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Biomedical subjects

R Krishnamoorthy

Publications and source records attributed to R Krishnamoorthy.

At least 109 records · Page 6Linked to original sources
PubMed1994

Reduced variability in Y-chromosome-specific haplotypes for some Central African populations.

Only 7 nonrare haplotypes concerning Y-chromosome-specific TaqI fragments recognized by DNA probes p49f and p49a were encountered in 433 individuals belonging to 7 populations living in central Africa. Most of the haplotypes (77.7%) are characterized by the allelic combination A1,C0,D0,F1,I1 (haplotype IV). The degree of variability displayed by these sequences is low in the African population studied, compared with Europeans, probably because of the recent common origin of the Bantu-speaking populations studied and the common practice of male polygamy in Africa.

Africa, Central↗
PubMed1994

Sequence correction and reassignment of the TaqI polymorphic site in the human inter-gamma-globin gene region, an African-specific marker.

We report here that the assignment of nt 37504 in the human inter-gamma-globin gene region of the HUMHBB locus sequence as a C is incorrect and should be replaced by a T. Accordingly, the polymorphic TaqI site, originally described at position 37503 as an African-specific marker, is actually located at position 37992. A PCR-based assay for this anthropologically important genetic marker is described.

Black People↗
PubMed1993

Inter-ethnic polymorphism of the beta-globin gene locus control region (LCR) in sickle-cell anemia patients.

Sequence polymorphisms within the 5'HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major beta s haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.

Algeria↗
PubMed1993

DNA haplotype dependency of debrisoquine 4-hydroxylase (CYP2D6) expression among extensive metabolisers.

Deficient debrisoquine/sparteine type oxidation is inherited as an autosomal recessive trait. Of all Caucasians, 5-10% are poor metabolisers, due to the absence of cytochrome P4502D6. Extensive metabolisers (EMs) exhibit highly variable metabolic activity. We investigated the relationship between CYP2D6 activity and genotypes of the CYP2D locus in a large set of French Caucasian families. Genotypes concern both common mutations affecting the enzyme activity and linked BamHI polymorphisms of the locus. We found, like other authors, that in EMs part of the heterogeneity is explained by a subgroup of individuals heterozygous for a mutant allele. However, a second level of heterogeneity was detected among individuals not carrying mutations, and this was related to a polymorphic BamHI-defined DNA haplotype. Different combinations of haplotypes are associated with differences in CYP2D6 metabolic activity. This finding might help to clarify the conflicting data on the relation between CYP2D6 activity and susceptibility to lung cancer.

Adolescent↗
PubMed1993

Differential effects of anionic, cationic, nonionic, and physiologic surfactants on the dissociation, alpha-chymotryptic degradation, and enteral absorption of insulin hexamers.

Various surfactants were investigated to compare their effects on insulin dissociation, alpha-chymotryptic degradation, and rat enteral absorption. With a circular dichroism technique, sodium dodecyl sulfate (SDS) at a 5 mM concentration was found to completely dissociate porcine-zinc insulin hexamers (0.5 mg/ml) into monomers. The catalytic activity of alpha-chymotrypsin (0.5 microM) was also abolished by 5 mM SDS. When insulin was injected into the distal jejunum/proximal ileum segment of the rat, 5 mM SDS greatly enhanced its pharmacological availability, from a negligible value to 2.8%. Being a cationic surfactant, hexadecyl trimethylammonium bromide (CTAB) also efficiently dissociated insulin hexamers at concentrations of 1-5 mM. However, extensive charge-charge interaction was observed below a CTAB concentration of 0.6 mM, leading to insulin precipitation at a molar CTAB:insulin ratio of 1:1 to 2:1. An alpha-chymotryptic degradation study also revealed near-complete dissociation of insulin hexamers at 1 mM CTAB. Above 1 mM, however, CTAB acted as an enzyme inhibitor, most likely by means of charge repulsion. Enteral absorption studies showed a much lower pharmacological availability, only 0.29%. Nonionic surfactants such as Tween 80 and polyoxyethylene 9 lauryl ether were ineffective in dissociating insulin hexamers. Tween 80, at 5 mM, neither significantly altered the alpha-chymotryptic degradation pattern nor enhanced the enteral absorption of insulin. The relative effectiveness of different species of bile salts on insulin hexamer dissociation appeared to be similar. Sodium glycocholate at a 30 mM concentration also significantly increased insulin pharmacological availability, to 2.3%. A morphological study did not reveal any significant alteration of the rat intestinal mucosal integrity after exposure to 5 mM SDS for 30 min.2+ transport.

Administration, Oral↗
PubMed1993

Effect of acylation on the ocular disposition of acyclovir. I: Synthesis, physicochemical properties, and antiviral activity of 2'-esters.

