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Biomedical subjects

P Rajagopal

Publications and source records attributed to P Rajagopal.

At least 19 recordsLinked to original sources
PubMed2003

Clinicopathological study of thymomas--correlation of histologic subtype to myasthenia gravis and prognosis.

Thymoma is the most common primary tumor of anterior superior mediastinum. Sixty cases of thymomas over a 12 year period were analysed and the histologic subtype, according to Marino and Muller-Hermilink, classification was correlated with presence or absence of myasthenia gravis (MG) and capsular invasion. Thirty four patients had myasthenia gravis associated with thymoma and there was one case of pure red cell aplasia. There were 3 (1) predominantly cortical, 28 (20) cortical, 12 (9) mixed, 16 (4) medullary thymomas and 1 (0) thymic carcinoma (Figures in parenthesis indicate number of cases associated with MG). Capsular invasion was seen in 25 cases. Association with myasthenia gravis and capsular invasion were seen predominantly in cortical and mixed thymomas which were also associated with aggressive behaviour.

Adolescent↗
PubMed2001

Structure of a BRCA1-BARD1 heterodimeric RING-RING complex.

The RING domain of the breast and ovarian cancer tumor suppressor BRCA1 interacts with multiple cognate proteins, including the RING protein BARD1. Proper function of the BRCA1 RING domain is critical, as evidenced by the many cancer-predisposing mutations found within this domain. We present the solution structure of the heterodimer formed between the RING domains of BRCA1 and BARD1. Comparison with the RING homodimer of the V(D)J recombination-activating protein RAG1 reveals the structural diversity of complexes formed by interactions between different RING domains. The BRCA1-BARD1 structure provides a model for its ubiquitin ligase activity, illustrates how the BRCA1 RING domain can be involved in associations with multiple protein partners and provides a framework for understanding cancer-causing mutations at the molecular level.

Amino Acid Sequence↗
PubMed1998

Prediction and structural characterization of an independently folding substructure in the src SH3 domain.

Previous studies of the conformations of peptides spanning the length of the alpha-spectrin SH3 domain suggested that SH3 domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identified a seven residue peptide in the src SH3 domain predicted to adopt a native-like structure, a type II beta-turn bridging unpaired beta-strands, that was not contained intact in any of the SH3 domain peptides studied earlier. NMR characterization confirmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3JNHalpha coupling constant patterns were indicative of a type II beta-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-confidence I-sites predictions identify protein segments that are likely to form native-like structures early in folding.

Amino Acid Sequence↗
PubMed1998

Solvent exchange rates of side-chain amide protons in proteins.

Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr. The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons. Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8. An NOE between a side-chain amide proton and a bound water molecule was also observed.

Bacterial Proteins↗
PubMed1997

NMR chemical shift perturbation mapping of DNA binding by a zinc-finger domain from the yeast transcription factor ADR1.

Mutagenesis studies have revealed that the minimal DNA-binding domain of the yeast transcription factor ADR1 consists of two Cys2-His2 zinc fingers plus an additional 20 residues proximal and N-terminal to the fingers. We have assigned NMR 1H, 15N, and 13C chemical shifts for the entire minimal DNA-binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single-finger constructs, and 13C alpha chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein-DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N-terminus of the finger 2 helix is affected. Furthermore, residues N-terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein-DNA interface. A striking correspondence is observed between the protein-DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis.

Amino Acid Sequence↗
PubMed1997

Phosphorylation on histidine is accompanied by localized structural changes in the phosphocarrier protein, HPr from Bacillus subtilis.

The histidine-containing protein (HPr) of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) serves a central role in a series of phosphotransfer reactions used for the translocation of sugars across cell membranes. These studies report the high-definition solution structures of both the unphosphorylated and histidine phosphorylated (P-His) forms of HPr from Bacillus subtilis. Consistent with previous NMR studies, local conformational adjustments occur upon phosphorylation of His 15, which positions the phosphate group to serve as a hydrogen bond acceptor for the amide protons of Ala 16 and Arg 17 and to interact favorably with the alpha-helix macrodipole. However, the positively charged side chain of the highly conserved Arg 17 does not appear to interact directly with phospho-His 15, suggesting that Arg 17 plays a role in the recognition of other PTS enzymes or in phosphotransfer reactions directly. Unlike the results reported for Escherichia coli P-His HPr (Van Nuland NA, Boelens R, Scheek RM, Robillard GT, 1995, J Mol Biol 246:180-193), our data indicate that phosphorylation of His 15 is not accompanied by adoption of unfavorable backbone conformations for active site residues in B. subtilis P-Ser HPr.

