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Biomedical subjects

S Trivedi

Publications and source records attributed to S Trivedi.

At least 19 recordsLinked to original sources
PubMed2006

Protein thermostability in Archaea and Eubacteria.

In order to survive at high temperatures, thermophilic prokaryotes (Archaea and Eubacteria) adopt different strategies. Among several important contributing factors for stability of proteins are CG-rich codons, the ratio of charged amino acids compared to uncharged amino acids, ionic interactions, amino acid preferences and their distribution, post-translational modifications, and solute accumulation. However, these factors may differ from taxon to taxon, both within and between species depending upon the composition of proteins found in these organisms. This is exemplified in the case of differences in strategies adopted by soluble proteins and membrane proteins. Therefore, it appears that no single factor or combination of factors together can be universally attributed to the provision of thermal stability in proteins.

Adaptation, Biological↗
PubMed2006

Comparison of simple sequence repeats in 19 Archaea.

All organisms that have been studied until now have been found to have differential distribution of simple sequence repeats (SSRs), with more SSRs in intergenic than in coding sequences. SSR distribution was investigated in Archaea genomes where complete chromosome sequences of 19 Archaea were analyzed with the program SPUTNIK to find di- to penta-nucleotide repeats. The number of repeats was determined for the complete chromosome sequences and for the coding and non-coding sequences. Different from what has been found for other groups of organisms, there is an abundance of SSRs in coding regions of the genome of some Archaea. Dinucleotide repeats were rare and CG repeats were found in only two Archaea. In general, trinucleotide repeats are the most abundant SSR motifs; however, pentanucleotide repeats are abundant in some Archaea. Some of the tetranucleotide and pentanucleotide repeat motifs are organism specific. In general, repeats are short and CG-rich repeats are present in Archaea having a CG-rich genome. Among the 19 Archaea, SSR density was not correlated with genome size or with optimum growth temperature. Pentanucleotide density had an inverse correlation with the CG content of the genome.

Archaea↗
PubMed2006

A decade's experience with vesicovaginal fistula in India.

A retrospective analysis of 252 cases of vesicovaginal fistulae was done to analyse its etio-pathology and management in an Indian population. After a thorough evaluation, different techniques of fistula closure were used for repair and the results were listed. The main outcome measures were the etiology of the fistula, need for tissue interposition and cure rate per repair as well as the overall cure rate. We compared our results with literature and concluded that simple and small fistulae should be repaired with layered closure. All complicated fistulae should be repaired with tissue interposition or tissue graft. This is the first study from India compiling 10 years of experience on vesicovaginal fistula.

Adolescent↗
PubMed2006

Post caesarean vesicouterine fistulae-- Youssef syndrome: our experience and review of published work.

OBJECTIVE: To analyse the incidence, symptomatology, diagnosis and therapeutic aspects of Youssef syndrome (post caesarean vesicouterine fistula), and to review relevant published work. METHODS: A retrospective study from the urosurgical unit of a tertiary care referral hospital was carried out. In a retrospective analysis of urogenital fistulae over 10 years, we identified 14 patients with uterovesical fistulae, resulting from caesarean section. All the patients were evaluated by history, physical examination, radiological tests and cystoscopy. All patients underwent transperitoneal repair of these fistulae with omental interposition. Results of surgery were evaluated by absence of cyclic haematuria, stoppage of urinary incontinence, and achievement of fertility. RESULTS: A total of 12 patients who had minimum follow up was included in the present study. The results showed that 50% of the fistulae resulted from emergency caesarean operation with 58% of patients presenting after their second caesarean section. The mean age of the patients was 19 years (range 15-29) and mean duration of symptoms was 7 months (range 3-16). Menouria and amenorrhoea were predominant presenting symptoms. The results of surgical treatment were excellent with good continence and resolution of the cyclic haematuria. Three pregnancies (37.5%) which resulted in elective caesarean section were recorded. CONCLUSION: Vesicouterine fistulae, despite being infrequent, are no longer a rare diagnosis and are most commonly secondary to lower segment caesarean section. With patient history and selected investigations diagnosis is relatively easy. The surgical repair of these fistulae is standard treatment, especially with delayed fistulae with achievement of total continence, and complete resolution of cyclic haematuria. Meticulous practice of obstetric and surgical principles during caesarean section can prevent the formation of these fistulae.

