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At least 631 records · Page 35Linked to original sources
PubMed2005

Proteomic and metabolomic analyses of atherosclerotic vessels from apolipoprotein E-deficient mice reveal alterations in inflammation, oxidative stress, and energy metabolism.

OBJECTIVE: Proteomics and metabolomics are emerging technologies to study molecular mechanisms of diseases. We applied these techniques to identify protein and metabolite changes in vessels of apolipoprotein E(-/-) mice on normal chow diet. METHODS AND RESULTS: Using 2-dimensional gel electrophoresis and mass spectrometry, we identified 79 protein species that were altered during various stages of atherogenesis. Immunoglobulin deposition, redox imbalance, and impaired energy metabolism preceded lesion formation in apolipoprotein E(-/-) mice. Oxidative stress in the vasculature was reflected by the oxidation status of 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12-month-old apolipoprotein E(-/-) mice. Nuclear magnetic resonance spectroscopy revealed a decline in alanine and a depletion of the adenosine nucleotide pool in vessels of 10-week-old apolipoprotein E(-/-) mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, which provides reducing equivalents for lipid synthesis and glutathione recycling, and successful replenishment of the vascular energy pool. CONCLUSIONS: Our study provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so far and highlights potential associations of immune-inflammatory responses, oxidative stress, and energy metabolism.

Animals↗
PubMed1993

Dominance of the mutant ALDH2(2) allele in the expression of human stomach aldehyde dehydrogenase-2 activity.

About half of Chinese individuals lack mitochondrial aldehyde dehydrogenase-2 (ALDH2) activity, which is responsible for the oxidation of acetaldehyde produced during ethanol metabolism. The ALDH2 deficiency in Chinese has been implicated in alcohol flush reaction and reported to be a negative risk factor for development of alcohol dependence. To assess the effects of inactive ALDH2 subunits, encoded by the mutant ALDH2(2) allele, on the catalytic activity of tetrameric enzyme molecules, we have phenotyped ALDH2 from 30 gastroendoscopic biopsies by using agarose isoelectric focusing and determined the genotypes from leukocytes of the same individuals by using polymerase-chain-reaction amplification and hybridization with allele-specific oligonucleotide probes. Sixteen subjects were homozygous for the ALDH2(1) allele, one was homozygous for ALDH2(2), and thirteen were the heterozygous genotype. None of the subjects with the mutant homozygotic and the heterozygotic genotypes exhibited the ALDH2 activity band or intermediate bands between ALDH2 and ALDH1 on isoelectric focusing gels. Our results support the notion that the mutant allele is dominant and that the heterotetrameric ALDH2 molecules containing the mutant subunits are enzymatically inactive or far less active.

Aldehyde Dehydrogenase↗
PubMed2004

[Clinical analysis of 50 cases of cytomegalovirus disease].

OBJECTIVES: To investigate the clinical features, diagnostic methods, therapeutic approaches, outcomes and the alterations of peripheral lymphocyte subsets in cytomegalovirus (CMV) diseases. METHODS: From August 2000 to October 2002, 50 cases of CMV diseases were treated in Peking Union Medical College Hospital. The diagnosis were made by their symptoms and positive CMV pp65 antigen or/and CMV specific IgM. Of the 50 patients, 15 were male, and 35 female. Their age was (35.5 +/- 16.2) years. Their clinical data were collected, and their peripheral lymphocyte subsets were measured at the time when they were first diagnosed as CMV disease. The lymphocyte subsets of 51 healthy blood donors were tested as normal controls. The lymphocyte subsets were compared between those who had an immunocompromising underlying disease and those who had not. RESULTS: (1) CMV disease occurred in 39 patients who had immunocompromising underlying diseases, and in 11 patients who had not; (2) CMV disease caused various symptoms, most of them were nonspecific. Fever (100%), hematological abnormalities (96%) and abnormal liver function test (30%) were the most common manifestations. 3 cases of CMV retinitis were acquired immune deficiency syndrome patients with CD4+ T cell count less than 50/ microl; 2 patients developed multiple organ failure; (3) The positive rates of CMV pp65 antigen and specific IgM assays were 85.3% and 68.3%, respectively; (4) Comparing with normal controls, all patients with CMV disease had decreased B cell and nature killer cells, increased CD8+ T cell percentage and a reversed CD4+/CD8+ ratio; further more, the patients with underlying diseases had lower CD4+ T-cell and CD8+ T-cell counts than those without underlying diseases. CONCLUSIONS: CMV disease occurred in patients with or without underlying diseases. Their clinical manifestations were heterogeneous and non-specific, therefore, special laboratory tests were necessary to confirm the diagnosis. pp65 antigen assay had more diagnostic value than IgM assay. The dramatic changes in lymphocyte subsets suggest CMV disease has a immunological pathogenesis.

