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Biomedical subjects

A Parikh

Publications and source records attributed to A Parikh.

At least 37 records · Page 2Linked to original sources
PubMed2000

Carotid artery distensibility and cardiac function in adolescents with type 1 diabetes.

OBJECTIVE: To determine the presence and correlates of early heart and blood vessel dysfunction in adolescents with type 1 diabetes mellitus (DM) of relatively short duration. STUDY DESIGN: A total of 33 patients with DM (20 male, mean age 15.8 +/- 1.3 years, mean DM duration 9.3 +/- 3.9 years) and 16 healthy subjects in a nondiabetic control group (7 male, mean age 17.4 +/- 1.7 years) underwent (1) ultrasonography of the right carotid artery to assess distensibility, compliance, and intimal-medial thickness (IMT), (2) echocardiographic assessment of systolic and diastolic ventricular function, (3) lipid profile and hemoglobin A(1c), and (4) overnight timed urine collections for albumin excretion rate. RESULTS: Ultrasonography showed significantly lower carotid artery distensibility in the DM group (38.5 +/- 8.2 x 10(-3) vs 46.5 +/- 11.7 x 10(-3)/kPa, P =.01) but no difference in compliance (14.0 +/- 3.4 x 10(-7) vs 15.8 +/- 2.9 x 10(-7)m(2)/kPa, P =.08) or IMT (0.061 +/- 0.013 vs 0.060 +/- 0.014 cm, P =.77). Left ventricular (LV) end-diastolic diameter, LV posterior wall thickness, end-systolic wall stress, shortening fraction, ejection fraction, LV mass, and diastolic function were similar in both groups. Total cholesterol, low density lipoprotein, high density lipoprotein, triglycerides, and blood pressure were also similar. The median albumin excretion rate was 4.8 microg/min in the DM group (range 1.1 to 19.2) and 3.0 microg/min in the control group (range 1.4 to 5.8) (P =.03). Hemoglobin A(1c) correlated inversely with both distensibility (r = -.43, P =.02) and compliance (r = -.39, P =.032). CONCLUSIONS: This study indicates that early changes in macrovascular function, namely lower carotid artery distensibility, may precede abnormalities in cardiac function or in arterial IMT in adolescents with short duration type 1 DM. It also supports a relationship between hyperglycemia and carotid artery dysfunction.

Adolescent↗
PubMed1999

Inter-individual differences in the metabolism of environmental toxicants: cytochrome P450 1A2 as a prototype.

Cytochrome P450 (P450) 1A2 provides an interesting paradigm for inter-individual differences in the metabolism of pro-carcinogens. The enzyme is known to vary 40-fold among individuals and may contribute to cancers caused by heterocyclic amines and other chemicals. Rat and human P450 1A2 are known to be 75% identical and were compared for several catalytic activities. The human enzyme was an order of magnitude more efficient in the N-hydroxylation of two heterocyclic amines. Further, the levels of P450 1A2 expressed in human livers show a 40-fold variation, with some as high as 0.25 nmol P450 1A2 per milligram microsomal protein. Some human liver samples are more active (than those isolated from polychlorinated biphenyl-treated rats) in the activation of heterocyclic amines. A bacterial genotoxicity assay has been developed in which human P450 1A2 and NADPH-P450 reductase are expressed within Escherichia coli and bacterial mutants can be assayed using reversion to lac prototrophy. A random mutagenesis strategy for human P450 1A2 has been developed and used to examine the changes in catalytic activity seen with many single-amino acid substitutions. These results may be of relevance in consideration of genetic polymorphisms. Further, the findings pose a challenge to molecular epidemiology effort in that results with one substrate do not necessarily predict those for others. Some dinitropyrenes are P450 1A2 substrates but others are not. 6-Nitrochrysene can be activated by human P450 1A2 but the (mono) nitropyrenes examined were not; these were oxidized by P450 3A4 instead.

Amino Acid Substitution↗
PubMed1999

Selection and characterization of human cytochrome P450 1A2 mutants with altered catalytic properties.

