Synthesis of naturally occurring diene and trienes by Te/Li exchange on (1Z,3Z)-butyltelluro-4-methoxy-1,3-butadiene

(1Z,3Z)-Butyltelluro-o-4-methoxy-1,3-butadiene 2 was obtained by the hydrotelluration of(Z)-1-methoxy-but-1-en-3-ynes 1. The butadienyllithium 3 obtained by the Te/Li exchange reaction in the (1Z,3Z)-1-butyltelluro-4-methoxy-1.3-butadiene 2 reacted with aldehydes to form the corresponding alcohols 4a-d with total retention of configuration. The alcohols formed undergo hydrolysis, resulting in the alpha,beta,gamma,delta-unsaturated aldehydes of (E,E) configuration, which are precursors of trienes obtained from natural sources. The products of this reaction were employed in the synthesis of methyl-(2E,4E)-decadienoate 7, which is a component of the flavor principles of ripe Bartlett pears. Performing the Wittig reaction of the methyl triphenylphosphorane with the deca-(2E,4E)-dienal 5a, we were able to synthesize the undeca-(1,3E,5E)-triene 6a. This compound is a sex-pheromone component of the marine brown algae Fucus serratus, Dictyopteris plagiograma, and Dictyopteris australis. Performing the Wittig reaction of methyl triphenylphosphorane with the octa-(2E,4E)-dienal 5c, the nona-(1,3E,5E)-triene 6b was synthesized. The compound obtained is a sex-pheromone component of the marine brown alga Sargassum horneri. The octa-( 1,3E,5E)-triene 6c was easily obtained from hepta-(2E,4E)-dienal 5d by the Wittig reaction with methyl triphenylphophorane. This compound is a sex-pheromone component of the marine brown alga Fucus serratus. (C) 2010 Elsevier Ltd. All rights reserved.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5`-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5`-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5`-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1- containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1- containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

The interaction of bothropstoxin-I (Lys49-PLA(2)) with liposome membranes

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which permeabilizes biological and artificial membranes by a mechanism independent of lipid hydrolysis. This mechanism has been investigated by studying the interaction of nine single tryptophan BthTx-I mutants with negatively charged phospholipid membranes. Changes in the solvent exposure of the tryptophan in each mutant were evaluated comparing the rate of chemical modification (k(mod)) by bromosuccinamide with the maximum intrinsic tryptophan fluorescence emission wavelength (lambda(max)) in buffer and in the presence of 10% DMPA/90% DPPC liposomes. No changes in lambda(max). were observed, whereas k(mod) values for tryptophans at positions 7, 10, 31 and 125 were significantly reduced in the presence of lipids, suggesting that bound phospholipid decreases solvent accessibility at these positions. Since the half-lives of the fluorescence and chemical modification effects differ by at least six orders of magnitude, these results suggest that the bound phospholipid may interact with multiple locations on the protein surface over micro- to millisecond timescales. (C) 2009 Elsevier Ltd. All rights reserved.

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K (m) and V (max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 A mu mol/min/mg, respectively. PG was found to have temperature optimum at 65 A degrees C and was totally stable at 45 A degrees C for 90 min. Half-life at 55 A degrees C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

CD4+T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes

P>Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4+ and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

H-1 NMR spectroscopic studies of the stability of an oxazolidine condensation product

Condensation of (-)-norephedrine with excess formaldehyde under mild conditions leads to formation of the 2:1 condensation product N,N'-methylenebis(4-methyl-5-phenyl)oxazolidine compared with the reaction with 1 mol of formaldehyde, which leads to 4-methyl-5-phenyloxazolidine. H-1 and C-13 NMR spectroscopy was used to monitor the stability of this compound and its decomposition products. The 2:1 condensation product is found to be stable in CDC1(3) but breaks down rapidly in CD3OD to yield a 50:50 mixture of 4-methyl-5-phenyloxazolidine and 3-hydroxymethyl-4-methyl-5-phenyloxazolidine. Upon addition of D2O to this equimolar mixture, the latter compound decomposes to norephedrine and formaldehyde, whereas the former compound is stable. (C) 1997 by John Wiley & Sons, Ltd.

