Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.     

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.     

Sequence homology between human alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III

alpha 1-Antichymotrypsin mRNA was isolated by specific polysome immunoprecipitation from turpentine-treated baboon liver. The highly enriched mRNA was used for synthesis and cloning of the corresponding cDNA. Baboon alpha 1-antichymotrypsin cDNA clones were identified by hybrid-selected translation, and the insert DNA fragment from one of the putative clones was used as a probe to screen a human liver cDNA library comprised of 40 000 independent transformants. One of the human cDNA clones was unambiguously identified to contain alpha 1-antichymotrypsin DNA sequences by comparison of its 5'-terminal nucleotide sequence with the N-terminal amino acid sequence of the protein. This cDNA clone, designated phACT235, contains 1524 base pairs of human DNA, which was sequenced in its entirety. The inserted DNA codes for a 25 amino acid signal peptide sequence and the entire mature alpha 1-antichymotrypsin of 408 amino acid residues. Comparison of the amino acid sequence of alpha 1-antichymotrypsin with that of the human alpha 1-antitrypsin has revealed a homology level similar to that between chymotrypsin and trypsin.     

Maternal morbidity associated with in utero transfer.

OBJECTIVE--To determine the extent of maternal morbidity associated with in utero transfer. DESIGN--Retrospective study of 190 consecutive cases over two years. SETTING--Liverpool Maternity Hospital. PATIENTS--190 Pregnant women were transferred to the hospital under the in utero transfer arrangements from district general hospitals both within and outside the Mersey region. The women admitted were divided into two categories: those in threatened or established uncomplicated preterm labour and those who may or may not have been in threatened or established preterm labour but who had coexisting complicating factors affecting the mother or fetus, or both. INTERVENTIONS--Planned delivery of the fetus if indicated and arrangements for appropriate postpartum care of the mother. MAIN OUTCOME MEASURE--Assessment of the progress of labour and, if appropriate, resuscitation of the mother. RESULTS--Women who were transferred with no coexisting disease (124) had relatively uncomplicated deliveries whereas those transferred with coexisting diseases (66) exhibited considerable morbidity and 17 of these required prolonged intensive monitoring after delivery. CONCLUSIONS--In utero transfer in healthy mothers may have benefits for babies born very prematurely. If mothers have coexisting disease, however, the desirability of transfer should be reviewed urgently in the light of the considerable maternal morbidity associated with these problems. In these cases transfer may introduce an additional hazard.     

Dietary zinc-methionine enhances mononuclear-phagocytic function in young turkeys

The ability of dietary zinc-methionine (Zn-Met) to enhance mononuclear-phagocytic function againstSalmonella arizona andenteritids was investigated in young turkeys. Feed/gain and body wt gain at 21 d of age were not affected by Zn-Met. The addition of 30 or 45 ppm Zn from Zn-Met to a Zn adequate diet significantly increased cutaneous basophil hypersensitivity to phytohemagglutinin-P. The clearance of intravenously administeredS. enteritidis from blood was not affected by 30 ppm of supplemental Zn from Zn-Met. However, 30 ppm Zn from Zn-Met increased the reduction of intravenously administeredS. arizona from spleen. Percentages of myeloid and mononuclear-phagocytic cells before and afterS. enteritidis infection were not affected by supplemental Zn-Met. Turkeys supplemented with Zn-Met showed enhanced in vitro phagocytosis ofS. enteritidis by Sephadex-elicited abdominal exudate cells. The phagocytosis ofS. arizona was unaffected by Zn-Met.     

