Association of a polymorphism of the angiotensin I-converting enzyme gene with essential hypertension.

Angiotensin I-converting enzyme (ACE) is responsible for production of angiotensin II and breakdown of kinins, leading to increased blood pressure (BP). Furthermore, ACE inhibitors are effective antihypertensive agents. A 287 bp insertion/deletion polymorphism in intron 16 of the ACE gene (ACE) was examined by PCR in a cross-sectional study of 80 hypertensive (HT) and 93 normotensive (NT) subjects whose parents had a similar BP status at age greater than or equal to 50. The frequency of the insertion allele was 0.56 in HTs and 0.41 in NTs, and the difference between observed alleles in all subjects in each group was significant (chi 2 = 7.6, P less than 0.01). The data thus provide evidence in favour of an association of HT with a polymorphism at the ACE locus (17q23), so implicating this locus, and possibly a genetic variant of ACE itself, in human essential hypertension.     

Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites.

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Iron-regulated membrane proteins and bacterial virulence

The amount of iron that might be readily available to bacteria in body fluids is extremely small. This iron restricted environment induces phenotypic changes in the metabolism and in the composition of the membrane proteins of bacteria growingin vivo. These changes are now providing a fresh insight into the capabilities of bacteria to multiply in host tissues and are suggesting new possibilities for targetting therapeutic and prophylactic measures.     

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Hormonal stimulation of tyrosinase activity in human foreskin organ cultures

Summary A human foreskin organ culture system has been developed to study the response of human skin to hormonal stimulation. Foreskins are maintained in culture on floating plastic supports which allows the epidermal surface to be exposed to air while the dermis is bathed in nutrient medium. Both black and white human foreskins can be maintained in organ culture for at least 1 wk with no change in the tissue structure or cell viability as determined by histochemical staining and by dopa reaction staining. Tyrosinase activity in both black and white human foreskin cultures decays markedly during the first 2 d of culture to a new steady state level which remains stable throughout the culture period. Both black and white foreskin cultures consistently demonstrate 2- to 10-fold increases in tyrosinase activity when treated with theophylline (1 mM). Approximately 90% of all skin cultures examined showed an increase in enzyme activity when treated with this phosphodiesterase inhibitor. Dibutyryl cAMP (0.1 mM) and [Nle⁴, D-phe⁷]-alpha MSH (10⁻⁸ M), were also found to markedly stimulate tyrosinase activity in some skin cultures, whereas alpha-MSH and prostaglandin E₁ produced only an inconsistent and small increase in the activity of the enzyme. Histamine (1 μM), vitamin D₃ (1 μM), and retinoic acid (1μM) failed to stimulate tyrosinase activity in either white or black foreskin cultures. This hormone-responsive organ culture system can be utilized to characterize the molecular processes responsible for the regulation of tyrosinase and pigmentation in human skin. This work was supported by a research contract from the Oklahoma Center for the Advancement of Science and Technology (OCAST) and by a research grant from the Presbyterian Health Foundation.     

Broad-scale approaches to the determination of soil microbial community structure: Application of the community DNA hybridization technique

Broad-scale approaches seek to integrate information on whole microbial communities. It is widely recognized that culture techniques are too selective and unrepresentative to allow a realistic assessment of the overall structure of microbial communities. Techniques based on fatty acid or metabolic profiles determine the phenotypic composition of the community. Complementary information about the genotypic structure of soil microbial communities necessitates analysis of community DNA. To determine broad-scale differences in soil microbial community structure (i.e., differences at the whole community level, rather than specific differences in species composition), we have applied a community hybridization technique to determine the similarity and relative diversity of two samples by cross hybridization. In previous studies this assay failed with whole-soil community DNA. Usable hybridization signals were obtained using whole-soil DNA, in this study, by digesting the DNA with restriction enzymes before the labeling with a random-primer reaction. The community hybridization technique was tested using a graded series of microbial fractions, increasing in complexity, all isolated from the same soil sample. This demonstrated that single bacterial species and a mixture of cultivable bacteria were less complex and only 5% similar to whole-community DNA or bacteria directly extracted from the soil. Extracted bacterial and whole-community DNA were 75% similar to each other and equally complex. When DNA was extracted from four different agricultural soils, their similarities ranged from 35 to 75%. The potential usefulness of community hybridization applied to soil microbial communities is discussed.