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Biomedical subjects

H A Nash

Publications and source records attributed to H A Nash.

At least 55 records · Page 3Linked to original sources
PubMed1991

Effect of tarnish on copper release.

The rate of copper loss from bright and tarnished collars from Copper T Model TCu 380A IUDs has been investigated in amino acid solutions of pH 5.5 and 7.4 and in serum. In all three media, the tarnished collars quickly became bright and lost copper at the same rate as the initially bright collars. The single exception was when a high ratio of copper surface to serum was used. Under those conditions the tarnished collars initially became bright but after two days a black precipitate appeared on both the initially bright and tarnished collars and weight loss ceased. When a higher ratio of serum to copper surface was used, the pattern was one of continuing loss although at a lower rate than in the amino acid solutions. It is concluded that tarnish does not compromise the oxidation and dissolution of copper even in serum. Serum is considered a surrogate for uterine fluid.

Amino Acids↗
PubMed1990

Bending and supercoiling of DNA at the attachment site of bacteriophage lambda.

Integration of the DNA of bacteriophage lambda into the chromosome of E. coli depends on the formation of a complex nucleoprotein array at a specific locus on the phage genome, the attachment site. Recent work shows how bending of this DNA (induced by a specific DNA-binding protein), and strain in this DNA (induced by supercoiling) contribute to the formation of the nucleoprotein structure. Further, there are new insights into the way this structure directs critical events during recombination.

Attachment Sites, Microbiological↗
PubMed1990

A genetic study of the anesthetic response: mutants of Drosophila melanogaster altered in sensitivity to halothane.

In an attempt to identify genes that control or encode the targets of general anesthetics, we have chemically mutagenized fruit flies and selected four lines that show an abnormal response to the volatile anesthetic halothane. Specifically, about 2-fold higher concentrations of halothane are required to induce the loss of motor control in the mutant flies. Fine mapping of two isolates indicates that they alter a previously uncharacterized gene of Drosophila. In the absence of anesthetics, these mutants display alterations of behavior that imply changes in the adult and the larval neuromuscular system.

Animals↗
PubMed1989

Functional replacement of a protein-induced bend in a DNA recombination site.

In recent years the capacity of proteins to bend DNA by binding to specific sites has become a widely appreciated phenomenon. In many cases, the protein-DNA interaction is known to be functionally significant because destruction of the DNA site or the protein itself results in an altered phenotype. An important question to be answered in these cases is whether bending of DNA is important per se or is merely a consequence of the way a particular protein binds to DNA. Here we report direct evidence from the bacteriophage lambda integration system that a bend introduced by a protein is intrinsically important. We find that a binding site for a specific recombination protein known to bend DNA can be successfully replaced by two other modules that also bend DNA; related modules that fail to bend DNA are ineffective.

Bacterial Proteins↗
PubMed1989

The interaction of E. coli IHF protein with its specific binding sites.

We have used two kinds of footprinting techniques, dimethylsulfate interference and hydroxyl radical protection, to explore the way that IHF recognizes its specific target sequences. Our results lead us to conclude that IHF recognizes DNA primarily through contacts with the minor groove, an unprecedented mode for a sequence-specific binding protein. We have also determined that, although IHF is a small protein that protects a large region of DNA, only a single IHF protomer is present at each binding site. IHF bends the DNA to which it binds. We have combined this fact plus our footprinting and stoichiometry data together with the crystal structure of a related protein, the nonspecific DNA binding protein HU, to propose a model for the way in which IHF binds to its DNA target.

Bacterial Proteins↗
PubMed1989

Heteroduplex substrates for bacteriophage lambda site-specific recombination: cleavage and strand transfer products.

Lambda's Int protein acts as a specific topoisomerase at attachment sites, the DNA segments that are required for site-specific recombination. Int cleaves each strand of an attachment site at a unique place and creates strand exchanges by joining broken ends from two different parents. To study the action of Int topoisomerase in more detail, heteroduplex attachment sites were made by annealing strands that are complementary except for a few base pairs that lie in the region between the points of top and bottom strand exchange in the attachment site core. These heteroduplexes appear to interact normally with Int and its accessory proteins IHF and Xis. Although the heteroduplex sites are specifically cleaved by Int topoisomerase, rejoining of the broken DNA is hindered by the lack of Watson--Crick complementarity adjacent to the break. Because of this, heteroduplexes accumulate broken intermediates which are then processed in novel ways. We have used this feature to provide new information about functional differences between attachment sites, to investigate the way Xis protein controls directionality of site-specific recombination, and to demonstrate that Int protein can join strands indiscriminately and can therefore generate recombinants with either of two genetic polarities.

Bacteriophage lambda↗
PubMed1988

Bacteriophage lambda site-specific recombination proceeds with a defined order of strand exchanges.

