Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

Ａｎｏｒｇａｎｉｓｍ,Ｓ.ｃｅｒｅｖｉｓｉａｅｗｉｄｅｌｙｕｓｅｄｉｎｂｒｅｗｉｎｇ,ｂａｋｉｎｇａｎｄｉｎｅｔｈａｎｏｌｐｒｏｄｕｃｔｉｏｎｐｒｏｃｅｓｓｅｓｉｓｎｏｔａｂｌｅｔｏｈｙｄｒｏｌｙｓｅｓｔａｒｃｈ.ＴｈｕｓｔｈｅｔｒａｄｉｔｉｏｎａｌｃｏｎｖｅｒｓｉｏｎｏｆｓｔａｒｃｈｉｎｔｏｅｔｈａｎｏｌａｎｄＣＯ2ｄｅｐｅｎｄｓｏｎｔｈｅａｄｄｉｔｉｏｎｏｆｔｈｅｅｎｚｙｍｅｓｐｒｉｏｒｔｏｆｅｒｍｅｎｔａｔｉｏｎ,ｗｈｉｃｈｌｅａｄｓｔｏｌｉｑｕｉｆｉｃａｔｉｏｎａｎｄｓａｃｃｈａｒｉｆｉｃａｔ…     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

Ｔｒａｎｓｃｒｉｐｔｉｏｎａｎｄｔｒａｎｓｌａｔｉｏｎａｒｅｔｗｏｓｔｅｐｓｏｆｇｅｎｅｅｘｐｒｅｓｓｉｏｎ.Ｔｒａｎｓｃｒｉｐｔｉｏｎ,ａｓａｃｏｎｔｒｏｌｓｔｅｐ,ｉｓａｍａｊｏｒａｓｐｅｃｔｉｎｒｅｇｕｌａｔｉｏｎｏｆｇｅｎｅｅｘｐｒｅｓｓｉｏｎ.Ｈｏｗｅｖｅｒ,ｔｈｅｒｅａｒｅａｎｕｍｂｅｒｏｆｅｘａｍｐｌｅｓｏｆｔｒａｎｓｌａｔｉｏｎａｌｃｏｎｔｒｏｌ.ＴｈｅｓｅｑｕｅｎｃｅｏｆＴＩＲ(ｔｒａｎｓｌａｔｉｏｎａｌｉｎｉｔｉａｔｉｏｎｒｅｇｉｏｎ)ｏｎｍＲＮＡｃａｎａｆｆｅｃｔｔｈｅｅｆｆｉｃｉ…     

大肠杆菌核糖体蛋白质L24和L27突变对λN和λQ基因表达的影响

核糖体是生物体翻译遗传密码的唯一场所。核糖体各组份的确切功能目前仍不清楚。本实验室的研究已证实：有些核糖体蛋白质突变能在翻译水平上影响核糖体翻译的特异性。本文使用转座子Tn10将核糖蛋白质L24（rpmA）突变从A19的衍生株转到T83菌株上,并研究这2个突变对λN和λQ基因及对N-lacZ和Q-lacZ融合基因表达的不同影响,为核糖体蛋白质能影响核糖体翻译的特异性提供了又一个实例。     

Analysis of leaky viral translation termination codons in vivo by transient expression of improved β-glucuronidase vectors

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of -glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Zinc transporter gene expression is regulated by pro-inflammatory cytokines: a potential role for zinc transporters in beta-cell apoptosis?

Background

Numerous studies have reported that age-induced increased parathyroid hormone plasma levels are associated with cognitive decline and dementia. Little is known about the correlation that may exist between neurological processing speed, cognition and bone density in cases of hyperparathyroidism. Thus, we decided to determine if parathyroid hormone levels correlate to processing speed and/or bone density.

Methods

The recruited subjects that met the inclusion criteria (n = 92, age-matched, age 18-90 years, mean = 58.85, SD = 15.47) were evaluated for plasma parathyroid hormone levels and these levels were statistically correlated with event-related P300 potentials. Groups were compared for age, bone density and P300 latency. One-tailed tests were used to ascertain the statistical significance of the correlations. The study groups were categorized and analyzed for differences of parathyroid hormone levels: parathyroid hormone levels <30 (n = 30, mean = 22.7 ± 5.6 SD) and PTH levels >30 (n = 62, mean = 62.4 ± 28.3 SD, p ≤ 02).

Results

Patients with parathyroid hormone levels <30 showed statistically significantly less P300 latency (P300 = 332.7 ± 4.8 SE) relative to those with parathyroid hormone levels >30, which demonstrated greater P300 latency (P300 = 345.7 ± 3.6 SE, p = .02). Participants with parathyroid hormone values <30 (n = 26) were found to have statistically significantly higher bone density (M = -1.25 ± .31 SE) than those with parathyroid hormone values >30 (n = 48, M = -1.85 ± .19 SE, p = .04).

