Biomedical subjects

V K Parnaik

Publications and source records attributed to V K Parnaik.

At least 19 recordsLinked to original sources

PubMed2005

Rescue of heterochromatin organization in Hutchinson-Gilford progeria by drug treatment.

Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment.

Cell Nucleus↗

PubMed2001

Novel Sp family-like transcription factors are present in adult insect cells and are involved in transcription from the polyhedrin gene initiator promoter.

We earlier documented the involvement of a cellular factor, polyhedrin (polh) promoter-binding protein, in transcription from the Autographa californica nuclear polyhedrosis virus polh gene promoter. Sequences upstream of the polh promoter were found to influence polh promoter-driven transcription. Analysis of one such region, which could partially compensate for the mutated polh promoter and also activate transcription from the wild-type promoter, revealed a sequence (AcSp) containing a CACCC motif and a loose GC box resembling the binding motifs of the transcription factor Sp1. AcSp and the consensus Sp1 sequence (cSp) specifically bound factor(s) in HeLa and Spodoptera frugiperda (Sf9) insect cell nuclear extracts to generate identical binding patterns, indicating the similar nature of the factor(s) interacting with these sequences. The AcSp and cSp oligonucleotides enhanced in vivo expression of a polh promoter-driven luciferase gene. In vivo mopping of these factor(s) significantly reduced transcription from the polh promoter. Recombinant viruses carrying deletions in the upstream AcSp sequence confirmed the requirement of these factor(s) in polh promoter-driven transcription in the viral context. We demonstrate for the first time DNA-protein interactions involving novel members of the Sp family of proteins in adult insect cells and their involvement in transcription from the polh promoter.

Animals↗

PubMed2001

Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis.

We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.

Animals↗

PubMed2001

SP3 and AP-1 mediate transcriptional activation of the lamin A proximal promoter.

Lamin A is a major component of the nuclear lamina that is expressed in various types of differentiated cells. We have analysed previously the putative promoter sequences of the gene and shown that the rat lamin A proximal promoter contains two essential motifs, a GC box that can bind to Sp1 and Sp3, and an AP-1 motif that can bind to c-Jun and c-Fos. In this study we have investigated the role of Sp1 and Sp3 in transactivation of the promoter. Functional analysis of the promoter in Drosophila SL2 cells has demonstrated that it is inactive in the absence of Sp proteins. Activation by expression of Sp3 is more pronounced than that by Sp1 although both proteins can bind to the GC box in vitro; activation clearly depends on an intact GC box as deduced from mutant analysis. Promoter activity in SL2 cells also requires an intact AP-1 motif, which can bind to endogenous Drosophila Jun and Fos proteins. Furthermore, overexpression of c-Jun and c-Fos results in fourfold activation of the promoter in PCC-4 embryonal carcinoma cells. Our demonstration that activation of the lamin A proximal promoter is mediated by Sp3 and AP-1 transcription factors affords a basis for further studies on the regulation of this important gene during development and disease.

Animals↗

PubMed2001

Distinct changes in intranuclear lamin A/C organization during myoblast differentiation.

Intranuclear lamin foci or speckles have been observed in various cell types. In order to explore the possibility of changes in internal lamin organization during muscle differentiation, we have examined the appearance of A-type lamin speckles that associate with RNA splicing factor speckles in C2C12 myoblasts and myotubes. Lamin speckles were observed in dividing myoblasts but disappeared early during the course of differentiation in postmitotic myocytes, and were absent in myotubes and muscle fibers. However, no changes were seen in the typical peripheral organization of lamins A/C or B1 or in RNA splicing factor speckles. Lamin speckles were also absent in quiescent myoblasts but reappeared as cells were reactivated to enter the cell cycle. These changes were not observed in other quiescent cell types. Immunoblot analysis indicated that the abundance and migration of lamins A and C was not altered in differentiated myoblasts. When myotube or quiescent myoblast nuclei were extracted with nucleases and detergent, a uniformly stained internal lamina was revealed, indicating that lamins A/C were antigenically masked in these cells, probably owing to structural reorganization of the lamina during differentiation or quiescence. Our results suggest that muscle cell differentiation is accompanied by regulated rearrangements in the organization of the A-type lamins.

Animals↗

PubMed2000

Phosphorylation of NPA58, a rat nuclear pore-associated protein, correlates with its mitotic distribution.

