Cathepsin E. The best laid plans of mice and men.
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Amino Acid Sequence↗
Biomedical subjects
J Kay
Publications and source records attributed to J Kay.
Fractal dimension and approximate entropy of heart period and heart rate: awake versus sleep differences and methodological issues.
1. Investigations that assess cardiac autonomic function include non-linear techniques such as fractal dimension and approximate entropy in addition to the common time and frequency domain measures of both heart period and heart rate. This article evaluates the differences in using heart rate versus heart period to estimate fractal dimensions and approximate entropies of these time series.2. Twenty-four-hour ECG was recorded in 23 normal subjects using Holter records. Time series of heart rate and heart period were analysed using fractal dimensions, approximate entropies and spectral analysis for the quantification of absolute and relative heart period variability in bands of ultra low (<0.0033 Hz), very low (0. 0033-0.04 Hz), low (0.04-0.15 Hz) and high (0.15-0.5 Hz) frequency.3. Linear detrending of the time series did not significantly change the fractal dimension or approximate entropy values. We found significant differences in the analyses using heart rate versus heart period between waking up and sleep conditions for fractal dimensions, approximate entropies and absolute spectral powers, especially for the power in the band of 0.0033-0.5 Hz. Log transformation of the data revealed identical fractal dimension values for both heart rate and heart period. Mean heart period correlated significantly better with fractal dimensions and approximate entropies of heart period than did corresponding heart rate measures.4. Studies using heart period measures should take the effect of mean heart period into account even for the analyses of fractal dimension and approximate entropy. As the sleep-awake differences in fractal dimensions and approximate entropies are different between heart rate and heart period, the results should be interpreted accordingly.
Adult↗
Natural processing sites for human cathepsin E and cathepsin D in tetanus toxin: implications for T cell epitope generation.
Cathepsin E is an aspartic proteinase that has been implicated frequently in Ag processing for presentation on class II MHC molecules, but no information exists on its cleavage specificity within Ags in relation to known T cell epitopes. We have analyzed the processing by cathepsin E of a large C-terminal domain of tetanus toxin (residues 872-1315), and we have compared the processing products with those liberated by cathepsin D, a related aspartic proteinase also thought to be involved in class II MHC-restricted Ag processing. Processing products were analyzed by N-terminal Edman degradation and mass spectrometry following reverse-phase HPLC separation of peptides. A total of 28 cleavage sites was identified, 11 of which were recognized by both cathepsins E and D. Most, although not all, sites were between pairs of hydrophobic residues and were located within the 200-amino-acid C terminal region known to contain several human T cell epitopes. Previously described T cell epitopes, for example, between residues 1273 and 1284, were flanked by cathepsin E and D cleavage sites. These data are consistent with an important role for cathepsins E and/or D in Ag processing in the human immune system.
Amino Acid Sequence↗
Fine structure of the murine leptin receptor gene: splice site suppression is required to form two alternatively spliced transcripts.
The fine structure of the murine leptin receptor gene (Lepr) is described. Duplicated ligand binding domains (conserved among cytokine receptors) are found in eight exons (coding exons 3 to 6 and 8 to 11). Thus, it is possible that a single leptin receptor molecule could have two functional ligand binding domains. The transmembrane region of Lepr is in coding exon 16 while the juxtamembrane JAK docking site is in coding exon 17. For all membrane-bound forms, the transcript must include 17 invariant exons and 1 alternatively spliced 3' terminal exon. The transcript encoding the soluble receptor (Re) includes 14 coding exons and an alternatively spliced 3' terminal exon. We have identified two splice variants (Rc and Re) for which there are no intervening sequences between the two final exons. This unusual juxtaposition of exons requires that splice donor sites at the 5' end of the respective terminal exons be ignored in the production of these splice variants. We suggest that splice site suppression is responsible for the formation of two of the alternatively spliced forms of the mouse Lepr gene. The juxtaposition of two coding exons separated by a consensus splice donor sequence is the structural substrate for this mode of alternative splicing. We present evidence that the Rc form is expressed in human tissues while the Re form, the soluble receptor, is not expressed.
Alternative Splicing↗
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↗
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↗
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↗
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↗
Effect of age on long-term heart rate variability.
OBJECTIVE: Previous studies on short-term time series of heart rage suggest an inverse relationship between age and spectral powers of heart rate variability in various frequency bands. In this study, we examined the relationship between age (6-61 years) and long-term heart rate variability. METHODS: We obtained 24-h Holter ECG in 33 healthy human subjects (11 children and 22 adults). The heart rate data were analyzed by using spectral analysis and fractal dimensions of the time series. RESULTS: We found a significant negative correlation between age and very low frequency (VLF, 0.0033-0.04 Hz), low frequency (LF, 0.04-0.15 Hz) and high frequency (HF, 0.15-0.5 Hz) powers and fractal dimensions during awake as well as sleep periods, and a positive correlation between age and LF/HF ratios. Age and ultra-low frequency (ULF, < 0.0033 Hz) were modestly and negatively correlated only during the awake period. CONCLUSIONS: Sleep ULF power is not significantly affected by age, whereas VLF, LF and HF powers and fractal dimensions of heart rate significantly decrease with age during awake as well as sleep periods.
Adolescent↗
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↗
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↗
The course of alcohol withdrawal in a general hospital.
We conducted an observational study of 539 episodes of alcohol withdrawal in a general hospital, to determine the natural history, the incidences of seizures, hallucinations and delirium, and the risk factors for these events. The reaction began soon after arrival, at a median time of 5 h, and resolved at a median time of 22 h. Patients with a blood alcohol level of zero were in withdrawal on arrival, and only four patients had reactions lasting 120 h or longer. Complications were observed in 113 patients (21%) during the admission. Seizures occurred on arrival, hallucinations usually in the first 24 h and delirium in the first 48 h. No mortality was associated with alcohol withdrawal itself, but complications did extend length of stay by a median of 4 days, with delirium contributing most to the increase. Patients over 70 years of age or admitted with seizures had an increased risk of complication, but the greatest risk was associated with a delay in assessment of > 24 h. We conclude that in general hospitals, the alcohol withdrawal reaction becomes established very early, and detection and monitoring of patients within the first 24 h is the most important element in management.
Adult↗
Characterization of the proteinase specified by varicella-zoster virus gene 33.
Varicella-zoster virus (VZV) genes 33 and 33.5 are predicted to encode the VZV proteinase and its substrate (the assembly protein) respectively. These genes were expressed in insect cells using recombinant baculovirus and it was confirmed that gene 33 encodes a proteinase capable of autoproteolytic processing at two positions. When VZV gene 33.5 was co-expressed with the VZV proteinase, processing of the VZV33.5 gene product was observed. A polyclonal antiserum to the VZV assembly protein domain highlighted a set of proteins in VZV infected HEL cells identical to those identified in insect cells expressing VZV genes 33 and 33.5. To facilitate further characterization of the VZV proteinase the enzyme was purified by affinity chromatography from an E. coli expression system and in vitro activity was observed.
Animals↗
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↗
Disputing psychiatry's redefinition.
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Career Choice↗
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↗
Vital signs: spotting liver disease in babies and children.
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Child↗
Brief psychodynamic psychotherapies. Past, present, and future challenges.
The future educational and research needs of brief psychodynamic psychotherapy are discussed. Specific shortcomings of current education and training in brief dynamic therapies are elucidated. Many of these are relevant to psychotherapy instruction in graduate medical education in nondynamic treatments as well. Educational and technological recommendations are suggested to remedy these shortcomings.
Forecasting↗