Biomedical subjects

L Tian

Publications and source records attributed to L Tian.

At least 19 recordsLinked to original sources

PubMed2001

Alternative splicing determines sensitivity of murine calcium-activated potassium channels to glucocorticoids.

1. Large-conductance Ca(2+)- and voltage-activated potassium (BK) channels are important regulators of cellular excitability. Here, we present a patch-clamp electrophysiological analysis of splice-variant-specific regulation by the synthetic glucocorticoid dexamethasone (DEX) of BK channels consisting of cloned STREX or ZERO alpha-subunit variants expressed in human embryonic kidney (HEK 293) cells. 2. STREX channels in isolated membrane patches were inhibited by protein kinase A (PKA) and this was blocked on pre-treatment of intact cells with DEX (100 nM) for 2 h. 3. The effect of DEX required the synthesis of new mRNA and protein. Furthermore, it required protein phosphatase 2A (PP2A)-like activity intimately associated with the channels, as it was blocked by 10 nM okadaic acid but not by the specific protein phosphatase-1 inhibitor peptide PPI-2. 4. ZERO variant channels that lack the STREX insert were activated by PKA but were not influenced by DEX. ZERO channels containing a mutant STREX domain (S4(STREX)A) were also activated by PKA. Importantly, DEX blocked PKA activation of S4(STREX)A channels in a PP2A-dependent manner. 5. Taken together, the STREX domain is crucial for glucocorticoid regulation of BK channels through a PP2A-type enzyme. Moreover, glucocorticoids appear to induce a generic set of proteins in different types of cells, the actions of which depend on the expression of cell-specific targets.

Alternative Splicing↗

PubMed2001

Selective inhibition of IL-2 gene expression by IL-2 antisense oligonucleotides blocks heart allograft rejection.

PURPOSE: We tested the effects of selective inhibition of interleukin (IL)-2 gene expression by IL-2 antisense oligonucleotide (oligo) with phosphorothioate (PS)/phosphodiester (PD)/2'-methoxyethyl (ME) modifications (17359) on T-cell function and the survival of heart allografts in mice. METHODS: The PS- (17328) or PS/PD/ME- (17359) IL-2 oligo was electroporated to mouse T cell lymphoma cells (TIB 155) stimulated with concanavalin A (Con A). Expression of IL-2 was analyzed by an ELISA spot assay and a reverse transcript polymerase chain reaction method. C3H (H-2k) mice transplanted with BALB/c (H-2d) heart grafts were treated i.v. with a 7-day osmotic pump with 20 mg/kg 17359 alone or in combination with sirolimus (SRL). RESULTS: In comparison with untreated controls, 500 to 2000 nM 17328 inhibited IL-2 protein production by 21.8% to 47.2%, whereas 500 to 2000 nM 17359 did so by 35.5% to 83.5% (both P<0.001). In vivo, 20 mg/kg 17359 prolonged survivals to a mean survival time (MST) of 18.3+/-2.6 days (P<0.001) in comparison with only 8.2+/-0.8 days in untreated controls. Although 0.2 mg/kg SRL alone produced a MST of 18.8+/-6.0 days (P<0.01), addition of 20 mg/kg 17539 synergistically extended survivals to 54.3+/-12.1 days (P<0.001). As expected, IL-2 mRNA, but not IL-7, IL-9, or IL-15 mRNA, was reduced in allografts from recipients treated with 17359 compared with untreated controls. Lymph node cells from the same recipients displayed reduction in proliferative response to donor alloantigen and in generation of alloantigen-specific cytotoxic T cells. CONCLUSION: Selective inhibition of IL-2 mRNA in vivo inhibits T-cell function and extends allograft survival.

Animals↗

PubMed2001

Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen.

