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Biomedical subjects

K Ray

Publications and source records attributed to K Ray.

At least 91 records · Page 5Linked to original sources
PubMed1998

Evaluation of an educational game for health sciences students.

An educational game was developed to promote student problem solving in the context of small group, problem-based learning (PBL). This study evaluated the game's effects on hypothesis and issue generation in PBL. Students in the Year III PBL course of a BScN program (N = 131) were stratified by program status and then randomly assigned to groups of 9 to 11 people per group. Half of the groups were randomly assigned to use the game (G); the remaining groups used the conventional method (CM) of generating issues and hypotheses. The G and CM groups crossed over for Term II. A Term I posttest demonstrated G groups had a higher proportion of accurate responses (85%) than CM groups (74%). A Term II posttest demonstrated no important differences between G and CM groups, suggesting the educational impact of the game persisted after students stopped using it. Strengths and limitations of the game as a learning aid also are addressed.

Adult↗
PubMed1998

Periodic syphilis profile in a New Delhi hospital.

The study reports the prevalence of syphilis determined at intervals of ten years amongst attendees of the Sexually Transmitted Diseases Centre (STD), in New Delhi, from 1954, to 1994. It was observed that the number of STD cases increased by about eight times in 1984 as compared to 1954. The prevalence of STDs had risen from 5.5 per cent in 1964 to 14.7 per cent in 1994. However, the syphilis load has been decreasing since 1974. Childhood syphilis has decreased from 12.6 per cent in 1954 to 0.5 per cent in 1994. However in adult males it increased from 62.3 per cent in 1954 to 80.4 per cent in 1984. Cardiovascular syphilis was last reported in 1954. The prevalence of syphilis was approximately three times higher in males than in females.

Adult↗
PubMed1998

Molecular diagnostic tests for ascertainment of genotype at the mucopolysaccharidosis type VII locus in dogs.

OBJECTIVE: To develop a molecular diagnostic test to ascertain genotype of the mucopolysaccharidosis type VII (MPS VII) locus. SAMPLE POPULATION: Blood samples from 45 mixed-breed (German Shepherd Dog X Beagle) dogs that were phenotypically normal or affected with MPSVII. PROCEDURE: The canine beta-glucuronidase gene (exon 3) was amplified by polymerase chain reaction (PCR), using 2 pairs of primers to determine the genotype of the MPSVII locus by 2 independent methods. For the first method, PCR products were used for heteroduplex analysis, using conformation-sensitive gel electrophoresis. In the second method, an allele-specific restriction site was created by use of a mismatch primer in PCR. The amplified DNA fragment was digested with a restriction enzyme (Eag I) to enable identification of the wild-type and mutant alleles. RESULTS: Conformation-sensitive gel electrophoresis resulted in a single DNA band representing homoduplex when the sample contained a wild-type or MPS VII allele, but 2 bands representing hetero- and homoduplexes when both alleles were in the sample. Restriction digestion of the DNA fragment obtained by use of a mismatch primer was cleaved only when the template was a wild-type allele. Thus, samples from phenotypically normal carrier dogs that contained wild-type and MPS VII alleles were partially digested by the enzyme. CONCLUSIONS: The diagnostic test used 2 strategies for independently ascertaining the wild-type or mutant MPS VII alleles in dogs. Thus, test results can distinguish phenotypically normal MPS VII-carrier dogs from homozygous normal dogs.

Alleles↗
PubMed1997

Construction of a panel of canine-rodent hybrid cell lines for use in partitioning of the canine genome.

We have constructed a collection of canine-rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescence in situ hybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.

Animals↗
PubMed1997

A linkage map of the canine genome.

A genetic linkage map of the canine genome has been developed by typing 150 microsatellite markers using 17 three-generation pedigrees, composed of 163 F2 individuals. One hundred and thirty-nine markers were linked to at least one other marker with a lod score > or = 3.0, identifying 30 linkage groups. The largest chromosome had 9 markers spanning 106.1 cM. The average distance between markers was 14.03 cM, and the map covers an estimated 2073 cM. Eleven markers were informative on the mapping panel, but were unlinked to any other marker. These likely represent single markers located on small, distinct canine chromosomes. This map will be the initial resource for mapping canine traits of interest and serve as a foundation for development of a comprehensive canine genetic map.

Animals↗
PubMed1997

The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction.

