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Biomedical subjects

J Clarke

Publications and source records attributed to J Clarke.

At least 127 records · Page 7Linked to original sources
PubMed1998

HIV/AIDS patients' perspectives on adhering to regimens containing protease inhibitors.

OBJECTIVE: To gather qualitative data regarding HIV/AIDS patients' perspectives about HIV-1 protease inhibitors (PIs), and about their experiences taking and adhering to regimens containing PIs. DESIGN: Six focus groups of persons under care for HIV were conducted between September and November 1996 regarding participants' knowledge, awareness, experiences when taking, and adherence to antiretroviral regimens containing PIs. An identical discussion guide was used to facilitate all six groups. Focus group proceedings were audiotaped, transcribed, coded for themes, and analyzed qualitatively. SETTING: HIV/AIDS practices of three teaching hospitals and two community health centers. PATIENTS/PARTICIPANTS: Fifty-six patients with HIV disease: 28 men and 28 women. MEASUREMENTS AND MAIN RESULTS: Knowledge and positive impressions of PIs were prevalent among this diverse group of persons with HIV, and did not differ by race/ethnicity or gender. Most knew that these were new, potent medications for treating HIV/AIDS. Networks of persons with HIV and medical providers were the most important information sources. Those taking PIs were aware that adherence to the regimen is important, and most were using special strategies to maximize their own adherence, but expressed considerable frustration about the central role these medication regimens had assumed in their life. A subset who did not believe they would adhere to these regimens had declined treatment with them. Motivating factors for taking and adhering to these complex regimens were improving CD4 counts and viral loads and the patient-provider relationship. CONCLUSIONS: Among those with HIV/AIDS, awareness of PIs and their effectiveness is substantial, owing to the impact of informal networks and medical providers. This early positive "reputation" of PIs may enhance motivation for adherence. Those who are taking PIs invest substantial effort adhering to these complex regimens, but resent the need to make medications the focus of their lives.

Adult↗
PubMed1998

Binding of insulin-like growth factor (IGF)-binding protein-5 to smooth-muscle cell extracellular matrix is a major determinant of the cellular response to IGF-I.

Insulin-like growth factor-binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I's effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Animals↗
PubMed1998

Screening for cognitive impairment in general practice: toward a consensus.

We considered whether general practitioners should examine all older patients over a certain age for cognitive impairment in screening for early dementia. We invited presentations from key experts, selectively reviewed the literature, and developed a consensus statement. The efficacy of and benefits from unselective use of cognitive testing and informant questionnaires for detecting early dementia in older patients attending general practice are limited. Positive predictive values of cognitive screening for dementia are less than 50%, even for older patient populations. Higher values may be obtained by testing patients who have a relevant history of cognitive or functional decline. Whatever procedures are adopted for screening older general practice attenders for cognitive impairment or early dementia, investigation is still required into the relative merits of different health professionals performing the screening, the positive and negative effects on patients and their families, and the cost-benefit ratio. The majority view of workshop participants was that cognitive testing should occur for older patients when there is a reason to suspect dementia. Testing may occur in an individual considered to be at risk because of an informant history of cognitive or functional decline, clinical observation, or, sometimes, very old age. No single instrument for cognitive screening is suitable for global use. Screening programs must be supported by training and supplemented by education for professionals and families in management of dementia.

Age of Onset↗
PubMed1998

Maintaining safe oxygen levels in semiclosed underwater breathing apparatus.

Semiclosed circuit underwater breathing apparatus (UBAs) are far more conserving of limited gas supplies than either steady-flow, open circuit or demand, open circuit systems. Semiclosed systems recirculate most of the diver's breathing gases, while injecting only a small percentage of the total circuit flow with a fresh makeup supply. The fresh, injected gas supply delivers the necessary makeup to compensate for the oxygen that the diver consumes during each pass through the lungs. This gas-conserving feature makes the utility for semiclosed UBAs for long-duration, deep diving applications more practical than either of the open circuit designs. However, although more gas conservative, these systems maintain variable oxygen levels during a dive that can fluctuate widely with changes in the diver's activity level, depth of the dive, makeup gas composition, or variations in the total circuit volume. It is essential that the oxygen levels in the circuit always remain within physiologically safe limits; neither too low to avoid the onset of hypoxia, nor too high to risk oxygen toxicity. This article presents an analytical assessment of the oxygen levels in semiclosed UBAs. This model allows the designers of semiclosed circuits to predict the effects of UBA circuit volumes, makeup gas compositions, injection rates for the makeup gas, diver depths, and initial circuit oxygen levels on the time-dependent levels of oxygen in these circuits. By using this analytical tool, judicious selection of these design variables can result in the most efficient utilization of the fresh gas supply carried by the diver, and ensure the safe operation of these UBA designs.

