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Biomedical subjects

W Ashraf

Publications and source records attributed to W Ashraf.

At least 19 recordsLinked to original sources
PubMed2001

The immunological dissection of the Escherichia coli molecular chaperone DnaJ.

In Escherichia coli, one of the main molecular chaperones is DnaJ (hsp40), which mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes. DnaJ is characterised by the presence of four distinct domains: the J-domain, glycine/phenylalanine-rich (G/F), cysteine-rich (Zn-finger) and C-terminal regions. Truncated DnaJ polypeptides (DnaJ 1-108, DnaJ Delta1-108, DnaJ Delta1-199) representing these domains were over-produced and used as a source of immunogens for the generation of sequence-specific polyclonal antibodies. Epitope mapping was achieved by Western blotting, which demonstrated the presence of antibodies directed against these domains. These characterised affinity-purified antibodies were then used to assess the role of DnaJ in the protection of firefly luciferase from irreversible heat-inactivation. In this study we have demonstrated the involvement of J-, G/F and Zn-finger domains in the protection of luciferase from heat-inactivation. The C-terminal region had only partial involvement in luciferase protection.

Amino Acid Motifs↗
PubMed1998

Purification and functional analysis of the DnaK homologue from Prevotella intermedia OMZ 326.

This study examined heat shock proteins (hsps) of the periodontal pathogen Prevotella intermedia and the closely related species, Prevotella nigrescens and Prevotella corporis. After heat shock at 45 degrees C for 5 min, cell-free extracts were analysed by SDS-PAGE and Western blotting with polyclonal antibodies against Escherichia coli hsps. P. intermedia, P. nigrescens and P. corporis all expressed a DnaK homologue. The P. nigrescens DnaK was of a similar molecular mass to E. coli DnaK (70 kDa), whilst those of P. intermedia and P. corporis were approximately 69 kDa. DnaJ homologues were expressed in each species; however, no homologue of GrpE was detected. P. intermedia DnaK was purified to homogeneity by ion-exchange and affinity-chromatography, and was shown to restore activity of denatured luciferase. This molecular chaperone activity was enhanced by E. coli DnaJ and GrpE which are components of the Hsp70 molecular chaperone machine.

Antibodies, Bacterial↗
PubMed1998

Physiological consequences of the over-production of E. coli truncated molecular chaperone DnaJ.

In Escherichia coli one of the main molecular chaperones is DnaJ (hsp40) which mediates in a variety of highly conserved cellular process including protein folding reactions and assembly/disassembly of protein complexes. In this study we have investigated the toxicity of the over-production of DnaJ and two truncated polypeptides by examining growth rates, cell morphology and cell viability. Full-length DnaJ (1-375 amino acids) and truncated polypeptides, corresponding to the last 176 (containing the substrate binding domain) and 266 (containing the zinc finger-like domain) amino acids of the C-terminus of DnaJ, DnaJ delta 1-199 and delta 1-108 respectively, were over-produced via IPTG induction. High levels of synthesis were determined by SDS-PAGE and Western blotting using anti-DnaJ antibodies. The over-production of full-length DnaJ resulted in a low degree of filamentation and a decrease in cell viability. However, over-production of DnaJ truncated polypeptides, especially DnaJ delta 1-108, was bactericidal and resulted in a loss of viability and defective septation.

Escherichia coli↗
PubMed1997

Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum.

A member of the AAA family of Mg2(+)-ATPases from the archaeon Thermoplasma acidophilum has been cloned and expressed in Escherichia coli. The protein, VCP-like ATPase of Thermoplasma acidophilum (VAT), is a homologue of SAV from Sulfolobus acidocaldarius and CdcH of Halobacterium salinarium, and belongs to the CDC48/VCP/p97 subfamily. The deduced product of the vat gene is 745 residues long (Mr 83,000), which has an optimal Mg2(+)-ATPase activity at 70 degrees C. Electron microscopy shows the purified protein to form single and double homo-hexameric rings. Although the symmetry is different, the appearance of the complexes formed of two rings resembles the 20S proteasome and Hsp60/GroEL.

Adenosine Triphosphatases↗
PubMed1997

Constipation in Parkinson's disease: objective assessment and response to psyllium.

