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Biomedical subjects

P North

Publications and source records attributed to P North.

At least 19 recordsLinked to original sources
PubMed2000

Immunogenic and antigenic dominance of a nonneutralizing epitope over a highly conserved neutralizing epitope in the gp41 envelope glycoprotein of human immunodeficiency virus type 1: its deletion leads to a strong neutralizing response.

The Kennedy peptide, (731)PRGPDRPEGIEEEGGERDRDRS(752), from the cytoplasmic domain of the gp41 transmembrane envelope glycoprotein of HIV-1 contains a conformationally dependent neutralizing epitope (ERDRD) and a linear nonneutralizing epitope (IEEE). No recognized murine T cell epitope is present. The peptide usually stimulates virus-specific antibody, but this is not always neutralizing. Here we show that IEEE (or possibly IEEE plus adjacent sequence) is immunogenically and antigenically dominant over the ERDRD neutralizing epitope. Thus rabbits immunized in a variety of routes, doses, and adjuvants with a chimeric cowpea mosaic virus (CPMV) expressing the Kennedy peptide on its surface (CPMV-HIV/1) synthesized IEEE-specific serum antibody but no ERDRD-specific or HIV-1-neutralizing antibody. To test if this resulted from immunodominance or from a hole in the antibody repertoire, we immunized rabbits with chimera CPMV-HIV/29, which expresses the GERDRDR part of the Kennedy sequence. This chimera readily stimulated ERDRD-specific, neutralizing antibody. In mice the situation was less extreme, but individual animals with low neutralizing titers had a high ratio of IEEE-specific:ERDRD-specific antibody. Data are consistent with immunodominance of IEEE over ERDRD in the Kennedy peptide. IEEE-specific antibody was also antigenically dominant and prevented ERDRD-specific antibody from binding to its epitope and from neutralizing HIV-1. It may be that HIV-1 has evolved a nonneutralizing immunodominant epitope that allows it to possess a neutralizing epitope without suffering the consequences, and this idea is supported by the covariance of both epitope sequences. To our knowledge this is the first example of a defined sequence that controls the activity of an adjacent epitope.

Amino Acid Sequence↗
PubMed1999

Evidence of residual disease in ossicles of patients undergoing cholesteatoma removal.

For the past several years there has been much debate regarding the advisability of reusing the incus for ossicular reconstruction in cases involving cholesteatoma. There appears to be some evidence that microscopic foci of cholesteatoma in the incus could lead to reimplantation of the cholesteatoma should the incus be used in the reconstruction phase. In an effort to elucidate the incidence of microscopic residual cholesteatoma, the incudes of patients with cholesteatoma were examined both grossly in the operating room and microscopically in the laboratory for erosion and residual cholesteatoma. Our examination showed that a number of specimens apparently free of cholesteatoma after macroscopic examination had microscopic evidence of cholesteatoma. Likewise, microscopic examination of an incus that appeared to be free of residual cholesteatoma revealed epithelial cells deeply invading the bone. Macroscopic examination consistently underestimated the amount of erosion that was clearly evident upon histologic examination. In light of these findings, gross examination of the incus after removal of cholesteatoma is not reliably predictive of invasive microscopic disease. Reusing the ossicles in this situation creates the potential of reimplanting the disease.

Cholesteatoma, Middle Ear↗
PubMed1995

Clenbuterol: a substitute for anabolic steroids?

Clenbuterol is a recently popular drug used by athletes in many sports for its purported anabolic effects and reduction of subcutaneous fat. It is a beta-2 (beta 2) agonist prescribed overseas as a bronchodilator, but not approved for use in this country. It is on the banned substance list of the United States Olympic Committee. To avoid any erosion of confidence, physicians caring for athletes need accurate information regarding clenbuterol. Such information is unavailable within the routine medical environs. A review of the literature of animal husbandry reveals that this drug, when administered in doses far greater than those required for bronchodilation, does indeed increase the deposition rate of lean mass and retard adipose gain. There are no human studies available. Animal studies were conducted on laboratory and slaughter stock. No investigation into long-term cardiovascular side effects has been undertaken. The rate of extrapolation from animal studies to unsupervised human usage is alarming. If this category of drugs does preserve lean mass in humans, there are legitimate medical applications. Trials of efficacy and safety are needed.

Adipose Tissue↗
PubMed1995

Cyclic AMP-dependent protein kinase type I is involved in hypersensitivity of human breast cells to topoisomerase II inhibitors.

