Stabilization of Endophytic Fungus Cercospora kikuchii Lipase by Spray Drying in the Presence of Maltodextrin and beta-Cyclodextrin

In this study the effects of spray-drying conditions on the retention of enzyme activity of lipase produced by the endophytic fungus Cercospora kikuchii have been investigated. Drying runs were carried out in a bench-top spray dryer with a concurrent flow regime. The influence of the variables inlet temperature of drying gas, Tgi (86.4 to 153.6 degrees C); mass flow rate of the enzymatic extract fed to the dryer, Ws (2.63 to 9.36g/min); and concentration of the drying adjuvant added to the extract, ADJ (1.95 to 12.05%), on the spray-drying performance and on product quality was evaluated through experimental planning and regression analysis. The use of maltodextrin, as a stabilizing agent, slightly improved the retention of enzyme activity compared to -cyclodextrin. Statistical optimization of the experimental results allowed the determination of the processing conditions that maximized the retention of the enzymatic activity (RAE), namely, concentration of drying adjuvants of 12.05%, inlet temperature of the drying gas of 153.6 degrees C, and flow rate of the enzymatic extract fed to the dryer of 9.36g/min for the both drying adjuvants investigated.

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders. (C) 2009 Elsevier GrnbH. All rights reserved.

Neutralization of pharmacological and toxic activities of Bothrops snake venoms by Schizolobium parahyba (Fabaceae) aqueous extract and its fractions

The aqueous extract prepared from Schizolobium parahyba (Sp) leaves, a native plant from Atlantic Forest (Brazil), was tested to analyse its ability to inhibit some biological and enzymatic activities induced by Bothrops alternatus (BaltCV) and Bothrops moojeni (BmooCV) snake venoms. Sp inhibited 100% of lethality, blood incoagulability, haemorrhagic and indirect haemolytic activities at a 1:10 ratio (venom/extract, w/w), as well as coagulant activity at a 1:5 ratio (venom/extract, w/w) induced by both venoms. BaltCV fibrinogenolytic activity was also neutralized by Sp at a 1:10 ratio, resulting in total protection of fibrinogen B beta chain and partial protection of A alpha chain. Interaction tests have demonstrated that, at certain extract/proteins ratios, Sp precipitates proteins non-specifically suggesting the presence of tannins, which are very likely responsible for the excellent inhibiting effects of the analysed ophidian activities. Sp aqueous extract chromatography on Sephadex LH-20 was carried out aiming at the separation of these compounds that mask the obtained results. Thus, the fractionation of Sp resulted in three fractions: F1 (methanolic fraction); F2 (methanol:water fraction, 1:1 v/v); and F3 (aqueous fraction). These fractions were analysed for their ability to inhibit the BaltCV fibrinogenolytic activity. F1 inhibited 100% the venom fibrinogenolytic activity without presenting protein precipitation effect; F2 showed only partial inhibition of this venom activity. Finally, F3 did not inhibit fibrinogen proteolysis, but presented strong protein precipitating action. We conclude that Sp aqueous extract, together with tannins, also contains other compounds that can display specific inhibitory activity against snake venom toxins.

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

A proteinase, named BmooMP alpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the B beta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMP alpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMP alpha-I activity. Since the BmooMP alpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis. (C) 2007 Elsevier Ltd. All rights reserved.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene bla(CTX-M-2) was identified in 3 different enterobacteria (E. coli. Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), hut not as part of the resistance cassettes region. Multi locus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coil isolates in this study and strains associated with meningitis in the E. coil MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coil in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa. which is purified by affinity, with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright (C) 2009 John Wiley & Sons. Ltd.

Predominance of CTX-M-type extended-spectrum beta-lactamase genes among enterobacterial isolates from outpatients in Brazil

Two hundred fifty-seven nalidixic acid-resistant enterobacterial isolates were collected in a Brazilian community from January 2000 to May 2005 to determine the prevalence of plasmid-encoded extended-spectrum beta-lactamases. The bla(CTX-M) genetic environment was determined by polymerase chain reaction and sequencing. Eleven isolates (4.2%) harbored a bla(CTX-M-2) gene, 3 isolates bla(CTX-M-9), 2 isolates bla(CTX-M-8), and 6 isolates bla(SHV-5). Two novel bla(CTX-M-2) variants, namely, bla(CTX-M-74) and bla(CTX-M-75), were identified. (C) 2009 Elsevier Inc. All rights reserved.

