Search PubMed⌕ Search

Biomedical subjects

M S Patel

Publications and source records attributed to M S Patel.

At least 91 records · Page 5Linked to original sources
PubMed1994

Identification of a sequence motif upstream of the Drosophila dopa decarboxylase gene that enhances heterologous gene expression.

In this paper we have examined the role that element S, a DNA sequence motif found approximately 215 bp upstream of the Dopa decarboxylase (Ddc) gene, might play in regulating Ddc expression. Nearly identical versions of the element are present upstream of four other Drosophila genes. For two of these, the element appears to be an important component of the upstream regulatory region, since mutations in it reduce expression of the downstream gene. Because an element S polymorphism differentiates the Ddc+ allele of an inbred laboratory strain from the Ddc+4 allele present in a strain isolated from the wild, we decided to test the activity of both forms. Oligonucleotides containing Ddc+ or Ddc+4 versions of element S were synthesized and their ability to drive the expression of an heterologous (Adh) reporter gene at the second molt was examined. Transgenic larvae carrying the element S-Adh fusion constructs consistently exhibited Adh levels that were elevated 1.5-fold above those seen in control organisms. We have also determined the effects of element S in white prepupae and once again, ADH expression levels were significantly above controls in both groups of transformants carrying the element S construct. The results point to a functional role for element S. Since reporter gene expression in third instar larvae was restricted to tissues where ADH is normally found, we conclude that element S is not involved in directing the tissue specificity of Ddc expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1993

Identification of the essential cysteine residue in the active site of bovine pyruvate dehydrogenase.

Pyruvate dehydrogenase (E1), the first catalytic component of the bovine pyruvate dehydrogenase complex, is composed of two nonidentical subunits in a tetrameric alpha 2 beta 2 form. The sulfhydryl-specific reagent N-ethylmaleimide (NEM) was used to identify the reactivities and function of cysteinyl residues and subsequent identification of these residues in the active site of bovine E1. Treatment of E1 with 0.2 mM NEM resulted in loss (90%) of enzymatic activity; the inactivation followed bimolecular reaction kinetics. The inactivation was almost entirely prevented by thiamin pyrophosphate (TPP) and pyruvate; protection is probably due to formation of the hydroxyethylidene-TPP intermediate. To identify the reactive cysteinyl residues in the active site region, the nonessential SH groups in E1 were first modified with NEM in the presence of TPP and pyruvate. After quenching with dithiothreitol and removal of the substrate and cofactor by dialysis, the modified E1 was treated with [14C]NEM to label the exposed cysteinyl residue(s) in or near the active site region. The data indicate that NEM reacted in the active site region of the E1 component with a stoichiometry of 2 mol of [14C]NEM bound per mol of E1 tetramer. The initial rapid labeling of E1 with [14C]NEM established that incorporation was predominantly into the alpha subunit. A single radiolabeled peptide was isolated following V8 protease digestion of radiolabeled E1 by [14C]NEM. Sequence analysis of the labeled peptide derived from bovine E1 demonstrated that the labeled cysteinyl residue was equivalent to Cys-62 in the alpha subunit (mature form) of human E1.

Amino Acid Sequence↗
PubMed1993

Identification of two missense mutations in a dihydrolipoamide dehydrogenase-deficient patient.

The molecular basis of dihydrolipoamide dehydrogenase (E3; dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) deficiency in an E3-deficient patient was studied. Fibroblasts cultured from the patient contained only approximately 6% of the E3 activity of cells from a normal subject. Western and Northern blot analyses indicated that, compared to control cells, the patient's cells had a reduced amount of protein but normal amounts of E3 mRNA. Direct sequencing of E3 cDNA derived from the patient's RNA as well as each of the subclones of the cDNA revealed that the patient had two substitution mutations in the E3 coding region. One mutation changed a single nucleotide from A to G, resulting in substitution of Glu (GAA) for Lys-37 (AAA). The other point mutation was a nucleotide change from C to T, resulting in the substitution of Leu (CTG) for Pro-453 (CCG). These mutations appear to be significant in that they alter the active site and possibly the binding of FAD.

Amino Acid Sequence↗
PubMed1993

Multiple protein-binding domains and functional cis-elements in the 5'-flanking region of the human pyruvate dehydrogenase alpha-subunit gene.

