Biomedical subjects

K D Mehta

Publications and source records attributed to K D Mehta.

At least 19 recordsLinked to original sources

PubMed2005

Adenosine deaminase activity in sera of patients with visceral leishmaniasis in Nepal.

This longitudinal study was conducted in BP Koirala Institute of Health Sciences (BPKIHS), a Medical University situated in eastern Nepal, between May 2001 and December 2001. The main objective of the study was to identify the role of adenosine deaminase (ADA) activity in patients with visceral leishmaniasis (VL) for management. There was a significant increase in mean ADA activity in sera of 49 patients with VL (323.71+/-184.51 IU/L) compared with 50 samples of control groups (47.11+/-24.94 IU/L) from the same endemic area (P < 0.001). ADA activities were found to be significantly decreased (50.35+/-41.35 IU/L) in follow-up cases (n = 19) after 30 days with sodium stibogluconate treatment at a dose of 20 mg/kg/day intramuscularly. The fall in the level of ADAF (after treatment) in follow-up cases correlated with the cure of disease, as evident from improvement of vital signs and symptoms and the absence of Leishmania donavani bodies in the sera. The study therefore suggests the possibility of using human serum ADA as a convenient marker to evaluate the diagnosis of VL to support the clinical findings, especially in those settings where there is a lack of highly qualified personnel and diagnostic facilities.

Adenosine Deaminase↗

PubMed1999

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Inhibition of stress-activated p38 mitogen-activated protein kinase induces low-density lipoprotein receptor expression.

We have recently shown that different signal transduction pathways initiated by a variety of agents converge on growth-responsive p42/44MAPK signaling cascade to induce low-density lipoprotein (LDL) receptor expression. Our recent demonstration that stress-activated p38MAPK negatively regulates LDL receptor expression in an isoform-specific manner via modulation of p42/44MAPK cascade represents a new dimension of complexity in the molecular communication that governs LDL receptor expression. The suggested one-way communication between p38MAPK and p42/44MAPK provides a potential mechanism for fine-tuning cellular levels of cholesterol in response to a diverse set of environmental cues, including stress. Cross talk between MAPKs opens new avenues toward understanding a variety of pathogenic processes; this makes them tempting targets for therapeutic interventions in cardiovascular diseases.

Animals↗

PubMed1999

Critical role of p42/44(MAPK) activation in anisomycin and hepatocyte growth factor-induced LDL receptor expression: activation of Raf-1/Mek-1/p42/44(MAPK) cascade alone is sufficient to induce LDL receptor expression.

The protein synthesis inhibitor anisomycin activates stress-related mitogen-activated protein kinases (MAPKs), namely, c-jun NH(2)-terminal kinase (p46/54(JNK)) and p38(MAPK) in mammalian cells. In this paper, we show that although exposure to anisomycin resulted in rapid and strong activation of p46/54(JNK) and p38(MAPK), with a delayed low level dual-phosphorylation of mitogen/extracellular protein kinase (p42/44(MAPK)), low density lipoprotein (LDL) receptor induction depends solely on the mild activation of p42/44(MAPK) signaling cascade in HepG2 cells. Unlike hepatocyte growth factor (HGF) which caused LDL receptor induction via rapid, strong, and Ras-dependent p42/44(MAPK) activation, anisomycin-induced p42/44(MAPK) activity and increased LDL receptor expression in a Ras-independent manner. Finally, we examined the role of the p42/44(MAPK) signaling cascade in LDL receptor induction by activating this kinase independently of anisomycin or HGF. By using estrogen-dependent human Raf-1 protein kinase in transient transfection assays, we show that the exclusive activation of the Raf-1/MEK-1/p42/44(MAPK) signaling cascade with antiestrogen ICI 182, 780 caused induction of LDL receptor expression to the same level as observed with either HGF or anisomycin. Consistent with the role of p42/44(MAPK), induction was strongly inhibited by pretreatment with the MEK-1/2 inhibitor PD98059. Our observation that anisomycin can use p42/44(MAPK) signaling cascade is a departure from established thinking, and the results presented shows that activation of the p42/44(MAPK) alone is sufficient to fully induce LDL receptor transcription.

