Anticipation of pain enhances the nociceptive transmission and functional connectivity within pain network in rats

Background: Expectation is a very potent pain modulator in both humans and animals. There is evidence that pain transmission neurons are modulated by expectation preceding painful stimuli. Nonetheless, few studies have examined the influence of pain expectation on the pain-related neuronal activity and the functional connectivity within the central nociceptive network. Results: This study used a tone-laser conditioning paradigm to establish the pain expectation in rats, and simultaneously recorded the anterior cingulate cortex (ACC), the medial dorsal thalamus (MD), and the primary somatosensory cortex (SI) to investigate the effect of pain expectation on laser-induced neuronal responses. Cross-correlation and partial directed coherence analysis were used to determine the functional interactions within and between the recorded areas during nociceptive transmission. The results showed that under anticipation condition, the neuronal activity to the auditory cue was significantly increased in the ACC area, whereas those to actual noxious stimuli were enhanced in all the recorded areas. Furthermore, neuronal correlations within and between these areas were significantly increased under conditions of expectation compared to those under non-expectation conditions, indicating an enhanced synchronization of neural activity within the pain network. In addition, information flow from the medial (ACC and MD) to the lateral (SI cortex) pain pathway increased, suggesting that the emotion-related neural circuits may modulate the neuronal activity in the somatosensory pathway during nociceptive transmission. Conclusion: These results demonstrate that the nociceptive processing in both medial and lateral pain systems is modulated by the expectation of pain.

REPRODUCTIVE BIOLOGY AND FUNCTIONAL MATURITY IN FEMALES OF AEGLA FRANCA (DECAPODA: ANOMURA: AEGLIDAE)

We describe the reproductive period. fecundity, and average size at the onset of functional maturity of female Aegla franca, the northernmost distributed aeglid species. The reproductive period is markedly seasonal and takes place front May (austral mid-autumn) to August (late winter). Ovigerous females appear quite abruptly in the population by May, and this condition is observed in all adult females sampled regardless of their size. The average size at the onset of functional maturity in females, at which 50% of the females sampled during the reproductive period were considered adults, was 12.75 mm CL. The smallest post-ovigerous female measured 12.06 mm carapace length (CL). Mean fecundity (+/- S.D.) from 41 females bearing early and intermediate eggs was 129.1 +/- 32.2 and corresponded to a mean female CL of 14.11 mm. The elliptical-shaped eggs exhibited significant increase in size along the development stages. The third pair of pleopods bore higher number of eggs than the others. Compiled information regarding the reproductive period reported for aeglids revealed all increase in the breeding period length with latitude. The reproductive period tends to be shorter in localities under larger rainfall variation and smaller temperature variability than in sites with opposite climate conditions. Eggs tend to be fewer in number and larger in size towards lower latitudes. We present an hypothesis that stream water velocity might act as a major selective pressure during the early life history of fluvial aeglids with direct effect on the reproductive pattern.

Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome c

The alternative low-spin states of Fe3+ and Fe2+ cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met 80 by another strong field ligand at the sixth heme iron coordination position, Fe3+ ALSScytc exhibited 1-nm Soret band blue shift and e enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe3+ and Fe2+ ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe3+ ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential similar to 200 mV lower than the wild-type protein (1220 mV) and was more susceptible to the attack of free radicals.

Analysis of genomic and functional RFLP derived markers associated with sucrose content, fiber and yield QTLs in a sugarcane (Saccharum spp.) commercial cross

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.

Rheological and functional properties of flours from banana pulp and peel

Unripe banana flour can be an alternative to minimize post-harvest loss and to increase the aggregate value of banana fruit. Flour from unripe banana is rich in phytosterols and resistant starch, being proposed as health food. Flours from unripe banana peel and pulp were evaluated on their composition, phytosterols content, thermal and rheological properties, and pasting profiles. High amounts of beta-sitosterol, campesterol, and stigmasterol were found in flour from banana peel. These samples showed lower viscosity values of pasting profiles, lower energy enthalpy on gelatinization, and higher temperature of gelatinization than those ones from pulp. Anti-oxidant treatment of fruits with citric acid does not change pasting profiles of flours from pulp, but resulted in slight increase in viscosity, suggesting that structure of starch could be modified by acidification.

