Biomedical subjects
Publications and source records attributed to K Ray.
The study of fluorescence energy transfer from the phenyl groups of the micellar triton X-100 (TX-100) to solubilised 1-pyrene butyric acid (PBA) has been carried out. Through the analysis of the donor fluorescence quenching energy transfer efficiency has been determined. The observed donor-acceptor separation suggests that pyrene molecules are distributed uniformly in the micellar core.
Interleukin-1 (IL-1) is a proinflammatory cytokine that elicits its pleiotropic effects through activation of the transcription factors NF-kappaB and AP-1. Binding of IL-1 to its receptor results in rapid assembly of a membrane-proximal signalling complex that consists of two different receptor chains (IL-1Rs), IL-1RI and IL-1RAcP, the adaptor protein MyD88, the serine/threonine kinase IRAK and a new protein, which we have named Tollip. Here we show that, before IL-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with IL-1RAcP. Co-recruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and IL-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the IL-1R signalling pathway.
We aim to monitor the trends of antimicrobial resistance in Neisseria gonorrhoeae and to compare the results of antimicrobial sensitivity by disc diffusion and minimum inhibitory concentration (MIC). Two hundred and eleven confirmed strains of N. gonorrhoeae were subjected to antimicrobial sensitivity testing by disc diffusion using penicillin, tetracycline, ciprofloxacin and ceftriaxone from 1995 to June 1999. Penicillinase-producing Neisseria gonorrhoeae (PPNG) were detected by lodometric method. Minimum inhibitory concentration was determined by E test. A low level of penicillin resistance and PPNG detected in 1996 was maintained over the years. Significant increasing trend of tetracycline and ciprofloxacin resistance with high MIC i.e. 2-96 microg/ml and 1-32 microg/ml respectively were found. Ceftriaxone was found to be the drug of choice, being 100% sensitive. Comparison of resistance pattern by the 2 tests showed satisfactory agreement. Emergence of penicillin, quinolone and tetracyline resistance in N. gonorrhoeae isolates from a major STD centre at New Delhi indicates the need for increased awareness, prudent use of antimicrobials, and evaluation of new antimicrobials for the treatment of gonorrhoea.
Naturally occurring mutations identified in subjects with autosomal dominant hypocalcemia (ADH) and the calcimimetic compound, R-568, have both been reported to increase Ca2+ sensitivity of the Ca2+ receptor (CaR). To gain insight into their mechanism of action, we studied interactions between four different ADH mutations located in the amino-terminal extracellular domain (ECD) and R-568. We found that R-568 increased the sensitivity of three of the ADH mutant receptors, but the Leu125Pro mutant appeared to be maximally left-shifted in that neither R-568 addition nor combining other ADH mutations with Leu125Pro gave increases in sensitivity comparable to those seen with the three other ADH mutations studied. We also made use of truncation and deletion mutants of the CaR and CaR/metabotropic glutamate receptor type 1 (mGluR1) chimeras to study both the site of action of R-568 and the effect of the Leu125Pro activating mutation. R-568 was effective in receptor constructs containing the seven transmembrane domain (7TM) of the CaR, but not in those containing the mGluR1 7TM. R-568, moreover, imparted Ca2+ responsiveness to CaR constructs lacking all or part of the CaR ECD. The Leu125Pro mutation in contrast conferred no or minimal increase in Ca2+ responsiveness to CaR constructs lacking part of the CaR ECD but showed a striking increase in basal activity in the context of chimeras containing an mGluR1 7TM. Our results localize the site of action of NPS-568 specifically to the CaR 7TM. Our results with the Leu125Pro mutant, furthermore, suggest that the mGluR1 7TM domain may be more permissive for activation than the 7TM domain of the CaR.
We studied the activity of mutants involving the aminoterminal extracellular, seven-transmembrane (7TM) and carboxy-terminal tail domains of the human Ca2+ receptor to gain insight into the functional interactions between these domains during receptor activation. Missense mutations of highly conserved residues, D190 and E297, in the extracellular domain (ECD), and a mutation within part of the proximal carboxyterminal tail, A877-880E, resulted in receptors with severely reduced response to Ca2+ despite adequate cell surface expression. Coexpression of either D190A or E297K mutants with A877-880E led to significant reconstitution of function. No such reconstitution occurred when D190A or E297K mutants were coexpressed with a truncation mutant possessing an intact amino-terminal extracellular and first transmembrane domain, despite evidence for heterodimerization and cell surface expression of the respective mutant receptors. In addition, no reconstitution of function was observed when D190A was coexpressed with a deletion Ca2+ receptor mutant lacking only a cysteine-rich region located in the ECD of the Ca2+ receptor (Ca-//-Ca). Moreover, coexpression of this Ca-//-Ca with A877-880E did not recover function. The results show that Ca2+ receptor extracellular and 7TM domains are discrete entities that can communicate within the context of a heterodimer composed of complementary mutant receptors. Two intact 7TM domains and two intact cysteine-rich regions appear to be required for such communication to occur. The results are discussed in the context of a speculative model of receptor structure and function.
KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.
We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor.
cGMP-phosphodiesterase (PDE) is composed of two catalytic (alpha and beta) and two identical inhibitory (gamma) subunits. The human gene (PDE6D) encoding a new subunit (delta) has been characterized and mapped to the long arm of chromosome 2 (HSA2q35-q36) where a new autosomal recessive retinitis pigmentosa (arRP) locus (RP26) has been localized. Characterization of the canine PDE6D shows the gene is about 4.2kb containing four exons interrupted by three introns; the size of the cDNA is 1059bp with an open reading frame (ORF) of 453bp. A single transcript of identical size (1.43kb) was detected in all tissues examined (liver, lung, spleen, kidney, heart, brain and retina), with the highest abundance in the retina. Canine PDE6D has been localized to canine radiation hybrid group 14-a, which extends conserved synteny between the dog, human chromosome 2q and mouse chromosome 1. The characterization of the canine PDE6D gene and its mapping provide important information for testing causal association of the gene with canine retinal degenerations, in particular rod-cone dysplasia 2 (rcd2) in collie dogs. This disease is characterized by abnormal retinal cGMP metabolism due to a deficiency in cGMP-PDE activity, yet the alpha, beta and gamma subunits of PDE have been excluded as candidate gene loci.
Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the beta-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13-q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits.
We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.
Upon interleukin 1 (IL-1) stimulation, the IL-1-receptor (IL-1R)-associated kinase (IRAK) is rapidly recruited to the IL-1R complex and undergoes phosphorylation. Here we demonstrate that recombinant wild-type IRAK (IRAK-WT), but not a kinase-defective mutant with Asp340 replaced by an asparagine residue (IRAK-Asp340Asn), is highly phosphorylated and is capable of auto-phosphorylation in vitro. Overexpression of both IRAK-WT and IRAK-Asp340Asn caused activation of nuclear factor kappaB, suggesting that the kinase activity of IRAK is not required outside of the IL-1R complex.
Electron transmission experiments demonstrate a large asymmetry in the scattering probability of polarized electrons by thin organized films of chiral molecules. This large asymmetry results from the interaction of the electron's wavefunction with many scatterers (molecules) in the organized monolayer structure and represents a manifestation of quantum interference on the scale of supramolecular lengths.
Rod-cone dysplasia types 1 (rcd1; Irish setter) and 2 (rcd2; collie) in dogs are early onset forms of progressive retinal atrophy (PRA) which serve as models of retinitis pigmentosa (RP) in humans. As both rcd1 and rcd2 result from abnormal retinal cGMP metabolism associated with a deficiency in cGMP-phosphodiesterase (PDE) activity, and a nonsense mutation in the PDE6B subunit gene has been shown to cause rcd1, the genes encoding the four subunits of the PDE complex (PDE6A, PDE6B, PDE6G and PDE6D) make compelling candidates for the rcd2 locus. We adopted diverse strategies to evaluate causal association of the four PDE subunit genes with the rcd2 phenotype. Identification in an informative pedigree of obligate recombinations between intragenic polymorphisms within PDE6A and PDE6D and the rcd2 locus unequivocally excludes these two genes. PDE6B was excluded by a breeding strategy demonstrating nonallelism of rcd1 and rcd2. Direct sequencing of PDE6G from an rcd2 -homozygous collie dog revealed no abnormality in the entire genomic sequence. To evaluate cosegregation between PDE6G and rcd2, advantage was taken of prior knowledge that PDE6G and Galactokinase 1 (GALK1) localize to the same canine-rodent somatic hybrid cell line. Linkage analysis using a single nucleotide polymorphism (SNP) in the PDE6G gene, and a (CA)n repeat polymorphism in the GALK1 gene, which were both segregating in an unrelated pedigree, established close linkage of these two genes (theta = 0; Z = 4.21). Identification of obligate recombinations between GALK1 and the rcd2 locus in an informative rcd2 pedigree thus excluded PDE6G as a candidate gene for rcd2; the exclusion distance between GALK1 and rcd2 is at least 0.35 cM. These results therefore exclude the entire set of genes coding for the rod PDE complex as candidates for rcd2.
We report two cases of hyperkalaemia related to the use of the salt substitute 'Lo Salt' in hypertensive patients on treatment with ACE inhibitors. In each case serum potassium returned to the normal range after cessation of the salt substitute. Without vigilance the contribution of the salt substitute to hyperkalaemia would have been overlooked and an ACE inhibitor erroneously withdrawn.