Anisomycin↗

PubMed1998

Differential roles of extracellular signal-regulated kinase-1/2 and p38(MAPK) in interleukin-1beta- and tumor necrosis factor-alpha-induced low density lipoprotein receptor expression in HepG2 cells.

The inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF), elevated in inflammatory, malignant, and infectious diseases, induce low density lipoprotein (LDL) receptor transcription in HepG2 cells, and such an induction can account for hypocholesterolemia associated with these states. However, the signaling mechanisms of cytokine-mediated LDL receptor induction are largely unexplored. In the present studies, we examined the potential involvement of different mitogen-activated protein kinase (MAPK) pathways. Northern analysis demonstrated that IL-1beta or TNF significantly increased LDL receptor transcript in HepG2 cells, whereas expression of another tightly regulated sterol-responsive squalene synthase gene was unaffected. IL-1beta treatment resulted in transient activation of three MAPK cascades, namely p46/54(JNK), p38(MAPK), and ERK-1/2, with maximal activation of 20-, 25-, and 3-fold, respectively, occurring 15-30 min after cytokine addition. PD98059, a specific inhibitor of MAPK kinase activity, inhibited IL-1beta-induced LDL receptor expression. In contrast, SB202190, a specific inhibitor of p38(MAPK), enhanced IL-1beta-induced LDL receptor expression, with a concomitant increase in ERK-1/2 activity. Similarly, TNF induced LDL receptor expression also required ERK-1/2 activation. Finally, sterols repressed IL-1beta induced receptor expression, without affecting ERK-1/2 activation. These results show that IL-1beta- or TNF-induced LDL receptor expression requires ERK-1/2 activation, that the p38(MAPK) pathway negatively regulates LDL receptor expression, and that sterols inhibit induction at a point downstream of ERK-1/2 in HepG2 cells.

Animals↗

PubMed1998

Genetic complexity of the human geranylgeranyltransferase I beta-subunit gene: a multigene family of pseudogenes derived from mis-spliced transcripts.

Geranylgeranyltransferase I controls the function of a variety of cellular proteins by attaching a geranylgeranyl group to the carboxy-terminus of proteins. The purified enzyme from rat brain is comprised of two polypeptides, a catalytic alpha-subunit (GGTalpha) and a substrate-binding beta-subunit (GGTbeta). The present paper demonstrates the existence of a GGTbeta multigene family in humans by describing the presence and characterization of at least 13 pseudogenes related to this protein. Sequencing of numerous PCR-derived clones, obtained following amplification of human genomic DNA, revealed multiple, distinct but highly related sequences. All clones had a common deletion of 99-bp that conforms to the GT-AG rule of splicing in eukaryotes, and differed from the human GGTbeta cDNA sequence by multiple nucleotide substitutions. PCR amplification from mRNA, however, yielded only the sequence expected for the expressed GGTbeta protein. This apparent paradox was resolved by cloning and sequencing a complete GGTbeta-specific pseudogene. Multiple features of the cloned gene, in particular the absence of introns, presence of flanking direct repeats, and the lack of sequence similarity with the untranscribed region of the gene, indicate that this clone represents a processed pseudogene possibly resulting from a mis-spliced transcript. Multiple GGTbeta-specific pseudogenes appear to have resulted from more than one retroposition event. These results suggest a potential role for mis-splicing in the evolutionary diversity of pseudogenes.

Alkyl and Aryl Transferases↗

PubMed1997

Identification of essential nucleotides of the FP1 element responsible for enhancement of low density lipoprotein receptor gene transcription.

