Disruption of bovine sperm functions in the presence of aplastic midpiece defect.

BACKGROUND: Bulls are of great importance in the productive chain and for this reason they should have a good semen quality. There is no doubt that sperm morphology is very important to bull fertility, although little is known about how exactly the abnormal morphologies may affect sperm functions. OBJECTIVES: To detail the morphological description of the aplastic midpiece defect (AMD), as well as to understand its consequences for male fertility based on membrane and acrosome status, mitochondrial membrane potential and DNA integrity parameters. MATERIALS AND METHODS: The bulls were divided into two groups: control, consisting of satisfactory potential breeders (n = 3); and AMD, consisting of unsatisfactory potential breeders with a high percentage of AMD (n = 3). Bulls were evaluated by the breeding soundness evaluation; five ejaculates were collected from each animal and analyzed by flow cytometry. RESULTS: Spermatozoa from AMD group exhibited lower sperm motility and vigor (p < 0.05). In addition, it also exhibited lower mitochondrial membrane potential (p < 0.05), a higher percentage of spermatozoa with DNA fragmentation (p < 0.05), lower acrosome and plasma membrane integrity (p < 0.05), and higher lipid bilayer sperm membrane disorganization (p < 0.05) in comparison with control bulls. DISCUSSION: These findings may be due to oxidative stress and a reduction of the energy production capacity in addition to an alteration in the structural composition of the sperm cell. Moreover, semen with a high percentage of AMD may also be undergoing apoptosis. CONCLUSION: Bulls with a high percentage of AMD in their semen are not suitable for reproduction. Furthermore, it suggests there is a putative genetic basis for this sperm defect.

The Past and Current Potential Distribution of the Fruit Fly Anastrepha obliqua (Diptera: Tephritidae) in South America.

Anastrepha obliqua (Macquart) is an agricultural pest of wide geographic distribution infesting a vast list of host species in America. However, little is known about the past geographic distribution of this species. In this study, we investigated the potential past and current distribution of species in South America. In this sense, the MaxEnt algorithm was used to model the ecological niche of the species in the past (Last Interglcial Maximum; Last Glacial Maximum) and current periods. The results suggested that under the current climatic conditions, A. obliqua showed high environmental suitability to become established in most South American countries, especially in Brazil. The lowest suitability indices were observed in Chile, Argentina, and Uruguay. The past analysis for Last Glacial Maximum revealed that there was no significant change in the distribution potential of the species when compared to the current model; however, in the Last Interglacial Maximum period, there was a large reduction in the areas of suitability for the species when compared to the current and Last Glacial Maximum distribution models. The analysis also revealed vast areas of refuges for the species mainly on the coast of Brazil, as well as Venezuela, Bolivia, Guyana, and Surinam. The results presented here may be useful for future phylogeographical studies in order to test if the refuge areas concentrate greater genetic diversity for this species. In addition, our study provides important information for understanding the current dynamics of A. obliqua, which may be useful for control programs in places where this species can become a pest.

Validation of EST-derived microsatellite markers for two Cerrado-endemic Campomanesia (Myrtaceae) species.

We assessed the transferability of 120 EST-derived Eucalyptus microsatellite primers to Campomanesia adamantium and C. pubescens. Both species are berry trees native to the Brazilian Cerrado, and population genetic information is poor. Twelve markers were used to analyze the genetic variability of four sampled populations. Regarding DNA extraction, we sampled leaf tissues from two populations of each species (80 individuals). Of the 120 primers evaluated, 87 did not amplify any PCR products, and 21 rendered nonspecific amplification. Twelve primers were successfully transferred, providing a low combined probability of genetic identity for both species (5.718 x 10(-10) for C. adamantium; 1.182 x 10(-11) for C. pubescens) and a high probability of paternity exclusion (0.99939 for C. adamantium; 0.99982 for C. pubescens). The average number of alleles in the polymorphic loci was 6.8 for C. adamantium and 7.8 for C. pubescens, ranging from 2 to 16 alleles per locus. The observed heterozygosity values for C. adamantium and C. pubescens were 0.504 and 0.503, respectively, and the expected heterozygosity values for C. adamantium and C. pubescens were 0.517 and 0.579, respectively. The populations exhibited structured genetic variability with qP values of 0.105 for C. adamantium and 0.249 for C. pubescens. Thus, we concluded that these 12 microsatellite markers, transferred from Eucalyptus, were efficient for population genetic studies of C. adamantium and C. pubescens.

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona.

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.

