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Biomedical subjects

J Kay

Publications and source records attributed to J Kay.

At least 19 recordsLinked to original sources
PubMed1997

Cloning, expression and characterisation of murine procathepsin E.

The cDNA encoding murine procathepsin E was isolated and sequenced and recombinant enzyme was produced in Escherichia coli. The activity of the purified recombinant mouse cathepsin E was characterised quantitatively using two synthetic peptide substrates and naturally occurring inhibitors. The majority of the recombinant enzyme was present as a homodimer (Mr approximately 80) in which the two monomers were linked by an intermolecular disulfide bond. By analogy to previous studies with human cathepsin E, this is most likely a consequence of the presence of a unique cysteine residue near the N-terminus of the mature proteinase. The availability of (i) recombinant murine enzyme in reasonable quantities and (ii) a full-length cDNA now enables structural investigations and attempts to generate 'knock-out' mice deficient in this important aspartic proteinase to be undertaken.

Amino Acid Sequence↗
PubMed1997

Escape mutants of HIV-1 proteinase: enzymic efficiency and susceptibility to inhibition.

Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases containing mutations of single residues (e.g., G48V, V82F, I84V and L90M) were purified and their catalytic efficiencies relative to that of wild-type proteinase were examined using a polyprotein (recombinant HIV-1 gag) substrate and several series of synthetic peptides based on the -Hydrophobic * Hydrophobic-, -Aromatic * Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic * Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The K(i) values determined for one inhibitor (Ro31-8959) showed that its potency towards the V82F, L90M, I84V and G48V mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor.

Anti-HIV Agents↗
PubMed1997

Expression and characterisation of plasmepsin I from Plasmodium falciparum.

Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5-10-fold lower k(cat)/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.

Animals↗
PubMed1997

Changes in HIV-related behaviors among heterosexual alcoholics following addiction treatment.

In order to measure changes in HIV-related behaviors among heterosexual alcoholics following treatment, we conducted a prospective cohort study of 700 self-identified alcoholics recruited from five public alcohol treatment centers, all of which included HIV risk-reduction counseling. Respondents underwent an HIV antibody test and interviewer-administered questionnaire at entry to alcohol treatment and after a mean of 13 months later. Compared to baseline, at follow-up there was an overall 26% reduction in having sex with an injection-drug-using partner (23% versus 32%, P < .001) and a 58% reduction in the use of injection drugs (15% versus 37%, P < .001), along with smaller improvements in other behaviors. Respondents also showed a 77% improvement in consistent condom use with multiple sexual partners (35% versus 20%, P < .01) and a 23% improvement in partner screening (71% versus 57%, P < .001). Respondents who remained abstinent showed substantially greater improvement than those who continued to drink.

Adult↗
PubMed1997

Monitoring fluid absorption using 1% ethanol-tagged glycine during operative hysteroscopy.

STUDY OBJECTIVE: To assess the value of expired breath ethanol as a marker of irrigating fluid absorption during hysteroscopic surgery using 1% ethanol-tagged 1.5% glycine. DESIGN: Prospective analysis. SETTING: Endoscopy training center of a university hospital. PATIENTS: Forty-eight women undergoing major hysteroscopic surgery for menorrhagia (40 transcervical endometrial resections, 8 rollerball endometrial ablations). INTERVENTIONS: Expired breath ethanol and venous blood samples were taken before and at 10-minute intervals during surgery. Volumetric absorption of irrigating fluid was checked at the same time. MEASUREMENTS AND MAIN RESULTS: Expired breath ethanol concentration, serum ethanol, several biochemical variables, and volume of absorbed irrigating fluid (direct and indirect) were measured. There was a linear positive correlation (r = 0.86, p <0.001) between direct vascular absorption of the irrigating fluid and expired breath ethanol concentration. Prediction can be given with 95% confidence that if the alcolmeter reading is below 0.45%, the volume of irrigating fluid absorbed is below 2000 ml. No significant correlation was seen between expired breath ethanol and indirect fluid absorption. CONCLUSIONS: As it is not possible to distinguish direct and indirect fluid absorption during hysteroscopic surgery, measuring expired breath ethanol is insufficient to assess overall fluid balance, and continuous volumetric assessment is still required.

Absorption↗
PubMed1997

Interaction between beta 2-microglobulin and advanced glycation end products in the development of dialysis related-amyloidosis.