Topoisomerase II (Topo II) is an essential enzyme that catalyzes the breakage of double-strand DNA and is the target of several effective anticancer drugs, including the epipodophyllotoxins. The regulatory subunits of the cyclic AMP-dependent protein kinase are differentially expressed in normal and cancer cells. The RIalpha subunit is overexpressed in cells transformed by transforming growth factor-alpha (TGF-alpha) or Ha-ras oncogene. It has been shown that murine cells transformed by Ha-ras become hypersensitive to Topo II-targeting anticancer drugs. In this report we have tested whether any correlation exists between the expression of RIalpha protein and cellular sensitivity of Topo II-targeting drugs. Normal human breast MCF-10A cells and their derivatives overexpressing TGF-alpha, Ha-ras, or the different protein kinase subunits were treated with either Topo II inhibitors, such as etoposide, teniposide, or amsacrine, or with drugs which act independently of Topo II, such as bleomycin. Here we show that MCF-10A TGF-alpha and MCF-10A Ha-ras cells overexpress the RIalpha protein and become hypersensitive to epypodophyllotoxins and amsacrine but not to bleomycin. Direct introduction of the RIalpha gene into MCF-10A induces hypersensitivity to Topo II inhibitor drugs. In contrast, the overexpression of the other protein kinase subunits, RIIbeta or Calpha, does not modify the drug sensitivity of MCF-10A cells. No differences in the mRNA/protein content or in the activity of Topo II were found between hypersensitive cells and parental MCF-10A cells, suggesting that RIalpha may influence drug sensitivity via modulation of events downstream of the Topo II-DNA cleavable complex.

Amsacrine↗
PubMed1993

Repair and misrepair of site-specific DNA double-strand breaks by human cell extracts.

The rejoining by human cell extracts of a double-strand break induced by endonuclease treatment at one of several sites within a small DNA molecule was studied. Rejoining was found at each of 8 sites tested, but the rejoin efficiency varied with the nature of the break (e.g., breaks with cohesive ends were rejoined more efficiently than blunt-ended breaks). Extracts from primary and immortalized cell lines, as well as those from individuals with ataxia telangiectasia (A-T), showed the same pattern of relative rejoin efficiencies. However, mis-rejoining varied with the cell extract used, and was particularly elevated with two immortalized A-T cell lines. Mixing experiments showed that the mis-rejoining property of extracts could act in a semi-dominant fashion, depending on the individual efficiencies of the component extracts. The mis-rejoin mechanism involved deletion at sites of short direct repeats at various distances from the initial break site. A model of deletion formation (the strand-exposure and repair model) is restated to explain the sequence repeat dependence found, and is compared to models of homologous DNA recombination.

Ataxia Telangiectasia↗
PubMed1992

A mechanism for deletion formation in DNA by human cell extracts: the involvement of short sequence repeats.

DNA molecules carrying a site-specific double-strand break were exposed to nuclear extracts from human cell lines. It was shown previously that breaks could be rejoined correctly by human extracts, but that a proportion of the rejoined molecules had suffered deletions and insertions. The 'mis-rejoined' proportion was higher with cell extracts from an individual with the disorder ataxia-telangiectasia than with normal cell extracts. We now show by sequence analysis that deletions in extract-treated molecules occur exclusively between short direct repeats (2-6 base pairs). A mis-rejoined molecule containing an insertion of 300 bp also had a repeat-based deletion at the same site. A number of different direct repeats are involved; however, some clustering of these occurs especially on the upstream side of the initial breakpoint. These data are most simply interpreted in terms of a model of deletion formation involving single-strand exposure and repair, perhaps with the action of other DNA-metabolising enzymes influencing the frequency with which some repeats are involved.

Ataxia Telangiectasia↗
PubMed1992

Sexual activity and condom use in high school students.

OBJECTIVE: To determine the level of sexual experience and condom use among Year 9 high school students. DESIGN: Self-administered questionnaire to all Year 9 students attending a personal development workshop. SETTING: The study was carried out in two regional high schools in New South Wales, one in an urban area and the other in a rural area. PARTICIPANTS: A total of 253 students in Year 9 at the two high schools; only two students declined to participate. RESULTS: Seventy-one students (28.1%) had had intercourse at least once. There was no statistically significant difference between the percentage of male and female students or rural and urban students who had had intercourse. Thirty students (42.3%) always used condoms, 35.2% used them sometimes and the other sexually active students had never used them. The commonest reasons for non-use were unplanned sex, unavailability or no time. CONCLUSIONS: The unpredictability of teenage sexual activity may mean that condoms are not available at the crucial time. Girls appear to have more difficulty in asking for condoms to be used, but the numbers are two small in this survey to draw definite conclusions. If both pregnancy and sexually transmissible diseases are to be prevented among adolescents, sex education must encourage condom use as the first method of contraception for this group and emphasise the need for consistent availability and use.

