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Arun Malhotra

Publications and source records attributed to Arun Malhotra.

At least 19 recordsLinked to original sources
PubMed2007

Dual ectopic thyroid: case series and review of the literature.

Ectopic thyroid tissue is an uncommon congenital aberration. It is further unusual for ectopic thyroid to be present at 2 different sites simultaneously. Only 19 cases of dual ectopic thyroid have been reported in the English literature. Most of the patients were adolescents and presented with anterior neck swelling with or without altered metabolic status. Lingual/sublingual thyroid was the most common ectopic location. Subhyoid was the most common site of a second ectopic thyroid in these patients. The thyroid scan has been used successfully to diagnose ectopic thyroid tissue. We report 4 such cases in which ectopic thyroid tissue was simultaneously present at 2 different locations using thyroid scanning. The literature of already reported cases is reviewed in detail.

Adolescent↗
PubMed2006

The molecular basis for ligand specificity in a mouse olfactory receptor: a network of functionally important residues.

Sequence differences between members of the mouse olfac-tory receptor MOR42 subfamily (MOR42-3 and MOR42-1) are likely to be the basis for variation in ligand binding preference among these receptors. We investigated the specificity of MOR42-3 for a variety of dicarboxylic acids. We used site-directed mutagenesis, guided by homology modeling and ligand docking studies, to locate functionally important residues. Receptors were expressed in Xenopus oocytes and assayed using high throughput electrophysiology. The importance of the Val-113 residue, located deep within the receptor, was analyzed in the context of interhelical interactions. We also screened additional residues predicted to be involved in ligand binding site, based on comparison of ortholog/paralog pairs from the mouse and human olfactory receptor genomes (Man, O., Gilad, Y., and Lancet, D. (2004) Protein Sci. 13, 240-254). A network of 8 residues in transmembrane domains III, V, and VI was identified. These residues form part of the ligand binding pocket of MOR42-3. C12 dicarboxylic acid did not activate the receptor in our functional assay, yet our docking simulations predicted its binding site in MOR42-3. Binding without activation implied that C12 dicarboxylic acid might act as an antagonist. In our functional assay, C12 dicarboxylic acid did indeed act as an antagonist of MOR42-3, in agreement with molecular docking studies. Our results demonstrate a powerful approach based on the synergy between computational predictions and physiological assays.

Animals↗
PubMed2006

Structural basis for processivity and single-strand specificity of RNase II.

RNase II is a member of the widely distributed RNR family of exoribonucleases, which are highly processive 3'-->5' hydrolytic enzymes that play an important role in mRNA decay. Here, we report the crystal structure of E. coli RNase II, which reveals an architecture reminiscent of the RNA exosome. Three RNA-binding domains come together to form a clamp-like assembly, which can only accommodate single-stranded RNA. This leads into a narrow, basic channel that ends at the putative catalytic center that is completely enclosed within the body of the protein. The putative path for RNA agrees well with biochemical data indicating that a 3' single strand overhang of 7-10 nt is necessary for binding and hydrolysis by RNase II. The presence of the clamp and the narrow channel provides an explanation for the processivity of RNase II and for why its action is limited to single-stranded RNA.

Binding Sites↗
PubMed2006

Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli.

Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: calcitonin like receptor (CLR), receptor activity modifying protein (RAMP1) and receptor component protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here, we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a maltose binding protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While His tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function.

Amino Acid Sequence↗
PubMed2006

Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing.

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3' to 5' exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT-PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.

Dystrophin↗
PubMed2006

Left ventricular ejection fraction and volumes on rest gated 201Tl perfusion SPECT: comparison with two-dimensional echocardiography.

