Biomedical subjects

R Peng

Publications and source records attributed to R Peng.

At least 19 recordsLinked to original sources

PubMed2005

Dexamethasone induces IL-10-producing monocyte-derived dendritic cells with durable immaturity.

It is highly desirable that immature dendritic cells (DC) used for tolerance induction maintain steady immature state with predominant interleukin (IL)-10 production. In this study, we attempted to develop DC with durable immaturity and other tolerogenic features by using dexamethasone (Dex). We found DC derived from human monocytes in the presence of 10(-7) m Dex were negative for CD1a. Compared with control transduced DC (Ctrl-DC), Dex-DC expressed lower CD40, CD80 and CD86 but equivalent human leucocyte antigen-DR. Both immature Dex- and Ctrl-DC did not express CD83. Nevertheless, upon stimulation of lipopolysaccharide (LPS) or CD40 ligand, the expression of CD40, CD80, CD83 and CD86 was upregulated on Ctrl-DC but not on Dex-DC. The immaturity of Dex-DC was durable following Dex removal. Interestingly, Dex-DC maintained production of large amount of IL-10 and little IL-12 five days after Dex removed. Further study indicated that high-level IL-10 production by Dex-DC was associated with high-level phosphorylation of extracellular signal-regulated kinase (ERK) as blockade of this enzyme markedly attenuated IL-10 production. Furthermore, Dex-DC sustained the capability of high phosphorylation of ERK and IL-10 production 5 days after Dex removal. In addition, Dex-DC had significantly lower activity in stimulating T-cell proliferation. Neutralization of IL-10, to some extent, promoted DC maturation activated by LPS, as well as T-cell stimulatory activity of Dex-DC. The above findings suggest that IL-10-producing Dex-DC with durable immaturity are potentially useful for induction of immune tolerance.

Antigens, CD↗

PubMed2005

Long duration asymmetrical postural tremor is likely to predict development of Parkinson's disease and not essential tremor: clinical follow up study of 13 cases.

BACKGROUND: Patients presenting with asymmetrical postural tremor with or without mild rest tremor may be diagnosed as having essential tremor (ET), although there is considerable diagnostic uncertainty as to the long term outcome of these patients. OBJECTIVE: In this study, retrospective observations were made on 13 patients presenting originally with asymmetrical postural tremor, initially thought to have ET based on tremor characteristics, alcohol responsiveness, and family history but who subsequently met the criteria of Parkinson's disease (PD). METHODS: The patients were observed and followed up clinically with ancillary imaging using dopamine transporter SPECT scan or levodopa challenge tests in some cases. The diagnosis at original presentation with postural tremor was made with retrospective case note review. RESULTS: After a variable and long latent period all patients developed additional signs suggesting a clinical diagnosis of PD although picking up an initial label of ET. CONCLUSIONS: We suggest exercising caution regarding a diagnosis of ET in patients presenting with late onset asymmetrical postural tremor even if there is no rest tremor. Alcohol sensitivity of tremor, family history of tremor, or responsiveness to beta blockers may not be helpful in diagnosing ET in these cases and some may develop PD in the long term.

Aged↗

PubMed2003

A delta-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity.

The short field-life of Bacillus thuringiensis (Bt) insecticidal crystal protein has limited its use. When the Bt toxin is produced in Pseudomonas fluorescens it can be encapsulated and retain its effectiveness for two to three times longer than other Bt formulations. In order to improve Bt expression, we have synthesized cryIA(c) Bt delta-endotoxin encoding region (GenBank AF537267) according to the usage codon of P. fluorescens and transformed the Bt toxin expression cassette into P. fluorescens strains. T7 RNA polymerase and the T7 promoter system were used to control expression of Bt toxin. SDS-PAGE and Western blotting assay revealed that the delta-endotoxin was expressed as 8% of the total protein in P. fluorescens. In in vitro tests, release of toxin from dead bacteria was demonstrated. Supplementation of diets with Bt toxin-containing Pseudomonas bacterium resulted in high mortality of cabbage butterfly ( Pieris brassicae) larvae.

Animals↗

PubMed2001

Notch1IC partially replaces EBNA2 function in B cells immortalized by Epstein-Barr virus.

