Methyl-β-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue

This study was designed to determine whether methyl-β-cyclodextrin (MCD) can substitute for albumin in incubation medium for neonatal swine adipose tissue explants, or whether MCD affects metabolism and cytokine expression. Subcutaneous adipose tissue explants (100 ± 10 mg) were prepared from 21-day-old pigs. Explants were incubated in medium 199 supplemented with 25 mM HEPES, 1.0 nM insulin at 37°C. The medium also contained bovine serum albumin (BSA) or MCD at 0%, 0.05%, 0.1%, 0.2% or 0.3%. Tissue explants were treated with these media for 1 h and then switched to the same basal incubation medium containing 0.05% BSA. Explants were removed from basal medium at 2 or 8 h of incubation, and real-time PCR was performed to assess expression of tumor necrosis α (TNF) and interleukin 6 (IL6), acetyl CoA carboxylase (ACAC) and fatty acid synthase (FASN). Alternatively, rates of ¹⁴C-glucose oxidation and lipogenesis were monitored ± insulin (100 nM), following MCD treatment. Incubation with BSA had minimal effects on gene expression or adipose tissue metabolism, only producing a doubling in TNF mRNA abundance (P < 0.01). Treatment with MCD increased TNF mRNA abundance by eightfold (P < 0.009), whereas IL6 gene expression increased a 100-fold (P < 0.001) with a suppression in ACAC and FASN expression (P < 0.01). This was paralleled by MCD inhibition of insulin-stimulated glucose oxidation and lipogenesis (P < 0.001). Addition of a TNF antibody to the incubation medium alleviated this inhibition of insulin-stimulated glucose metabolism by ∼30% (P < 0.05).     

Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase Ⅱ (EG Ⅱ) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR.It was cloned into the expression vector pGAPZαA.The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being lin-earized by BspHI digestion.The recombinant Pichia pas-toris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG Ⅱ were also explored.     

Cells previously desensitized to type 1 interferons display different mechanisms of activation of stat-dependent gene expression from naïve cells

Over the past decade, a wealth of knowledge has been obtained concerning the mechanisms by which interferons (IFNs) and other cytokines activate or down-regulate immediate early genes via the Jak/Stat pathway. In contrast, little information is available on interferon-activated gene expression in na?ve cells compared with cells that have been desensitized and subsequently resensitized to the actions of these cytokines. In na?ve cells, the ISG54 gene is activated via IFN beta-stimulated formation of ISGF3, a heterotrimeric DNA binding complex consisting of p48 (IRF9) and tyrosine-phosphorylated Stat1 and Stat2. In contrast, in previously desensitized cells IFN beta weakly stimulates the assembly of an ISGF3-like complex that lacks Stat1, even though ISG54 mRNA induction is the same as in na?ve cells. The lack of Stat1 tyrosine phosphorylation and DNA binding is due to increased activity of a protein-tyrosine phosphatase. In cells that do not express the tyrosine phosphatase Tc-PTP, the rate of Stat1 dephosphorylation is the same in na?ve and previously desensitized cells. These results implicate Tc-PTP in a novel role in the regulation of type 1 interferon-stimulated gene expression.     

Neuregulin 1-β regulates cell adhesion molecule L1 expression in the cortex and hippocampus of mice

Neuregulin 1 (Nrg1) functions in neuronal migration, survival and differentiation as well as synaptogenesis during ontogenetic development and maintenance of synaptic functions in the adult mammalian brain. The neural adhesion molecule L1 (L1CAM) functions in similar overlapping, but also non-overlapping roles in the nervous system. In the present study, we therefore investigated some aspects of the functional relationship between Nrg1 and L1 in mammalian neural cells. Nrg1 regulates the expression of L1 in cultures of both human neuroblastoma SK-N-SH cells and mouse cortical and hippocampal neurons. To analyze the role of Nrg1 on L1 expression in vivo, young adult male mice received intraperitoneal injections of Nrg1 or PBS (vehicle control). The correlation between Nrg1 and L1 expression was tested by qPCR, Western blot analysis, and immunocytology. Our data indicate that neuregulin 1-β (Nrg1β) increases L1 expression in neurons of the cerebral cortex, and decreases expression in neurons of the hippocampus in vitro and in vivo. In addition, Nrg1 induces phosphorylation of its receptors, ErbB2 and ErbB4, the predominant ErbB receptors in the nervous system. These results show that Nrg1β affects expression of L1 in the central nervous system and in parallel activates the ErbB receptors for Nrg1, suggesting a crosstalk between molecules that are of prime importance for nervous system functions.     

Alteration of gene expression in reactive astrocytes induced by Aβ1-42 using low dose of methamphetamine

Molecular Biology Reports - Alzheimer's disease (AD) is a degenerative brain disorder. Due to the relationship between the functional loss of astrocytes and AD, the present study aims to...     

Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase II (EG II) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR. It was cloned into the expression vector pGAPZαA. The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being linearized by BspHI digestion. The recombinant Pichia pastoris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG II were also explored. __________ Translated from Microbiology, 2006, 33(6): 68273 [译自: 微生物学 通报]     

Muscle gene expression of CGRP-α, CGRP receptor,nAchR-β, and GDNF in response to different endurance training protocols of Wistar rats

Molecular Biology Reports - The neuromuscular junction underwent adaptations to meet the demands of muscles following increased muscle activity. This study aimed to investigate the effects of...     

α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers

Introduction

Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.

Methods

All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.

Results

In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.

Conclusions

The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.     

Effects of methylation of deiodinase 3 gene on gene expression and severity of Kashin–Beck disease

Kashin–Beck disease (KBD) is a complex endemic osteoarthropathy, which mainly occurs in the northeast to southwest China. Iodothyronine deiodinases 3 (DIO3) is one of the selenoproteins, which is closely related to bone metabolism and unclear to KBD. This study aims to investigate the role and associated mechanisms of methylation and expression of DIO3 with disease severity in patients with KBD. We performed a bioinformatics analysis first to identify the biological mechanisms involved in selenoproteins. The methylation status of the DIO3 gene and DIO3 gene expression, as well as DIO3-related regulatory genes in patients with KBD, were analyzed. We found that 15 CpG sites of six selenoproteins were hypomethylated with 5-azacytidine treatment. DIO3 hypermethylation was associated with an increased risk of KBD and may lead to downregulation of DIO3 gene expression as well as be an indicator of the severity of KBD, which may provide a new insight for gene–environment correlations and interactions in etiology and pathogenesis of KBD.     

Are data from different gene expression microarray platforms comparable?

Many commercial and custom-made microarray formats are routinely used for large-scale gene expression surveys. Here, we sought to determine the level of concordance between microarray platforms by analyzing breast cancer cell lines with in situ synthesized oligonucleotide arrays (Affymetrix HG-U95v2), commercial cDNA microarrays (Agilent Human 1 cDNA), and custom-made cDNA microarrays from a sequence-validated 13K cDNA library. Gene expression data from the commercial platforms showed good correlations across the experiments (r = 0.78-0.86), whereas the correlations between the custom-made and either of the two commercial platforms were lower (r = 0.62-0.76). Discrepant findings were due to clone errors on the custom-made microarrays, old annotations, or unknown causes. Even within platform, there can be several ways to analyze data that may influence the correlation between platforms. Our results indicate that combining data from different microarray platforms is not straightforward. Variability of the data represents a challenge for developing future diagnostic applications of microarrays.