We have characterized the 5'-flanking region of the alpha-subunit gene of the human pyruvate dehydrogenase (E1). DNase I footprinting with rat liver nuclear extracts identified 7 major protein-binding domains termed P1 through P7 in a 796 base pair DNA fragment (base pairs -763 to +33). P1 through P4 are clustered in the -221/+33 region. These protein-binding domains contain several known consensus sequences such as a TATA box, CAAT box, Sp1, and CRE, which all have previously been implicated in the constitutive transcription of several genes. Oligonucleotide competition studies indicate that oligonucleotides specific for CTF/NF-1 and Sp1 displaced the nuclear proteins bound to the CAAT box (within P3) and an Sp1 site (within P4), respectively. Several other well-characterized and purified transactivators (c-Fos, c-Jun, C/EBP, AP-2, and Sp1) have been shown to bind to the -221/+33 region. Other elements located upstream of the -221/+33 region, which includes nuclease protection domains P5-P7, are required for enhanced promoter activity of the 796 bp sequence. Promoter activity was measured by transient expression of a chloramphenicol acetyltransferase gene ligated to deletion fragments of the 5'-flanking region. Crucial element(s) for promoter activity and complex DNA-nuclear protein interactions were confined within a region spanning -221/+33. This region also retained more than 75% of the promoter activity of the 796 bp sequence. Additionally, this promoter region shows characteristics of both facultative and housekeeping gene promoters, suggesting complex transcriptional regulation.

Base Sequence↗
PubMed1993

An outbreak of epidemic polyarthritis (Ross River virus disease) in the Northern Territory during the 1990-1991 wet season.

OBJECTIVE: To describe the epidemiology of a large outbreak of epidemic polyarthritis in the Northern Territory during the wet season of 1990-1991. DESIGN, SETTING AND PARTICIPANTS: Arbovirus cases notified to the Northern Territory Department of Health and Community Services by general practitioners and local laboratories between 1 July 1990 and 30 June 1991. MAIN OUTCOME MEASURES: Date and place of infection, age, sex and symptoms. RESULTS: Doctors in the Northern Territory notified 368 cases; another 14 were infected interstate. The epidemic started in September, peaked in January and tailed off in April. The highest attack rates occurred in the rural areas of Jabiru, Litchfield Shire and Katherine. Those most affected were 30-34 year olds. Children, the elderly and Aboriginal people were under-represented. CONCLUSIONS: Epidemic polyarthritis is a wet season problem in the Northern Territory, affecting the rural towns and districts more than the cities. Pre-planned mosquito control measures (effective water drainage and larval control) limited the extent of the 1990-1991 epidemic in Darwin City and Palmerston. The low attack rate in children reflects asymptomatic and less clinically severe infections. The under-representation of Aboriginal people may be the result of infection occurring earlier in life. A related cross-sectional seroprevalence survey has shown that rural Aboriginal people across all age groups have a significantly higher seropositive rate than urban non-Aboriginal residents.

Adolescent↗
PubMed1993

Epidemic meningococcal meningitis in central Australia, 1987-1991.

OBJECTIVE: To describe an outbreak of meningococcal meningitis and the impact of rifampicin chemoprophylaxis on secondary attack rates among Aboriginal people in central Australia. DESIGN: Prospective study of patients admitted to hospital between September 1987 and May 1991. SETTING: The Alice Springs Health Region of the Northern Territory and the Anangu Pitjantjatjara Lands of South Australia, covering a population of 13,228 Aboriginal people. SUBJECTS: Patients admitted to the Alice Springs Hospital with clinical signs or autopsy findings of meningococcal disease. Rifampicin chemoprophylaxis was given to close contacts of all cases. Mencevax AC vaccine was offered to children aged 1 to 15 years in the Region. MAIN OUTCOME MEASURES: Blood or cerebrospinal fluid (CSF) with Neisseria meningitidis, or a positive result of latex agglutination testing on CSF. Positive isolates were serogrouped. RESULTS: Seventy-seven cases of meningococcal disease were diagnosed in Aboriginal people over four years compared with one to two cases per year previously; of these, 60 were definite, 7 probable and 10 suspected cases. Seventy-six subjects had meningitis, of whom one also had the clinical features of meningococcal septicaemia; one other subject had positive blood cultures with a mild febrile illness without features of meningitis. The annual attack rate of meningococcal disease in the Aboriginal population was 1.6/1000. The relative risk for secondary cases was estimated to be between 0.3 (95% confidence interval [CI], 0.09-0.92) and 0.5 (95% CI, 0.15-1.53). The annual attack rate in the non-Aboriginal population was 0.04/1000. CONCLUSIONS: The epidemic closely resembled those in sub-Saharan Africa, and in socioeconomically marginalised groups in developed countries. The relative risk for secondary cases was lower than generally reported, and was attributed to chemoprophylaxis for close contacts and the mass vaccination program for children. Until there are major improvements in living conditions, infectious diseases such as those transmitted by airborne droplets will continue to occur in Aboriginal communities.

