A lipocalin sequence signature modulates cell survival

Lipocalins are beta-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Determination of eight fatty acid ethyl esters in meconium samples by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A number of fatty acid ethyl esters (FAEEs) have recently been detected in meconium samples. Several of these FAEEs have been evaluated as possible biomarkers for in utero ethanol exposure. In the present study, a method was optimized and validated for the simultaneous determination of eight FAEEs (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate and ethyl arachidonate) in meconium samples. FAEEs were extracted by headspace solid-phase microextraction. Analyte detection and quantification were carried out using GC-MS operated in chemical ionization mode. The corresponding D5-ethyl esters were synthesized and used as internal standards. The LOQ and LOD for each analyte were <150 and <100 ng/g, respectively. The method showed good linearity (r(2)>0.98) in the concentration range studied (LOQ -2000 ng/g). The intra- and interday imprecision, given by the RSD of the method, was lower than 15% for all FAEEs studied. The validated method was applied to 63 authentic specimens. FAEEs could be detected in alcohol-exposed newborns ( >600 ng/g cumulative concentration). Interestingly, FAEEs could also be detected in some non-exposed newborns, although the concentrations were much lower than those measured in exposed cases.

Hypoxia Inducible Factor-Dependent Regulation of Angiogenesis by Nitro-Fatty Acids

Objective-Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. Methods and Results-The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 mu mol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1 alpha (HIF-1 alpha) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1 alpha target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1 alpha expression. Short hairpin RNA-mediated knockdown of HIF-1 alpha attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. Conclusion-Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1 alpha. (Arterioscler Thromb Vasc Biol. 2011;31:1360-1367.)

Ascorbic acid concentration is reduced in the secondary aqueous humour of glaucomatous patients

P>Background: We aimed to evaluate the ascorbic acid concentration in secondary aqueous humour (AH) from glaucomatous patients and to compare it with primary AH from primary open-angle glaucoma patients and non-glaucomatous patients. Methods: Primary AH samples were prospectively obtained from clinically uncontrolled primary open-angle glaucoma patients and senile cataract patients (controls) prior to trabeculectomy and cataract surgery. Secondary AH samples were obtained from eyes with previous intraocular surgery, prior to trabeculectomy or cataract surgery. AH (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber just before surgery and then immediately stored at -80 degrees C. The ascorbic acid concentration was determined in a masked fashion by high-pressure liquid chromatography. Results: A total of 18 patients with senile cataract, 16 glaucomatous patients with primary AH (no previous intraocular surgery) and 11 glaucomatous patients with secondary AH (previous intraocular surgery) were included. There was no difference in mean age between groups (P = 0.15). The mean +/- standard deviation concentration of ascorbic acid in the secondary AH from glaucomatous patients (504 +/- 213 mu mol/L [95% confidence interval {CI}, 383-624]) was significantly lower than the concentration of ascorbic acid found in the primary aqueous of primary open-angle glaucoma (919 +/- 427 mu mol/L [95% CI, 709-1128]) and control patients (1049 +/- 433 mu mol/L [95% CI, 848-1249]; P < 0.01, Kruskal-Wallis test). Conclusions: The ascorbic acid concentration in secondary AH of glaucomatous patients was approximately twofold lower in comparison with primary AH of glaucomatous and cataract patients. The implications of a reduced concentration of ascorbic acid in the secondary AH deserve further investigation.

Relationships between gene polymorphisms of folate-related proteins and vitamins and metabolites in pregnant women and neonates

Background: The methylenetetrahydrofolate reductase (MTHFR), glutamate carboxypeptidase II (GCPII) and reduced folate carrier (RFC1) gene polymorphisms were associated with folate status. We investigated the effects of these polymorphisms on serum folate (SF) and folate-related metabolites in mothers and their neonates. Methods: Cobalamin (Cbl), SF, total homocysteine (tHcy), methylmalonic acid (MMA), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in 275 healthy women and their neonates. MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms were determined by PCR-RFLP. Results: Maternal tHcy was affected individually by MTHFR C677T and GCPII C1561T polymorphisms and by combined genotypes MTHFR 677TT/GCPII 1561CC and MTHFR 677TT/RFC1 80AG. The MTHFR and RFC1 polymorphisms were not associated with variations in vitamins or SAM, SAH and MMA in neonates. Neonatal tHcy was predicted directly by maternal tHcy and inversely by maternal SF, neonatal Cbl and neonatal RFC1 80G allele (AG+GG genotypes). Maternal MMA and SAM/SAH were predicted by creatinine and Cbl, respectively. Neonatal MMA was predicted by maternal MMA and GCPII 1561T allele (CT+TT genotypes) and by neonatal Cbl. Conclusions: Maternal tHcy was affected by MTHFR C677T, RFC1 A80G and GCPII C1561T polymorphisms. Maternal GCPII C1561T variant was associated with neonatal MMA. Neonatal RFC1 A80G polymorphism influenced tHcy in neonates. (C) 2008 Elsevier B.V. All rights reserved.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women

Objectives: To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/ S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status. Subjects/ Methods: Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism ( RFLP). Results: Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l. Conclusions: Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

Synthesis of benzophenones from geminal biaryl ethenes using m-chloroperbenzoic acid

The oxidation of geminal biaryl ethenes 3 and 1,3-enynes 5 using m-chloroperbenzoic acid in dichloromethane at room temperature presents a catalyst-free approach for the synthesis of functionalized benzophenones 4 and ynones 6, respectively. (C) 2009 Elsevier Ltd. All rights reserved.

Extractive Fermentation of Clavulanic Acid by Streptomyces DAUFPE 3060 Using Aqueous Two-Phase System

The influence of four variables, specifically PEG molar mass (400, 1,000, and 8,000 g/mol), concentrations of PEG and phosphate salts (15, 20, and 25% for both), and agitation intensity (110, 150, and 200 rpm), on clavulanic acid (CA) extraction by extractive fermentation with PEG/phosphate salts aqueous two-phase system was investigated in shaken flasks using a 2(4-1)-fractional factorial design. After selection of the two most significant variables (agitation intensity and PEG molar mass), an optimization study conducted according to a 2(2)-central composite design revealed that 25% PEG 8,000 g/mol and phosphate salts at 240 rpm (run 6) were the best conditions for the extractive fermentation, leading to the best results in terms of partition coefficient (k = 8.2), yield of CA in the PEG-rich phase (eta(T) = 93%) and productivity (P = 5.3 mg/Lh). As a first attempt to make a scale-up of these results, the effectiveness of the extractive fermentation was then checked in a bench-scale bioreactor under conditions as close as possible to the optimum ones determined in flasks. The highest CA concentration obtained in the PEG-rich phase (691 mg/L) was 30% higher than in flasks, thus demonstrating the potential of such a new process, integrating the production and extraction steps, as a promising, low-cost tool to obtain high yields of this and similar products. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 95-103, 2011

Screening of Variables Influencing the Clavulanic Acid Production by Streptomyces DAUFPE 3060 Strain

Clavulanic acid (CA) is a beta-lactam antibiotic, which has a potent beta-lactamase inhibiting activity. The influence of five variables, namely pH (6.0, 6.4, and 6.8), temperature (28A degrees C, 30A degrees C, and 32A degrees C), agitation intensity (150, 200, and 250 rpm), glycerol concentration (5.0, 7.5, and 10 g/L) and soybean flour concentration (5.0, 12.5, and 20 g/L), on CA production by a new isolate of Streptomyces (DAUFPE 3060) was investigated in 250-mL Erlenmeyer flasks using a fractional factorial design. Temperature and soybean flour concentration were shown to be the two variables that exerted the most important effects on the production of CA at 95% confidence level. The highest CA concentration (494 mg/L) was obtained after 48 h at 150 rpm, 32A degrees C, pH 6.0, 5.0 g/L glycerol, and 20 g/L soybean flour concentrations. Under these conditions, the yields of biomass and product on consumed substrate were 0.26 g(X)/g(S) and 64.3 mg(P)/g(S), respectively. Fermentations performed in 3.0-L bench-scale fermenter allowed increasing the CA production by about 60%.

Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060

Clavulanic acid (CA) is a potent inhibitor of beta-lactamases, produced by some resistant pathogenic microorganisms, which allows efficient treatment of infectious diseases. The kinetic and thermodynamic parameters of CA production by a new isolate of Streptomyces DAUFPE 3060 and its degradation were evaluated. The effect of temperature on the system was investigated in the range 24-40 degrees C adopting an overall model accounting for (a) the Arrhenius-type formation of CA by fermentation, (b) the hypothetical reversible unfolding of the enzyme limiting the overall metabolism, and (c) the irreversible first-order degradation of CA. The higher rates of CA formation (k(CA) = 0,107 h(-1)) and degradation (k(d) = 0.062 h(-1)) were observed at 32 and 40 degrees C, respectively. The main thermodynamic parameters of the three above hypothesized events were estimated. In particular, the activation parameters of degradation (activation energy = 39.0 kJ/mol; Delta H(d)* = 36.5 kJ/mol; Delta S(d)* = -219.7 J/(mol K); Delta G(d)* = 103.5 kJ/mol) compare reasonably well with those reported in the literature for similar system without taking into account the other two events. (C) 2009 Elsevier Inc. All rights reserved.

Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach

Clavulanic acid (CA) is a beta-lactam antibiotic that alone exhibits only weak antibacterial activity, but is a potent inhibitor of beta-lactamases enzymes. For this reason it is used as a therapeutic in conjunction with penicillins and cephalosporins. However, it is a well-known fact that it is unstable not only during its production phase, but also during downstream processing. Therefore, the main objective of this study was the evaluation of CA long-term stability under different conditions of pH and temperature, in the presence of variable levels of different salts, so as to suggest the best conditions to perform its simultaneous production and recovery by two-phase polymer/salt liquid-liquid extractive fermentation. To this purpose, the CA stability was investigated at different values of pH (4.0-8.0) and temperature (20-45 degrees C), and the best conditions were met at a pH 6.0-7.2 and 20 degrees C. Its stability was also investigated at 30 degrees C in the presence of NaCl, Na(2)SO(4), CaCl(2) and MgSO(4) at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 6.5). All salts led to increased CA instability with respect to the buffer alone, and this effect decreased in following sequence: Na(2)SO(4) > MgSO(4) > CaCl(2) > NaCl. Kinetic and thermodynamic parameters of CA degradation were calculated adopting a new model that took into consideration the equilibrium between the active and a reversibly inactivated form of CA after long-time degradation. (C) 2009 Elsevier B.V. All rights reserved.

Liquid-liquid extraction by mixed micellar systems: A new approach for clavulanic acid recovery from fermented broth

This work is the first attempt to apply aqueous two-phase mixed micellar systems (ATPMS) of the nonionic surfactant Triton X-114 and the anionic one AOT to extract clavulanic acid (CA) from broth fermented by Streptomyces clavuligerus. Cloud points were determined in McIlvane buffer pH 6.5 with or without NaCl, and diagram phases/coexistence curves were constructed. CA partition was investigated following a 2(4)-full factorial design in which AOT (0.022, 0.033 and 0.044% w/w), Triton X-114 (1.0, 3.0 and 5.0% w/w) and NaCl (0, 2.85 and 5.70% w/w) concentrations and temperature (24,26 and 28 degrees C) were selected as independent variables, and CA partition coefficient (K(CA)) and yield in the top phase (eta(CA)) as responses. CA partitioned always to the top, micelle-poor phase. The regression analysis pointed out that NaCl concentration and interaction between temperature and Triton X-114 concentration had statistically significant effects on K(CA), while eta(CA) was mainly influenced by temperature, Triton X-114 concentration and their interaction. Different ATPMS compositions were then needed to maximize these responses, specifically 0.022% (w/w) AOT, 5% (w/w) Triton X-114 for K(CA) (2.08), and 0.044% (w/w) AOT, 1% (w/w) Triton X-114 for eta(CA) (98.7%), both at 24 degrees C without NaCl. Since at 0.022% (w/w) AOT, 1% (w/w) Triton X-114 and 28 degrees C without NaCl the system was able to ensure satisfactory intermediate results (K(CA) = 1.48; eta(CA) = 86.3%), these conditions were selected as the best ones. These preliminary results are of concern for possible industrial application, because CA partition to the dilute phase can simplify the subsequent purification protocol. (C) 2011 Elsevier B.V. All rights reserved.

Influence of total solids contents of milk whey on the acidifying profile and viability of various lactic acid bacteria

The main objectives of the present study were (a) to study the effects of the different combinations of Lactobacillus delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), and Bifidobacterium animalis subsp. lactis (BI) in co-culture with Streptococcus thermophilus (St) on the rate of acid development in milk and milk-whey mixture, and (b) the effect of the level of the total solids of the different bases on the acidification profile and viability of potential health-promoting microorganisms. The co-culture of St-Lr showed the lowest values V(max) in all bases; while the co-culture St-Bl had high t(Vmax) in milk and whey bases (12 and 10 g/100 g, respectively). Co-cultures St-La and St-Lb reached V(max) at pH 5.5, while St-Lr and St-Bl at pH 5.91. Fermentation time to reach pH 4.5 was longer when St-Lr co-culture was used, while St-Lb had the lowest value. All the products had slight development of acid during the storage period, and lowest values were observed when the St-Bl co-culture was employed. Lb, BI and St cultures had high counts at pH 4.5 in the three bases. The total solids affected the viability of Lb and La. The technological interest of these combinations is discussed in this article. (C) 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. AM rights reserved.

Extraction of clavulanic acid using aqueous two-phase micellar system

An investigation of clavulanic acid behavior in an aqueous two-phase micellar system employing the surfactants n-decyltetraethylene oxide (C(10)E(4)) and dodecyldimethylamine oxide (DDAO) was carried out. According to the results, clavulanic acid partitions evenly between the two phases of DDAO micellar system, mixed DDAO C(10)E(4) micellar system, as well as C10E4 micellar system. Therefore, electrostatic interactions between positively charged DDAO-containing micelles and negatively charged drug were not strong enough to influence the partitioning. Nevertheless, clavulanic acid extraction from Streptomyces clavuligerus fermentation broth in C(10)E(4) micellar system employing a previous protein denaturation step provided recovery of 52% clavulanic acid with removal of 70% of the contaminant proteins, which is already promising as a purification strategy. (C) 2011 International Union of Biochemistry and Molecular Biology, Inc. Volume 58, Number 2, March/April 2011, Pages 103-108. E-mail: corangel@usp.br

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese

Lactic acid bacteria ( LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n = 2) and Lactococcus lactis ssp. lactis (n = 18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.