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Biomedical subjects

A J Hunt

Publications and source records attributed to A J Hunt.

At least 19 recordsLinked to original sources
PubMed2000

20-Hydroxyecdysone release from biodegradable devices: the effect of size and shape.

20-Hydroxyecdysone (20-OH) is a natural compound with many demonstrated effects on the physiological functions of vertebrates, particularly increased protein synthesis. Our study sought a suitable dosage form with continuous release of the drug lasting several weeks for implantation into agricultural animals. Biodegradable microparticles and implants of poly(L-lactic) and poly(DL-lactic) acids were prepared. Oligomers of these materials were synthesized, and a method of melting the binary mixture of the oligomer and 20-OH was employed. The particles were prepared simply by grinding the solidified block of the melt and sieving. Implants were prepared by extruding the melt into silicone tubes, removing the solidified content, and cutting into cylinders of 2 mm diameter and various lengths. A new method of preparation of hollow cylinders by aspirating air into silicone tubes filled with the melt was developed. The experiments demonstrated stability of 20-OH during heat treatment. Release of the active ingredient was tested in static in vitro conditions, analogous to those at the site of implantation, and prolonged drug release was obtained with both types of implant. The hollow implants gave release rates nearest to ideal zero-order kinetics and would appear most appropriate for testing in vivo.

Absorbable Implants↗
PubMed1998

Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen.

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311-1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.

3T3 Cells↗
PubMed1998

The dynamic behavior of individual microtubules associated with chromosomes in vitro.

Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which they are bound. The lengths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-time assay that displays directly the movements of individual chromosome-associated microtubules as they elongate and shorten. Chromosomes isolated from cultured Chinese hamster ovary cells were adhered to coverslips and then allowed to bind labeled microtubules. In the presence of tubulin and GTP, these microtubules could grow at their chromosome-bound ends, causing the labeled segments to move away from the chromosomes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. The link between a microtubule and a chromosome was mechanically strong; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore-microtubule junction. The behavior of the microtubules in vitro was regulated by the chromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of depolymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each microtubule interacts with an increasing number of chromosome-associated binding sites as it approaches the kinetochore.

Adenosine Triphosphate↗
PubMed1996

Patterning of functional antibodies and other proteins by photolithography of silane monolayers.

We have demonstrated the assembly of two-dimensional patterns of functional antibodies on a surface. In particular, we have selectively adsorbed micrometer-scale regions of biotinylated immunoglobulin that exhibit specific antigen binding after adsorption. The advantage of this technique is its potential adaptability to adsorbing arbitrary proteins in tightly packed monolayers while retaining functionality. The procedure begins with the formation of a self-assembled monolayer of n-octadecyltrimethoxysilane (OTMS) on a silicon dioxide surface. This monolayer can then be selectively removed by UV photolithography. Under appropriate solution conditions, the OTMS regions will adsorb a monolayer of bovine serum albumin (BSA), while the silicon dioxide regions where the OTMS has been removed by UV light will adsorb less than 2% of a monolayer, thus creating high contrast patterned adsorption of BSA. The attachment of the molecule biotin to the BSA allows the pattern to be replicated in a layer of streptavidin, which bonds to the biotinylated BSA and in turn will bond an additional layer of an arbitrary biotinylated protein. In our test case, functionality of the biotinylated goat antibodies raised against mouse immunoglobulin was demonstrated by the specific binding of fluorescently labeled mouse IgG.

Animals↗
PubMed1994

Gay men as victims of nonconsensual sex.

Incidents of nonconsensual sexual activity among 930 homosexually active men living in England and Wales are analyzed. Of these men, 27.6% said they had been sexually assaulted or had sex against their will at some point in their lives; one third had been forced into sexual activity (usually anal intercourse) by men with whom they had previously had, or were currently having, consensual sexual activity. The contention that male rape is usually committed by heterosexually identified men, primarily as an expression of power and control, is not supported. Recognition that gay men rape other gay men is needed, both by the gay community and support services for victims.

