Involvement of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase 1/2 in EGF‐induced angiogenesis

Angiogenesis is a process during which endothelial cells divide and migrate to form new capillaries from the preexisting blood vessels. The present study was designed to investigate whether MAPKs (mitogen‐activated protein kinases) play crucial roles in regulating EGF (epidermal growth factor)‐induced endothelial cell angiogenesis. Our results showed that EGF stimulated HUVEC (human umbilical vein endothelial cells) proliferation in a concentration‐dependent manner, of which the maximum effective concentration of EGF was 10 ng/ml. Western blot analysis showed that EGF at 10 ng/ml significantly induced the phosphorylation of ERK1/2 (extracellular signal‐regulated kinase 1 and 2) and p38 kinase at 5 min, while it induced the phosphorylation of JNK/SAPK (c‐Jun N‐terminal kinase/stress‐activated protein kinase) at 15 min. Further results showed that a JNK/SAPK inhibitor, SP600125, and a specific siRNA JNK/SAPK could both significantly inhibit EGF‐induced tube formation in HUVEC cells, and an ERK1/2 inhibitor PD098059 could also block the tube formation in some content, while a p38 inhibitor SB203580 failed to do so. Furthermore, only SP600125 significantly inhibited EGF‐induced HUVEC cell proliferation under no cytotoxic concentration, so did JNK/SAPK siRNA. In conclusion, JNK/SAPK and ERK1/2 signals therefore play critical roles in EGF‐mediated HUVEC cell angiogenesis.     

Increased ethanol resistance in <Emphasis Type="Italic">Ethanolic Escherichia coli</Emphasis> by Insertion of heat-shock genes BEM1 and SOD2 from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ethanol is generally toxic to microorganisms, and intracellular and extracellular accumulation of ethanol inhibits cell growth and metabolism. In this study, pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) were cloned into pET-32a vector and then introduced into E. coli BL21 to produce ethanol. Heat shock genes (BEM1 and SOD2) from Saccharomyces cerevisiae were inserted into recombinant ethanolic E. coli using pET28_a vector to improve ethanol shock resistance. Three different strains were constructed: Ethanolic E. coli (adhB and pdc genes inserted using pET32_a vector), BEM1 gene-inserted E. coli (BEM1 inserted using pET_28a), and SOD2-inserted E. coli (SOD2 inserted using pET28_a). Construction of these three different strains allowed comparison of the functions of these heat shock genes as well as their roles in ethanol tolerance. The toxicity of ethanol in recombinant ethanolic E. coli was tested by measuring cell growth in response to various ethanol concentrations. The results show that SOD2-inserted E. coli showed higher ethanol resistance than ethanolic E. coli.     

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).     

JH biosynthesis by reproductive tissues and corpora allata in adult longhorned beetles, Apriona germari

We report on juvenile hormone (JH) biosynthesis from long‐chain intermediates by specific reproductive tissues and the corpora allata (CA) prepared from adult longhorned beetles, Apriona germari. The testes, male accessory glands (MAGs), ovaries, and CA contained the long‐chain intermediates in the JH biosynthetic pathway, farnesoic acid (FA), methyl farnesoate (MF), and JH III. The testes and ovaries, but not CA, produced radioactive JH III after the addition of ³H‐methionine and, separately, unlabeled methionine, to the incubation medium. We inferred that endogenous FA is methylated to MF in the testes and ovaries. Addition of farnesol led to increased amounts of FA in the testes, MAGs, ovaries, and CA, indicating oxidation of farnesol to FA. Addition of FA to incubation medium yielded increased JH III, again indicating methylation of FA to MF in the testes, MAGs, ovaries, but not CA. Addition of MF to incubation medium also led to JH III, from which we inferred the epoxidation of MF to JH III. JH biosynthesis from farnesol in the testes, MAGs, and ovaries of A. germari proceeds via oxidation to FA, methylation to MF, and epoxidation to JH III. This is a well‐known pathway to JH III, described here for the first time in reproductive tissues of longhorned beetles. © 2010 Wiley Periodicals, Inc.     

Influence of the acetylcholinesterase active site protonation on omega loop and active site dynamics

Existence of alternative entrances in acetylcholinesterase (AChE) could explain the contrast between the very high AChE catalytic efficiency and the narrow and long access path to the active site revealed by X-ray crystallography. Alternative entrances could facilitate diffusion of the reaction products or at least water and ions from the active site. Previous molecular dynamics simulations identified side door and back door as the most probable alternative entrances. The simulations of non-inhibited AChE suggested that the back door opening events occur only rarely (0.8% of the time in the 10ns trajectory). Here we present a molecular dynamics simulation of non-inhibited AChE, where the back door opening appears much more often (14% of the time in the 12ns trajectory) and where the side door opening was observed quite frequently (78% of trajectory time). We also present molecular dynamics, where the back door does not open at all, or where large conformational changes of the AChE omega loop occur together with alternative passage opening events. All these differences in AChE dynamical behavior are caused by different protonation states of two glutamate residues located on bottom of the active site gorge (Glu202 and G450 in Mus musculus AChE). Our results confirm the results of previous molecular dynamics simulations, expand the view and suggest the probable reasons for the overall conformational behavior of AChE omega loop.     

Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells

Curcumin is considered a pharmacologically safe agent that may be useful in cancer chemoprevention and therapy. Here, we show for the first time that curcumin effectively induces paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells, by promoting vacuolation that results from swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). Inhibition of protein synthesis by cycloheximide blocked curcumin-induced vacuolation and subsequent cell death, indicating that protein synthesis is required for this process. The levels of AIP-1/Alix protein, a known inhibitor protein of paraptosis, were progressively downregulated in curcumin-treated malignant breast cancer cells, and AIP-1/Alix overexpression attenuated curcumin-induced death in these cells. ERK2 and JNK activation were positively associated with curcumin-induced cell death. Mitochondrial superoxide was shown to act as a critical early signal in curcumin-induced paraptosis, whereas proteasomal dysfunction was mainly responsible for the paraptotic changes associated with ER dilation. Notably, curcumin-induced paraptotic events were not observed in normal breast cells, including mammary epithelial cells and MCF-10A cells. Taken together, our findings on curcumin-induced paraptosis may provide novel insights into the mechanisms underlying the selective anti-cancer effects of curcumin against malignant cancer cells.     

Synthesis and biological activity of oxytocin analogues containing unnatural amino acids in position 9: structure activity study

We report the solid phase synthesis and some pharmacological properties of 24 oxytocin (OT) analogues. Basic modifications at position 9 (introduction of l- or d-β-(2-thienyl)-alanine [L- or D-Thi], or l- or d-3-Pyridylalanine [l- or d-3-Pal]) were combined with d-tyrosine(OEthyl) [d-Tyr(Et)] or d-1-naphthylalanine [d-1-Nal] in position 2 and β-mercaptopropionic acid (Mpa) in position 1 modifications in altogether 14 analogues. Additionally, 8 analogues having α-aminoisobutyric acid [Aib] or d-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (d-Tic) or diethylglycine (Deg) in position 9 and d-Tyr(Et) or d-1-Nal or d-Tic in position 2 and Mpa or Pen (ββ-dimethylcysteine) in position 1 were prepared. Two of these analogues have one more modification in position 6, i.e. Pen. Furthermore, two analogues having Mpa in position 1 and d-Tyr(Et) or d-1-Nal in position 2 were prepared for comparison purposes. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest anti-oxytocic activity was [Mpa¹, d-Tyr(Et)², Deg⁹]OT (pA₂ = 8.68 ± 0.26); this analogue was also selective.     

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH₂) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O₂) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.