Adolescent↗
PubMed2003

TGF-beta 1 regulates lymphocyte homeostasis by preventing activation and subsequent apoptosis of peripheral lymphocytes.

TGF-beta1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-beta1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-beta1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1(-/-) mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-gamma and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1(-/-) T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1(-/-) T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4(+) T cells have elevated intracellular Ca(2+) levels. However, upon subsequent stimulation in vitro, increases in Ca(2+) levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1(-/-) splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca(2+) levels.

Animals↗
PubMed2004

Mutation spectrum in Taiwanese patients with phenylalanine hydroxylase deficiency and a founder effect for the R241C mutation.

The spectrum of phenylalanine hydroxylase (PAH) gene mutations was determined in 25 families of hyperphenylalaninemia identified by a neonatal screening program in Taiwan. The coding sequence and exon-flanking intron sequences of PAH gene were amplified and sequenced. Mutations were identified in forty-five of the 50 chromosomes. R241C was the most common mutation (36% of the chromosomes), followed by R408Q (14% of the chromosomes). The remaining mutations were rare and seven mutations have not been reported before: p.F233L (c.697T>C), p.R252Q (c.756G>A), p.E286K (c.856G>A), p.G312V (c.935G>T), p.P314T (c.940C>A), p.I95del (c.284_286delTCA), and p.T81fsX6 (c.241_256del). Both p.R241C and p.R408Q are classified as mild phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) mutation, which may explain the fact that classical PKU is very rare in Taiwan (n=4, or one in 413,035). This strong founder effect for the p.R241C mutation has been described neither in the Caucasian populations, nor in other reports from Chinese. Since most of the populations in Taiwan are derived from Southeastern China, the spectrum of PAH gene mutations in Southeastern China should be different from other Chinese populations. This report not only disclose a specific spectrum of PAH gene mutation in Taiwan, but may also give clues to the movement of populations in Mainland China.

Amino Acid Metabolism, Inborn Errors↗
PubMed2006

Swiss patients with variegate porphyria have unique mutations.

BACKGROUND: Variegate porphyria (VP), also known as South African porphyria, is a low-penetrance, autosomal dominant disorder as the result of a partial deficiency of protoporphyrinogen oxidase (PPOX). Clinically, VP is characterised by photosensitivity and neurovisceral attacks whereby the two symptoms can appear separately or together in patients. VP is little known in Switzerland. In this study, we report a clinical, biochemical and mutational study of eight Swiss VP patients and their families. RESULTS: Six of the eight index patients presented with only skin symptoms, and one with only neurological symptoms. Another patient had both skin and neurological symptoms. Faecal porphyrin excretion was elevated in all patients thus enabling diagnosis. Four different mutations including three novel mutations (G11D, 1041-1042 ins T and 1262-1263 ins 22bp) were identified in this cohort. Mutation 1082-1083 ins C, which had been reported in the French VP population, was shared by five apparently unrelated patients of this study. CONCLUSION: The novel PPOX gene mutations are apparently unique to the Swiss population. Both clinical and biochemical presentations varied considerably even among those patients who carried an identical mutation, which does not favour the existence of a genotype-phenotype correlation in VP.

Adolescent↗
PubMed2000

Regulation of apoptosis by phosphatidylinositol 4,5-bisphosphate inhibition of caspases, and caspase inactivation of phosphatidylinositol phosphate 5-kinases.