Bacillus subtilis↗
PubMed1997

Demonstration of protein-protein interaction specificity by NMR chemical shift mapping.

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.

Bacillus subtilis↗
PubMed1996

Influence of N-cap mutations on the structure and stability of Escherichia coli HPr.

This paper describes the effect of N-capping substitutions on the structure and stability of histidine-containing protein (HPr). We have used NMR spectroscopy and conformational stability studies to quantify changes in local and global free energy due to mutagenesis at Ser46, the N-cap for helix B in HPr. Previous NMR studies suggested that helix B of Escherichia coli HPr is dynamic as judged by the rate of exchange of amide protons with solvent. Ser46 was chosen because it is the site of regulatory phosphorylation in HPrs from Gram-positive bacteria, and mutation of this residue to an aspartic acid (S46D) in E. coli HPr (Gram-negative) also makes it a poor substrate in the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Therefore, to understand the mechanism of inactivation of E. coli S46D HPr, as well as the effect of mutagenesis on protein stability, we have characterized three mutants of E. coli HPr: Ser46 has been mutated to an Asp, Asn, and Ala in S46D, S46N, and S46A HPrs, respectively. The results indicate that these N-cap replacements have a marked influence on helix B stability. The effect of mutagenesis on local stability is correlated to global unfolding of HPr. The ability of amino acids to stabilize helix B is Asp > Asn > Ser > Ala. In addition, since there are neither large-scale conformational changes nor detectable changes in the active site of S46D HPr, it is proposed that the loss of phosphotransfer activity of S46D HPr is due to unfavorable steric and/or electrostatic interactions of the Asp with enzyme I of the PTS.

Bacterial Proteins↗
PubMed1995

Phosphorylation of serine-46 in HPr, a key regulatory protein in bacteria, results in stabilization of its solution structure.

The serine-phosphorylated form of histidine-containing protein (HPr), a component of the phosphoenolpyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy and solvent denaturation studies. The results indicate that phosphorylation of Ser 46, the N-cap of alpha-helix-B, does not cause a conformational change but rather stabilizes the helix. Amide proton exchange rates in helix-B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaturation, with a delta delta G of 0.7-0.8 kcal mol-1. A mutant in which Ser 46 is replaced by aspartic acid shows a similar stabilization, indicating that an electrostatic interaction between the negatively charged groups and the helix macrodipole contributes significantly to the stabilization.

Amino Acid Sequence↗
PubMed1994

Structural consequences of histidine phosphorylation: NMR characterization of the phosphohistidine form of histidine-containing protein from Bacillus subtilis and Escherichia coli.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system involves a series of reactions in which phosphoprotein intermediates are formed. Histidine-containing protein (HPr) is phosphorylated on the N delta 1 position of the imidazole ring of His15 by enzyme I and acts as a phosphoryl donor to the sugar-specific enzymes IIA. The structure of phosphorylated HPr from Bacillus subtilis, primarily, and from Escherichia coli has been studied by nuclear magnetic resonance (NMR) spectroscopy. Phosphorylation of His15 results in large downfield shifts in amide proton and nitrogen resonances for residues 16 and 17 but results in only modest or no shifts in other backbone resonances. The exchange rates of these two amide groups are decreased more than 10-fold upon phosphorylation. Analysis of the coupling constants 3JNH alpha revealed no significant changes throughout the protein, indicating that backbone phi dihedral angles do not change detectably. 3J alpha beta and 3JN beta patterns determined from P.E.COSY and HNHB spectra, respectively, revealed a change in one side chain, that of conserved Arg17. Analysis of NOESY spectra revealed a limited number of changes in NOEs involving protons in Ser12, His15, Arg71, and Pro18 in B. subtilis HPr. The NMR results indicate that the Arg17 side chain becomes limited in its conformational range in the phosphorylated protein, taking on a conformation that points its guanidinium group toward the phosphoryl group on His15. In addition, the tautomeric and ionization states of His15 have been determined using 15N and 31P NMR. At neutral pH, the imidazole is predominantly in the protonated form and the phosphoryl group is in the dianionic form in P-His15. Altogether, the results indicate that phosphorylation of His15 yields only a local effect on the protein's structure. The data are consistent with a small change in the disposition of the histidine side chain, allowing phosphoryl group oxygens to serve as hydrogen bond acceptors for the amide protons of residues Ala16 and Arg17, which constitute the first two residues of an alpha-helix. Thus, similar to many proteins that bind phosphoryl moieties noncovalently, the phosphoryl group in P-His15-HPr is situated to allow for a favorable electrostatic interaction at the N-terminal end of an alpha-helix.