Conclusion

Our findings of a statistically lower bone density and prolonged P300 in patients with high parathyroid hormone levels may suggest that increased parathyroid hormone levels coupled with prolonged P300 latency may become putative biological markers of both dementia and osteoporosis and warrant intensive investigation.     

The gene expression of the myocardial lipid droplet protein is highly regulated by PPARγ in adipocytes differentiated from MEFs or SVCs

We demonstrate that expression of the myocardial lipid droplet protein (MLDP) and ERα observed in adipose tissues is undetectable in 3T3-L1 cells but detectable in mouse embryonic fibroblasts (MEFs) and stromal-vascular cells (SVCs) during adipocyte differentiation. MLDP gene expression in MEFs or SVCs is induced by treatment with a PPARγ agonist or forced expression of PPARγ, indicating that PPARγ enhances MLDP expression during adipogenesis. PCR analyses reveal the dual expression of SREBP-1a and SREBP-1c in MEFs and SVCs as well as white adipose tissues unlike the predominant expression of SREBP-1a in 3T3-L1 cells. These results suggest that MEFs and SVCs are useful model cells for examining function of MLDP in lipid droplet formation and adipocyte differentiation.     

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for -glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3 flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5 expression control sequences.     

Up-regulation of interferon-stimulated gene 15 and its conjugation machinery,UbE1L and UbcH8 expression by tumor necrosis factor-α through p38 MAPK and JNK signaling pathways in human lung carcinoma

Exosomes are nanometer-sized vesicles involved in intercellular communication, and they are released by various cell types. To learn about exosomes produced by Schwann cells (SCs) and to explore their potential function in repairing the central nervous system (CNS), we isolated exosomes from supernatants of SCs by ultracentrifugation, characterized them by electron microscopy and immunoblotting and determined their protein profile using proteomic analysis. The results demonstrated that Schwann cell-derived exosomes (SCDEs) were, on average, 106.5 nm in diameter, round, and had cup-like concavity and expressed exosome markers CD9 and Alix but not tumor susceptibility gene (TSG) 101. We identified a total of 433 proteins, among which 398 proteins overlapped with the ExoCarta database. According to their specific functions, we identified 12 proteins that are closely related to CNS repair and classified them by different potential mechanisms, such as axon regeneration and inflammation inhibition. Gene Oncology analysis indicated that SCDEs are mainly involved in signal transduction and cell communication. Biological pathway analysis showed that pathways are mostly involved in exosome biogenesis, formation, uptake and axon regeneration. Among the pathways, the neurotrophin, PI3K-Akt and cAMP signaling pathways played important roles in CNS repair. Our study isolated SCDEs, unveiled their contents, presented potential neurorestorative proteins and pathways and provided a rich proteomics data resource that will be valuable for future studies of the functions of individual proteins in neurodegenerative diseases.

     

Baked corn (Zea mays L.) and bean (Phaseolus vulgaris L.) snack consumption lowered serum lipids and differentiated liver gene expression in C57BL/6 mice fed a high-fat diet by inhibiting PPARγ and SREBF2

The aim was to determine the effect of consuming a baked white corn/bean snack (70/30% blend) on improving diet-induced dyslipidemia and liver differential gene expression in mice fed a high-fat diet (HFD). C57BL/6 mice were randomized into six groups and different doses of the snack (0.5–2.0 g/d) supplemented to a basal HFD for 12 weeks. Unsupplemented HFD and a standard diet were used as positive and negative controls, respectively. Groups receiving HFD1.0, HFD1.5 and HFD2.0 showed attenuation in body weight gain (20%). Serum cholesterol and triglycerides were reduced (P<.05), 29% and 31%, respectively. Blood glucose was also reduced (P<.05) in all groups receiving the snack. Histological analysis showed a reduction in adipocyte diameters (P<.05) suggesting an attenuation of lipid accumulation. Snack consumption induced differential expression of 529 genes in the liver; RGS16 was the highest up-regulated molecule (+15-fold change). Increased expression of this gene could have improved glucose metabolism in HFD2.0. Ingenuity Pathway Analysis downstream analysis showed a predicted inhibition of target genes of peroxisome PPARγ and key regulators of lipogenic genes in the liver. The results suggest that consumption of a white corn/bean snack (70%/30% blend) attenuates weight gain, fat mass accumulation, adipocyte size and nonalcoholic fatty liver disease in HFD-fed mice by inhibiting PPARγ and SREBF2. The study proposes that this type of product might be beneficial by preventing dyslipidemia, obesity and hepatic steatosis.