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L Rao

Publications and source records attributed to L Rao.

At least 37 records · Page 2Linked to original sources
PubMed1997

Bcl-2 and the ICE family of apoptotic regulators: making a connection.

Apoptosis, a genetically programmed mechanism of eliminating cells in response to a variety of stimuli, provides protection against cancer and viral infections as well as maintenance of homeostasis in living organisms. Two classes of molecules, the Bcl-2 family of regulators and the ICE family of proteases, have emerged from different vertebrate, invertebrate and viral systems that have been used to elucidate the pathways leading to apoptosis. However, no connection between these two disparate families of apoptotic regulators has been convincingly established. In reviewing the recent advances pertaining to the Bcl-2 and ICE-related protein families, one can address the question of a functional relationship between the two classes of proteins.

Animals↗
PubMed1997

Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NF-kappaB family members.

The expression of keratins K5 and K14 is restricted to the basal layers of the healthy epidermis, whereas the expression of K6 and K17 is induced in response to proliferative and inflammatory signals, respectively. The control of keratin expression occurs primarily at the transcriptional level. We studied the effects of transcription factors of the AP-1 and NF-kappaB families on the expression of those four keratin genes. We chose AP-1 and NF-kappaB proteins because they are activated by many extracellular signals, including those in hyperproliferative and inflammatory processes. DNA constructs expressing the transcription factors were, in various combinations, cotransfected with constructs containing keratin gene promoters and the CAT reporter gene into HeLa cells or keratinocytes. We found that the K5 and K14 promoters, which are coexpressed in vivo, are regulated in parallel by the cotransfected genes. Both were activated by the c-Fos and c-Jun components of AP-1, but not by Fra1. On the other hand, the NF-kappaB proteins, especially p65, suppressed these two promoters. The K17 promoter was specifically activated by c-Jun, whereas the other transcription factors tested had no significant effect. In contrast, the K6 promoter was very strongly activated by all AP-1 proteins, especially by the c-Fos + c-Jun and Fra1 + c-Jun combinations. It was also strongly activated by the p65 NF-kappaB protein. AP-1 and NF-kappaB acted synergistically in activating the K6 promoter, although the AP-1 and the NF-kappaB responsive sites could be separated physically. These results suggest that the interplay of AP-1 and NF-kappaB proteins regulates epidermal gene expression and that the activation of these transcription factors by extracellular signaling molecules brings about the differential expression of keratin genes in epidermal differentiation, cutaneous diseases, and wound healing.

Base Sequence↗
PubMed1996

Epitope mapping using histidine-tagged protein fragments: application to Escherichia coli RNA polymerase sigma 70.

We present a simple, rapid, and inexpensive method for mapping epitopes of monoclonal antibodies using His-tagged protein fragments. In essence, four steps are involved: (i) purify overproduced His-tagged protein from inclusion body; (ii) fragment the protein by partial chemical or enzymatic hydrolysis and isolate the His-tagged peptides with a Ni(2+)-chelate column to generate a "ladder" containing a distribution of peptide fragment sizes; (iii) fractionate the isolated peptides by SDS-polyacrylamide gel electrophoresis and probe the ladder by immunoblot analysis; (iv) determine the size of the fragments that do and do not bind the monoclonal antibody using size markers specific to the His-tagged protein being studied. We have applied this method successfully to map the epitope positions of known and new monoclonal antibodies to Escherichia coli sigma 70. We believe this method will find broad application.

Antibodies, Monoclonal↗
PubMed1996

The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein.

