Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.     

Preliminary characterization and grouping of Candida species by numerical analysis of protein profiles obtained by polyacrylamide gel electrophoresis

Whole-cell proteins from isolates of five Candida species (Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida guilliermondii) were separated by SDS-PAGE and the profiles obtained were converted into a binary data matrix that produced a cophenetic correlation phenogram. The analysis of the phenogram allowed detection of the cophenetic correlation levels existing among these species.     

Purification, and biochemical and biophysical characterization of cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha- helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.     

Identifying cardiovascular neurocircuitry involved in the exercise pressor reflex in humans using functional neurosurgery

Groups III and IV afferents carry sensory information regarding the muscle exercise pressor reflex, although the central integrating circuits of the reflex in humans are still poorly defined. Emerging evidence reports that the periaqueductal gray (PAG) could be a major site for integrating the "central command" component that initiates the cardiovascular response to exercise, since this area is activated during exercise and direct stimulation of the dorsal PAG causes an increase in arterial blood pressure (ABP) in humans. Here we recorded local field potentials (LFPs) from various "deep" brain nuclei during exercise tasks designed to elicit the muscle pressor reflex. The patients studied had undergone neurosurgery for the treatment of movement or pain disorders, thus had electrodes implanted stereotactically either in the PAG, subthalamic nucleus, globus pallidus interna, thalamus, hypothalamus, or anterior cingulate cortex. Fast Fourier transform analysis was applied to the neurograms to identify the power of fundamental spectral frequencies. Our PAG patients showed significant increases in LFP power at frequencies from 4 to 8 Hz (P < 0.01), 8 to 12 Hz (P < 0.001), and 12 to 25 Hz (P < 0.001). These periods were associated with maintained elevated ABP during muscle occlusion following exercise. Further increases in exercise intensity resulted in corresponding increases in PAG activity and ABP. No significant changes were seen in the activity of other nuclei during occlusion. These electrophysiological data provide direct evidence for a role of the PAG in the integrating neurocircuitry of the exercise pressor reflex in humans.     

RAPD and ISSR markers in the evaluation of genetic divergence among accessions of elephant grass

Considering the expected genetic variability of elephant grass (Pennisetum purpureum), due to its cultivation in different continents, we characterized and estimated the genetic divergences between 46 accessions of elephant grass with different edaphoclimatic adaptations, using RAPD and ISSR markers. We evaluated, comparatively, the consistency of the information achieved with these markers. Twenty-six RAPD and 25 ISSR primers were employed. The RAPD markers produced 185 bands, 72% of which were polymorphic, with a mean of 5.11 polymorphic bands per primer. The 25 ISSR starters produced 216 bands; 76% were polymorphic, with a mean of 6.56 polymorphic bands per primer. The correlation between the genetic distances achieved by the RAPD and ISSR markers was 0.76, which is highly significant by the Mantel test. Based on UPGMA grouping, considering the point of sudden change, five and six groups were formed for the data from the RAPD and ISSR markers, respectively. These markers provided partially concordant groups, indicating that these techniques can provide consistent information and consequently could be used in studies of genetic diversity among accessions.     

Carvedilol Prevents Ovariectomy-Induced Myocardial Contractile Dysfunction in Female Rat

Carvedilol has beneficial effects on cardiac function in patients with heart failure but its effect on ovariectomy-induced myocardial contractile dysfunction remains unclear. Estrogen deficiency induces myocardial contractile dysfunction and increases cardiovascular disease risk in postmenopausal women. Our aim was to investigate whether carvedilol, a beta receptor blocker, would prevent ovariectomy-induced myocardial contractile dysfunction. Female rats (8 weeks old) that underwent bilateral ovariectomy were randomly assigned to receive daily treatment with carvedilol (OVX+CAR, 20 mg/kg), placebo (OVX) and SHAM for 58 days. Left ventricle papillary muscle was mounted for isometric tension recordings. The inotropic response to Ca²⁺ (0.62 to 3.75 mM) and isoproterenol (Iso 10⁻⁸ to 10⁻² M) were assessed. Expression of calcium handling proteins was measured by western blot analysis. Carvedilol treatment in the OVX animals: prevented weight gain and slight hypertrophy, restored the reduced positive inotropic responses to Ca²⁺ and isoproterenol, prevented the reduction in SERCA2a expression, abolished the increase in superoxide anion production, normalized the increase in p22^phox expression, and decreased serum angiotensin converting enzyme (ACE) activity. This study demonstrated that myocardial contractile dysfunction and SERCA2a down regulation were prevented by carvedilol treatment. Superoxide anion production and NADPH oxidase seem to be involved in this response.     

Assessment of the Relationship between Self-Declared Ethnicity,Mitochondrial Haplogroups and Genomic Ancestry in Brazilian Individuals

In populations that have a high degree of admixture, such as in Brazil, the sole use of ethnicity self-declaration information is not a good method for classifying individuals regarding their ethnicity. Here, we evaluate the relationship of self-declared ethnicities with genomic ancestry and mitochondrial haplogroups in 492 individuals from southeastern Brazil. Mitochondrial haplogroups were obtained by analyzing the hypervariable regions of the mitochondrial DNA (mtDNA), and the genomic ancestry was obtained using 48 autosomal insertion-deletion ancestry informative markers (AIM). Of the 492 individuals, 74.6% self-declared as White, 13.8% as Brown and 10.4% as Black. Classification of the mtDNA haplogroups showed that 46.3% had African mtDNA, and the genomic ancestry analysis showed that the main contribution was European (57.4%). When we looked at the distribution of mtDNA and genomic ancestry according to the self-declared ethnicities from 367 individuals who self-declared as White, 37.6% showed African mtDNA, and they had a high contribution of European genomic ancestry (63.3%) but also a significant contribution of African ancestry (22.2%). Of the 68 individuals who self-declared as Brown, 25% showed Amerindian mtDNA and similar contribution of European and African genomic ancestries. Of the 51 subjects who self-declared as black, 80.4% had African mtDNA, and the main contribution of genomic ancestry was African (55.6%), but they also had a significant proportion of European ancestry (32.1%). The Brazilian population had a uniform degree of Amerindian genomic ancestry, and it was only with the use of genetic markers (autosomal or mitochondrial) that we were able to capture Amerindian ancestry information. Additionally, it was possible to observe a high degree of heterogeneity in the ancestry for both types of genetic markers, which shows the high genetic admixture that is present in the Brazilian population. We suggest that in epidemiological studies, the use of these methods could provide complementary information.     

Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA

The formamidopyrimidine-DNA glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60 degrees bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.     

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml^?1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml^?1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.