RNA segment 2 of influenza A/NT/60/68 P1 protein.

A DNA copy of influenza A/NT/60/68 viral RNA segment 2, corresponding to protein P1, has been cloned in the E.coli plasmid pBr322. The clone is 2341 nucleotides long and represents a full-length copy of the viral RNA. In the viral complementary (plus sense) strand there is an open reading frame that is 2271 nucleotides long. The predicted primary gene product is a basic 86,300 dalton protein with a net charge at neutral pH of +23. A 29 amino acid stretch of the protein (coded by nucleotide residues 583-669) is highly basic and contains 7 lysine and 8 arginine residues. Other smaller clusters of basic amino acids are also present in the protein.     

Incorporation of 3H from delta-(L-alpha-amino (4,5-3H)adipyl)-L-cysteinyl-D-(4,4-3H)valine into isopenicillin N.

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.     

A mutational analysis of active site residues in trans-3-chloroacrylic acid dehalogenase

trans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations.