Genistein inhibits differentiation of primary human adipocytes

Genistein, a major soy isoflavone, has been reported to exhibit antiadipogenic and proapoptotic potential in vivo and in vitro. It is also a phytoestrogen which has high affinity to estrogen receptor beta. In this study, we determined the effect of genistein on adipogenesis and estrogen receptor (ER) alpha and beta expression during differentiation in primary human preadipocytes. Genistein inhibited lipid accumulation in a dose-dependent manner at concentrations of 6.25 microM and higher, with 50 microM genistein inhibiting lipid accumulation almost completely. Low concentrations of genistein (3.25 microM) increased cell viability and higher concentrations (25 and 50 microM) decreased it by 16.48+/-1.35% (P<.0001) and 50.68+/-1.34% (P<.0001). Oil Red O staining was used to confirm the effects on lipid accumulation. The inhibition of lipid accumulation was associated with inhibition of glycerol-3-phosphate dehydrogenase activity and down-regulation of expression of adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, glycerol-3-phosphate dehydrogenase, adipocyte fatty acid binding protein, fatty acid synthase, sterol regulatory element-binding protein 1, perilipin, leptin, lipoprotein lipase and hormone-sensitive lipase. These effects of genistein during the differentiation period were associated with down-regulation of ERalpha and ERbeta expression. This study adds to the elucidation of the molecular pathways involved in the inhibition of adipogenesis by phytoestrogens.     

Evaluation of the compact high-field orbitrap for top-down proteomics of human cells

Mass spectrometry based proteomics generally seeks to identify and fully characterize protein species with high accuracy and throughput. Recent improvements in protein separation have greatly expanded the capacity of top-down proteomics (TDP) to identify a large number of intact proteins. To date, TDP has been most tightly associated with Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Here, we couple the improved separations to a Fourier-transform instrument based not on ICR but using the Orbitrap Elite mass analyzer. Application of this platform to H1299 human lung cancer cells resulted in the unambiguous identification of 690 unique proteins and over 2000 proteoforms identified from proteins with intact masses <50 kDa. This is an early demonstration of high throughput TDP (>500 identifications) in an Orbitrap mass spectrometer and exemplifies an accessible platform for whole protein mass spectrometry.     

Mouse allergen, lung function, and atopy in Puerto Rican children

Objective

To examine the relation between mouse allergen exposure and asthma in Puerto Rican children.

Methods

Mus m 1, Der p 1, Bla g 2, and Fel d 1 allergens were measured in dust samples from homes of Puerto Rican children with (cases) and without (controls) asthma in Hartford, CT (n = 449) and San Juan (SJ), Puerto Rico (n = 678). Linear or logistic regression was used for the multivariate analysis of mouse allergen (Mus m 1) and lung function (FEV₁ and FEV₁/FVC) and allergy (total IgE and skin test reactivity (STR) to ≥1 allergen) measures.

Results

Homes in SJ had lower mouse allergen levels than those in Hartford. In multivariate analyses, mouse allergen was associated with higher FEV₁ in cases in Hartford (+70.6 ml, 95% confidence interval (CI) = 8.6–132.7 ml, P = 0.03) and SJ (+45.1 ml, 95% CI =  −0.5 to 90.6 ml, P = 0.05). In multivariate analyses of controls, mouse allergen was inversely associated with STR to ≥1 allergen in non-sensitized children (odds ratio [OR] for each log-unit increment in Mus m 1 = 0.7, 95% CI = 0.5–0.9, P<0.01). In a multivariate analysis including all children at both study sites, each log-increment in mouse allergen was positively associated with FEV₁ (+28.3 ml, 95% CI = 1.4–55.2 ml, P = 0.04) and inversely associated with STR to ≥1 allergen (OR for each log-unit increment in Mus m 1 = 0.8, 95% CI = 0.6–0.9, P<0.01).

Conclusions

Mouse allergen is associated with a higher FEV₁ and lower odds of STR to ≥1 allergen in Puerto Rican children. This may be explained by the allergen itself or correlated microbial exposures.     

Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.     

Differences in rat dorsal striatal NMDA and AMPA receptors following acute and repeated cocaine-induced locomotor activation

Sprague-Dawley rats can be classified as low or high cocaine responders (LCRs or HCRs, respectively) based on their locomotor activity induced by an acute low dose of cocaine. Upon repeated cocaine exposure, LCRs display greater locomotor sensitization, reward, and reinforcement than HCRs. Altered glutamate receptor expression in the brain reward pathway has been linked to locomotor sensitization and addiction. To determine if such changes contribute to the differential development of locomotor sensitization, we examined protein levels of total, phosphorylated, and cell surface glutamate N-methyl D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors (Rs) following acute or repeated cocaine (10 mg/kg, i.p.) in LCRs, HCRs and saline controls. Three areas involved in the development and expression of locomotor sensitization were investigated: the ventral tegmental area (VTA), nucleus accumbens (NAc) and dorsal striatum (dSTR). Our results revealed differences only in the dSTR, where we found that after acute cocaine, GluN2B(Tyr-1472) phosphorylation was significantly greater in LCRs, compared to HCRs and controls. Additionally in dSTR, after repeated cocaine, we observed significant increases in total GluA1, phosphorylated GluA1(Ser-845), and cell surface GluA1 in all cocaine-treated animals vs. controls. The acute cocaine-induced increases in NMDARs in dSTR of LCRs may help to explain the more ready development of locomotor sensitization and susceptibility to addiction-like behaviors in rats that initially exhibit little or no cocaine-induced activation, whereas the AMPAR increases after repeated cocaine may relate to recruitment of more dorsal striatal circuits and maintenance of the marked cocaine-induced locomotor activation observed in all of the rats.     

Telomerase regulation

The intimate connection between telomerase regulation and human disease is now well established. The molecular basis for telomerase regulation is highly complex and entails multiple layers of control. While the major target of enzyme regulation is the catalytic subunit TERT, the RNA subunit of telomerase is also implicated in telomerase control. In addition, alterations in gene dosage and alternative isoforms of core telomerase components have been described. Finally, telomerase localization, recruitment to the telomere and enzymology at the chromosome terminus are all subject to modulation. In this review we summarize recent advances in understanding fundamental mechanisms of telomerase regulation.     

Novel small-molecule inhibitors of hepatitis C virus entry block viral spread and promote viral clearance in cell culture

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection.