PubMed Health⌕ Search

Biomedical subjects

R Radhakrishnan

Publications and source records attributed to R Radhakrishnan.

At least 37 records · Page 2Linked to original sources
PubMed1997

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.

Amino Acid Sequence↗
PubMed1997

Refined structures of three crystal forms of toxic shock syndrome toxin-1 and of a tetramutant with reduced activity.

The structure of toxic shock syndrome toxin-1 (TSST-1), the causative agent in toxic shock syndrome, has been determined in three crystal forms. The three structural models have been refined to R-factors of 0.154, 0.150, and 0.198 at resolutions of 2.05 A, 2.90 A, and 2.75 A, respectively. One crystal form of TSST-1 contains a zinc ion bound between two symmetry-related molecules. Although not required for biological activity, zinc dramatically potentiates the mitogenicity of TSST-1 at very low concentrations. In addition, the structure of the tetramutant TSST-1H [T69I, Y80W, E132K, I140T], which is nonmitogenic and does not amplify endotoxin shock, has been determined and refined in a fourth crystal form (R-factor = 0.173 to 1.9 A resolution).

Bacterial Toxins↗
PubMed1997

Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions.

The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single-domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.

Amino Acid Sequence↗
PubMed1997

Current status of filariasis in Chavakad taluk, Trichur district, Kerala.

A sample survey using parasitological, clinical and entomological indicators was carried out in all the 18 administrative units of Chavakad taluk, Trichur district, Kerala, India to assess the current filariasis situation. Cluster sampling procedure was followed to screen individuals. Both Wuchereria bancrofti and Brugia malayi species were found to be prevalent in this taluk. Microfilaria (mf) carriers with W. bancrofti were detected in eight areas while B. malayi was recorded only from one area. The two species were found to co-exist in another area. The highest infection rate registered for W. bancrofti was 1.51 while it was 0.3 for B. malayi. Infection due to W. bancrofti constituted 87.88% of the total 33 microfilaria cases. Prevalence of B. malayi was very low. Cases with clinical manifestation of filariasis were recorded in all the four areas surveyed. The present trend in the prevalence of infection (mf) and disease showed a decline in both the species when compared to earlier surveys of 1960s. At least 11 areas are still endemic for filariasis in this taluk. Although prevalence of mf was recorded for the first time in one of the areas viz., Elavalli, the rate was only 0.16%. Entomological surveys revealed the presence of 14 mosquito species, of which Culex quinquefasciatus contributed 84.85% and Mansonia 0.77%. While C. quinquefasciatus was recorded in all the 18 areas, Mansonia spp were found only in 8 areas. Only C. quinquefasciatus was found to harbour different developmental stages of W. bancrofti, with overall infection and infectivity rates of 1.94 and 0.97 respectively. The possible reason for the decline in vector density and infection in man are postulated.

Adolescent↗
PubMed1996

Delayed postprandial retinyl palmitate and squalene removal in a patient heterozygous for apolipoprotein A-IFIN mutation (Leu 159-->Arg) and low HDL cholesterol level without coronary artery disease.

A low HDL cholesterol level is frequently but not consistently associated with inefficient postprandial fat clearance. We studied triglycerides, retinyl palmitate and squalene and apolipoprotein B-48 after a fat loading test in one subject heterozygous for a novel point mutation of apolipoprotein A-I (A-IFIN, Leu 159-->Arg) and low HDL cholesterol level without coronary artery disease, and in 16 healthy controls with the same apolipoprotein E phenotype, 3/3, as the proband. HDL cholesterol and apolipoprotein A-I levels were 0.32 mmol/l and 57 mg/dl in the proband, and 1.29 +/- 0.12 mmol/l (mean +/- S.E.) and 126 +/- 4 mg/dl in the controls. The peak concentration for triglycerides in plasma, chylomicrons and VLDL occurred at 4 h both in the case and controls. However, the peak concentrations for retinyl palmitate and squalene in chylomicrons and VLDL were delayed to 12 h in the proband compared with 4 and 9 h in the controls. The peak of apolipoprotein B-48 occurred at 6 h in the proband and at 4 h in the controls, so that triglycerides, apolipoprotein B-48 and retinyl palmitate and squalene peaked differently. After 24 h, retinyl palmitate, squalene, and apolipoprotein B-48 had returned to the baseline levels. The results show for the first time an impaired postprandial lipoprotein removal in a case heterozygote with moderately low HDL cholesterol due to an apolipoprotein A-1 mutation not associated with coronary artery disease.