A series of aliphatic 2'-esters of acyclovir ([9-(2-hydroxyethoxymethyl)guanine]) were synthesized by direct acylation in a pyridine-N,N-dimethylformamide solution. The prodrugs were characterized as to their aqueous solubility in phosphate buffer (pH 7.4), partition coefficients in 1-octanol/phosphate buffer (pH 7.4), bioreversion kinetics by the soluble ocular esterases, and in vitro effectiveness against Herpes group viruses. The compounds exhibit an expected decrease in aqueous solubility upon esterification with a corresponding increase in the 1-octanol/water partition coefficient. The butyrate ester shows good aqueous stability in the neutral pH ranges. The apparent first order rate constants of bioreversion varied with the steric nature and polarity of the acyl substituent. The butyrate and pivalate esters were evaluated for their anti-herpesvirus activity and cellular toxicity. The butyrate ester possesses similar anti-herpesvirus activity to acyclovir and has a very high selectivity index. The pivalate ester shows poor anti-herpes simplex virus activity; however, it is unique in its effectiveness against the Epstein-Barr virus.

Acyclovir↗
PubMed1993

Molecular heterogeneity of the XbaI defined 44kb allele of the CYP2D locus within the Caucasian population.

1. Cytochrome P450 debrisoquine (CYP2D6) activity is polymorphic and under genetic control. Most Caucasians are extensive metabolizers, but 5%-10% are poor metabolizers. 2. Restriction fragment length polymorphism analysis of the CYP2D6 locus identifies a 29kb XbaI fragment, either normal (D6-wt) or mutated, and three mutated XbaI alleles (44kb, 11.5kb and 16 + 9kb). The 44kb allele was initially considered as a poor metabolizer allele owing to a D6-B mutation, but cases of 44kb allele not carrying the D6-B, and therefore potentially functional, have been found. The degree of molecular heterogeneity of this allele was investigated by phenotype and genotype analysis of families. 3. Thirty-one French Caucasian families, representing 117 individuals, possessing at least one 44kb allele in each family were selected. Phenotypes were determined using dextromethorphan, and the XbaI, NcoI and BamH1 RFLPs of 42 independent chromosomes were analyzed. 4. 80% of the XbaI 44kb alleles carried the CYP2D6-B mutation and had an additional NcoI fragment (12.5kb or 4.8kb). The remaining 20% did not carry the CYP2D6-B or A mutations and had no extra NcoI fragment. 5. Information on three families demonstrated that 44kb alleles not carrying the CYP2D6-B mutation were associated with the extensive metabolizer phenotype. 6. We conclude that a substantial percentage of XbaI 44kb alleles is associated with a functional CYP2D gene, and therefore, that the XbaI 44kb allele is not consistently a poor metaboliser allele.

Alleles↗
PubMed1993

Rapid analysis of -alpha 3.7 thalassaemia and alpha alpha alpha anti 3.7 triplication by enzymatic amplification analysis.

In this report we describe a PCR-based method for the diagnosis of the most common form of alpha thalassaemia, the -alpha 3.7 deletion which occurs throughout all tropical and subtropical regions of the world. The same procedure also identifies the reciprocal recombinant chromosome (alpha alpha alpha anti 3.7). Restriction mapping of the PCR products has enabled us to distinguish between the type I (-alpha 3.7 I), type II (-alpha 3.7 II) and type III (-alpha 3.7 III) deletions. This strategy will be very useful in screening programmes of alpha thalassaemia occurring on its own or in association with beta thalassaemia and sickle cell disease.

Globins↗
PubMed1993

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

Amino Acid Sequence↗
PubMed1993

DNA typing with digoxigenin-11-dideoxyuridinetriphosphate-labelled oligonucleotide probes enables non-radioactive analysis using a dual detection system: application for screening HLA-DQA1 polymorphisms.

We describe a standardized, highly sensitive, nonradioactive detection procedure for HLA class-II typing of DQA1 alleles and suballeles which has important and cost-effective application in studying HLA disease associations. The procedure involves polymerase chain reaction-based target DNA sequence amplification and dot blotting followed by stringent hybridization with digoxigenin-11-dideoxyuridine triphosphate 3'-end-labelled allele-specific oligonucleotide probes. The dual detection system described here makes this procedure very versatile.

Alleles↗
PubMed1993

[Dysregulation of in vitro TNF-beta production in insulin-dependent diabetes mellitus].

The possible role of tumor necrosis factors (TNF) in autoimmunity led us to explore the relationship between TNF production, polymorphism of the TNF-beta gene and type one diabetes. In the diabetic group we found a low production of TNF-beta. This abnormality appeared not to be exclusively related to the TNF-beta gene and could be also due to polymorphic regulatory sequences in the major histocompatibility complex region.

Adolescent↗
PubMed1992

DNA sequence variation in a negative control region 5' to the beta-globin gene correlates with the phenotypic expression of the beta s mutation.