Amides↗
PubMed1994

Percutaneous balloon valvuloplasty compared with open surgical commissurotomy for mitral stenosis.

BACKGROUND: Percutaneous balloon mitral valvuloplasty has been proposed as an alternative to open surgical commissurotomy for the treatment of rheumatic mitral-valve stenosis. METHODS: We enrolled 60 patients with severe mitral stenosis and favorable valvular anatomy in a prospective, randomized trial comparing the two procedures. All patients underwent cardiac catheterization before the procedure and one week, six months, and three years thereafter. Hemodynamic data were analyzed by investigators who were blinded to the patients' treatment assignments. RESULTS: Mitral-valve areas improved initially in both groups, from a mean (+/- SD) of 0.9 +/- 0.3 cm2 to 2.1 +/- 0.6 cm2 in the balloon-valvuloplasty group (30 patients; P < 0.01) and from 0.9 +/- 0.3 cm2 to 2.0 +/- 0.6 cm2 in the surgical group (30 patients; P < 0.001). Although improvement was maintained in both groups, mitral-valve areas were greater in the patients in the balloon-valvuloplasty group at three years (2.4 +/- 0.6 cm2, vs. 1.8 +/- 0.4 cm2 in the surgery group, P < 0.001). Restenosis occurred in three patients in the balloon-valvuloplasty group and four in the surgery group. One patient in the balloon-valvuloplasty group died of an apparent stroke after 2.5 years; four patients in the balloon-valvuloplasty group had residual atrial septal defects, and three patients (two in the balloon-valvuloplasty group and one in the surgery group) were judged to have severe mitral regurgitation. Seventy-two percent of the patients who underwent balloon valvuloplasty and 57 percent of the surgically treated patients were in New York Heart Association functional class I (i.e., they had no cardiovascular symptoms) at three years. No patient was lost to follow-up. CONCLUSIONS: In the treatment of mitral stenosis, balloon valvuloplasty and open surgical commissurotomy have comparable initial results and low rates of restenosis, and both produce good functional capacity for at least three years. The potential complications associated with balloon valvuloplasty should be noted. The better hemodynamic results at three years, lower cost, and elimination of the need for thoracotomy suggest that balloon valvuloplasty should be considered for all patients with favorable mitral-valve anatomy.

Adolescent↗
PubMed1994

Thrombolysis in left-sided prosthetic valve occlusion: immediate and follow-up results.

Thrombolytic therapy is used increasingly for prosthetic valve thrombosis. Fear of peripheral embolism has limited its use in left-sided valve occlusions. Thirty-eight patients with prosthetic valve occlusion were treated with thrombolytic agents on 44 occasions. Duration of thrombolytic therapy was individualized. Patients were followed up with clinical, echocardiographic, and cine-fluoroscopic evaluation. Average time from onset of symptoms to presentation was 9.7 days. The anticoagulation status was inadequate in 70% of instances. The majority (75%) were in functional class IV, 8 of them being in shock. Overall success was seen in 88.6%: immediate complete success in 18 and partial in 21. Nine of those with partial success had delayed opening of the leaflets. There were nine instances of rethrombosis and one instance of re-rethrombosis; 6 patients received repeat thrombolysis with success in 5. Seventy-six percent (29/38) of the patients were surviving at the time of reporting. In conclusion, the present study demonstrates the feasibility of thrombolytic therapy for left-sided prosthetic valve occlusion. Delayed opening is common in patients with initial partial success. Rethrombosis remains a major problem.