The G-protein-coupled calcium receptor plays a key role in extracellular calcium homeostasis. To examine the role of the membrane-spanning domains and the approximately 200-residue cytoplasmic carboxyl terminus of the calcium receptor in cell-surface expression and signal transduction, we transfected HEK-293 cells with a series of truncation and carboxyl-terminal missense mutants and analyzed expression by immunoblotting, glycosidase digestion, intact cell immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Two truncation mutants terminating at residues 706 and 802 within the second and third intracellular loops, respectively, were not properly glycosylated, failed to reach the cell-surface, and showed no calcium response, indicating that mutant receptors with the full extracellular domain but only three or five transmembrane domains are improperly folded and/or processed. Truncation mutants terminating at residues 888 and 903 within the carboxyl terminus were equivalent to the wild type in all assays, whereas mutants truncated at residues 865 and 874 showed no response to calcium, despite only approximately 25% reduction in cell-surface expression. Mutants with a full-length carboxyl terminus but with residues between positions 874 and 888 replaced with alanines showed either no (Ala875, Ala876, and Ala879) or significantly reduced (Ala881-Ala883) calcium response at levels of cell-surface expression equivalent to those of the wild-type receptor. These results indicate that deletion of the majority of the carboxyl terminus is compatible with normal processing, cell-surface expression, and signal transduction of the receptor. The truncation and alanine substitution mutants identify a small region between residues 874 and 888 critical for normal signal transduction by the receptor.

Amidohydrolases↗
PubMed1997

The IL1 receptor accessory protein is responsible for the recruitment of the interleukin-1 receptor associated kinase to the IL1/IL1 receptor I complex.

Following interleukin-1 (IL1) stimulation, an IL1 receptor associated kinase (IRAK) is rapidly recruited to the receptor complex. However, it is not understood if IRAK is able to interact directly with the intracellular portion of the IL1-RI or if its recruitment is mediated by a different molecule. Using the yeast two-hybrid system, we have analysed possible protein-protein interactions between IRAK, IL1-RI and IL1-RAcP. We found that IRAK is able to interact with the equivalent cytoplasmic region of the IL1-RAcP but is unable to interact with the cytoplasmic region of the IL1-RI. Immunoprecipitation of the IL1-RAcP followed by Western blot analysis using anti-IRAK antibodies revealed that IRAK co-precipitated with the IL1-RAcP. We propose that, in non-stimulated cells, IRAK is bound to the IL1-RAcP and therefore, following IL1 stimulation, both molecules are recruited simultaneously to the ILI-RI complex.

3T3 Cells↗
PubMed1997

Complete deletion of factor IX gene and inhibition of factor IX activity in a labrador retriever with hemophilia B.

Hemophilia B is a heritable bleeding disorder caused by mutations in the gene coding for coagulation factor IX. The defect has been identified in purebred and mixed-breed dogs. Management of affected dogs requires transfusion of canine blood products supplying active factor IX. Production of inhibitors to factor IX is a complication of transfusion therapy that has been documented as affecting human patients. Risk for producing coagulation inhibitors is greatest for patients having large factor IX gene deletions. To our knowledge, this is the first report of canine factor IX inhibitor production in dogs. The affected dog had clinically severe hemophilia B caused by complete deletion of the factor IX gene and developed resistance to transfusion. Comprehensive evaluation of hemophilic dogs, including assays of specific factor activity, concentration, and factor inhibition, enhances diagnosis and management of this bleeding disorder. Characterization of the molecular defect causing hemophilia is useful for genetic counseling and identifying individuals at highest risk for producing coagulation inhibitors.

Animals↗
PubMed1997

Structure and analysis of the transducin beta3-subunit gene, a candidate for inherited cone degeneration (cd) in the dog.

The cDNA for the beta3-subunit of cone-specific transducin (Tbeta3) was cloned and characterized from wild type dogs, and used in linkage studies as a candidate gene for cone degeneration. Sequence analysis of the Tbeta3 cDNA revealed an open reading frame of 1020 bp, potentially coding for a protein of 340 amino acids (aa). The deduced aa sequence of canine Tbeta3 shares 97% identity with the previously identified human Tbeta3, and 82% identity with bovine rod-specific transducin (Tbeta1). RT-PCR and sequencing of the amplified products demonstrated that the retinal canine Tbeta3 gene is expressed in two different transcripts which can be generated by alternative splicing of the intron in the 3'-untranslated region (UTR). The short and the long mRNAs differ in the length of their 3'-UTR by 456 nt. We have also determined the genomic organization of the canine Tbeta3 gene; it consists of ten exons and the first exon is in the 5'-UTR. The cDNA encoding Tbeta3 from cd-affected dogs was also cloned and sequenced. We found no differences at the nucleotide level between the cDNAs isolated from normal and diseased retinas. The level of transcription of Tbeta3 mRNA in the cd dog retina appeared to be normal. Linkage analysis of a crossbred informative pedigree showed five obligate recombinants out of nine informative offspring. These results suggest that Tbeta3 is not a candidate gene for the cone degeneration of the cd mutant.