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3. PIP2 inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP2 levels in mixed lipid vesicles. Caspase 3 binding to PIP2 was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase alpha (PIP5KIalpha), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIalpha was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP. Protection was not mediated through PIP3 or Akt activation. An anti-apoptotic role for PIP(2) is further substantiated by our finding that PIP5KIalpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIalpha in vitro. Mutation of the P(4) position (D279A) of the PIP5KIalpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIalpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KIalpha inactivation contributes to the progression of apoptosis. This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.

Apoptosis↗
PubMed2003

Suppression of oxidative stress after transient focal ischemia in interleukin-1 knock out mice.

Interleukin-1 (IL-1) contributes to ischemic neurodegeneration. However, the mechanisms regulating action of IL-1 are still poorly understood. In order to clarify this central issue, mice that were gene deficient both IL-1alpha and beta (IL-1 KO) and wild-type mice were subjected to 1 hour transient middle cerebral artery occlusion (tMCAO). The concentration of 8-hydroxy deoxyguanosine (8OHdG) which is considered to be a reliable oxidative DNA damage by superoxide anion, in brain and of total nitric oxide (NO) in plasma were determined by use of HPLC. Twenty-four hours after tMCAO, the ratio of 8OHdG to dG in the ipsilateral hemisphere of wild-type mice were 2.24 x 10(-3) and 4.41 x 10(-3) in the neocortex and striatum, respectively. The concentration of 8OHdG in the ipsilateral hemisphere of the wild-type mice was higher than that of the IL-1 KO mice. The concentration of total NO in the plasma of IL-1 KO mice was also lower than that of the wild-type 24 hours after tMCAO. These results strongly suggest that IL-1 is participated in generating reactive oxygen spices and it aggravates and induces the ischemic neuronal cell death.(183 words).

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Molecular basis for type 1 antithrombin deficiency: identification of two novel point mutations and evidence for a de novo splice site mutation.

Inherited type 1 antithrombin (AT) deficiency is characterized by a reduction in both immunologically and functionally detectable protein. The disorder is associated with a high risk of thromboembolic disease. We have investigated the molecular basis of type 1 AT deficiency in three unrelated families. We have used the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and the intron-exon junctions of the AT gene. Two novel point mutations were identified. A T to C single-base substitution was found in codon 421 in exon 6 (nucleotide position 13380), leading to an AT 421 isoleucine to threonine substitution. In another kindred, one of three Cs at nucleotide (nt) positions 5448 to 5450 in exon 3A (codon 151 or 152) was deleted, resulting in a frameshift mutation and predicting premature termination of protein translation at codon 251. In a third family, a previously reported G to A substitution, at nt position 9788 in intron 4, 14 bp in front of exon 5, was found. We have demonstrated the creation of a de novo exon 5 splice site by ectopic transcript analysis of lymphocyte mRNA. In all cases, the affected individuals were heterozygous for the mutation and no variant AT protein was detected.

Adolescent↗
PubMed1996

Induction of apoptosis by c-Fos protein.

The role of c-Fos in apoptosis was examined in two Syrian hamster embryo cell lines (sup+I and sup-II) and a human colorectal carcinoma cell line (RKO), using the chimeric Fos-estrogen receptor fusion protein c-FosER. As previously reported, contrasting responses were observed when these two cell lines were placed under growth factor deprivation conditions; sup+I cells were highly susceptible to apoptosis, whereas sup-II cells were resistant. In this report, we show that the activated c-FosER protein induces apoptosis in sup-II preneoplastic cells in serum-free medium, indicating that c-Fos protein can induce apoptotic cell death in these cells. c-Fos-induced apoptosis was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that the c-Fos transcriptional activation activity is not involved. This conclusion was further supported by the observation that overexpression of v-Fos, which is highly proficient in transcriptional activation but deficient in the transcriptional repression activity associated with c-Fos, did not induce apoptosis. Constitutively expressed Bcl-2 delayed the onset of low-serum-induced apoptosis in sup+I cells and enhanced survival in sup-II cells. Further, coexpression of Bcl-2 and c-FosER in sup+I or sup-II cells protected the cells from c-FosER-induced apoptosis. The possibility that c-FosER-induced apoptosis requires a p53 function was examined. Colorectal carcinoma RKOp53+/+ cells, which do not normally undergo apoptosis in serum-free medium, showed apoptotic DNA fragmentation upon expression and activation of c-FosER. Further, when the wild-type p53 protein was diminished in the RKO cells by infection with the papillomavirus E6 gene, subsequent c-FosER-induced apoptosis was blocked. The data suggest that c-Fos protein plays a causal role in the activation of apoptosis in a p53-dependent manner. This activity does not require new protein synthesis and is blocked by overexpression of Bcl-2 protein.