Immortalization of B cells by Epstein-Barr virus (EBV) depends on the virally encoded EBNA2 protein. Although not related by sequence, the cellular Notch protein and EBNA2 share several biochemical and functional properties, such as interaction with CBF1 and the ability to activate transcription of a number of cellular and viral genes. Whether these similarities are coincidental or exemplify EBNA2 mimicry of evolutionarily conserved cellular signaling pathways is unclear. We therefore investigated whether activated forms of Notch could substitute for EBNA2 in maintaining the immortalized phenotype of EBV-infected B cells. To address this question, we devised a transcomplementation system using EREB2.5 cells. EREB2.5 cells are immortalized by EBV expressing a conditional estrogen receptor EBNA2 fusion protein (EREBNA2), and cellular proliferation is dependent on the availability of estrogen. Withdrawal of estrogen results in inactivation of EREBNA2, leading to growth arrest and eventually to cell death. Transduction of EREB2.5 cells with a lentiviral vector expressing wild-type EBNA2 rescued EREB2.5 cells from the growth-inhibitory effects of estrogen deprivation, in contrast to transduction with the lentivirus vector alone. EREB2.5 cells were also rescued by enforced expression of human Notch1IC after estrogen starvation, but this effect was restricted to cells expressing high levels of the transcription factor. Compared to wild-type EBNA2-expressing EREB2.5 cells, the Notch-expressing cells expanded more slowly after estrogen starvation, and once established, they continued to display a lower proliferation rate. Analysis of viral and cellular gene expression from transduced EREB2.5 cells after estrogen withdrawal indicated that both wild-type EBNA2- and Notch1IC-positive cells expressed c-Myc at levels similar to those found in parental EREB2.5 cells. However, the latter cells expressed LMP-1 far less efficiently than cells transduced with the wild-type EBNA2 gene. Cells rescued by either wild-type EBNA2 or Notch1IC expressed surface CD21 and CD23 proteins, but not CD10, indicating that induction of relevant type III latency markers was maintained. The data imply that both Notch and EBNA2 activate an important subset of cellular genes associated with type III latency and B-cell growth, while EBNA2 more efficiently induces important viral genes, such as LMP-1. Thus, exploitation of conserved Notch-related signaling pathways may represent a key mechanism by which EBNA2 contributes to EBV-induced cell immortalization.

B-Lymphocytes↗

PubMed2001

GM-CSF and IL-2 induce specific cellular immunity and provide protection against Epstein-Barr virus lymphoproliferative disorder.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is a potentially life-threatening complication in immune-deficient patients. We have used the severe combined immune deficient (SCID) mouse engrafted with human leukocytes (hu-PBL-SCID) to evaluate the use of human cytokines in the prevention of EBV-LPD in vivo. Daily low-dose IL-2 therapy can prevent EBV-LPD in the hu-PBL-SCID mouse, but protection is lost if murine natural killer (NK) cells are depleted. Here we demonstrate that combined therapy with human GM-CSF and low-dose IL-2 is capable of preventing EBV-LPD in the hu-PBL-SCID mouse in the absence of murine NK cells. Lymphocyte depletion experiments showed that human NK cells, CD8(+) T cells, and monocytes were each required for the protective effects of GM-CSF and IL-2 combination therapy. This treatment resulted in a marked expansion of human CD3(+)CD8(+) lymphocytes in vivo. Using HLA tetramers complexed with EBV immunodominant peptides, a subset of these lymphocytes was found to be EBV-specific. These data establish that combined GM-CSF and low-dose IL-2 therapy can prevent the immune deficiencies that lead to fatal EBV-LPD in the hu-PBL-SCID mouse depleted of murine NK cells, and they point to a critical role for several human cellular subsets in mediating this protective effect.

Animals↗

PubMed2001

[Modulation of angelica sinensis polysaccharides on the expression of nitric oxide synthase and Bax, Bcl-2 in liver of immunological liver-injured mice].