Adolescent↗
PubMed2004

Three-dimensional finite element analysis of residual stress in arteries.

Calculation of residual stress in arteries, using the analytical approach has been quite valuable in our understanding of its critical role in vascular mechanics. Stresses are calculated at the central section of an infinitely long tube by imposing a constant axial stretch while deforming the artery from the stress-free state to its unloaded state. However, segments used to perform opening-angle measurements have finite lengths. Further, the stress-free artery configuration is assumed to be circular. Experiments show that they are slightly noncircular. The numerical approach to residual stress calculation can allow us to study both these issues. Using 3D cylindrical geometries and an isotropic material model, we investigated how segment length can affect residual stress calculations and identified the appropriate segment length for experiments. Further, we recorded and used the true noncircular stress-free state of an artery segment, computed the residual stress distribution, and compared it to that from a similar, but circular segment. Our findings suggest that segment length must be ten times the wall thickness for it to be "long" enough. We also found that the circularity assumption may be a reasonable approximation for typical arteries.

Animals↗
PubMed2002

Laparoscopic ureterolithotomy: technical considerations and long-term follow-up.

OBJECTIVE: To analyse the technical details and the long-term results of laparoscopic ureterolithotomy. PATIENTS AND METHODS: Laparoscopic ureterolithotomy was undertaken in 101 patients between 1991 and 2001; in only one patient with retroperitoneal fibrous adhesions was the procedure transperitoneal, being retroperitoneal in all the others, using Gaur's balloon retroperitoneoscopy. The mean (range) stone size was 16 (10-47) mm, and the stones were in the upper ureter in 75, mid-ureter in 11 and lower ureter in 15 patients. Nine patients had more than one stone, the maximum being six, in a megaureter. Most were impacted for > 2 months, the maximum being 240 months. RESULTS: Laparoscopic ureterolithotomy was successful in 93 patients, with the eight failures being mostly early in the series. The mean operative duration was 79 min (66 min when the ureter was left open and 92 min when it was sutured). The overall mean duration of urinary leakage was 5.5 days, which was reduced to 3.2 days by stenting and suturing the ureter. The mean (range) blood loss was 25 (5-100) mL. The overall complication rate was high (31%) because of prolonged urinary leakage in 20 patients. No patient required morphine for pain relief and the mean for oral analgesic use was 2.5 days. The mean hospital stay was 3.5 days and that for resuming work 14 (7-28) days. CONCLUSIONS: Laparoscopic ureterolithotomy by the retroperitoneal approach is a safe and reliable minimally invasive procedure. Although its role as a salvage procedure for failed extracorporeal shock wave lithotripsy and ureteroscopy is undisputed, in selected patients with large chronically impacted ureteric stones and particularly with solitary kidneys, it may be considered the first-line treatment.

Adolescent↗
PubMed2002

Retroperitoneal laparoscopic pyelolithotomy for staghorn stones.

PURPOSE: To establish the feasibility of the retroperitoneal laparoscopic approach for treatment of patients with staghorn renal calculi who are under consideration for an open pyelolithotomy. PATIENTS AND METHODS: Retroperitoneal laparoscopic pyelolithotomy was performed in three patients with impacted staghorn renal calculi between 22 and 45 mm in largest diameter. One of the patients also had multiple caliceal stones. These patients wanted an open procedure but had agreed to have their stone removal done laparoscopically in view of our experience. The previously described retroperitoneal laparoscopic approach was used, and the handling of some of the specific problems in such patients is described in the text. RESULTS AND CONCLUSION: The stones were removed successfully in all the three patients, and there were no complications. Although no definitive conclusion can be drawn from this small series, the paper shows the feasibility of the retroperitoneal laparoscopic approach in a select group of patients with staghorn renal calculi.

Adult↗
PubMed2000

Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor.

