Fibrinogen and fibrin in strong magnetic fields. Complementary results and discussion

When fibrin polymerizes in a strong magnetic field, it can be highly oriented. The structural diffraction study of the oriented polymer becomes thus possible. The magnetic birefringence can also be used to study the development of the polymer Fibrinogen in solution is weakly oriented in high magnetic fields. In this work we present complementary results and discussion. The validity of the comparison of the orientation parameters of fibrinogen and fibrin with those of other orientable known biological structures is discussed. The orientation of fibrin formed from fibrin monomer solution is compared to that of fibrin formed by the action of thrombin on fibrinogen. The conditions to obtain highly oriented fibrin gels suitable for three dimensional structure studies are also briefly discussed.     

Sapatoxins,aliphatic ester tigliane diterpenes from Sapium indicum

From the unripe fruits of Sapium indicum, three aliphatic esters of the tigliane nucleus were isolated. These compounds were derivatives of 4-deoxyphorbol. Sapatoxin A was identified as 12-O-[n-deca-2,4,6-trienoyl]-4-deoxyphorbol-13-acetate, B as 12-O-[n-deca-2,4,6-trienoyl]-4-deoxy-5-hydroxyphorbol-13-acetate and C as 12-O-[n-deca-2,4,6-trienoyl]-4,20-dideoxy-5-hydroxyphorbol-13-acetate, by spectroscopic analysis and hydrolysis reactions.     

Organic acids from the anaerobic decomposition of Agropyron repens rhizomes

Phytotoxicity originating from the anaerobic decomposition of couch grass rhizomes has been studied. Short chain aliphatic (acetic, propionic and butyric) acids appear to be mainly responsible but hexanoic, succinic, phenylacetic, cinnamic, p-coumaric, 4-hydroxyphenylpropionic and 3,4-dihydroxyphenylpropionic acids are also present in the phytotoxic solutions formed during the decomposition.     

Enzymatic synthesis of new aromatic and aliphatic esters of flavonoids using Candida antarctica lipase as biocatalyst

Enzymatic acylation of rutin and esculin with aromatic, aliphatic and aryl-aliphatic acids using Candida antarctica lipase in tert amyl alcohol as solvent was investigated under low water content. Whatever the acyl donor used, the conversion yields and initial rates for esculin were higher than for rutin. For a given flavonoid, the performance of these reactions depended on the acyl donor structures. For aliphatic acids, conversion yields and initial rates of both flavonoids were respectively in the ranges of 68–90% and of 9.5×10^?2–72×10^?2 mmol l^?1 h^?1. For aromatic acids, the reaction occurred only with the aryl subgroup (cinnamic, hydrocinnamic, 3,4-dihydroxyhydrocinnamic and 4-hydroxyphenyl acetic acids) and was drastically influenced by the presence of side chain and substitution patterns of the aromatic ring. Except for hydrocinnamic acid (75%, 23.4×10^?2 mmol l^?1 h^?1), with these acids the conversion yields and initial rates were lower and in the range of 10–45% and of 0.7×10^?2 to 12.1×10^?2 mmol l^?1 h^?1. Unsaturation of the side chain of the hydrocinnamic acid decreased the esculin conversion rate from 75 to 13% and initial rate from 23.4 to 1.76×10^?2 mmol l^?1 h^?1. The presence of hydroxyl or nitro-groups on the aromatic ring of the aryl aliphatic acid also reduced conversion yields and initial rates. Even without a spacer, the non-phenolic ring acid (quinic acid) was reactive and lead to conversion yields of about 20 and 23% respectively for rutin and esculin.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

Synthesis and biological evaluation of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives as potential anticancer agents

A series of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives 5, 6 and 7 were prepared starting from 6-phenyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-3-amine 3 via selective N-alkylation, followed by reaction with different primary aliphatic amines, cyclic secondary amines or l-amino acids under different set of conditions. All the synthesized compounds 5, 6 and 7 were screened for anticancer activity against four cancer cell lines such as A549—Lung cancer (CCL-185), MCF7—Breast cancer (HTB-22), DU145—Prostate cancer (HTB-81) and HeLa—Cervical cancer (CCL-2). The compounds 5i and 6e are found to have promising bioactivity at micro molar concentration.     

