Search PubMed⌕ Search

Biomedical subjects

H A Nash

Publications and source records attributed to H A Nash.

At least 73 records · Page 4Linked to original sources
PubMed1985

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). II. Use of compounds of the muramyl dipeptide (MDP) family as adjuvants.

Earlier tests of an antipregnancy vaccine consisting of the beta-subunit of human chorionic gonadotropin (beta-hCG) linked by reaction with a carbodiimide reagent to tetanus toxoid (TT) and adsorbed on Al(OH)3 resulted in antibody responses that were judged inadequate in some women. Experiments were therefore conducted to evaluate the effectiveness of additional adjuvants in increasing the antibody response. Muramyl dipeptide (MDP) and several of its analogs were formulated with the vaccine and tested in rabbits. Some of the analogs, and notably N-acetyl-normuramyl-L-alanyl-D-isoglutamine, elicited substantial increments in the ability of the antisera to bind [125I]hCG and in its ability to neutralize hCG in the rat uterine weight assay. The effectiveness of these peptides was greatest when formulated in a water-in-oil emulsion. Increments of 10 fold were attained using a vegetable oil as the oil component. The MDP analogs were much less effective as adjuvants when formulated in oil-in-water emulsions or in aqueous suspensions of the antigen. It is concluded that selected MDP analogs incorporated in a water-in-vegetable oil emulsion can markedly increase the circulating antibody response to the beta-hCG-TT vaccine.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1985

Formulation of a potential antipregnancy vaccine based on the beta-subunit of human chorionic gonadotropin (beta-hCG). III. Evaluation of various vehicles and adjuvants.

Rabbits were used to test the efficacy of several materials as supplementary adjuvants when administered as part of a vaccine formulation consisting of the beta-subunit of human chorionic gonadotropin linked to tetanus toxoid (beta-hCG-TT) and adsorbed on Al(OH)3. In the amounts used, Corynebacterium parvum, levamisole, thymic factor, and N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propanediamine exhibited little adjuvant activity although the latter material elicited marginal increments when incorporated in liposomes. A Salmonella lipopolysaccharide preparation (SPLPS) and a streptococcal preparation (OK-432) each gave approximately 7-fold increments in titer. The SPLPS preparation was pyrogenic at the doses used. OK-432 was nonpyrogenic and did not cause other evident undesirable effects. It may therefore prove to be a useful adjuvant. It gave a nearly flat dose response curve over the range of 0.5 to 4.0 mg per rabbit. Incorporation of beta-hCG-TT on Al(OH)3 in a water-in-oil emulsion caused a moderate increase in titers. Incorporation into liposomes or an oil-in-water emulsion was not effective.

Adjuvants, Immunologic↗
PubMed1985

Genetic rearrangement of DNA induces knots with a unique topology: implications for the mechanism of synapsis and crossing-over.

We have determined the topological sign of the knots produced by a cycle of phage lambda integrative recombination. To insure that these knots reflect intrinsic features of the reaction mechanism, the substrate was constructed so that random interwrapping of segments of DNA played a minimal role in the topological outcome. The knotted DNA was coated with the bacteriophage T4 uvsX gene product and examined in the electron microscope to determine the nature of each crossing point or node. All of the knots were identical; they were trefoils with three nodes of positive sign. We interpret this result to mean that one recombination site, which previous work had indicated is organized into a nucleosome-like structure, is wrapped with a handedness identical to that found in nucleosomes. Therefore, this wrapping may explain the dependence of recombination on supercoiling of the substrate DNA. Moreover, we show that the topological result sharply limits acceptable mechanisms for the details of strand exchange.

Bacteriophage lambda↗
PubMed1984

Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda.