Random mutagenesis is an approach that has the potential to provide useful information about cytochrome P450 (P450) enzymes but has not been extensively used to date. We applied our previously developed systems for generation of random libraries of human P450 1A2 with the putative substrate recognition sequences mutated (individual residues) and an Escherichia coli genotoxity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). A total of 27 mutants were screened from 6000 clones, a small portion of the library. The sequence changes were identified, and E. coli membranes containing each P450 (with NADPH-P450 reductase expressed using a bicistronic vector) were used to determine kcat and Km values for 7-ethoxyresorufin and phenacetin O-deethylation and the (in vitro) activation of MeIQ with another bacterial genotoxicity system (Salmonella typhimurium umu). Within each assay, the values of kcat/Km varied by 2 orders of magnitude, and in some cases these parameters were 3-4-fold higher than for the native enzyme. The profiles of the mutants varied considerably for the three different reactions. Some of the mutants in the Asp-320 region may be compared with site-directed mutants of rat P450 1A2 already reported, with several differences noted. Other mutants have not been studied before in human P450 1A2 or homologues and are of interest; i.e., all Phe-226 mutants showed considerably reduced activity but Glu-225 mutants had increased catalytic activities. In principle, this approach may be applied to random mutagenesis of any enzyme that converts chemicals to mutagens and can be expressed in bacteria.

Amino Acid Sequence↗
PubMed1999

Nasal douching as a valuable adjunct in the management of chronic rhinosinusitis.

The effect of nasal douching in 40 patients with chronic rhinosinusitis was tested, and two different preparations compared: 19 receiving traditional alkaline nasal douche and 21 receiving a sterile sea water spray, in addition to their regular treatment. Douching per se improved endoscopic appearances (p = .009), and quality of life scores (p = .008). These measures did not change in a control group (n = 22) who received standard treatment for chronic rhinosinusitis, but no douche. There were significant differences between the two douching preparations in that the alkaline nasal douche improved endoscopic appearances but not quality of life, whereas the opposite was true for the spray.

Adolescent↗
PubMed1998

Metabolic activation of aromatic amine mutagens by simultaneous expression of human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and N-acetyltransferase in Escherichia coli.

We describe the construction of a new strain of Escherichia coli designed to bioactivate aromatic amines and to detect their mutagenicity with high sensitivity. Strain DJ4309 bears two plasmids, a pACYC184-derived plasmid which expresses Salmonella typhimurium acetyl CoA:arylamine N-acetyltransferase (NAT) and a pBR322-derived plasmid which expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase. The combined actions of these enzymes convert aromatic amines into reactive, mutagenic N-acetoxy esters. The strain also carries a mutated copy of the lacZ gene (on an F' factor) which reverts to the wild-type gene by a -(GpC) frameshift mutation. Strain DJ4309 expresses high levels of NAT and cytochrome P450 1A2 and is very sensitive to mutagenesis induced by representative aromatic amines. Mutagenicity of 2-aminoanthracene in strain DJ4309 is higher than can be obtained by rat liver homogenate 9000g supernatant (S9) activation in the parent strain lacking the P450 expression vector. Strain DJ4309 provides a useful system for detecting mutagenic aromatic amines and for studying their metabolism by human P450 1A2.

Arylamine N-Acetyltransferase↗
PubMed1998

In vivo crushing of an aortic stent enables endovascular repair of a large infrarenal aortic pseudoaneurysm.

PURPOSE: To report an unusual approach to endovascular exclusion of a large aortic pseudoaneurysm. METHODS AND RESULTS: A 63-year-old male had an unsuccessful endovascular repair of an aortic anastomotic pseudoaneurysm that left an expanded, uncovered Palmaz stent lying obliquely within the aorta. After nearly 3 years, the pseudoaneurysm enlarged to 7 cm, and the patient became symptomatic. Repair of the pseudoaneurysm was accomplished by crushing the indwelling stent to allow placement of a stent-graft. CONCLUSIONS: Malpositioned stents that are hindering an endoluminal procedure may be crushed against the arterial wall in vivo to facilitate passage of endovascular instruments or devices.

