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J Shah

Publications and source records attributed to J Shah.

At least 19 recordsLinked to original sources
PubMed1995

Comparison of characteristics of Q beta replicase-amplified assay with competitive PCR assay for Chlamydia trachomatis.

In order to study infections due to Chlamydia trachomatis, we have compared semiquantitative PCR and Q beta replicase-amplified assays for detection of this organism. The PCR assay was directed against the C. trachomatis 16S rRNA gene. Quantitation was accomplished by adding known amounts of a plasmid containing a truncated segment of the 16S rRNA gene target to chlamydia-containing samples and then amplifying with a common primer set. The Q beta replicase assay consisted of reversible target capture of C. trachomatis 16S rRNA, which was followed by amplification of an RNA detector probe in the presence of the enzyme Q beta replicase. In a clinical matrix, the lower limit of detection of both the PCR and Q beta replicase assays was five elementary bodies. The Q beta replicase and PCR assays were quantitative over 10,000- and 1,000-fold ranges of organisms, respectively. Analysis of the effects of endocervical matrix on amplification was accomplished by examining 94 endocervical specimens by each technique. Both assays detected five of six culture-confirmed specimens as well as three culture-negative specimens. PCR inhibitors were detected in 13 specimens. The Q beta replicase assay, in contrast, showed no evidence of sample inhibition. The Q beta replicase and PCR assays should allow quantitative investigation of infections due to C. trachomatis. In addition, because it targets highly labile RNA, the Q beta replicase assay may facilitate investigations into the role of active persisting infection in culture-negative inflammatory conditions.

Base Sequence↗
PubMed1995

Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis.

Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear-negative samples. The results of the Q beta replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested. Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effects in all but two specimens, one of which was culture positive. The decreased sample utilization also resulted in a false-negative result with a third specimen which was culture positive for M. tuberculosis. Quantitative smear results and QB replicase assay estimates of the number of organisms present in these specimens were in close agreement. The QB replicase assay performed well in comparison with both culture and PCR and should offer a rapid means for detecting and controlling infection due to M. tuberculosis.

Bacteriological Techniques↗
PubMed1995

Age-dependent alterations in Na+, K(+)-ATPase activity in the central nervous system of spontaneously hypertensive rats: relationship to the development of high blood pressure.

We have previously demonstrated that cerebroventricular administrations (i.c.v) of potassium chloride solutions (KCl; 0.375-1.25 mumoles/5 microliters) elicit ouabain-sensitive, concentration-dependent decreases in the blood pressure and heart rates of anesthetized, normotensive Sprague-Dawley (SD) rats. These studies have suggested an inverse relationship between Na(+)-pump activity in the central nervous system (CNS) and central sympathetic outflow. Such a view is further supported by the present studies showing that i.c.v. injections of KCl failed to produce any alterations in the blood pressures of rats pretreated with an autonomic ganglionic blocker, chlorisondamine. In the present studies, depressor responses to i.c.v. potassium chloride were considered as functional indices for evaluation of neuronal Na(+)-pump activity in 8 and 12 week old (8 wk and 12 wk) SHR, WKY and Sprague-Dawley (SD) rats. Basal arterial blood pressures of 8 wk-old SD and SHR, and the responsiveness of these two groups to i.c.v. potassium chloride solutions are similar and they both are significantly greater than that of age matched WKY. However, in the 12 wk-old groups, arterial pressure of SHR was significantly greater than that of WKY as well as SD, whereas the depressor responses to KCl in SHR were significantly greater than that of only WKY. Pretreatment of the rats with i.c.v. ouabain abolished the differences in the hypotensive responses to i.c.v. potassium chloride that existed between various groups but not the differences in the basal blood pressures. Evaluation of these data suggest that a) the centrally mediated hypotensive responses to K+ in various groups could depend upon Na+, K(+)-pump activity in C.N.S. and/or on basal central sympathetic discharge; b) central sympathetic activity is greater in SHR only when compared to WKY but not to SD; c) since the central Na(+)-pump activity and sympathetic tone appears to be similar in SHR and SD, mechanisms other than the increases in sympathetic activity must play a prominent role in the development of spontaneous hypertension; d) attenuation of neuronal Na(+)-pump activity cannot account for greater sympathetic tone in SHR and SD-rats when compared to WKY.

Aging↗
PubMed1995

The effect of patient controlled analgesia and continuous epidural infusion on length of hospital stay after total knee or total hip replacement.

