Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.     

Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape

Background  

Universally accepted landmark stages are necessary to highlight key events in plant reproductive development and to facilitate comparisons among species. Domestication and selection of tomato resulted in many varieties that differ in fruit shape and size. This diversity is useful to unravel underlying molecular and developmental mechanisms that control organ morphology and patterning. The tomato fruit shape gene SUN controls fruit elongation. The most dramatic effect of SUN on fruit shape occurs after pollination and fertilization although a detailed investigation into the timing of the fruit shape change as well as gene expression profiles during critical developmental stages has not been conducted.     

Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.     

Sequence features involved in the mechanism of 3' splice junction wobbling

Background  

Alternative splicing is an important mechanism mediating the diversified functions of genes in multicellular organisms, and such event occurs in around 40-60% of human genes. Recently, a new splice-junction wobbling mechanism was proposed that subtle modifications exist in mRNA maturation by alternatively choosing at 5'- GTNGT and 3'- NAGNAG, which created single amino acid insertion and deletion isoforms.     

Mite allergen Der-p2 triggers human B lymphocyte activation and Toll-like receptor-4 induction

Background

Allergic disease can be characterized as manifestations of an exaggerated inflammatory response to environmental allergens triggers. Mite allergen Der-p2 is one of the major allergens of the house dust mite, which contributes to TLR4 expression and function in B cells in allergic patients. However, the precise mechanisms of Der-p2 on B cells remain obscure.

Methodology/Principal Findings

We investigated the effects of Der-p2 on proinflammatory cytokines responses and Toll-like receptor-4 (TLR4)-related signaling in human B cells activation. We demonstrated that Der-p2 activates pro-inflammatory cytokines, TLR4 and its co-receptor MD2. ERK inhibitor PD98059 significantly enhanced TLR4/MD2 expression in Der-p2-treated B cells. Der-p2 markedly activated mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) and decreased p38 phosphorylation in B cells. MKP-1-siRNA downregulated TLR4/MD2 expression in Der-p2-treated B cells. In addition, Der-p2 significantly up-regulated expression of co-stimulatory molecules and increased B cell proliferation. Neutralizing Der-p2 antibody could effectively abrogate the Der-p2-induced B cell proliferation. Der-p2 could also markedly induce NF-κB activation in B cells, which could be counteracted by dexamethasone.

Conclusions/Significance

These results strongly suggest that Der-p2 is capable of triggering B cell activation and MKP-1-activated p38/MAPK dephosphorylation-regulated TLR4 induction, which subsequently enhances host immune, defense responses and development of effective allergic disease therapeutics in B cells.     

Rat brain aryl acylamidase: multiple forms and inhibition effects of LSD, serotonin and related compounds

At least two forms of aryl acylamidase (E.C.3.5.1.13, AAA) were separated from rat brain extracts by ammonium sulfate precipitation (33–60% saturation) and subsequent Bio-Gel column chromatography. Fraction AAA-1 showed pH optimum at 7.5 whereas AAA-2 showed a pH optimum at 5.5 AAA-1 activity was markedly inhibited at pH 7.5 by d-LSD and 2-Br-LSD, moderately inhibited by 5-HT and slightly inhibited by tryptamine but it was not affected by 1-LSD, at 0.1 mM concentration. AAA-2 was only moderately inhibited at pH 5.5 by d-LSD and 2-Br-LSD but not affected by 1-LSD, 5-HT or tryptamine at the same concentrations. Catecholamines and their structurally related drugs had no significant effects on either enzyme activity. Kinetic studies with AAA-1 indicated competitive inhibition by d-LSD with a K_i value of 4.90 ± 0.61 μM.     

EGF upregulates Na+/H+ exchanger NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness

Na+/H+ exchanger 1 (NHE1) is involved in cell migration but little is known about the signal pathways that regulate NHE1 activity and that are associated with tumor cell invasiveness. This study is to investigate the mechanisms by which epidermal growth factor (EGF) regulates NHE1 expression to promote cervical cancer cell invasiveness and the clinical significance in early-stage cervical cancer. NHE1 protein was scanty in normal or noncancerous cervical tissues of all surgical specimens examined (n = 92). Tumor tissues clearly expressed NHE1 protein with different amounts. The differential expression level of NHE1 is associated with the clinical outcome. NHE1 protein was also differentially expressed between normal cervical epithelial cells and two cervical cancer cell lines. Cervical cancer cells benefit some enhanced cellular functions from NHE1 abundance, such as cell volume regulation, migration, and invasion. Interestingly, NHE1 colocalized with EGF in cervical cancer tissues. Studies in cell culture systems indicated that EGF-stimulated NHE1 abundance in a time-dependent manner by post-translational regulation. This implies a likely autocrine or paracrine EGF stimulation of NHE1 production in vivo. In addition, the phosphoinositide 3-kinase pathway is the dominant signal controlling EGF-stimulated NHE1 abundance. Pharmacological inhibition of NHE1 activity markedly inhibited the basal and EGF-stimulated cervical cancer cell migration. Image studies and immunoprecipitaion experiments suggest that EGF-induced NHE1 translocation to the leading-edge lamellipodia, where NHE1 interacted with actin-associated protein Ezrin, thereby remodeling cytoskeleton and stimulating cervical cancer cell migration. In conclusion, EGF upregulates NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.     

Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis

A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant γ-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6–8 and 40 °C, respectively. The chloride salt of metal ions Mg²⁺, K⁺, and Na⁺ can activate BLrGGT, whereas that of Pb²⁺ dramatically inhibited it. The substrate specificity study showed that l-γ-glutamyl-p-nitroanilide (l-γ-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k _cat of 105 s⁻¹ and a K _m of 21 μM when using l-γ-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important γ-glutamyl compounds.     

Effect of red mold rice supplements on serum and meat cholesterol levels of broilers chicken

Monacolin K is a secondary metabolite produced by Monascus species. It was found that it is able to decrease cholesterol levels. In this study, red mold rice (RMR) was added to the diet of Arbor Acres broiler chickens, and the cholesterol level in meat, as well as the concentration of triglyceride, the high-density lipoprotein cholesterol (HDL-C), and the low-density lipoprotein cholesterol (LDL-C) in the serum were evaluated. Four-week-old broilers are studied and divided into four groups in that each group contains 15 subjects. A 3-week experimental feeding trial was conducted in which three groups of broilers were fed 2.0, 5.0, and 8.0% of RMR (RMR groups) within their diet, respectively, and the result was compared to the control group. The results indicated that for each RMR group, the cholesterol content was significantly lower than that of the control group; in addition, their meat products contain higher level of unsaturated fatty acids. Triglyceride and cholesterol concentration in serum was also found to be considerably lower in RMR groups when compared to control group. Finally, in RMR groups, HDL-C/LDL-C and HDL-C/cholesterol ratios were all higher than those of the control group. In short, the results demonstrated that the cholesterol levels could be lowered by adding RMR to the diet of chickens.