Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA2 activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA2 activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED50 of 0.270 microg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.     

Variation in the Distribution of Trace Elements in Renal Cell Carcinoma

The development of cancer is a complex, multistage process during which a normal cell undergoes genetic changes that result in phenotypic alterations and in the acquisition of the ability to invade other sites. Inductively coupled plasma optical emission spectroscopy was used to estimate the contents of Al, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, P, Pb, and Zn in healthy kidney and renal cell carcinoma (RCC), and significant differences were found for all elements. Along with the progression of the malignant disease, a progressive decrease of Cd and K was observed. In fact, for Cd, the concentration in stage T4 was 263.9 times lower than in stage T1, and for K, the concentration in stage T4 was 1.73 times lower than in stage T1. Progressive accumulation was detected for P, Pb, and Zn in stage T4. For P, the concentration in stage T4 was 11.1 times higher than in stage T1; for Pb, the concentration in stage T4 was 232.7 times higher than in T1; and for Zn, the concentration in T4 was 8.452 times higher than in T1. This study highlights the marked differences in the concentrations of selected trace metals in different malignant tumor stages. These findings indicate that some trace metals may play important roles in the pathogenesis of RCC.     

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5' extremity of each mRNA, supplying the 5'-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania (Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.     

Secondary forests on anthropogenic soils in Brazilian Amazonia conserve agrobiodiversity

Throughout Brazilian Amazonia anthropogenic soils that are the product of pre-Columbian settlements are called Terra Preta de índio (Indian Dark Earths, TPI). These soils are dramatically different from surrounding soils due to long-term human activity, but there is little information about how secondary forest succession is affected by these differences. We tested if community structure (density, richness and basal area), floristic composition and domesticated species’ richness and density were similar between TPI and non-anthropogenic soils (NAS) in 52 25 × 10 m secondary forest plots in different successional stages near three traditional communities along the middle Madeira River, Central Amazonia. We sampled 858 woody individuals on TPI (77 domesticated) and 1095 on NAS (27 domesticated); 550 understory palms on TPI (169 domesticated) and 778 on NAS (123 domesticated). We found 179 species on TPI (10 domesticated), 190 on NAS (8 domesticated), and 74 (25%) in both environments. Although community structure on TPI and NAS was fairly similar, they showed significantly distinctive floristic compositions, both for woody individuals and understory palms. The density and richness of domesticated species was significantly higher on TPI than on NAS for woody individuals, but not for palms. The intimate long-term association of TPI with human activity has lead to the formation of distinct secondary forests and has favored the concentration of domesticated populations of crop species. Hence, secondary forests on anthropogenic soils concentrate agrobiodiversity, offering advantages for in situ conservation of genetic resources, and are unique ecosystems that should be considered in conservation efforts.     

Surprising negative association between IgG1 allotype disparity and anti-adalimumab formation: a cohort study

Introduction

The human monoclonal antibody adalimumab is known to induce an anti-globulin response in some adalimumab-treated patients. Antibodies against adalimumab (AAA) are associated with non-response to treatment. Immunoglobulins, such as adalimumab, carry allotypes which represent slight differences in the amino acid sequences of the constant chains of an IgG molecule. Immunoglobulins with particular IgG (Gm) allotypes are racially distributed and could be immunogenic for individuals who do not express these allotypes. Therefore, we investigated whether a mismatch in IgG allotypes between adalimumab and IgG in adalimumab-treated patients is associated with the development of AAA.

Methods

This cohort study consisted of 250 adalimumab-treated rheumatoid arthritis (RA) patients. IgG allotypes were determined for adalimumab and for all patients. Anti-idiotype antibodies against adalimumab were measured with a regular radio immunoassay (RIA), and a newly developed bridging enzyme linked immunosorbent assay (ELISA) was used to measure anti-allotype antibodies against adalimumab. The association between AAA and the G1m3 and the G1m17 allotypes was determined. For differences between groups we used the independent or paired samples t-test, Mann-Whitney test or Chi square/Fisher's exact test as appropriate. To investigate the influence of confounders on the presence or absence of AAA a multiple logistic regression-analysis was used.