BACKGROUND: Rest gated 201Tl images are considered to be of poor count statistics due to lower energy and low photon flux of 201Tl in addition to increased attenuation and low dose that can be administered. We compared the left ventricular ejection fraction (LVEF), end diastolic (EDV) and end systolic volume (ESV) obtained on 4 h gated rest 201Tl myocardial perfusion single photon emission computed tomography (SPECT) with those obtained by two-dimensional echocardiography (2-D ECHO) in patients with known or suspected coronary artery disease (CAD). METHODS: Eighty-two consecutive patients who underwent gated 201Tl stress-rest myocardial perfusion SPECT and 2-D ECHO were studied. The gated thallium images were processed with Siemens e-soft autocardiac processor and LVEF, EDV and ESV were evaluated using Emory Cardiac Toolbox. The same parameters were also assessed on the 2-D ECHO using the modified Simpson method for comparison. RESULTS: Out of 82 rest gated images, one study was excluded because of poor count statistics. In 81 (99%) patients there was good linear correlation with 2-D ECHO values and rest gated 201Tl SPECT images for EDV, ESV and LVEF. Pearson's correlation co-efficient (r value) for EDV, ESV and LVEF between the two methods was 0.78, 0.79 and 0.88, respectively. A Bland-Altman plot showed close agreement with LVEF but not for EDV and ESV. CONCLUSION: These results suggest that the 4 h rest gated 201Tl study gives a reliable value for the LVEF compared to 2-D ECHO and can be used in routine clinical practice.

Adult↗
PubMed2005

Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors.

We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors.

Amino Acid Sequence↗
PubMed2005

Crystal structure of Escherichia coli RNase D, an exoribonuclease involved in structured RNA processing.

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.

Amino Acid Motifs↗
PubMed2005

Reflux in native kidneys mimicking urine leak postrenal transplant.

A 19-year man underwent live related renal transplantation for end-stage renal disease. He was referred for a baseline renal scan on day 4. The urine output was satisfactory and the serum creatinine showed a decreasing trend. Renal dynamic scintigraphy with technetium-99m ethylenedicysteine demonstrated radiotracer activity above the transplant kidney, suspected to be a urine leak. Postvoiding posterior, delayed images confirmed that the radiotracer activity in the anterior postvoiding images was the result of vesicoureteral reflux from the patient's native kidneys.

Adult↗
PubMed2004

Purification and crystallization of Escherichia coli oligoribonuclease.

Oligoribonuclease (Orn) is an essential 3'-to-5' hydrolytic exoribonuclease which degrades short oligoribonucleotides to 5' mononucleotides. Escherichia coli Orn has been crystallized under several different conditions using ammonium sulfate, sodium citrate and sodium acetate as precipitants. Both native and selenomethionine-labeled oligoribonuclease (SeMet-Orn) can be crystallized at room temperature in 1.4-1.55 M sodium citrate. The SeMet-Orn crystals diffract to 2.2 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.43, b = 72.87, c = 147.76 A, and two dimers in the asymmetric unit. When grown in the presence of manganese, a second crystal form (Mn-SeMet-Orn) was obtained containing a single dimer per asymmetric unit (P2(1)2(1)2(1); a = 63.74, b = 74.31, c = 74.19 A). Finally, a hexagonal crystal form was obtained using sodium acetate as a precipitant (a = 91.5, b = 91.5, c = 111.1 A). This crystal (Zn-ApUp-Orn) belongs to the P6(5) space group and has three oligoribonuclease molecules per asymmetric unit.

Cloning, Molecular↗
PubMed2004

Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors.

Alpha-conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat alpha3beta2 receptors, expressed in Xenopus oocytes, are blocked with an IC(50) of 3.7 +/- 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of alpha3beta2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and approximately 2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the alpha3beta2 receptor.

Alanine↗
PubMed2004

Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold.

TruD, a recently discovered novel pseudouridine synthase in Escherichia coli, is responsible for modifying uridine13 in tRNA(Glu) to pseudouridine. It has little sequence homology with the other 10 pseudouridine synthases in E. coli which themselves have been grouped into four related protein families. Crystal structure determination of TruD revealed a two domain structure consisting of a catalytic domain that differs in sequence but is structurally very similar to the catalytic domain of other pseudouridine synthases and a second large domain (149 amino acids, 43% of total) with a novel alpha/beta fold that up to now has not been found in any other protein.

Amino Acid Sequence↗
PubMed2004

Evaluation of dilated upper renal tracts by technetium-99m ethylenedicysteine F+O diuresis renography in infants and children.