The purpose of this study was to determine the correlation between patient controlled analgesia and continuous epidural analgesia after total knee or total hip replacement on the length of hospital stay. Stress responses to postoperative pain, including decreased mobility, compromised respiratory function, increased catecholamine release, and hypercoagulation, may adversely affect patient recovery, thus lengthening hospital stay. A retrospective chart review of 127 adult, American Society of Anesthesiology (ASA) I, II, or III, patients who had undergone total knee arthroplasty (TKA) or total hip replacement (THR) was obtained. One patient group received epidural anesthesia and postoperative analgesia (EAA) through continuous catheter infusion of bupivacaine or preservative free morphine. The second group underwent general anesthesia and postoperative patient controlled anesthesia (G-PCA) of meperidine hydrochloride or morphine. Length of stay (LOS) was defined as the time period beginning with admission to the post-anesthesia care unit (PACU) until 10 AM the day of discharge. The mean LOS, in hours, for EAA-morphine was 121; compared with EAA-bupivacaine, 142; G-PCA-meperidine, 134; and G-PCA-morphine, 142. These findings were not statistically significant at P = 0.054. LOS did not correlate with age, weight, height, type or surgery, or the ASA classification. Further research into the effectiveness of continuous infusion of epidural bupivacaine and epidural morphine and their impact on LOS may be warranted.

Analgesia, Epidural↗
PubMed1994

Cisplatin, fluorouracil, and leucovorin. Increased toxicity without improved response in squamous cell head and neck cancer.

OBJECTIVE: To evaluate the activity and toxicity of the drug combination cisplatin, fluorouracil by continuous infusion, and high-dose oral leucovorin calcium (PFL) as induction chemotherapy in patients with advanced and untreated squamous cell head and neck (SCHN) cancer. DESIGN: Nonrandomized, prospective trial. SETTING: Referral center (comprehensive cancer center). PATIENTS: Twenty-two patients with stage III (n = 7) and IV (n = 15) M0 SCHN cancer of the larynx (n = 13), hypopharynx (n = 7), and oropharynx (n = 2) whose standard treatment would have required total laryngectomy. INTERVENTIONS: Three cycles of PFL were administered prior to local-regional therapy (concomitant cisplatin and radiation and/or neck dissection, with total laryngectomy reserved for nonresponse or relapse). Chemotherapy included cisplatin (100 mg/m2) on day 1 by short intravenous infusion; fluorouracil (800 mg/m2) on days 1 through 5 by continuous infusion; and leucovorin (100 mg) every 4 hours by mouth for 30 doses. The PFL combination was administered every 21 days. MAIN OUTCOME MEASURES: Clinical response to chemotherapy and observed toxic effects during chemotherapy. RESULTS: Five patients were inevaluable for response, with three early deaths (infection in two and sudden death in one), one cerebrovascular accident, and one patient declining further chemotherapy. Of the remaining 17 patients, 10 had a major response to chemotherapy, but in only five patients (29%) was this complete (95% confidence interval, 8% to 51%). Other significant toxic effects included grade 3 to 4 mucositis in eight patients and grade 3 to 4 neutropenia in 10. CONCLUSIONS: While PFL is active in patients with SCHN cancer, we were unable to reproduce the high complete response rates reported by other centers. Its use can be associated with significant toxic effects. We do not recommend the use of PFL for the treatment of patients with SCHN cancer outside the context of a clinical trial until there is further critical assessment of its activity and toxicity.

Adult↗
PubMed1994

The relationship of loss of heterozygosity to tobacco exposure and early recurrence in head and neck squamous cell carcinoma.

BACKGROUND: Tobacco usage contributes to carcinomas of the lung, bladder, esophagus, uterine cervix, and head and neck, and can induce specific genetic lesions. Studies of the above tumor types have documented allelic deletions affecting 3p, 5q, 9p, 9q, 10q, 11p, 13q, 17p, and 18q. Relationships between genetic loss, tobacco exposure, and patient outcome have not been described. PATIENTS AND METHODS: To confirm and further define loss of heterozygosity in head and neck squamous cell carcinoma (HNSCC), and to examine relationships between loss of heterozygosity and both tobacco exposure and early recurrence, we undertook this study on previously untreated patients with HNSCC. We performed a Southern blot analysis using 11 probes specific for loci deleted in tobacco-associated cancers. We have investigated 42 prospectively collected, paired samples of HNSCC and peripheral blood. Demographic and follow-up data were collected on these patients. RESULTS: Significant loss of heterozygosity was observed in descending order of frequency at 11p, 9p, 17p, 3p, 10q, and 13q. All nonsmokers showed loss of heterozygosity on one or more loci compared with only 53% of smokers (P < 0.05). Furthermore, patients with multiple deletions had a significantly higher rate of early recurrence than those with fewer deletions (P < 0.05). CONCLUSION: Multiple deletions occurred more frequently in nonsmokers and predicted a higher risk of early recurrence.

