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Biomedical subjects

A Majumdar

Publications and source records attributed to A Majumdar.

At least 73 records · Page 4Linked to original sources
PubMed1999

Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein.

The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle. Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif. One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin. Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema. Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP. An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42. A carboxy-terminal beta-hairpin and alpha-helix pack against switch II. The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42. Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases. Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors.

Amino Acid Sequence↗
PubMed1999

Podocyte differentiation in the absence of endothelial cells as revealed in the zebrafish avascular mutant, cloche.

The physiological functions of the zebrafish pronephros are blood plasma filtration and osmoregulation. The pronephric glomerulus is vascularized through a capillary network sprouting from the dorsal aorta. Vascularization of the glomerulus, visualized by flk-1 expression and alkaline phosphatase reactivity, involves the intimate association between podocytes and endothelial cells and the formation of an intervening glomerular basement membrane (GBM). Cell-cell interactions between podocytes and endothelial cells are thought to play an important role in glomerular angiogenesis. In order to determine whether endothelial cell-derived signals were required for podocyte differentiation, we employed in situ hybridization and electron microscopy to investigate glomerulogenesis in the zebrafish mutant cloche (clo), where endothelial cell development is blocked at an early stage. In clo mutants, glomerular epithelial cells expressing the podocyte specific marker wt1 display well-formed foot processes and are able to form a GBM, suggesting podocytes are able to morphologically differentiate in the absence of endothelia or endothelial-derived signals. The presence of irregular aggregates in the clo GBM as well as the apparent effacement of podocyte foot processes implies a role for endothelial cells in the maintenance of the mature glomerular filtration barrier.

Animals↗
PubMed1999

RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.

Arginine↗
PubMed1999

Observation and measurement of internucleotide 2JNN coupling constants between 15N nuclei with widely separated chemical shifts.

Scalar coupling correlations between hydrogen bonded 15N nuclei in non Watson-Crick base pairs is a critical step in the structure determination of unusual nucleic acids. For observing the 2JNN coupling constant between far upfield N2,N6 (amino) nitrogens and far downfield (N1,N3,N7) nitrogens (separated by 150-160 ppm), the HNN-COSY experiment (Dingley and Grzesiek, 1998) is rather insensitive, due to technical difficulties associated with simultaneous excitation of both extremes of the 15N spectrum. These nuclei may be correlated by treating them in a pseudo-heteronuclear manner, using 15N selective pulses. The wide chemical shift separation allows accurate measurement of the 2JNN coupling constant using spin-echo difference methods. Pulse sequences for observation and measurement of 2JNN coupling constants between amino and N7 nuclei are presented and demonstrated on an A-A mismatch segment of the uniformly (15N,13C) labelled DNA sample, d(GGAGGAT)2.

Base Pairing↗
PubMed1999

Observation of internucleotide NH...N hydrogen bonds in the absence of directly detectable protons.

Several structural motifs found in nucleic acids involve N-H...N hydrogen bonds in which the donor hydrogens are broadened to extinction due to chemical or conformational exchange. In such situations, it is impossible to use the well-established HNN-COSY or soft HNN-COSY experiments, which report the presence of the hydrogen bond directly on the donor proton(s). We present a pulse sequence, H(CN)N(H), for alleviating this problem in hydrogen bonds of the type NdH...Na-CH, in which the donor Nd nitrogen is correlated with the corresponding non-exchangeable C-H proton associated with the acceptor Na nitrogen. In this way, missing NdH...Na correlations in an HNN-COSY spectrum may be recovered from CH-Nd correlations in the H(CN)N(H) spectrum. By correlating a different set of nuclei relative to the HNN-COSY class of experiments, the H(CN)N(H) experiment also serves to remove ambiguities associated with degeneracies in HNN-COSY spectra. The technique is demonstrated on d(GGAGGAG)4,a quadruplex containing a novel A. (G.G.G.G). A hexad and on d(GGGCAGGT)4, containing a G.G.G.C tetrad, in which missing NH2...N7 correlations are retrieved via H8-(N2,N6) correlations in the H(CN)N(H) spectrum.

Adenine↗
PubMed1999

Substance P-induced vasodilatation is mediated by the neurokinin type 1 receptor but does not contribute to basal vascular tone in man.