Adolescent↗
PubMed1993

Utilisation of health services by aboriginal Australians with diabetes.

Diabetes is a major public health problem for Aboriginal Australians. We wished to determine the extent and pattern of health service utilisation by Aboriginal people with diabetes in central Australia. Medical records of all Aboriginal people known to have diabetes (n = 374), identified by a previous study, were examined for attendance to health services in central Australia. All had non-insulin-dependent diabetes. Between January 1984 and December 1986, Aboriginal adults with diabetes were admitted to hospital on 694 occasions, accounting for 10.8% of adult Aboriginal admissions. The crude admission rates were 0.78 and 0.84 per diabetes-year for men and women, respectively. The age-adjusted relative risks for admission, compared with Aboriginal non-diabetic patients, were 2.93 (95% C.I., 2.62-3.26) for men and 2.46 (95% C.I., 2.28-2.66) for women. If admission for conditions associated with diabetes are excluded, the admission rates were similar for the two groups. Infection was the most common reason for attendance to a health service, representing 41.7% and 39.8% of male and female admissions, and 21.8% and 26.3% of male and female outpatient attendances. Aboriginal patients with diagnosed diabetes suffer high morbidity and contribute disproportionately to health system costs.

Adult↗
PubMed1993

Overview of pup in a cup model: hepatic lipogenesis in rats artificially reared on a high-carbohydrate formula.

The artificial rearing technique allows nutritional investigations to be conducted in rat pups during a critical period that previously has been inaccessible to researchers. The technique will be useful for identifying dietary components contributing to metabolic adaptations in the preweaning period as well as "metabolic imprinting" or permanent metabolic effects in adult rats resulting from early dietary intervention. Artificially reared rat pups fed a formula high in carbohydrate-derived energy in the preweaning period have the following characteristics compared with pups fed a high-fat formula or reared naturally: (i) a higher level of plasma insulin, (ii) an increased hepatic lipogenic capacity and (iii) precocious induction of hepatic malic enzyme. The results also show that early exposure to a high-carbohydrate diet in the preweaning period predisposes the rat to an increased lipogenic capacity in liver and adipose tissues and to the development of obesity later in adult life.

Animals↗
PubMed1993

Control of a community outbreak of measles which started in a poorly immunised high school population.

An outbreak of measles occurred in Darwin from February to March 1991. The first case was in a 13-year-old high school student who had returned from a holiday overseas. She was symptomatic on the second day of the new school term. She infected an infant while both waited in a doctor's surgery. Outbreak control measures were instituted 18 days later when the Communicable Diseases Centre was first alerted of cases through the laboratory notification scheme. Through active surveillance, we identified 76 cases of measles, of whom 92 per cent (70 cases) were under 20 years of age. Of these, 46 were students at the index high school in which the attack rate was 39.2 per 1,000. They transmitted the disease to six unvaccinated siblings aged 11 to 18 years, resulting in a secondary attack rate of 113 per 1,000 in this age group (relative risk of disease in siblings 2.8, 95 per cent confidence interval 1.2 to 6.2). The outbreak affected one other high school, a number of primary schools, one tertiary institution, and nine children under five years. Only four of the cases had a verified history of previous immunisation against measles. The outbreak was arrested within two weeks of instituting community-wide control measures. Inadequate immunisation coverage among school-aged children and delays in notification contributed to the severity of the outbreak. Improved measles surveillance systems, including telephone notification of clinical cases are needed so that control measures can be instituted immediately within the household and in the community.