Adolescent↗
PubMed1990

The rejoining of double-strand breaks in DNA by human cell extracts.

A double-strand DNA break was introduced at a specific site within the lacZ gene of plasmid pUC18 using one of several restriction enzymes, and the plasmid exposed to nuclear extracts from human cell lines. Physical rejoining of DNA was monitored by Southern analysis after gel separation, and the fidelity of rejoining by expression of the lacZ gene after bacterial transformation with the treated plasmid. Breaks at the SalI and EcoRI sites were rejoined by extracts to form circular monomers, but the efficiency of rejoining was much higher at the SalI site. Measurement of rejoining at several adjacent sites having different types of termini, consistently showed a range of efficiencies with 5' 4-base greater than 3' 4-base overhangs and 4-base greater than 2-base greater than no overhang. Similar efficiencies were found for nuclear extracts from transformed cell lines, both from a 'normal' individual and an ataxia-telangiectasia (A-T) patient, and from a non-transformed normal cell culture. In contrast at some sites, especially those with a low rejoin efficiency, the fidelity of rejoining was very much lower for the A-T extracts than for normal cell extracts. Mis-rejoining was, however, unrelated to rejoin efficiency at other sites, suggesting that factors such as the exact sequence at the break site on the molecule may also influence the fidelity of rejoining.

Base Sequence↗
PubMed1988

Influence of human immunodeficiency virus antibody testing on sexual behaviour in a "high-risk" population from a "low-risk" city.

A survey was undertaken of homosexual and bisexual men in Brisbane to establish whether knowledge of their human immunodeficiency virus (HIV)-antibody status had influenced any sexual behaviour that was likely to spread HIV type 1 (HIV-1). Of the 318 respondents, 123 respondents knew their HIV serological status, and 13 of these were HIV seropositive. Of the 195 respondents who previously had not been tested, 10 individuals proved to be HIV seropositive. Eighty-two per cent of subjects stated that they had reduced their sexual activity because of their awareness of the acquired immunodeficiency syndrome (AIDS); this reduction was equally common among those who had or had not previously had their HIV serological status checked. Anal intercourse was practised most frequently by those subjects who were HIV seropositive and were not aware of it; nevertheless, unprotected anal intercourse was common among subjects who knew their HIV serological status, including those who knew that they were HIV seropositive. Eighty-nine of 208 subjects who were practising anal intercourse had never used a condom. Usage of a condom was marginally more common among those subjects who previously had been tested for the presence of HIV antibodies (P = 0.06), and this was particularly so for those subjects who knew that they were HIV seropositive (P less than 0.01). Condom usage was no more common among those subjects who knew that they were HIV seronegative, when compared with those subjects who did not know their status. These data show that knowledge of a negative HIV-antibody test-result has no substantial association with safer sexual behaviour and suggest that whereas targeted information programmes have had some impact on behaviour in high-risk groups in Brisbane, by the end of July 1986, these programmes had not yet resulted in safer sexual practices by the majority of homosexual and bisexual men.

AIDS Serodiagnosis↗
PubMed1986

The analysis of multiple polymorphic loci on a single human chromosome to exclude linkage to inherited disease: cystic fibrosis and chromosome 4.

Classical linkage programs analyze the segregation of two markers in informative families. When several markers are available for one human chromosome, pairwise analysis can exclude linkage between each marker and an inherited disease. The identification of restriction fragment length polymorphisms has made many new informative markers, assigned to chromosomes, available. We have adapted the multipoint linkage program MLINK developed by Lathrop et al. in order to exclude linkage between cystic fibrosis and several markers known to be on human chromosome 4. The exclusion obtained is greater than that for a pairwise analysis.

Chromosome Mapping↗
PubMed1984

Protein-mediated exchange of synthetic phosphatidylcholines into synaptosomal membranes.