Previous work has established that integration of the genome of bacteriophage lambda into the chromosome of its bacterial host proceeds via two independent strand exchanges, which make and then resolve a Holliday-structure intermediate. We find that a phosphorothioate substitution at the site of exchange in one strand of a recombination site depresses the yield of Holliday structures much more than a similar substitution in the other strand. Furthermore, we show that the Holliday structures that accumulate in unblocked reactions have all been made by recombination of one particular pair of strands. We conclude that there is a strong bias in the choice of strands that initiate crossing-over. Excision, the recombination reaction that excises the integrated prophage, exhibits the same bias as integration. This proves, at least at the level of strand exchange, that excision is not the simple reversal of integration. We have altered the relative orientation of parts of the phage attachment site, attP, to demonstrate that the strand-exchange bias is determined not by the local environment around the point of exchange in the core of attP but by more distant elements in its arms. This suggests that the order of the strand exchanges is dictated by an asymmetry in the way that the nucleosome-like structure that forms at attP brings the bacterial site, attB, into juxtaposition prior to strand exchange. Finally, we use the altered attP to show that homology between attP and attB is most critical when it is adjacent to the point of strand exchange.

Attachment Sites, Microbiological↗
PubMed1988

An intermediate in the phage lambda site-specific recombination reaction is revealed by phosphorothioate substitution in DNA.

It has been proposed that phage lambda site-specific recombination proceeds via two independent strand exchanges: the first exchange forming a Holliday-structure which is then converted into complete recombinant products by the second strand exchange. If this hypothesis is correct, one should be able to trap the putative Holliday intermediate by preventing the second strand exchange. In this paper, we show that substitution of phosphorothioate for phosphate in one strand of a recombination site is an effective way to block recombination while permitting the accumulation of a novel structure. This effect is seen only when phosphorothioate is positioned at a point of potential cleavage by Int recombinase, demonstrating that the inhibition of strand exchange is highly specific. Analysis of the novel structure that accumulates in these reactions proves that it contains a Holliday joint. Holliday-structures can also be detected in unblocked recombinations but are present at very low levels. The characteristics of Holliday-structure formation that we describe substantiate the proposed recombination pathway.

Bacteriophage lambda↗
PubMed1988

Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor.

Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda. IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination. It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination. In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA. In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound. In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int. Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int. To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments. This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.

Bacteriophage lambda↗
PubMed1988

Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex.

During lambda integration, Int recombinase must specifically bind to and cut attachment sites on both the viral and host chromosomes. We show here by foot-printing and by a novel cleavage assay that the bacterial attachment site, attB, cannot stably bind Int in competition with other DNAs. Instead, during recombination reactions, attB obtains its Int by collision with the intasome, a nucleoprotein assembly that forms on the viral attachment site, attP. Our cleavage assay also shows that the capture of attB by the attP intasome does not depend on DNA homology between the two sites; synapsis is governed solely by protein-protein and protein-DNA interactions.

Attachment Sites, Microbiological↗
PubMed1987

Contraceptive steroids and coronary artery atherosclerosis in cynomolgus macaques.

The influence of two types of steroidal contraception on the extent of coronary, aortic, carotid, and iliaco-femoral atherosclerosis was assessed in 57 cynomolgus macaques with moderate diet-induced hyperlipoproteinemia. Thirteen animals were treated with an intravaginal ring that released 17 beta-estradiol and levonorgestrel. Fifteen females were treated with an oral contraceptive (OC) composed of ethinyl estradiol and norgestrel. Fifteen females received placebo vaginal rings, and 14 males were untreated. The contraceptive treatments resulted in similar large reductions in plasma high-density lipoprotein (HDL) cholesterol concentrations. Neither treatment influenced the prevalence of coronary artery atherosclerosis. However, treatment with the contraceptive vaginal ring was associated with increased extent of coronary artery atherosclerosis (plaque size) relative to untreated females, whereas treatment with the OC was not. The contrasting effects of the two treatments could not be explained by differences in total plasma cholesterol, HDL cholesterol, or blood pressure. The results suggest that the greater estrogenic influence associated with the ethinyl estradiol-containing OC resulted in inhibition of coronary artery atherosclerosis despite a pronounced progestin-induced lowering of plasma HDL cholesterol concentration and, further, that hormonal balance may have a marked influence on the relationship between plasma lipids and atherogenesis.

Animals↗
PubMed1987

Role of homology in site-specific recombination of bacteriophage lambda: evidence against joining of cohesive ends.