Air Conditioning↗
PubMed1997

Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: description of the folding pathway.

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of 10(4) structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix alpha1 (residues 13-17) and in helix alpha2 as well as a turn and nonnative hydrophobic clustering between beta3 and beta4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in alpha1 and the C-terminal portion of the beta-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from phi value analysis and NMR.

Amino Acid Sequence↗
PubMed1997

Folding and stability of a fibronectin type III domain of human tenascin.

The folding of an isolated fibronectin type III domain of human tenascin, a large extra-cellular matrix protein, has been characterised. The isolated module, which has no disulphide bonds, can be reversibly unfolded by chemical denaturant and temperature. Equilibrium unfolding, measured using a number of different probes, fits to a two-state transition, with consistent measures of DeltaGH2OD-N. Folding and refolding rate constants have been determined over a range of denaturant concentrations. The refolding kinetics are bi-phasic, and in the transition region the slow phase dominates refolding kinetics. Outside the transition region the folding of the fast-folding species fits to a two-state model. There is no evidence for significant accumulation of partially folded intermediates.

Cloning, Molecular↗
PubMed1997

Hepsin, a cell surface serine protease identified in hepatoma cells, is overexpressed in ovarian cancer.

Extracellular proteases mediate the digestion of neighboring extracellular matrix components in initial tumor growth, allow shedding or desquamation of tumor cells into the surrounding environment, provide the basis for invasion of basement membranes in target metastatic organs, and are required for release and activation of many growth and angiogenic factors. We identified overexpression of the serine protease hepsin gene in ovarian carcinomas and investigated the expression of this gene in 44 ovarian tumors (12 low malignant potential tumors and 32 carcinomas) and 10 normal ovaries. Quantitative PCR was used to determine the relative expression of hepsin compared to that of beta-tubulin. The mRNA expression levels of hepsin were significantly elevated in 7 of 12 low malignant potential tumors and in 27 of 32 carcinomas. On Northern blot analysis, the hepsin transcript was abundant in carcinoma but was almost never expressed in normal adult tissue, including normal ovary. Our results suggest that hepsin is frequently overexpressed in ovarian tumors and therefore may be a candidate protease in the invasive process and growth capacity of ovarian tumor cells.

Carcinoma, Hepatocellular↗
PubMed1997

Thermodynamics of denaturation of mutants of barnase with disulfide crosslinks.

We have measured the effects of disulfide crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At first sight, the data are consistent with the hypothesis that disulfide crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disulfide crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for denaturation (delta C(p)d), determined from the slope of the enthalpy of denaturation versus temperature, are not confined to mutants with disulfide crosslinks. The value of delta C(p)d is lower in four stabilised mutants than in wild-type barnase, irrespective of the presence of a disulfide crosslink, while the delta C(p)d remains unchanged in a destabilised mutant containing a disulfide. The variation in delta C(p)d may result from an inherent temperature-dependence of delta C(p)d, since it is measured for each mutant over a different temperature range. The thermodynamics of denaturation of the disulfide mutant with a crosslink between positions 70 and 92 change anomalously with pH but in a similar way to that of the D93N mutant of barnase, which lacks the D93-R69 salt-bridge present in the wild-type. This finding confirms initial observations in the X-ray structure of this disulfide mutant that the salt-bridge has been disrupted by the introduced crosslink.

Bacterial Proteins↗
PubMed1997

Expression of the farnesyltransferase beta-subunit gene in human ovarian carcinoma: correlation to K-ras mutation.