Adolescent↗
PubMed1992

Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy.

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.

Amino Acid Sequence↗
PubMed1991

Percutaneous balloon versus surgical closed commissurotomy for mitral stenosis. A prospective, randomized trial.

BACKGROUND: We performed a prospective, randomized trial comparing percutaneous balloon commissurotomy with surgical closed commissurotomy in 40 patients with severe rheumatic mitral stenosis. METHODS AND RESULTS: Data were analyzed by investigators who were masked to treatment assignment or phase of study. Patients randomized to balloon (n = 20) or surgical (n = 20) commissurotomy had severe mitral stenosis without significant baseline differences (left atrial pressure, 26.1 +/- 4.2 versus 27.6 +/- 6.2 mm Hg; mitral valve gradient, 18.0 +/- 4.2 versus 19.7 +/- 6.3 mm Hg; mitral valve area, 1.0 +/- 0.2 versus 1.0 +/- 0.4 cm2, respectively). At 1-week follow-up after balloon commissurotomy, pulmonary wedge pressure was 14.3 +/- 7.2 mm Hg; mitral valve gradient was 9.6 +/- 5.1 mm Hg; and mitral valve area was 1.6 +/- 0.6 cm2 (all p less than 0.0001). At 1-week follow-up after surgical closed commissurotomy, wedge pressure was 13.7 +/- 5.4 mm Hg; mitral valve gradient was 9.4 +/- 4.2 mm Hg (both p less than 0.0001); and mitral valve area was 1.6 +/- 0.7 cm2 (p less than 0.003). At 8-month follow-up, improvement occurred in both groups: Mitral valve area was 1.6 +/- 0.6 cm2 in the balloon commissurotomy group (p less than 0.002) and was 1.8 +/- 0.6 cm2 in the surgical closed commissurotomy group (p less than 0.0001). There was no difference between the groups at 1-week or 8-month follow-up (all p greater than 0.4). One case of severe mitral regurgitation occurred in each group; complications were otherwise related to transseptal catheterization. There was no death, stroke, or myocardial infarction. Cost analysis revealed that balloon commissurotomy may substantially exceed the cost of surgical commissurotomy in developing countries, whereas it may represent a significant savings in industrialized nations. CONCLUSIONS: We conclude that percutaneous balloon commissurotomy and surgical closed commissurotomy result in comparable hemodynamic improvement that is sustained through 8 months of follow-up.

Adult↗
PubMed1991

Mitral valvular stromal degeneration.

A 35-year-old female who presented with features of mitral valve disease showed mitral stromal deposits which were acidophilic in nature and brilliantly red by Masson trichrome. Stromal GAG (glycosaminoglycan) alterations are considered responsible for the deposits.

Adult↗
PubMed1989

NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4.

The complexes formed by the homopurine and homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4 have been investigated by one- and two-dimensional 1H NMR. Under appropriate conditions [low pH, excess d(TC)4 strand] the oligonucleotides form a triplex containing one d(GA)4 and two d(TC)4 strands. The homopurine and one of the homopyrimidine strands are Watson-Crick base paired, and the second homopyrimidine strand is Hoogsteen base paired in the major groove to the d(GA)4 strand. Hoogsteen base pairing in GC base pairs requires hemiprotonation of C; we report direct observation of the C+ imino proton in these base pairs. Both homopyrimidine strands have C3'-endo sugar conformations, but the purine strand does not. The major triplex formed appears to have four TAT and three CGC+ triplets formed by binding of the second d(TC)4 strand parallel to the d(GA)4 strand with a 3' dangling end. In addition to the triplexes formed, at least one other heterocomplex is observed under some conditions.

Base Composition↗