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Biomedical subjects

A Mukhopadhyay

Publications and source records attributed to A Mukhopadhyay.

At least 19 recordsLinked to original sources
PubMed2007

Epithelial-mesenchymal interactions in keloid pathogenesis modulate vascular endothelial growth factor expression and secretion.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis during the wound healing process. As epithelial-mesenchymal interactions have been shown to regulate a plethora of genes in wound healing, we hypothesized that these interactions might have a role in modulating VEGF expression and angiogenesis. A two chamber co-culture model was used, wherein normal and keloid keratinocytes and fibroblasts were physically separated by membrane inserts while allowing cytokine diffusion. Cell lysates obtained from keratinocytes co-cultured with fibroblasts demonstrated increased expression of VEGF. An enzyme-linked immunosorbent assay (ELISA) showed significant increase in VEGF expression in co-culture conditioned media compared with controls. Additionally, the conditioned medium from keloid keratinocyte and fibroblast co-cultures increased proliferation and formation of complex three-dimensional capillary-like structures in human umbilical vein endothelial cells, emphasising the importance of epithelial-mesenchymal interactions in the angiogenic process. Immunostaining of keloid tissue localized VEGF in the basal layer of the epidermis and also demonstrated higher blood vessel density than normal skin. Keloid tissue extract also demonstrated increased expression of VEGF compared with normal skin. It is likely that epidermal VEGF exerts significant paracrine control over the dynamics and expression profile of underlying dermal fibroblasts. Addition of the inhibitors WP631, mitoxantrone, and Rapamycin to keloid keratinocyte and fibroblast co-cultures, downregulated secreted VEGF expression in a dose-dependent manner, suggesting therapeutic potential for these compounds in the treatment of keloid scars.

Antibiotics, Antineoplastic↗
PubMed2006

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea.

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated '126-1', was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F(1)s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for '126-1' cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of '126-1' cytoplasm for hybrid seed production. The uniqueness of '126-1' has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.

Brassica napus↗
PubMed2006

The use of the Rusch balloon for management of severe postpartum haemorrhage.

Tamponade techniques using a uterine balloon in management of postpartum haemorrhage has been reported increasingly in recent years. The aim of this retrospective study is to evaluate the use of the Rusch hydrostatic balloon in the management of severe postpartum haemorrhage not controlled by medical measures. All women had risk factors for PPH. The Rusch balloon was used in all cases of PPH apart from traumatic PPH, which is considered as a contraindication for its use. The Rusch balloon was successful in seven out of the eight cases treated. We have introduced guidelines for using the Rusch balloon and they are provided in this paper.

Adult↗
PubMed2006

OCA1 in different ethnic groups of india is primarily due to founder mutations in the tyrosinase gene.

Oculocutaneous albinism (OCA) is a heterogeneous group of autosomal recessive disorders characterized by an abnormally low amount of melanin in the eyes, skin and hair, and associated with common developmental abnormalities of the eye. Defects in the tyrosinase gene (TYR) cause a common type of OCA, known as oculocutaneous albinism type 1 (OCA1). The molecular basis of OCA has been studied extensively in different population groups, but very little information is available on Indian patients. Our investigation covering thirteen ethnic groups of India, some representing >20 million people, revealed that among 25 OCA families 12 were affected with OCA1, and that these cases were primarily due to founder mutations in TYR. We detected nine mutations and eight SNPs in TYR, of which six mutations (five point mutations & one gross deletion) were novel. In contrast to most reports describing compound heterozygotes, the presence of homozygotes in 10 out of the 12 pedigrees underscores the lack of intermixing between these ethnic groups in India. Haplotype analysis suggested a few founder chromosomes causing the disease in the majority of the patients. Direct detection of the mutations prevalent in specific ethnic groups could be used for carrier detection and genetic counselling.

Albinism, Oculocutaneous↗
PubMed2006

Prospective study of a new serological test (ASSURE TB Rapid Test) for the diagnosis of pulmonary tuberculosis.