Amino Acid Sequence↗
PubMed1997

Cytosolic arylamine N-acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes.

The purpose of this study was to determine the molecular basis in the dog for an unusual and absolute deficiency in the activity of cytosolic N-acetyltransferase (NAT), an enzyme important for the metabolism of arylamine and hydrazine compounds. NAT activity towards two NAT substrates, p-aminobenzoic acid and sulfamethazine, was undetectable in dog liver cytosol, despite substrate concentrations ranging from 10 microM to 4 mM and a wide range of incubation times. Similarly, no protein immunoreactive to NAT antibody was evident on western blot analysis of canine liver cytosol. Southern blot analysis of genomic DNA from a total of twenty-five purebred and mixed bred dogs, and eight wild canids, probed with a full-length human NAT2 cDNA, suggested an absence of NAT sequences in all canids. Polymerase chain reaction amplification of genomic DNA using degenerate primers designed to mammalian NAT1 and NAT2 consensus sequences generated products of the expected size in human, mouse, rabbit, and cat DNA, but no NAT products in any dog or wild canids. These results support the conclusion that cytosolic NAT deficiency in the domestic dog is due to a complete absence of NAT genes, and that this defect is shared by other canids.

4-Aminobenzoic Acid↗
PubMed1997

Canine rod photoreceptor cGMP-gated channel protein alpha-subunit: studies on the expression of the gene and characterization of the cDNA.

Rod photoreceptor cyclic GMP gated-channel protein is a key component of the visual transduction cascade in the vertebrate retina. The protein is composed of at least two subunits (alpha and beta). Mutations in the alpha-subunit (CNGC1) have been shown to cause retinitis pigmentosa (RP) in humans. Several heterogeneous canine retinal diseases, which are clinically similar to RP, are known collectively as progressive retinal atrophy (PRA) and occur in dogs in a breed-specific manner. For the purpose of examining CNGC1 gene as a candidate for PRA, we report here the characterization of canine CNGC1 cDNA, and examine the expression of the gene in different tissues by northern analysis, reverse transcription and polymerase chain reaction (RT-PCR), and retinal immunocytochemistry. The characterized canine CNGC1 cDNA sequence contains 2717 nucleotides which include 211 bp 5"-untranslated region and 430 bp 3"-untranslated region including the poly A tail. It is predicted to encode a protein containing 691 amino acids which include six putative transmembrane domains, a pore loop and a cGMP binding domain as well as one potential extracellular site for N-linked glycosylation. Over the coding region, the canine CNGC1 shares 85-90% identity in the nucleotide sequence and 91-94% identity in the deduced amino acid sequence with its homologues in other mammalian species. However, the homology drops to only 71% and 78% of shared nucleotide and predicted amino acid sequences, respectively, when compared to the chicken CNGC1. Among all the tissues examined the gene is expressed at a much higher level in retina as a major transcript of 3.5 kb length. In addition, another minor transcript (9.8 kb) is consistently observed in the canine retinal RNA which may represent the canine homologue of the rod specific beta-subunit of the cyclic nucleotide-gated channel protein. Transcripts were detected only in retina by northern analysis but low level of expression of CNGC1 was detected in liver, kidney, heart and brain by RT-PCR. The expression of the CNGC1 protein was found to be localized specifically to the photoreceptor outer segment by immunocytochemistry.

Amino Acid Sequence↗
PubMed1997

Differential alterations in metabolic pattern of the six major UsnRNAs during development.