The ras signaling protein requires a posttranslational modification to localize it to the inner surface of plasma membrane. In this state it can behave as a signal transduction mediator. Farnesyltransferase plays an important role in this posttranslational processing of ras by attaching a farnesyl group to the cysteine of the ras C-terminal tetrapeptide. In this study, we investigated the relationship of K-ras expression and mutation with farnesyltransferase beta-subunit expression in 20 ovarian tumors (17 carcinomas and 3 low malignant potential tumors) and 4 normal ovaries. The expression level of mRNA was determined by using quantitative PCR and mutation analysis was performed by direct cDNA sequencing. K-ras mutations were found in 1 of 3 low malignant potential tumors and in 4 of 17 carcinoma cases. K-ras mRNA overexpression was found in 1 of 3 low malignant potential tumors (one with mutated ras) and in only 1 of 17 carcinoma cases. Farnesyltransferase beta-subunit mRNA overexpression was found in 2 of 3 low malignant potential tumors and in 7 of 17 carcinoma cases. Interestingly, all K-ras mutation cases showed farnesyltransferase beta-subunit overexpression. These findings suggest that there may be a direct relationship between K-ras (ras dysfunction) mutation and expression of farnesyltransferase beta-subunit gene.

Alkyl and Aryl Transferases↗
PubMed1997

Hydrogen exchange at equilibrium: a short cut for analysing protein-folding pathways?

Hydrogen exchange is an attractive method for observing small populations of partly unfolded states of proteins at equilibrium. It has been suggested that these represent folding intermediates so that hydrogen exchange can offer a short cut for studying protein-folding pathways. This cannot work in theory because it is not possible to tell whether they are intermediates or side reactions. Experimental studies of barnase and chymotrypsin inhibitor 2 show that there is no obvious relationship between hydrogen exchange at equilibrium and their folding pathways.

Hydrogen↗
PubMed1997

Cytochrome P450 1A-like proteins expressed in the islets of Langerhans and altered pancreatic beta-cell secretory responsiveness.

1. The cytochrome P450 (CYP) mixed-function oxidase system is widely distributed in body tissues and plays a key role in the metabolism of endogenous and exogenous compounds. Little attention has been paid to the expression of the system in the islets of Langerhans. The current study has examined the expression and potential role of the CYP1A family within the islets of Langerhans of control and 3-methylcholanthrene (3-MC)-induced Wistar rats. 2. CYP1A expression within pancreatic slices and islets from 3-MC-induced and control rats demonstrated that CYP1A-like protein levels were induced by 3-MC pretreatment (25 mg kg-1 day-1; i.p. for 3 days). 3. Effects of 3-MC-induction on beta-cell secretory responsiveness were investigated by use of rat collagenase-isolated islets. Insulin release from control islets incubated with 3 mM glucose (basal) was 1.4 +/- 0.2 ng/islet h-1 (mean +/- s.e.mean, n = 7). Incubation with 16.7 mM glucose, 25 mM KCl, 100 microM arachidonic acid, or 100 microM carbachol caused a 4.4, 7.0, 4.0 and 4.2 fold, respectively, increase in insulin release (P < 0.001). Forskolin (2 microM), or phorbol 12-myristic 13-acetate (10 nM) potentiated glucose-stimulated insulin release 1.2 and 1.6 fold (P < 0.01) whereas adenalin (1 microM) caused a 76% inhibition (P < 0.01). 4. Islets from 3-MC pretreated animals displayed similar responsiveness to all agents tested except arachidonic acid, carbachol and forskolin. Insulin release in response to arachidonic acid and carbachol was enhanced 5.2 and 5.0 fold, respectively, by 3-MC pretreatment (P < 0.001 compared to control islets incubated with 3 mM glucose); the effect of forskolin on insulin output was significantly decreased (20%; P < 0.01) compared to control islets. 5. 3-MC pretreatment did not cause any significant differences in food intake, plasma glucose or total islet insulin content. Incubation of islets with 3-MC in vitro (1 microM - 10 mM) did not affect basal or glucose-stimulated insulin release. 6. These data suggest that CYP1A-like protein expression within the pancreatic islets of Langerhans is inducible and may have a role in the alteration of pancreatic beta-cell secretory responsiveness.

Animals↗