Effects of different tannin-rich extracts and rapeseed tannin monomers on methane formation and microbial protein synthesis in vitro

Tannins, polyphenolic compounds found in plants, are known to complex with proteins of feed and rumen bacteria. This group of substances has the potential to reduce methane production either with or without negative effects on digestibility and microbial yield. In the first step of this study, 10 tannin-rich extracts from chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed, and four rapeseed tannin monomers (pelargonidin, catechin, cyanidin and sinapinic acid) were used in a series of in vitro trials using the Hohenheim gas test, with grass silage as substrate. The objective was to screen the potential of various tannin-rich extracts to reduce methane production without a significant effect on total gas production (GP). Supplementation with pelargonidin and cyanidin did not reduce methane production; however, catechin and sinapinic acid reduced methane production without altering GP. All tannin-rich extracts, except for tara extract, significantly reduced methane production by 8% to 28% without altering GP. On the basis of these results, five tannin-rich extracts were selected and further investigated in a second step using a Rusitec system. Each tannin-rich extract (1.5 g) was supplemented to grass silage (15 g). In this experiment, nutrient degradation, microbial protein synthesis and volatile fatty acid production were used as additional response criteria. Chestnut extract caused the greatest reduction in methane production followed by valonea, grape seed and sumach, whereas myrabolan extract did not reduce methane production. Whereas chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production. However, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry matter and organic matter was also reduced by tannin supplementation, and no differences were found between the tannin-rich extracts. CP degradation and ammonia-N accumulation in the Rusitec were reduced by tannin treatment. The amount and efficiency of microbial protein synthesis were not significantly affected by tannin supplementation. The results of this study indicated that some tannin-rich extracts are able to reduce methane production without altering microbial protein synthesis. We hypothesized that chestnut and valonea extract have the greatest potential to reduce methane production without negative side effects.     

Chiral separation of aliphatic primary amino alcohols as o‐phthaldialdehyde/mercaptoethanol derivatives on polysaccharide‐based chiral stationary phases

A sensitive chiral high performance liquid chromatography (HPLC) method for the determination of aliphatic primary amino alcohol isomers with o‐phthaldialdehyde/mercaptoethanol precolumn derivatization has been developed and validated. Seven chiral columns were tested in a reversed phase mode. Excellent enantioseparation with the resolution more than 2.0 was achieved on Chiralcel OJ‐3R. The effect of various chromatographic conditions including column temperature, acetonitrile content in the mobile phase, buffer pH, buffer concentration, and buffer type in the mobile phase on the retention and the selectivity was investigated. The final mobile phase consisted of binary mixture of 20mM ammonium formate solution with acetonitrile (75:25; v/v). The analyses were performed at mobile phase flow rate of 1.0 mL/min and the column temperature of 40°C. The fluorescence detection was performed at excitation wavelength of 345 nm and emission wavelength of 450 nm. The developed method was fully validated in terms of linearity, sensitivity, accuracy, precision, intermediate precision, and selectivity according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines using internal normalization procedure. The proposed chiral method was proved to be highly sensitive, simple, and rapid and was successfully applied to the determination of D‐Valinol content in commercially available samples of L‐Valinol.     

Participation of chaperonin GroEL in the folding of D-glyceraldehyde-3-phosphate dehydrogenase. An approach based on the use of different oligomeric forms of the enzyme immobilized on sepharose

The binding of denatured B. stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the E. coli chaperonin GroEL was investigated in two systems: (1) GroEL immobilized on Sepharose via a single subunit was titrated with urea-denatured soluble GAPDH and (2) a Sepharose-bound denatured GAPDH monomer was titrated with soluble GroEL. Similar apparent K _D values for the complex GroEL·GAPDH were obtained in both cases (0.04 and 0.03 M, respectively), the stoichiometry being 1.0 mol chaperonin per GAPDH subunit in the system with the immobilized GroEL and 0.2 mol chaperonin per Sepharose-bound GAPDH monomer. Addition of GroEL and Mg·ATP to a reactivation mixture increased the yield of reactivation of both E. coli and B. stearothermophilus GAPDHs. Incubation of the Sepharose-bound catalytically active tetrameric and dimeric GAPDH forms with the protein fraction of a wild-type E. coli cell extract resulted in the binding of GroEL to the dimer and no interaction with the tetrameric form. These data suggest that GroEL may be capable of interacting with the interdimeric contact regions of the folded GAPDH dimers.     

Enhanced Melanogenesis Induced by Tyrosinase Gene-Transfer Increases Boron-Uptake and Killing Effect of Boron Neutron Capture Therapy for Amelanotic Melanoma

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, ¹⁰B-p-boronophenylalanine (¹⁰B-BPA), has been established, but amelanotic melanoma is insufficiently responsive to ¹⁰B-BPA BNCT in comparison with actively melanin-producing melanoma. Although the accumulation mechanism of ¹⁰B-BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in ¹⁰B-BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase-related protein (TRP)-l and TRP-2 are well expressed. Thus, by using a similarly tyrosinase-lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase-cDNA into these cells. TA1059 cells acquired higher DOPA-oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher ^p-boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by ¹⁰B-BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene-transfer effectively improves the treatment of amelanotic melanoma by BNCT.