The uridylic acid rich nuclear RNAs (U1-U6 snRNAs) are involved mainly in the processing of pre-mRNA and pre-rRNA. So, any control of cell growth through pre-mRNA/pre-rRNA processing may have some regulation through altered UsnRNAs metabolism. With this idea, attempts have been made to see how the metabolism of the six major UsnRNAs' changed during the normal process of cellular proliferation associated with differentiation from pluripotent/totipotent stem cells of early embryonic stage to much more differentiated state of different cell/tissue lineages in different tissues/organs during the fetal and neonatal stages of growth. It has been seen that the levels of the six major UsnRNAs were high in day 8 embryo when the cells were mainly pluripotent/totipotent in nature, and during the progression of embryonic development the levels of these UsnRNAs gradually decreased (approximately 35-65%) up to the midgestational period (day 13) with some exception, when the organogenesis has already been started. However in the fetal life, the levels of these UsnRNAs were maximum or comparable around 18 +/- 2 days of gestation in comparison to that in day 8 embryo when the kinetics of the maturational status of the different organs were quite high. But, the levels of these UsnRNAs' became low during day 21 of fetal life or in day 0 of birth (perturation period) in all the tissues/organs except high UsnRNAs' level in spleen. In the neonatal life, around 3 +/- 1 days of birth these UsnRNAs' levels again became maximum in all the tissues/organs (except in thymus) followed by decrease up to 5/6 days, and to become steady with slight increase within one to two weeks, when the kinetics of the organ maturation reached to a steady state. In case of thymus, the levels of the U3-U6 snRNAs were high on day 0 of birth followed by decrease in their level on day 1/2 and then increased to become steady within 2-4 weeks; whereas the U1 and U2 snRNAs' levels were high on day 3 of birth and the subsequent changes were similar to that in other tissues/organs. Thus the different UsnRNAs' metabolism in the perturation period and in the early stages of neonatal life has indicated the differential cellular functions in these two stages of development. These alterations in the metabolism of these UsnRNAs might be due to the differential changes in the rate of synthesis of these UsnRNAs and/or with their differential turnover rate in the different stages of development. Also, the differential variations of these UsnRNAs' levels have been observed among the different tissues/organs at the respective stages of development indicating the differences in the UsnRNAs' metabolism among the different cell/tissue lineages. Thus, it can be concluded that the metabolism of these UsnRNAs were developmentally regulated with some cell/tissue lineage variations, which might have some role in the developmentally regulated cellular process of proliferation and differentiation, through altered RNA splicing and processing.

Animals↗
PubMed1997

Canine rod transducin alpha-1: cloning of the cDNA and evaluation of the gene as a candidate for progressive retinal atrophy.

PURPOSE: Progressive retinal atrophy (PRA) represents a heterogeneous group of retinal dystrophies, distinct forms of which occur in different canine breeds. The present study was undertaken to evaluate the gene for the alpha-1 subunit of the rod specific G-protein transducin (GNAT1), a member of the phototransduction pathway, as a candidate for progressive rod cone degeneration (pred) in poodles, early retinal degeneration (erd) in elkhounds, and rod cone dysplasia 2 (rcd2) in collies. METHODS: Oligonucleotide primers were designed from the consensus region of known cDNA sequences for GNAT1 from other species. Canine GNAT1 cDNA was cloned and sequenced after reverse transcription (RT) and polymerase chain reaction (PCR) of total retinal RNA, and PCR amplification of specific sequences from a canine retinal cDNA library. Large, intron containing fragments of the canine transducin alpha-1 subunit gene were amplified from genomic DNA of individuals in PRA informative pedigrees, using canine-specific primers. PCR products were digested with Nci I, to enable typing of individuals in the PRA affected pedigrees for a previously identified GNAT1 restriction fragment length polymorphism (RFLP). RESULTS: The sequence of canine GNAT1 cDNA is reported (GenBank accession no. U65376). Over the coding region, the canine GNAT1 cDNA sequence presented here shares 92-95% identity with human, bovine and murine sequences. The canine cDNA encodes a polypeptide of 350 amino acids; its theoretical translation is 98-99% identical with the corresponding GNAT1 sequence from each of the other 3 species and it has no unique amino acids. In rcd2 and erd pedigrees informative for both the disease locus and the GNAT1 Nci I RFLP, a minimum of 3 and 2 recombinants were identified, respectively. Similarly, in a prcd pedigree, 3 of 7 progeny informative for both prcd and this RFLP were obligate recombinants. CONCLUSIONS: The canine GNAT1 gene has been excluded as a candidate for prcd, erd and rcd2. Sequence information of canine GNAT1 gene will enable testing this locus as a candidate in other canine hereditary retinal degenerations.

Amino Acid Sequence↗