Adult↗
PubMed1996

Zinc mediated dimer of human interferon-alpha 2b revealed by X-ray crystallography.

BACKGROUND: The human alpha-interferon (huIFN-alpha) family displays broad spectrum antiviral, antiproliferative and immunomodulatory activities on a variety of cell types. The diverse biological activities of the IFN-alpha's are conveyed to cells through specific interactions with cell-surface receptors. Despite considerable effort, no crystal structure of a member of this family has yet been reported, because the quality of the protein crystals have been unsuitable for crystallographic studies. Until now, structural models of the IFN-alpha's have been based on the structure of murine IFN-beta (muIFN-beta). These models are likely to be inaccurate, as the amino acid sequence of muIFN-beta differs significantly from the IFN-alpha's at proposed receptor-binding sites. Structural information on a huIFN-alpha subtype would provide an improved basis for modeling the structures of the entire IFN-alpha family. RESULTS: The crystal structure of recombinant human interferon-alpha 2b (huIFN-alpha 2b) has been determined at 2.9 A resolution. HuIFN-alpha 2b exists in the crystal as a noncovalent dimer, which associates in a novel manner. Unlike other structurally characterized cytokines, extensive interactions in the dimer interface are mediated by a zinc ion (Zn2+). The overall fold of huIFN-alpha 2b is most similar to the structure of muIFN-beta. Unique to huIFN-alpha 2b is a 3(10) helix in the AB loop which is held to the core of the molecule by a disulfide bond. CONCLUSIONS: The structure of huIFN-alpha 2b provides an accurate model for analysis of the > 15 related type 1 interferon molecules. HuIFN-alpha 2b displays considerable structural similarity with muIFN-beta, interleukin-10 and interferon-gamma, which also bind related class 2 cytokine receptors. From these structural comparisons and numerous studies on the effects of mutations on biological activity, we have identified protein surfaces that appear to be important in receptor activation. This study also reveals the potential biological importance of the huIFN-alpha 2b dimer.

Amino Acid Sequence↗
PubMed1995

Actions of D-amino acid-substituted analogues of des-Asp-angiotensin I on the central pressor action of angiotensin III.

The ability of intracerebroventricularly (i.c.v.) administered D-amino acid-substituted analogues of des-Asp-angiotensin I to attenuate the central pressor action of angiotensin III in the rat was investigated. Of the 9 D-amino acid-substituted analogues, only D-tyrosine-des-Asp-angiotensin I was active. I.c.v. D-tyrosine-angiotensin I but not i.c.v. D-isoleucine-angiotensin I (when prevented from degradation by angiotensin converting enzyme with captopril) also attenuated the central pressor action of angiotensin III. In vitro incubation of angiotensin I, D-tyrosine-angiotensin I and D-isoleucine-angiotensin I with brain homogenate resulted in the formation of des-Asp-angiotensin I, D-tyrosine-des-Asp-angiotensin I and D-isoleucine-des-Asp-angiotensin I, respectively. This shows that i.c.v. angiotensin I and D-tyrosine-angiotensin I were converted by brain aminopeptidase to des-Asp-angiotensin I and D-tyrosine-des-Asp-angiotensin I, respectively, which then attenuated the pressor action of angiotensin III. When compared to the findings of similar D-substitution studies carried out with angiotensin II and [Sar1,Ile8]angiotensin II by other investigators, des-Asp-angiotensin I has a stringent structural-activity relationship. These findings suggest that, at the physiological level, des-Asp-angiotensin I is formed from angiotensin I and that the nonapeptide probably acts on a distinct subtype of angiotensin receptors.

Amino Acids↗
PubMed1995

MF59. Design and evaluation of a safe and potent adjuvant for human vaccines.

MF59 is a safe, practical, and potent adjuvant for use with human vaccines. The formulation is easily manufactured, may be sterilized by filtration, and is both compatible and efficacious with all antigens tested to date. MF59 has been shown to be a potent stimulator of cellular and humoral responses to subunit antigens in both animal models and clinical studies. Toxicology studies in animal models and Phase I-III studies in humans have demonstrated the safety of MF59 with HSV, HIV, and influenza vaccines.

Adjuvants, Immunologic↗
PubMed1994

Enhanced pressor response to angiotensin III in spontaneously hypertensive rats: effects of losartan.