Under physiological conditions, integration of lambda DNA into the Escherichia coli chromosome requires the direct participation of only two proteins, the viral int gene product and E. coli integration host factor (IHF). A variant of the int gene has been isolated that permits integrative recombination in cells mutant for one of the two subunits of IHF (Miller, H.I., Mozola, M.A., and Friedman, D.I. (1980) Cell 20, 721-729). In the present work, we have purified Int-h, the product of this variant gene. In contrast to the wild-type int gene product (Int+), which produces almost no recombinants in the absence of IHF, purified Int-h protein sponsors reduced but significant levels of integrative recombination in the absence of any E. coli supplement. This shows that the int gene encodes all the information necessary for the elementary steps in recombination and implies that IHF functions as an accessory protein. When supplemented by IHF, recombination promoted by Int-h resembles that promoted by Int+ in kinetics, stoichiometry of Int and IHF, and nature of the recombinant product. Under these conditions, Int-h uses supercoiled DNA more effectively than nonsupercoiled DNA as a substrate for recombination, as does Int+. However, in the absence of IHF, Int-h recombines supercoiled and nonsupercoiled substrates identically, indicating that IHF is an important part of the mechanism that senses the supercoiled state of the substrate DNA during recombination. A surprising difference in recombination carried out by Int-h in the presence or absence of IHF concerns the degree to which sites on the same circle recombine with one another as opposed to sites on sister molecules. In the presence of IHF, Int-h favors intramolecular recombination, as does Int+. However, in the absence of IHF, Int-h almost exclusively promotes intermolecular recombination.

Bacteriophage lambda↗
PubMed1984

E. coli integration host factor binds to specific sites in DNA.

E. coli integration host factor (IHF) both participates directly in phage lambda site-specific recombination and regulates the expression of phage and bacterial genes. Using protection from nuclease and chemical attack as an assay, we examined the interaction of IHF with DNA. We found that IHF is a specific DNA binding protein that interacts with three distinct segments of attP, the recombination site carried by phage lambda. We also found that specific IHF binding sites are located in non-att DNA. Several non-att IHF binding sites that we have identified are adjacent to genes whose expression is altered in IHF mutants. From comparison of the sequences protected by IHF, we suggest that the critical determinant in specific IHF-DNA interaction is contained in the sequence T.PyAA...PuTTGaT.A.PuTT...PyAACtA.

Bacteriophage lambda↗
PubMed1984

In vitro regulation of phage lambda cII gene expression by Escherichia coli integration host factor.

The effect of Escherichia coli integration host factor (IHF) on phage lambda gene expression has been examined in a simplified DNA-directed in vitro system that measures the formation of the first dipeptide of the gene product. Plasmid pKC30cII, which contains the phage lambda genes N, cII and O, under control of the PL promoter, was used as template to study the expression of the first dipeptide of the gene products--i.e., fMet-Asp for N protein, fMet-Val for cII, and fMet-Thr for O. Purified IHF stimulates the DNA-directed synthesis of fMet-Val (cII) and fMet-Thr (O) 2-3-fold but has no effect on the synthesis of fMet-Asp (N). In this in vitro system, the stimulation by IHF of cII and O gene expression is at the level of transcription. Phage lambda repressor completely inhibits dipeptide synthesis in the presence or absence of IHF. The results are consistent with a role of IHF as a transcription antiterminator, perhaps functioning at or near the tR1 site preceding the cII gene.

Bacteriophage lambda↗
PubMed1984

Lambda integrative recombination: supercoiling, synapsis, and strand exchange.

We have presented the first results of three ongoing experiments on the biochemical mechanism of lambda site-specific recombination. First, we have shown that under the tension induced by negative supercoiling, a segment of attP and attR undergoes a transition to a novel conformation. The basis for detecting this new structure implies that it contains unpaired bases, perhaps arising as a result of the transition of an inverted repeat to a cruciform structure. Since the segment undergoing transition lies outside the homologous core region, the unpairing of DNA that we have detected cannot be directly involved in synapsis. It will be of interest to learn whether recombination proteins alter this conformational change, possibly to extend its effect into the core region. Second, we have demonstrated that replacing phosphate groups in attachment site DNA with phosphorothioates interferes with recombination. Our first experiments indicate that the effect depends on locating phosphorothioates precisely at the site of recombination crossover. We take this to mean that interference is primarily at the step of breakage and reunion; we plan to exploit this inhibition in an attempt to trap synaptic intermediates in recombination. Finally, we have found that the rare cleavage of attP DNA by Int can involve either one or both strands of the duplex. This indicates that the degree of coordination between Int action at the two halves of the core region can be variable. Although we do not know whether this variability affects binding of Int, breakage of DNA, or resealing of broken strands, our data suggest that this variability can be regulated by sulfhydryl reagents.

Bacteriophage lambda↗
PubMed1983

Knotting of DNA caused by a genetic rearrangement. Evidence for a nucleosome-like structure in site-specific recombination of bacteriophage lambda.