Familial hyperparathyroidism (HPT), characterized by hypercalcemia and hypercalciuria, and familial benign hypocalciuric hypercalcemia (FHH) are the most common causes of hereditary hypercalcemia. The calcium-sensing receptor (CaR) regulates PTH secretion and renal calcium excretion. Heterozygous inactivating mutations of the gene cause FHH, whereas CaR gene mutations have not been demonstrated in HPT. In a kindred with 20 affected individuals, the hypercalcemic disorder segregated with inappropriately higher serum PTH and magnesium levels and urinary calcium levels than in unaffected members. Subtotal parathyroidectomy revealed parathyroid gland hyperplasia/adenoma and corrected the biochemical signs of the disorder in seven of nine individuals. Linkage analysis mapped the condition to markers flanking the CaR gene on chromosome 3q. Sequence analysis revealed a mutation changing phenylalanine to leucine at codon 881 of the CaR gene, representing the first identified point mutation located within the cytoplasmic tail of the CaR. A construct of the mutant receptor (F881L) was expressed in human embryonic kidney cells (HEK 293), and demonstrated a right-shifted dose-response relationship between the extracellular and intracellular calcium concentrations. The hypercalcemic disorder of the present family is caused by an inactivating point mutation in the cytoplasmic tail of the CaR and displays clinical characteristics atypical of FHH and primary HPT.

Adult↗
PubMed1999

Intermolecular interactions between dimeric calcium-sensing receptor monomers are important for its normal function.

We recently demonstrated that the G protein-coupled, extracellular calcium-sensing receptor (CaR) forms disulfide-linked dimers. The functional significance of dimerization of this receptor was suggested by our earlier observations that CaRs carrying certain point mutations exert dominant negative effects on the function of the coexpressed wild-type receptor both in vivo and when cotransfected in human embryonic kidney cells. In this study, we explored the functional consequences of CaR dimerization. Coexpression in human embryonic kidney cells of specific pairs of mutant CaRs, each with reduced or absent activity because of distinct loss-of-function mutations, results in the formation of heterodimers and partially reconstitutes extracellular calcium-dependent signaling. Moreover, our results suggest that the CaR has at least two functionally separable domains. However, the presence of an abnormal domain in each mutant monomer substantially impairs the function of the CaR heterodimer, resulting in the reconstituted CaRs having characteristics distinct from those of the wild-type CaR. Our study suggests that intermolecular interactions within the dimeric CaR are important for the receptor's function.

Calcium Signaling↗
PubMed1999

A family history study of male sexual orientation using three independent samples.

Available evidence suggests that male homosexuality is both familial and somewhat heritable and that some cases may be caused by an X-linked gene. However, most studies have recruited subjects in a relatively unsystematic manner, typically via advertisements, and hence suffer from the potential methodological flaw of ascertainment bias due to volunteer self-selection. In the present study we assessed the familiality of male homosexuality using two carefully ascertained samples and attempted to replicate findings consistent with X-linkage in three samples. The percentage of siblings of the probands rated as either homosexual or bisexual, with a high degree of certainty, ranged from 7 to 10% for brothers and 3 to 4% for sisters. These estimates are higher than recent comparable population-based estimates of homosexuality, supporting the importance of familial factors for male homosexuality. Estimates of lambda s for male homosexuality ranged from 3.0 to 4.0. None of the samples showed a significantly greater proportion of maternal than paternal homosexual uncles or homosexual male maternal first cousins. Although our results differed significantly with those of some prior studies, they do not exclude the possibility of moderate X-linkage for male sexual orientation.

Adolescent↗
PubMed1998

Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells.

The extracellular calcium (Ca2+o)-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface cross-linking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.

Biotinylation↗
PubMed1998

Protein kinase C phosphorylation of threonine at position 888 in Ca2+o-sensing receptor (CaR) inhibits coupling to Ca2+ store release.

Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+i dynamics in parathyroid cells.

Calcium↗
PubMed1998

Sodium and ionic strength sensing by the calcium receptor.