Results

Adalimumab carries the G1m17 allotype. No anti-allotype antibodies against adalimumab were detected. Thirty-nine out of 249 patients had anti-idiotype antibodies against adalimumab (16%). IgG allotypes of RA patients were associated with the frequency of AAA: patients homozygous for G1m17 had the highest frequency of AAA (41%), patients homozygous for G1m3 the lowest frequency (10%), and heterozygous patients' AAA frequency was 14% (P = 0.0001).

Conclusions

An allotype mismatch between adalimumab and IgG in adalimumab-treated patients did not lead to a higher frequency of AAA. On the contrary, patients who carried the same IgG allotype as present on the adalimumab IgG molecule, had the highest frequency of anti-adalimumab antibodies compared to patients whose IgG allotype differed from adalimumab. This suggests that the allotype of adalimumab may not be highly immunogenic. Furthermore, patients carrying the G1m17-allotype might be more prone to antibody responses.     

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.     

Exploiting the full power of temporal gene expression profiling through a new statistical test: Application to the analysis of muscular dystrophy data

Background  

The identification of biologically interesting genes in a temporal expression profiling dataset is challenging and complicated by high levels of experimental noise. Most statistical methods used in the literature do not fully exploit the temporal ordering in the dataset and are not suited to the case where temporal profiles are measured for a number of different biological conditions. We present a statistical test that makes explicit use of the temporal order in the data by fitting polynomial functions to the temporal profile of each gene and for each biological condition. A Hotelling T ²-statistic is derived to detect the genes for which the parameters of these polynomials are significantly different from each other.     

Effects of gamma-hexachlorocyclohexane and L-3,3',5-triiodothyronine on rat liver cytochrome P4502E1-dependent activity and content in relation to microsomal superoxide radical generation

Liver microsomal cytochrome P4502E1-dependent p-nitrophenol (PNP) hydroxylation and expression of cytochrome P4502E1 were studied in rats subjected to gamma-hexachlorocyclohexane (HCCH) or L-3,3,5-triiodothyronine (T3) administration as a possible mechanism contributing to superoxide radical (O2.-) generation. HCCH treatment (a single dose of 40 mg/kg body wt) produced a 43% increase in the content of total cytochrome P450, whereas T3 (daily doses of 0.1 mg/kg body wt for two consecutive days) led to a 37% decrease. NADPH-dependent O2.- generation was elevated by HCCH and T3, expressed as either per mg of protein or per nmol of cytochrome P450, with a 135% enhancement in the O2.- production/superoxide dismutase (SOD) activity ratios being observed in both conditions. This was partly due to depression of SOD activity. Concomitantly, the molecular activity of NADPH-cytochrome p450 reductase was enhanced by 90 and 69% by HCCH and T3, respectively. In these conditions, microsomal PNP hydroxylation showed increases of 58 and 45% in HCCH- and T3-treated rats over control values, respectively, with a parallel 31% (HCCH) and 41% (T3) enhancement in the content of cytochrome P4502E1 assessed by western immunoblotting. We conclude that HCCH and T3 enhance the expression and activity of cytochrome P4502E1 and that of NADPH-cytochrome P450 reductase in rat liver, regardless of the changes in total cytochrome P450 content, representing major contributory mechanisms to microsomal NADPH-dependent O2.- generation.     

Liver necrosis induced by acute intraperitoneal ethanol administration in aged rats

It is generally agreed that the deleterious pathophysiological effects of ethanol are caused, at least partially by an increase in free radical production. However, little attention has been directed to the effects of ethanol upon elderly organisms. Male Wistar rats at ages 3, 6, 12, 18 and 24 months were treated either with a single i.p. dose of 35% ethanol (v/v) at 3 g ethanol/kg body weight or an isovolumetric amount of 0.9% saline solution. We then assessed the plasma levels of transaminases and hepatic levels of oxidative stress-related parameters, followed by liver histological evaluation. The younger rats (3 months old) were not affected by the treatment with ethanol with respect to any of the studied parameters except for a lowering of total hepatic GSH and an increase in hepatic thiobarbituric acid reactants (TBARS) formation, while animals older than 3 months were increasingly more affected by the treatment. Acute ethanol treatment elicited the similar responses to those in the 3 months-old group, plus a decrease in the hepatic and plasma levels of beta-carotene and the plasma level of alpha-tocopherol, as well as an increase in the activity of plasma transaminases. In the 12,18 and 24 months old groups, there was increasing liver necrosis. These findings suggest that liver damage induced by acute ethanol administration in elderly rats may involve a lack of antioxidants.