Animals↗
PubMed1995

Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis.

The retinoblastoma susceptibility gene (Rb) participates in controlling the G1/S-phase transition, presumably by binding and inactivating E2F transcription activator family members. Mouse embryonic fibroblasts (MEFs) with no, one, or two inactivated Rb genes were used to determine the specific contributions of Rb protein to cell cycle progression and gene expression. MEFs lacking both Rb alleles (Rb-/-) entered S phase in the presence of the dihydrofolate reductase inhibitor methotrexate. Two E2F target genes, dihydrofolate reductase and thymidylate synthase, displayed elevated mRNA and protein levels in Rb- MEFs. Since absence of functional Rb protein in MEFs is sufficient for S-phase entry under growth-limiting conditions, these data indicate that the E2F complexes containing Rb protein, and not the Rb-related proteins p107 and p130, may be rate limiting for the G1/S transition. Antineoplastic drugs caused accumulation of p53 in the nuclei of both Rb+/+ and Rb-/- MEFs. While p53 induction led to apoptosis in Rb-/- MEFs, Rb+/- and Rb+/+ MEFs underwent cell cycle arrest without apoptosis. These results reveal that diverse growth signals work through Rb to regulate entry into S phase, and they indicate that absence of Rb protein produces a constitutive DNA replication signal capable of activating a p53-associated apoptotic response.

Animals↗
PubMed2006

Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g., G-to-T, G-to-A, -1 frameshifts, etc.) via its major adduct [+ta]-B[a]P-N2-dG. We recently showed that the dominant G-to-T mutation depends on DNA polymerase V (DNAP V), but not DNAPs IV or II, when studied in a 5'-TG sequence in E. coli. Herein we investigate what DNAPs are responsible for non-mutagenic bypass with [+ta]-B[a]P-N2-dG, along with its mirror image adduct [-ta]-B[a]P-N2-dG. Each adduct is built into a 5'-TG sequence in a single stranded M13 phage vector, which is then transformed into eight different E. coli strains containing all combinations of proficiency and deficiency in the three lesion-bypass DNAPs II, IV and V. Based on M13 progeny output, non-mutagenic bypass with [-ta]-B[a]P-N2-dG depends on DNAP IV. In contrast, non-mutagenic bypass with [+ta]-B[a]P-N2-dG depends on both DNAPs IV and V, where arguments suggest that DNAP IV is involved in dCTP insertion, while DNAP V is involved in extension of the adduct-G:C base pair. Numerous findings indicate that DNAP II has a slight inhibitory effect on the bypass of [+ta]- and [-ta]-B[a]P-N2-dG in the case of both DNAPs IV and V. In conclusion, for efficient non-mutagenic bypass (dCTP insertion) in E. coli, [+ta]-B[a]P-N2-dG requires DNAPs IV and V, [-ta]-B[a]P-N2-dG requires only DNAP IV, while DNAP II is inhibitory to both, and experiments to investigate these differences should provide insights into the mechanism and purpose of these lesion-bypass DNAPs.

Bacteriophage M13↗
PubMed2005

Tellimagrandin I enhances gap junctional communication and attenuates the tumor phenotype of human cervical carcinoma HeLa cells in vitro.

