Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the beta-lactam producer Penicillium chrysogenum

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of g-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA. (C) 2011 Elsevier Inc. All rights reserved.

Characterization of new polymorphic functional markers for sugarcane

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[02/01167-1]

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAC-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Elp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. (C) 2009 Elsevier Masson SAS. All rights reserved.

Functional and Evolutionary Insights from the Genomes of Three Parasitoid Nasonia Species

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.

EXISTENCE RESULTS FOR ABSTRACT DEGENERATE NEUTRAL FUNCTIONAL DIFFERENTIAL EQUATIONS

In this paper we discuss the existence of solutions for a class of abstract degenerate neutral functional differential equations. Some applications to partial differential equations are considered.

Existence results for a damped second order abstract functional differential equation with impulses

In this work we study the existence and regularity of mild solutions for a damped second order abstract functional differential equation with impulses. The results are obtained using the cosine function theory and fixed point criterions. (C) 2009 Elsevier Ltd. All rights reserved.

The existence of solutions for impulsive neutral functional differential equations

This work deals with the existence of mild solutions for a class of impulsive functional differential equations of the neutral type associated with the family of linear closed (not necessarily bounded) operators {A(t) : t is an element of 1}. (C) 2009 Elsevier Ltd. All rights reserved.

Approximate controllability of second-order distributed implicit functional systems

In this paper we study the approximate controllability of control systems with states and controls in Hilbert spaces, and described by a second-order semilinear abstract functional differential equation with infinite delay. Initially we establish a characterization for the approximate controllability of a second-order abstract linear system and, in the last section, we compare the approximate controllability of a semilinear abstract functional system with the approximate controllability of the associated linear system. (C) 2008 Elsevier Ltd. All rights reserved.

Existence of solutions for second order partial neutral functional differential equations

We establish existence of mild solutions for a class of abstract second-order partial neutral functional differential equations with unbounded delay in a Banach space.

On a class of abstract neutral functional differential equations

By using the theory of semigroups of growth a, we discuss the existence of mild solutions for a class of abstract neutral functional differential equations. A concrete application to partial neutral functional differential equations is considered. (C) 2011 Elsevier Ltd. All rights reserved.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Echocardiographic predictors of functional capacity in endomyocardial fibrosis patients

Endomyocardial fibrosis (EMF) is a restrictive cardiomyopathy manifested mainly by diastolic heart failure. It is recognized that diastole is an important determinant of exercise capacity. The purpose of this study was to determine whether resting echocardiographic parameters might predict oxygen consumption (VO(2p)) by ergoespirometry and the prognostic role of functional capacity in EMF patients. A total of 32 patients with biventricular EMF (29 women, 55.3 +/- 11.4 years) were studied by echocardiography and ergoespirometry. The relationship between the echocardiographic indexes and the percentage of predicted VO(2p) (%VO(2p)) was investigated by the `stepwise` linear regression analysis. The median VO(2p) was 11 +/- 3 mL/kg/min and the %VO(2p) was 53 +/- 9%. There was a correlation of %VO(2p) with an average of A` at four sites of the mitral annulus (A` peak, r = 0.471, P = 0.023), E`/A` of the inferior mitral annulus (r = -0.433, P = 0.044), and myocardial performance index (r = -0.352, P = 0.048). On multiple regression analysis, only A` peak was an independent predictor of %VO(2p) (%VO(2p)= 26.34 + 332.44 x A` peak). EMF patients with %VO(2p)< 53% had an increased mortality rate with a relative risk of 8.47. In EMF patients, diastolic function plays an important role in determining the limitations to exercise and %VO(2p) has a prognostic value.

Age-Related Metabolic Profiles in Cognitively Healthy Elders: Results from a Voxel-Based [(18)F]Fluorodeoxyglucose-Positron-Emission Tomography Study with Partial Volume Effects Correction

BACKGROUND AND PURPOSE: Functional brain variability has been scarcely investigated in cognitively healthy elderly subjects, and it is currently debated whether previous findings of regional metabolic variability are artifacts associated with brain atrophy. The primary purpose of this study was to test whether there is regional cerebral age-related hypometabolism specifically in later stages of life. MATERIALS AND METHODS: MR imaging and FDG-PET data were acquired from 55 cognitively healthy elderly subjects, and voxel-based linear correlations between age and GM volume or regional cerebral metabolism were conducted by using SPM5 in images with and without correction for PVE. To investigate sex-specific differences in the pattern of brain aging, we repeated the above voxelwise calculations after dividing our sample by sex. RESULTS: Our analysis revealed 2 large clusters of age-related metabolic decrease in the overall sample, 1 in the left orbitofrontal cortex and the other in the right temporolimbic region, encompassing the hippocampus, the parahippocampal gyrus, and the amygdala. The division of our sample by sex revealed significant sex-specific age-related metabolic decrease in the left temporolimbic region of men and in the left dorsolateral frontal cortex of women. When we applied atrophy correction to our PET data, none of the above-mentioned correlations remained significant. CONCLUSIONS: Our findings suggest that age-related functional brain variability in cognitively healthy elderly individuals is largely secondary to the degree of regional brain atrophy, and the findings provide support to the notion that appropriate PVE correction is a key tool in neuroimaging investigations.

Executive dysfunction correlates with impaired functional status in older adults with varying degrees of cognitive impairment

Background: Previous studies have reported an association between executive dysfunction and the ability to perform activities of daily living (ADL)s among older adults. This study aims to examine the association between executive functions and functional status in a cross-section of older adults with varying degrees of cognitive impairment. Methods: 89 individuals (mean age 73.8 years) were recruited at a memory clinic in Sao Paulo, Brazil. Subjects underwent evaluation, and were allocated into three diagnostic groups according to cognitive status: normal controls (NC, n = 32), mild cognitive impairment (MCI, n = 3 1) and mild Alzheimer`s disease (AD, n=26). Executive functions were assessed with the 25-item Executive Interview (EXIT25), and functional status was measured with the Direct Assessment of Functional Status test (DAFS-R). Results: Significantly different total DAFS-R scores were observed across the three diagnostic groups. Patients with AD performed significantly worse in EXIT25 compared with subjects without dementia, and no significant differences were detected between NC and MCI patients. We found a robust negative correlation between the DAFS-R and the EXIT25 scores (r=-0.872, p < 0.001). Linear regression analyses suggested a significant influence of the EXIT-25 and the CAMCOG on the DAFS-R scores. Conclusion: Executive dysfunction and decline in general measures of cognitive functioning are associated with a lower ability to undertake instrumental ADLs. MCI patients showed worse functional status than NC subjects. MCI patients may show subtle changes in functional status that may only be captured by objective measures of ADLs.