The clinical diversity of sickle cell anemia is strongly related to the degree of intracellular hemoglobin S (Hb S) polymerization, which in turn is dependent on the intracellular concentration of Hb S. We have recently defined a region of DNA approximately 500 bp 5' to the human beta-globin gene that acts as a silencer for the transcription of this gene and have shown that a polymorphism in this sequence is associated with a thalassemic phenotype of the beta-globin gene. In this work we have examined the correlation of DNA sequence polymorphisms in this silencer with binding of a previously identified putative repressor protein, BP1, and with the expression of Hb S in individuals heterozygous for the beta s allele. It was found that specific configurations of the motif, (AT)x(T)y, are homogeneous for the major haplotypes of the beta-globin gene cluster described on beta s chromosomes. Binding of BP1 was measured to DNA of three haplotypes: Indian, Benin, and Bantu. BP1 binds most tightly to DNA of the Indian haplotype, and these patients produce less beta s protein than Benin patients, whose DNA exhibits weaker affinity for BP1. Binding of BP1 is the weakest to DNA of the Bantu haplotype, which is associated with clinically more severe sickle cell symptoms. These data are consistent with the hypothesis that these polymorphisms may not be neutral and that the DNA sequence at this site may affect the expression of the beta s gene. Such an effect may be synergistic with other genetic variables, such as fetal hemoglobin levels, F-cell numbers, and the number of alpha-globin genes, in determining intracellular polymerization and, thus, the severity of the sickle cell syndromes.

Africa↗
PubMed1992

Genetic epidemiology of beta-thalassemia in Sicily: do sequences 5' to the G gamma gene and 5' to the beta gene interact to enhance HbF expression in beta-thalassemia?

The present epidemiological study of the molecular characteristics of beta-thalassemia in Sicily was prompted by the disparate phenotypic expression (in clinical status and absolute HbF level) observed in two beta-thalassemic homozygotes who were also homozygous for the beta-like globin gene cluster haplotype III. We suspected that polymorphisms within haplotype III could be the cause for the discrepancy. Based on the association of particular conformations of the (AT)xT(y) motif (-540 5' to the beta gene) with milder forms of thalassemia and sickle cell anemia, 38 homozygous beta-thalassemia patients were studied to define their haplotypes, the -158 site 5' to the G gamma gene (linked to haplotype III) and the structure of the (AT)xT(y) motif. We found that the patient who was phenotypically mild and homozygous for beta-thalassemia, haplotype III, and the -158 C----T mutation was homozygous for the rare (AT)9T5 motif. In contrast, the patient homozygous for beta-thalassemia, haplotype III, and the -158 mutation, but exhibiting a severe clinical course, was homozygous for the (AT)7T7 configuration. Others have suggested that (AT)9T5 is a negative regulatory protein binding sequence, and it is a silent carrier state for beta-thalassemia. The usual configuration (AT)7T7, has considerably less affinity for regulatory protein binding, and it is the most common configuration in Sicilian beta-thalassemics (67 of the 78 chromosomes studied). Within the 38 patients studied, seven were informative because they had various combinations of the (AT)9T5 and (AT)7T7 motif, and the -158 C----T mutation. The results in these patients suggest that only the co-presence of the (AT)9T5 configuration and a C----T change at -158 5' to the G gamma gene is associated with high HbF expression and a mild clinical phenotype. We postulate that these two regions of the beta-like globin gene cluster interact, when endowed with the proper sequences, to enhance the expression of HbF secondary to anemia.

Adolescent↗
PubMed1992

Presence of an African beta-globin gene cluster haplotype in normal chromosomes in Sicily.

African admixture in Sicily has been long suspected because of the presence of the sickle gene. Nevertheless, the degree of African admixture cannot be derived from the study of HbS frequency, since this gene was most likely expanded by the selective pressure of malaria, for a long time endemic to the region. We have examined 142 individuals from the Sicilian town of Butera (12% sickle trait) to search for other markers of the globin gene cluster less likely to be selected for by malaria. The TaqI polymorphism in the intervening sequences between the two gamma genes is informative. We have found only two instances of this African marker (TaqI(-)) among 267 normal chromosomes, demonstrating that the admixture occurred at a much lower level than previously thought.

Adolescent↗
PubMed1992

A novel sickle cell mutation of yet another origin in Africa: the Cameroon type.

The sickle cell mutation (beta s) arose as at least three independent events in Africa and once in Asia, being termed the Senegal, Benin, Bantu and Indian types respectively. An investigation in Cameroon was carried out to determine whether the atypical sickle genes observed in the neighboring countries are the result of recombination or the presence of a sickle cell mutation of a different genetic origin. It was conducted on 40 homozygous SS patients followed at the Blood Transfusion Center in the capital city of Yaoundé. On 80 beta s chromosomes, 13 exhibited a novel polymorphic pattern that was observed three times in the homozygous state. This chromosome contains an A gamma T gene. The restriction fragment length polymorphism haplotype is different from all the other beta s chromosomes in both the 5' and 3' regions, but has previously been reported in sporadic cases. The (AT)8(T)5 sequence in the -500 region of the beta gene is specific and different from that of the Senegal, Benin, Bantu or Indian beta s genes. All the carriers of this specific chromosome belong to the Eton ethnic group and originate from the Sanaga river valley. This observation strongly argues for yet another independent origin of the sickle cell mutation in Africa, here referred to as the "Cameroon type". The Benin haplotype and a Benin/Bantu recombinant haplotype have been observed in the other studied populations: Ewondo, Bamiléké, Bassa, Yambassa and Boulou.

Anemia, Sickle Cell↗