AIMS: Following intravenous administration of its prodrug, L-758,298, we assessed the pharmacodynamics of L-754,030, a novel and highly selective NK1 receptor antagonist, by examining systemic haemodynamics and the blood flow responses to intra-arterial substance P infusion. METHODS: Sixteen healthy male volunteers participated in a double-blind, randomised, placebo controlled crossover trial of L-758 298. Forearm blood flow was measured using venous occlusion plethysmography during intrabrachial substance P infusion (0.125-128 pmol min-1 ). In part 1, eight subjects received substance P infusions before and during placebo, 0.25 mg, 1 mg or 5 mg of L-758 298. In part 2, eight subjects received substance P infusions 24 h after placebo or 1.43 mg of L-758 298. RESULTS: L-758 298 caused dose dependent inhibition of substance P induced vasodilatation (P<0.001). Placebo adjusted differences (95% CI) in baseline forearm blood flow, mean arterial pressure and heart rate showed no relevant changes with 5 mg of L-758 298 (>1400-fold shift in substance P response): 0.00 (-0.49 to +0.49) ml 100 ml-1 min-1, 1. 0 (-3.2 to +5.2) mmHg and 1.9 (-5.9 to +9.7) beats min-1, respectively. Twenty-four hours after 1.43 mg of L-758,298, there was approximately 34-fold shift in response to substance P induced vasodilatation (P<0.008) at plasma L-754 030 concentrations of 2-3 ng ml-1. L-758 298 was generally well tolerated without serious adverse events. CONCLUSIONS: Substance P induced forearm vasodilatation is mediated by the endothelial cell NK1 receptor in man but endogenous substance P does not appear to contribute to the maintenance of peripheral vascular tone or systemic blood pressure.

Acetals↗
PubMed1998

Characterizing semilocal motions in proteins by NMR relaxation studies.

The understanding of protein function is incomplete without the study of protein dynamics. NMR spectroscopy is valuable for probing nanosecond and picosecond dynamics via relaxation studies. The use of 15N relaxation to study backbone dynamics has become virtually standard. Here, we propose to measure the relaxation of additional nuclei on each peptide plane allowing for the observation of anisotropic local motions. This allows the nature of local motions to be characterized in proteins. As an example, semilocal rotational motion was detected for part of a helix of the protein Escherichia coli flavodoxin.

Escherichia coli↗
PubMed1998

SIRE-1, a copia/Ty1-like retroelement from soybean, encodes a retroviral envelope-like protein.

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large, copia/Ty1-like retroelement designated SIRE-1. A copy of this element has been recovered from a Glycine max genomic library. DNA sequence analysis of two SIRE-1 subclones revealed that SIRE-1 contains a long, uninterrupted, ORF between the 3' end of the pol ORF and the 3' long terminal repeat (LTR), a region that harbors the env gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, alpha-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of SIRE-1 to the copia/Ty1 family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to copia/Ty1 retrotransposons.

Amino Acid Sequence↗
PubMed1998

Synthesis of a methylenebis(phosphonate) analogue of mycophenolic adenine dinucleotide: a glucuronidation-resistant MAD analogue of NAD.

Mycophenolic alcohol (MPAlc), obtained by reduction of the carboxylic group of mycophenolic acid (MPA), was coupled with 2',3'-O-isopropylideneadenosine 5'-methylenebis(phosphonate) (4) in the presence of diisopropylcarbodiimide (DIC) to give P1-(2',3'-O-isopropylideneadenosin-5'-yl)-P2-(mycophenolic alcohol-6'-yl)methylenebis(phosphonate) (8) in 32% yield. Deisopropy-lidenation of 8 with CF3COOH/H2O afforded the methylenebis(phosphonate) analogue 3 of mycophenolic adenine dinucleotide (MAD). Compound 3, beta-methylene-MAD, was found to be a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH) type II (Ki = 0.3 microM) as well as an inhibitor of growth of K562 cells (IC50 = 1.5 microM). In contrast to MPA and mycophenolic alcohol, beta-methylene-MAD was not converted into the glucuronide when incubated with uridine 5'-diphosphoglucuronyltransferase.

Adenine Nucleotides↗
PubMed1998

3D HCCH-COSY-TOCSY experiment for the assignment of ribose and amino acid side chains in 13C labeled RNA and protein.

A new 3D HCCH-COSY-TOCSY experiment is presented for the assignment of RNA sugar and protein side chains. The experiment, which combines COSY and TOCSY units, is more powerful than the sum of individual HCCH-COSY and HCCH-TOCSY pulse sequences. The experiment was applied to a 13C, 15N-labeled 26 mer RNA complexed with the antibiotic tobramycin, and a 12 kDa 13C, 15N-labeled FKBP12 protein sample. The power of HCCH-COSY-TOCSY is demonstrated through complete spin system assignments of sugars in the 26 mer RNA sample, which could not be assigned using a combination of HCCH-COSY, HCCH-TOCSY and 13C-edited NOESY experiments.

Amino Acids↗
PubMed1998

Characterization of the dose-dependent time of peak effect in indirect response models.