OBJECTIVE: To investigate the modulation of Angelica Sinensis Polysaccharides on the expression of nitric oxide synthase and Bax, Bcl-2 in the liver of immunological liver injured mice. METHODS: Immunological liver injury was induced by lipopolysaccharide (ip, 2mug/10g) in bacillus calmette-guerin (ip, 1mg/10g, qd/1 d) primed mice. Angelica Sinensis Polysaccharides was administrated to the mice with 30mg/kg, 60mg/kg, respectively. The effects of Angelica Sinensis Polysaccharides on the expression of nitric oxide synthase, Bcl-2, and Bax gene were assessed by immunohistochemistry, and the nitric oxide (NO) production, alanine transaminase (ALT) activity, glutathione S-transferase (GST) activity in serum were determined. RESULTS: The levels of NO, sALT, and sGST were significantly higher in immunological liver-injured mice than controls. The contents of iNOS and Bax were 17.8 times (P<0.001) and 1.31 times (P<0.05) of control values, respectively. cNOS expression was not obviously changed, and no Bcl-2 expression was found in immunological liver-injured mice. Angelica Sinensis Polysaccharides of 30mg/kg could reduce the levels of NO, sALT, and sGST by 24.6%, 40.8%, and 18.4%, respectively; the expression of iNOS and Bax decrease by 84.2% and 37.1%; and the expression of cNOS, Bcl-2 increase by 66.8% and 3.38 times, respectively. The influence of Angelica Sinensis Polysaccharides of 60mg/kg was not so obvious as that of 30mg/kg on sGST, NO, cNOS, Bax, and Bcl-2, but was more effective on sALT and iNOS. CONCLUSIONS: NO production may play a role in the LPS-induced hepatotoxicity. Angelica Sinensis Polysaccharides can alleviate the immune liver injury by modulating the expression of cNOS, iNOS, Bax, Bcl-2.

Angelica sinensis↗

PubMed2001

[A pathologic study on the bronchioles and lung tissues in guinea pig asthma models].

OBJECTIVE: To probe further the pathologic changes in peripheral airways and lung tissues and to observe the changes of surfactant in asthma. METHODS: (1) The guinea pig asthma models were established with ovalbumin challenge method. (2) 6 groups were studied: control group, asthma group of day 1, asthma group of day 4, asthma group of day 14, intraperitoneal dexamethasone group, and budesonide inhalation group. (3) The pathologic changes were observed under optical and electron microscope. (4) The total and differential white blood cells counts of bronchoalveolar lavage fluid(BALF) were carried out. (5) The total amounts of phospholipids which represent the levels of pulmonary surfactant were measured with phosphorus detection method. RESULTS: (1) Significant hyperplasia of mucous membrane, basement membrane, and smooth muscle layer of central and peripheral airways in the asthma groups were seen. The alveolar walls were significantly thickened and alveolar spaces diminished (P < 0.001, compared with the control group). Significant infiltration of eosinophils and lymphocytes were observed in airways and lung tissues. (2) The total cell number and the number eosinophils and lymphocytes in BALF were significantly increased in the asthma groups than those in the control group(P < 0.01). Neutrophils were dominant at early stage, but eosinophils and lymphocytes at late stage. (3) The ultrastructural changes of type II alveolar cells in asthma group included cell swelling, evacuation of lamellar bodies and partial cell desquamation. (4) The total amount of phospholipids in BALF was significantly decreased in the asthma groups as compared with that in the control group (P < 0.01). The above changes were significantly improved in both corticosteroid treated groups. CONCLUSION: Widespread and significant inflammation with eosinophilic infiltration exists in the bronchioles and alveolar walls in guinea pig asthma models. Not only there is structural change, but also the function of alveolar cells is influenced. Asthma is a disease involving the whole airway and lung system.

Animals↗

PubMed2001

[Serum glutathione S-transferase in bromobenzene-induced acute hepato-toxicity in mice].

The activity of serum glutathione S-transferase (GST) was tested in bromobenzene-induced acute hepatotoxicity in mice. The data showed that a lot of small local necrosis in liver biopsy was observed and serum GST activity was increased after 2 h of treatment with bromobenzene (150 mg/kg i.p.) in mice. Serum alanine transaminase (ALT) activity was also gradually enhanced after 8 hours of treatment. The most obvious changes were at 16 hours of bromobenzene treatment, and serum GST activity was firstly restored to normal level after 24 hours of treatment. There were good positive correlations between serum GST, ALT, as well as serum GST and the changes on liver biopsy. Evaluating analysis of the diagnosis test showed that the sensitivity, specificity and accuracy was 89%, 75% and 82% in serum GST test, and 39%, 92% and 64% in serum ALT test respectively. The ROC work curve also demonstrated that serum GST was more sensitive and specific than serum ALT.