The conscious spontaneously hypertensive rat (SHR), compared to its normotensive control Wistar Kyoto rat (WKY), exhibited a significantly greater pressor response to i.v. angiotensin II and angiotensin III. Losartan (10 mg/kg i.v.) lowered the basal blood pressure of the SHR but had no significant effect on that of the WKY. However, it attenuated the pressor response to both angiotensins in the SHR and WKY, the degree of attenuation being significantly greater with angiotensin III. In addition, the pressor responses induced by both angiotensin II and angiotensin III in the WKY, compared to those of the SHR, were more markedly inhibited by losartan. The results indicate a possible over-expression of angiotensin AT1 receptors in the SHR, and that both angiotensin II and angiotensin III contribute to the hypertension by acting on these receptors.

Angiotensin II↗
PubMed1994

Novel central action of des-Asp-angiotensin I.

Intracerebroventricularly administered des-Asp-angiotensin I, when prevented from degradation by prior administration of captopril, attenuated dose-dependently the central pressor actions of angiotensin II and angiotensin III in the spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats. This finding is the first demonstration of an intrinsic action of des-Asp-angiotensin I and, together with earlier finding of its increased production in the hypothalamus of the spontaneously hypertensive rat, may support the suggestion that the nonapeptide is a functional angiotensin that regulates the pressor action of angiotensin II and angiotensin III in the brain.

Angiotensin I↗
PubMed1994

Effect of pentobarbital and chlordiazepoxide on the central pressor action of angiotensins in normo- and hypertensive rats.

Pentobarbital and chlordiazepoxide administered intracerebroventricularly (i.c.v.) attenuated dose dependently the pressor action of i.c.v. angiotensin II and angiotensin III in the conscious spontaneously hypertensive rat (SHR) and the normotensive control Wistar Kyoto rat (WKY). Attenuation of the pressor action by the two drugs was more marked in the WKY than in the SHR. Based on the fact that both drugs act via the gamma-aminobutyric acid (GABA) receptors, the data support the suggestions (i) that a central GABAergic system regulates blood pressure, (ii) that there is probable dysfunction of the GABA receptors in SHR.

Angiotensins↗
PubMed1994

Preliminary crystallographic study of protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.

The enzyme protocatechuate 3,4-dioxygenase from the Gram positive organism Brevibacterium fuscum crystallizes in the triclinic space group P1 with unit cell dimensions a = 96.1 A, b = 97.2 A, c = 118.1 A and alpha = 113.9 degrees, beta = 90.7 degrees, gamma = 117.8 degrees. The rod-like crystals diffract to 2.4 A resolution. Rotation function analysis suggests that there are six promoters arranged with local 32 symmetry in the asymmetric unit rather than the previously proposed pentameric complex.

Brevibacterium↗
PubMed1994

Preliminary crystallographic analysis of methane mono-oxygenase hydroxylase from Methylosinus trichosporium OB3b.

The hydroxylase component of the enzyme methane mono-oxygenase from Methylosinus trichosporium OB3b has been crystallized in the orthorhombic space group C222(1) with unit cell dimensions a = 264.5 A, b = 71.2 A, c = 139.4 A. The crystals grow as square, thick plates and diffract to beyond 2 A resolution. There is one half of the hydroxylase dimer in the asymmetric unit.

Crystallization↗
PubMed1993

How the anti-(metal chelate) antibody CHA255 is specific for the metal ion of its antigen: X-ray structures for two Fab'/hapten complexes with different metals in the chelate.

Antibodies with bound metal-chelate haptens provide new means for exploiting the diverse properties of metallic elements. The murine monoclonal antibody CHA255 (IgG1 lambda) binds the metal-chelate hapten indium (III)-4-[N'-(2-hydroxyethyl)thioureido]-L-benzyl-EDTA (designated In-EOTUBE) with high affinity (K(a) = 1.1 x 10(10) M-1). Antibody binding is highly specific for the indium chelate; the affinity decreases as much as 10(4) with other metals, even those having ionic radii close to indium. To better understand this selectivity, the crystal structure of the antigen-binding fragment (Fab') of CHA255 complexed with its hapten, In(III)-EOTUBE, was determined by molecular replacement and refined at 2.2-A resolution. The structure of CHA255 Fab' complexed with Fe(III)-EOTUBE was also determined and refined at 2.8-A resolution. In both structures, the hapten's EDTA moiety is half-buried near the center of the complementarity-determining regions (CDR's). Five of the six CDR's on the Fab' interact with the hapten through protein side-chain atoms (but not main-chain atoms). A novel feature of the In-EOTUBE/Fab' complex is coordination of the indium by N epsilon of one histidine from the heavy chain's third CDR (distance = 2.4 A). The histidine coordination is not observed in the Fe-EOTUBE/Fab' complex, due mainly to a slightly different hapten conformation that reduces metal accessibility; this may partially explain the 20-fold lower affinity of CHA255 for iron hapten. An unexpected feature of the Fab' overall is an elbow angle of 193 degrees (the angle between the pseudodyad axes of the Fab's constant and variable domains).