The E1B 19K protein is a potent apoptosis inhibitor and the putative adenovirus Bcl-2 homolog. To investigate the mechanism of apoptosis regulation, 19K-interacting cellular proteins were identified using the yeast two-hybrid system, and Bax was one of seven 19-K interacting clones. Residues 50-78 of Bax containing a conserved region designated Bcl-2 homology region 3 (BH3) were sufficient for specific binding to both the E1B 19K and Bcl-2 proteins. The Bax-E1B 19K interaction was detectable in vitro and in lysates from mammalian cells, and Bax expression antagonized E1B 19K protein function. bax mRNA and protein levels were p53-inducible with kinetics identical to that of p21/Waf-1/Cip-1, and E1B 19K and Bcl-2 expression did not affect Bax or p21/Waf-1/Cip-1 accumulation. In cells where p53 was mutant, Bax expression induced apoptosis, suggesting that Bax was sufficient for apoptosis, and acted downstream of p53. p53 may simultaneously activate the transcription of genes required for both growth arrest (p21/Waf-1/Cip-1) and death (bax), and E1B 19K and Bcl-2 may act distally and function through interaction with and antagonism of Bax to prevent apoptosis. With the death pathway disabled, induction of growth arrest by p53 can then be manifested.

Adenovirus E1B Proteins↗
PubMed1996

Report of successful use of argatroban as an alternative anticoagulant during coronary stent implantation in a patient with heparin-induced thrombocytopenia and thrombosis syndrome.

Heparin-induced thrombocytopenia and thrombosis syndrome (HITTS) is a severe complication of heparin caused by an antibody response to the heparin-platelet factor 4 complex which results in severe thrombosis. Heparin rechallenge in HITTS patients carries a high risk of inducing thrombosis. Antithrombin agents represent treatment alternatives in HITTS patients who require anticoagulation. We report successful coronary stent implantation in a HITTS patient using the antithrombin agent argatroban.

Aged↗
PubMed1996

Lamin proteolysis facilitates nuclear events during apoptosis.

Expression of the adenovirus E1A oncogene stimulates both cell proliferation and p53-dependent apoptosis in rodent cells. p53 implements apoptosis in all or in part through transcriptional activation of bax, the product of which promotes cell death. The adenovirus E1B 19K product is homologous in sequence and in function to Bcl-2, both of which bind to and inhibit the activity of Bax and thereby suppress apoptosis. The E1B 19K protein also interacts with the nuclear lamins, but the role of this interaction in the regulation of apoptosis is not known. Lamins are, however, substrates for members of the interleukin-1 beta-converting enzyme (ICE) family of cysteine proteases that are activated during apoptosis and function downstream of Bcl-2 in the cell death pathway. lamins are degraded during E1A-induced p53-dependent apoptosis. Lamin A and C are cleaved into 47- and 37-kD fragments, respectively, and the site of proteolysis is mapped to a conserved aspartic acid residue at position 230. The cleavage of lamins during apoptosis is consistent with the activation of an ICE-related cysteine protease down-stream of p53. No lamin protease activity was detected in cells expressing the E1B 19K protein, indicating that 19K functions upstream of protease activation in inhibiting apoptosis. Substitution of the aspartic acid at the cleavage site produced a mutant lamin protein that was resistant to proteolysis both in vitro and in vivo. Expression of uncleavable mutant lamin A or B attenuated apoptosis, delaying cell death and the associated DNA fragmentation by 12 h. Mutant lamin expressing cells failed to show the signs of chromatin condensation and nuclear shrinkage typical of cell death by apoptosis. Instead, the nuclear envelope collapsed and the nuclear lamina remained intact. However, the late stage of apoptosis was morphologically unaltered and formation of apoptotic bodies was evident. Thus, lamin breakdown by proteolytic degradation facilitates the nuclear events of apoptosis perhaps by facilitating nuclear breakdown.

Adenovirus E1B Proteins↗
PubMed1995

Algorithms for reengineering 1991 census geography.

"The availability of GIS [geographic information systems] technology and digital boundaries of census output areas now makes it possible for users to design their own census geography. Three algorithms are described that can be used for this purpose. An Arc/Info implementation is briefly outlined and case studies presented to demonstrate some of the results of explicitly designing zoning systems for use with 1991 [U.K.] census data."

