A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

Background

Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells.

Methodology/Principal Findings

In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects.

Conclusions

3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism.     

Suppression of HIV-1 Replication by a Combination of Endonucleolytic Ribozymes (RNase P and tRNase ZL)

We examined the combinatorial action of RNase P and tRNase ZL-mediated specific inhibition of HIV-1 in cultured cells. We designed two short extra guide sequences (sEGS) that specifically recognize the tat and vif regions of HIV-1 mRNA and mediate the subsequent cleavage of hybridized mRNA by the RNase P and tRNase ZL components. We constructed an RNase P and tRNase ZL-associated vif and tat sEGS expression vector, which used the RNA-polymerase III dependent U6 promoter, as an expression cassette for EGS. Together, the RNase P and tRNase ZL-associated sEGS molecules allow more efficient suppression of HIV-1 mRNA production when separately applied. The possibilities offered by the vector to encode sEGS will provide a powerful tool for gene therapy.     

Suppression Of Hepatitis C Virus Core Protein By Short Hairpin Rna Expression Vectors In The Core Protein Expression Huh-7 Cells

Short hairpin RNAs (shRNAs) efficiently inhibit gene expression by RNA interference. Here, we report the efficient inhibition by DNA-based vector-derived shRNAs of core protein expression in Huh-7 cells. The shRNAs were designed to target the core region of the hepatitis C virus (HCV) genome. The core region is the most conserved region in the HCV genome, making it an ideal target for shRNAs. We identified an effective site on the core region for suppression of the HCV core protein. The HCV core protein in core protein-expressing Huh-7 cells was downregulated by core protein-shRNA expression vectors (core-shRNA-452, 479, and 503). Our results support the feasibility of using shRNA-based gene therapy to inhibit HCV core protein production.     

Antisense Phosphorothioate Oligonucleotides Targeted to the Human Chemokine Receptor CXCR4

Abstract

The CXC chemokine receptor CXCR4 is used as a major co-receptor for fusion and entry by syncytia-inducing T-tropic (X4) isolates of HIV-1. In the present study, we report the effects of an antisense oligodeoxyribonucleotide on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for Human Immunodeficiency Virus type 1 (HIV-1) infection. Antisense phosphorothioate oligodeoxyribonucleotides (anti-S-ODNs) corresponding to the sequence of bases 69 to 88 of the human CXCR4 mRNA gene were synthesized. When the naked anti-S-ODN was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor were reduced up to 50%, indicating sequence-specific inhibition. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for delivery of anti-S-ODNs. The anti-S-ODN encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN.     

Suppression of Human Immunodeficiency Virus Type 1 (HIV-1) Replication by an HIV-1-dependent Double Locked Vector with the Cre/loxP System

We previously demonstrated the function of an HIV-1-dependent ribozyme expression vector, with which the site-specific excision of loxP sequences can be achieved by using the Cre-loxP system (ON/OFF) as a molecular switch in an acute HIV-1 infection. However, this expression system also revealed the lower, non-specific expression of the anti-HIV-1 ribozyme in the absence of tat. To circumvent this problem, we used the more efficient HIV-1-dependent Cre recombinase gene expression vector, encoding the LTR-gag-p17 (extending from the 5′-LTR to the middle of the gag gene (pLTR-gag-p17-Cre)). Comparatively, the pLTR-gag-p17-Cre induces a higher Cre-protein expression level in an HIV-1 infection-dependent manner than the minimal pLTR-Cre. Furthermore, we constructed the ploxP-Rz-U5 and pLTR-gag-p17-Cre plasmids and also combined them into a single vector, pLTR-gag-p17-Cre/loxP-Rz-U5, for a comparison of their anti-HIV-1 activities. The resultant simultaneous expression of the Cre protein and the homologous recombination of the two loxP sequences induced a high level of HIV-1 replication inhibition (95%). Significantly, a high steady-state of ribozyme expression was observed in the RT-PCR analysis. These data imply that targeting the HIV-1 genes with the pLTR-gag-p17-Cre/loxP-Rz-U5 vector, which mediates HIV-1-dependent ribozyme expression, would be a useful tool for HIV-1 gene therapy applications.     

