Praziquantel and albendazole damaging action on in vitro developing Mesocestoides corti (Platyhelminthes: Cestoda)

Parasitic flatworms present several steps of body architecture rearrangement during their fast transition from one developmental stage to another, which are, at least in part, responsible for their evasion from host immune response. Besides, different developmental stages present different degrees of susceptibility to drug action, and the identification of more susceptible stages is of importance for the definition of therapeutical approaches. Mesocestoides corti (syn. Mesocestoides vogae) is considered a good model to study cestode biology because it can be easily manipulated both in vivo and in vitro and due to its relatively close relationship to cestodes of medical relevance, such as those from genera Echinococcus or Taenia. We have analyzed the damaging action of two broad spectrum anthelmintic drugs (praziquantel and albendazole) throughout the in vitro strobilization process of M. corti in order to identify developmental stages or body structures more susceptible to these drugs. Tetrathyridia (larval stage) and segmented-induced worms were cultivated and treated with praziquantel and albendazole. Whole mounted samples, taken from different developmental stages, were fixed and stained with fluorophore-labeled WGA lectin and phalloidin for the analysis of tegument and muscles, respectively. Confocal laser scanning microscopy was used to identify anatomical changes and lesions caused by each anthelmintic drug in a 3D view. We demonstrated that both praziquantel and albendazole cause extensive tissue damage, especially on tegument, and that adult forms were the most susceptible to drug exposure.     

Antimicrobial metabolites produced by an intertidal Acremonium furcatum

In a screening for antimicrobial metabolites, amides of D-allo- and L-isoleucine derivatives were isolated from the culture of a marine strain of Acremonium furcatum. Structural elucidation of these compounds was performed by analysis of spectroscopic data and confirmed by synthesis. All of the compounds, natural and synthetic intermediates, were bioassayed against bacteria and phytopathogenic fungi, with many showing remarkable antifungal activities.     

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.     

LOH at 6p21.3 region and HLA class altered phenotypes in bladder carcinomas

Alterations in HLA class I antigen expression have been frequently described in different epithelial tumors and are thought to favor tumor immune escape from T lymphocyte recognition. Multiple molecular mechanisms are responsible for these altered HLA class I tumor phenotypes. Some are structural defects that produce unresponsiveness to treatment with interferons. Others include alterations in regulatory mechanisms that can be switched on by treatment of tumor cells with different cytokines. One important mechanism belonging to the first group is loss of heterozygosity (LOH) at chromosome region 6p21.3, which can lead to HLA haplotype loss. In this investigation, the frequency of LOH at 6p21 chromosome region was studied in 69 bladder carcinomas. Short tandem repeat analysis showed that 35% of cases had LOH in this chromosome region. By considering these results together with immunohistological findings previously published by our group, we identified a distribution pattern of HLA class I altered phenotypes in bladder cancer. The most frequently altered phenotype in bladder carcinomas was total loss of HLA class I expression (17 cases, 25%), followed by phenotype II associated with HLA haplotype loss (12 cases, 17.5%), and HLA allelic loss (ten cases, 14.5%). Nine cases (13%) were classified as having a compound phenotype, five cases (7%) as having HLA locus loss, and in 16 cases (23%) no alteration in HLA expression was detected. An important conclusion of this report is that a combination of different molecular and immunohistological techniques is required to precisely define which HLA alleles are lost during tumor progression and to characterize the underlying mechanisms of these losses. These studies should be performed when a cancer patient is to be included in an immunotherapy protocol that aims to stimulate different immune effector mechanisms.     

