Suppression of ceramide-induced cell death by hepatitis C virus core protein

The hepatitis C virus (HCV) core protein is believed to be one of viral proteins that are capable of preventing virus-infected cell death upon various stimuli. But, the effect of the HCV core protein on apoptosis that is induced by various stimuli is contradictory. We examined the possibility that the HCV core protein affects the ceramide-induced cell death in cells expressing the HCV core protein through the sphingomyelin pathway. Cell death that is induced by C(2)-ceramide and bacterial sphingomyelinase was analyzed in 293 cells that constitutively expressed the HCV core protein and compared with 293 cells that were stably transfected only with the expression vector. The HCV core protein inhibited the cell death that was induced by these reagents. The protective effects of the HCV core protein on ceramide-induced cell death were reflected by the reduced expression of p21(WAF1/Cip1/Sid1) and the sustained expression of the Bcl-2 protein in the HCV core-expressing cells with respect to the vector-transfected cells. These results suggest that the HCV core protein in 293 cells plays a role in the modulation of the apoptotic response that is induced by ceramide. Also, the ability of the HCV core protein to suppress apoptosis might have important implications in understanding the pathogenesis of the HCV infection.     

Ubiquitin and Ubiquitin-like Modifiers in Plants

Posttranslational modifications of proteins by small polypeptides including ubiquitination, neddylation (related to ubiquitin (RUB) conjugation), and sumoylation are implicated in plant growth and development, and they regulate protein degradation, location, and interaction with other proteins. Ubiquitination mediates the selective degradation of proteins by the ubiquitin (Ub)/proteasome pathway. The ubiquitin-like protein RUB is conjugated to cullins, which are part of a ubiquitin E3 ligase complex that is involved in auxin hormonal signaling. Sumoylation, by contrast, is known for its involvement in guiding protein interactions related to abiotic and biotic stresses and in the regulation of flowering time. ATG8/ATG12-mediated autophagy influences degradation and recycling of cellular components. Other ubiquitin-like modifiers (ULPs) such as homology to Ub-1, ubiquitin-fold modifier 1, and membrane-anchored Ub-fold are also found in Arabidopsis. ULPs share similar three-dimensional structures and a conjugation system, including E1 activating enzymes, E2 conjugation enzymes, and E3 ligases, as well as proteases for deconjugation and recycling of the tags. However, each of the ULP posttranslational modifications possesses its own specific enzymes and modifies its specific targets selectively. This review discusses recent findings on ubiquitination and ubiquitin-like modifier processes and their roles in the posttranslational modification of proteins in Arabidopsis.     

Microbial flocculant from Arcuadendron sp. TS-49

Summary A flocculant was purified from the culture broth of Archuadendron sp. TS-49 by a series of precipitations with acetone, 60% ammonium sulfate-butanol, salting-out by dialysis, and cetylpyridinium chloride. The flocculating activity was observed most highly at pH 3.0 and markedly enhanced by the addition of salts, especially in the case of FeCl₃ or FeSO₄. This bioflocculant efficiently flocculated all tested solids, including various microorganisms and organic/ inorganic materials. Qualitative analyses of the bioflocculant showed that it might contain hexosamine, uronic acid, neutral sugar, and protein.     

Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

Time-calibrated phylogenomics of the porcine epidemic diarrhea virus: genome-wide insights into the spatio-temporal dynamics

Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea (PED), has led to tremendous economic losses in the global swine industry. Although the phylogeny of PEDV has been investigated extensively at the molecular level, there was no time-calibrated phylogenomic study on the virus. To improve insight into this topic, we analyzed 138 published genome sequences using the Bayesian coalescent analyses as well as Bayesian inferences and maximum likelihood methods. All of the global PEDV isolates were divided into six groups, except for one unclassified isolate. Of the six groups, Groups 1–5 comprised pandemic viruses while the remaining Group 6 contained classical isolates. Interestingly, the two clades, both pandemic and classical, consisted of clade-specific amino acid sequences in five genes: ORF1a, ORF1b, S, ORF3, and N. Within the pandemic clade, Group 1 and Group 2 originated from North America, whereas Group 3–Group 5 were derived from Asia. In Group 2, there was a common origin of S INDEL isolates. Within each group, there was no apparent association between temporal or geographic origin and heterogeneity of PEDVs. Our findings also showed that the PEDV virus evolved at a rate of 3.38?×?10^?4 substitutions/site/year, and the most recent common ancestor of the virus emerged 75.9 years ago. Our Bayesian skyline plot analysis indicated that the PEDV had maintained constant effective population size excluding only a short period, around 2012, when a valley shaped decline in the effective number of infections occurred.     

cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-β. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.     

Targeting of focal adhesion kinase by small interfering RNAs reduces chondrocyte redifferentiation capacity in alginate beads culture with type II collagen

Type II collagen is a major protein that maintains biological and mechanical characteristics in articular cartilage. Focal adhesion kinase (FAK) is known to play a central role in integrin signaling of cell–extracellular matrix (ECM) interactions, and chondrocyte–type II collagen interactions are very important for cartilage homeostasis. In this study, we focused on phosphorylation of FAK and MAP kinase in chondrocyte–type II collagen interaction and dedifferentiation, and the effects of FAK knockdown on chondrocyte‐specific gene expression and cell proliferation were determined. The addition of exogenous type II collagen to chondrocytes increased levels of tyrosine phosphorylation, p‐FAK_Y397, and p‐ERK1/2. In contrast, expression levels of p‐FAK_Y397 and p‐ERK1/2, but not p‐Smad2/3, were decreased in dedifferentiated chondrocytes with loss of type II collagen expression. Type II collagen expression was significantly increased when dedifferentiated chondrocytes were transferred to alginate beads with TGF‐β1 or type II collagen, but transfected cells with small interfering RNA for FAK (FAK‐siRNA) inhibited mRNA expression of type II collagen and SOX‐6 compared to the control. These FAK‐siRNA‐transfected cells could not recover type II collagen even in the presence of TGF‐β1 or type II collagen in alginate beads culture. We also found that FAK‐siRNA‐transfected cells decreased cell proliferation rate, but there was no effect on glycosaminoglycans (GAGs) secretion. We suggest that FAK is essentially required in chondrocyte communication with type II collagen by regulating type II collagen expression and cell proliferation. J. Cell. Physiol. 218: 623–630, 2009. © 2008 Wiley‐Liss, Inc.     

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol‐butanol‐ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1083–1088, 2013