Blotting, Southern↗
PubMed1994

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).

Biophysical Phenomena↗
PubMed1993

Physiological significance of Na+/K(+)-ATPase activity in the central nervous system and endogenous sodium-pump inhibitors in the neural regulation of arterial blood pressure.

In anesthetized Sprague-Dawley rats, cerebrolateral ventricular administration of potassium chloride solutions (KCl, 0.375-1.25 mumol, i.c.v.) produced concentration-dependent reductions in the arterial blood pressure and heart rate. These responses were significantly attenuated by prior i.c.v.-administration ouabain, a selective inhibitor of the Na+ pump, and by endothelin (ET-1), an endogenous peptide that is present in the CNS, suggesting that this peptide may participate in the neural regulation of arterial pressure via modulation of Na(+)-pump activity. Although both acute fluid volume expansion and/or osmotic stimulus have been shown to facilitate the release of the endogenous Na(+)-pump inhibitor(s) into the circulation, only volume expansion significantly attenuated the cardiovascular effects of i.c.v. potassium chloride. These observations collectively suggest that the Na+, K(+)-ATPase activity in CNS and Na(+)-pump inhibitors may play a significant role in the central regulation of arterial pressure under certain physiological conditions.

Animals↗
PubMed1993

Migration of Drosophila germ cells: analysis using enhancer trap lines.

Cell migration is a common feature of development. In order to understand more about the factors that control these movements we have embarked on further analysis of the migration of Drosophila germ cells. This process involves passage of the germ cells across the gut primordium and migration toward the mesoderm where the somatic gonad forms. We are particularly interested in the early phase of this migration when the germ cells interact with the amnioproctodeal invagination, the developing gut, before entering into association with the mesoderm. We will summarize the results of our and other studies of these events before describing a number of enhancer trap lines which show expression in the amnioproctodeal invagination during the early phase of germ cell migration. These reveal more about the complexity of this tissue and suggest this tissue is capable of guiding the early phase of germ cell migration.

Animals↗
PubMed1992

Circular dichroic studies of protein kinase C and its interactions with calcium and lipid vesicles.

Circular dichroism was used to study the secondary structure of protein kinase C (PKC) in aqueous solution and the conformational changes resulting due to the presence of its regulatory cofactors (e.g. Ca2+, phosphatidylserine (PS) and phorbol 12-myristate 13-acetate (PMA)). Computer analysis of the CD data for the estimates of secondary structure showed that PKC maintains a highly ordered structure containing 36% alpha-helix, 57% beta-sheet and 7% beta-turn. PKC displays a minor conformational change upon addition of Ca2+. However, a larger change is observed on adding phosphatidylserine vesicles in the presence of Ca2+. In this case, the alpha-helix content is decreased by approx. 35% and beta-sheet increased by approx. 16%. The protein does not experience further significant changes in conformation on adding PMA.

Adenosine Triphosphate↗
PubMed1992

Antagonism by endothelin of centrally mediated cardiovascular effects of potassium in anaesthetized rats.

1. We have previously demonstrated that cerebroventricular administration of potassium chloride (KCl) solutions produces dose-dependent reductions in blood pressure and heart rate in anaesthetized rats and that these effects are significantly attenuated by ouabain, a selective inhibitor of the Na(+)-pump. These observations suggest an important relationship between Na+,K(+)-ATPase activity in the central nervous system (CNS) and neural mechanisms involved in the regulation of cardiovascular function. 2. Since endothelin-1 (ET-1) has been shown to affect various ion transport mechanisms, including the Na(+)-pump, the present studies were conducted to evaluate whether this peptide would antagonize central effects of KCl. 3. The present studies demonstrate that cumulative doses of ET-1 (0.8-3.2 pmol, intracerebrolateral ventricular administration, i.c.v.) produced significant attenuation of hypotension and bradycardia produced by i.c.v. injections of KCl (0.75 mumol/5 microL, i.c.v.); in a separate series, a single high dose of ET-1 (4.0 pmol, i.c.v.) significantly reduced cardiovascular responses to various doses of KCl (0.375, 0.75, 1.25 mumol/5 microL, i.c.v.). 4. These studies suggest that endothelin may be involved in the regulation of arterial pressure since it is present in CNS and possesses a ouabain-like effect. However, it is not conclusive that ET-1 inhibits neuronal Na(+)-pump, since alternative mechanisms can also account for the efficacy of the peptide to antagonize central effects of potassium chloride.