Tellimagrandin I and chebulinic acid, two hydrolysable tannins, have been shown to exert anti-tumor properties. Dysfunctional gap junctional communication (GJIC) has been recognized as being involved in carcinogenesis. The human cervical carcinoma HeLa cells have been reported to be deficient in functional GJIC. In present study, we investigated whether tellimagrandin I and chebulinic acid might restore functional GJIC in HeLa cells. Both compounds could inhibit the growth of HeLa cells. Either Lucifer yellow transfer assay or calcein transfer assay demonstrated that tellimagrandin I improved GJIC in HeLa cells while chebulinic acid showed no effect on GJIC. The GJIC enhancement by tellimagrandin I occurred along with an increase of Cx43 gene expression at mRNA and protein levels. Exposure to tellimagrandin I also led to inhibition of proliferation and anchorage-independent growth of HeLa cells. In addition, tellimagrandin I decreased the percentage of cells in the G0/G1 and G2/M phases coinciding with an increase in the percentage of cells in the S phase. The accumulation of cells in S phase was coupled with a decreased expression of cyclin A that was critical to the progression of S phase. These results suggested that restoring GJIC might be one explanation for tellimagrandin I antitumor effects, whereas chebulinic acid exerted antitumor action through other pathways.

Antineoplastic Agents↗
PubMed2006

[Screen and identification of Streptocococcus pneumoniae genes specifically induced in host].

To identify in vivo-induced genes of S. pneumoniae and search new potential drug targets and vaccine candidates, a selection system was developed based on the in vivo expression technology (IVET). Promoter galU gene which is critical for the capsular polysaccharide biosynthesis and lacZY gene which encodes bea-galactosidase were employed as dual reporter genes. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose and synthesizing capsular polysaccharide, therefore can't survival in the host. Firstly, the random pieces of S. pneumoniae chromosomal DNA (200-500 bp), obtained by partial Sau3Al restriction digestion, were subcloned into the Bgl II site of pEVP3-galU. Transformation by this plasmid library yielded promoter-trap library in S. pneumoniae. Then, the library was used to infect animals. Bacteria were harvested from lung tissue. White clones on TSA agar containing X-gal were used to reinfect animals. The sequence of infection and sorting was performed twice, 165 white clones harvested from the final round of infection were analyzed. A total of 15 unique sequences were obtained through in vivo screen. The bioinformatics analysis showed that these ivi genes involved in colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism and cell wall synthesis. And there were some hypothetical proteins have unknown functions. Part of these genes may be related with virulence and can be used as vaccine candidates and drug targets.

Animals↗
PubMed2004

Mutation hotspots in the human porphobilinogen deaminase gene: recurrent mutations G111R and R173Q occurring at CpG motifs.

Acute intermittent porphyria (AIP) is an inherited disorder in the haem biosynthetic pathway caused by a partial deficiency of porphobilinogen (PBG) deaminase. To date, more than 200 different mutations have been identified in the PBG deaminase gene (PBGD) in AIP patients from various countries and ethnic groups. While the majority of the PBGD gene mutations, including most of the mutations occurring at CpG dinucleotides, are family-specific, a few CpG mutations have been observed in a number of AIP patients of European origin. To study the origin of these common CpG mutations, eight intragenic single-nucleotide polymorphisms (SNPs) in the PBGD gene, as well as eight microsatellites flanking the gene in chromosome 11 were used to construct haplotypes in six AIP families of German, Polish and Swiss origins who carried either G111R (4707G>A) or R173Q (6391G>A) mutations. Among the three R173Q families, three distinct haplotypes were found to be cosegregated with the mutation. One Swiss and one German G111R family shared partially an intragenic and its extended microsatellite haplotype, whereas the Polish G111R family showed a unique haplotype. These results indicated that the recurrent CpG mutations that exist in the European AIP population can be either of ancestral origins or derived from de novo events.

Chromosome Mapping↗
PubMed2002

IL-10 regulates murine lupus.