Synthesis, identification, characterization, stability, solubility, and protein binding of ester derivatives of salicylic acid and diflunisal

O-Acyl esters were prepared from salicylic acid and diflunisal by esterification with the appropriate acyl anhydride (in the presence of sulfuric acid at 80 degrees C) or acyl chloride (in the presence of pyridine at 0 degrees C). Synthesis, identification and characterization of these compounds is described. In vitro hydrolysis, solubility and protein binding studies of these O-acyl esters were performed. For the diflunisal esters, the melting points fell as the side chain was increased from ethyl to pentyl. The melting points showed no significant difference as the length of the side chain was increased from pentyl to heptyl. The aspirin analogues showed a similar trend, The relationship between solubility and carbon chain length agreed closely with that for the melting points with carbon chain length. In vitro non-enzymatic hydrolysis studies concluded that: (1) hydrolysis rate constants generally decreased with carbon chain length; (2) the diflunisal esters have shorter half lives compared with their salicylate counterparts; and (3) the in vitro hydrolysis of these compounds was retarded by the presence of bovine serum albumin. Protein binding experiments showed that the strength of binding of the aspirin and diflunisal analogues to bovine serum albumin increased with carbon chain length. (C) 1997 Elsevier Science B.V.

The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by Xanthomonas albilineans. A gene (albD) from Pantoea dispersa has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin, The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases, The AlbD protein, purified to homogeneity by means of a glutathione S-transferase gene fusion system, showed strong esterase activity on p-nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of p-nitrophenyl butyrate and detoxification of albicidins required no complex cofactors, Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor, These data strongly suggest that AlbD is an albicidin hydrolase, The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35 degrees C, Expression of albD in transformed X. albilineans strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane, The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.

Oral Azithromycin for Treatment of Posterior Blepharitis

Purpose: To evaluate the effects of oral azithromycin in patients with posterior blepharitis. Methods: Twenty-six eyes of 13 patients with posterior blepharitis diagnosed by a qualified ophthalmologist were enrolled in this study. Patients were instructed to use oral azithromycin 500 mg per day for 3 days in 3 cycles with 7-day intervals. Subjective clinical outcomes were graded and scored 1 day before and 30 days after the end of the treatment (53 days after initiating the treatment) based on severity scores of: (1) eyelid debris; (2) eyelid telangiectasia; (3) swelling of the eyelid margin; (4) redness of the eyelid margin; and (5) ocular mucus secretion. For the assessment of global efficacy, patients were asked by the investigator to rate the subjective symptoms (eyelid itching, ocular itching, eyelid hyperemia, ocular hyperemia, ocular mucus secretion, photophobia, foreign body sensation, and dry eye sensation) on a scale of 0 (no symptoms) to 5 (severe symptoms). Break-up time, Schirmer I test, corneal fluorescein staining score, and rose bengal staining score were also performed in all patients. Results: All clinical outcomes scoring showed statistically significant improvement after oral azithromycin, except for eyelid swelling. Average subjective symptom grading improved statistically after treatment with oral azithromycin, except for eyelid hyperemia, photophobia, and foreign body sensation. Average tear film break-up time values showed statistically significant improvement after the treatment with oral azithromycin. No statistically significant improvement was observed on average values of Schirmer I test, corneal fluorescein staining score, and rose bengal staining score. Conclusions: The combination of multiple clinical parameters shown in this study supports the clinical efficacy of pulsed oral azithromycin therapy for the management of posterior blepharitis.

Two-Year Outcome of Partial Lacrimal Punctal Occlusion in the Management of Dry Eye Related to Sjogren Syndrome

Purpose: To analyze the influence of thermal partial punctal occlusion on the ocular surface of dry eye related to Sjogren syndrome. Material and Methods: Thirty-seven eyes of 19 patients (3 male and 16 female; 49.11 +/- 14.33 years old) with keratoconjunctivitis sicca were enrolled in this study. Superior and inferior partial occlusion were performed in both eyes under topical anesthesia using thermal cautery with a sterile tip to obtain lacrimal punctum smaller than 0.5 mm. Schirmer I, break-up-time, diameter of lacrimal puncta, corneal fluorescein, and rose Bengal staining scores were analyzed before and after 24 weeks and after 24 months of the procedure. All measurements were performed under controlled climate. Results: The average lacrimal punctum diameter before the procedure was 0.65 +/- 0.134 mm. All lacrimal puncta were successfully reduced to less than 0.5 mm after 4 weeks of the procedure. The average Schirmer I test values improved statistically after 24 weeks and maintained stable after 24 months. Average break-up-time, rose Bengal, and fluorescein staining score values improved statistically after 24 weeks and improved even more after 24 months. Average Schirmer I test, break-up-time, rose Bengal, and fluorescein staining scores showed significant improvement (p < 0.0001) after 24 months of partial thermal punctal occlusion. Conclusion: Our study showed that reducing the punctum diameter to 0.5 mm can improve vital staining scores, break-up-time, and Schirmer I test in dry eye related to Sjogren syndrome.

