Antiviral effect of hepatitis B virus S/C gene loci antisense locked nucleic acid on transgenic mice in vivo.

We investigated the effects of hepatitis B virus (HBV) S/C double gene loci antisense locked nucleic acid on replication and expression of HBV in hepatitis transgenic mice. HBV mice (N = 30) were randomly divided into five groups of six mice: 5% glucose solution control, empty liposome control, single-target S, single-target C, and dual-target SC groups. An antisense locked nucleic acid fragment was injected into the mice. Serum HBsAg, serum HBV DNA, HBV C-mRNA expression in liver tissue, HbsAg and HbcAg expression in hepatocytes, serum albumin, alanine transaminase (ALT), urea nitrogen, and creatinine were detected. Liver and kidney sections were examined for the effects of antisense locked nucleic acid. The expression of HBsAg was markedly inhibited; the inhibition rates of the S, C, and SC target groups were 36.63, 31.50, and 54.87%, respectively; the replication of HBV DNA was also inhibited: 23.97, 21.13, and 35.83%, respectively. After injection at 1, 3, and 5 days, the corresponding rates for HBsAg inhibition were 14.40, 25.61, and 31.33%, and for HBV DNA inhibition they were 11.04, 19.24, and 24.13%. Compared with the control group, the differences in serum albumin, ALT, urea nitrogen, and creatinine in each group were not statistically significant, and the number of HbsAg- and HBcAg-positive cells in the mouse liver was significantly reduced. The liver and kidney tissues were normal. The gene therapy had significant inhibitory effects on the replication and expression of HBV in transgenic mice, and double-gene targeting was better than single-gene targeting.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41 percent in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity.

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG(2) cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG(2) cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG(2) cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G(1) phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf discs exposed to paraquat.

In the present work, the response of tobacco (Nicotiana tabaccum L.) wild-type SR1 and transgenic CAT1AS plants (with a basal reduced CAT activity) was evaluated after exposure to the herbicide paraquat (PQ). Superoxide anion (O (2) (.-) ) formation was inhibited at 3 or 21 h of exposure, but H(2)O(2) production and ion leakage increased significantly, both in SR1 or CAT1AS leaf discs. NADPH oxidase activity was constitutively 57% lower in non-treated transgenic leaves than in SR1 leaves and was greatly reduced both at 3 or 21 h of PQ treatment. Superoxide dismutase (SOD) activity was significantly reduced by PQ after 21 h, showing a decrease from 70% to 55%, whereas catalase (CAT) activity decreased an average of 50% after 3 h of treatment, and of 90% after 21 h, in SR1 and CAT1AS, respectively. Concomitantly, total CAT protein content was shown to be reduced in non-treated CAT1AS plants compared to control SR1 leaf discs at both exposure times. PQ decreased CAT expression in SR1 or CAT1AS plants at 3 and 21 h of treatment. The mechanisms underlying PQ-induced cell death were possibly not related exclusively to ROS formation and oxidative stress in tobacco wild-type or transgenic plants.

The hobo transposon and hobo-related elements are expressed as developmental genes in Drosophila.

Transposable elements comprise a significant part of genomes and are involved in their evolvability. The hobo element is found as an active class II transposable element in Drosophila melanogaster that is able to induce gonadal dysgenesis. Some hobo-related sequences (hRSs) are thought to be relics of old "hobo" invasions, and are therefore ancient genomic constituents. However, some of these hRSs are still mobile. The present study analyzed the expression pattern of hobo and a particular type of hRSs, hobo(VAHS). Both elements were shown to be expressed as sense and antisense mRNA transcripts. Expression analysis in whole mount embryos revealed a pattern similar to that of some developmental regulatory genes. Here we suggest that cis-regulatory sequences similar to those in developmental genes exist in hobo sequences. Therefore, hobo mobilization may contribute to the development of new regulatory networks during genomic evolution.

Isolation, structure, and antibacterial activities of lucensimycins D-G, discovered from Streptomyces lucensis MA7349 using an antisense strategy.

Bacterial resistance to existing antibiotics continues to grow, necessitating the discovery of new compounds of this type. Antisense-based whole-cell target-based screening is a new and highly sensitive antibiotic discovery approach that has led to a number of new natural product antibiotics. Screening with a rpsD-sensitized strain led to the discovery of a number of natural product polyketides from Streptomyces lucensis. Complete workup of the fermentation extract of this strain allowed for the isolation of seven new compounds, lucensimycins A-G (1-3, 4a, 5-7), with varying degrees of antibacterial activities. Lucensimycin E (5) exhibited the best activity and showed MIC values of 32 microg/mL against Staphylococcus aureus and 8 microg/mL against Streptococcus pneumoniae. The isolation, structure elucidation, and antibacterial activities of four new members, lucensimycins D-G, are described. Lucensimycins D (4a) and E (5) are N-acetyl-l-cysteine adducts of lucensimycin A (1). Semisynthesis of lucensimycins D and E from lucensimycin A has also been described. Lucensimycins F and G are myo-inositolyl-alpha-2-amino-2-deoxy-l-idosyl amide derivatives of lucensimycins D and E, respectively. The relative configuration of these compounds was determined, in part, by molecular dynamics simulations.

C8orf4/TC-1 (thyroid cancer-1) gene expression in thyroid cancer and goiter.

BACKGROUND: The expression of the thyroid cancer-1(TC-1) gene seems to be related with malignant transformation in the thyroid tissue. OBJECTIVE: We evaluated the potential use of TC-1 gene expression as a marker of malignancy in thyroid nodules. METHODS: A total of 92 frozen thyroid samples were studied, including 46 samples from thyroid nodules (19 papillary carcinomas, 1 follicular carcinoma, 24 adenomatous goiters, and 2 follicular adenomas) and 46 samples from normal surrounding thyroid tissue. Total RNA was extracted and TC-1 expression was assessed by semiquantitative Multiplex PCR. Results were verified using real-time RT-PCR in some of the samples. RESULTS: Overall mean TC-1 gene expression (normalized by the ABL gene) was 1.73 +/- 1.67 (0.33-9.33). There was a significant difference (p < 0.001) between TC-1 gene expression in benign thyroid lesions (1.07 +/- 0.10) and carcinomas (2.73 +/- 0.51). CONCLUSION: Our results suggest that TC-1 gene expression may be useful in the differential diagnosis of goiters and thyroid papillary carcinomas.

Apical distribution of HFE-beta2-microglobulin is associated with inhibition of apical iron uptake in intestinal epithelia cells.

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276-1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, beta-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and beta-2 microglobulin (beta2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE-beta2m to the apical membrane. The amount of HFE-beta2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or beta2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE-beta2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.

Reduction of G protein-coupled receptor kinase 2 expression in U-937 cells attenuates H2 histamine receptor desensitization and induces cell maturation.

Histamine and H2 agonists transiently induce an important cAMP response in promonocytic U-937 cells but fail to induce monocytic differentiation because of a rapid receptor desensitization mediated by G protein-coupled receptor kinases (GRKs). The aims of the present study were to investigate the participation of GRK2 in the desensitization mechanism of the H2 receptor in U-937 cells by reducing GRK2 levels through antisense technology and to evaluate the differentiating capacity of cells expressing lower GRK2 level, stimulated by H2 agonists. By stable U-937 cell transfection with a GRK2-antisense cDNA, we obtained D5 and A2 cell clones exhibiting a reduction in GRK2 expression and an H3 clone with no significant difference in GRK2 expression from control cells. The cAMP response induced by the H2 agonist in D5 and A2 but not in H3 cells was higher than in U-937 and persisted for a longer period of time, although the number of H2 receptors in D5 and A2 cells was lower than in U-937. Furthermore, D5 and A2 cells treated with H2 agonist showed patterns of c-Fos and CD88 expression consistent with monocytic differentiated cells. Overall, these results indicate a direct correlation between the expression of GRK2 and the desensitization of natively expressed H2 receptors in U-937 cells, suggesting that GRK2 plays a major role in the regulation of these receptors' response. In turn, desensitization process is a key component of H2 receptor signaling, determining the differentiation capability of promonocytic cells.

Rol del gen SPARC en la capacidad tumorigénica de células de melanoma humano / The role of SPARC gene in tumorigenic capacity of human melanoma cells

En un estudio previo demostramos que líneas celulares y tumores de melanoma humano expresan altos niveles de la proteína de matriz extracelular SPARC. Para determinar su rol en la progresión del melanoma humano, la línea IIB-MELLES fue transfectada con el cDNA de SPARC anti-sentido. Se aislaron tres clones con expresión disminuida de SPARC. Ninguno de ellos mostró cambios en la cinética de crecimiento in vitro comparado con las células control. La inyección s.c. de células control en ratones atímicos mostró desarrollo tumoral en el 100 por ciento de los animales, mientras que ninguno de los clones dio origen a tumores. Estos estudios demuestran que SPARC podría jugar un rol central en la progresión del melanoma humano. (AU)

Rol del gen SPARC en la capacidad tumorigénica de células de melanoma humano / The role of SPARC gene in tumorigenic capacity of human melanoma cells

En un estudio previo demostramos que líneas celulares y tumores de melanoma humano expresan altos niveles de la proteína de matriz extracelular SPARC. Para determinar su rol en la progresión del melanoma humano, la línea IIB-MELLES fue transfectada con el cDNA de SPARC anti-sentido. Se aislaron tres clones con expresión disminuida de SPARC. Ninguno de ellos mostró cambios en la cinética de crecimiento in vitro comparado con las células control. La inyección s.c. de células control en ratones atímicos mostró desarrollo tumoral en el 100 por ciento de los animales, mientras que ninguno de los clones dio origen a tumores. Estos estudios demuestran que SPARC podría jugar un rol central en la progresión del melanoma humano.

[The role of SPARC gene in tumorigenic capacity of human melanoma cells]. / Rol del gen SPARC en la capacidad tumorigénica de células de melanoma humano.

Previous studies from our laboratory have demonstrated that human melanoma cell lines and tumors expressed high levels of the extracellular protein SPARC. In order to demonstrate its role in human melanoma progression, IIB-MEL-LES human melanoma cells were transfected with SPARC full length c-DNA in the antisense orientation. In vivo studies demonstrated that all the control mice injected with parental cells developed tumors, while none of the mice injected with cells obtained from three different clones with diminished levels of SPARC expression, developed tumors. These studies suggest that SPARC may play a key role in human melanoma progression.