MRL/MpJ-Tnfrsf6(lpr) (MRL/MpJ-Fas(lpr); MRL-Fas(lpr)) mice develop a spontaneous lupus syndrome closely resembling human systemic lupus erythematosus. To define the role of IL-10 in the regulation of murine lupus, IL-10 gene-deficient (IL-10(-/-)) MRL-Fas(lpr) (MRL-Fas(lpr) IL-10(-/-)) mice were generated and their disease phenotype was compared with littermates with one or two copies of an intact IL-10 locus (MRL-Fas(lpr) IL-10(+/-) and MRL-Fas(lpr) IL-10(+/+) mice, respectively). MRL-Fas(lpr) IL-10(-/-) mice developed severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomerulonephritis, and higher mortality than their IL-10-intact littermate controls. The increased severity of lupus in MRL-Fas(lpr) IL-10(-/-) mice was closely associated with enhanced IFN-gamma production by both CD4(+) and CD8(+) cells and increased serum concentration of IgG2a anti-dsDNA autoantibodies. The protective effect of IL-10 in this lupus model was further supported by the observation that administration of rIL-10 reduced IgG2a anti-dsDNA autoantibody production in wild-type MRL-Fas(lpr) animals. In summary, our results provide evidence that IL-10 can down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses. Modulation of the level of IL-10 may be of potential therapeutic benefit for human lupus.

Animals↗
PubMed2005

Influence of intron and exon splicing enhancers on mammalian cell expression of a truncated spike protein of SARS-CoV and its implication for subunit vaccine development.

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. A truncated S protein of the TW1 strain, STR2 (88 kDa), carrying three S fragments (S74-253, S294-739, and S1129-1255) was investigated to study the influences of intron and exon splicing enhancers to improve STR2 protein expression in mammalian cells. Our results showed that STR2 protein expression with the use of an 138 base-pair intron addition increased by 1.9-, 2.5-, and 4.1-fold in Vero E6, QBI-293A cells, and CHO/dhFr- cells (dihydrofolate reductase [dhfr] gene deficient CHO cells), respectively. Using the exon splicing enhancers, including a bidirectional splicing enhancer (BSE) or an exon splicing enhancer derived from the EDA alternative exon of the fibronectin gene (EDA ESE), were also found to increase STR2 protein expression in CHO/dhFr- cells by 1.7- and 2.6-fold. Nevertheless, combination of the intron and the exon splicing enhancers resulted in suppressing the intron-enhancing e STR2 protein expression in in CHO/dhFr- cells. Our studies also demonstrated the STR2 protein was mainly as the Endo H-sensitive glycoprotein (115 kDa) expressed in Vero E6, QBI-293A, and CHO/dhFr- cells. However, only a minor form of the Endo H-resistant glycoproteins ( approximately 130 kDa) was detected in CHO/dhFr- cells. Taken together, our results indicated that intron had a better enhancing effect on STR2 protein expression than exon splicing enhancers, and the expression of approximately 130 kDa STR2 glycoprotein was enhanced by the intron addition into the expression vector construct. Results of the present study can provide an optimal strategy to enhance SARS-CoV S protein expression in mammalian cells and may contribute to the development of SARS-CoV subunit vaccine.

Animals↗
PubMed2006

Sustained activation of Rac1 in hepatic stellate cells promotes liver injury and fibrosis in mice.

Rac, a small, GTP-binding protein in the Rho family, regulates several cellular functions, including the activation of NADPH oxidase, a major intracellular producer of reactive oxygen species (ROS). Hepatic stellate cells (HSCs) isolated from mice that are genetically deficient in NADPH oxidase produce less ROS, and their activation during chronic liver injury is abrogated, resulting in decreased liver fibrosis. Therefore, we hypothesized that HSC ROS production and activation would be enhanced, and fibrosis worsened, by increasing Rac expression in HSCs. To achieve this, we used transgenic mice that express constitutively active human Rac1 under the control of the alpha-smooth muscle actin (alpha-sma) promoter, because alpha-sma expression is induced spontaneously during HSC activation. Transgene expression was upregulated progressively during culture of primary Rac-transgenic HSCs, and this increased HSC ROS production as well as expression of activation markers and collagen. Similarly, Rac mice treated with carbon tetrachloride (CCl(4)) accumulated greater numbers of activated HSCs and had more liver damage, hepatocyte apoptosis, and liver fibrosis-as well as higher mortality-than CCl(4)-treated wild-type mice. In conclusion, sustained activation of Rac in HSCs perpetuates their activation and exacerbates toxin-induced liver injury and fibrosis, prompting speculation that Rac may be a therapeutic target in patients with cirrhosis.

Actins↗