Adolescent↗
PubMed1994

The force exerted by a single kinesin molecule against a viscous load.

Kinesin is a motor protein that uses the energy derived from the hydrolysis of ATP to power the transport of organelles along microtubules. To probe the mechanism of this chemical-to-mechanical energy transduction reaction, the movement of microtubules across glass surfaces coated with kinesin was perturbed by raising the viscosity of the buffer solution. When the viscosity of the solution used in the low density motility assay was increased approximately 100-fold through addition of polysaccharides and polypeptides, the longer microtubules, which experienced a larger drag force from the fluid, moved more slowly than the shorter ones. The speed of movement of a microtubule depended linearly on the drag force loading the motor. At the lowest kinesin density, where dilution experiments indicated that the movement was caused by a single kinesin molecule, extrapolation of the linear relationship yielded a maximum time-averaged drag force of 4.2 +/- 0.5 pN per motor (mean +/- experimental SE). The magnitude of the force argues against one type of "ratchet" model in which the motor is hypothesized to rectify the diffusion of the microtubule; at high viscosity, diffusion is too slow to account for the observed speeds. On the other hand, our data are consistent with models in which force is a consequence of strain developed in an elastic element within the motor; these models include a different "ratchet" model (of the type proposed by A. F. Huxley in 1957) as well as "power-stroke" models.

Adenosine Triphosphate↗
PubMed1993

Kinesin swivels to permit microtubule movement in any direction.

Kinesin is a motor protein that uses the energy derived from ATP hydrolysis to transport organelles along microtubules. By analyzing the thermal fluctuation of microtubules tethered to glass surfaces by single molecules of kinesin, we have measured the torsional flexibility of the motor protein. The torsional stiffness of kinesin, (117 +/- 19) x 10(-24) N.m.rad-1 (mean +/- SEM), is so low that one kT of energy (approximately 4.1 x 10(-21) J at room temperature) is sufficient to twist a kinesin molecule through more than 360 degrees from its resting orientation. Consistent with this flexibility, motility assays show that one or more kinesin molecules can move a microtubule equally well in any direction. These results explain how a motor on the surface of an organelle can rapidly bind to and capture a microtubule irrespective of the organelle's orientation. Furthermore, the flexibility ensures that several motors can efficiently work together even though they are randomly oriented on the surface of an organelle rather than being in precise arrays like the motors of muscle and cilia.

Cilia↗
PubMed1993

Changes in condom use by gay men.

Cross sectional and longitudinal data on condom use among homosexually active men is presented. Data pertain to two waves of interviewing (1987/8 and 1991) from Project SIGMA, a large longitudinal study of male homosexual behaviour in the light of HIV. Aggregate changes across the time-period indicate an increase in condom use for both insertive and receptive anal intercourse, particularly with casual partners. Condom use is still much more prevalent with casual rather than regular partners. Longitudinal analysis show considerable changes at the individual level, with almost a third of men changing their practices. Half of those decreased their risk behaviours, whilst the other half increased them. Less than 1% used condoms for fellatio in the year preceding the 1991 interview, but 8.3% had used other barriers during sex, primarily for hygiene and fun reasons, rather than as disease prophylaxis. Some implications are drawn for condom promotion and HIV prevention.

Adolescent↗
PubMed1993

No connection between alcohol use and unsafe sex among gay and bisexual men.

OBJECTIVE: To investigate the relationship between alcohol use and unsafe sexual behaviour. METHODS: The paper discusses data collected from 461 gay and bisexual men interviewed in England and Wales by Project SIGMA during 1991-1992. These data were collected during face-to-face interviews using retrospective weekly diary techniques and include details of all sexual sessions and alcohol use. The 819 reported sexual sessions with other men are divided into those that involved alcohol use (30.6%) and those that did not. RESULTS: Differences in the incidence of HIV risk behaviours between sexual sessions that involved alcohol use and those that did not are small, and none are statistically significant. Furthermore, for those men who engaged in sexual behaviour whilst under the influence of alcohol, the quantity of alcohol consumed had no effect on sexual behaviour. CONCLUSIONS: Among gay and bisexual men, sex under the influence of alcohol is no more likely to be unsafe than sex among men who have not consumed alcohol.