Estimation of the Fraction of Cancer Cells in a Tumor DNA Sample Using DNA Methylation

Contamination of normal cells is almost always present in tumor samples and affects their molecular analyses. DNA methylation, a stable epigenetic modification, is cell type-dependent, and different between cancer and normal cells. Here, we aimed to demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample, using esophageal squamous cell carcinoma (ESCC) as an example. First, by an Infinium HumanMethylation450 BeadChip array, we isolated three genomic regions (TFAP2B, ARHGEF4, and RAPGEFL1) i) highly methylated in four ESCC cell lines, ii) hardly methylated in a pooled sample of non-cancerous mucosae, a pooled sample of normal esophageal mucosae, and peripheral leukocytes, and iii) frequently methylated in 28 ESCCs (TFAP2B, 24/28; ARHGEF4, 20/28; and RAPGEFL1, 19/28). Second, using eight pairs of cancer and non-cancer cell samples prepared by laser capture microdissection, we confirmed that at least one of the three regions was almost completely methylated in ESCC cells, and all the three regions were almost completely unmethylated in non-cancer cells. We also confirmed that DNA copy number alterations of the three regions in 15 ESCC samples were rare, and did not affect the estimation of the fraction of cancer cells. Then, the fraction of cancer cells in a tumor DNA sample was defined as the highest methylation level of the three regions, and we confirmed a high correlation between the fraction assessed by the DNA methylation fraction marker and the fraction assessed by a pathologist (r=0.85; p<0.001). Finally, we observed that, by correction of the cancer cell content, CpG islands in promoter regions of tumor-suppressor genes were almost completely methylated. These results demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample.     

Biochemical Features of a Catalytic Antibody Light Chain, 22F6, Prepared from Human Lymphocytes

Human antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.     

Site-specific aspartic acid isomerization regulates self-assembly and neurotoxicity of amyloid-β

Amyloid-β (Aβ) proteins, which consist of 42 amino acids (Aβ_1–42), are the major constituent of neuritic plaques that form in the brains of senile patients with Alzheimer’s disease (AD). Several reports state that three aspartic acid (Asp) residues at positions 1, 7, and 23 in Aβ_1–42 in the plaques of patients with AD are highly isomerized from the l- to d-form. Using biophysical experiments, the present study shows that simultaneous d-isomerization of Asp residues at positions 7 and 23 (d-Asp^7,23) enhances oligomerization, fibril formation, and neurotoxic effect of Aβ_1–42. In addition, d-isomerization of Asp at position 1 (d-Asp¹) suppresses malignant effects induced by d-Asp^7,23 of Aβ_1–42. These results provide fundamental information to elucidate molecular mechanisms of AD pathogenesis and to develop potent inhibitors of amyloid aggregates and Aβ neurotoxicity.     

Xylem exudate composition and root-to-shoot nickel translocation in Alyssum species

Aims

An improved understanding of the Ni root-to-shoot translocation mechanism in hyperaccumulators is necessary to increase Ni uptake efficiency for phytoextraction technologies. It has been presumed that an important aspect of Ni translocation and storage involves chelation with organic ligands. It has been reported that exposing several Ni hyperaccumulator species of Alyssum to Ni elicited a large increase in the histidine level of the xylem sap. In later studies it was shown that as time progressed the histidine:Ni ratio dropped considerably. Moreover, previous studies analyzed the relationship between Ni and ligands in plants that were exposed to Ni only for a few hours and therefore obtained results that are unlikely to represent field soils where plants are at steady-state Ni uptake. The aim of this study was to understand the quantitative relationship between Ni and organic ligands in the xylem sap of various Alyssum genotypes or species that reached steady-state Ni uptake after being exposed to Ni in either nutrient solution or serpentine soil for up to 6 weeks.

Methods

Total Ni concentration, 17 amino acids, 9 organic acids, and nicotianamine were measured in xylem sap of 100-day old plants of Alyssum.

Results

Results showed that the concentration of Ni in xylem sap of various Alyssum genotypes was 10–100 fold higher than the concentration of histidine, malate, citrate, and nicotianamine, which were the predominant Ni ligands measured in the sap.

Conclusion

When the physiology of the whole plant is taken into account, our results indicate that the concentration of organic chelators is too low to account for the complexation of all the Ni present in the xylem sap of Alyssum at steady-state Ni hyperaccumulation, and suggest that most of the Ni in xylem sap of this species is present as the hydrated cation.