OBJECTIVE: To prospectively compare a rapid tuberculosis serological test, ASSURE TB Rapid Test, with traditional smear and culture methods for the diagnosis of pulmonary tuberculosis (PTB). DESIGN: All consecutive in-patients aged > or = 18 years suspected of having active PTB and admitted between June 2001 and March 2003 were tested with three sputum samples for smear and culture of Mycobacterium tuberculosis and serology (done within 3 days). RESULTS: Of 238 patients initially enrolled (male: female 2.5:1, mean age 56.6 years), the final analysis included 216 patients. For the final diagnosis of PTB, the sensitivity and specificity of the serological test were respectively 60.2% (95%CI 50.5-69.1) and 82.3% (95%CI 74.2-88.2) compared to 53.4% (95%CI 43.8-62.7) and 98.2% (95%CI 93.8-99.5) for the smear test. A combination of smear and serology provided an increased sensitivity of 74.8% (95%CI 65.6-82.2), but a lower specificity of 80.5% (95%CI 72.3-86.8). CONCLUSION: The new serological test showed a moderate increase in sensitivity but a decrease in specificity compared to smear examination. The combination (smear + serology) test further increased the sensitivity while maintaining a moderate specificity.

Adolescent↗
PubMed2005

Mapping and tagging of seed coat colour and the identification of microsatellite markers for marker-assisted manipulation of the trait in Brassica juncea.

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607-614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes (BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene (BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm.

Chromosome Mapping↗
PubMed2005

Smad3 signalling plays an important role in keloid pathogenesis via epithelial-mesenchymal interactions.

Smad signalling plays important roles in developmental and cancer biology as well as in fibropathogenesis. Its role in keloid biology is not known. Epithelial-mesenchymal interactions, originally described in normal skin, have recently been established to play a significant role in keloid pathogenesis, and demonstrate the important influence of keratinocyte paracrine factor signalling on fibroblast behaviour. The present study investigated the role of downstream Smad cascade induction in this interaction. Normal fibroblasts (NF) and keloid fibroblasts (KF) were co-cultured in serum-free medium with normal keratinocytes (NK) or keloid keratinocytes (KK) for 5 days, after which fibroblast cell lysates were subjected to western blot and immunoprecipitation analysis to quantify the levels of Smad and Smad2/3/4 binding complex. In another set of experiments, wild-type (wt), Smad2-null (Smad2-/-) and Smad3-null (Smad3-/-) mouse embryonic fibroblasts (MEF) were assayed for cell proliferation and collagen production after serum-free co-culture with KK or exposure to conditioned media collected from serum-free KK/KF co-culture. Compared to normal skin, keloids expressed high basal levels of TGFbetaR1 and TGFbetaR2, Smad2, 3 and 4 and phospho-Smad2. Upregulation of TGFbetaR1 and TGFbetaR2, Smad3 and p-Smad2 was observed in KF co-cultured with KK, together with enhanced Smad3 phosphorylation and Smad2/3/4 binding complex production. When MEF-wt, MEF-Smad2-/- or MEF-Smad3-/- were co-cultured with KK or exposed to KK/KF co-culture conditioned media, enhanced proliferation and collagen production were seen in MEF-wt and MEF-Smad2-/- but not in MEF-Smad3-/- cells. The activation of Smad signalling, importantly that of Smad3, appears to be one facet of the complex epithelial-mesenchymal interactions in keloid pathogenesis, resulting in active KF proliferation and collagen-ECM production in co-culture with KK. This finding suggests the suppression of Smad signalling as a novel approach in keloid therapy.

Adolescent↗
PubMed2005

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence.

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli beta-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.

Amino Acid Sequence↗
PubMed2005

Glutathione S-transferase M1 and T1 null genotype frequency in chronic myeloid leukaemia.