Overcoming immune tolerance of the growth factors associated with tumor growth should be a useful approach to cancer therapy by active immunity. We used vascular endothelial growth factor (VEGF) as a model antigen to explore the feasibility of the immunogene tumor therapy with a vaccine based on a single xenogeneic homologous gene, targeting the growth factors associated with angiogenesis. To test this concept, we constructed a plasmid DNA encoding Xenopus homologous VEGF (XVEGF-p) and control vectors. We found that immunogene tumor therapy with a vaccine based on XVEGF was effective at both protective and therapeutic antitumor immunity in several tumor models in mice. VEGF-specific autoantibodies in sera of mice immunized with XVEGF-p could be found in Western blotting analysis and ELISA assay. The purified immunoglobulins were effective at the inhibition of VEGF-mediated endothelial cell proliferation in vitro, and at antitumor activity and the inhibition of angiogenesis by adoptive transfer in vivo. The elevation of VEGF in the sera of the tumor-bearing mice could be abrogated with XVEGF-p immunization. The antitumor activity and production of VEGF-specific autoantibodies, significantly elevated IgG1 and IgG2b, could be abrogated by the depletion of CD4(+) T lymphocytes. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factors associated with tumor growth in a cross reaction with single xenogeneic homologous gene and may be of importance in the further exploration of the applications of other xenogeneic homologous genes identified in human and other animal genome sequence projects in cancer therapy.

Animals↗

PubMed2001

A-fibres sprouting from lamina I into lamina II of spinal dorsal horn after peripheral nerve injury in rats.

We have examined the labeling pattern in the spinal dorsal horn by an intra-sciatic nerve injection of cholera toxin B subunit conjugated horseradish peroxidase (HRP) after transection of the posterior cutaneous nerve and inferior gluteal nerve, and found that the cholera toxin B subunit conjugated HRP labeling in lamina I was expanding into lamina II and there was a shrinking gap between lamina I and lamina III. This result suggests that A-fibre sprouting arise after peripheral nerve injury, but mainly from small calibre Adelta-fibres which terminate in lamina I.

Afferent Pathways↗

PubMed2001

Alternative splicing switches potassium channel sensitivity to protein phosphorylation.

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.

Alternative Splicing↗

PubMed2001

Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development.

Histone acetylation and deacetylation play essential roles in eukaryotic gene regulation. Reversible modifications of core histones are catalyzed by two intrinsic enzymes, histone acetyltransferase and histone deacetylase (HD). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation correlates with gene activation. We produced transgenic plants expressing the antisense Arabidopsis HD (AtHD1) gene. AtHD1 is a homolog of human HD1 and RPD3 global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones, notably tetraacetylated H4. Reduction in AtHD1 expression and AtHD1 production and changes in acetylation profiles were associated with various developmental abnormalities, including early senescence, ectopic expression of silenced genes, suppression of apical dominance, homeotic changes, heterochronic shift toward juvenility, flower defects, and male and female sterility. Some of the phenotypes could be attributed to ectopic expression of tissue-specific genes (e.g., SUPERMAN) in vegetative tissues. No changes in genomic DNA methylation were detected in the transgenic plants. These results suggest that AtHD1 is a global regulator, which controls gene expression during development through DNA-sequence independent or epigenetic mechanisms in plants. In addition to DNA methylation, histone modifications may be involved in a general regulatory mechanism responsible for plant plasticity and variation in nature.

Acetylation↗

PubMed2001

A case of X-linked agammaglobulinemia diagnosed in adulthood.

X-linked agammaglobulinemia (XLA), caused by mutations in Bruton's tyrosine kinase (BTK), typically presents in early childhood. We report here the case of a male diagnosed at age 23 years with hypogammaglobulinemia, originally classified as common variable immunodeficiency (CVID). On further analysis at age 40, flow cytometric analysis of lymphocytes showed only 0.1% B cells and Western blot analysis showed a deficiency of BTK protein in peripheral blood mononuclear cells, indicating the patient has XLA. BTK cDNA and genomic DNA analysis revealed a splice site mutation at the 3' end of intron 13. Multiple abnormally spliced mRNA species were identified, one of which was predicted to produce a protein with a 24-amino-acid insertion between the SH2 and kinase domains. In vitro kinase assay of this product showed weak kinase activity, perhaps resulting in milder than usual disease. XLA can present in adult males, and sporadic cases may be misdiagnosed as CVID.

Adult↗

PubMed2001

Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP.