We evaluated the reliability of patient history and the effect of psyllium on symptoms and colorectal function in 12 patients with Parkinson's disease (PD) and constipation. In all but two, constipation anteceded the development of parkinsonian symptoms. A comparison with prospectively obtained stool diaries confirmed the patients' reported constipation in 7 of the 12 patients. Those patients with confirmed constipation had lower stool weights and reported more straining at stool. Measures of colonic and anorectal function were similar in those who were truly constipated and those who were not. Among those PD subjects with confirmed constipation, psyllium increased stool frequency and weight but did not alter colonic transit or anorectal function. We conclude that prospectively obtained stool diaries should be employed to confirm constipation in PD and that psyllium produces both subjective and objective improvements in constipation related to PD.

Aged↗
PubMed1996

An examination of the reliability of reported stool frequency in the diagnosis of idiopathic constipation.

OBJECTIVES: To investigate the reliability of reported stool frequency in the diagnosis of constipation. METHODS: Our criterion for the diagnosis of idiopathic constipation was < or = 3 stools per week for > or = 6 months. Subjects who believed that they met this criterion were invited to participate. Daily stool frequency was recorded over a 4-wk period, and all stools were weighed during the final week. A colon transit study and anorectal manometry were performed at the beginning and at the end of the study. RESULTS: On review of stool diaries from 45 subjects who described chronic constipation and who completed the 4-wk study, only 22 (49%) actually satisfied our criterion for idiopathic constipation. The remaining 23 (51%) subjects, although describing constipation, had, on average, 6 stools/wk. A history of psychiatric illness was 5 times more frequent among those whose bowel symptoms correlated poorly with objective measures. Measures of difficult defecation were similar in the two groups. Mean colon transit time was significantly longer and correlated closely with stool weight only in subjects who truly were constipated (p < 0.05). Anorectal manometry was not helpful in discriminating between those who satisfied our criterion for constipation and those who did not. CONCLUSIONS: Defining constipation on the basis of a patient's reported stool frequency may prove misleading; the diagnosis of idiopathic constipation should be supported by the use of stool diaries and a colon transit study.

Adult↗
PubMed1995

Dopaminergic defect of enteric nervous system in Parkinson's disease patients with chronic constipation.

Clinical studies suggest that gut disorders are common in Parkinson's disease, but the morphological basis is unknown. Depletion of dopamine-containing neurons in the central nervous system is a basic defect in Parkinson's disease. We compared colonic tissue from 11 patients with advanced Parkinson's disease, 17 with adenocarcinoma (normal tissue was studied), and five who underwent colectomy for severe constipation. Immunohistochemistry was used to stain myenteric and submucosal neurons for dopamine, tyrosine hydroxylase, and vasoactive intestinal polypeptide (VIP). Each class of neurons was quantified as a percentage of the total neuronal population stained for the marker protein gene product 9.5. Nine of the 11 Parkinson's disease patients had substantially fewer dopaminergic myenteric neurons than the other subjects (mean 0.4 [SE 0.2] vs 6.9 [2.3] in controls and 5.7 [2.0] in constipated subjects). There was very little difference between the groups in numbers of tyrosine-hydroxylase and VIP neurons. Two Parkinson's disease patients had similar distributions of all types of neurons, including dopaminergic myenteric neurons, to the controls. High-performance liquid chromatography showed lower levels of dopamine in the muscularis externa (but not mucosa) in four Parkinson's disease patients than in four controls (7.3 [5.1] vs 24.2 [4.6] nmol per g protein), but levels of dopamine metabolites were similar in the two groups. The identification of this defect of dopaminergic neurons in the enteric nervous system in Parkinson's disease may lead to better treatment of colorectal dysfunction in this disease.

Adenocarcinoma↗
PubMed1995

Utilization of scalp hair for evaluating epilepsy in male and female groups of the Pakistan population.

Concentrations of Fe, Cu, Zn, Cr, Mg and Ca are determined in scalp hair of adult epileptic and normal male and female subjects by atomic absorption spectroscopy. A total of 85 hair samples for normal male/female subjects and 78 of male/female epileptic subjects were analyzed within the respective age groups of 28 to 30 and 24 to 27 years. The study showed higher average concentration of Zn, Fe and Cu in normal male subjects compared with the epileptic subjects. Levels of Ca, Mg and Cr were lower in normal male adults than those found in epileptic subjects. Epileptic females showed higher average concentrations of Zn, Ca and Cu. The correlation coefficient study indicated significant correlation (r > 0.589 at P = 0.001) between Fe and Cr, Mg, Ca and Cu for epileptic females only. For normal male subjects Ca showed a strong correlation for Zn and Fe and for Cr and Ca. The regression analysis showed the significant role of Zn, Ca, Mg and Cr in scalp hair of normal and epileptic subjects. Accordingly, the metal-to-metal ratio analysis revealed critical Mg/Zn ratios as indicators of healthy (< 1) and epileptic (> 1) subjects. The ratio data are used for evaluation of the status of epileptic patients based on their ailment history.