Polymorphisms associated with genes coding for glutathione S-transferase enzymes are known to influence metabolism of different carcinogens and have been associated with incidence of various types of cancer. We have determined the GST M1 and GST T1 'null' genotype frequency in 81 patients with chronic myeloid leukaemia (CML) and 123 racially and geographically matched control individuals by multiplex polymerase chain reaction (PCR). GST M1 null genotype frequencies in CML and controls were 28.4% and 27.7%, respectively. GST T1 null genotype frequencies in CML and controls were 19.8% and 7.3%, respectively. The GST T1 null genotype frequency in CML patients is significantly different from that in controls (odds ratio (OR) 3.12, 95% confidence interval (CI) 1.3-7.45, P=0.008).

Adolescent↗
PubMed2005

Conditioned medium from keloid keratinocyte/keloid fibroblast coculture induces contraction of fibroblast-populated collagen lattices.

BACKGROUND: Keloid scars represent a pathological response to cutaneous injury. Overproliferation of fibroblasts and overproduction of collagen characterize these abnormal scars. The pathology of these scars remains poorly understood. The role of epithelial-mesenchymal interactions in keloid pathogenesis and scar contracture has recently been explored. OBJECTIVES: To test our hypothesis that epithelial-mesenchymal interactions play a major role in modulating keloid scar contracture. METHODS: A coculture model was employed wherein keloid and normal keratinocytes were cocultured with keloid or normal fibroblasts, and the conditioned media from day 5 cocultures were collected to study the effect of the paracrine secretions on contraction of an in vitro fibroblast-populated collagen lattice (FPCL) model. RESULTS: Keloid keratinocyte/keloid fibroblast coculture conditioned media brought about increased contraction of the collagen lattice compared with non-cocultured conditioned media. When keloid fibroblasts populated the collagen lattice, significantly increased lattice contraction was induced compared with lattices populated by normal fibroblasts. The addition of antitransforming growth factor (TGF)-beta neutralizing antibody to the conditioned media produced an attenuation of the contraction of the FPCLs. When keloid and normal fibroblasts were cultured on chamber slides and treated with conditioned media from coculture and non-coculture series, immunohistochemical analysis demonstrated an increased expression of alpha-smooth muscle actin (a marker for fibroblast differentiation into myofibroblasts) in fibroblasts exposed to conditioned media from coculture. CONCLUSIONS: These data indicate that epithelial-mesenchymal interactions are likely to play a major role in scar contracture and scar pathogenesis, and underscore the role of TGF-beta1 as a key player in keloid pathogenesis.

Actins↗
PubMed2005

Myocilin gene implicated in primary congenital glaucoma.

Primary congenital glaucoma (PCG) has been associated with CYP1B1 gene (2p21), with a predominantly autosomal recessive mode of inheritance. Our earlier studies attributed CYP1B1 mutations to only 40% of Indian PCG cases. In this study, we included 72 such PCG cases where CYP1B1 mutations were detected in only 12 patients in heterozygous condition, implying involvement of other gene(s). On screening these patients for mutations in myocilin (MYOC), another glaucoma-associated gene, using denaturing high-performance liquid chromatography followed by sequencing, we identified a patient who was double heterozygous at CYP1B1 (c.1103G>A; Arg368His) and MYOC (c.144G>T; Gln48His) loci, suggesting a digenic mode of inheritance of PCG. In addition, we identified the same MYOC mutation, implicated for primary open angle glaucoma, in three additional PCG patients who did not harbor any mutation in CYP1B1. These observations suggest a possible role of MYOC in PCG, which might be mediated via digenic interaction with CYP1B1 and/or an yet unidentified locus associated with the disease.

Aryl Hydrocarbon Hydroxylases↗
PubMed2005

A prospective audit of referrals for breathlessness in patients hospitalised for other reasons.