The general conceptual model for non-steady state pharmacokinetic/pharmacodynamic data includes a distribution phase between the plasma and the biophase compartments, which can be expressed by a link model, and inhibition or stimulation of the production or removal of a mediator, which can be expressed by an indirect response model. The inhibition or the stimulation step modeled by an indirect response model generates dose-dependent time of peak effect. This report provides a mathematical expression for this time of peak effect which is then used to determine how this time depends on dose, the endogenous elimination rate of the mediator, and the pharmacokinetic parameters of the drug. The report then uses this time of peak effect to find the response versus time curve. The mathematical relationship for the time of peak effect and the response vs. time curve are then developed for a cascade of two indirect steps. The approach presented here is easily implemented on a spreadsheet and does not require numerically solving nonlinear differential equations. The approach should help to analyze various issues related to fitting indirect response models to non-steady state pharmacokinetic/pharmacodynamic data, especially, when one is trying to fit data to a cascade of indirect steps.

Humans↗
PubMed1998

Targeted gene knockout mediated by triple helix forming oligonucleotides.

Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.

Animals↗
PubMed1998

Solution structure of P22 transcriptional antitermination N peptide-boxB RNA complex.

We have determined the solution structure of a 15-mer boxB RNA hairpin complexed with a 20-mer basic peptide of the N protein involved in bacteriophage P22 transcriptional antitermination. Complex formation involves adaptive binding with the N peptide adopting a bent alpha-helical conformation that packs tightly through hydrophobic and electrostatic interactions against the major groove face of the boxB RNA hairpin, orienting the open opposite face for potential interactions with host factors and/or RNA polymerase. Four nucleotides in the boxB RNA hairpin pentaloop form a stable GNRA like tetraloop structural scaffold on complex formation, allowing the looped out fifth nucleotide to make extensive hydrophobic contacts with the bound peptide. The guanidinium group of a key arginine is hydrogen-bonded to the guanine in a loop-closing sheared G.A mismatch and to adjacent backbone phosphates. The identified intermolecular contacts account for the consequences of N peptide and boxB RNA mutations on bacteriophage transcriptional antitermination.

Amino Acid Sequence↗
PubMed1998

A two-dimensional 1H detected 13C NMR investigation of pyruvate metabolism in Halobacterium salinarium.

Two-dimensional 1H detected 13C NMR spectroscopy has been used to study the intracellular metabolism of [3-(13)C]pyruvate in Halobacterium salinarium. The method, resulting in considerable improvement in spectral resolution and signal-to-noise ratio, is well suited for studying transient metabolic intermediates. Pyruvate utilization by the bacterium is a double exponential function with rate constants of 49.13 and 4.67x10(-3) per min. The relative 13C enrichment is the fastest for C-3 glutamate. Glutamate C-4 labeling decreases initially and increases later on during incubation, while glutamine C-3 is high to begin with and exhibits a declining trend. The glutamate labeling indicates a high initial flux through pyruvate carboxylase and extensive randomizing of the label in the tricarboxylic acid cycle.

Citric Acid Cycle↗
PubMed1998

Gastric rupture secondary to successful Heimlich manoeuvre.

A fatal case of gastric rupture following the Heimlich manoeuvre is reported. This life-threatening complication has only been reported previously in seven patients with a high mortality rate. All patients should be assessed immediately following this manoeuvre for any potentially life-threatening complications.

Airway Obstruction↗
PubMed1998

Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.

The zebrafish pronephric kidney provides a simplified model of nephron development and epithelial cell differentiation which is amenable to genetic analysis. The pronephros consists of two nephrons with fused glomeruli and paired pronephric tubules and ducts. Nephron formation occurs after the differentiation of the pronephric duct with both the glomeruli and tubules being derived from a nephron primordium. Fluorescent dextran injection experiments demonstrate that vascularization of the zebrafish pronephros and the onset of glomerular filtration occurs between 40 and 48 hpf. We isolated fifteen recessive mutations that affect development of the pronephros. All have visible cysts in place of the pronephric tubule at 2-2.5 days of development. Mutants were grouped in three classes: (1) a group of twelve mutants with defects in body axis curvature and manifesting the most rapid and severe cyst formation involving the glomerulus, tubule and duct, (2) the fleer mutation with distended glomerular capillary loops and cystic tubules, and (3) the mutation pao pao tang with a normal glomerulus and cysts limited to the pronephric tubules. double bubble was analyzed as a representative of mutations that perturb the entire length of the pronephros and body axis curvature. Cyst formation begins in the glomerulus at 40 hpf at the time when glomerular filtration is established suggesting a defect associated with the onset of pronephric function. Basolateral membrane protein targeting in the pronephric duct epithelial cells is also severely affected, suggesting a failure in terminal epithelial cell differentiation and alterations in electrolyte transport. These studies reveal the similarity of normal pronephric development to kidney organogenesis in all vertebrates and allow for a genetic dissection of genes needed to establish the earliest renal function.

Animals↗