Adult↗
PubMed1995

Anorectal function in fluctuating (on-off) Parkinson's disease: evaluation by combined anorectal manometry and electromyography.

Anorectal dysfunction and constipation are well recognized in Parkinson's disease and may reflect the direct involvement of the gastrointestinal tract by the primary Parkinson's disease process. We hypothesized, therefore, that anorectal function would alter in parallel with fluctuations in motor function related to on- and off-periods in Parkinson's disease, and employed combined anorectal manometry and electromyography to investigate anorectal function during both on- and off-periods in patients with Parkinson's disease. Manometric recordings revealed a deterioration in voluntary sphincter squeeze during off-periods (squeeze index, on versus off, mean +/- SEM: 46.4 +/- 11.1 versus 29.6 +/- 7.9 mm Hg, p < 0.05); correspondingly, simultaneous electromyographic (EMG) recordings showed poor recruitment of external anal sphincter and puborectalis muscles during off-periods. A hypercontractile ("paradoxical") rectosphincteric reflex response occurred during both on- and off-periods, and was associated with an increase in EMG activity in the external sphincter and/or the puborectalis muscle. These changes in manometric and EMG parameters paralleled changes in overall motor function. These findings provide further support for the involvement of the pelvic floor musculature in the Parkinson's disease process and also provide EMG correlates for some of the manometric abnormalities described in Parkinson's disease.

Age of Onset↗
PubMed1995

Age- and sex-based comparative distribution of selected metals in the scalp hair of an urban population from two cities in Pakistan.

Concentrations of Cu, Zn, Pb and Ni in the scalp hair of male and female donors, with an age range of 6-60 years, were determined by ICP atomic emission spectroscopy. The donors were drawn from the densely populated city of Lahore and the relatively less-populated capital city of Islamabad for comparative evaluation of the metal levels in relation to age, sex and location. Linear regression analyses and correlation between paired metals indicated a positive correlation between Cu and Zn for both sexes ( [Formula: see text] ) and between Pb and Ni ( [Formula: see text] ) for males and females of Lahore. Metal concentrations varied between the two cities and the two sexes. The highest mean concentration was found for Zn at 180.5 microg g(-1) for males and 202.4 microg g(-1) for females from Lahore, while for counterparts from Islamabad the values were 105.2 microg g(-1) and 206.6 microg g(-1). Copper showed an identical mean concentration (20.8 microg g(-1)) in the hair of both males and females from Lahore; however, relatively lower mean concentrations (7.7 and 10.8 microg g(-1)) were observed for donors from Islamabad. Mean Pb concentrations in hair of male donors from Lahore and Islamabad were 9.4 and 7.0 microg g(-1), respectively; in female groups the concentrations were 14.3 and 5.7 microg g(-1), respectively. Ni showed the lowest concentration range (4.3-4.5 microg g(-1)) of all the four metals for subjects from Lahore, and this was higher than the corresponding range (2.0-3.2 microg g(-1)) for subjects from Islamabad. The findings are discussed in relation to the available data from the literature.

Journal Article↗
PubMed1995

Effects of psyllium therapy on stool characteristics, colon transit and anorectal function in chronic idiopathic constipation.

BACKGROUND: Psyllium is widely used in the symptomatic therapy of constipation. Its effects on colonic function and their correlation with symptomatic response have not been defined. METHODS: After a 4-week baseline, placebo, run-in phase, 22 subjects with idiopathic constipation confirmed by prospectively administered stool diaries were randomly assigned to receive either psyllium (5 g b.d., 11 patients) or placebo (11 patients) for 8 weeks, followed by another 4-week wash-out, placebo phase. A colon transit study and anorectal manometry were performed at the beginning and at the end of each study phase. Subjects recorded, in diaries, their daily stool frequency, difficulty with defecation and weekly stool weight. RESULTS: Stool frequency increased significantly after 8 weeks of psyllium treatment (3.8 +/- 0.4 vs. 2.9 +/- 0.1 stools/week, P < 0.05) as did stool weight (665.3 +/- 95.8 g vs. 405.2 +/- 75.9 g, P < 0.05). Subjects also reported an improvement in stool consistency (stool consistency score: 3.2 +/- 0.2 vs. 3.8 +/- 0.2, P < 0.05) and pain on defecation (pain score: 2.0 +/- 0.4 vs. 2.6 +/- 0.5, P < 0.05) on psyllium. Colon transit and anorectal manometry parameters were unchanged on psyllium. Subjects treated with placebo did not show any change in either subjective or objective measures of constipation. CONCLUSIONS: Psyllium increases stool frequency and weight and improves stool consistency in idiopathic constipation. These effects are not associated with significant changes in either colonic or rectal motor function. We suggest that the beneficial effects of psyllium in constipation are primarily related to a facilitation of the defecatory process.

