Biomedical subjects

J Kay

Publications and source records attributed to J Kay.

At least 19 recordsLinked to original sources

PubMed1992

Intrinsic activity of precursor forms of HIV-1 proteinase.

The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.

Amino Acid Sequence↗

PubMed1992

Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobichydrophobic- or -aromaticpro- cleavage sites.

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Kinetic parameters for the generation of endothelins-1,-2 and -3 by human cathepsin E.

The specific conversion of human endothelin (ET) precursors big ET-1, big ET-2 and big ET-3 into their respective ET by cathepsin E was examined. Comparable pH optima were obtained for ET-1 and ET-2 generation, whereas effective conversion of big ET-3 into ET-3 necessitated a lower pH value. Determination of kinetic parameters (Km, kcat.) for all three conversions indicated that the precursors were efficiently bound by cathepsin E. The significance of the values obtained for the catalytic-centre activities and the effect of a specific inhibitor are discussed.

Amino Acid Sequence↗

PubMed1992

Sequestration of cadmium and copper by recombinant rainbow trout and human metallothioneins and by chimeric (mermaid and fishman) proteins with interchanged domains.

A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.

Amino Acid Sequence↗

PubMed1992

Musculoskeletal manifestations in beta 2-microglobulin amyloidosis. Case discussion.

The cases presented illustrate some of the typical (case 1) and less common (case 2) clinical features of beta 2m amyloidosis. The accumulation of beta 2m amyloid in tissues is a potentially severe complication of dialysis-treated chronic renal failure. Beta 2m amyloidosis has been shown to have distinct clinical, radiologic, and pathologic features. The pathogenesis of this condition is not yet clearly understood, and recommendations for the clinical management of these patients at present are limited to recognition of the disease and symptomatic treatment. Further insights into the biology of this disease should lead to new strategies for prevention and treatment.

Aged↗

PubMed1992

Utility of high-resolution ultrasound for the diagnosis of dialysis-related amyloidosis.

OBJECTIVE: To evaluate the utility of real-time, high-resolution ultrasound of the shoulder in the diagnosis of dialysis-related amyloidosis. METHODS: We performed a case series study of 2 groups of patients seen at a referral-based clinic in a tertiary care hospital. The shoulders of 13 patients with normal renal function and of 38 patients receiving long-term hemodialysis were studied by real-time, high-resolution ultrasound. All hemodialysis patients were evaluated clinically for the presence of dialysis-related amyloidosis. Surgical specimens of joints were available for all 13 patients with normal renal function and for 17 of the 38 hemodialysis patients. These specimens were evaluated for the presence of beta 2-microglobulin (beta 2m) amyloid by Congo red and immunohistochemical staining. RESULTS: Two ultrasonographic findings were selectively observed in the dialysis patients with clinical and histologic evidence of beta 2m amyloid in comparison with patients with normal renal function and no evidence of amyloid: rotator cuffs greater than 8 mm in thickness and echogenic pads between muscle groups of the rotator cuff. The presence of at least 1 of these 2 findings corresponded to the presence of clinically and histologically evident beta 2m amyloid with a sensitivity of 79% and a specificity of 100%. When additional patients without surgical specimens for histologic confirmation of amyloidosis were included, the sensitivity of these 2 sonographic findings was 72% and the specificity was 97%. CONCLUSION: Real-time, high-resolution ultrasound is a relatively sensitive and highly specific noninvasive adjunct to the clinical diagnosis of beta 2m amyloidosis in patients receiving long-term hemodialysis.

Adult↗

PubMed1992

Antigen processing for presentation by class II major histocompatibility complex requires cleavage by cathepsin E.

Proteolytic degradation (processing) of antigen by antigen-presenting cells is a major regulatory step in the activation of a T lymphocyte immune response. However, the enzymes responsible for antigen processing remain largely undefined. In this study we show that cathepsin E, and not the ubiquitous lysosomal cathepsin D, is the major aspartic proteinase in a murine antigen-presenting cell line, A20. This enzyme is localized to a non-lysosomal compartment of the endosomal system in these cells. Functional studies using a highly specific inhibitor of cathepsin E show that this enzyme is essential for the processing of ovalbumin by this cell line. Thus, cathepsin E, whose function was hitherto unknown, may play a major role in antigen processing.

