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1.
Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.  相似文献   
2.
A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.  相似文献   
3.
The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.  相似文献   
4.
Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.  相似文献   
5.
Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer.  相似文献   
6.
Electroantennograms (EAGs) were recorded from unmated, laboratory-reared, male and female oriental fruit flies, Dacus dorsalis, in response to a range of between C1 and C12 carbon chain-length saturated and unaturated aliphatic alcohols and aldehydes, most all of which are known host-plant volatiles. Only two of the 35 compounds tested elicited significantly larger EAGs from female than male antennae. For the two functional-group series tested, aldehydes elicited responses greater than or equal to the responses to the alcohols. In general, the unsaturated alcohols did not elicit responses significantly different from the saturated alcohols. However, the unsaturated aldehydes, (E)-2-hexenal and 10-undecenal, elicited larger amplitude EAGs than their saturated analogs. EAGs were significantly greater for a particular carbon chain-length, with responsiveness to primary alcohols peaking at C6 and aldehydes peaking at C7. The (E)-2- monoenic alcohols peaked at C6, while the (E)-3-alcohols plateaued between C5 and C8. The greatest EAG responses of all compounds tested were elicited by the saturated and unsaturated C6 alcohols and aldehydes which are constitutents of the general green-leaf volatile complex that emanates from most plants. The potential adapative benefit of selective sensitivity to green-leaf volatiles is discussed in regards to foraging behaviors of oriental fruit flies.
Résumé Des électroantennogrammes (EAG) ont enregistré les réponses, en élevages de femelles et mâles vierges de Dacus dorsalis, à une gamme de chaînes de carbones de C1 à C12 saturés et non-saturés d'alcools aliphatiques et d'aldéhydes, dont beaucoup sont connus comme substances volatiles des végétaux. Seulement 2 des 35 composés examinés ont provoqué des EAG significativement plus importants chez les femelles que chez les mâles. Pour les séries des deux groupes fonctionnels examinés, les aldéhydes ont provoqué des réponses supérieures ou égales aux alcools. En général, les réponses aux alcools nonsaturés n'étaient pas significativement différentes des réponses aux alcools saturés. Cependant, les aldéhydes non-saturés, (E)-2-hexénal et 10-undécénal, ont induit des EAG de plus grande ampleur que leurs analogues saturés. Les EAG étaient significativement les plus importants pour une chaîne de longueur particulière, la réponse aux alcools primaires culminant en C6 et les aldéhydes en C7. Les alcools monoéniques (E)-2- culminaient en C6, tandis que les alcools (E)-3- étaient étales entre C5 et C8. Les EAG les plus importants ont été obtenus pour tous les composés examinés avec les alcools et aldéhydes en C6 qui appartiennent à l'odeur verte complexe émise par beaucoup de plantes. Le bénéfice adaptatif potentiel de la sensibilité sélective à l'odeur verte des feuilles est examinée en fonction du comportement de prospection de D. dorsalis.
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7.
8.
Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.  相似文献   
9.
Isolated microspores of rice (Oryza saliva L.) cultivars, IR36and IR43, belonging to the recalcitrant indica subspecies werecultured. Two types of microspores were observed after isolationfrom the fresh anthers and from pre-cultured anthers—onetype consisted of vacuolated, larger-sized grains, while theother was composed of microspores of smaller sizes with densecytoplasm. Within few days in culture, all the smaller sizedgrains were dead, and only the large grains were viable andproduced pollen embryos. After 30 days from culture, microcalliwere transferred to semisolid modified Murashige and Skoog mediumcontaining 1 mg/liter each of kinetin and naphthaleneaceticacid and kept under continuous light at 25?C. IR36 showed onlycell division while IR43 gave 32 green plants from these experiments. (Received January 18, 1990; Accepted July 4, 1990)  相似文献   
10.
Expression vectors that yield mono-, di-, and tricistronic mRNAs upon transfection of COS-1 cells were used to assess the influence of the 5' nontranslated regions (5'NTRs) on translation of reporter genes. A segment of the 5'NTR of encephalomyocarditis virus (EMCV) allowed translation of an adjacent downstream reporter gene (CAT) regardless of its position in the mRNAs. A deletion in the EMCV 5'NTR abolishes this effect. Poliovirus infection completely inhibits translation of the first cistron of a dicistronic mRNA that is preceded by the capped globin 5'NTR, whereas the second cistron preceded by the EMCV 5'NTR is still translated. We conclude that the EMCV 5'NTR contains an internal ribosomal entry site that allows cap-independent initiation of translation. mRNA containing the adenovirus tripartite leader is also resistant to inhibition of translation by poliovirus.  相似文献   
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