Secretion of the STA3 heat-stable enterotoxin of Escherichia coli: extracellular delivery of Pro-STA is accomplished by either Pro or STA

The methanol-soluble, heat-stable enterotoxin of Escherichia coli is a protease-resistant extracellular peptide which is synthesized as a 72-amino-acid precursor Pre-Pro-STA3. The specific roles of Pre (19 amino acids), Pro (34 amino acids) and STA3 (19 amino acids) in the secretion process were studied by functionally deleting each of the three domains. Deletion of the Pre signal sequence resulted in a short-lived cell-associated molecule with an M(r) equivalent to that of Pro-STA3. Deletion of Pro (i.e., Pre-STA3) resulted in the rapid extracellular accumulation of STA3; the periplasmic intermediate found in the secretion of the wild-type toxin was undetected. Deletion of the STA3 domain resulted in a cell-associated Pre-Pro peptide; with time this form converted to periplasmic Pro which later became extracellular. When DNA encoding either STA3, by itself, or Pro-STA3 (lacking the signal peptide) was expressed, these peptides were degraded intracellularly, with no periplasmic or extracellular forms detected. The results presented demonstrate that the signal peptide (Pre) is essential even for the export of small peptides to the periplasm, and that its absence causes the STA3 domain to become susceptible to intracellular proteases. The rapid degradation of intracellular STA3 indicates that its proteolytic resistance is acquired in a compartment other than the cytoplasm. The results also show that after the Pre domain is proteolytically cleaved from Pre-STA3 and Pre-Pro, the STA3 and Pro peptides can exit to the culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H-¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.     

Molecular phylogeny of three ambiguous ciliate genera: Kentrophoros,Trachelolophos and Trachelotractus (Alveolata,Ciliophora)

Gao, S., Strüder‐Kypke, M.C., Al‐Rasheid, K.A.S., Lin, X. & Song, W. (2010). Molecular phylogeny of three ambiguous ciliate genera: Kentrophoros, Trachelolophos and Trachelotractus (Alveolata, Ciliophora).—Zoologica Scripta, 39, 305–313. Very few molecular studies on the phylogeny of the karyorelictean ciliates have been carried out because data of this highly ambiguous group are extremely scarce. In the present study, we sequenced the small subunit ribosomal RNA genes of three morphospecies representing two karyorelictean genera, Kentrophoros, Trachelolophos, and one haptorid, Trachelotractus, isolated from the South and East China Seas. The phylogenetic trees constructed using Bayesian inference, maximum likelihood, maximum parsimony and neighbor‐joining methods yielded essentially similar topologies. The class Karyorelictea is depicted as a monophyletic clade, closely related to the class Heterotrichea. The generic concept of the family Trachelocercidae is confirmed by the clustering of Trachelolophos and Tracheloraphis with high bootstrap support; nevertheless, the order Loxodida is paraphyletic. The transfer of the morphotype Trachelocerca entzi Kahl, 1927 to the class Litostomatea and into the new haptorid genus Trachelotractus, as suggested by previous researchers based on morphological studies, is consistently supported by our molecular analyses. In addition, the poorly known species Parduczia orbis occupies a well‐supported position basal to the Geleia clade, justifying the separation of these genera from one another.     

Small diameter blood vessels with controllable micropore structure induced by centrifugal force for improved endothelialization

The micropore structure is prerequisite for fast and durable endothelialization of artificial small diameter blood vessels (ASDBVs). Although some methods, such as salt leaching, coagulation, and electrospinning, have been developed to construct micropores for ASDBVs, the uncontrollability of the structure and the complicated procedures of the process are still the issues to be concerned about. In this study, a compact device based on the principle of centrifugal force is established and used to prepare polyurethane (PU) ASDBVs with micropore structures by blasting different porogens. It is found that the glass beads could construct micropores with regular round shape, uniform distribution, and controllable size (60–350 µm), which significantly improves the endothelialization of PU‐based ASDBVs, especially when the pore size is about 60 µm. This method is easy‐accessible and wide‐applicable, which provides a new pathway for the research and development of ASDBVs.     

Separation and Purification of Antioxidant Peptides from Enzymatically Prepared Scorpion (Buthus martensii Karsch) Protein Hydrolysates

International Journal of Peptide Research and Therapeutics - The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein...     