Screening of plant extracts from the Brazilian Cerrado for their in vitro trypanocidal activity

In this study we report the screening of the in vitro trypanocidal activity of 20 extracts obtained from 10 different plant species growing in the Brazilian Cerrado: Aspidosperma macrocarpum Mart. (Apocynaceae), Aegiphila sellowiano Cham. (Verbenaceae), Byrsonima intermedia Juss. (Malpighiaceae), Cyperus rotundus L. (Cyperaceae), Leandra lacunosa Cogn. (Melastomataceae), Miconia ligustroides (DC.) Naudin. (Melastomataceae), Miconia sellowiana Naudin.(Melastomataceae),Myrcia variabilis Mart.ex DC. (Myrtaceae), Solanum lycocarpum St. Hil. (Solanaceae), and Tibouchina stenocarpa Cogn. (Melastomataceae). The most active extracts were submitted to phytochemical analyses. High-resolution gas chromatography analysis of the n-hexane extract of T. stenocarpa (IC(50) = 23.6 mu g/mL), the most active extract amongst all the tested samples, allowed the identification of beta-amyrin, alpha-amyrin, lupeol, friedelin, beta-friedelanol, campesterol, stigmasterol, and beta-sitosterol. Oleanolic and ursolic acids were isolated from the methylene chloride extract of T stenocarpa (IC(50) = 51.5 mu g/mL), while ursolic acid was isolated from the methylene chloride extract of M. variabilis (IC(50)=38.4 mu g/mL). Solasonine and solamargine were identified as major compounds by mass spectrometry analysis in the hydroalcoholic extract of the fruits of S. lycocarpum (IC(50)=57.1 mu g/mL).The results showed that the trypanocidal activity may be related to the major compounds identified in the crude active extracts.

Nosocomial outbreak by imipenem-resistant metallo-beta-lactamase-producing Pseudomonas aeruginosa in an adult intensive care unit in a Brazilian teaching hospital

Objective: To describe an outbreak of imipenem-resistant metallo-beta-lactamase-producing Pseudomonas aeruginosa, enzyme type bla, by horizontal transmission in patients admitted to a mixed adult ICU. Methods: A case-control study was carried out, including 47 patients (cases) and 122 patients (control) admitted to the mixed ICU of a university hospital in Minas Gerais. Brazil from November 2003 to July 2005. The infection site, risk factors, mortality, antibiotic susceptibility, metallo-beta-lactamase (MBL) production, enzyme type, and clonal diversity were analyzed, Results: A temporal/spatial relationship was detected in most patients (94%), overall mortality was 55.3%, and pneumonia was the predominant infection (85%). The majority of isolates (95%) were resistant to imipenem and other antibiotics, except for polymyxin, and showed MBL production (76.7%). Only bla SPM-1 (33%) was identified in the 15 specimens analyzed. In addition, 4 clones were identified, with a predominance of clone A (61.5%) and B (23.1%). On multivariate analysis, advanced age, mechanical ventilation, tracheostomy, and previous imipenem use were significant risk factors for imipenem-resistant P. aeruginosa infection. Conclusions: Clonal dissemination of MBL-producing P. aeruginosa strains with a spatial/temporal relationship disclosed problems in the practice of hospital infection control, low adherence to hand hygiene, and empirical antibiotic use. (C) 2008 Elsevier Espana, S.L. All rights reserved.

Synthesis of (2S)-isopropyl-5-alkynylpyrimidin-2-ones: precursors of beta-aminoacids

The efficient palladium-catalyzed Suzuki-Miyaura cross-coupling reaction of (2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-one with, arylethynyl-, heteroarylethynyl-, and alkylethynyltrifluoroborate salts is reported. The standard protocol was evaluated and optimized in order to gain access to suitable precursors of enantiopure 2-substituted beta-amino acids. The scope and limitations of this methodology are discussed. (C) 2010 Elsevier Ltd. All rights reserved.

Functionalization of (2S)-Isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones by a Suzuki-Miyaura Cross-Coupling Reaction Using Aryltrifluoroborate Salts: Convenient Enantioselective Preparation of alpha-Substituted beta-Amino Acids

A simple protocol for the Pd(OAc)(2)-catalyzed cross-coupling reaction of 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones with potassium aryltrifluoroborates was developed. The reaction is performed at 110 degrees C with a ligand-free catalyst. In all cases, complete conversion of the 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones and aryltrifluoroborates into the C-C coupling products was observed within 30-360 min. It is noteworthy that a large variety of groups present in the potassium aryltrifluoroborates (-CF(3), -OMe, -SEt, -CN, -CHO, -Cl, -Cbz, -NCbz, -OH, -CO(2)H) could be tolerated. Hydrogenation of the endocyclic double bonds in the Suzuki-Miyaura products followed by acid hydrolysis afforded highly enantioenriched alpha-aryl-substituted beta-amino acids.

