Search PubMed⌕ Search

Biomedical subjects

K Roy

Publications and source records attributed to K Roy.

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

Alterations in total iron, zinc, and calcium levels and their influence on the hepatic activities of gamma-glutamyl transferase and glucose-6-phosphatase in the host bearing transplantable murine lymphoma.

The levels of iron, zinc, and calcium in liver as well as serum, together with the enzymatic activities of gamma-glutamyl transferase (GGT, EC 2.3.2.2) and glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9) in liver, were critically monitored over various periods in male Swiss albino mice bearing Dalton's lymphoma (DL), a transplantable ascites-producing tumor. Both hepatic and serum contents of iron, zinc, and calcium were found to be maximally elevated (p < 0.001) on day 15 after tumor transplantation as compared with their contents in normal animals. There was a gradual increase in the activity of GGT in liver in lymphoma-bearing mice in comparison with their normal counterparts, which showed a maximum peak (p < 0.001) on day 15, followed by a continuous and sharp fall. Hepatic G-6-Pase activity was found to decrease continuously throughout the progression of lymphoma as compared with its levels to normal animals. Tumor-cell counts in peritoneal lymph fluids of mice containing DL yielded a maximum count of 155.7 x 10(3) cells/mm3 on day 15. A significant correlation was observed among the levels of different metals, enzymatic activities, and tumor-cell counts at different periods of study. From these results, it can be concluded that the metals studied may have a role in initiating and controlling cellular proliferations, through their effects on modulating the activities of the possibly preneoplastic and neoplastic marker enzymes named above.

Animals↗
PubMed1997

RFC-1 gene expression regulates folate absorption in mouse small intestine.

Mediated folate compound transport inward in isolated luminal epithelial cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensitivity to the stilbene inhibitor, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid. pH dependence was manifested as higher maximum capacity (Vmax) for influx of l, L-5-CH3-H4folate at acidic pH compared with neutral or alkaline pH with no effect on saturability (Km). The pH-dependent component was relatively insensitive to inhibition by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and highly saturable (Km or Ki = 2 to 4 microM) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors. The non-pH-dependent component was highly sensitive to 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and poorly and variably saturable (Km or Ki = 20 to >2000 microM) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l,L-5-CH3-H4folate influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was associated with an increase in RFC-1 gene expression in the form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane protein detected by folate-based affinity labeling and with anti-mouse RFC-1 peptide antibodies. The size of this protein was the same as that encoded by RFC-1 mRNA. The treatment of mature absorptive cells with either the affinity label or the anti-RFC-1 peptide antibodies inhibited influx of l, L-[3H]-5-CH3-H4folate in a concentration-dependent manner. These results strongly suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Organization, structure and alternate splicing of the murine RFC-1 gene encoding a folate transporter.

The structural organization of the murine RFC-1 gene encoding a folate transporter has been determined. The entire nucleotide sequence of the L1210 cell RFC-1 cDNA, the 3'- and 5'-untranslated regions and the coding sequence were found to be distributed in eight exons, including six primary exons and alternates to exon 1 and exon 5, spanning 10.4 kb. Splice variants were identified in an L1210 cell cDNA library. The most common incorporates exons 1 through 6, encoding a 58-kDa polypeptide. The two least common incorporate exons 1 and 2, a truncated version of exon 3 and exons 4 through 6; or exons 1 through 4, an alternate to exon 5, and exon 6, encoding polypeptides of 53.6 and 43.4 kDa, respectively. A fourth variant reported earlier (GenBank/EMBL accession No. L36539) by others incorporates what we have found to be an alternate of exon 1 and exons 2 through 6. A relatively GC-rich region of the genome just 5' of exon 1 as well as exon 1a appears to be distinctly promoter-like and encodes a number of putative cis-acting elements. The findings pertaining to alternates of exon 1 suggest that the transcription of RFC-1 variants results from two different promoters.