Aneurysm, False↗
PubMed1998

Random mutagenesis by whole-plasmid PCR amplification.

Random mutagenesis has become a powerful means of studying the effects of a large number of permutations of a particular DNA sequence. As a prime example, libraries of randomized cDNA clones, when translated into their corresponding proteins, can be useful in investigating the functional contributions of a mutagenized region to the overall properties of a protein. Existing molecular cloning techniques for random mutagenesis are tedious and frequently plagued with high levels of background from wild-type (nonmutagenized) template. We report a PCR-based method involving amplification of an entire plasmid containing a gene sequence of interest with partially complementary degenerate oligonucleotides for randomization of up to 12 consecutive nucleotide residues. Sequential treatment of the PCR product with Dpn/and a second specific restriction endonuclease and T4 DNA ligase followed by high-efficiency electroporation permits the generation of libraries with very low background. This technique should prove useful for studies on enzyme structure-function relationships as well as for other diverse applications.

Codon↗
PubMed1998

Twenty years of biochemistry of human P450s: purification, expression, mechanism, and relevance to drugs.

Today cytochrome P450 (P450) research is accepted as an integral part of drug development and discovery. Work leading to this point included biochemical studies on P450 in experimental animal models and application to human systems. The development of recombinant expression systems has been an important part of the progress, and in this article we describe some recently developed bacterial systems that can be used for the production of metabolites, genotoxicity testing, and screening in random mutagenesis work. Rate-limiting aspects of P450 reactions vary with particular systems, and further investigations are in order. Non-ionic detergents have been utilized widely in P450 purification work; these compounds are now shown to be substrates for P450s. These oxidations are not only of fundamental interest in expanding the repertoire of P450 substrates but have significance in light of human exposure to these compounds.

Cytochrome P-450 Enzyme System↗
PubMed1997

Temporary arterial stenting: comparison to permanent stenting and conventional balloon injury in a rabbit carotid artery model.

The objective was to assess the arterial wall response to temporary stenting with a removable nitinol stent in comparison with permanent stenting and balloon injury at 28 days in the rabbit carotid artery. Restenosis remains an important limiting factor after the implantation of permanent metallic stents and balloon angioplasty. We have developed a temporary nitinol stent that uses a bolus injection of warmed saline to collapse the stent for percutaneous removal. Vascular changes related to the thermal saline bolus injection required to remove a nitinol implanted stent were assessed in 12 rabbit carotid arteries at 7 and 28 days postinjection. Nitinol stents, inflated to 3.0 mm diameter, were implanted for 3 days (n = 6) and histology and quantitative histomorphometry examined at 28 days. Results were compared with permanently implanted stents (n = 5) and balloon injury (n = 9). Dual bolus injection of 10 ml at 70 degrees C created an acute necrotizing injury and chronic neointimal proliferation, whereas injections of 5 ml at 63 degrees C were minimally injurious. Temporary stenting resulted in the least neointimal proliferation measured by the intima to media ratio (0.22 +/- 0.10 vs. 1.59 +/- 0.31 for permanent stenting and; 0.49 +/- 0.14 for balloon injury; P < 0.001). Temporary stenting maintained a significantly larger lumen than balloon (1.53 +/- 0.72 mm2 vs. 0.64 +/- 0.14 mm2; P < 0.001), which could not be explained by absolute changes in intimal cross sectional area (0.14 +/- 0.07 mm2 vs. 0.21 +/- 0.06 mm2 respectively; P = 0.33). Temporary stenting resulted in a relatively larger vessel area within the external elastic lamina than with balloon (2.28 +/- 1.06 mm2 vs. 1.30 +/- 0.18 mm2; P = 0.007). The thermal stent recovery process can create necrotizing vascular injury and neointimal proliferation at higher temperatures and injectate volumes. Stent removal after 3 days using 63 degrees C saline bolus injection results in less neointimal proliferation than with permanent stents or balloon injury. In comparison to balloon injury, temporary stenting also may have a long-lasting beneficial effect on vessel recoil and remodeling, resulting in larger lumen size after stent removal.