Alanine Transaminase↗

PubMed2001

[Effects of estradiol and isoflavoid on the expression of adhesion molecules on neutrophils].

OBJECTIVE: To elucidate the effects of estradiol and isoflavoid on the expression of adhesion molecules on neutrophils. METHODS: Neutrophils of healthy subjects and ischemic stroke patients were treated with tumor necrosis factor alpha (TNF alpha), in the presence or absence of different concentrations of isoflavoid (WZ1, WZ2) and estradiol (WZ3, WZ4). Flow cytometry was used to detect the expression of CD18 and CD62L on neutrophil surface. RESULTS: 1. 10 ng/ml TNF alpha could activate neutrophils of healthy subjects; it increased the expression of CD18 by 10% on neutrophil surface and shed CD62L from the surface as shown by a 15% decrease of the fluorescence intensity and a 30% decrease of the percentage of positive cell. 2. Isoflavoid (WZ1, WZ2) had no significant effect on the expression of CD18 and CD62L on neutrophils. 3. Pretreatment of neutrophils with estradiol (WZ3, WZ4) could inhibit the activation of neutrophils by TNF alpha, which decreased the fluorescence intensity of CD18 by 8%, increased the fluorescence intensity of CD62L by 15% and increased the percentage of CD62L positive cell by 20%. 4. TNF alpha could activate the neutrophils of ischemic stroke patients strikingly; it increased the fluorescence intensity of CD18 by 20% and decreased the fluorescence intensity and percentage of positive cell of CD62L by 30%, and there was a significant difference when the patients were compared with the healthy subjects. Estradiol had the same effect on the expression of CD18 and CD62L as on those of healthy subjects. CONCLUSION: 1. TNF alpha is a strong activator of neutrophils; it plays an important role in the occurrence and development of ischemic stroke. 2. Isoflavoid has no obvious effect on the expression of adhesion molecules on neutrophils, so its role in protecting the cardiovascular system may come into play not by the way of affecting adhesion molecules expression. 3. Pretreatment of neutrophils with estradiol could protect them from activation by TNF alpha, thus decreasing the expression of adhesion molecules, the adhesion of neutrophils -endothelial cells, and hence the risk of ischemic stroke. However, in case that the neutrophils have been activated by TNF alpha, estradiol has no effect on the expression of adhesion molecules; this implies that estradiol possibly has no valid anti-adhesion therapeutic effect on ischemic stroke.

Adult↗

PubMed2001

[Direct determination of Pb, Zn and Cd in environmental and biological samples by FAAS].

This paper describes interference and background correction problem of direct determination of Pb, Zn and Cd in soil, sediment, plants, halobios and sea water by FAAS. With the improvement of limit of detection(L.O.D) of instrument, Pb, Zn and Cd in same environmental and biological samples can be analyzed directly by FAAS, but it is worth taking attention that there are quantities of elements which lead to background interference in air-ethyne flame, such as Al, Na, K Ca, Mg and Fe. Compare with no background correction, limit of detection using D2 lamp background correction is improved by 1.05-4.5 times, the recoveries and the relative standard deviation(R.S.D) are better, standard samples were also analyzed with satisfactory results.

Animals↗

PubMed2000

Evidence for overlapping and distinct functions in protein transport of coat protein Sec24p family members.

Budding of transport vesicles from the endoplasmic reticulum in yeast requires the formation, at the budding site, of a coat protein complex (COPII) that consists of two heterodimeric subcomplexes (Sec23p/Sec24p and Sec13p/Sec31p) and the Sar1 GTPase. Sec24p is an essential protein and involved in cargo selection. In addition to Sec24p, the yeast Saccharomyces cerevisiae expresses two non-essential Sec24p-related proteins, termed Sfb2p (product of YNL049c) and Sfb3p/Lst1p (product of YHR098c). We here show that Sfb2p and, less efficiently, Sfb3p/Lst1p are able to bind, like Sec24p, the integral membrane cargo protein Sed5p. We also demonstrate that Sfb2p, like Sec24p and Sfb3p/Lst1p, forms a complex with Sec23p in vivo. Whereas the deletion of SFB2 did not affect transport kinetics of various proteins, the maturation of the glycolipid-anchored plasma membrane protein Gas1p was differentially impaired in sfb3 knock-out cells. We generated several conditional-lethal sec24 mutants that, combined with null alleles of SFB2 and SFB3/LST1, led to a complete block of transport between the endoplasmic reticulum and the Golgi (sec24-11/Deltasfb2) or to cell death (sec24-11/Deltasfb3). Of the Sec24p family members, Sfb2p is the least abundant at steady state, but high intracellular concentrations of Sfb2p can rescue sec24 mutants under restrictive conditions. The data presented strongly suggest that the Sec24p-related proteins function as COPII components.