Bacteriophage lambda integration and excision take place at specific loci called attachment sites. Earlier work has shown that efficient recombination requires the identical sequence to be present in both attachment sites throughout the seven-base-pair region between the points of strand exchange. A plausible model for the role of homology postulates that Int, the site-specific recombinase, makes double-strand breaks at attachment sites such that each broken end has a short single-strand protrusion. Recombination would then depend upon the capacity of these protrusions to form Watson-Crick helices--i.e., to anneal--a process that might require perfect complementarity between the cohesive ends. To test this model, we have studied Int-promoted crosses in which one attachment site is a heteroduplex. Specifically, we constructed sites in which the seven-base-pair region between the points of strand exchange contains one or more noncomplementary pairs. The double-strand break and annealing mechanism predicts that crosses with these heteroduplex sites should yield one completed recombinant and one broken site. We find that such nonreciprocal recombination is uncommon and that the typical outcome of crosses involving a heteroduplex site is a reciprocal recombinant in which both products are resealed. Moreover, the occasional appearance of nonreciprocal products can be explained by our finding that Int can cleave heteroduplex attachment sites after recombination is completed. Taken together, our data strongly indicate that bacteriophage lambda recombination does not proceed by the homology-dependent annealing of cohesive ends; acceptable alternatives for the role of homology are discussed.

Bacteriophage lambda↗
PubMed1987

Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits.

Integration host factor (IHF) is a small, basic protein that is needed for efficient recombination of bacteriophage lambda, as well as for other host and viral functions. We have constructed strains in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter. Separate overexpression of himA and hip led to the production of unstable and insoluble peptides, respectively. In contrast, the overexpression of both genes conjointly led to the accumulation of large amounts of active IHF. Extracts of such cells provided the starting material for a rapid purification procedure that results in milligram quantities of apparently homogeneous IHF.

Bacterial Proteins↗
PubMed1986

The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination.

Lambda integrative recombination depends on supercoiling of the phage attachment site, attP. Using dimethylsulfate protection and indirect end-labeling, the interaction of the recombination proteins Int and IHF with supercoiled and linear attP has been studied. Supercoiling enhances the binding of Int to attP, but not if a truncated attP site is employed or if IHF is omitted. We reason that the altered affinity reflects the formation of a higher-order nucleoprotein structure, an "attP intasome," that involves Int and IHF assembly of both arms of attP into a wrapped configuration. The good correlation between the degree and sign of supercoiling needed to promote recombination and that needed for the "attP intasome" indicates that the primary role of supercoiling is to drive the formation of the wrapped structure.

Bacterial Proteins↗
PubMed1986

Role of Escherichia coli IHF protein in lambda site-specific recombination. A mutational analysis of binding sites.

The phage lambda attachment site, attP, contains three binding sites for an Escherichia coli protein, IHF, that is needed for efficient integrative recombination. We have used synthetic oligodeoxyribonucleotides to direct multiple base changes at each of these three sites. Alteration by two base-pairs of the consensus sequence for the leftmost binding site specifically interferes with IHF binding to that site and modestly depresses recombination in vitro. For each of the three binding sites, alteration of the consensus sequence by four base-pairs strongly depresses recombination in vitro, indicating that all three sites are important for attP function. The mutated attP sites are also depressed for recombination in vivo but some of the mutants are less affected than they are in vitro. The disparity between effects in vivo and in vitro for some mutants but not others suggests that the three binding sites are not functionally equivalent and that at some sites additional E. coli factors may replace or assist IHF. The non-equivalence of the three IHF sites is also indicated by the behavior of prophage attachment sites carrying mutations in the binding sites.

Attachment Sites, Microbiological↗
PubMed1986

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). IV. The role of adjuvants in booster injections.

Experiments were conducted in rabbits to determine the effect of adjuvant use on the antibody response following booster injections. The antigen used was in all cases the beta-subunit of human chorionic gonadotropin linked to tetanus toxoid (beta-hCG-TT). Adjuvants used were Al(OH)3, MDP analogs, and a streptococcus preparation, OK432. Primary vaccinations included Al(OH)3 adjuvant with or without supplementary adjuvants. In general, the greater the antibody response following primary vaccination, the greater the response following booster vaccination whether or not adjuvant was used in the booster. No increment in antibody titers was found by reason of including MDP analogs in booster vaccinations. OK432, in contrast, gave increased responses in booster injections which were in several cases statistically significant. The value of including Al(OH)3 in booster injections is not clear from the experimental data. In no case was the increment due to its inclusion large.

Adjuvants, Immunologic↗
PubMed1985

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). I. Alternative macromolecular carriers.

The antibody response obtained after vaccinating rabbits with the beta-subunit of human chorionic gonadotropin (beta-hCG) linked to several protein and polysaccharide carriers was measured. In all but one preparation, carbodiimide was used to couple the beta-hCG to the carrier. Tetanus toxoid (TT) and cholera vaccine proved the most effective carriers among those examined. TT from different manufacturers proved to be greatly different in free amino group content and differed in ability to participate in the coupling reaction. Reasonably good replication of the coupling reaction was obtained with different production lots from the same manufacturer. Inferior antigenic response was obtained with the products of coupling beta-hCG to H. pertussis, influenza vaccine, polylysine, pneumococcus polysaccharide, or E. coli polysaccharide. The findings indicate TT and cholera vaccine to be especially effective in enhancing the antigenicity of a weakly antigenic peptide but point to significant differences in the TT from different manufacturers.

Adjuvants, Immunologic↗