INTRODUCTION: This prospective audit examines the diagnostic and clinical outcomes of consecutive adult inpatients who were admitted to a university hospital for other reasons and referred for breathlessness to respiratory physicians. METHODS: We enrolled all adult inpatients referred for breathlessness from May 2000 to October 2001. We evaluated the clinical features and utility of routine investigations, such as blood tests and radiology. Subsequent investigations were undertaken at the discretion of the physician. RESULTS: Of the 105 patients, 49 were men and 56 were women. Their mean age was 66 plus or minus 18 years. Surgical departments and cardiology were the main referring departments. Respiratory infection (31 percent) was the most common diagnosis. Acute pulmonary embolism (PE) was diagnosed in four patients postoperatively. Chest radiographs were helpful in making a diagnosis in 66 percent of patients. Computed tomography pulmonary angiogram of the thorax was performed in 31 of the 34 patients who were investigated for acute PE. More postoperative than non-postoperative patients were tested for PE (p-value less than 0.0001). CONCLUSION: In hospitalised patients referred for breathlessness, respiratory infections were the most common diagnosis and the chest radiograph was the most useful initial investigation. Computed tomography pulmonary angiogram was the preferred investigation for acute PE and clinicians were more inclined to investigate for PE in postoperative patients.

Aged↗
PubMed2004

Molecular pathology of haemophilia B: identification of five novel mutations including a LINE 1 insertion in Indian patients.

Heterogeneous mutations in factor IX (FIX) gene cause haemophilia B and a large number of mutations have been characterized. However, reports on gene defects among Indian haemophilia B patients are rare despite a high estimate of such patients in the country. We report identification of 22 independent mutations including five novel mutations in 24 unrelated patients. The novel gene defects include two point mutations, two deletions and one insertion of a LINE 1 element. Majority of the mutations (14 of 24) occurred on the same haplotype background, but do not suggest any founder effect. Direct identification of mutations can be utilized to perform the carrier detection and prenatal diagnosis, especially in families with isolated patients.

Base Sequence↗
PubMed2003

Molecular tagging of erucic acid trait in oilseed mustard (Brassica juncea) by QTL mapping and single nucleotide polymorphisms in FAE1 gene.

Molecular mapping and tagging of the erucic acid trait (C22:1) in Brassica juncea was done by a candidate gene approach. Two QTLs underlying the variation of seed erucic acid content were assigned to two linkage groups of a B. juncea map using a doubled haploid (DH) mapping population derived from high x low erucic acid F(1) hybrid. Two consensus primers corresponding to the full-length Fatty Acid Elongase 1 ( FAE1) gene, reported to be involved in the elongation of C18:1 to C22:1, were designed. PCR amplification and subsequent cloning and sequencing identified two FAE1 genes ( FAE1.1 and FAE1.2) in both high and low erucic acid mustard lines. Sequence alignment of corresponding FAE1 genes between high and low erucic acid mustard lines identified four substitution type single nucleotide polymorphisms (SNPs) in FAE1.1 and three in FAE1.2. Using the SNuPE method of SNP genotyping, these two genes were mapped to two independent loci that co-segregated with the two QTLs governing the erucic acid trait. Association of wild ( E1E2) and mutant ( e1e2) haplotypes of two FAE1 genes with erucic acid variation in two segregating populations revealed that the e1e1e2e2 genotype identified low erucic acid individuals (<2%) and E1E1E2E2 identified individuals with highest erucic acid content (>40%). The E1e1E2e2 heterozygote was found to be intermediate in phenotype. The applicability of these SNPs in marker-assisted manipulation of the erucic acid trait was verified by genotyping a set of contrasting germplasm of B. juncea belonging to two distinct gene pools (Indian and east European) and other oil-yielding Brassica species.

Acetyltransferases↗
PubMed2003

Analysis of haemophilia B database and strategies for identification of common point mutations in the factor IX gene.