Amino Acid Sequence↗
PubMed1991

A comparative study of the bronchodilator effect and duration of action of liposome encapsulated beta-2 adrenergic agonists in the guinea-pig.

The abilities of the adrenergic beta-2 agonists metaproterenol and albuterol in free and liposome encapsulated forms to prevent the bronchoconstrictor response to histamine (10 micrograms/kg i.v.) were compared in anesthetized guinea-pigs. Intratracheal instillation of the unencapsulated adrenergic agonists prevented the histamine bronchoconstriction in a dose dependent fashion. Liposomal metaproterenol was less effective in this regard than the same doses of free drug. In contrast, liposomal albuterol produced an initial bronchodilator effect which was similar to comparable doses of free albuterol but the effect was sustained for a longer duration. These data suggest that intratracheal liposome encapsulated bronchodilators were releashed slowly to the bronchi. This slow release rate reduced the available concentrations of metaproterenol below that required for bronchodilator effects, but the more potent albuterol achieved an active concentration and showed a more sustained duration of action. Further, studies showed that liposome encapsulated material could be aerosolized using a standard air blast nebulizer without significant difference (compared to free drug) in the mass of drug delivered or alteration in the percentage of the drug encapsulated within the liposomes.

Adrenergic beta-Agonists↗
PubMed1989

Crystallographic analysis of the inhibition of porcine pancreatic elastase by a peptidyl boronic acid: structure of a reaction intermediate.

The crystal structure of porcine pancreatic elastase (PPE) complexed to carbobenzoxy-alanylisoleucine-boronic acid (ZAIB) is reported to 2.09-A resolution and refined to an R factor of 0.15. This is the first reported structural analysis of PPE with an isoleucine residu in the primary specificity pocket. The results include (1) marked displacement of the inhibitor out of the active site leading to (2) a close (2.2 A) direct contact between B (boron atom of the inhibitor) and N epsilon of His-57 and also (3) covalent bonding (1.5 A) to O gamma of Ser-195. A scheme for the mechanism of inhibition of PPE by ZAIB is proposed. A comparison with a peptidyl difluoromethyl ketone-PPE complex (Ki = 9.5 microns) is made to explain the strong inhibition of PPE by ZAIB (Ki = 0.3 micron). These results lead us to characterize this structure as a time- and space-averaged reaction intermediate, providing fresh insight into the cramped dimensions available in enzymatic catalyses.

Animals↗
PubMed1989

X-ray studies on crystalline complexes involving amino acids and peptides. Part XX. Crystal structures of DL-arginine acetate monohydrate and DL-lysine acetate and a comparison with the corresponding L-amino acid complexes.

Crystals of DL-arginine acetate monohydrate, C6H15N4O2+C2H3O2-.H2O, are monoclinic, P2(1)/c, with a = 13.552(2), b = 5.048(2), c = 18.837(3) A, beta = 101.34(2) degrees and Z = 4, and those of DL-lysine acetate, C6H15N2O2+.C2H3O2- are triclinic, P1, with a = 5.471(2), b = 7.656(2), c = 12.841(2) A, alpha = 94.48(1), beta = 94.59(2), gamma = 98.83(2) degrees and Z = 2. The structures have been solved by direct methods and refined to R = 0.058 and 0.077 for 1522 and 1259 observed reflections respectively. The difference in the number and the nature of proton donors leads to a difference in hydrogen bond density in the two structures. The basic elements of aggregation in both the structures are pairs of amino acid molecules, each pair stabilized by two centrosymmetrically related hydrogen bonds involving alpha-amino and alpha-carboxylate groups, stacked along the shortest dimension to form columns. The pairs are held together in each column by head-to-tail sequences. The columns stack along a crystallographic axis to form layers. Adjacent layers are bridged by acetate ions. The amino acid-acetate interactions are primarily through side chains and involve specific interactions and characteristic interaction patterns. The gross features of molecular aggregation are nearly the same in DL-arginine acetate monohydrate and L-arginine acetate whereas they are substantially different in the lysine complexes. In both cases, one of the two head-to-tail sequences in the L complex is replaced by a hydrogen bonded loop involving alpha-amino and alpha-carboxylate groups, in the DL complex. This may have implications for prebiotic condensation during chemical evolution.

Acetates↗