Low density lipoprotein (LDL) receptor gene is regulated at the transcriptional level by the intracellular level of sterols in animal cells. We have recently identified a 20 bp long region (-145 to -126), designated Footprint 1 (FP1), participating in maximal expression of the human LDL receptor gene in the absence of sterols in HepG2 cells [Mehta, K. D., Chang, R., Underwood, J., Wise, J. and Kumar, A. (1996) J. Biol. Chem ., 271, 33616-33622]. To determine the minimal FP1 sequence and to define the critical nucleotides required for function, a series of single nucleotide substitutions were introduced in the FP1 region. Twenty-three independent mutations were analyzed by transfection into HepG2 cells. These studies localize the regulatory region to 14 bp and demonstrate the requirement for essential guanine nucleotides at positions -135 and -136 for FP1 function. Furthermore, transfection studies suggest that the FP1-dependent increase in reporter gene expression is possibly mediated through interaction with the sterol-regulatory element. UV cross-linking and Southwestern blot analysis identified FP1-binding factors of approximately 50 and 125 kDa, which we have denoted p50 and p125. Mutations of the critical guanine residues (-135/-136) decreased the formation of the specific protein-DNA complex with the FP1 sequence and abolished its binding to the p125. We conclude that direct interaction of the p125 factor with these nucleotides of the FP1 element potentially contributes to FP1-dependent induction of LDL receptor gene expression.

Animals↗

PubMed1997

p21ras farnesyltransferase alpha- and beta-subunits are phosphorylated in PC-12 cells: TGF-beta signaling pathway independent phosphorylation.

Farnesyltransferase (FTase) catalyzes the transfer of a farnesyl isoprenoid to the conserved carboxyl-terminal cysteine residue of proteins terminating with the CAAX sequence. Rat brain FTase is a heterodimer consisting of a 49 kDa alpha-subunit and a 46 kDa beta-subunit. In this report, we show, for the first time, that the beta-subunit of FTase is phosphorylated in vivo and the FTase heterodimer contains phosphorylated alpha/beta-subunits in rat adrenal medulla pheocytochroma PC-12 cells. The presence of the phosphorylated FTase subunits as heterodimer in PC-12 cells which are known to be deficient in TGF-beta signaling pathways argues against the involvement of this pathway in their phosphorylation and heterodimerization.

Alkyl and Aryl Transferases↗

PubMed1997

Expression of the farnesyltransferase beta-subunit gene in human ovarian carcinoma: correlation to K-ras mutation.

The ras signaling protein requires a posttranslational modification to localize it to the inner surface of plasma membrane. In this state it can behave as a signal transduction mediator. Farnesyltransferase plays an important role in this posttranslational processing of ras by attaching a farnesyl group to the cysteine of the ras C-terminal tetrapeptide. In this study, we investigated the relationship of K-ras expression and mutation with farnesyltransferase beta-subunit expression in 20 ovarian tumors (17 carcinomas and 3 low malignant potential tumors) and 4 normal ovaries. The expression level of mRNA was determined by using quantitative PCR and mutation analysis was performed by direct cDNA sequencing. K-ras mutations were found in 1 of 3 low malignant potential tumors and in 4 of 17 carcinoma cases. K-ras mRNA overexpression was found in 1 of 3 low malignant potential tumors (one with mutated ras) and in only 1 of 17 carcinoma cases. Farnesyltransferase beta-subunit mRNA overexpression was found in 2 of 3 low malignant potential tumors and in 7 of 17 carcinoma cases. Interestingly, all K-ras mutation cases showed farnesyltransferase beta-subunit overexpression. These findings suggest that there may be a direct relationship between K-ras (ras dysfunction) mutation and expression of farnesyltransferase beta-subunit gene.

Alkyl and Aryl Transferases↗

PubMed1997

Phorbol ester-induced low density lipoprotein receptor gene expression in HepG2 cells involves protein kinase C-mediated p42/44 MAP kinase activation.

