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Biomedical subjects

A Majumdar

Publications and source records attributed to A Majumdar.

At least 55 records · Page 3Linked to original sources
PubMed2000

Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase.

The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophage PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight enzyme:inhibitor complex. To create human cells that are impaired for UDG activity, the human glioma U251 cell line was engineered to produce active Ugi protein. In vitro assays of crude cell extracts from several Ugi-expressing clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell lines were characterized for their ability to conduct in vivo uracil-DNA repair. Whereas transfected plasmid DNA containing either a U:G mispair or U:A base pairs was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi. Experiments using a shuttle vector to detect mutations in a target gene showed that Ugi-expressing cells exhibited a 3-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions. The growth rate and cell cycle distribution of Ugi-expressing cells did not differ appreciably from their parental cell counterpart. Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate Ugi-insensitive excision of uracil bases from DNA was not detected in the parental U251 cells. However, a Ugi-insensitive UDG activity of unknown origin that recognizes U:G mispairs and to a lesser extent U:A base pairs in duplex DNA, but which was inactive toward uracil residues in single-stranded DNA, was detected under assay conditions previously shown to be efficient for detecting TDG.

Bacillus Phages↗
PubMed2000

A two-stranded template-based approach to G.(C-A) triad formation: designing novel structural elements into an existing DNA framework.

We have designed a DNA sequence, d(G-G-G-T-T-C-A-G-G), which dimerizes to form a 2-fold symmetric G-quadruplex in which G(syn). G(anti).G(syn).G(anti) tetrads are sandwiched between all trans G. (C-A) triads. The NMR-based solution structural analysis was greatly aided by monitoring hydrogen bond alignments across N-H...N and N-H...O==C hydrogen bonds within the triad and tetrad, in a uniformly ((13)C,(15)N)-labeled sample of the d(G-G-G-T-T-C-A-G-G) sequence. The solution structure establishes that the guanine base-pairs with the cytosine through Watson-Crick G.C pair formation and with adenine through sheared G.A mismatch formation within the G.(C-A) triad. A model of triad DNA was constructed that contains the experimentally determined G.(C-A) triad alignment as the repeating stacked unit.

Base Pair Mismatch↗
PubMed2000

The zebrafish floating head mutant demonstrates podocytes play an important role in directing glomerular differentiation.

In zebrafish, the pronephric glomerulus occupies a midline position underneath the notochord and is vascularized through angiogenic capillary ingrowth from the dorsal aorta. The midline mutants floating head (flh), sonic you (syu), and you-too (yot) provide the opportunity to study glomerular differentiation in the absence of the notochord and vascularization from the dorsal aorta. In flh, syu, and yot mutants, glomeruli differentiate at ectopic lateral positions within the embryo and contain morphologically identifiable podocyte and endothelial cell types. In the absence of the dorsal aorta, endothelia from an alternate source are recruited by podocytes during glomerular vascularization to make functional glomeruli. Our results suggest that midline signals are required for proper glomerular morphogenesis but not for the differentiation of podocytes. Podocytes appear to play an important role in directing cellular recruitment events leading to glomerular differentiation. Furthermore, we find defects in sclerotomal development that correlate with defects in glomerular morphogenesis suggesting a possible link between the formation of these embryonic structures.

Animals↗
PubMed2000

A dimeric DNA interface stabilized by stacked A.(G.G.G.G).A hexads and coordinated monovalent cations.