Alternative Splicing↗
PubMed1997

Posttranscriptionally mediated decreases in folylpolyglutamate synthetase gene expression in some folate analogue-resistant variants of the L1210 cell. Evidence for an altered cognate mRNA in the variants affecting the rate of de novo synthesis of the enzyme.

L1210 cell variants resistant to edatrexate (EDX) were isolated by selection in vivo during therapy with this folate analogue. Among the variants selected, seven (L1210/EDX-4 to -7 and L1210/EDX-12 to -14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthetase (FPGS) activity compared with parental L1210 cells. Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Vmax with no change in apparent Km. The results of an analysis of FPGS activity in mixtures of variant and parental cell extract suggested that no endogenous inhibitors in the variant cells or stimulatory factors in parental cells accounted for the differences observed. Also, FPGS from variant and parental cells showed no difference in thermostability. Decreases in a 60-61-kDa protein as shown by immunoblotting with anti-FPGS peptide antibody were found to occur commensurately with the decrease in FPGS activity in cell extract from the variants compared with parental cells. However, no evidence was obtained for a difference in turnover of FPGS protein during measurement of the decay of FPGS activity in cycloheximide-treated variant and parental cells. In addition, Northern blotting of poly(A)+ RNA did not reveal any difference in the size or level of FPGS mRNA among these various cell types. Studies of in vitro translation of hybridization-selected FPGS mRNA from L1210 cells showed that both mitochondrial and cytosolic forms of FPGS were generated during the reaction. Moreover, FPGS mRNA from the variant cells was significantly less effective in mediating formation of the FPGS peptide product in a manner correlating with FPGS activity and protein found in the cytosol of the various cell types. These results suggest that FPGS gene expression in these variants is posttranscriptionally altered at the level of the cognate mRNA itself and that this alteration constitutively down-regulates the steady-state level of FPGS in these variants.

Aminopterin↗
PubMed1997

Additional organizational features of the murine folylpolyglutamate synthetase gene. Two remotely situated exons encoding an alternate 5' end and proximal open reading frame under the control of a second promoter.

Nucleotide sequence analysis of independently isolated clones from a mouse liver cDNA library identified two additional splice variants of folylpolyglutamate synthetase (FPGS) mRNA with novel sequence at the 5' end. These variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2. Exon A1a encodes most of the 5'-untranslated region. Exon A1b encodes a downstream segment of the 5'-untranslated region, two ATG start codons, and a unique mitochondrial leader peptide as well as 15 additional amino acids of cytosolic FPGS not encoded by all previously identified (Roy, K., Mitsugi, K., and Sirotnak, F. M. (1996) J. Biol. Chem., 271, 23820-23827) splice variants. It was also found by direct sequencing of genomic fragments that although exon A1b is spliced to exon 2, these new alternatives (i.e. exons A1a and A1b) to exon 1 are found approximately 9.5 kilobases upstream from exons B1a, B1b, and B1c. Exons A1a and A1b are separated from each other by a 124-nucleotide intron. Sequencing of the region 5' to exon A1a revealed a nucleotide sequence that was promoter-like and different from the downstream promoter region in the content of putative cis-acting elements. Primer extension analysis identified a number of potential transcription start sites within the more 3' end of this region. FPGS RNA transcripts incorporating exons A1a and A1b were detected in both normal mouse tissues, particularly, liver and kidney, and also to a varying extent in tumors; FPGS RNA transcripts incorporating exons B1a, B1b, and B1c were detected mainly in tumors. Thus, transcription of the FPGS gene in this tissue-specific manner appears to reflect the different usage of alternates to exon 1 under the control of different promoters. An unusual splice variant identified infrequently in a mouse liver cDNA library was 2.67 kilobases in size and incorporated exons A1a and A1b and a segment of the downstream promoter region along with exons B1c and B1b and exons 2-15.

Alternative Splicing↗
PubMed1997

Autofluorescence characteristics of oral mucosa.

