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Biomedical subjects

S K Sharma

Publications and source records attributed to S K Sharma.

At least 19 recordsLinked to original sources
PubMed1992

Immunodetection of recombinant proteins based on antibodies directed against a metal binding peptide engineered for purification by immobilized metal affinity chromatography.

Recently we reported (Evans, D.B., Tarpley W.G. and Sharma, S.K. (1991) Expression and characterization of chimeric rDNA proteins engineered for purification and cleavage. Protein Expr. Purif. 2, 205-213) a genetically engineered metal binding peptide (mbp) for the purification of recombinant proteins by immobilized metal affinity chromatography (IMAC). Therefore, we have been interested in developing mbp-based immunodetection methods for these engineered proteins. To this end, the following linker peptide containing the mbp (His-Asp-His-Asp-His) was designed, synthesized and conjugated to porcine thyroglobulin: Ac-Cys-Gly-Glu-Glu-His-Asp-His-Asp-His-Pro-Phe-His-Leu. Rabbits immunized with this conjugate developed antibodies that cross-react with peptides containing the mbp sequence. A number of chimeric recombinant proteins, expressed in E. coli, with and without the mbp portion (His-Asp-His-Asp-His) of the fusion peptide (His-Asp-His-Asp-His-Pro-Phe-His-Leu) were analyzed by ELISA and immunoblotting. Results from these studies show that the anti-mbp antibodies detect chimeric proteins containing the mbp, while chimeric proteins lacking this pentapeptide were negative in both immunodetection techniques. The usefulness of this approach has also been demonstrated in following IMAC purification and enzymatic cleavage of the mbp. These immunodetection techniques utilizing anti-mbp antibodies should be applicable to other proteins engineered to contain the mbp for IMAC purification.

Amino Acid Sequence↗
PubMed1992

Enzymatic kinetic studies with the non-nucleoside HIV reverse transcriptase inhibitor U-9843.

The polymer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT (reverse transcriptase) and the drug possesses excellent antiviral activity at nontoxic doses in HIV-infected lymphocytes grown in tissue culture. The drug also inhibits RTs isolated from other species such as AMV and MLV retroviruses. Enzymatic kinetic studies of the HIV-1 RT catalyzed RNA-directed DNA polymerase function, using synthetic template:primers, indicate that the drug acts generally noncompetitively with respect to the template:primer binding site but the specific inhibition patterns change somewhat depending on the drug concentration. The inhibitor acts noncompetitively with respect to the dNTP binding sites. Hence, the drug inhibits this RT polymerase function by interacting with a site distinct from the template:primer and dNTP binding sites. In addition, the inhibitor also impairs the DNA-dependent DNA polymerase activity of HIV-1 RT and the RNase H function. This indicates that the drug interacts with a target site essential for all three HIV RT functions addressed (RNA- and DNA-directed DNA polymerases, RNase H).

Antiviral Agents↗
PubMed1992

An ultrasensitive human immunodeficiency virus type 1 protease radioimmuno rate assay with a potential for monitoring blood levels of protease inhibitors in acquired immunodeficiency disease syndrome patients.

The angiotensin I-based peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser yields angiotensin I (Ang I) and Leu-Glu-Glu-Ser upon hydrolysis by the human immunodeficiency virus type 1 (HIV-1) protease, but not by human renin. N-terminal sequencing of the reaction products showed that the HIV-1 protease cleaved exclusively at the Leu-Leu bond. The rate of Ang I formation can be measured by a radioimmunoassay, since the parent peptide has minimal cross reactivity in this assay. The rate of enzymatic hydrolysis is maximal at pH 4.5-5.0 and at an ionic strength of 1 M. At 37 degrees C, 0.1 M Na acetate buffer, pH 5.0, 1 M NaCl, 10% glycerol, 5% ethylene glycol, 1 mg/ml bovine serum albumin, and 3 mM EDTA, the reaction obeys Michaelis-Menten type kinetics with Km = 17.2 +/- 3.5 microM and kcat = 2.30 +/- 0.33 min-1. The activity assay readily quantitates as little as 0.25 nM of HIV-1 protease. The production of Ang I by the HIV-1 protease is inhibited in the presence of a HIV-1 protease inhibitor. The newly discovered substrate is relatively insensitive to human or monkey serum. Therefore, the effect of sera from 20 patients with advanced acquired immunodeficiency disease syndrome (AIDS) on Ang I production in the above assay system was examined. Results of this study indicate that it may be possible to adapt the above Ang I-based system to determine blood levels of HIV-1 protease inhibitors in AIDS patients during clinical trials.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Adjunctive thrombolytic therapy for angioplasty in ischemic rest angina: results of a double-blind randomized pilot study.