Adult↗
PubMed1994

Bacterial oxidation of propane.

The bacterial metabolism of propane and the pathway(s) involved are poorly understood, as the relative importance of terminal versus subterminal oxidation of propane, via propan-1-ol and propan-2-ol, respectively, is still unclear. In the case of bacteria, the ability to oxidize propane appears to be confined mainly to the Gram-positive Corynebacterium - Nocardia - Mycobacterium - Rhodococcus complex. Studies on propane oxidation have been hampered by a lack of firm enzymological data; for example, to date there are no reports of a purified propane oxygenase system. However, oxygenase activity has been confirmed by the production of propan-1-ol and/or propan-2-ol, and more recently by the co-oxidation of propene to 1,2-epoxypropane in cell extracts of propane-grown cells. Here, we review the use of genetic, biochemical and immunological techniques to assess the role(s) of terminal and subterminal oxidation in the metabolism of propane by Rhodococcus rhodochrous PNKb1 and present a general overview of the topic.

Alcohol Oxidoreductases↗
PubMed1994

Age and sex dependence of selected trace metals in scalp hair of urban population of Pakistan.

Concentrations of ten metals (Ag, Al, Cd, Cr, Cu, Fe, Mn, Sr, Ca and Mg) in scalp hair of urban donors from the city of Lahore, Pakistan were determined by the nitric acid/perchloric acid digestion based method by ICP-AES. The age and sex dependence of metal levels was investigated for the age groups 10-47 and 7-55 years, for females and males, respectively. In total 45 female and 42 male samples were analysed in triplicate. The maximum concentration in the male group was that of Fe, between 45 and 330 mg/kg, followed by Sr between 4.5 and 109 mg/kg. On average, the following order of metal concentration in male hair was observed: Ca > Mg > Fe > Sr > Al > Cu > Mn > Cr > Ag > Cd. For female samples Cu was found to have a maximum concentration (210 mg/kg) followed by Fe (170 mg/kg) and Sr (140 mg/kg). In this group the concentration order was: Ca > Mg > Cu > Fe > Sr > Al > Mn > Ag/Cr > Cd. A metal to metal correlation study showed that different metals were mutually correlated in male and female hair. Regression equations illustrated a distinct dependence of metal concentrations on the age of donors. The data are compared with corresponding data reported in the literature.

Adolescent↗
PubMed1994

Anorectal manometry in the assessment of anorectal function in Parkinson's disease: a comparison with chronic idiopathic constipation.

We investigated the role of anorectal manometry in evaluating constipation and anorectal function in 15 patients with Parkinson's disease (PD) and compared results with those of 9 patients with idiopathic constipation (IC) and 8 control (C) subjects. Anal sphincter pressures on voluntary squeeze were lower in the PD patients. Sustained squeeze pressures (mm Hg C versus IC versus PD: 46.8 +/- 5.2 versus 31.2 +/- 3.6 versus 26.6 +/- 3.9; p < 0.05 PD versus C), squeeze duration (seconds: 53.6 +/- 2.5 versus 48.5 +/- 4.1 versus 33.6 +/- 9; p < 0.05 PD versus C) and squeeze index (area under the squeeze curve: 44.0 +/- 2.9 versus 34.5 +/- 3.3 versus 21.4 +/- 2.9; p < 0.001 PD versus C) were significantly lower in the PD group in comparison to the control group. In contrast, none of the parameters of anorectal manometry differed between controls and patients with idiopathic constipation. Some Parkinson's disease patients demonstrated an abnormal, hypercontractile response on testing of the rectoanal inhibitory reflex. Anal sphincter length, basal sphincter pressures, maximal squeeze pressures, extent of relaxation on rectoanal inhibitory reflex and threshold volume for rectal sensation were similar in the three groups. We conclude that an impaired squeeze response is a specific feature of anorectal function in Parkinson's disease. This may indicate direct involvement of the pelvic floor musculature by the parkinsonian disease process.

Aged↗