Adolescent↗
PubMed1993

Long-term effects on pancreatic function of feeding a HC formula to rats during the preweaning period.

We have investigated pancreatic changes associated with hyperinsulinemia and an insulin secretory response to an oral glucose load in a new rat model for obesity. Male Sprague-Dawley pups were reared on a high-carbohydrate (HC) or high-fat (HF) formula by gastrostomy during the suckling period and were weaned onto a stock diet. These animals remained either nutritionally unchallenged or challenged with a high-sucrose diet during the postweaning period. The HC formula-fed animals showed increased insulin concentrations in the plasma and pancreas and also showed impaired insulin secretory response compared with mother-fed control or HF animals in adult life. Immunocytochemical and morphometric studies revealed that hyperinsulinemia in the HC animals during the preweaning period and also in adult life was associated with hypertrophy of beta-cells in the pancreas. The results show that consumption of a HC formula during the suckling period influences pancreatic islet morphology resulting in hyperinsulinemia which eventually leads to the development of obesity later in adult life.

Animals↗
PubMed1993

Chromosome localization and RFLP analysis of PDC-E2: the major autoantigen of primary biliary cirrhosis.

Patients with primary biliary cirrhosis are well known for the presence of titer antibodies against dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex. We have taken advantage of a cDNA probe for dihydrolipoamide acetyltransferase to explore the possibility of polymorphism of the E2 subunit by probing genomic DNA from 38 patients with primary biliary cirrhosis and 26 healthy controls. To detect restriction fragment length polymorphism, DNA was digested with ten specific restriction enzymes that often detect polymorphism, including Bam HI, Bgl II, Eco RI, Hind III, Hinf I, Msp I, Pst I, Pvu II, Rsa I and Taq I. A Taq I polymorphism was found in 19 of 38 patients with PBC and 6 of 26 normal controls. In addition, using fluorescence in situ hybridization, the gene for dihydrolipoamide acetyltransferase was mapped on human chromosome 11 band q23.1. Interestingly, this region of the long arm of chromosome 11 is often associated with cytogenetic abnormalities, including translocations.

Autoantigens↗
PubMed1993

Long-term effects of feeding high carbohydrate diet in pre-weaning period by gastrostomy: a new rat model for obesity.

In the present study, we report the long-term metabolic consequences of feeding a milk substitute formula that is high in carbohydrate-derived calories during the suckling period. Male Sprague-Dawley rat pups were raised by gastrostomy on a high carbohydrate (HC) formula or a high fat (HF) formula (which mimicked rat milk composition in macronutrients) during the pre-weaning period (day 4 to 24). These rats were then weaned to a laboratory stock diet and subsequently challenged with a high sucrose diet to augment the development of obesity. The pups receiving the HC formula developed obesity in later life, even though there was no change in the body weight of this group compared to mother-fed (MF) controls or HF formula fed animals during the pre-weaning period. The HC rats were hyperinsulinemic and their growth rates were greater than MF rats starting at day 55. The lipogenic capacity of liver as well as adipose tissues (epididymal and omental) was higher in HC animals compared to MF control animals, as reflected by increases in two key lipogenic enzymes (fatty acid synthase and glucose-6-phosphate dehydrogenase) and in vitro synthesis of lipids. An analysis of adipose tissue morphology in adult rats showed an increase in cell size in epididymal adipose tissue of HC rats compared to the MF group. However, there was no significant difference in cell size in omental adipose tissue between the MF and HC rats. The HF group behaved similarly to the MF control group in growth pattern and measured metabolic parameters.

Adipose Tissue↗
PubMed1992

Characterization of the transcriptional regulatory region of the human dihydrolipoamide dehydrogenase gene.

Dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4) is the common component of the three mammalian alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. To study regulation of E3 gene expression, a 12-kilobase clone from a human leukocyte genomic library was isolated, and a 1.8-kilobase fragment containing part of the first intron, the first exon, and 1.5 kilobases of the 5' flanking region of the E3 gene was sequenced. The nucleotide sequence of the E3 promoter region revealed consensus sequences for several DNA binding proteins but no apparent TATA box or Sp1 sites. Although the 1.6-kilobase 5' flanking region has a low percentage of G+C (44%), the nucleotide sequence between +1 and -150 base pairs has a G+C content of 67%. Primer extension analysis showed a major transcriptional start site located 95 nucleotides upstream from the translation initiation codon. A series of 5' deletions from the E3 promoter-regulatory region were ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting constructs were transfected into HepG2 cells. The longest E3 promoter-CAT construct had a relatively high level of CAT enzyme activity, and deletion of a promoter element between -769 and -1223 base pairs resulted in a 3-fold increase in reporter gene expression. These results suggest that the human E3 promoter has characteristics of housekeeping and facultative promoters and that a negative regulatory element is present between 769 and 1223 base pairs upstream from the transcription start site.

Analysis of Variance↗
PubMed1992

Characterization of two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln).

Two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln) were expressed in pyruvate dehydrogenase complex-deletion mutant Escherichia coli JRG1342. The expressed mutant E3s were purified to near homogeneity using DEAE-Sephacel and hydroxyapatite columns. The initial velocity measurements in the absence of products for the Gln-452 mutant E3 in the direction of NAD+ reduction showed parallel lines in double-reciprocal plots, indicating that the mutant E3, like wild-type enzyme, catalyzed E3 reaction via a ping-pong mechanism. The specific activity of the Gln-452 mutant E3 was about 0.2% of that of wild-type enzyme. Its Km for dihydrolipoamide was dramatically increased by 63-fold. The substitution of His-452 to Gln resulted in a destabilization of the transition state of human E3 catalysis by about 6.4 kcal mol-1. The Gln-457 mutant E3, unlike wild-type enzyme, catalyzed E3 reaction via a sequential mechanism in the direction of NAD+ reduction based on the intersecting lines shown on double-reciprocal plots. Its specific activity decreased to 28% of that of wild-type enzyme. Its Km for dihydrolipoamide increased about 4.3-fold. The substitution of Glu-457 to Gln resulted in a destabilization of the transition state by about 1.7 kcal mol-1. These results indicate that His-452, which is a possible proton acceptor/donor in human E3 reaction, is critical to human E3 catalysis and that the local environment around His-452 and Glu-457, which are suggested to be hydrogen-bonded, is important in the binding of dihydrolipoamide to the enzyme.

Amino Acid Sequence↗
PubMed1992

Mutations and polymorphisms in the pyruvate dehydrogenase E1 alpha gene.

We present an update on mutations and polymorphisms in the human X chromosome located pyruvate dehydrogenase E1 alpha gene. A total of 20 different mutations are tabulated. The mutations include deletions, insertions, and point mutations. Certain sequences seem particularly prone to mutation. Most of the mutations are found in exons 10 and 11. Furthermore, four of the mutations are seen in unrelated patients. Little is known about how the mutations affect the structure or function of the pyruvate dehydrogenase complex.

Amino Acid Sequence↗
PubMed1992

A structural model for human dihydrolipoamide dehydrogenase.

The hypothesis that dihydrolipoamide dehydrogenases (E3s) have tertiary structures very similar to that of human glutathione reductase (GR) was tested in detail by three separate criteria: (1) by analyzing each putative secondary structural element for conservation of appropriate polar/nonpolar regions, (2) by detailed comparison of putative active site residues in E3s with their authentic counterparts in human GR, and (3) by comparison of residues at the putative dimeric interface of the E3s with the authentic residues in GR. All three criteria are satisfied in a convincing way for the 7 E3s that were considered, supporting the conclusion that the structural scaffolding and the overall tertiary structure (which determines the location of functional sites and residues) are remarkably similar for the E3s and for GR. These analyses together with the crystal structures of human erythrocyte GR formed the basis for construction of a molecular model for human E3. The cofactor FAD and the substrates NAD and lipoic acid were also included in the model. Unexpectedly, the surface residues in the cleft that holds the lipoamide were found to be highly charged and predominantly acidic, allowing us to predict that the region around the lipoamide in the subunit should be basic in nature. The molecular model can be tested by site-directed mutagenesis of residues predicted to be in the dihydrolipoamide acetyltransferase subunit binding cleft.

Amino Acid Sequence↗