OBJECTIVES: A multicenter pilot study was instituted to assess the role of intracoronary thrombolytic therapy during angioplasty for ischemic rest angina. BACKGROUND: Acute thrombotic coronary occlusion is increased during angioplasty for unstable angina, and intracoronary thrombolytic agents have been used to maintain patency. Prophylactic use of intracoronary thrombolytic agents has been advocated in certain high risk subgroups, although no studies have randomized therapy. METHODS: Ninety-three patients with either unstable angina and pain at rest (trial A, 66 patients) or postinfarction pain at rest (trial B, 27 patients) were randomized in double-blind fashion to administration of either intracoronary urokinase, 150,000 U, or saline solution placebo given immediately before angioplasty. Cineangiograms of the culprit lesion were recorded and analyzed in blinded fashion by a core laboratory for definite or possible (haziness) filling defects 15 min after angioplasty or after acute closure. RESULTS: Urokinase decreased filling defects at 15 min after angioplasty in comparison with placebo (14% vs. 29%, respectively, p = 0.08). Four patients in each treatment group developed acute vessel closure. However, although urokinase significantly reduced the incidence of filling defects in trial A (3% vs. 23%, p = 0.03), the drug had no effect at the selected dose in trial B (42% vs. 43%, respectively). Acute vessel closure occurred significantly more frequently in trial B than in trial A, and urokinase at the selected dose also had no effect. Ischemic events after angioplasty appeared to be related more to dissection than to thrombosis, although redilation, which was more frequent after placebo administration, may have reduced their incidence as well as that of acute closure. CONCLUSIONS: These data suggest a possible role for intracoronary urokinase during angioplasty for unstable angina. The lack of effect after infarction may represent a greater thrombus burden or degree of plaque disruption. A trial utilizing higher doses of urokinase in a larger patient group is in progress.

Angina, Unstable↗
PubMed1992

Clinical, angiographic and anatomic findings in acute severe ischemic mitral regurgitation.

Severe mitral regurgitation (MR) due to coronary artery disease unfavorably alters prognosis for medical therapy and is also associated with increased surgical mortality. In this report, the clinical, angiographic and pathoanatomic findings in 50 consecutive patients with severe ischemic MR were characterized. Forty-two patients (84%) either presented with acute myocardial infarction or a well-documented prior infarction. Eleven patients (22%) were in cardiogenic shock at the time of catheterization. Forty patients (80%) had greater than 70% stenosis of the right and left circumflex coronary arteries with or without left anterior descending coronary artery stenosis. Segmental asynergy of the left ventricular wall was present in 48 patients (96%) and involved the inferior wall in 43 (86%). Mean ejection fraction for the group was 51 +/- 7%. A total of 15 patients had direct inspection of the mitral valve apparatus at surgery or autopsy. Posteromedial papillary muscle involvement was found in 14 patients, fibrosis or necrosis in 10 and rupture in 4, with anterolateral papillary muscle rupture in 1 patient. Thus, acute severe ischemic MR is usually associated with significant narrowing of both right and left circumflex coronary arteries, and posteromedial papillary muscle involvement.

Acute Disease↗
PubMed1992

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.

Animals↗
PubMed1992

On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Amino Acid Sequence↗
PubMed1992

Case report: traumatic loss of the entire urethra and bladder neck in a girl--reconstruction by modified Flocks bladder tube.