Influence of Plasma Protein Binding on Pharmacodynamics: Estimation of In Vivo Receptor Affinities of beta Blockers Using a New Mechanism-Based PK-PD Modelling Approach

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic-pharmacodynamic studies with four beta blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist-antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K(B),(vivo)). These values were compared with in vitro affinities (K(B),(vitro)) on the basis of both total and free drug concentrations. For the total drug concentrations, the K(B),(vivo) estimates were 26, 13, 6.5 and 0.89 nM for S(-)-atenolol, S(-)-propranolol, S(-)-metoprolol and timolol. The K(B),(vivo) estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K(B),(vivo)-K(B),(vitro) correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(-)-propranolol (ratio K(B),(vivo)/K(B),(vitro) similar to 6.8). For the free drug, the correlation approximated the line of identity. Using this model, for beta-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics. (C) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3816-3828, 2009

[beta]-Hydroxy-F128b-Methylbutyrate (HMB) Supplementation and the Promotion of Muscle Growth and Strength

[beta]-Hydroxy [beta]-methylbutyrate (HMB), a metabolite of the essential amino acid leucine, is one of the latest dietary supplements promoted to enhance gains in strength and lean body mass associated with resistance training. Unlike anabolic hormones that induce muscle hypertrophy by increasing muscle protein synthesis, HMB is claimed to influence strength and lean body mass by acting as an anticatabolic agent, minimising protein breakdown and damage to cells that may occur with intense exercise. Research on HMB has recently tested this hypothesis, under the assumption that it may be the active compound associated with the anticatabolic effects of leucine and its metabolites. While much of the available literature is preliminary in nature and not without methodological concern, there is support for the claims made regarding HMB supplementation, at least in young, previously untrained individuals. A mechanism by which this may occur is unknown, but research undertaken to date suggests there may be a reduction in skeletal muscle damage, although this has not been assessed directly. The response of resistance trained and older individuals to HMB administration is less clear. While the results of research conducted to date appear encouraging, caution must be taken when interpreting outcomes as most manuscripts are presented in abstract form only, not having to withstand the rigors of peer review. Of the literature reviewed relating to HMB administration during resistance training, only 2 papers are full manuscripts appearing in peer reviewed journals. The remaining 8 papers are published as abstracts only, making it difficult to critically review the research. There is clearly a need for more tightly controlled, longer duration studies to verify if HMB enhances strength and muscular hypertrophy development associated with resistance training across a range of groups, including resistance trained individuals.

The effect of repeated prepulse and reflex stimulus presentations on startle prepulse inhibition

The magnitude of a startle reflex is inhibited if the reflex-eliciting stimuli is preceded by a prepulse stimulus at a short lead interval. Previous research in humans has shown that the extent of prepulse inhibition decreases over repeated presentations of reflex stimuli and prepulse-reflex stimulus pairings. The present study (N=70) investigated the effect of repeated presentations of prepulse stimuli, reflex stimuli, or prepulse-reflex stimulus pairings on prepulse inhibition. Five groups of subjects were presented during habituation training with either (a) reflex stimuli, (b) prepulse-reflex stimulus pairings, (c) a random sequence of prepulse and reflex stimuli, (d) prepulse stimuli, or (e) experimentally irrelevant light stimuli. Prepulse inhibition was reduced if startle stimuli were presented during habituation ((a), (b), (c)), but not after repeated presentation of the prepulse or the light stimulus ((d), (e)). The reduction in prepulse inhibition was abolished after dishabituation of the startle reflex. The present results indicate that habituation of the startle reflex can result in a reduction of prepulse inhibition. (C) 1998 Elsevier Science B.V.

Synthesis, NMR studies and conformational analysis of oxazolidine derivatives of the beta-adrenoreceptor antagonists metoprolol, atenolol and timolol

Formaldehyde-derived oxazolidine derivatives 4-7 of the beta-adrenoreceptor antagonists metoprolol 1, atenolol 2 and timolol 3 have been synthesised. Conformational analysis of 1-3 and the oxazolidine derivatives 4-7 has been performed using H-1 NMR spectroscopy and computational methods. The H-1 NMR studies show that for the aryloxypropanolamine beta-adrenoreceptor antagonists there is a predominance of the conformer in which the amine group is approximately antiperiplanar or trans to the aryloxymethylene group. Both H-1 NMR data and theoretical studies indicate that the oxazolidine derivatives 4-7 and the aryloxypropanolamine beta-adrenoreceptor antagonists 1-3 adopt similar conformations around the beta-amino alcohol moiety. Thus, oxazolidine ring formation does not dramatically alter the preferred conformation adopted by the beta-amino alcohol moiety of 1-3. Oxazolidine derivatives of aryloxypropanolamine beta-adrenoreceptor antagonists may therefore be appropriate as prodrugs, or semi-rigid analogues, when greater lipophilicity is required for drug delivery.