We report on the identification of an A.(G.G.G.G).A hexad pairing alignment which involves recognition of the exposed minor groove of opposing guanines within a G.G.G.G tetrad through sheared G.A mismatch formation. This unexpected hexad pairing alignment was identified for the d(G-G-A-G-G-A-G) sequence in 150 mM Na(+) (or K(+)) cation solution where four symmetry-related strands align into a novel dimeric motif. Each symmetric half of the dimeric "hexad" motif is composed of two strands and contains a stacked array of an A.(G.G.G.G).A hexad, a G.G.G.G tetrad, and an A.A mismatch. Each strand in the hexad motif contains two successive turns, that together define an S-shaped double chain reversal fold, which connects the two G-G steps aligned parallel to each other along adjacent edges of the quadruplex. Our studies also establish a novel structural transition for the d(G-G-A-G-G-A-N) sequence, N=T and G, from an "arrowhead" motif stabilized through cross-strand stacking and mismatch formation in 10 mM Na(+) solution (reported previously), to a dimeric hexad motif stabilized by extensive inter-subunit stacking of symmetry-related A.(G.G.G.G).A hexads in 150 mM Na(+) solution. Potential monovalent cation binding sites within the arrowhead and hexad motifs have been probed by a combination of Brownian dynamics and unconstrained molecular dynamics calculations. We could not identify stable monovalent cation-binding sites in the low salt arrowhead motif. By contrast, five electronegative pockets were identified in the moderate salt dimeric hexad motif. Three of these are involved in cation binding sites sandwiched between G.G.G. G tetrad planes and two others, are involved in water-mediated cation binding sites spanning the unoccupied grooves associated with the adjacent stacked A.(G.G.G.G).A hexads. Our demonstration of A.(G. G.G.G).A hexad formation opens opportunities for the design of adenine-rich G-quadruplex-interacting oligomers that could potentially target base edges of stacked G.G.G.G tetrads. Such an approach could complement current efforts to design groove-binding and intercalating ligands that target G-quadruplexes in attempts designed to block the activity of the enzyme telomerase.

Adenine↗
PubMed2000

Zebrafish wnt4b expression in the floor plate is altered in sonic hedgehog and gli-2 mutants.

The floor plate of the neural tube serves an important function as a source of signals that pattern cell fates in the nervous system as well as directing proper axon pathfinding. We have cloned a novel zebrafish wnt family member, wnt4b, which is expressed exclusively in the floor plate. To place wnt4b in the context of known regulators of midline development, its expression was analyzed in the zebrafish mutants cyclops (cyc), floating head (flh), you-too (yot), and sonic you (syu). wnt4b expression in the medial and lateral floor plate are shown to be regulated independently: medial floor plate expression occurs in the absence of a notochord, while lateral floor plate expression requires a functional notochord, sonic hedgehog and gli-2.

Amino Acid Sequence↗
PubMed2000

Glomerular prolapse as precursor of one type of segmental sclerosing lesions.

A distinctive segmental glomerular abnormality is confined to the region of the tubular opening. The hypothesis was that this followed prolapse of the tuft into the tubule. Analysis was made of 39 renal biopsy specimens with acute postinfective glomerulonephritis, later material from ten cases, four specimens from three women with pre-eclampsia, and 21 control specimens, with morphometry of glomeruli and immunohistological examination for immunoproteins and monocytes/macrophages. Prolapse was found in 14 specimens with acute postinfective glomerulonephritis, associated in eight with adhesion to Bowman's capsule and local alterations in the tuft, which together constitute early tip changes. Another three had early tip changes only and eight others had thin adhesions between the tuft and capsule next to the tubular opening. Later material confirmed this order of development and showed another late change, with sclerosed and hyaline material in the tuft and adhesion at the tubular origin. Findings in pre-eclampsia were comparable. Glomeruli were significantly larger in acute postinfective glomerulonephritis than in controls and were shown by others to be larger in pre-eclampsia than in normal pregnancy. Immunohistology showed IgM and a few foamy monocytes/macrophages in early tip changes but not in prolapsed loops. Glomerular prolapse appears to be a temporary consequence of acute enlargement of the tuft, probably causes mechanical damage to epithelial cells, and is a precursor of permanent structural changes near the tubular origin. This gives a unifying hypothesis to explain how these changes can be seen in acute postinfective glomerulonephritis, pre-eclampsia, and many other human and experimental renal disorders.

Acute Disease↗
PubMed2000

Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins.

In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or.an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in 13C/15N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen-carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond 3hJ(NC') couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.

Carbon Isotopes↗
PubMed2000

Detection of very weak side chain-main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins by sensitivity-enhanced NMR spectroscopy.

We describe the direct observation of very weak side chain-main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation. between the hydrogen acceptor side chain carboxylate carbon 13CO2delta of glutamate 54 and the hydrogen donor backbone amide 15N of methionine 49 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond 3hJ(NCO2delta) coupling by employing a novel sensitivity-enhanced HNCO-type experiment, CPD-HNCO. The 3hJ(NCO2delta) coupling constant appears to be even smaller than the average value of backbone 3hJ(NC') couplings, consistent with more extensive local dynamics in protein side chains.