Censuses↗
PubMed1995

In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus.

The RNA polymerase (RNAP) holoenzyme of Staphylococcus aureus was purified by DNA affinity, gel filtration, and ion-exchange chromatography. This RNAP contained four major subunits with apparent molecular masses of 165, 130, 60, and 47 kDa. All four subunits of the RNAP were serologically related to the subunits of Escherichia coli E sigma 70 holoenzyme by Western immunoblot analysis. The 60-kDa subunit was subsequently isolated and found to react with a monoclonal antibody specific to the E. coli sigma 70 subunit. This sigma 70-related protein allowed E. coli core RNAP promoter-specific initiation and increased transcription by S. aureus RNAP that is unsaturated with sigma. We therefore suggest that this 60-kDa protein is a sigma factor. Purified S. aureus RNAP transcribed from the promoters of several important S. aureus virulence genes (sea, sec, hla, and agr P2) in vitro. The in vitro transcription start sites of the sea, sec, and agr P2 promoters, mapped by primer extension, were similar to those identified in vivo. The putative promoter hexamers of these three genes showed strong sequence similarity to the E. coli sigma 70 consensus promoter, and transcription by E sigma 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli sigma 70-dependent promoters. Taken together, our results indicate that the promoter sequences recognized by purified S. aureus RNAP are similar but not identical to those recognized by E. coli E sigma 70.

Bacterial Proteins↗
PubMed1995

Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein.

BRK cell lines that stably express adenovirus E1A and a murine temperature-sensitive p53 undergo apoptosis when p53 assumes the wild-type conformation. Expression of the E1B 19,000-molecular-weight (19K) protein rescues cells from this p53-mediated apoptosis and diverts cells to a growth-arrested state. As p53 likely functions as a tumor suppressor by regulating transcription, the ability of the E1B 19K protein to regulate p53-mediated transactivation and transcriptional repression was investigated. In promoter-reporter assays the E1B 19K did not block p53-mediated transactivation but did alleviate p53-mediated transcriptional repression. E1B 19K expression permitted efficient transcriptional activation of the p21/WAF-1/cip-1 mRNA by p53, consistent with maintenance of the growth arrest function of p53. The E1B 19K protein is thereby unique among DNA virus-transforming proteins that target p53 for inactivation in that it selectively modulates the transcriptional properties of p53. The E1B 19K protein also rescued cells from apoptosis induced by inhibitors of transcription and protein synthesis. This suggests that cell death may result from the inhibition of expression of survival factors which function to maintain cell viability. p53 may induce apoptosis through generalized transcriptional repression. In turn, the E1B 19K protein may prevent p53-mediated apoptosis by alleviating p53-mediated transcriptional repression.

Adenovirus E1B Proteins↗
PubMed1994

Localization of the intrinsically bent DNA region upstream of the E.coli rrnB P1 promoter.

DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.

Base Sequence↗
PubMed1994

Factor independent activation of rrnB P1. An "extended" promoter with an upstream element that dramatically increases promoter strength.

The extraordinary strength of the Escherichia coli rRNA promoter rrnB P1 derives primarily from sequences upstream of the core (-10, -35) region. We find that sequences between -40 and -60 increase the activity of this promoter at least 30-fold in vitro and in vivo. This region, which we refer to as the upstream (UP) element, is located between the -35 consensus hexamer and the previously characterized binding sites for the rRNA transcription factor Fis. The effect of the UP element is independent of Fis in vivo, and independent of any other proteins besides RNA polymerase (RNAP) in vitro. The UP element increases the overall second-order rate constant for association of RNAP with the promoter (ka) and probably the apparent overall first-order isomerization constant (ki). Together with the previously reported protection of the UP element region by RNAP in footprinting experiments, these results indicate that rrnB P1 has an "extended" promoter structure, consisting of the UP element and the core promoter region. We find that the UP element is a separable promoter module that can function to increase the activity of the lac core promoter in an rrnB P1-lac hybrid promoter construct. A functional UP element is not absolutely essential for stimulation of rrnB P1 by the Fis protein.