Physical-chemical and functional properties of maca root starch (Lepidium meyenii Walpers)

The starch of maca (Lepidium meyenii Walpers) presented oval and irregular morphology, with granule size between 7.4 and 14.9 mu m in length and 5.8 and 9.3 mu m in diameter. The isolated starch showed the following features: purity of 87.8%, with 0.28% lipids, 0.2% fibre and 0.12% fixed mineral residue, and no protein detected; the ratio between the amylose and amylopectin contents were 20:80: the solubility at 90 degrees C was 61.4%, the swelling power was 119.0g water/g starch and the water absorption capacity was 45.9 g water/g starch; the gel turbidity rose 44% during the storing time; the gelatinization temperature was 47.7 degrees C and the transition enthalpy 6.22 J/g; the maximum viscosity reached 1260 UB at 46.4 degrees C, with breakdown, setback and consistence of 850, 440 and -410 UB, respectively. The low gelling temperature and the stability during gel refrigeration could be adequate for foods requiring moderate temperature process, but not for frozen food. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning, characterization and functional analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly -2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81 in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

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)).

Quercetin in lyotropic liquid crystalline formulations: Physical, chemical and functional stability

The purpose of this study was to develop a lyotropic liquid crystalline formulation using the emulsifier vitamin E TPGS and evaluate its behavior after incorporation of a flavonoid, quercetin. The physical (macro and microscopic), chemical (determination of quercetin content by the HPLC method) and functional (determination of quercetin antioxidant activity by DPPH center dot assay) stability of the lamellar liquid crystalline formulation containing flavonoid was evaluated when stored at 4+/-2 degrees C; 30+/-2 degrees C/70+/-5% RH (relative humidity) and 40+/-2 degrees C/70+/-5% RH during 12 months. The lamellar liquid crystalline structure of the formulation was maintained during the experiment, however chemical and functional stability results showed a great influence of the storage period in all conditions tested. A significant decrease in quercetin content (approximately 40%) was detected during the first month of storage and a similar significant loss in antioxidant activity was detected after 6 months. The remaining flavonoid content was unchanged during the final 6 months of the experimental period. The results suggest possible interactions between quercetin and the liquid crystalline formulation, which could inhibit or reduce the quercetin activity incorporated in the system. In conclusion, the present study demonstrated that incorporation of quercetin (1%) did not affect the liquid crystalline structure composed of vitamin E TPGS/IPM/PG-H2O (1:1) at 63.75/21.25/15 (w/w/w). Nevertheless, of the total quercetin incorporated in the system only 60% was free to act as an antioxidant.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M(r) = 34,000 under reducing conditions and pI similar to 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (-70 to 37 degrees C), pH values (3-9) or in the presence of divalent metal ions (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom. (C) 2009 Elsevier Ltd. All rights reserved.

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.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Structural and functional characterisation of human sulfotransferases

The human aryl sulfotransferases HAST4 and HAST4v vary by only two amino acids but exhibit markedly different affinity towards the sulfonate acceptor p-nitrophenol and the sulfonate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). To determine the importance of each of these amino acid differences, chimeric constructs were made of HAST4 and HAST4v. By attaching the last 120 amino acids of HAST-4v to HAST4 (changing Thr235 to Asn235) we have been able to produce a protein that has a K-m for PAPS similar to HAST4v. The reverse construct, HAST4v/4 produces a protein with a K-m for PAPS similar to HAST4. These data suggests that the COOH-terminal of sulfotransferases is involved in co-factor binding. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes

Arylamine N-acetyltransferase (NAT) was first identified as the inactivator of the anti-tubercular drug isoniazid, The enzyme was shown to catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of the hydrazine drug. The rate of inactivation of isoniazid was polymorphically distributed in the population and was one of the first examples of pharmacogenetic variation, NAT was identified recently in Mycobacterium tuberculosis and is a candidate for; modulating the response to isoniazid, Genome sequences have revealed many homologous members of this unique family of enzymes. The first three-dimensional structure of a member of the NAT family identifies a catalytic triad consisting of aspartate, histidine and cysteine proposed to form the activation mechanism. So far, all procaryotic NATs resemble the human enzyme which acetylates isoniazid (NAT2), Human NAT2 is characteristic of drug-metabolizing enzymes: it is found in liver and intestine, In humans and other mammals, there are up to three different isoenzymes. If only one isoenzyme is present, it is like human NAT1. Human NAT1 and its murine equivalent specifically acetylate the folate catabolite p-amino-benzoylglutamate. NAT1 and its murine homologue each have a ubiquitous tissue distribution and are expressed early in development at the blastocyst stage, During murine embryonic development, NAT is expressed in the developing neural tube. The proposed endogenous role of NAT in folate metabolism, and its multi-allelic nature, indicate that its role in development should be assessed further.