The signaling pathway involved in low density lipoprotein (LDL) receptor gene expression induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated in the human hepatoma HepG2 cell line. Treatment of HepG2 cells with 100 nM TPA resulted in an approximately 20-fold increase in LDL receptor mRNA level, as determined by RT-PCR, which peaked at 2-4 h of treatment and subsequently declined. The protein kinase C (PKC) inhibitors calphostin C and staurosporine prevented TPA-mediated LDL receptor mRNA induction. In contrast, TPA did not affect squalene synthase mRNA expression. Immunoblotting of cell extracts with isozyme-specific PKC antibodies revealed that HepG2 cells expressed PKC alpha, which was mainly cytosolic, and PKC beta, PK epsilon, and PKC zeta, all of which were present in both the cytosolic and particulate fractions. Treatment of HepG2 cells with 100 nM TPA resulted in translocation of cytosolic PKC alpha to the particulate fraction, with a maximum at 30 min-2 h of treatment, but was without effect on the subcellular distribution of the other isozymes. TPA treatment also led to activation of the mitogen-activated protein kinase (MAPK) ERK cascade. The specific MAPK pathway inhibitor PD98059 blocked TPA-induced ERK activation. Furthermore, pretreatment of cells with PD98059 inhibited TPA-induced LDL receptor mRNA induction. Moreover, pretreatment of cells with calphostin C inhibited TPA-mediated ERK activation and LDL receptor mRNA induction in a dose-dependent fashion. Based on a close kinetic correlation between PKC alpha translocation and ERK activation, and the effects of specific inhibitors, these findings suggest that translocation/activation of PKC alpha, and subsequent activation of the Raf-1/MEK/ERK MAPK cascade, represent key events in the transcriptional induction of LDL receptor gene by TPA in HepG2 cells.

Blotting, Northern↗

PubMed1996

Identification of a novel cis-acting element participating in maximal induction of the human low density lipoprotein receptor gene transcription in response to low cellular cholesterol levels.

In this paper, we present both in vivo and in vitro evidence for the presence of a novel cis-acting regulatory element that is required for maximal induction of the human low density lipoprotein (LDL) receptor gene following depletion of cellular sterols in HepG2 cells. First, in vivo dimethyl sulfate footprinting of the human LDL receptor promoter before and after transcriptional induction in HepG2 cells revealed protection from -145 to -126, 5'-GAGCTTCACGGGTTAAAAAG-3' (referred to as FP1 site). Second, transient transfections of HepG2 cells with promoter luciferase reporter constructs containing the FP1 site resulted in significant enhancement (approximately 375%) of reporter gene expression in response to low levels of sterols compared with parallel plasmid without the FP1 site. In addition, this response was markedly attenuated on nucleotide substitutions within the FP1 site. Third, by electrophoretic mobility shift assays, the FP1 sequence was found to bind protein(s) from HepG2 nuclear extracts in a sequence-specific manner. In vitro binding of the FP1 mutants paralleled the results obtained for their in vivo transcription. On the basis of competition profiles, the FP1-binding factor is different from the known transcription factors binding to the AT-rich CArG and GArC motifs. Furthermore, the FP1-binding protein is not specific to HepG2 cells because nuclear factor(s) with the same specificity was observed in nuclear extracts of non-hepatic HeLa cells. We conclude that transcriptional induction of the LDL receptor gene in response to sterol depletion is mediated, in part, by an highly conserved novel cis-acting element through the binding of specific nuclear protein(s).

Base Sequence↗

PubMed1996

Alpha-subunit of farnesyltransferase is phosphorylated in vivo: effect of protein phosphatase-1 on enzymatic activity.

Farnesyltransferase is a heterodimer consisting of a 49 kDa alpha-subunit and a 46 kDa beta-subunit. In this report, we demonstrate that the endogenous heterodimeric farnesyltransferase protein is phosphorylated at the alpha-subunit in vivo and phosphorylation plays a role in the regulation of farnesyltransferase activity. In vivo 32P-labeling of PC-12 cells followed by immunoprecipitation with specific anti rat alpha-subunit IgG showed a labeled alpha-subunit protein band at an expected molecular mass of 49 kDa. Treatment of PC-12 cells with protein phosphatase inhibitor, Calyculin A, resulted in a decrease in FTase activity, and phophoserine/phosphothreonine-specific protein phosphatase-1 treatment of PC-12 and GM37 cell extracts resulted in 100% and 375% increase in farnesyltransferase activity, respectively, compared to untreated extracts.