Cryptic gene regulatory elements are sequences that are inactive at their native locations in the genome but have the ability to become functional when positioned adjacent to genes. We have recently isolated such a cryptic sequence from tobacco, tCUP, that can act as a promoter. A 135-bp fragment spanning extending from position -197 to -62, relative to the transcription start site, was found to promote GUS expression in all of the major organs of transgenic Arabidopsis plants. Furthermore, this 135-bp fragment complemented the -46 minimal promoter of CaMV 35S and conferred constitutive expression on transgenic Arabidopsis plants. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from tobacco leaves interact with the 135-bp fragment. tCUP has a core promoter that lacks the TATA consensus sequence but addition of a TATA-box sequence increased the core promoter activity by three-fold. The sequence surrounding the transcription start site of tCUP has sequence similarity with the initiator element (Inr), and deletion of this sequence significantly reduced promoter activity, suggesting that an essential Inr element may exist in the tCUP core promoter. Fusion of the GCC-box enhancer element from pathogenesis-related genes to the core promoter elevated tCUP core promoter activity. Our study indicates that cryptic promoters are similar in composition and organization to promoters associated with expressed genes and that their promoter elements can be combined to create composite promoters that are fully functional. This data provides direct evidence that the expression pattern of plant genes can be influenced by cryptic gene regulatory elements when they are brought into juxtaposition with genes through DNA rearrangements.

Arabidopsis↗

PubMed2001

Analysis of carotenoid biosynthetic gene expression during marigold petal development.

Marigold (Tagetes erecta L.) flower petals synthesize and accumulate carotenoids at levels greater than 20 times that in leaves and provide an excellent model system to investigate the molecular biology and biochemistry of carotenoid biosynthesis in plants. In addition, marigold cultivars exist with flower colors ranging from white to dark orange due to >100-fold differences in carotenoid levels, and presumably similar changes in carbon flux through the pathway. To examine the expression of carotenoid genes in marigold petals, we have cloned the majority of the genes in this pathway and used these to assess their steady-state mRNA levels in four marigold cultivars with extreme differences in carotenoid content. We have also cloned genes encoding early steps in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all isoprenoids, including carotenoids, as well as two genes required for plastid division. Differences among the marigold varieties in the expression of these genes suggest that differences in mRNA transcription or stability underlie the vast differences in carotenoid synthesis and accumulation in the different marigold varieties.

Blotting, Northern↗

PubMed2001

Characterization of a second carotenoid beta-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus.

Xanthophylls are oxygenated carotenoids that perform critical roles in plants. Beta-carotene hydroxylases (beta-hydroxylases) add hydroxyl groups to the beta-rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis. The lut1 mutation of Arabidopsis disrupts epsilon-ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on epsilon-ring structures. We have used library screening and genomics-based approaches to isolate a second beta-hydroxylase genomic clone and its corresponding cDNA from Arabidopsis. The encoded protein is 70% identical to the previously reported Arabidopsis beta-hydroxylase 1. Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent. Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, beta-hydroxylase 1 mRNA is always present at higher levels. Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of beta-ring containing carotenes and are only weakly active towards epsilon-ring containing carotenes. Neither beta-hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase. These data indicate that the LUT1 locus encodes a protein necessary for epsilon-ring hydroxylation but unrelated to beta-hydroxylases at the level of amino acid sequence.

Amino Acid Sequence↗

PubMed2001

Role of CD4+ and CD8+ T cells in early and late acute rejection of small bowel allograft.

