Preliminary X-ray crystallographic studies of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

Crotoxin B is a basic phospholipase A(2) found in the venom of Crotalus durissus terrificus and is one of the subunits that constitute crotoxin. This heterodimeric toxin, which is the main component of C. d. terrificus venom, is completed by an acidic, nontoxic and non-enzymatic component (crotoxin A) and is involved in important envenomation effects, such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, no crystal structure is currently available for crotoxin, crotoxin A or crotoxin B. In this work, the crystallization, X-ray diffraction data collection to 2.28 angstrom resolution and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2) is presented.

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Cratalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

Partial cDNA sequence analysis of myosin Va from rainbow trout (Oncorhynchus mykiss) and its relationship to myosin v isoforms from other vertebrates.

Partial cDNA sequences of myosin V from rainbow trout Oncorhynchus mykiss were analyzed and showed high similarity to MVa from other vertebrates. Phylogenetic analysis has shown that events resulting in the formation of paralogous copies of myosin Va, Vb, and Vc occurred before the divergence of vertebrates into different classes. Expression analysis of myosin Va, Vb, and Vc in different O. mykiss tissues revealed MVa exclusively expressed in hypophysis and brain whereas Vb and Vc were expressed in practically all tissues analyzed. The nucleotide sequence for myosin V was explored in a fish species for the first time and these results represent an important start in understanding the organization, evolution, and expression of myosins in early vertebrates. The data presented here represent contributions to the knowledge of rainbow trout genome. A better understanding of this economically important species could assist in development of improved strains of this fish for aquaculture.

Partial molecular characterization of the Nile tilapia (Oreochromis niloticus) alpha-cardiac muscle actin gene and its relationship to actin isoforms of other fish species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lipoperoxidation and cyclooxygenase enzyme inhibitory piperidine alkaloids from Cassia spectabilis green fruits

Phytochernical work in the search for bioactive metabolites from the methanolic extract of Senna spectabilis green fruits led to the isolation of a new piperidine alkaloid, (+)-3-O-feruloylcassine (1), in addition to the known (-)-spectaline (2) and (-)-3-O-acetylspectaline (3). The isolates were submitted to in vitro evaluation of lipoperoxidation (LPO) and cyclooxygenase enzymes (COX-1 and -2) inhibitory properties and showed moderate antioxidant activities (40-70%) at 100 ppm when compared to commercial standards BHT and vitamin E and moderate inhibition of COX-1 (ca. 40%) and marginal inhibition of COX-2 enzymes (< 10%) at 100 ppm when compared to nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, rofecoxib, and celecoxib, respectively.

Differences in reactivity of paracoccidioidomycosis sera with gp43 isoforms

The glycoprotein gp43 from Paracoccidioides brasiliensis is the main antigenic component in paracoccidioidomycosis (PCM) because it is recognized by 100% of PCM patients. It has also been shown that different fungal strains produce gp43 with at least four isoform profiles. In this study, different isoform profiles from gp43, with pIs ranging from 5.8 to 8.5, were affinity purified from various P. brasiliensis (B-339, S.S., 1925 and I-9) exoantigens. Because of the isoform heterogeneity, we questioned whether those isoform profiles could be similarly recognized by acute or chronic PCM patients. By using a specific and sensitive method for detection of human IgG anti-gp43 antibodies, the monoclonal antibody capture immunoassay, we report that not all gp43 isoform profiles are equally recognized in PCM sera when anti-gp43 MAb 17c was employed as capturing antibody. Our result showed that recognition of pI 8.5 gp43 isoform was significantly lower for both acute (56%) and chronic patients (71%), compared with gp43 isoforms from the standard strain B-339. on the other hand, when anti-gp43 MAb 8a, which recognizes a different antigenic epitope was used to capture the different gp43 isoform profiles, all patient's sera reacted similarly. The results described suggest that not all the antigenic epitopes expressed by gp43 are equally present in all P. brasiliensis strains.

Isolation, characterization and biological activity of acidic phospholipase A(2) isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIIB diffracted beyond 1.8 Angstrom resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 Angstrom, b = 54.2 Angstrom and c = 90.7 Angstrom. The crystal structure has been refined to a crystallographic residual of 16.1% (R-free = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Treatment of experimental periodontal disease by a selective inhibitor of cyclooxygenase-2 with scaling and root planing (SRP)

Lumiracoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) approved for the relief of symptoms of chronic inflammatory conditions. The aim of this study was to evaluate the effects of this specific inhibitor of COX-2 as adjunctive treatment on induced periodontitis in rats. Periodontal disease was induced at the first mandibular molar of 60 rats. After 7 days, the ligature was removed and all animals were submitted to scaling and root planing (SRP) along with local irrigation with saline solution and were divided into 2 groups: SRP (n = 30)-received subcutaneous injections of 1 mg/kg of body weight/day of saline solution for 3 days and; SRP + L (n = 30)-received subcutaneous injections of 1 mg/kg of body weight/day of Lumiracoxib for 3 days. Ten animals in each group were killed at 7, 15, and 30 days. The histological description was performed and the histometric values were statistically analyzed. In Group SRP + L, the histometric analysis (0.58 ± 0.08, 0.64 ± 0.06, and 0.56 ± 0.10 mm 2) showed less bone loss (p < 0.05) than Group SRP (1.52 ± 0.08, 1.55 ± 0.09, and 1.49 ± 0.24 mm 2) at 7, 15, and 30 days, respectively. Within the limits of this study it can be concluded that subcutaneous application of specific inhibitor of COX-2 was a beneficial adjunctive treatment for periodontal diseases induced in rats. © 2010 Springer Basel AG.