Animals↗
PubMed1991

Effect of cadmium on Ca2+ transport in brain microsomes.

The effect of Cd2+ on Ca2+ transport properties (uptake/release) in rat brain microsomes is examined by the tracer method using 45Ca2+. Cadmium ion (Cd2+) shows a dose-dependent inhibition of Ca(2+)-ATPase activity and consequently, exhibits a reduction in ATP-dependent Ca2+ uptake. In addition to this, Cd2+ also stimulates a rapid release of Ca2+ (t1/2 = 0.5 min) from the microsomes in a dose-dependent manner. The effect of Cd2+ is reversible by 1 mM cysteine or dithiothreitol (DTT). It is suggested that Cd2+ plays an important role in regulating the transmembrane flux of the cations in the microsomes. This effect is dramatically modulated by DTT suggesting a role of sulfhydryl groups in Ca(2+)-transport.

Animals↗
PubMed1991

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.

Acetylation↗
PubMed1991

Polymerase chain reaction-mediated gene synthesis: synthesis of a gene coding for isozyme c of horseradish peroxidase.

The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory. In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target. The PCR facilitates synthesis and purification of the gene simultaneously. The gene for HRP was synthesized by ligating all 40 oligonucleotides in a single step followed by PCR amplification. The gene was also synthesized from its fragments by using an overlap extension method similar to the procedure as described [Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. (1989) Gene 77, 61-68]. A method for combining different DNA fragments, in-frame, by using the PCR was also developed and used to synthesize the HRP gene from its gene fragments. This method is applicable to the synthesis of even larger genes and to combine any DNA fragments in-frame. After the synthesis, preliminary characterization of the HRP gene was also carried out by the PCR to confirm the arrangement of oligonucleotides in the gene. This was done by carrying out the PCR with several sets of primers along the gene and comparing the product sizes with the expected sizes. The gene and the fragments generated by PCR were cloned in Escherichia coli and the sequence was confirmed by manual and automated DNA sequencing.

Base Sequence↗
PubMed1991

A dose-ranging study of the pharmacokinetics of codeine phosphate following intravenous administration to rats.

The linearity of the pharmacokinetics of codeine was examined in male Sprague-Dawley rats given iv bolus doses of 1, 1.5, 3, and 4 mg/kg of codeine phosphate. Codeine and morphine were determined in serial blood samples utilizing HPLC with electrochemical detection. Codeine exhibits characteristics consistent with a two-compartment pharmacokinetic model. The kinetics of codeine are linear in the iv dose range 1-4 mg/kg. The ratio AUCmorphine:AUCcodeine increases disproportionately with increasing doses of codeine.

Animals↗
PubMed1991

Role of Na+,K(+)-ATPase in the centrally mediated hypotensive effects of potassium in anaesthetized rats.

Several investigators have demonstrated the antihypertensive properties of potassium in various models of hypertension. The present studies were conducted to determine whether central mechanisms contribute to these salutary effects of potassium. In Inactin-anaesthetized rats, intracerebroventricular administration of KCl solutions (0.375, 0.75 and 1.25 mumol/5 microliters) produced concentration-dependent reductions in arterial pressure and heart rate. These effects were significantly attenuated by prior central administration of ouabain, a selective inhibitor of the sodium pump. In a separate series of experiments, prior central administration of alpha 1- and alpha 2-antagonist phentolamine, or the dopamine receptor (DA1 and DA2) antagonist RS-sulpiride, was also effective in inhibiting the hypotensive and bradycardiac effects of intracerebroventricular administration of potassium. Thus, these data suggest that activation of Na+,K(+)-ATPase and central noradrenergic and dopaminergic mechanisms are involved in the central actions of potassium and these central mechanisms may contribute to the salutary effects of a potassium-rich diet in hypertensive subjects. The present studies demonstrate a potentially important relationship between Na+,K(+)-ATPase activity in the central nervous system and neural regulation of arterial blood pressure.

Anesthesia, General↗
PubMed1991

Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA.

The phylogenetic interrelationships of members of the genus Listeria were investigated by using reverse transcriptase sequencing of 16S rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeria monocytogenes group of species (including Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) and (ii) the species Listeria grayi and Listeria murrayi. At the intergeneric level a specific phylogenetic relationship between the genera Listeria and Brochothrix was evident. The sequence data clearly demonstrated that the genus Listeria is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobacillaceae.

Base Sequence↗