At the onset of mitosis in higher eukaryotic cells, the nuclear envelope and its components including subunits of the nuclear pore complexes are disassembled, and these are reassembled toward the end of mitosis. We have studied the role of protein phosphorylation in this process, by investigating the phosphorylation status of a specific pore-associated protein during mitosis. Using a monoclonal antibody, mAb E2, earlier shown to inhibit nuclear protein import in rat fibroblast cells, we have identified a 58-kDa protein termed NPA58 that is partially associated with nuclear pores based on a high degree of coincident immunofluorescence in dual labeling experiments with mAb 414, a well-studied pore-complex-reactive antibody. NPA58 is specifically phosphorylated during mitosis and dephosphorylated upon release from metaphase arrest. Confocal microscopy analysis shows that NPA58 is dispersed in the cytoplasm early in mitosis when it is phosphorylated, while its relocalization in the reforming nuclear envelope during telophase temporally correlates with its dephosphorylation upon release from metaphase arrest. Our data provide in vivo evidence that the modifications mediated by phosphorylation and dephosphorylation are required for regulating the mitotic localization of a nuclear-pore-associated protein.

Animals↗

PubMed1999

Colocalization of intranuclear lamin foci with RNA splicing factors.

The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.

Animals↗

PubMed1999

Identification of altered DNA-protein interactions at the lamin A proximal promoter in quiescent hepatocytes.

The nuclear lamina of differentiated cells is composed of A-type and B-type lamins. The expression of the A-type lamins has been shown to be primarily under transcriptional control in proliferating cells. In order to gain insights into the factors involved in downregulation of the promoter in quiescent cells, we have compared the DNA-protein interactions at the rat lamin A proximal promoter in fetal (dividing) hepatocytes and adult (quiescent) hepatocytes by DNase I footprinting analysis, electrophoretic mobility shift assays and UV crosslinking experiments. We have identified proteins from adult hepatocyte nuclear extracts that are bound to sequences within 28 bp downstream of the transcription initiation site (62 and 48 kDa proteins) and within 20 bp upstream of the initiation site (54 kDa protein), which may function as transcriptional repressors of the lamin A proximal promoter.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Functional analysis of the 5' promoter region of the rat lamin A gene.

The A-type lamins are constituents of the nuclear lamina in differentiated cells and have been proposed to play an important role in nuclear organization. In this study, we isolated and characterized a genomic clone containing the putative promoter region of the rat lamin A gene. Sequence analysis of about 2 kb of this region combined with primer extension data revealed the presence of a TATA box at -33, a GC box at -101, and AP1 motifs at -7, -424, and -1677. Deletion analysis of the promoter fragments in three mammalian cell lines indicated that a 221-bp segment of the proximal promoter containing the GC box and AP1 motif at -7 was sufficient to give high levels of luciferase activity in reporter gene assays. Mutations in these two motifs resulted in considerable loss of reporter gene activity. Analysis by electrophoretic mobility shift assays (EMSAs) has provided evidence for specific binding of the AP1 and Sp1 family of transcription factors to the promoter, a conclusion supported by DNase I footprinting data. This characterization of the 5' promoter region of the lamin A gene should afford a basis for the further clarification of the mechanism of regulation of this important gene during growth and development.

Animals↗

PubMed1996

An improved method to distinguish micrococcal nuclease sensitivity of chromatin.

The resolution of the standard micrococcal nuclease assay for sensitivity of active chromatin has been enhanced by the inclusion of an additional step of digesting nuclease-digested DNA with a suitable restriction enzyme prior to Southern hybridization. The improved assay has been used to analyze the chromatin structure of the lamin A, albumin and alpha-fetoprotein genes during rat liver development.

Animals↗

PubMed1996

Activation of the lamin A gene during rat liver development.

We have studied the regulation of expression of the A-type lamins, which are constituents of the nuclear lamina. During rat liver development, high levels of lamin A and C mRNAs were observed in 15-day fetal liver but were barely detectable in the adult. The chromatin conformation of the lamin A gene was sensitive to DNase I in 15-day fetal liver but became mostly insensitive in the adult. Lamin A and C proteins could be detected in fetal liver and persisted in the adult. Our evidence suggests that the lamin A gene is actively transcribed early in liver differentiation and its activity declines considerably in adult liver.

Animals↗

PubMed1996

Nucleocytoplasmic transport of proteins and control of cellular function.

Nucleocytoplasmic transport of proteins occurs through pores embedded in the nuclear envelope. Recent studies have defined the cytoplasmic factors required for signal-mediated import of nuclear proteins. Considerable progress has been made in understanding the mechanism of nuclear export of proteins by the identification of specific signal sequences needed for export. Regulatory molecules have been shown to adopt novel mechanisms to control their entry into the nucleus and thereby regulate their functions.