Complete loss of the urethra and bladder neck secondary to pelvic fracture in a female patient is rare. We report a case of pelvic fracture resulting from a traffic collision leading to complete loss of the urethra including the bladder neck in an adolescent girl. A new urethra was reconstructed from a modified Flocks bladder tube and wrapped with omentum. At 2 years follow-up, the patient is fully continent and voiding satisfactorily. The problems of female urethral reconstruction are discussed.

Adolescent↗
PubMed1992

Congenital tracheo-oesophageal fistula in a young adult.

Congenital tracheo-oesophageal fistula (TEF) is rare in adults. Patients who present with repeated attacks of chest infection since birth or cough, choking and cyanosis during feeding should be investigated for TEF. It should be possible to detect all cases of tracheo-oesophageal fistulae using bronchoscopy, oesophagoscopy and CT either singly or in combination. These investigations also help in deciding on the route of exploration and the type of surgery. Disconnection of the abnormal fistulous tract brings dramatic relief of symptom and prevents further pulmonary damage.

Adult↗
PubMed1992

Chemodissolution of urinary uric acid stones by alkali therapy.

Experience with chemodissolution of uric acid stones in 30 patients is presented. Chemodissolution was achieved either with infusion of 0.16 M i.v. lactate or oral sodium bicarbonate, in addition to liberal fluid intake and allopurinol wherever indicated. In some cases direct chemodissolution by in situ irrigation with sodium bicarbonate solution was done after an initial percutaneous nephrostomy. Seven patients presented with acute obstructive anuria. In this group, 5 of them had bilateral obstructive calculi, while 2 had unilateral obstruction in a solitary kidney. The latter 2 had complete recovery following intravenous lactate therapy. Of the 5 presenting with bilateral obstruction, 2 patients had complete response to chemodissolution, whereas the remaining 3 had only a partial response requiring surgery for ultimate salvage. In this group I, 6 patients are doing well with a normal serum creatinine at 3 months to 4 years follow-up, while 1 patient has a serum creatinine, stabilised at 3.2 mg%. In the second group, 23 patients presented with non-obstructing urinary stones. Flank pain was the commonest complaint and a concomitant history of gout was present in 6 patients. Hyperuricaemia was detected in 12 and hyperuricosuria in 19. All cases were managed by high fluid intake and oral sodium bicarbonate, with self-monitoring of urine pH, which was kept between 6.5 and 7.0. Allopurinol was administered in cases having hyperuricaemia and/or hyperuricosuria. Systemic alkali therapy in the form of intravenous molar lactate or sodium bicarbonate is effective and safe both in obstructive anuria and non-obstructive urinary uric acid stones.

Aged↗
PubMed1992

Giant calculus in anterior urethral diverticulum.

The case of probably the largest stone in a giant anterior urethral diverticulum presenting with septicemia and renal failure is reported. Stage surgical reconstruction had an excellent result.

Diverticulum↗
PubMed1992

Pulmonary function and immunologic abnormalities in miliary tuberculosis.

In a group of 33 patients with miliary tuberculosis (MTB), pulmonary function testing and bronchoalveolar lavage (BAL) were each performed in 31. The arterial blood gas analysis was done in 13 patients. Mild restrictive ventilatory defect, hypoxemia, and hypocapnia were observed. BAL revealed lymphocytic alveolitis. The lymphocyte subsets were measured in the BAL fluid and peripheral blood in 10 patients. The helper lymphocytes were decreased in the peripheral blood and increased in the BAL fluid. The immunoglobulins (IgG, IgA, and IgM) were significantly increased in the peripheral blood and BAL fluid in 18 patients. Serum complement (C3) (n = 23) and BAL fluid fibronectin (n = 22) were also significantly increased. Lymphocytic alveolitis and the raised levels of IgG and IgA in the peripheral blood and BAL fluid persisted at the end of 9 months of standard chemotherapy for tuberculosis. Long-term studies with larger numbers of patients are required to monitor the course of alveolitis in MTB. An addition of corticosteroids to the antituberculosis drug regimen may be considered in the treatment of these patients.

Adult↗
PubMed1992

Characterization of rat gastric inhibitory peptide cDNA.