BACKGROUND: The fluorescence characteristics of tissues depend upon their biochemical composition and histomorphological architecture, both of which undergo a change during malignant transformation. These changes are detectable as an alteration in the fluorescence spectral profile of the tissues. METHODS: Biopsy specimens from clinically suspicious lesions and normal-appearing oral mucosa were obtained from patients. Fluorescence spectroscopic measurements were obtained to study the differences between normal and dysplastic tissues and to determine the most appropriate excitation wavelength(s) for exploiting these differences. RESULTS: Fluorescence spectra from a total of 12 histologically normal (healthy mucosa or benign lesions) and ten abnormal (dysplastic or malignant) tissue samples were compared. Significant spectral differences were seen between the two groups. These differences were most marked at the excitation wavelength of 410 nm. Using this wavelength, fluorescence correctly diagnosed 20 of 22 samples studied. CONCLUSIONS: This technique accurately differentiates normal from abnormal tissues in vitro and has the potential applications for in vivo use as a noninvasive diagnostic tool.

Biopsy↗
PubMed1997

I wish I'd live.

Explore the source record for details and available documents.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Organization and alternate splicing of the murine folylpolyglutamate synthetase gene. Different splice variants in L1210 cells encode mitochondrial or cytosolic forms of the enzyme.

The organization of the murine folylpolyglutamate synthetase (FPGS) gene has been determined by sequence analysis that also revealed an interesting complexity in the case of exon 1. The entire nucleotide sequence of the L1210 FPGS cDNA, the 3'- and 5'-untranslated regions, the mitochondrial leader sequence, and the coding region were found to be distributed on 15 exons with an overall length of 10.358 kilobases. Two splice variants of exon 1 were identified by screening of an L1210 cell cDNA library. Variant I (exons 1a + 1b plus 2-15) incorporates all of the sequence homologous to the recently reported (Taylor, S. M., Freemantle, S. J., and Moran, R. G. (1995) Cancer Res. 55, 6030-6034) human exon 1, including two ATG start codons at positions +1 and +126, and encodes both mitochondrial and cytosolic form of FPGS. The most prevalent variant, Variant II (exons 1b plus 2 to 15), incorporates only a portion (92 nucleotides at the 3' end) of this sequence, incorporates only one ATG start codon at position +126, and encodes only cytosolic FPGS. The existence of this variant is consistent with the identification of an appropriately situated internal donor/acceptor site in what was believed to be exon 1. A third related variant (Variant III) with a novel 5' termini was originally identified by screening of a mouse liver cDNA library. This variant, which occurs at moderately low frequency in the L1210 cell cDNA library, incorporates an alternate to exon 1a (exon 1c) spliced to exon 1b plus exons 2-15 and encodes a different mitochondrial leader peptide than Variant I. The identification of these variants suggests another possible mechanism, i.e. at the level of precursor mRNA splicing, for regulating synthesis of mitochondrial versus cytosolic forms of FPGS in the cell. Exon 1c is positioned in the gene upstream of exon 1a separated by an intron of 56 nucleotides within a region of DNA sequence that like the homologous human sequence is distinctly promoter-like. However, the sequence of this region differs from the human sequence in terms of the number, position, and type of putative regulatory elements, particularly in regard to the number of SP-1 binding sites and the position of multiple transcription start sites as determined by enzymatic primer extension.

Alternative Splicing↗
PubMed1996

Cross-screening: a new method to assemble clones rapidly and unambiguously into contigs.

We have developed a new procedure that relies on an array of cross-hybridization tests to order a set of random clones into a contig. The method, called cross-screening, uses each clone as a target and its end sequences as probes, in a matrix of reciprocal cross-hybridization tests performed on a single blot. The relationships among the clones are determined rapidly from the pairwise tests, allowing clone order to be determined directly. We have applied this technique to DNAs from a set of overlapping lambda clones from Drosophila chromosome 4. The location and orientation of each clone derived from the cross-screening data was that expected from the map assembled from overlapping restriction sites and chromosomal walking. The procedure provided additional information on a previously unknown, internally repeated DNA sequence. To demonstrate the general utility of the procedure, we have applied it to a previously described clone set within a contig in region 22q12 of human chromosome 22. The correct relative position and orientation of these clones were derived from the cross-screening data without knowledge of, or reference to, any nucleotide sequence or restriction site analysis of the DNA concerned. The cross-screening procedure is fast, economical, and robust and allows clone overlaps to be determined efficiently, with minimal interference from repeated DNA sequences. This new procedure is specifically designed for small groups of overlapping clones (tens to hundreds) and should facilitate the ordering of subclone libraries derived from small chromosomes or the large cloned inserts carried in YAC, BAC, and P1 vectors.

Animals↗
PubMed1996

Role of vitamin D3 on the activity patterns of hepatic drug metabolizing enzymes in transplantable murine lymphoma.

Vitamin D3 (D3) has been found to exert varied pharmacological actions including restriction of cell growth of a number of malignant cell lines in vitro and inhibition of the promotion of chemical carcinogenesis in mouse skin. In an attempt to confirm the efficacy of D3 as an antineoplastic agent, the present investigation aims at characterizing the importance of D3 in modulating hepatic drug metabolizing enzymes, namely, cytosolic glutathione S-transferase (GSHT), microsomal UDP glucuronyl transferase (UDPGT), and cytochrome P-450, which have been reported by us in recent literature as significant neoplastic markers in mice bearing Dalton's lymphoma (DL). Results show that D3 causes a 150% elevation of GSHT activity and the maintenance of normal, near-control UDPGT activity and cytochrome P-450 content, up to almost 30 days following tumor transplantation, along with bringing about a twofold increase in survival of the host mice. In conclusion, we confirm the definite and significant antitumorigenic role of D3 and its involvement with the discussed hepatic tumor markers in monitoring the processes that lead to cell survival.

Animals↗
PubMed1995

Ligand-directed immunoaffinity purification and properties of the one-carbon, reduced folate transporter. Interspecies immuno-cross-reactivity and expression of the native transporter in murine and human tumor cells and their transport-altered variants.

Almost complete purification (> 95%) of the 46-kDa murine, one-carbon, reduced folate transporter (RFT) at a recovery of 20% was obtained by ligand-directed immunoaffinity fractionation from transporter overproducing L1210/R83 cells. These cells were labeled with the N-hydroxysuccinimide ester of [3H]aminopterin (AMT), the isolated plasma membrane alkaline washed to remove nonintegral membrane proteins, detergent-solubilized, and RFT-separated on an anti-AMT antibody-protein G-Sepharose column followed by preparative SDS-polyacrylamide gel electrophoresis. Anti-RFT antibody, subsequently derived, differentially blotted (L1210/R83 >> L1210/0) a 46-kDa protein during SDS-polyacrylamide gel electrophoresis of plasma membrane from L1210/R83 and L1210 cells and in L1210/R83 cells after trichloroacetic acid precipitation. In contrast to that reported for human tumor cells, glycosidase treatment of RFT revealed no common N- or O-linked core oligosaccharides associated with this protein. The same 46-kDa protein at different relative levels was revealed in a Western blot of plasma membrane from other murine tumors. Blotting of plasma membrane from methotrexate resistant, transport defective L1210 cell variants exhibited wild-type levels of a less electrophoretically mobile RFT or greater levels of the same 46-kDa RFT which could not be affinity labeled with N-hydroxysuccinimide-[3H]AMT. The same antibody differentially blotted a 83-kDa plasma membrane protein from human HL-60 and CCRF-CEM cells with different levels of reduced folate transport and affinity labeling of RFT, verifying the conserved nature of this protein consistent with earlier functional studies.

Aminopterin↗
PubMed1995

Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription.

L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H. (1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of 1,L5-CHO-folateH4 (natural diastereoisomer of 5-formyl-tetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constitutively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.

Amino Acid Sequence↗