AIM: To evaluate the feasibility of modifying diuresis renography by the simultaneous administration of Tc-99m ethylenedicysteine and furosemide in the investigation of hydronephrosis and hydroureteronephrosis in infants and children. Parameters assessed were the diuretic response in normal kidneys and the ability of the F+0 study to differentiate between renal obstruction and nonobstruction. METHODS: One hundred and thirty-three children (93 males, 40 females; mean age 35.2 months) with sonographic diagnoses of hydronephrosis or hydroureteronephrosis underwent F+0 diuresis renography. Tc-99m ethylenedicysteine (3.7 MBq/kg body weight) and furosemide at an appropriate dose were administered intravenously at the start of the study. Posterior imaging of the kidneys and bladder was performed for 20 min followed by imaging after voiding. All patients were followed-up for 12 months, and the results of the initial F+0 diuresis renography were compared with the final diagnoses. Final diagnosis was based on the pediatric urologist's decision of either surgery or conservative management. RESULTS: A renal unit was defined as a kidney and its ureter. There were 262 renal units with 4 patients having a solitary kidney. 90 normal and 172 abnormal renal units on sonography were assessed by F+0 diuresis renography. The furosemide clearance half time for the 90 normal renal units was 5.8 +/- 1.4 min. Of the 172 abnormal renal units, 100 were classified as nonobstructed and 72 as obstructed on diuresis renography. All 100 nonobstructed renal units were correctly classified with no false-negative studies; of the 72 renal units classified as obstructed, there were 43 true-positive studies and 29 false-positive studies. The sensitivity was 100%, specificity was 78% and accuracy was 83%. CONCLUSION: Tc-99m ethylenedicysteine F+0 diuresis renography is a valid method for the investigation of hydronephrosis and hydroureteronephrosis in infants and children.

Child, Preschool↗
PubMed2004

Ephrins and their receptors: binding versus biology.

Ephrins and Eph receptors play important roles in the development of the central nervous system and peripheral tissues by orchestrating cellular movements, resulting in events such as axonal growth cone guidance, tissue segmentation, and angiogenic remodeling. To understand the role of specific ephrin and Eph receptor interactions, it is important to identify the binding specificity between individual ligand-receptor complexes. To date, a dogma in the field suggests that there may be promiscuous binding within the subclasses of the ephrin family. However, this overlooks and contradicts several binding studies that suggest specificity within each subclass. Although binding studies only provide evidence on the dynamics and strength of protein interactions, they do not indicate whether particular interactions are physiologically relevant. Thus, distribution and gene targeted mutations of ephrins and their receptors can provide critical insights into the relevance of specific ligand-receptors interactions. This review mainly focuses on the B-class family and will evaluate the differences between binding affinities and biological functions, importance of oligomeric interactions, and structural differences and similarities between classes.

Animals↗
PubMed2004

Reproducibility of regional left ventricular wall thickening obtained by gating resting and redistribution 201Tl myocardial SPECT studies.

BACKGROUND: We have assessed the reproducibility of the estimates of regional left ventricular wall thickening between resting and redistribution Tl gated single photon emission computed tomography (SPECT) studies. METHODS: Thirty patients (28 males, two females) aged between 38 and 72 years (57.0 +/- 8.4 years) underwent resting and redistribution Tl gated SPECT. Perfusion was assessed semi-quantitatively using scores of 0-4. The assessment of wall thickening was performed both visually and by using automated software using scores of 0-3. The assessment of reproducibility was performed in 20 individual segments and 37 pairs of contiguous segments in each patient. RESULTS: In resting studies, mean global left ventricular ejection fraction (LVEF), end diastolic volume and end systolic volume were 34.2 +/- 11.7%, 180.5 +/- 70.6 ml and 123.3 +/- 60.0 ml, respectively. The corresponding values for the redistribution studies were 32.7 +/- 10.1%, 179.7 +/- 70.6 ml and 125.5 + 63.0 ml, respectively. There was good correlation between the resting and redistribution LVEFs (r=0.88), end diastolic volumes (r=0.90) and end systolic volumes (r=0.933). Reproducibility of visual wall thickening was 66% in individual segments and 82% in pairs of contiguous segments, and that of automated wall thickening was 48.0% in individual segments and 68% in pairs of contiguous segments. CONCLUSION: In conclusion, global left ventricular functional parameters obtained by resting Tl gated SPECT studies are highly reproducible in patients with impaired resting left ventricular function and large areas of scarred myocardium. Wall thickening and wall motion data are more reproducible in contiguous segments than in individual segments.

Adult↗