Base Sequence↗
PubMed1994

Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.

Expression of the adenovirus E1A oncogene induces apoptosis which impedes both the transformation of primary rodent cells and productive adenovirus infection of human cells. Coexpression of E1A with the E1B 19,000-molecular-weight protein (19K protein) or the Bcl-2 protein, both of which have antiapoptotic activity, is necessary for efficient transformation. Induction of apoptosis by E1A in rodent cells is mediated by the p53 tumor suppressor gene, and both the E1B 19K protein and the Bcl-2 protein can overcome this p53-dependent apoptosis. The functional similarity between Bcl-2 and the E1B 19K protein suggested that they may act by similar mechanisms and that Bcl-2 may complement the requirement for E1B 19K expression during productive infection. Infection of human HeLa cells with E1B 19K loss-of-function mutant adenovirus produces apoptosis characterized by enhanced cytopathic effects (cyt phenotype) and degradation of host cell chromosomal DNA and viral DNA (deg phenotype). Failure to inhibit apoptosis results in premature host cell death, which impairs virus yield. HeLa cells express extremely low levels of p53 because of expression of human papillomavirus E6 protein. Levels of p53 were substantially increased by E1A expression during adenovirus infection. Therefore, E1A may induce apoptosis by overriding the E6-induced degradation of p53 and promoting p53 accumulation. Stable Bcl-2 overexpression in HeLa cells infected with the E1B 19K- mutant adenovirus blocked the induction of the cyt and deg phenotypes. Expression of Bcl-2 in HeLa cells also conferred resistance to apoptosis mediated by tumor necrosis factor alpha and Fas antigen, which is also an established function of the E1B 19K protein. A comparison of the amino acid sequences of Bcl-2 family members and that of the E1B 19K protein indicated that there was limited amino acid sequence homology between the central conserved domains of E1B 19K and Bcl-2. This domain of the E1B 19K protein is important in transformation and regulation of apoptosis, as determined by mutational analysis. The limited sequence homology and functional equivalency provided further evidence that the Bcl-2 and E1B 19K proteins may possess related mechanisms of action and that the E1B 19K protein may be the adenovirus equivalent of the cellular Bcl-2 protein.

Adenovirus E1A Proteins↗
PubMed1994

Bcl-2 blocks p53-dependent apoptosis.

Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity.

Animals↗
PubMed1993

Enhancing factor protein from mouse small intestines belongs to the phospholipase A2 family.

Enhancing factor (EF), a growth regulatory molecule, isolated from mouse small intestines, has been well characterized in this laboratory. It increases the binding of epidermal growth factor in a unique manner via its own receptor. In the first 20 N-terminal amino acids sequenced, EF showed 50% homology to human Group II phospholipase A2 (PLA2). Here we propose that EF is yet another, unidentified isoform of PLA2 which regulates cell proliferation via modulation of EGF binding. To our knowledge, this is the first report implicating PLA2-II-like molecules in growth regulation.

Amino Acid Sequence↗
PubMed1992

The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.

Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis.

Adenoviridae↗
PubMed1992

Carcinoma of the rectal pouch following restorative proctocolectomy. Report of a case.

A case of adenocarcinoma developing in the pouch following restorative proctocolectomy is presented. This seems to be the third reported in the literature. The carcinoma developed from the remnants of precancerous rectal mucosa left in the muscular rectal cuff. The patient had been suffering from ulcerative colitis for 17 years prior to the development of the malignancy. He presented with features of subacute intestinal obstruction. Diagnosis was by sigmoidoscopic examination of the pouch and biopsy. He was treated with abdominoperineal resection of the pouch and rectum, followed by chemotherapy.

Adenocarcinoma↗