BACKGROUND/PURPOSE: Results of small bowel transplantation remain unsatisfactory because of severe immune rejection. The current study aims to elucidate the role of activation of CD4+ and CD8+ T cells in early and late acute rejection of small bowel allograft and, hence, provide the immunologic basis for developing new therapeutic strategies. METHODS: We used an MHC fully mismatched (DA to Lewis) heterotopic rat small bowel transplant model and a unique FK506-based immunosuppressive regimen, which suppresses early acute rejection but does not prevent late acute rejection. Flow cytometric analysis was used to quantitate the number of activated CD4+ and CD8+ T cells in graft and host mesenteric lymph nodes. RESULTS: The survival (mean +/- SD) of intestinal allograft was significantly prolonged, from 6.6 +/- 0.84 days for the untreated group to 40.7 +/- 14.1 days for the FK506-treated group. Activation of CD4+ cells was suppressed significantly in the FK506-treated group on postoperative day 7 compared with the untreated group (29.4% +/- 3.55% v 52.83% +/- 11.9%; P <.01). Activation of CD8+ cells was similarly suppressed (31.5 +/- 10.34% v 48.53 +/- 14.34%; P <.05). Interestingly, at late acute rejection, activated CD4+ and CD8+ T cells remained at almost the same low levels as those on postoperative day 7 in the FK506-treated group. The spleen to body weight ratio was significantly increased in the untreated group (0.53 +/- 0.07), and slightly increased in the FK treated group (0.27 +/- 0.07, on postoperative day 7; 0.24 +/- 0.07 at late acute rejection) compared with the syngeneic group (0.18 +/- 0.02). CONCLUSION: The activation of CD4+ and CD8+ T cells was suppressed effectively by early potent immunosuppressive treatment resulting in prolonged survival of intestinal allograft. At late acute rejection, the CD4+ and CD8+ T cells remained at low-level activation status, in contrast to the surge of CD4+ and CD8+ activation during early acute rejection. This suggests that persistent T cell activation even at low level is sufficient to cause the late acute rejection eventually. A therapeutic strategy targeting these cells is needed for long-term engraftment.

Animals↗

PubMed2001

p53 gene expression in relation to indoor exposure to unvented coal smoke in Xuan Wei, China.

Lung cancer mortality rates in Xuan Wei County, which are among the highest in China, have previously been associated with exposure to indoor emissions from burning smoky coal. To determine if this association is stronger among lung cancer patients with abnormal expression of p53, we performed a population-based case-control study. Ninety-seven newly diagnosed lung cancer patients and 97 controls, individually matched by age, sex, and home fuel type, were enrolled. We used immunocytochemical methods to assess p53 protein accumulation in exfoliated tumor cells isolated from sputum samples. As expected, the amount of lifetime smoky coal use was associated with an overall increase in lung cancer risk. Compared with subjects who used less than 130 tons of smoky coal during their lifetime, the odds ratios (OR) for lung cancer were 1.48 (95% confidence interval [CI], 0.73 to 3.02) for subjects exposed to 130 to 240 tons, and 3.21 (95% CI, 1.23 to 9.03) for subjects who used more than 240 tons of smoky coal (P for trend 0.01). The effect was due almost exclusively to the pattern in women, almost all of whom were nonsmokers. Further, among highly exposed women, the association was substantially larger and achieved statistical significance only among patients with sputum samples that were positive for p53 overexpression (OR, 18.72; 95% CI, 1.77 to 383.38 vs OR, 4.80; 95% CI, 0.66 to 43.87 for p53-negative cases). This study suggests that exposure to the combustion products of smoky coal in Xuan Wei is more strongly associated with women who have lung cancer accompanied by p53 protein overexpression in exfoliated tumor cells.

Air Pollution, Indoor↗

PubMed2001

On estimating medical cost and incremental cost-effectiveness ratios with censored data.

Medical cost estimation is very important to health care organizations and health policy makers. We consider cost-effectiveness analysis for competing treatments in a staggered-entry, survival-analysis-based clinical trial. We propose a method for estimating mean medical cost over patients in such settings. The proposed estimator is shown to be consistent and asymptotically normal, and its asymptotic variance can be obtained. In addition, we propose a method for estimating the incremental cost-effectiveness ratio and for obtaining a confidence interval for it. Simulation experiments are conducted to evaluate our proposed methods. Finally, we apply our methods to a clinical trial comparing the cost effectiveness of implanted cardiac defibrillators with conventional therapy for individuals at high risk for ventricular arrhythmias.

Arrhythmias, Cardiac↗

PubMed2001

Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family.