Amino Acid Sequence↗

PubMed2000

Presence of human beta- and gamma-herpes virus DNA in Hodgkin's disease.

Herpes viruses have been implicated in the etiology of Hodgkin's disease (HD). We studied the prevalence of human cytomegalovirus (CMV), human herpes viruses type-6 (HHV-6), type-7 (HHV-7) and type 8 (HHV-8) DNA in up to 88 Hodgkin's disease biopsies in comparison to Epstein-Barr virus (EBV) DNA by polymerase chain reaction (PCR). Non-Hodgkin lymphomas (NHL) and reactive lesions served as controls. CMV and HHV-6 were found in 8/86 (9%) and 11/88 (13%) HD cases, respectively, by nested primer PCR. Except for three cases harbouring HHV-6 type-B, only HHV-6 type-A was detected in HD. HHV-7 was observed by nested PCR in 33/88 (38%) HD cases and was already detectable in 15/88 (17%) HD cases by a single-round PCR indicating elevated virus copy numbers. Seven of these cases showed co-infection with HHV-6, and 11 cases were found to contain EBV DNA. 7/8 CMV-positive HD cases also harboured EBV DNA. HHV-8 DNA was not detected by single round or nested PCR in any HD case investigated. Thus, CMV, HHV-6, and HHV-7 were present in small proportions of HD cases, with frequent co-infection of HHV-6 and HHV-7, and frequent association with EBV. In contrast to EBV, beta-herpes viruses are therefore unlikely to have a role in the aetiology of HD. Rather, the presence of these viruses seems to reflect impaired immunological surveillance.

Betaherpesvirinae↗

PubMed2000

Rating the quality of the family environment in different cultures.

OBJECTIVE: To examine the extent to which clinicians from different cultures agree when rating the quality of a child's family environment and the effect of country, language, and training factors on these ratings. METHOD: Eighty-seven health professionals from Malaysia, Spain, Australia, Indonesia, the United States, Denmark, and Singapore rated 7 case vignettes using the Global Family Environment Scale. Half (54%) were psychiatrists. One quarter (26%) performed the ratings after attending a training session, the rest (n = 64, 74%) after reading a training manual. RESULTS: Overall, interrater agreement (intraclass r) was 0.84 (95% confidence interval: 0.68-0.96). There were no significant differences in agreement according to country, language, training, or professional group, although there were country differences in the ratings given to 2 vignettes. The majority of raters found the description of the anchor points (86%), training manual (95%), and case vignettes (97%) clear. CONCLUSIONS: Clinicians from different cultures seem to be able to make global ratings of the quality of the family environment that are reliable and consistent when using case vignettes. This can be achieved with little training. Global ratings of the quality of the family environment may be a useful tool in mental health research and clinical work.

Adult↗

PubMed2000

Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses.

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.

Amino Acid Sequence↗

PubMed2000

Regulation of the Epstein-Barr virus C promoter by AUF1 and the cyclic AMP/protein kinase A signaling pathway.

EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the expression of viral and cellular genes required for EBV-driven B-cell immortalization. Elucidating the mechanisms by which EBNA2 regulates viral and cellular gene expression is necessary to understand EBV-induced B-cell immortalization and viral latency in humans. EBNA2 targets to the latency C promoter (Cp) through an interaction with the cellular DNA binding protein CBF1 (RBPJk). The EBNA2 enhancer in Cp also binds another cellular factor, C promoter binding factor 2 (CBF2), whose protein product(s) has not yet been identified. Within the EBNA2 enhancer in Cp, we have previously identified the DNA sequence required for CBF2 binding and also determined that this element is required for efficient activation of Cp by EBNA2. In this study, the CBF2 activity was biochemically purified and microsequenced. The peptides sequenced were identical to the hnRNP protein AUF1. Antibodies against AUF1 but not antibodies to related hnRNP proteins reacted with CBF2 in gel mobility shift assays. In addition, stimulation of the cellular cyclic AMP (cAMP)/protein kinase A (PKA) signal transduction pathway results in an increase in detectable CBF2/AUF1 binding activity extracted from stimulated cells. Furthermore, the CBF2 binding site was able to confer EBNA2 responsiveness to a heterologous promoter when transfected cells were treated with compounds that activate PKA or by cotransfection of plasmids expressing a constitutively active catalytic subunit of PKA. EBNA2-mediated stimulation of the latency Cp is also increased in similar cotransfection assays. These results further support an important role for CBF2 in mediating EBNA2 transactivation; they identify the hnRNP protein AUF1 as a major component of CBF2 and are also the first evidence of a cis-acting sequence other than a CBF1 binding element that is able to confer responsiveness to EBNA2.

Amino Acid Sequence↗

PubMed2000

Conserved regions in the Epstein-Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2.

Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus DeltaIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.

Amino Acid Sequence↗

PubMed2000

The C-514T polymorphism in the human hepatic lipase gene promoter diminishes its activity.

Four common polymorphisms in the promoter of the hepatic lipase gene (LIPC) that are in almost complete linkage disequilibrium have been described in different ethnic groups. The T and A alleles at positions -514 and -250, respectively, were observed to be associated with decreased hepatic lipase (HL) activity and increased triglyceride content of HDL and LDL particles. We investigated whether these polymorphisms have any effect on transcriptional activity of the proximal promoter (-639 to +29) in transient transfection assays. We found that the promoter with T at position -514 had approximately 30% lower activity than the one with C at the same position (P < 0.0005) regardless of the genotype at position -250. In conclusion, these data indicate that the -514T allele may contribute significantly to decreased HL activity and the resultant increase in plasma levels of triglyceride-rich HDL(2) and large buoyant LDL particles. In addition, this promoter variant may explain the finding that its presence attenuates the increase in HL activity with increasing intra-abdominal fat in women.

Base Sequence↗

PubMed1999

Mutation analysis of BRCA1, TP53, and KRAS2 in ovarian and related pelvic tumors.

Cancer may be viewed as a genetic disease resulting from critical mutations that disrupt normal cell growth. To characterize the involvement of the BRCA1 and TP53 tumor suppressor genes and of the KRAS2 protooncogene in gynecologic cancer, mutation analysis of these genes was conducted in pelvic tumors of 85 patients that included 49 epithelial ovarian carcinoma cases. The 85 pelvic tumors contained 5 tumors with BRCA1 mutations, 33 with TP53 mutations, and 1 with a KRAS2 mutation. Each of the BRCA1 and KRAS2 mutations, and 25 of the TP53 mutations, were in ovarian carcinomas. Four of the BRCA1 mutations were germline and 1 was somatic. The 4 patients with germline BRCA1 mutations had an early age of disease onset (33-48 years) relative to the mean age of onset (58 years) of all 49 ovarian carcinoma patients, and 3 of these 4 patients had a family history of ovarian or breast cancer. None of the 4 tumors with germline BRCA1 mutations had a KRAS2 mutation or a TP53 mutation, despite a 51% frequency of TP53 mutations in the 49 ovarian carcinomas. Three of the 4 tumors with germline BRCA1 mutations retained a wild-type BRCA1 allele. The tumor with the somatic BRCA1 mutation contained a TP53 mutation and had no evidence for wild-type BRCA1 and TP53 alleles. These data suggest that both BRCA1 and TP53 were inactivated in 1 of 49 ovarian carcinomas. Moreover, mutational inactivation of both BRCA1 and TP53 did not occur in 4 tumors with a germline BRCA1 mutation. It has been proposed that tumorigenesis in cells with a heterozygous BRCA1 mutation requires inactivation of the wild-type BRCA1 and TP53 alleles, which results in genomic instability and acquisition of mutations in protooncogenes. Clearly, mutational inactivation of TP53 and the wild-type BRCA1 allele in ovarian tumors with a heterozygous, germline BRCA1 mutation is not an absolute requirement for tumor formation. It is possible that these alleles may be inactivated by nonmutational mechanisms or that other tumor formation pathways exist.

Adult↗