NFAT isoforms regulate muscle fiber type transition without altering can during aerobic training

The purpose of this study was to determine whether the aerobic training-induced fiber-type transition in different muscles is associated with alterations in NFAT isoforms gene expression. We hypothesized that the aerobic training-induced fiber-type transition would be mediated by NFATc1-c3 isoforms without altering the CaN expression. Male Wistar rats (80 days old) were divided into a trained group (T; n=8) that underwent an 8-wk swimming endurance training program (5 days/week) and a control group (C; n=8). After the experimental period, the animals were sacrificed, and the soleus (SOL) and plantaris (PL) muscles were collected for morphometrical, histochemical and molecular analyses. Aerobic training induced a type I-to-type IIA fiber transition in the SOL muscle and a type IIB-to-type IIA fiber transition in the PL muscle, which were concomitant with a significant (p<0.05) increase in NFATc1-c3 gene expression in both the SOL and PL muscles. In contrast, the expression levels of calcineurin (CaN) and NFATc4 remained unchanged. Therefore, our results showed that fiber type switching induced by aerobic training is mediated by NFATc1-c3 isoforms without altering the CaN expression. © Georg Thieme Verlag KG Stuttgart. New York.

Pharmacological Evaluation and Preparation of Nonsteroidal Anti-Inflammatory Drugs Containing an N-Acyl Hydrazone Subunit

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Temporal analysis of prostaglandin F2 alpha receptor, caspase 3, and cyclooxygenase 2 messenger RNA expression and prostaglandin F2 alpha receptor and cyclooxygenase 2 protein expression in endometrial tissue from multiparous Nelore (Bos taurus indicus) cows treated with cloprostenol sodium during puerperium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Increased cyclooxygenase-2 and vascular endothelial growth factor protein expression is associated with canine prostatic carcionogenesis process

Background: COX-2 is one of the most important prostaglandin involved in urologic cancer and seems to be associated with tumor progression, invasion, and metastasis. In addition, several effects have been reported for VEGF, including inducing angiogenesis, promoting cell migration, and inhibiting apoptosis. COX2 and VEGF up-regulation have been reported in human prostate cancer. Due to the importance of canine natural model for prostate cancer, the aim of this study was to evaluate COX-2 and VEGF protein expression in canine carcinogenic process. Material and Methods: Seventy-four prostatic tissues from dogs were selected to be evaluated for protein expression by immunohistochemistry (IHC), including: 10 normal prostatic tissues, 20 benign prostatic hyperplasias (BPH), 25 proliferative inflammatory atrophies (PIA) and 20 prostatic carcinomas (PCa). COX-2 and VEGF were detected using the monoclonal antibody CX-294 (1:50 dilution, Dako Cytomation and sc-53463 (1:100 dilution, Santa Cruz), respectively. The immunolabelling was performed by a polymer method (Histofine, Nichirei Biosciences). All reaction included negative controls by omitting the primary antibody. The percentage of C-MYC, E-cadherin, and p63- positive cells per lesion was evaluated according to Prowatke et al. (2007). The samples were scored separately according to staining intensity and graded semi-quantitatively as negative, weakly positive (1), moderately positive, and strongly positive. The score was done in one 400 magnification field, considering only the lesion, since this was done in a TMA core of 1 mm. For statistical analyses, the immunostaining classifications were reduced to two categories: negative and positive. The negative category included negative and weakly positive staining. Chi-square or Fisher exact test was used to determine the association between the categorical variables. Results: The COX-2 protein expression was elevated in the cytoplasm of the canine PCa and PIA compared to normal prostate (p=0.002). VEGF protein expression was increased in 94.75% of the PCa and 100% of the PIA compared with to normal prostate (p = 0.001). No difference was found when compared normal prostate with BPH. Conclusions: This study has demonstrated that the carcinogenesis of canine prostatic tissue may be related to gain of COX-2 and VEGF protein expression.

Pharmacological evaluation and preparation of nonsteroidal anti-inflammatory drugs containing an N-Acyl hydrazone subunit

A series of anti-inflammatory derivatives containing an N-acyl hydrazone subunit (4a–e) were synthesized and characterized. Docking studies were performed that suggest that compounds 4a–e bind to cyclooxygenase (COX)-1 and COX-2 isoforms, but with higher affinity for COX-2. The compounds display similar anti-inflammatory activities in vivo, although compound 4c is the most effective compound for inhibiting rat paw edema, with a reduction in the extent of inflammation of 35.9% and 52.8% at 2 and 4 h, respectively. The anti-inflammatory activity of N-acyl hydrazone derivatives was inferior to their respective parent drugs, except for compound 4c after 5 h. Ulcerogenic studies revealed that compounds 4a–e are less gastrotoxic than the respective parent drug. Compounds 4b–e demonstrated mucosal damage comparable to celecoxib. The in vivo analgesic activities of the compounds are higher than the respective parent drug for compounds 4a–b and 4d–e. Compound 4a was more active than dipyrone in reducing acetic-acid-induced abdominal constrictions. Our results indicate that compounds 4a–e are anti-inflammatory and analgesic compounds with reduced gastrotoxicity compared to their respective parent non-steroidal anti-inflammatory drugs.