Dialysis related amyloidosis (DRA) is a progressive debilitating complication of long-term dialysis. beta 2-microglobulin (beta 2m) amyloid deposition occurs preferentially in older patients and initially is located in collagen-rich osteo-articular tissues. Since an age-dependent increase in the formation of advanced glycation end products (AGE) has been observed in collagen-containing structures, we hypothesized that AGE-modified beta 2m in the amyloid of DRA may be formed locally in osteo-articular structures as a subsequent event of its binding to collagen-AGE. Based on this hypothesis, we investigated the binding between beta 2m and AGE-modified collagen (collagen-AGE) in vitro. Significantly larger amounts of human beta 2m were bound to types I to IV of immobilized collagen-AGE than to unmodified collagens (P < 0.0001). The quantity of beta 2m bound to collagen-AGE was dependent on the concentrations of both beta 2m and of AGE contained in collagen (P < 0.01). Unmodified beta 2m was more avidly bound to collagen-AGE or collagen in comparison to AGE-modified beta 2m (P < 0.0001). beta 2m bound to collagen-AGE could be modified further by nonenzymatic glycosylation during three weeks of incubation with physiologic concentrations of glucose. Similar processes in vivo may be important in the pathobiology of DRA.

Amyloidosis↗
PubMed1997

Analysis of 24-hour ECG in patients with panic disorder.

Previous evidence suggests an increased cardiovascular morbidity in patients with panic disorder. In this study, we compared 24-hour ECG in patients with panic disorder (n = 22; age: 36.1 +/- 7.6 years) and healthy controls (n = 21; age: 34.6 +/- 10.0 years). The QTc intervals during the day or night were not significantly different between patients and controls. Ventricular ectopic beats were also not significantly different between the two groups. These results do not suggest any overt cardiac arrhythmias in this age group of patients with panic disorder.

Adult↗
PubMed1997

The EM algorithm in medical imaging.

This article outlines the statistical developments that have taken place in the use of the EM algorithm in emission and transmission tomography during the past decade or so. We discuss the statistical aspects of the modelling of the projection data for both the emission and transmission cases and define the relevant probability models. This leads to the use of the method of maximum likelihood as a means of estimating the relevant unknown parameters within a given region of a patient's body and to the use of the EM algorithm to compute the reconstruction. Various different types of EM algorithm are discussed, including the SAGE algorithms of Fessler and Hero. The limitations of the EM algorithm, per se, are covered and the need for regularization is stressed. A number of different methods for penalizing the likelihood are described and a number of algorithms for the computation of the penalized EM reconstruction are discussed.

Algorithms↗
PubMed1996

Expression, characterisation and mutagenesis of the aspartic proteinase from equine infectious anaemia virus.

The gene encoding the proteinase from equine infectious anaemia virus (EIAV) was cloned and expressed in Escherichia coli. The recombinant EIAV proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (HIV) origin with an efficiency that can approach that exhibited by HIV proteinase. EIAV proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of HIV-1 proteinase, including those which have been licenced as anti-AIDS drugs. In this respect, EIAV proteinase behaves like an extreme case of a drug-resistant mutant of HIV-1 proteinase that has arisen under selective drug pressure. Only one potent inhibitor (HBY-793) of HIV-1 proteinase showed comparable efficiency against the EIAV enzyme; the compounds A-77003 and A-76889, which differ only in their stereochemistry and which are otherwise structurally identical to HBY-793 from residues P2 to P2' [nomenclature of Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162], were not effective inhibitors of EIAV proteinase. Mutant forms of EIAV proteinase (Thr30-->Asp and Ile54-->Gly) were generated and their ability to interact with substrates and inhibitors was characterised. HBY-793 inhibited [Gly54]proteinase as effectively as the wild-type proteinase but was tenfold less potent against [Asp30]proteinase. Data interpretations are presented, based on the structure solved for the complex between HBY-793 and EIAV [Gly54]proteinase [Gustchina A., Kervinen, J., Powell, D. J., Zdanov, A., Kay, J. & Wlodawer, A. (1996) Protein Sci. 5, 1453-1465].

Amino Acid Sequence↗
PubMed1996

Regulation of cathepsin E expression during human B cell differentiation in vitro.

Cathepsin E is an aspartic proteinase which has been implicated in antigen processing in the class II major histocompatibility complex pathway. In this study we show that cathepsin E, measured at both the protein and message level, is up-regulated late in human B cell activation. The implications of this observation in terms of cathepsin E function are discussed.