Intramolecular recombination between two attachment sites on a circular substrate can invert one segment of the circle with respect to the other. We have studied the topological form of the products of such site-specific inversion as a function of two parameters of the substrate circle: the degree of supercoiling and the distance between the recombining sites. For both integrative and excisive recombination, supercoiled substrates produced knotted recombinants; the complexity of the knots reflects the distance separating the sites. This confirms and extends earlier observations and supports the hypothesis that random interwrapping of segments of the double-helical substrate persists during recombination. For integrative recombination, we find that even at conditions that should limit random interwrapping, absence of supercoiling and very short separation between attachment sites, only about one-half of the recombinant products are simple circles and the rest are knotted. Under the same conditions, excisive recombination yields only simple circular inverted recombinants. We propose that the excess knotting that characterizes integrative recombination reflects the requirement for wrapping of one attachment site, presumably attP, into a nucleosome-like structure. This hypothesis accounts for both the frequency of knots and the observation that the extra knots are trefoils rather than more complex forms.

Attachment Sites, Microbiological↗
PubMed1983

Site-specific recombination of bacteriophage lambda. The change in topological linking number associated with exchange of DNA strands.

The changes in supercoiling that accompany site-specific recombination have been measured. In each experiment, the substrate was a circle that contained two attachment sites oriented as an inverted repeat; recombination between the sites inverts one segment of the circle with respect to the other. Using conditions developed in the accompanying work, a measurable amount of the recombinant is in the form of unknotted, simple circles. The difference between the topological linking number of this product relative to that of the substrate can be determined directly from the change in mobility during agarose gel electrophoresis. With partially supercoiled substrates, both integrative and excisive recombination are characterized by a unique change in linking number, a relaxation of two topological turns. For excisive recombination, it has been possible to study closed circular substrates that lack supercoils. In this case, changes in linking number of both +2 and -2 are observed. These results are used to evaluate various proposals for synapsis and strand exchange in bacteriophage lambda site-specific recombination.

Attachment Sites, Microbiological↗
PubMed1983

Release rates of levonorgestrel from Silastic capsules, homogeneous rods and covered rods in humans.

Three forms of subdermal implants containing levonorgestrel are described. These are: capsules, in which the powdered drug is sealed inside of lengths of Medical Grade Silastic tubing; homogeneous rods, in which the drug is uniformly dispersed in Silastic 382 Medical Grade Elastomer; and covered rods, in which a core rod of drug and filler-free polydimethylsiloxane polymer (50:50, Wt:Wt) are sealed inside thin-walled Silastic tubing. Long-term in vivo release rates from human subjects are presented; 6.5 years for capsules, 3.6 years for homogeneous rods and 4 years for covered rods. Sets of six capsules release a decreasing amount of drug through the first few hundred days in situ and after 500 days a fairly constant rate of about 35 micrograms per day is released (2 micrograms/cm). Homogeneous rods deliver a continuously declining amount of drug during the entire time studied. In the first 100 days the release averages 136 micrograms per day from a set of three 3-cm rods (15 micrograms/cm), gradually declining to 30 micrograms per day (3.3 micrograms/cm) from day 800 to day 1300. The covered rods deliver at a constant rate of 17.5 micrograms per day for a 3-cm rod (5.83 micrograms/cm) through 4 years.

Capsules↗
PubMed1983

The mechanism of phage lambda site-specific recombination: site-specific breakage of DNA by Int topoisomerase.

We demonstrate that the topoisomerase activity of bacteriophage lambda Int protein introduces single-strand breaks into duplex DNA at specific sites. Strand breakage is accompanied by the covalent linkage of Int to DNA. The linkage connects a residue in Int to the 3' phosphate of DNA at the site of breakage; the other breakage product has a 5' OH terminus. Int is the first procaryotic topoisomerase shown to break DNA in this manner. We find that in att sites, Int breaks DNA within the 15 bp homologous core. These sites of Int topoisomerase action result from the interaction of Int with "junction-type" recognition sequences (CAACTTNNT), and Int topoisomerase acts between the 7th and 8th bases of this sequence. The sites of breakage within the cores of attP and attB coincide exactly with positions where breakage and reunion occur during Int-dependent recombination. These results indicate that Int topoisomerase executes strand exchange during recombination.

Bacteriophage lambda↗
PubMed1981

Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination.