Animals↗

PubMed1992

Inhibition of aspartic proteinases by synthetic peptides derived from the propart region of human prorenin.

1. Five synthetic peptides which together spanned the propart segment of human prorenin were tested for their ability to interact with human renin, pepsin, gastricsin, cathepsin D, cathepsin E, calf chymosin and the aspartic proteinase from Endothia parasitica. 2. While two peptides showed no significant effect with any of the enzymes, a further two were cleaved by several enzymes. 3. Only one (corresponding to the 32P-43P residues in the propart sequence) acted as a weak competitive inhibitor of most of the enzymes.

Amino Acid Sequence↗

PubMed1992

Does letter-by-letter reading involve the spelling system?

Warrington and Shallice (Brain 103, 99-112, 1980) proposed that letter-by-letter readers can no longer access the visual word-form system, and read by using the spelling system 'in reverse'. Contrary to their suggestion, this paper presents the case of a letter-by-letter reader who appears to use quite different strategies in word reading and spelling. Reading and spelling are both impaired, but they appear to be impaired in different ways. Most strikingly, the patient's spelling errors consist largely of phonological regularizations--indeed there is little evidence that he has any lexical spelling knowledge available--whereas there are virtually no regularizations amongst his reading errors. These are largely visual paralexias and letter misidentifications. It is suggested that this pattern is more easily explained in terms of compensatory mechanisms that access the reading lexicon than by use of the spelling system 'in reverse'.

Adult↗

PubMed1992

Childhood migraine in general practice: clinical features and characteristics.

Previous studies have evaluated the clinical features and characteristics of migraine in selected children attending hospital clinics. There have, however, been no community studies on the prevalence and characteristics of migraine in children aged 3-11 years in British general practice. In this study, a total of 1,083 children (from 1,104 registered with a general practice) and their parents were interviewed. Possible migraine sufferers took part in an extended interview that assessed various characteristics of the patients and their attacks. The survey showed that migraine attacks started in infancy, and migrainous children were more likely to have mothers with migraine. Some clinical features were found to be age-dependent. Compared with similar hospital surveys there was a lower frequency of attacks recorded but the prevalence of aura was similar to that found in previous studies.

Age Factors↗

PubMed1992

Developmental surface dyslexia and dysgraphia: an orthographic processing impairment.

This study presents a detailed investigation of a young man in his early twenties who has suffered from a severe spelling impairment since childhood, and currently has a spelling age of only 9 years and 2 months. In contrast with the developmental phonological dyslexics reported by Campbell and Butterworth (1985) and Funnell and Davison (1989), his performance on tests of phonological awareness is good. In addition, he can read and spell non-words competently and, unlike normal 9-year-old children, virtually all of his spelling errors are phonologically appropriate. Further analysis of these errors reveals that he has knowledge of many of the different ways in which a given phoneme can be written, and that he uses phoneme-to-grapheme correspondences at the end of a word that are different from those he uses earlier in a word. However, he finds it difficult to spell words that contain uncommon phoneme-to-grapheme correspondences, which is compatible with the view that he has not developed an orthographic spelling lexicon. Although his oral reading of words is prompt and generally accurate, analysis of his lexical decision performance and the way that he defines homophones indicate that he does not have fully specified lexical entries available for reading either. We suggest that he suffers from a general orthographic processing deficit, and relies instead upon the combination of sub-lexical phonology and a lexicon that contains only partial information about the way in which words are spelt. This leads to reasonably competent reading, even of many irregular words, but produces very poor spelling. It is argued that qualitatively different types of developmental dyslexia do genuinely exist, but that reading impairments are likely to be much more pronounced in children who have a phonological rather than an orthographic processing deficit.

Adult↗

PubMed1992

The influence of isoflurane on the vascular reflex response to lung inflation in dogs.