Osthole stimulates bone formation,drives vascularization and retards adipogenesis to alleviate alcohol‐induced osteonecrosis of the femoral head

Characteristic pathological changes in osteonecrosis of the femoral head (ONFH) include reduced osteogenic differentiation of bone mesenchymal stem cells (BMSCs), impaired osseous circulation and increased intramedullary adipocytes deposition. Osthole is a bioactive derivative from coumarin with a wide range of pharmacotherapeutic effects. The aim of this study was to unveil the potential protective role of osthole in alcohol‐induced ONFH. In vitro, ethanol (50 mmol/L) remarkably decreased the proliferation and osteogenic differentiation of BMSCs and impaired the proliferation and tube formation capacity of human umbilical vein endothelial cell (HUVECs), whereas it substantially promoted the adipogenic differentiation of BMSCs. However, osthole could reverse the effects of ethanol on osteogenesis via modulating Wnt/β‐catenin pathway, stimulate vasculogenesis and counteract adipogenesis. In vivo, the protective role of osthole was confirmed in the well‐constructed rat model of ethanol‐induced ONFH, demonstrated by a cascade of radiographical and pathological investigations including micro‐CT scanning, haematoxylin‐eosin staining, TdT‐mediated dUTP nick end labelling, immunohistochemical staining and fluorochrome labelling. Taken together, for the first time, osthole was demonstrated to rescue the ethanol‐induced ONFH via promoting bone formation, driving vascularization and retarding adipogenesis.     

Effect of c‐Ski on atrial remodelling in a rapid atrial pacing canine model

Atrial fibrosis is an important factor in the initiation and maintenance of atrial fibrillation (AF); therefore, understanding the pathogenesis of atrial fibrosis may reveal promising therapeutic targets for AF. In this study, we successfully established a rapid atrial pacing canine model and found that the inducibility and duration of AF were significantly reduced by the overexpression of c‐Ski, suggesting that this approach may have therapeutic effects. c‐Ski was found to be down‐regulated in the atrial tissues of the rapid atrial pacing canine model. We artificially up‐regulated c‐Ski expression with a c‐Ski–overexpressing adenovirus. Haematoxylin and eosin, Masson's trichrome and picrosirius red staining showed that c‐Ski overexpression alleviated atrial fibrosis. Furthermore, we found that the expression levels of collagen III and α‐SMA were higher in the groups of dogs subjected to right‐atrial pacing, and this increase was attenuated by c‐Ski overexpression. In addition, c‐Ski overexpression decreased the phosphorylation of smad2, smad3 and p38 MAPK (p38α and p38β) as well as the expression of TGF‐β1 in atrial tissues, as shown by a comparison of the right‐atrial pacing + c‐Ski‐overexpression group to the control group with right‐atrial pacing only. These results suggest that c‐Ski overexpression improves atrial remodelling in a rapid atrial pacing canine model by suppressing TGF‐β1–Smad signalling and p38 MAPK activation.     

Synthesis of Novel Aryloxyethylamine Derivatives and Evaluation of Their in Vitro and in Vivo Neuroprotective Activities

A series of aryloxyethylamine derivatives were designed, synthesized and evaluated for their biological activity. Their structures were confirmed by ¹H‐NMR, ¹³C‐NMR, FT‐IR and HR‐ESI‐MS. The preliminary screening of neuroprotection of compounds in vitro was detected by MTT, and the anti‐ischemic activity in vivo was tested using bilateral common carotid artery occlusion in mice. Most of these compounds showed potential neuroprotective effects against the glutamate‐induced cell death in differentiated rat pheochromocytoma cells (PC12 cells), especially for (4‐fluorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}(4‐methoxyphenyl)methanone, (4‐bromophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐chlorophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone, (4‐chlorophenyl)(1‐{2‐[(naphthalen‐2‐yl)oxy]ethyl}piperidin‐4‐yl)methanone, (4‐chlorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone and {1‐[2‐(4‐bromophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone, which exhibited potent protection of PC12 cells at three doses (0.1, 1.0, 10 μM). Compounds (4‐fluorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, (4‐fluorophenyl){1‐[2‐(naphthalen‐2‐yloxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}(4‐methoxyphenyl)methanone and {1‐[2‐(4‐chlorophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone possessed the significant prolongation of the survival time of mice subjected to acute cerebral ischemia and decreased the mortality rate at all five doses tested (200, 100, 50, 25, 12.5 mg/kg) and had significant neuroprotective activity. In addition, (4‐fluorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}(4‐methoxyphenyl)methanone and {1‐[2‐(4‐chlorophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone possessed outstanding neuroprotection in vitro and in vivo. These compounds can be used as a promising neuroprotective agents for future development of new anti‐ischemic stroke agents. Basic structure–activity relationships are also presented.     

Synthesis of n-butyl acetate via reactive distillation column using Candida Antarctica lipase as catalyst

Bioprocess and Biosystems Engineering - The reactive distillation process for the synthesis of n-butyl acetate via transesterification of ethyl acetate with n-butyl alcohol catalyzed by immobilized...