Carbon Isotopes↗
PubMed2000

NMR detection of side chain-side chain hydrogen bonding interactions in 13C/15N-labeled proteins.

We describe the direct observation of side chain-side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen 15Nepsilon of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon 13CO2gamma of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond 3h JNepsilonCO2gamma coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The 3h JNepsilonCO2gamma coupling constant appears to be even smaller than the average value of backbone 3h JNC' couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.

Animals↗
PubMed2000

Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells.

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.

Animals↗
PubMed2000

Helix rim advancement for reconstruction of marginal defects of the pinna.

Helical rim defects commonly result from excision of neoplasms or from injury. Various techniques for reconstruction of such defects have been described. The wedge excision technique that is commonly described in texts causes butterfly deformity, webbing and cupping of the pinna and the authors believe that it should be abandoned. Helix rim advancement flaps were first described 30 years ago and offer a simple and versatile method for reconstruction with excellent cosmetic results. Line drawings and photographs show the operative sequence and the end results of such flaps.

Aged↗
PubMed2000

Renal pathological findings in infective endocarditis.

BACKGROUND: Accounts of renal pathological findings in infective endocarditis are mostly based on studies from many years ago. We reviewed a group of patients with infective endocarditis in the light of modern concepts of renal pathology, including the largest reported series of renal biopsies in this condition. METHODS: Renal tissue was available for retrospective study from 62 patients with confirmed infective endocarditis out of 354 diagnosed with the disease between 1981 and 1998 inclusive. Twenty patients had a renal biopsy and 42 a necropsy. RESULTS: Common renal lesions noted were localized infarcts in 31%, noted only in necropsy material, and acute glomerulonephritis in 26%, noted in biopsy and necropsy material. The commonest type of glomerulonephritis was vasculitic, without deposition of immunoproteins in glomeruli. Of the renal infarcts over half were due to septic emboli, mostly in patients infected with Staphylococcus aureus. Acute interstitial nephritis was found in 10% but was more common in biopsy material and seemed attributable to antibiotics. Renal cortical necrosis found in 10% was apparent only at necropsy. There were various other findings in the kidney. CONCLUSIONS: The kidneys are commonly affected in infective endocarditis by a variety of complications of clinical significance. The commonest type of glomerulonephritis does not appear to be attributable to deposition of immune complexes. A renal biopsy may be helpful in the investigation of renal impairment in a patient with infective endocarditis.

Adult↗
PubMed2000

N-terminal region of P protein of Chandipura virus is responsible for phosphorylation-mediated homodimerization.

The phosphoprotein P of Chandipura (CHP) virus, an Indian isolate of rhabdovirus, was found to support transcription upon phosphorylation by casein kinase II (CKII). A phosphorylation-induced change in the protein conformation was found to occur at the N-terminal region of the protein. Biochemical studies for further characterization of this phosphorylation-based conformational alteration demonstrated that phosphorylation leads to the transition from an 'open' to 'closed' structure of the protein. The phosphate group introduced by CKII was found to be resistant to phosphatases. This phosphorylation-based structural alteration changes the accessible hydrophobic surface area of the protein and also the available digestion sites of different proteases. The phosphorylated form of P protein was found to be a dimer by His-tag dilution assay. Using the same approach it was found that the N-terminal 46 amino acids are responsible for P-P dimerization, only after phosphorylation.

Binding Sites↗
PubMed2000

Zebrafish no isthmus reveals a role for pax2.1 in tubule differentiation and patterning events in the pronephric primordia.

Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, we have used the zebrafish pax2.1 mutant no isthmus to investigate the role for pax2.1 in development of the pronephros. We demonstrate a requirement for pax2.1 in multiple aspects of pronephric development including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi(- )larvae specifically lack pronephric tubules while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na(+)/K(+) ATPase (&agr;)1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron.

Animals↗
PubMed1999

Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples.