Linkage mapping of serotonin transporter protein gene SLC6A4 on chromosome 17

Abnormalities in monoamine metabolism, including serotonin metabolism, have been implicated in the pathophysiology of affective disorders, schizophrenia, suicide, and other psychiatric disorders. Serotonin transporter protein (SERT) allows neurons to retrieve serotonin that has been released into a synapse. SERT is a site of action for several drugs with CMS effects, including both therapeutic agents (e.g., antidepressants) and drugs of abuse (e.g., cocaine). This gene had previously been physically mapped to chromosome 17. We used a PCR product corresponding to the 3 untranslated region of the gene as a probe to identify restriction fragment length polymorphism (RFLP), which we then used to establish that the SLC6A4, genetic locus for SERT, is near 17q12 and probably flanked by D17S58 and D17S73 (a location consistent with observed crossovers). These data should be useful for linkage studies of neuropsychiatric disorders. (Joyce et al. 1993). Neurotransmitter reuptake sites (including also the norepinephrine transporter protein and the dopamine transporter protein) are logical candidate genes for susceptibility to psychiatric illness. We have previously (Gelernter et al. 1993) mapped the norepinephrine transporter protein to chromosome 16q21. We describe here linkage mapping of the serotonin transporter protein gene (gene symbol SLC6A4, for solute carrier family 6 (neurotransporter, serotonin), member 4), which was cloned in 1991 (Blakely et al. 1991; Hoffman et al. 1991) and previously assigned to chromosome 17, most likely to band 17q11.2, by in situ hybridization (Ramamoorthy et al. 1993). Our linkage results confirm the initial mapping of SLC6A4 and place it in the linkage map of proximal 17q.     

Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants

Two-dimensional tryptic mapping of ¹²⁵I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species.     

Studies of three Amerindian populations using nuclear DNA polymorphisms.

Three Amerindian populations, two from Rond?nia, Brazil (Karitiana and Rond?nia Suruí), and one from Campeche, Mexico (Mayan), were typed for up to 30 nuclear restriction fragment length polymorphisms (RFLPs). Heterozygosities, both observed and expected, were compared with those of Europeans. Average heterozygosity is reduced among these Amerindians (relative to that of Europeans) by 7.0% (Mayan) to 27.1% (Karitiana). This amount of heterozygosity in the nuclear DNA is nevertheless high enough that it is unlikely that there was a severe or prolonged bottleneck.     

Phycobilins of cryptophycean algae. Occurrence of dihydrobiliverdin and mesobiliverdin in cryptomonad biliproteins.

Structures of the open-chain tetrapyrrole (bilin) prosthetic groups of the cryptophycean biliproteins phycocyanin 645 (Cr-PC 645; from strain UW374), phycoerythrin 566 (Cr-PE 566; from strain Bermani) and phycoerythrin 545 (Cr-PE 545; from Proteomonas sulcata Hill & Wetherbee) were examined by absorption, 1H NMR spectroscopy, and mass spectrometry. These biliproteins carry the following covalently attached bilins: Cr-PC 645 (alpha subunit) has one mesobiliverdin, (beta subunit), two phycocyanobilins and a doubly linked 15,16-dihydrobiliverdin; Cr-PC 566 (alpha), bilin 584, (beta), phycoerythrobilin and two bilin 584 chromophores (Wedemayer, G.J., Wemmer, D.E., and Glazer, A.N. (1991) J. Biol. Chem. 266, 4731-4741); Cr-PE 545 (alpha) has one 15,16-dihydrobiliverdin and (beta), only phycoerythrobilins. This is the first report of naturally occurring biliproteins carrying either 15,16-dihydrobiliverdin or mesobiliverdin chromophores. Native cryptomonad phycobiliproteins have been classified on the basis of the position of their long wavelength absorption maxima. However, comparison of the bilins of Cr-PE 566 from strain Bermani with those of Cr-PE 566 of strain CBD shows that the two proteins carry different bilins on the alpha subunit. Consequently, the identity of the bilin prosthetic groups on cryptophycean phycobiliproteins cannot be unambiguously inferred from simple inspection of the visible absorption spectra.     

A review of methods for determining dietary range in adult parasitoids

Despite the potential importance of adult nutrition in the ecology of parasitoids, information on the range of natural foods they exploit is confined to a relatively small number of species. We provide some simple guidelines for determining dietary range in adult parasitoids, both to facilitate and to encourage further research into this aspect of parasitoid biology.