Gastric inhibitory peptide (GIP) is a 42 amino acid gastrointestinal peptide which inhibits gastric acid secretion and stimulates pancreatic insulin secretion in the presence of glucose. Here we report the sequence of the cDNA encoding the rat GIP precursor. PreproGIP was 144 amino acids in length and comprised the GIP peptide itself, N- and C-terminal flanking peptides of 22 and 59 amino acids respectively and a typical hydrophobic signal peptide. The sequence indicated that GIP is released from its precursor by cleavage at single arginine residues. The C-terminal flanking peptide may have an important function since it was well conserved and contained a region of 16 amino acids with only a single, conservative replacement. Rat GIP mRNA was found in the duodenum and jejunum. Levels of GIP mRNA in the duodenum were increased twofold after a period of 2 days of starvation. There was no detectable expression of the GIP gene in other parts of the gastrointestinal tract or in other endocrine tissues. However, in pancreatic mRNA preparations, a larger mRNA was detected after low stringency hybridization. This could represent a further member of this gene family.

Amino Acid Sequence↗
PubMed1992

Haematological and coagulation profile in acute falciparum malaria.

The haematological and coagulation profile of 30 cases of acute falciparum malaria were studied. Anaemia, mostly normocytic, normochromic, was observed in 86.7% of cases majority of whom had complications. Severe anaemia (HB < 6gm.) observed in 10% of cases was associated with 100% mortality. Leucocytosis and leucopenia were observed in 13.3% and 6.6% of cases respectively. 90% of cases had thrombocytopenia, the lowest count recorded being 26,000/- cmm. 16.7% of cases had evidence of intravascular coagulation but manifested as generalised bleeding in only one case. Bone marrow aspiration done in 10 cases revealed no abnormality, except for falciparum parasites observed in 2 cases.

Adult↗
PubMed1992

Wegener's granulomatosis in northern India.

Till recently, Wegener's granulomatosis (WG) was considered a rare disease in India. Over the last 5 years we studied 13 proven cases of WG. This paper describes the details of the disease as seen in these patients, and response to corticosteroid and cyclophosphamide therapy, and compares these observations with a Western and an Indian study.

Adult↗
PubMed1992

Oak (Quercus incana) leaf poisoning in cattle.

Oak poisoning occurred in crossbred cattle due to eating immature tender oak (Quercus incana) leaves. Mortality was 70%. The animals exhibited anorexia, severe constipation and brisket edema. The feces were hard, pelleted and coated with blood and mucous. Significant reductions in blood hemoglobin and mean corpuscular hemoglobin, and significant elevations in serum bilirubin were observed. Serum urea nitrogen and creatinine were greatly increased. There was bilirubinuria, proteinuria, hypoproteinemia and hypocalcemia, and greatly increased activities of serum aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase. The levels of tannins and condensed tannins were 97.7 mg tannic acid equivalent and 5.8 mg catechin equivalent/g of dry leaves. There was extensive nephro- and hepatotoxicity in the affected cattle due to hydrolysable tannins and simple phenols in the oak leaves.

Anemia, Hemolytic↗
PubMed1991

A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active.

We report here a human immunodeficiency virus type 1 (HIV-1) recombinant ribonuclease H (RNase H) domain engineered to contain an N-terminal tag for its isolation by affinity chromatography. The purified protein is active in hydrolyzing RNA-DNA hybrids in two separate in vitro assay systems. In light of recent reports of similar HIV-1 RNase H domains which were enzymatically inactive (Becerra, S. P., Clore, G. M., Gronenborn, A. M., Karlstrom, A. R., Stahl, S. J., Wilson, S.M., and Wingfield, P.T. (1990) FEBS Lett. 270, 76-80; Hostomsky, Z., Hostomska, Z., Hudson, G. O., Moomaw, E. W., and Nodes, B. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1148-1152), our results suggest that a stretch of 20-30 residues immediately upstream of the polymerase-RNase H junction (residues 440-441 of HIV-1 reverse transcriptase) may be required for productive binding and alignment of the hybrid RNA-DNA substrate. The active HIV-1 RNase H domain is suitable for structural analysis, thereby providing a unique active molecule to better understand the structural basis for the functional organization of RNase associated with the HIV-1 reverse transcriptase.

Amino Acid Sequence↗