Adolescent↗
PubMed1996

Structure of equine infectious anemia virus proteinase complexed with an inhibitor.

Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second alpha-helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate-binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme-ligand interactions at the periphery of the active site as well.

Amino Acid Sequence↗
PubMed1996

Reading speed in pure alexia.

This study investigated possible causes of differences in reading speed between two alexic patients who read words letter by letter. As both patients appeared to rely on serial left-to-right processing of letters within words, the difference in reading speed did not seem to be related to any differences in the extent to which the patients could recognize letters in words in parallel or 'ends-in'. Differences in reading speed also seemed to be unrelated to the patients ability to identify individual letters since their letter recognition accuracy was very similar. Furthermore, although patient PD was significantly slower at reading words aloud than patient DC, PD was in fact significantly quicker than DC on a test that has previously been used to assess letter recognition skills in letter-by-letter readers. It is therefore concluded that PD reads words more slowly because of an additional impairment at the level of the word form system. The results therefore reinforce the distinction between Type 1 and Type 2 letter-by-letter readers that was first drawn by Patterson and Kay.

Adult↗
PubMed1996

The Safety of Ethanol-Tagged Glycine for Monitoring Fluid Absorption During Hysteroscopy

We assessed the value of expired breath ethanol as a marker of 1% ethanol-tagged 1.5% glycine irrigating fluid absorption during hysteroscopic surgery in a prospective analysis of 48 women. Expired breath ethanol and venous blood samples were taken before and at 10-minute intervals during surgery. Volumetric absorption of irrigating fluid was checked at the same time. There was a linear positive correlation (r = 0.86, p <0.001) between direct absorption of the irrigating fluid and expired breath ethanol concentration. Prediction can be given with 95% confidence that if the alcohol meter reading is below 0.45 mg/ml, the volume of irrigating fluid absorbed is below 2000 ml. There was no significant correlation between expired breath ethanol and indirect fluid absorption. As it is not possible to distinguish direct and indirect fluid absorption during hysteroscopic surgery, the measurement of expired breath ethanol is insufficient to assess overall fluid balance, and continuous volumetric assessment is still required.

Journal Article↗
PubMed1996

Cloning, expression and characterization of the proteinase from human herpesvirus 6.

After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.

Amino Acid Sequence↗
PubMed1996

Performance characteristics of a dialysis-related amyloidosis questionnaire.

To evaluate the effects of beta-2 microglobulin amyloidosis on functional status, a 19-item self-administered questionnaire exploring two major domains-symptoms and disability-was developed as part of this study. Fifteen patients with dialysis-related amyloidosis (DRA) were identified and compared with 15 age-matched control subjects who had been on hemodialysis for less than 24 months. Demographic data, Charlson comorbidity scores, and other clinical and laboratory variables were recorded. Total dialysis-related amyloidosis questionnaire (DRAQ) score (52.1 +/- 16.3 versus 24.4 +/- 6.2, P < 0.0001) and the scores of the symptom (24.5 +/- 7.0 versus 11.5 +/- 2.3, P < 0.0001) and disability (27.5 +/- 10.8 versus 12.9 +/- 4.0, P < 0.0001) subscales were markedly increased among patients with DRA. Baseline characteristics among case and control subjects were similar, except for serum creatinine concentration, which tended to be lower among patients with DRA (8.9 +/- 2.6 versus 11.5 +/- 2.3 mg/dL, P = 0.07). Instrument reliability and internal consistency were high. With a total DRAQ score of 30 or more, the sensitivity and specificity of the instrument were 93% and 80%, respectively. The DRAQ is a reliable, internally consistent, and valid instrument that may be suitable for population screening and clinical practice.

Activities of Daily Living↗
PubMed1996

New challenges to the faculty in the education of psychiatrists.

Significant social and economic pressures on academic medicine to become more cost conscious and scientifically accountable have led to new tensions in psychiatric residency training. Failure to distinguish between changes in practice derived from scientific advances and those derived from other sources has major implications for the education of future psychiatrists. After providing a conceptual framework for understanding current forces that affect residency training, the author proposes a new role for the psychiatric faculty, who should protect the curriculum and the trainee from shortsighted demands that could lower the quality of residency education and make psychiatry a less attractive specialty.

Career Choice↗