Amino Acid Sequence↗

PubMed1995

Essential role of protein phosphorylation in nuclear transport.

We have investigated a possible role for protein phosphorylation in nuclear transport in semi-intact cells, prepared by digitonin permeabilization of rat F-111 fibroblasts. Treatment of semi-intact cells with alkaline phosphatase abolished the import of nuclear transport substrates, namely, signal peptide-albumin conjugates, as well as their signal-dependent binding at the nuclear pores, but did not affect the morphology of the cells, in particular their cytoskeletal network. Authentic transport and functional binding of the karyophilic protein at the nuclear envelope could be restored by incubation of phosphatase-treated cells with cytosol enriched in protein kinase C or with purified protein kinase A (catalytic subunit). Restoration of transport was blocked by specific inhibitors of these kinases. Since the protein phosphorylation required for nuclear transport appeared to be a reasonably stable modification, characterization of the phosphorylated proteins was attempted in kinase reactions with radiolabeled ATP. Two proteins of 60-62 kDa were the predominant substrates phosphorylated by both protein kinase C and protein kinase A under conditions wherein nuclear transport was restored. Our results suggest a requirement for phosphorylation of one or more proteins for binding of a karyophilic protein at the nuclear envelope.

Albumins↗

PubMed1994

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins.

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.

Amino Acid Sequence↗

PubMed1994

The coat protein of Indian peanut clump virus: relationships with other furoviruses and with barley stripe mosaic virus.

The 5'-most open reading frame of the c.4kb RNA-2 of Indian peanut clump furovirus (IPCV) encodes a protein of 208 amino acids. This protein is thought to be the coat protein of IPCV because its amino acid composition and M(r) closely resemble those reported for IPCV coat protein and because its amino acid sequence is 61% identical to that of the coat protein of peanut clump virus (PCV) from West Africa. The extent of the sequence identity between IPCV and PCV coat proteins confirms previous conclusions that the viruses are distinct rather than strains of one virus. The sequences of the coat proteins of IPCV and PCV were between 18% and 26% identical to those of other furoviruses and those of unrelated tobamoviruses and tobraviruses. In contrast, the coat protein sequences were 37% (IPCV) and 36% (PCV) identical to that of the coat protein of barley stripe mosaic hordeivirus (BSMV). This similarity between the coat proteins of viruses from different groups (= genera) is unusual but is consistent with previous reports of sequence relatedness in various genes between certain furoviruses and BSMV.

Amino Acid Sequence↗

PubMed1994

A conserved epitope on nuclear pore phosphoproteins reflects cell division status.

The nuclear pore complexes mediate the selective nuclear import of proteins in a signal- and energy-dependent process. We have earlier reported the characterization of a monoclonal antibody, Mab E2, that recognizes a novel class of nuclear pore phosphoproteins involved in signal-binding and protein transport. In the present study, we have analyzed the pattern of immunoreactivity of Mab E2 in cultured rat fibroblasts and have observed significant differences in the expression of epitopes in proliferating and quiescent cells. Furthermore, the common epitope recognized by Mab E2 is conserved across species, consistent with its essential role in nuclear protein import.

Amino Acid Sequence↗

PubMed1994

Effect of nuclear localization signal-receptor interaction on nuclear envelope-associated ATPase activity.

The possibility that interaction of the nuclear localization signal (NLS) with its pore receptor may directly stimulate nuclear envelope-associated ATPase activity and consequently provide energy for protein translocation across the pore has been studied. ATPase activity was assayed after cross-linking of the prototype NLS peptide with its pore receptor, or after preincubation of envelopes with NLS-albumin conjugates. Neither treatment enhanced enzyme activity. A more complex series of events may be required for energy-generation at the nuclear pore.

Adenosine Triphosphatases↗

PubMed1992

Incomplete splicing of simian virus 40 large T antigen transcripts in a transformed mouse cell line.

The SV40 large T antigen transcripts from an SV40-transformed mouse embryo fibroblast cell line (215) have been analyzed by Northern blots and by mapping and sequencing of the corresponding cDNAs. We have observed that T antigen mRNA is highly overexpressed in the 215 cells, and is mostly unspliced, but there are no sequence changes at the splice sites. However, normal amounts of wild-type T antigen are produced, suggesting that the splicing and translation of T antigen RNA is tightly controlled in these cells.

Animals↗