A purified preparation of the Escherichia coli integration host factor (IHF) displays two polypeptides of apparent molecular weight 11,000 and 9,500 when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Under nondenaturing conditions, IHF appears to exist as a 1:1 complex of these two polypeptides. Integrative recombination takes place in vitro when purified IHF and purified Int, a product of a bacteriophage lambda gene, are the only proteins added to reaction mixtures. No recombination is detected in the absence of either protein. The characteristics of the recombination reaction carried out by these two purified proteins are described. Purified IHF binds to DNA; in the presence of Int, a ternary complex is formed at one of the specific recombination sites. IHF hs no detectable endonuclease or topoisomerase activity. Several possibilities for the role of IHF in recombination are considered.

Amino Acids↗
PubMed1981

Direct role of the himA gene product in phage lambda integration.

The integration of phage lambda into the Escherichia coli chromosome is accomplished by a site-specific recombination between two unique DNA sequences (attB on the bacterial genome and attP on the phage; reviewed in refs 2, 3) and requires proteins encoded by both the bacterium and the phage. Genetic and biochemical studies have shown that bacterial strains mutant in the himA gene, located at 38 min on the E. coli map, are defective in the activity of the host-encoded component. They are, moreover, defective for the growth of bacteriophage Mu, for precise excision of transposable antibiotic resistance determinants and for the synthesis of the lambda int gene product. We now show that the himA gene product (phimA) is not solely a regulator of genes involved in integration but is one of two host polypeptides required for integrative recombination.

Bacteriophage lambda↗
PubMed1981

A multicenter study of levonorgestrel-estradiol contraceptive vaginal rings. II-Subjective and objective measures of effects. An international comparative trial.

Comparative clinical trials of 2 sizes of contraceptive vaginal rings and of an oral contraceptive were undertaken at 8 investigational sites (9 clinics). More than 500 women enrolled on each of the 3 study regimens. Side effects of the rings and of Nordette, the oral contraceptive, were evaluated by noting spontaneous complaints, by recording medications taken and by physical examination. Inquiries about changes in the frequency of specific conditions were made at the end of the subjects participation in the first year of the study. The incidence of spontaneous complaints was similar among users of the 2 different-sized rings and of the pill.

Adolescent↗
PubMed1981

A multicenter study of levonorgestrel-estradiol contraceptive vaginal rings. III-Menstrual patterns. An international comparative trial.

Menstrual events among users of contraceptive vaginal rings (CVRs), releasing levonorgestrel and estradiol, were studied in comparison with a combined oral contraceptive, Nordette, in multicentered trials. CVRs were made with outside diameters of 50 and 58 mm and released about 250 micrograms and 290 microgram of levonorgestrel and 150 microgram and 180 microgram of estradiol per day, respectively. The CVRs were used continuously for 3 weeks and then removed for 1 week. Both the CVRs and Nordette were perceived to reduce menstrual flow and days of menstrual bleeding. Twenty to 25% of CVR users perceived increased intermenstrual bleeding or spotting. Diaries indicate, however, that on average, CVR users experienced about 1 day per month of bleeding or spotting with the ring in place. CVRs and Nordette produced approximately the same total number of bleeding and spotting days during 6 cycles of use, 27-29, but the small (50-mm OD) ring was associated with somewhat more spotting. This ring was also associated with somewhat more prolonged bleeding and spotting runs and with more prolonged nonbleeding intervals than reported by users of the larger (58-mm OD) ring or of Nordette. Differences among regimens, however, tended to be small even when statistically significant. Evidence from menstrual diaries indicates that these CVRs, and in particular 58-mm ring, provide control over the menstrual cycle comparable to the Nordette.

Adolescent↗
PubMed1981

Strand exchange in lambda integrative recombination: genetics, biochemistry, and models.

We have asked, "What is the mechanism of strand exchange during site-specific recombination of phage lambda?" Crosses carried out in vivo have shown that the recombination joint can be extended rather than flush and that the four-strand breaks and rejoinings needed to from a recombinant can occur asynchronously. Crosses carried out in vitro have shown that all the nucleotides at the site of crossover are conserved during recombination, as are most or all of the superhelical turns present in the substrate molecules. We have presented new data showing that topoisomerase activity of Int protein relaxes DNA by making transient single-strand, rather than double-strand, breaks in the phosphodiester back-bone. These findings are incorporated into a model for strand exchange that has as its central intermediate a four-strand structure.

Bacteriophage lambda↗