Alloys↗
PubMed1997

Expression, purification, and characterization of a catalytically active human cytochrome P450 1A2:rat NADPH-cytochrome P450 reductase fusion protein.

An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression in Escherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5' end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3' end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser-Thr. This fusion plasmid was expressed in E. coli and the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP-agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochrome c reduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase in E. coli enhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.

Animals↗
PubMed1997

Expression of drug-metabolizing enzymes.

A principal advance in the production of drug-metabolizing enzymes has been the development of catalytically self-sufficient cytochrome P450 systems, including additional P450-reductase fusion proteins and Escherichia coli and baculovirus coexpression constructs. Continuing work with glutathione transferases has resulted in the identification of important residues by random mutagenesis screening techniques, as well as in the engineering of model Salmonella typhimurium strains for genotoxicity analysis.

Animals↗
PubMed1997

Drug metabolism by Escherichia coli expressing human cytochromes P450.

The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.

Catalysis↗
PubMed1997

Heterologous expression of human drug-metabolizing enzymes.

This article is a report on a symposium held at the March 1997 meeting of the American Society for Pharmacology and Experimental Therapeutics in San Diego. Current developments in the heterologous expression of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione transferase, and UDP-glucuronosyltransferase enzymes are described. Systems include bacteria, insect cells, and transient and stable mammalian cells. Uses of the products are described for discernment of which enzymes are involved in metabolism of drugs, genotoxicity assays, mutagenesis (for structure-activity relationships), large scale production of enzyme products, antibody production, and production of proteins for biophysical studies.

Enzymes↗
PubMed1996

The holmium: YAG laser for ureteric stones.

OBJECTIVE: To assess the efficacy of the pulsed holmium: YAG laser for the fragmentation of ureteric stones. PATIENTS AND METHODS: One hundred patients (72 males and 28 females, age range 14 months-85 years) underwent 114 ureteroscopic procedures using either a 7.2 F semi-rigid or 9.5 F flexible ureteroscope. A holmium: YAG laser (Sunrise Technologies. Fremont, Ca, USA) was used for laser lithotripsy at a maximum energy of 1.0 J/pulse at 5 Hz. Most of the stones (46%) were located in the upper third of the ureter. The mean size of the stones was 9 x 8 mm and the mean duration of the procedure was 73 min (including anaesthesia) with a mean hospital stay of 2.7 days. RESULTS: All the stones were accessed successfully using miniaturized endoscopes either retrogradely or antegradely. The holmium laser effectively fragmented all types of stones. Total clearance of all stones fragments was achieved in 87% of cases, with the best results obtained for stones in the lower third of the ureter (96%). The complications attributed directly to the laser included three strictures and three perforations of the ureteric wall. CONCLUSION: The holmium: YAG laser was effective in fragmenting ureteric stones irrespective of their hardness. However, it has the potential to damage the ureteric wall and must be used with caution.

Adolescent↗
PubMed1994

Management of middle ear myoclonus.

Tinnitus produced by synchronous repetitive contraction of the middle ear muscles (middle ear myoclonus) is a rare condition. We present six cases of middle ear myoclonus in whom different management regimes were successful. In two patients, the tinnitus was controlled by conservative measures. In one patient, whose tinnitus was associated with blepharospasm, significant improvement occurred following botulinum toxin injection into the ipsilateral orbicularis oculi. Three patients were cured by tympanotomy with stapedial and tensor tympani tendon section. The aetiology of this type of myoclonus remains unclear. The diagnosis is based on the history of involuntary and rhythmic clicking or buzzing tinnitus which is invariably unilateral. The primary differential diagnosis is palatal myoclonus whilst other local aural pathologies must be excluded by careful clinical assessment. Surgical section of these muscles via tympanotomy brings guaranteed relief when conservative measures fail.

Adult↗
PubMed1994

Pyocele of the middle turbinate.

Mucoceles are uncommon and the majority occur in the fronto-ethmoidal complex. We present the first case of a secondarily infected mucocele arising from a concha bullosa of the middle turbinate. It was excised intranasally with complete resolution of symptoms.

Aged↗