Alkyl and Aryl Transferases↗

PubMed1996

In vivo role of the Sp1 site neighboring sterol-responsive element-1 in controlling low-density lipoprotein receptor gene expression.

The in vivo role of the crucial Sp1 site neighboring sterol-responsive element-1 (SRE-1) in controlling LDL receptor gene expression in the presence or absence of sterols was examined. For this purpose the Xenopus laevis system was utilized as there are two different genes for LDL receptors in frogs which differ in their promoter region in the Sp1-binding sequence of repeat 3 present immediately adjacent to SRE-1. DNase I footprinting of promoters of both receptors showed differences in the affinity of this Sp1 site to purified transcription factor Sp1. Transcript levels of both LDL receptors were measured in livers of frogs on normal and cholesterol-enriched diets. Basal levels and extent of repression of LDL receptor gene on sterol administration were found to be dependent on the nature of the Sp1 site of repeat 3 under in vivo conditions. We conclude that this Sp1 site acts as a constitutive positive transcriptional element that forms a part of the active transcription complex irrespective of cellular sterol levels.

Animals↗

PubMed1996

Chiloscyllium plagiosum low-density lipoprotein receptor: evolutionary conservation of five different functional domains.

All five functional domains of the low-density lipoprotein (LDL) receptor were assembled in their modern form more than 450 million years ago, as revealed from the cloning and sequencing of an LDL receptor cDNA from Chiloscyllium plagiosum (banded cat shark). The shark LDL receptor has the same overall architecture as the mammalian and amphibian counterparts. Each of the seven cysteine-rich repeats in the ligand binding domain resembles its counterpart in the human LDL receptor more than it does the other repeats in the shark receptor as suggested by the presence of unique "signature" sequences, indicating that these repeats had already acquired their independent structures by the time of shark development. Furthermore, amino acid sequences of the entire ligand binding domain of shark LDL receptor show 35% identity over a stretch of 294 residues with a Lymnaea stagnalis G-protein-linked receptor (LSGLR). The region of homology between these unrelated proteins includes conservation of most of the unique characteristics of the cysteine-rich repeats of LDL receptor at the expected positions in LSGLR. The results presented are consistent with the hypothesis that all seven repeats in the ligand binding domain of LDL receptor may have been lifted directly from an ancestral gene instead of being evolutionary duplications of a single repeat recruited by the primitive LDL receptor from another gene.

Amino Acid Sequence↗

PubMed1991

The low density lipoprotein receptor in Xenopus laevis. I. Five domains that resemble the human receptor.

All five functional domains of the low density lipoprotein (LDL) receptor were assembled in their modern form more than 350 million years ago, as revealed from the sequence of two cloned cDNAs from the frog Xenopus laevis. The two cDNAs appear to represent duplicated copies of the LDL receptor gene that arose when the entire genome of Xenopus duplicated approximately 30 million years ago. Both frog LDL receptors bound Xenopus LDL with high affinity and human LDL with lower affinity when expressed in monkey COS cells. The receptors also showed high affinity for rabbit beta-migrating very low density lipoprotein and canine apoE-HDLc, both of which contain apolipoprotein E. Each of the seven cysteine-rich repeats in the ligand binding domain of the Xenopus receptors resembles its counterpart in the human, indicating that these repeats had already acquired their independent structures by the time of amphibian development. The cytoplasmic tail of both Xenopus receptors is 86% identical to the human, including the FDNPVY sequence necessary for internalization in coated pits. The attainment of a fully developed receptor structure in Xenopus suggests that earlier forms of the receptor may exist in animals that are older than amphibians. An accompanying paper demonstrates that expression of both Xenopus receptor genes is controlled by a sterol regulatory element that closely resembles the human sequence (Mehta, K.D., Brown, M.S., Bilheimer, D.W., and Goldstein, J.L. (1991) J. Biol. Chem. 266, 10415-10419).

