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Biomedical subjects

G Sarkar

Publications and source records attributed to G Sarkar.

At least 37 records · Page 2Linked to original sources
PubMed1998

PIG-B: a homemade monophasic cocktail for the extraction of RNA.

An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent are Phenol, Isoamyl alcohol, Guanidinium isothiocyanate, and Beta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.

Guanidines↗
PubMed1998

Artificial cavitation nuclei significantly enhance acoustically induced cell transfection.

The efficiency of ultrasound-mediated gene transfection was enhanced three- to fourfold, compared to previous results, through the use of green fluorescent protein reporter gene, cultured immortalized human chondrocytes and artificial cavitation nuclei in the form of Albunex. Cells were exposed to 1.0-MHz ultrasound transmitted through the bottom of six-well culture plates containing immortalized chondrocytes, media, DNA at a concentration of 40 micrograms/mL and Albunex at 50 x 10(6) bubbles/mL. Transfection efficiency increased linearly with ultrasound exposure pressure with a transfection threshold observed at a spatial average peak positive pressure (SAPP) of 0.12 MPa and reaching about 50% of the living cells when exposed to 0.41 MPa SAPP for 20 s. Adding fresh Albunex at 50 x 10(6) bubbles/mL prior to sequential 1-s, 0.32- or 0.41-MPa exposures increased transfection with each exposure, reaching 43% transfection after four exposures. Efficient in vitro and in vivo transfection now appear possible with these enhancements.

Albumins↗
PubMed1997

Glutathione S-transferase pi expression in matched human normal and malignant oral mucosa.

Over-expression of glutathione S-transferase pi (GST-pi) class isozyme is often associated with malignant transformation and/or drug resistance. To determine whether GST-pi is implicated in betel and tobacco related oral carcinogenesis and/or drug resistance, its expression was studied in oral untreated primary squamous cell carcinomas (SCCs), recurrent tumours and matched normal oral tissues by immunohistochemical and immunoblotting techniques. The GST-pi expression in primary tumours predominantly varied from mild to moderate levels and no significant difference in its expression was observed in the matched normal tissue surrounding these lesions. Mild to moderate levels of GST-pi expression in the oral mucosa of consumers of betel and tobacco observed in the matched normal tissues may support the physiological role of this detoxification enzyme in the metabolism of xenobiotics and elimination of toxic constituents of tobacco. The hallmark of the study is the significant increase in GST-pi expression in recurrent oral SCCs, compared to the matched normal tissues, as well as primary oral tumours, suggesting its potential role as an indicator of prolonged exposure to carcinogens, or prognosis of the disease.

Adolescent↗
PubMed1997

Osteoblasts express types I and II activin receptors during early intramembranous and endochondral bone formation.

Increasing evidence suggests a potential role for activin in bone formation. However, the cognate receptors through which activins function with respect to skeletal tissues have not yet been identified. Identification and regulation of expression of these receptors are necessary prerequisites to understanding the role of activins in bone metabolism. We detected mRNAs for three activin receptors, type I (ActRI), type II (ActRII), and type IIB (ActRIIB), in multiple skeletal tissues in rat, including tibia and costochondral growth plate, and also in cultured osteoblasts. To gain information about the relationship between receptor expression and different skeletal cell functions, we evaluated expression of the three receptors in a semiquantitative manner during the early stages of fracture healing, a model for rapid bone formation. Relatively high levels of ActRI and ActRII expression were detected in the callus at 7, 10, and 14 days after fracture, times that correlate with the interval of rapid intramembranous bone formation and the initiation of endochondral bone formation. Expression of the ActRIIB in the fracture callus was strikingly lower than either ActRI or ActRII. Immunostaining of the fracture callus and the newborn rat femur with an anti-ActRII antibody localized the receptor to osteoblasts at regions of membranous and endochondral bone formation. No staining of osteoblasts in fracture callus or bone was seen with an anti-ActRIIB antibody. These results provide strong evidence of the identification of the principal receptors through which activins could function in the skeletal system and further shed light on activin's mechanism of action in bone formation.

Activin Receptors↗
PubMed1996

Identification of a dual leucine zipper kinase involved in rat fracture repair.

Complete cDNA sequence was obtained for a gene found to be expressed at a high level during fracture healing in the rat. Comparison of the amino acid sequence (deduced from the cDNA sequence) with known sequences showed 99% identity with mouse Dual Leucine Zipper Kinase (mDLK) and 96% identity with human Zipper Protein Kinase (hZPK). Relative quantitation by RT-PCR indicates abundant expression of this gene in cultured chondrocytes, growth plate and brain tissue. Further, regulated expression of this gene has been observed during fracture healing in rat femur fracture model; maximum gene expression in this model appears to coincide with chondrogenesis and bone formation. Since DLK has been shown to function in the mitogen activated signaling (MAP) pathway, these results provide the first evidence for the involvement of this signaling pathway in fracture healing.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of osteoblast precursor cells from human bone marrow.