BACKGROUND: The Rex protein of the human T cell leukemia virus type 1 (HTLV-1) belongs to a family of proteins that use arginine-rich motifs (ARMs) to recognize their RNA targets. Previously, an in vitro selected RNA aptamer sequence was identified that mediates mRNA transport in vivo when placed in the primary binding site on stem-loop IID of the Rex response element. We present the solution structure of the HTLV-1 arginine-rich Rex peptide bound to its RNA aptamer target determined by multidimensional heteronuclear NMR spectroscopy. RESULTS: The Rex peptide in a predominantly extended conformation threads through a channel formed by the shallow and widened RNA major groove and a looped out guanine. The RNA aptamer contains three stems separated by a pair of two-base bulges, and adopts an unanticipated fold in which both junctional sites are anchored through base triple formation. Binding specificity is associated with intermolecular hydrogen bonding between guanidinium groups of three non-adjacent arginines and the guanine base edges of three adjacent G.C pairs. CONCLUSIONS: The extended S-shaped conformation of the Rex peptide, together with previous demonstrations of a beta-hairpin conformation for the bovine immunodeficiency virus (BIV) Tat peptide and an alpha-helical conformation for the human immunodeficiency virus (HIV) Rev peptide in complex with their respective RNA targets, expands our understanding of the strategies employed by ARMs for adaptive recognition and highlights the importance of RNA tertiary structure in accommodating minimalist elements of protein secondary structure.

Amino Acid Sequence↗
PubMed1999

Interlocked mismatch-aligned arrowhead DNA motifs.

BACKGROUND: Triplet repeat sequences are of considerable biological importance as the expansion of such tandem arrays can lead to the onset of a range of human diseases. Such sequences can self-pair via mismatch alignments to form higher order structures that have the potential to cause replication blocks, followed by strand slippage and sequence expansion. The all-purine d(GGA)n triplet repeat sequence is of particular interest because purines can align via G.G, A.A and G.A mismatch formation. RESULTS: We have solved the structure of the uniformly 13C,15N-labeled d(G1-G2-A3-G4-G5-A6-T7) sequence in 10 mM Na+ solution. This sequence adopts a novel twofold-symmetric duplex fold where interlocked V-shaped arrowhead motifs are aligned solely via interstrand G1.G4, G2.G5 and A3.A6 mismatch formation. The tip of the arrowhead motif is centered about the p-A3-p step, and symmetry-related local parallel-stranded duplex domains are formed by the G1-G2-A3 and G4-G5-A6 segments of partner strands. CONCLUSIONS: The purine-rich (GGA)n triplet repeat sequence is dispersed throughout the eukaryotic genome. Several features of the arrowhead duplex motif for the (GGA)2 triplet repeat provide a unique scaffold for molecular recognition. These include the large localized bend in the sugar-phosphate backbones, the segmental parallel-stranded alignment of strands and the exposure of the Watson-Crick edges of several mismatched bases.

Base Pair Mismatch↗
PubMed1999

Saccharide-RNA recognition in a complex formed between neomycin B and an RNA aptamer.

BACKGROUND: Aminoglycoside antibiotics can target RNA folds with micromolar affinity and inhibit biological processes ranging from protein biosynthesis to ribozyme action and viral replication. Specific features of aminoglycoside antibiotic-RNA recognition have been probed using chemical, biochemical, spectroscopic and computational approaches on both natural RNA targets and RNA aptamers identified through in vitro selection. Our previous studies on tobramycin-RNA aptamer complexes are extended to neomycin B bound to its selected RNA aptamer with 100 nM affinity. RESULTS: The neamine moiety (rings I and II) of neomycin B is sandwiched between the major groove floor of a 'zippered-up' G.U mismatch aligned segment and a looped-out purine base that flaps over the bound antibiotic. Specific intermolecular hydrogen bonds are observed between the charged amines of neomycin B and base mismatch edges and backbone phosphates. These interactions anchor 2-deoxystreptamine ring I and pyranose ring II within the RNA-binding pocket. CONCLUSIONS: The RNA aptamer complexes with tobramycin and neomycin B utilize common architectural principles to generate RNA-binding pockets for the bound aminoglycoside antibiotics. In each case, the 2-deoxystreptamine ring I and an attached pyranose ring are encapsulated within the major groove binding pocket, which is lined with mismatch pairs. The bound antibiotic within the pocket is capped over by a looped-out base and anchored in place through intermolecular hydrogen bonds involving charged amine groups of the antibiotic.

Anti-Bacterial Agents↗