Haemophilia B is an X-linked recessively inherited bleeding disorder caused by heterogeneous mutations spanning the entire factor IX gene. As spontaneous germ-line mutations are known to occur mostly at CpG dinucleotides in the FIX gene, control of the disease would require continuous carrier detection and mutation screening. Identification of point mutations, the most common type of mutation in FIX gene, is more challenging compared with deletion and insertion mutations. We examined the haemophilia B database to identify specific nucleotides in the FIX gene that are mutated in relatively large number of patients and the variability (if any) in the mutational hotspots at CpG dinucleotides. It was found that while mutations responsible to account for all 2348 haemophilia B patients covered 20% of the FIX cDNA, only 1% of the cDNA involving mostly CpG dinucleotides accounted for mutation in 42.41% of the patient pool. Thus, only 27 nucleotides need to be investigated to identify the common point mutations, among which 15 are predicted to undergo change in restriction sites on mutation. It is interesting to note that seven nucleotides occurring in CpG dinucleotides do not have any reported mutation despite each of those being predicted to harbour mutation as a result of transition and having mutations recorded in the database for the neighbouring nucleotides. Strikingly large number of mutation in codon 296 causing T to M change in catalytic domain originally proposed to be the result of the founder effect also contains largest number of haplotype suggesting recurrence of de novo mutation.

DNA, Complementary↗
PubMed2003

Colony-stimulating activity of fetal liver cells: synergistic role of stem cell factor in bone marrow recovery from aplastic anemia.

Previously, we and others have shown that fetal liver infusion (FLI) leads to autologous hematopoietic improvement in 40-54% of patients with aplastic anemia. However, whether this recovery was spontaneous or the effect of the infused liver cells was not clear. To dissect the role of FLI in autologous hematopoietic recovery, the colony-supporting potential of fetal liver-conditioned medium (FLCM) was evaluated in bone marrow (BM) cells of normal adult and aplastic anemia patients. In both cases, each sample of FLCM supported the growth of colony-forming cells in semi solid culture medium. The FLCM was assayed for the presence of four principal colony-stimulating cytokines, namely stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). While GM-CSF, IL-3, and Epo were present in insignificant amounts or were altogether absent, 50-635 pg/ml of SCF was found in 8 of the 13 FLCM samples tested. Preliminary results of bioneutralization assay indicated the possible role of SCF, secreted by the FL cells, in colony-supporting activity of aplastic anemia and normal BM cells. Overall, our in vitro study implicates the paracrine role of infused FL cells in regenerating autologous hematopoiesis in aplastic anemia patients.

Anemia, Aplastic↗
PubMed2003

DNA linkage based diagnosis of Wilson disease in asymptomatic siblings.

BACKGROUND & OBJECTIVES: Wilson disease (WD) is an autosomal recessive disorder caused by defects in ATP7B gene located in chromosome 13q14, and manifested as hepatolenticular degeneration as a result of accumulation of copper. No information on the mutation in the ATP7B gene and haplotypes using linked markers is available for WD patients in India. Hence, the present study was undertaken to identify, by a PCR-based molecular diagnostic test, presymptomatic siblings of WD affected individuals in families with multiple offspring. METHODS: Genomic DNA was prepared from the peripheral blood of the patients, siblings and his/her first degree relatives. The repeat-markers flanking WD locus were amplified by PCR using fluorescent labeled primers. Amplified DNA fragments were analyzed by polyacrylamide gel electrophoresis in ABI 377 DNA sequencing system. Genotypes of the samples were determined using Genescan software. Haplotypes were determined based on segregation of the alleles in the families under study. RESULTS: Among 15 WD affected families with multiple children, 4 cases were identified where younger siblings shared same genotype as the patient at all three markers analyzed. Further, eight different haplotypes were detected in the four patients. INTERPRETATION & CONCLUSION: The siblings of the WD patients carrying the same genotype at the markers linked to WD locus were presymptomatically diagnosed individuals. Presence of eight different haplotypes in the four patients suggested mutational heterogeneity at the WD locus. The test helps clinicians for therapeutic intervention in suspect WD cases by copper chelating agents prior to manifestation of overt clinical symptoms.

Adolescent↗