Alcohol Drinking↗
PubMed1993

Sex role separation in sexual diaries of homosexual men.

OBJECTIVE: To measure types of sex role prevalence in common and risk-related behaviours among gay men for modelling HIV transmission. DESIGN: Cohort study of 385 homosexually active men recording sexual diaries over 1-month periods. METHODS: Measures of incidence of behavioural sex roles for masturbation, fellatio, anal intercourse and anilingus by relationship type, derived from 1-month sexual diary data. RESULTS: Low behavioural role rigidity for masturbation and fellatio, but higher rigidity for anal intercourse and anilingus. Participants with no regular partner showed a relatively low frequency of anal intercourse, whereas those in closed relationships showed a high frequency. CONCLUSION: Although anal intercourse shows a certain degree of behavioural role rigidity, this rigidity is not large enough to conclude that gay men exclusively engage in either an active or a passive role. Typical rates for exclusive active and passive roles for anal intercourse during the month the diaries were recorded were in the range of 12-15%; the dual role was significantly higher.

Adolescent↗
PubMed1993

The testing of saliva samples for HIV-1 antibodies: reliability in a non-clinic setting.

AIMS: To assess the reliability of saliva samples as a means of testing for HIV-antibodies outside clinic settings. METHODS: Men taking part in a non-clinic longitudinal study of homosexually active men provided samples of saliva and blood. Sera were screened using a competitive ELISA (Wellcozyme) and positive sera were confirmed by an indirect ELISA (Abbott). Saliva samples were screened either using an IgG captive radioimmunoassay or an amplified ELISA. RESULTS: A total of 534 paired saliva and blood samples were tested. Overall sensitivity was 96.2% and specificity was 100%. None of the saliva tests were falsely positive for HIV-1 antibodies. CONCLUSIONS: HIV-1 saliva tests can reliably be used in a non-clinic or field setting. However, if results are to be given to respondents, it is necessary to offer adequate counselling and consider the mechanisms for referral and follow-up for those that are found to be HIV-1 antibody positive.

HIV Antibodies↗
PubMed1992

Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.

Chromatography, Gel↗
PubMed1992

The sexual behaviour of young gay men in England and Wales.

Much research has concluded that young gay men are at disproportionately higher risk of HIV infection through unsafe sexual behaviour. This paper is the first report on this particular group in the UK. As part of a larger cohort study, 111 young (less than 21) gay and bisexual men were interviewed about their sexual behaviour, knowledge of AIDS and HIV, particularly safer sexual practices and other factors. Three per cent had not had sex and 13% had not had anal intercourse with a man at the time of interview. Forty-five per cent had sexual experience with a woman and 34% had engaged in vaginal intercourse. The average age at first homosexual experience was just under 15 years and this is higher than other age groups in the cohort. Median number of male partners in the year before interview was 5, with 1 of whom anal intercourse occurred. Sexual repertoire was relatively restricted compared to older men. Condom use was slightly more consistent and levels of knowledge not significantly different. From these findings, the contention that young gay men are at higher risk of HIV infection through risky sexual behaviour is not supported.

Adolescent↗
PubMed1992

Maintenance of open gay relationships: some strategies for protection against HIV.

The role of sexual exclusivity (monogamy) in relation to HIV, and the use of rule making in non-exclusive (open) relationships, are discussed. Data from interviews with 387 homosexually active men are presented. The most common sexual/relational configuration amongst these men is that of open relationships. Sexual non-exclusivity was found to be associated with longer relationships, and a greater age difference between partners. The strategies some of these men are using to maintain sexually non-exclusive relationships are outlined. These rules pertain both to interpersonal dynamics and HIV prevention. Differing epidemiological significance of the rules and some implications for health education are discussed.

Adolescent↗