Evidence for a catalytic Mg2+ ion and effect of phosphate on the activity of Escherichia coli phosphofructokinase-2: regulatory properties of a ribokinase family member

Phosphofructokinase-2 (Pfk-2) from Escherichia coli belongs to the ribokinase family of sugar kinases. One of the signatures observed in amino acid sequences from the ribokinase familiy members is the NXXE motif, which locates at the active site in the ribokinase fold. It has been suggested that the effect of Mg2+ and phosphate ions on enzymatic activity, observed in several adenosine kinases and ribokinases, would be a widespread feature in the ribokinase family, with the conserved amino acid residues in the NXXE motif playing a role in the binding of these ions at the active site [Maj, M. C., et al. (2002) Biochemistry 41, 4059-4069]. In this work we study the effect of Mg2+ and phosphate ions on Pfk-2 activity and the involvement of residue E190 from the NXXE motif in this behavior. The kinetic data are in agreement with the requirement of a Mg2+ ion, besides the one present in the metal-nucleotide complex, for catalysis in the wild-type enzyme. Since the response to free Mg2+ concentration is greatly affected in the E190Q mutant, we conclude that this residue is required for the proper binding of the catalytic Mg2+ ion at the active site. The E190Q mutant presents a 50-fold decrease in the kcat value and a 15-fold increment in the apparent Km for MgATP(2-). Inorganic phosphate, typically considered an activator of adenosine kinases, ribokinases, and phosphofructokinases (nonhomologous to Pfk-2) acted as an inhibitor of wild-type and E190Q mutant Pfk-2. We suggest that phosphate can bind to the allosteric site of Pfk-2, producing an inhibition pattern qualitatively similar to MgATP(2-), which can be reversed to some extent by increasing the concentration of fructose-6-P. Given that the E190Q mutant presents alterations in the inhibition by MgATP(2-) and phosphate, we conclude that the E190 residue has a role not only in catalysis but also in allosteric regulation.     

The influence of natural rubber/Au nanoparticle membranes on the physiology of Leishmania brasiliensis

The development of nanotechnology has generated new means of disease diagnosis and treatment. Infectious diseases, including leishmaniasis, malaria, etc., have benefited from the advent of new nanomaterials and/or nanodevices capable of detecting specific antigens and antibodies with high specificity and low cost. In this paper, we present an investigation on a single-celled protozoan Leishmaniasis parasite, a disease considered of standard infectivity, given the high degree of immunological specificity. Natural rubber (NR) membranes incorporating gold nanoparticles (GNPs) were placed in the culture medium and the physiological behavior of Leishmania brasiliensis promastigotes was evaluated. The natural rubber membranes containing GNPs decreased the population growth rate, showing a lower index of living promastigotes (attached to the membrane surface) depending on the amount of nanoparticles deposited in the membrane surface. Such membranes may be used to develop a flexible band-aid for skin lesions from degenerative infection state, inhibiting the population growth of parasites in the lesions. In addition, natural rubber membranes would also stimulate angiogenesis in damaged tissues.     

Sperm viability of canine and caprine semen samples preserved in a dry shipper

This study assessed the efficacy of a dry shipper to preserve canine and caprine semen samples. After equilibration, semen straws from six Majorera bucks and five dogs were frozen and stored in liquid nitrogen (LN). Thirty days after freezing, half of the frozen straws were transferred from LN to a dry shipper (DS). Then, thawing was performed at 1, 2, 3, 5 and 7 days and the percentages of motile spermatozoa, acrosome intact spermatozoa and abnormal spermatozoa were determined. The sperm motility (total and progressive) of canine semen samples preserved with DS was quite similar to those preserved in LN, and no significant differences were observed throughout the experimental period. In addition, no differences were observed in the number of abnormal spermatozoa (range: 13.2-19.0%) or intact acrosome (range 91.3-95%) between both storage protocols. Buck semen samples showed equivalent levels of progressive motility (between 50% and 60%) and intact acrosome membrane (around 70%) during the first 3 days of storage in both procedures; however, from the fifth day of storage onwards, a notable decrease in semen quality was observed in the samples preserved in DS, showing a dramatic fall in the semen viability after 7 days of preservation (12.3% and 36.8%, progressive fast spermatozoa and acrosome integrity, respectively). In dog samples, the present study confirmed that seminal quality did not show modifications for the preservation period (7 days), confirming the efficacy of the dry shipper to preserve frozen samples for a short time. However, under the circumstances reported in this study, the sperm quality of buck samples preserved in the dry shipper only held during the first 3 days of storage, and therefore, its practical application could be more limited.