Arginase-1 A New Immunohistochemical Marker of Hepatocytes and Hepatocellular Neoplasms

The distinction of hepatocellular carcinoma (HCC) from metastatic tumor in the liver often presents a diagnostic challenge that carries significant impact on prognostication and therapy. The number of diagnostically useful immunohistochemical markers of hepatocytes is limited to hepatocyte paraffin antigen (HepPar-1), polyclonal carcinoembryonic antigen, and CD10, with alpha-fetoprotein and glypican-3 labeling HCCs. Arginase-1 (Arg-1) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of arginine to ornithine and urea. We used immunohistochemistry to compare the sensitivity of Arg-1 to that of HepPar-1 in 151 HCCs. We found that the overall sensitivities of Arg-1 and HepPar-1 are 96.0% and 84.1%, respectively. The sensitivities of Arg-1 in well, moderately, and poorly differentiated HCCs are 100%, 96.2%, and 85.7%, respectively, whereas, in comparison, HepPar-1 demonstrated sensitivities of 100%, 83.0%, and 46.4% for well, moderately, and poorly differentiated tumors, respectively. There were no HCCs in our study that were reactive for HepPar-1 but nonreactive for Arg-1. We also examined Arg-1 expression in nonhepatocellular tumors, including many that are potential mimics of HCC (renal cell carcinomas, neuroendocrine tumors, melanomas, gastric adenocarcinomas, and adrenocortical carcinomas) and found that only 2 non-HCC tumors were reactive for Arg-1. Arg-1 represents a sensitive and specific marker of benign and malignant hepatocytes that may ultimately prove to be a useful diagnostic tool in routine surgical pathology practice.

Phenolics and Antioxidant Properties of Fruit Pulp and Cell Wall Fractions of Postharvest Banana (Musa acuminata Juss.) Cultivars

Banana fruits are important foods, but there have been very few studies evaluating the phenolics associated with their cell walls. In the present study, (+) catechin, gallocatechin, and (-) epicatechin, as well as condensed tannins, were detected in the soluble extract of the fruit pulp; neither soluble anthocyanidins nor anthocyanins were present. In the soluble cell wall fraction, two hydroxycinnamic acid derivatives were predominant, whereas in the insoluble cell wall fraction, the anthocyanidin delphinidin, which is reported in banana cell walls for the first time, was predominant. Cell wall fractions showed remarkable antioxidant capacity, especially after acid and enzymatic hydrolysis, which was correlated with the total phenolic content released after the hydrolysis of the water-insoluble polymer, but not for the posthydrolysis water-soluble polymer. The acid hydrolysis released various monosaccharides, whereas enzymatic hydrolysis released one peak of oligosaccharides. These results indicate that banana cell walls could be a suitable source of natural antioxidants and that they could be bioaccessible in the human gut.

In vivo degradation of banana starch: Structural characterization of the degradation process

This work reports the first ultrastructural investigation into the degradation process that starch granules isolated from bananas (cv. Nanicao) undergo during ripening. Starch granules from green bananas had a smooth surface, while granules from ripe bananas were more elongated with parallel striations, as revealed by CSLM and SEM. AFM images revealed that the first layer covering the granule surface is composed of a hard material and, as degradation proceeds, hard and soft regions seem to be repeated at regular intervals. WAXD patterns of banana starches were C-type, and the crystalline index was reduced during ripening. The B-/A-type ratio was increased, indicating the preferential degradation of the A-type allomorph. The branch-chain length distribution showed predominantly short chains of amylopectin (A and B1-chain). The fa/fb ratio was reduced during degradation, while amylose content was increased. The results allowed a detailed understanding of the changes that starch granules undergo during banana ripening. (C) 2010 Elsevier Ltd. All rights reserved.