The calcium-sensing receptor (CaR) is activated by small changes in extracellular calcium [Ca2+]o) in the physiological range, allowing the parathyroid gland to regulate serum [Ca2+]o; however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and charged residues in the extracellular domain of the CaR appear critical for receptor activation through electrostatic interactions, suggesting that ionic strength could modulate CaR activation by polycationic agonists. Changes in the concentration of external NaCl potently altered the activation of the CaR by external Ca2+ and spermine. Ionic strength had an inverse effect on the sensitivity of CaR to its agonists, with lowering of ionic strength rendering the receptor more sensitive to activation by [Ca2+]o and raising of ionic strength producing the converse effect. Effects of osmolality could not account for the modulation seen with changes in NaCl. Other salts, which differed in the cationic or anionic species, showed shifts in the activation of the CaR by [Ca2+]o similar to that elicited by NaCl. Parathyroid cells were potently modulated by ionic strength, with addition of 40 mM NaCl shifting the EC50 for [Ca2+]o inhibition of parathyroid hormone by at least 0.5 mM. Several CaR-expressing tissues, including regions of the brain such as the subfornical organ and hypothalamus, could potentially use the CaR as a sensor for ionic strength and NaCl. The Journal guidelines state that the summary should be no longer than 200 words.

Adenosine Triphosphate↗
PubMed1998

4-iodotamoxifen aziridine, a new affinity labeling agent for the rapid detection of estrogen receptor isoforms.

We describe the simple and fast preparation of a new radioiodinated probe for the detection of the estrogen receptor (ER) and its isoforms. Iodotamoxifen aziridine was labeled with iodine 125 ([125I]TAZ) in position 4 of the alpha aromatic ring. The yield was high (>75%), the label was stable and the specific activity was near optimal (1900-2170 Ci/mmol). The apparent relative binding affinity of the probe to a recombinant human ER (hER) was high (RBA = 35 vs estradiol = 100). Electrophoretic studies (SDS-PAGE) with this hER indicated the high potency of [125I]TAZ at very low concentration (<1 nM) to reveal ER bands after a short exposure time (1-4 days). Competition between this probe and various compounds as well as chemical treatments of the ER with SH-reactive chemicals, demonstrated the labeling specificity. Analysis of cytosols from a panel of cell lines and various rat reproductive organs displayed characteristic ER bands (67, 50 and 37 kDa) suppressed by unlabeled E2. Detection in nonreproductive organs of 43 kDa E2-nondisplaceable peptide raised the question upon the presence of altered and/or variant ERs in many tissues. Data concerning human breast cancer cytosols were in complete accordance with those established with [3H]TAZ: high ER polymorphism in most ER-positive samples and peculiar forms (mainly 43 kDa) in ER-negative samples. Hence, [125I]TAZ appears especially useful for the detection of altered ER or related peptides in breast cancers.

Affinity Labels↗
PubMed1998

Evolution towards hormone independence of the MXT mouse mammary tumor is associated with a gradual change in its estrogen receptor molecular polymorphism.

Using a method based on [3H]tamoxifenaziridine ([3H]TAZ) labeling, sequential immunoadsorption with anti-ER monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and fluorography, we observed a striking change inthe estrogen receptor (ER) electrophoresis pattern of the transplantable MXT mouse mammary tumor. Early, ER "rich" tumors (approximately 100 fmol/mg prot) displayed classical cytosolic 67 and 50 KDa bands. These bands disappeared in favor of a "cytosolic" 35 KDa band during progression towards undifferentiated ER "poor" tumors (approximately 25 fmol/mg prot). Although we can not rule out that this 35 KDa peptide results from in vivo ER proteolysis, it seems unique in view of the following: 1. It is immunoadsorbed not only by an anti-ER monoclonal antibody (H-222) directed to the hormone-binding domain, but also by an anti-ER monoclonal antibody (H-226) which interacts with an epitope in the A/B region close to the DNA-binding domain and is mainly exposed under activation conditions. 2. It does not bind [3H]estradiol([3H]E2) and a tentative to restore its [3H]E2 binding capacity with calmodulin and ATP was unsuccessful. The observation of similar approximately 35 KDa ERs in the nuclear fraction of early tumor transplants and in control uterus suggests that this peptide is already in an activated form. Structural alterations of ER and/or associated "anchorage" nuclear proteins may beat the origin of its cytosolic localization. Moreover, the fact that the addition of calmodulin and ATP to late MXT transplants cytosols fails to increase their [3H]E2 binding capacity indicates that the low ER content of these tumors does not result from a deficiency in the phosphorylation status of the receptor.