A phosphatidylcholine (PC) exchange protein from bovine liver was used to exchange endogenous synaptosomal membrane PC's with PC's of defined fatty-acid composition from phospholipid vesicles. Up to 50% of the total synaptosomal PC could be exchanged during a 3 h incubation with PC's which were in the liquid-crystalline state at the temperature of incubation (dimyristoyl-, dioleoyl- and dielaidoyl-PC). The biphasic kinetics of the exchange of 14C-labeled 1-palmitoyl-2-oleoyl-PC into isolated synaptic plasma membrane vesicles indicated that the half-time for transbilayer equilibrium of PC in these membranes was about 10 h. Hence, the observed 50% exchange of total synaptosomal PC probably represented nearly complete exchange of PC in the outer face of the synaptosomal plasma membrane. This extensive exchange was accomplished without apparent loss of synaptosomal function, including membrane potential and high-affinity uptake of choline and gamma-aminobutyric acid. PC's in the gel state (dipalmitoyl- and distearoyl-PC) could not be exchanged extensively into the synaptosomal membranes. However, from within gel-state distearoyl-PC liposomes, a trace amount of fluid 1-palmitoyl-2-oleoyl-PC (Tm less than 10 degrees C) could be preferentially exchanged into the synaptosomes at 32 degrees C with little transfer of the saturated PC.

Androgen-Binding Protein↗
PubMed1984

Inhibition of (Na,K)-ATPase by tetravalent vanadium.

Vanadyl, the tetravalent state of vanadium and a divalent cation, VO2+, was a relatively powerful inhibitor of highly purified membrane-bound sodium and potassium ion transport adenosine triphosphatase. The sensitivity of the ATPase activity to vanadyl characteristically correlated positively with the specific activity of the enzyme preparation. Inhibition ranged from nearly complete inhibition at less than 5 microM vanadyl for some of the purest fractions (specific activity approximately 45 mumol/min/mg of protein) to no observable inhibition at 300 microM vanadyl in one crude preparation of the enzyme with a specific activity of 10 mumol/min/mg of protein. The level of free vanadyl was reduced by incubation with these membranes, but this reduction was not sufficient to account for the low sensitivity to vanadyl observed in crude preparations. A reduction in specific activity by partial inactivation of a sensitive preparation by treatment with FeCl3 and ascorbate reduced its sensitivity to vanadyl. Anionic ligands of the enzyme, vanadate or ATP, increased the rate of recovery from inhibition after chelation of free vanadyl. At pH 6.1, the inhibition was characteristically fully reversible (t1/2 approximately 10 min), whereas at pH 8.1 it was stable for hours. The degree and stability of enzyme inhibition by vanadyl increased for several hours during incubation of the vanadyl-enzyme mixture, and at pH 6.1 the properties of the inhibitor itself also changed with time. Preincubation of the ion at that pH for 5 h before addition of the enzyme produced a more stable inhibition. The time- and pH-dependent changes in the degree and stability of enzyme inhibition probably relate to the complex chemistry of the vanadyl ion in solution.

Animals↗
PubMed1983

Alteration of synaptic membrane cholesterol/phospholipid ratio using a lipid transfer protein. Effect on gamma-aminobutyric acid uptake.

A procedure was developed to vary the cholesterol-to-phospholipid (Ch/PL) ratio of synaptic plasma membranes and synaptosomes using a nonspecific lipid transfer protein so that membrane lipid composition could be correlated with presynaptic function. In synaptic plasma membranes, Ch/PL molar ratios from 0.21 to 1.19 were produced from a normal value of 0.52 +/- 0.01 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 32 degrees C. In synaptosomes, Ch/PL ratios from 0.16 to 0.81 were similarly produced from a normal value of 0.38 +/- 0.04. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively in the phospholipid-to-protein ratio. The fluidity of the synaptic plasma membrane, as estimated by 1,6-diphenylhexatriene anisotropy measurements, was increased by lowering the Ch/PL ratio and decreased by raising the Ch/PL ratio. Decreasing the Ch/PL ratio of synaptosomes and synaptic plasma membrane vesicles resulted in loss of sodium-dependent gamma-aminobutyric acid (GABA) uptake (70-100% loss at Ch/PL ratios decreased to 40% of normal) and reduction in the number of accessible GABA-binding sites. Choline uptake was not affected in these same preparations. GABA uptake was restored by reinserting cholesterol into the membrane. Synaptosomal membrane potential and synaptic plasma membrane sodium permeability were not affected by changing the Ch/PL ratio. Increase in the Ch/PL ratio above normal had no effect on either choline or GABA uptake. Both the decrease in the Ch/PL ratio and the increase in the lipid-to-protein ratio increase membrane "fluidity," lipid-to-protein ratio increase membrane "fluidity," which may modulate the vertical displacement and motional characteristics of the GABA transporter.

Animals↗