Positive pressure ventilation can affect hemodynamic stability by neuroreflex-mediated activity. Inhalational anesthesia is known to attenuate the arterial baroreflex function; however, little information is known about the effect of volatile anesthetics on the lung inflation reflex. The influence of isoflurane on static lung inflation reflex-induced changes in venous capacitance and systemic resistance was investigated in dogs. After controlling carotid sinus pressure at 50 mmHg and initiating total cardiopulmonary bypass, the lungs were inflated to tracheal pressures of 10 and 20 mmHg. The systemic vascular resistance index (SVRI) decreased by 0.04 +/- 0.03 and 0.13 +/- 0.03 mmHg.kg.min.ml-1 during tracheal inflation pressures of 10 and 20 mmHg, respectively. There as an accompanying change in systemic vascular capacitance index (SVCI) by 1.0 +/- 0.65 and 3.3 +/- 0.82 ml.kg-1 during tracheal inflation pressures of 10 and 20 mmHg. The addition of isoflurane decreased the reflex vascular response to lung inflation in a dose-dependent manner. A concentration of 1 MAC isoflurane administered via the cardiopulmonary bypass machine attenuated the change in SVRI to tracheal inflation pressures of 10 and 20 mmHg by 75% and 67%, respectively. Isoflurane at 1 MAC also reduced the reflex capacitance response to tracheal pressures of 10 and 20 mmHg by 36% each. Lung inflation-induced changes in SVRI and SVCI were abolished at isoflurane concentrations of 2 MAC. We conclude that under the conditions of this study, 1 MAC isoflurane was shown to attenuate lung reflex-induced changes in SVRI and SVCI and that at higher isoflurane concentrations (2 MAC) these reflex-induced changes were not seen.

Animals↗

PubMed1992

Epidemiology of headache and childhood migraine in an urban general practice using Ad Hoc, Vahlquist and IHS criteria.

In a survey conducted by a UK urban general practice, 98.1 per cent (1083) of the children registered aged three to 11 years took part in an interview. Possible migraine subjects took part in an extended interview, the results of which were recorded using three different sets of diagnostic criteria: Vahlquist, Ad Hoc and International Headache Society. The prevalence of migraine ranged from 3.7 to 4.9 per cent, depending on the criteria used. The prevalence of migraine with aura (1.5 per cent) was similar by all criteria, whereas that for migraine without aura ranged from 2.2 to 3.4 per cent. The prevalence of migraine increased with age. The IHS criteria appeared less sensitive than the Ad Hoc for migraine without aura. Operational criteria using three attacks lasting longer than one hour may be more specific for the diagnosis of migraine in young children.

Age Factors↗

PubMed1992

Does a history of maternal migraine or depression predispose children to headache and stomach-ache?

The incidence of the life time history of both maternal migraine and depression were assessed in children with headache, migraine, recurrent abdominal pain and abdominal migraine. An epidemiological survey of 1,104 children registered with a general practice was undertaken. The incidence of maternal migraine and depression agreed with previous estimates. Children with migraine had a greater proportion of mothers with a history of migraine than those who experienced headache alone compared with controls. The survey showed that mothers with depression predisposed their children to headache but not specifically migraine. A history of maternal depression and migraine was significantly more common and proportionately higher in children with abdominal migraine and recurrent abdominal pain.

Abdominal Pain↗

PubMed1992

Substrate specificity and inhibitors of aspartic proteinases.

Aspartic proteinases of vertebrate, fungal and retroviral origin have been characterised by utilisation of chromogenic peptide substrates and synthetic inhibitors as probes. By this means, the molecular topography of sub-sites within the active site cleft of individual enzymes has been elucidated. With suitable selection of residues to occupy each sub-site, the design of effective inhibitors specifically targetted against individual aspartic proteinases has become feasible.

Amino Acid Sequence↗

PubMed1991

A plant metallothionein produced in E. coli.

A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of approximately 6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

Amino Acid Sequence↗

PubMed1991

Elucidation of cDNA sequences for metallothioneins from rainbow trout, stone loach and pike liver using the polymerase chain reaction.

Metallothionein cDNAs were generated from the livers of three fish species and amplified by PCR. Two distinct coding sequences (A and B) were elucidated for rainbow trout metallothioneins but single isoforms were encoded by genes isolated from the stone loach and pike. Different sized transcripts were observed both with stone loach and pike but these were accounted for by altered lengths of 3' untranslated regions.

Amino Acid Sequence↗

PubMed1991

Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase.

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.

Base Sequence↗