Animals↗
PubMed1997

Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Missense mutations have been identified in the coding region of the extracellular calcium-sensing receptor (CASR) gene and cause human autosomal dominant hypo- and hypercalcemic disorders. The functional effects of several of these mutations have been characterized in either Xenopus laevis oocytes or in human embryonic kidney (HEK293) cells. All of the mutations that have been examined to date, however, cause single putative amino acid substitutions. In this report, we studied a mutant CASR with an Alu-repetitive element inserted at codon 876, which was identified in affected members of families with the hypercalcemic disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), to understand how this insertion affects CASR function. After cloning of the Alu-repetitive element into the wild-type CASR cDNA, we transiently expressed the mutant receptor in HEK293 cells. Expression of mutant and wild-type receptors was assessed by Western analysis, and the effects of the mutation on extracellular calcium (Ca2+(o)) and gadolinium (Gd3+(o)) elicited increases in the cytosolic calcium concentration (Ca2+(i)) were examined in fura-2-loaded cells using dual wavelength fluorimetry. The insertion resulted in truncated receptor species that had molecular masses some 30 kD less than that of the wild-type CASR and exhibited no Ca2+(i) responses to either Ca2+(o) or Gd3+(o). A similar result was observed with a mutated CASR truncated at residue 876. However, the Alu mutant receptor had no impact on the function of the coexpressed wild-type receptor. Interestingly, the Alu mutant receptor demonstrated decreased cell surface expression relative to the wild-type receptor, whereas the CASR (A877stop) mutant exhibited increased cell surface expression. Thus, like the missense mutations that have been characterized to date in families with FHH, the Alu insertion in this family is a loss-of-function mutation that produces hypercalcemia by reducing the number of normally functional CASRs on the surface of parathyroid and kidney cells. In vitro transcription of exon 7 of the CASR containing the Alu sequence yielded the full-length mutant product and an additional shorter product that was truncated due to stalling of the polymerase at the poly(T) tract. In vitro translation of the mutant transcript yielded three truncated protein products representing termination in all three reading frames at stop codons within the Alu insertion. Thus sequences within the Alu contribute to slippage or frameshift mutagenesis during transcription and/or translation.

Animals↗
PubMed1997

In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2+-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia.

We characterized the in vivo, cellular and molecular pathophysiology of a case of neonatal hyperparathyroidism (NHPT) resulting from a de novo, heterozygous missense mutation in the gene for the extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR). The female neonate presented with moderately severe hypercalcemia, markedly undermineralized bones, and multiple metaphyseal fractures. Subtotal parathyroidectomy was performed at 6 wk; hypercalcemia recurred rapidly but the bone disease improved gradually with reversion to an asymptomatic state resembling familial benign hypocalciuric hypercalcemia (FBHH). Dispersed parathyroid cells from the resected tissue showed a set-point (the level of Ca2+(o) half maximally inhibiting PTH secretion) substantially higher than for normal human parathyroid cells (approximately 1.8 vs. approximately 1.0 mM, respectively); a similar increase in set-point was observed in vivo. The proband's CaR gene showed a missense mutation (R185Q) at codon 185, while her normocalcemic parents were homozygous for wild type (WT) CaR sequence. Transient expression of the mutant R185Q CaR in human embryonic kidney (HEK293) cells revealed a substantially attenuated Ca2+(o)-evoked accumulation of total inositol phosphates (IP), while cotransfection of normal and mutant receptors showed an EC50 (the level of Ca2+(o) eliciting a half-maximal increase in IPs) 37% higher than for WT CaR alone (6.3+/-0.4 vs. 4.6+/-0.3 mM Ca2+(o), respectively). Thus this de novo, heterozygous CaR mutation may exert a dominant negative action on the normal CaR, producing NHPT and more severe hypercalcemia than typically seen with FBHH. Moreover, normal maternal calcium homeostasis promoted additional secondary hyperparathyroidism in the fetus, contributing to the severity of the NHPT in this case with FBHH.

Calcium↗