Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Outcome of breech delivery at term.

OBJECTIVE--To compare neonatal mortality and morbidity in term infants presenting by the breech and delivered vaginally or by caesarean section. DESIGN--Population based comparison of outcomes. Data derived from the St Mary''s maternity information system. SETTING--North West Thames Regional Health Authority, 1988-90. SUBJECTS--3447 singleton fetuses presenting by the breech at term. MAIN OUTCOME MEASURES--Intrapartum and neonatal mortality, low Apgar scores, intubation at birth, and admission to special care baby units. RESULTS--After the exclusion of babies with congenital anomalies the incidence of intrapartum and neonatal death associated with vaginal birth was 8/961 (0.83%) compared with 1/2486 (0.03%) in babies born by caesarean section (relative risk 20, 95% confidence interval 2.5 to 163). The numbers of low Apgar scores and neonatal intubation were doubled in babies born vaginally or by emergency caesarean section compared with those delivered by elective operation. CONCLUSIONS--The good neonatal outcome associated with elective caesarean delivery of the term breech fetus may influence the decision of women and their obstetricians about mode of delivery.     

Studies of the induction of dominant lethals and translocations in male mice after chronic exposure to microwave radiation

Male C3H mice were exposed to 100 W m-2 of 2.45 GHz continuous-wave microwave radiation for 6 h per day for a total of 120 h over an 8-week period. The exposure level was chosen so that the specific energy absorption rate (SAR) would be approximately equal to the level of 4 W kg-1 which is considered by a number of organizations to be a threshold for adverse biological effects. At the end of the treatment period the mice were mated with a different group of (C3H x 101) F1 hybrid females each week for the following 8 weeks. There was no significant reduction in pregnancy rate, preimplantation survival or postimplantation survival in the exposed group compared to sham-exposed controls. At the end of the mating period a cytogenetic analysis was carried out of meiotic chromosome preparations of testicular tissue, thus sampling cells that were stem cell spermatogonia during the treatment regime. The results showed no difference in the frequency of reciprocal translocations between the sham and treated groups, or in the frequency of cells with autosome or sex chromosome univalents. Low levels of fragments and exchanges were found in both groups. It is concluded that there is no evidence in this experiment to show that chronic exposure of male mice to 2.45 GHz microwave radiation induces a mutagenic response in male germ cells. This conclusion is in agreement with the observations of Berman et al. (1980), who reported a lack of male germ cell mutagenesis after repetitive or chronic exposure of rats to 2.45 GHz.     

Chromosomal localization of the human alpha 1-antitrypsin gene (PI) to 14q31-32.

In situ hybridization of a recombinant cDNA probe containing the human alpha 1-antitrypsin gene to metaphase chromosomes demonstrated significant hybridization to chromosomal segment 14q31-32. A high percentage of cells analyzed (31%) displayed labeling on chromosome 14. Of all labeled sites on chromosome 14, 60% were found on segment 14q31-32. These results refine the previous assignment of the human alpha 1-antitrypsin gene to segment 14q24.1-32.1.     

A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis

A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis.     

Efficient extraction of RNA from mammalian tissue

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.