Musashi expression in ��-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic β-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates β-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of β-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and β-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and β-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory β-cell proliferation and the loss of β-cell gene expression seen in specific phases of the progression to type 2 diabetes.     

Fungal β-glucosidase expression in Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae strains expressing β-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) were constructed and compared to S. cerevisiae Y294[SFI], previously identified as the best β-glucosidase-producing strain. The PcbglB was also intracellularly expressed in combination with the lac12 lactose permease of Kluyveromyces lactis in S. cerevisiae Y294[PcbglB?+?Lac12]. The recombinant extracellular β-glucosidases indicated maximum activity in the pH range 4-5 and temperature optima varying from 50 to 75?°C. The S.?cerevisiae Y294[Pccbgl1] strain performed best under aerobic and anaerobic conditions, producing 2.6 times more β-glucosidase activity than S. cerevisiae Y294[SFI] and an ethanol concentration of 4.8?g?l(-1) after 24?h of cultivation on cellobiose as sole carbohydrate source. S. cerevisiae Y294[Tabgl1] was unable to grow on cellobiose (liquid medium), whereas S. cerevisiae Y294[PcbglB?+?Lac12] exhibited limited growth.     

A common element in epidermal expression?

Members of an epidermally expressed gene cluster on human chromosome 21 each contain a short sequence similar to an element that can drive ectoderm-specific gene expression.     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.     

Sex-associated difference in estrogen receptor β expression in N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric cancers in rats

Epidemiologic studies indicate that the incidence of gastric cancer is higher in males than in females. Although the mechanisms mediating this difference are unclear, a role for estrogens has been proposed. We used Western blotting to evaluate the role of estrogen receptor (ER) subtypes ERα and ERβ and proliferating cell nuclear antigen (PCNA) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats; ERα and ERβ mRNA levels also were analyzed by quantitative real-time RT-PCR analysis. The incidence of gastric cancer was significantly higher in male than female rats. In both sexes, ERα expression was similar in MNNG-treated cancerous and noncancerous tissues and normal gastric tissue. However, ERβ expression in MNNG-treated cancerous and noncancerous tissues was significantly lower in male rats and higher in female rats than that in normal gastric tissue; MNNG-induced cancerous tissue showed the highest ERβ expression. PCNA expression in MNNG-treated cancerous tissues was higher than that in noncancerous tissues, and was higher in male rats than female rats. Western blotting results were consistent with the mRNA changes determined by quantitative real-time RT-PCR. The present study provides evidence of a sex-associated difference in ERβ and PCNA expression in MNNG-induced gastric cancers in Wistar rats.     

Α-syntrophin modulates myogenin expression in differentiating myoblasts

Background

α-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor.

Methods and Findings

The absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression.

Conclusions

We conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression.     

Developmental expression of β-glucosidase in olive leaves

Plant β-glucosidases catalyze the hydrolysis of glycosidic linkages and play a vital role in defense against pathogens and stress. The present work investigated the relationship between leaf development and β-glucosidase protein content in Olea europea L. (cv. Picual) leaves. The total chlorophyll content increased with leaf age in current-season leaves. Immunoblot analysis revealed that the content of 61 kD protein of β-glucosidase also increased with leaf age, and that the enzyme existed in three isoforms (pI 5.8–6.2). Statistical analysis indicated a strong correlation between chlorophyll and β-glucosidase protein contents.     

Genotype- and promoter-induced variability in transient β-glucuronidase expression in pea protoplasts

Summary Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of -glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 F; and using 125 g of calf thymus carrier DNA and 75 of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.NRCC No. 30910     

TGF-β polymorphism and its expression correlated with CXCR4 expression in human breast cancer

The role of chemokines and the growth factors has been extensively analyzed both in cancer risk and tumor progression. The transforming growth factor beta (TGF-β) and chemokine (C-X-C motif) receptor 4 (CXCR4) genes are implicated in several diseases, including breast cancer. Genomic DNA was obtained from 21 samples of peripheral blood or from normal tissue, previously fixed in formalin and embedded in paraffin for TGF-β T869C polymorphism analyses. Total cellular RNA was extracted from the same 21 patients, but from fresh tissue (tumor and adjacent healthy from the same breast) for expression analysis by Real Time PCR. No significant differences were observed in genotype distribution according to clinicopathological characteristics. Transforming growth factor beta (TGF-β) mRNA expression was assessed according to T869C polymorphism and CC patients presented a higher TGF-β expression but not significant when compared to other genotypes (p?=?0.064). A positive correlation was observed in relative mRNA expressions of CXCR4 and TGF-β (p?=?0.020). It is known that overexpression of TGF-β by both tumor and stromal tissue can facilitate the development of metastases, mainly by TGF-β stimulated angiogenesis and increased tumor cell motility. Our findings suggested a role of these genes as progression markers for breast carcinoma.     

Muscarinic receptor-mediated increase in ζ-PKC expression in SK-N-SH human neuroblastoma cells

Anti-peptide antibodies specific for each protein kinase C (PKC) isozyme were used to screen SKN-SH human neuroblastoma cells. These cells were found to express only - and -PKC. Stimulation of these cells with phorbol esters caused -but not -PKC to translocate from cytosolic to membrane fractions. Stimulation of these cells with carbachol, which releases inositol trisphosphate and diacylglycerol, caused a transient translocation of -PKC but not of -PKC. Carbachol did, however, cause a gradual increase in immunoreactive -PKC which reached maximal values 10–20 min after stimulation. These results implicate -PKC in a receptor-mediated signalling event.     

Direct regeneration in vitro and transient GUS expression in Mentha×piperita

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient -glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l^–1) and 6-benzylaminopurine (2 mg l^–1), followed by selection of regenerated shoots with 200 mg 1^–1 kanamycin.Abbreviations BA 6-benzylaminopurine - GUS -glucuronidase - MS Murashige and Skoog (1962) - NAA -naphthalenacetic acid - PIG particle inflow gun - SEM scanning electron microscope     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

Resveratrol induces insulin gene expression in mouse pancreatic α-cells

Background

Type 1 and type 2 diabetes are characterized by loss of β-cells; therefore, β-cell regeneration has become one of the primary approaches to diabetes therapy. Resveratrol, a naturally occurring polyphenolic compound, has been shown to improve glycaemic control in diabetic patients, but its action on pancreatic α-cells is not well understood.

Findings

Using mouse α-cells (αTC9), we showed that resveratrol induces expression of pancreatic β-cell genes such as Pdx1 and Ins2 in a SirT1-dependent manner. The mRNA and protein levels of insulin were further increased by histone deacetylase (HDAC) inhibition.

Conclusion

In summary, we provide new mechanistic insight into the anti-diabetic action of resveratrol through its ability to express β-cell genes in α-cells.