Amino Acid Sequence↗

PubMed1991

The low density lipoprotein receptor in Xenopus laevis. II. Feedback repression mediated by conserved sterol regulatory element.

The 5'-flanking regions of the two low density lipoprotein (LDL) receptor genes in Xenopus laevis contain three repeat sequences that are virtually identical to the repeats that mediate sterol-regulated transcription of the human LDL receptor gene. Like their human counterparts, Xenopus repeats 1 and 3, but not repeat 2, bind the transcription factor Sp1 and thus probably function as positive transcription elements. Xenopus repeat 2, like human repeat 2, contains all of the nucleotides that are required for sterol regulation. Administration of sterols repressed Xenopus LDL receptor mRNA in cultured A6 kidney cells and in the liver of intact frogs. In frogs this repression was associated with a 2-fold increase in plasma LDL levels. Xenopus LDL contains a protein corresponding in size to human apoB-100, a ligand for the LDL receptor. We found no evidence that frog plasma contains B-48, nor did we observe a clear-cut protein corresponding to apoE. We conclude that the structural gene for the LDL receptor has been under sterol-mediated regulation at least since the time of amphibian development more than 350 million years ago.

Animals↗

PubMed1990

The ANB1 locus of Saccharomyces cerevisiae encodes the protein synthesis initiation factor eIF-4D.

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. We have shown recently that an upstream repressor site located in the 5'-flanking region of this gene controls its expression (Mehta, K.D., and Smith, M. (1989) J. Biol. Chem. 265, 8670-8675). In this paper, we present the complete genomic sequence of the ANB1 locus of S. cerevisiae. The ANB1 locus encodes a protein of 157 residues with an Mr of 17, 134. The deduced amino acid sequence of the ANB1 gene product shows strikingly extensive sequence and structural homology (63.5% identical residues and an additional 15% conservative substitutions) to the 154-amino-acid-long human and rabbit eukaryotic translation initiator factor (eIF)-4D. Factor eIF-4D is the only known mammalian protein that undergoes a unique post-translational modification of Lys-50 to the amino acid hypusine, and interestingly the same lysine is also present in the ANB1 gene product. Results presented provide strong evidence that the ANB1 locus that encodes a transcript, tr-2, and a second locus encoding a transcript, tr-1, together encode two forms of yeast eIF-4D. Interestingly, heme regulates both the loci in an opposite manner; as a result it can dictate the isoform available under conditions of high and low oxygen tension. The ROX1 locus of S. cerevisiae is known to regulate CYC1, COXVb, and ANB1 genes at the transcriptional level; the ROX1 locus thus regulates all known anaerobically expressed genes that are involved in different cellular functions such as respiration and protein synthesis.

Amino Acid Sequence↗

PubMed1989

Identification of an upstream repressor site controlling the expression of an anaerobic gene (ANB1) in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. A 299-base pair-long fragment from the 5'-flanking region of the ANB1 gene was found to confer oxygen-mediated negative regulation to an heterologous CYC1-LacZ hybrid gene. Studies with deletions of predefined length in this fragment demonstrated the presence of separate elements that comprise an upstream promoter that is active in the absence or presence of oxygen, and an upstream repressor site (URS) which confers strong repression upon the promoter element when oxygen is present. The promoter element is located 5' to the URS in the ANB1 gene. Mixed oligonucleotide-directed mutagenesis was used to obtain nucleotide substitutions in the URS which partially or completely inactivated this sequence without affecting the promoter activity. The URS region has three short direct repeats which seem to be important for function, as nucleotide substitutions within the repeats and not outside them, inactivated URS function. A model to explain the negative regulation of the ANB1 gene by oxygen and heme is proposed.

Anaerobiosis↗