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow. Because of the lack of a practical procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow, previous studies of marrow stromal cells have been made in confluent cultures of bone marrow when the osteoblast (OB) precursors are already differentiated. Also these studies utilized cultures containing mixed populations of cells including hematopoietic cells. Thus we have employed a negative immunoselection procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived from the plastic-adherent mononuclear marrow cells cultured in the presence of serum. By reverse transcriptase polymerase chain reaction (RT-PCR) analysis for mRNA, and by immunocytochemical study for protein, we studied the sequential expression in culture of multiple markers of the osteoblast phenotype--alkaline phosphatase, osteopontin, parathyroid hormone receptor, types I and III procollagen, and osteocalcin--as well as lipoprotein lipase (LPL), a marker of the adipocyte phenotype. At an early stage of culture (7-9 days), human OB precursors formed colonies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on growth to a confluent monolayer (approximately 14 days). LPL mRNA was expressed at high levels in the colony stage, and its level decreased upon confluency, suggesting a loss of potential for commitment to the adipocyte lineage. Interestingly, treatment with dexamethasone at 10(-8) M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid droplets in confluent cultures. Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR). Thus these immunoselected marrow stromal cells have the characteristics of authentic human osteoblast precursor cells which also are capable of differentiating into adipocytes.

Alkaline Phosphatase↗
PubMed1995

Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum.

Estrogen deficiency is well recognized as a cause of bone loss in rats and humans. Likewise, treatment with estrogen results in prevention of this loss. Initially, this effect was thought to be indirectly mediated but, more recently, estrogen receptors (ER) have been reported in osteosarcoma cells and primary cultures originating from surgical waste, suggesting a direct effect of this steroid hormone. Detection of ER in skeletal tissues, however, has remained elusive. The purpose of this investigation was to establish the efficacy of the highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) technique to detect ER in a well defined skeletal tissue (calvarial periosteum) that is responsive to the hormone. Primers were made specific to rat ER sequences. Total RNA was extracted from rat uterus, liver, spleen, and the periosteum using an organic solvent method. cDNA was synthesized from 2 micrograms total RNA. cDNA corresponding to 40 ng total RNA/sample produced intense PCR products for ER. In descending order of intensity were uterus, liver, bone, and spleen. Importantly, a similar time-course for estrogen-induced down-regulation of steady-state mRNA levels for alkaline phosphatase and osteonectin was observed in calvarial periosteum and tissues known to express estrogen receptors. These data provide in vivo evidence of ER mRNA in bone and suggest that at least some of estrogen's action on bone is directly modulated.

Animals↗
PubMed1994

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves' ophthalmopathy.

Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves' disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, we have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. We suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties.

Adult↗
PubMed1993

Absence of chromosome fragility at 19q13.3 in patients with myotonic dystrophy.

Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by a trinucleotide (AGC) amplification at 19q13.3. The degree of trinucleotide amplification may increase in successive generations and generally correlates with severity of the disorder. Because amplification of a trinucleotide repeat is also associated with the observation of a fra(X)(q27.3) in the fragile X syndrome, we investigated whether chromosome fragility at 19q13.3 might be inducible in patients with DM. Using 3 different culture stress systems (medium 199, RPMI 1640 with excess TdR, and RPMI 1640 with FudR) and high resolution chromosome analyses, we studied 6 individuals with DM and 5 unaffected relatives representing two unrelated families. Molecular studies were done on two of the most affected patients and showed AGC repeat sequences of 970 and 1,260 at 19q13.3. The normal range of AGC repeat is 5 to 30. We found no indication of fragility at 19q13.3 in any of these individuals.

Adolescent↗
PubMed1993

A yeast antiviral protein, SKI8, shares a repeated amino acid sequence pattern with beta-subunits of G proteins and several other proteins.

SKI8 is a yeast antiviral gene, essential for controlling the propagation of M double-stranded RNA (dsRNA) and thus for preventing virus-induced cytopathology. Our DNA sequence of SKI8 shows that it encodes a 397 amino acid protein containing two copies of a 31 amino acid repeat pattern first identified in mammalian beta-transducin and Cdc4p of yeast. There are also four copies of this repeat in yeast Mak11p, necessary for M dsRNA propagation, and three copies in the putative product of the Dictyostelium AAC3 gene. Analysis of 36 cases of the repeat unit shows they have a consensus predicted structure: N-helix-sheet-turn-sheet-turn-sheet-helix-C.

Amino Acid Sequence↗
PubMed1993

Absence of prohibitin gene mutations in human epithelial ovarian tumors.

Multiple loss of heterozygosity (LOH) studies of ovarian cancers have found a high incidence of chromosome 17 loss in these tumors. Several authors have suggested that the region commonly deleted encompasses 17q12-21. In addition, this region has recently been reported to be linked to the familial breast/ovarian cancer syndrome. Recently the human prohibitin gene was mapped to region 17q12-22. Prohibitin causes arrest of DNA synthesis by fibroblast and HeLa cells and prohibitin shows significant homology to a gene (Cc) thought to be important for the regulation of development of Drosophila melanogaster. These findings have led many to consider the prohibitin gene a potential tumor suppressor gene. In addition, sequence analysis of exon 4 of human prohibitin gene revealed mutations in 4 of 23 sporadic breast carcinomas. Because of the proposed function for prohibitin, its alterations in breast cancers, and the fact that its location on 17q falls within a commonly deleted region in ovarian cancers, we have undertaken an analysis of the sequence of prohibitin in epithelial ovarian cancers. Using several polymorphic DNA probes, we identified 20 epithelial ovarian tumors which demonstrated LOH for the region that contains the prohibitin gene. To evaluate whether mutations of prohibitin may be important in ovarian carcinogenesis, we have sequenced exons 4 and 5 of this gene using the technique of genomic amplification with transcript sequencing. Only normal exon 4 and 5 sequence was observed among the 20 tumors screened. These results demonstrate that this region of the prohibitin gene is not mutated in epithelial ovarian cancers and suggest that the prohibitin gene does not play a role in ovarian carcinogenesis. Sequencing of further exons and introns are needed to confirm this latter hypothesis.

Base Sequence↗