Purebred strains, pronounced phenotypic variation, and a high incidence of heritable disease make the domestic dog uniquely suited to complement genetic analyses in humans and mice. A comprehensive genetic linkage map would afford many opportunities in dogs, ranging from the positional cloning of disease genes to the dissection of quantitative differences in size, shape, and behavior. Here we report a canine linkage map with the number of mapped loci expanded to 276 and 10-cM coverage extended to 75-90% of the genome. Most of the 38 canine autosomes are likely represented in the collection of 39 autosomal linkage groups. Eight markers were sufficiently informative to detect linkage at distances of 10-13 cM, yet remained unlinked to any other marker. Taken together, the results suggested a genome size of about 27 M. As in other species, the genetic length varied between sexes, with the female autosomal distance being approximately 1.4-fold greater than that of male meioses. Fifteen markers anchored well-described genes on the map, thereby serving as landmarks for comparative mapping in dogs. We discuss the utility of the current map and outline steps necessary for future map improvement.
Familial hypoparathyroidism is an unusual and genetically heterogeneous group of disorders that may be isolated or may be associated with congenital or acquired abnormalities in other organs or glands. We have evaluated a family with a novel syndrome of autosomal dominant hypoparathyroidism, short stature, and premature osteoarthritis. A 74-yr-old female (generation I) presented with hypoparathyroidism, a movement disorder secondary to ectopic calcification of the cerebellum and basal ganglia, and a history of knee and hip replacements for osteoarthritis. Two members of generation II and one member of generation III were also documented with hypoparathyroidism, short stature, and premature osteoarthritis evident as early as 11 yr. Because of the known association between autosomal dominant hypoparathyroidism and activating mutations of the calcium-sensing receptor (CaR) gene, further studies were performed. Sequencing of PCR-amplified genomic DNA revealed a leucine to valine substitution at position 616 in the first transmembrane domain of the CaR, which cosegregated with the disorder. However, this amino acid sequence change did not affect the total accumulation of inositol phosphates as a function of extracellular calcium concentrations in transfected HEK-293 cells. In conclusion, a sequence alteration in the coding region of the CaR gene was identified, but is not conclusively involved in the etiology of this novel syndrome. The cosegregation of hypoparathyroidism, short stature, and osteoarthritis in this kindred does suggest a genetic abnormality involving a common molecular mechanism in parathyroid, bone, and cartilage.
PURPOSE: Progressive rod-cone degeneration (prcd) is an autosomal recessive retinal degeneration of dogs characterized by abnormalities in lipid metabolism. It has recently been mapped to the centromeric region of canine chromosome 9, homologous to human 17q, which contains the apolipoprotein H (apoH, protein; APOH, gene) gene involved in lipid metabolism and regulation of triglycerides. The present study was undertaken to evaluate APOH as a positional candidate for prcd. METHODS: Expression of APOH in the retina was examined by reverse transcription-polymerase chain reaction (RT-PCR) and by immunocytochemistry in normal and prcd-affected dogs. The level of apoH in the plasma was determined by western blot analysis. Intragenic polymorphic markers were identified and typed in the prcd pedigree. Canine-rodent hybrid cell lines were analyzed to detect canine APOH. RESULTS: ApoH has been localized to the photoreceptor outer segment layer by immunocytochemistry. Its expression in the retina of normal and prcd-affected dogs was confirmed by RT-PCR. The levels of antihuman apoH cross-reacting material in plasma were similar in all dogs, regardless of disease status. Finally, linkage analysis of the APOH gene with the disease locus in the prcd pedigree detected 3 recombinants among 70 informative offsprings (lod score 15.09 at 0 = 4.3 centimorgan [cM]). CONCLUSIONS: APOH is expressed in the retina and tightly linked to the prcd locus. However, despite its potential role in phenotypes of abnormal lipid metabolism associated with prcd, the gene has been excluded as a primary candidate for prcd by linkage analysis.
The molecular defects causing severe factor IX deficiency were identified in two distinct canine breed-variants. Both defects were associated with an absence of plasma factor IX coagulant activity and antigen. A large deletion mutation was found in 1 breed variant, spanning the entire 5' region of the factor IX gene extending to exon 6. An approximately 5 kb insertion disrupted exon 8 of the second breed-variant. This insertion was associated with alternative splicing between a donor site 5' and acceptor site 3' to the normal exon 8 splice junction, with introduction of a new stop codon. The resultant transcript lacked most of the factor IX catalytic domain and 3' untranslated region. Molecular analyses of canine hemophilia B define an experimental model for study of inhibitor formation and gene therapy strategies, and provide insight into spontaneous mutation mechanisms in the factor IX gene and on the X chromosome of mammalian species.