Inhibitor of apoptosis protein (IAP)-like protein-1 (ILP-1) (also known as X-linked IAP [XIAP] and mammalian IAP homolog A [MIHA]) is a potent inhibitor of apoptosis and exerts its effects, at least in part, by the direct association with and inhibition of specific caspases. Here, we describe the molecular cloning and characterization of a human gene related to ILP-1, termed ILP-2. Despite high homology to ILP-1, ILP-2 is encoded by a distinct gene, which in normal tissues is expressed solely in testis. In contrast to ILP-1, overexpression of ILP-2 had no protective effect on apoptosis mediated by Fas (also known as CD95) or tumor necrosis factor. However, ILP-2 potently inhibited apoptosis induced by overexpression of Bax or by coexpression of caspase 9 with Apaf-1, and preincubation of cytosolic extracts with ILP-2 abrogated caspase activation in vitro. A processed form of caspase 9 could be coprecipitated with ILP-2 from cells, suggesting a physical interaction between ILP-2 and caspase 9. Thus, ILP-2 is a novel IAP family member with restricted specificity for caspase 9.

Amino Acid Sequence↗

PubMed2001

Violence, alcohol, and completed suicide: a case-control study.

OBJECTIVE: Violent behavior may represent a risk factor for suicide. The authors tested the hypothesis that violent behavior in the last year of life is associated with completed suicide, even after controlling for alcohol use disorders. METHOD: The authors analyzed data from the 1993 National Mortality Followback Survey, a nationally representative survey conducted by telephone interview with decedents' next of kin. Data on 753 victims of suicide were compared with data on 2,115 accident victims. Decedents ranged in age from 20 to 64. Dichotomous measures of violent behavior in the past year and history of alcohol misuse were derived by using the four-item CAGE questionnaire. Multiple logistic regression was used to evaluate the interactions of violent behavior with alcohol misuse, gender, and age, respectively, in predicting suicide versus accidental death. Education and race were included as covariates. RESULTS: Violent behavior in the last year of life was a significant predictor of suicide; the relationship was especially strong in individuals with no history of alcohol misuse, those who were younger, and women. CONCLUSIONS: Violent behavior distinguished suicide victims from accident victims, and this finding is not attributable to alcohol use disorders alone. Given that violent behavior increases the risk of suicide, violence prevention initiatives may serve to decrease the risk of suicide as well.

Accidents↗

PubMed2001

Kaplan-Meier analysis of dental implant survival: a strategy for estimating survival with clustered observations.

The study's purposes were to estimate dental implant survival in a statistically valid manner and to compare three models for estimating survival. We estimated survival using three different statistical models: (1) randomly selecting one implant per patient; (2) utilizing all implants, assuming independence among implants from the same subject; and (3) utilizing all implants, assuming dependence among implants from the same subject. The cohort was composed of 660 patients who had 2286 implants placed. Due to the high success rates of implants, the five-year survival point and standard error estimates varied little among the three models. Patients at high risk for implant failure (smokers) manifested greater variation in the standard error estimates among the three models, 8.2%, 4.0%, and 5.6%, respectively. To obtain statistically valid survival confidence intervals when performing Kaplan-Meier survival analyses, we recommend adjusting for dependence when there are multiple observations within the same subject.

Dental Implantation, Endosseous↗

PubMed2001

[Progress in targeting gene therapy].

In recent years the research on targeting in gene therapy has made much exciting progress. Many new technologies show great promising prospects such as the delivery of therapeutic gene to targeted cells by some antibodies or ligands, the use of specific gene promoter to tightly control gene expression within disease microenvironment, the use of some orally taken drugs for regulating expression time and expression level of DNA, and the targeted correction of deleted or mutated gene using RNA/DNA oligonucleotides. Yet, researchers still have a long way to go in order to reach the goal of applying the targeting gene therapy in clinical practice.

Gene Expression↗

PubMed2000

Inhibition of C-raf expression by antisense oligonucleotides extends heart allograft survival in rats.

BACKGROUND: C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival. METHODS: Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2'-methoxyethyl (ME) substitutions at the 2'-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides. RESULTS: Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos. CONCLUSIONS: C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity.

Animals↗