A critical contribution of both CD28 and ICOS in the adjuvant activity of Neisseria meningitidis H44/76 LPS and lpxL1 LPS.

The development of novel vaccines against Neisseria meningitidis recently gained momentum by the generation of penta-acylated lpxL1 LPS which has similar adjuvant activity, but reduced endotoxic activity as compared to hexa-acylated wild type (H44/76) LPS. We investigated the costimulation requirements for the adjuvant activity of both forms of LPS by immunizing CD28-, ICOS- and B7.1/2/ICOS-deficient mice. Both ICOS and CD28 appeared essential for optimal adjuvant activity of H44/76 LPS or lpxL1 LPS. Interestingly, ICOS-mediated costimulation predominates in the adjuvant activity of lpxL1 LPS, while both ICOS and CD28 are required for H44/76 LPS adjuvant activity.

CD28 and ICOS: similar or separate costimulators of T cells?

Numerous studies have revealed that the B7.1/B7.2-CD28 and B7RP-1-ICOS (Inducible COStimulator) pathways provide crucial costimulatory signals to T cells. We have compared the contribution of these pathways during primary and effector responses, in vitro and in vivo, molecularly as well as functionally. This comparison between CD28 an ICOS after initiation of T cell activation demonstrates that both CD28 and ICOS function similarly during expansion, survival and differentiation of T cells and that both CD28 and ICOS are necessary for proper IgG responses. The major differences between CD28 and ICOS are differences in expression of both receptors and ligands, and the fact that CD28 induces IL-2 production, whereas ICOS does not. In addition, ICOS is more potent in the induction of IL-10 production, a cytokine important for suppressive function of T regulatory cells. All data available at present indicate that both molecules are very suitable candidates for immunotherapy, each in their own unique way.

ICOS contributes to T cell expansion in CTLA-4 deficient mice.

Both CD28 and ICOS are important costimulatory molecules that promote Ag-specific cellular and humoral immune reactions. Whereas CD28 is generally thought to be the most important molecule in the initiation of a T cell response, ICOS is considered to act during the effector phase. We have investigated the contribution of ICOS to T cell responses in the absence of CTLA-4-mediated inhibition. Mice lacking CTLA-4, which show spontaneous CD28-mediated CD4(+) T cell activation, expansion and differentiation, were treated with antagonistic alphaICOS antibodies. Blocking the interaction between ICOS and its ligand B7RP-1 significantly reduced this aberrant T cell activation and caused a reduction in T cell numbers. In vitro analysis of CD4(+) T cells from treated mice revealed that ICOS blockade significantly reduced Th1 differentiation, while Th2 differentiation was only moderately inhibited. Further in vitro stimulation experiments demonstrated that ICOS is able to induce proliferation of murine CD4(+) and CD8(+) T cells but only in the presence of IL-2. These results indicate that ICOS is not only important for T cell effector function but also contributes to the expansion phase of a T cell response in the presence of CD28 signaling.

In vitro antimicrobial activity of the aminoglycoside arbekacin tested against oxacillin-resistant Staphylococcus aureus isolated in Brazilian hospitals.

Arbekacin is an aminoglycoside used in Japan for treating infections caused by gentamicin and oxacillin-resistant S. aureus (ORSA). The objective of this study was to determine the in vitro antimicrobial activity of arbekacin against 454 clinical isolates of ORSA. The isolates were consecutively collected between January and July, 2000, from patients hospitalized in 8 Brazilian medical centers. The antimicrobial susceptibility testing was performed by disk diffusion method according to NCCLS recommendations. The vast majority of the isolates, 453 strains (99.8%), were considered susceptible to arbekacin based on the criteria proposed by the Requirements for Antibiotic Products of Japan. Only 1 isolate (0.2%) was classified as resistant. On the other hand, high rates of resistance were demonstrated for other aminoglycosides, such as gentamicin (97.6% resistance) and amikacin (97.0% resistance). Resistance rate was also high for ciprofloxacin (98.0%). All isolates were considered susceptible to vancomycin. The excellent in vitro antimicrobial activity of arbekacin demonstrated in this study indicates that this antimicrobial agent may play an important role in the treatment of severe ORSA infections, especially those that show poor clinical response with vancomycin monotherapy. Since the aminoglycosides should not be used as monotherapy to treat Gram positive infections, further studies evaluating in vitro and in vivo synergistic activity of arbekacin combinations are necessary to clarify the clinical role of this aminoglycoside.

Antimicrobial susceptibility of coagulase-negative staphylococci and characterization of isolates with reduced susceptibility to glycopeptides.

The antimicrobial susceptibility of 239 coagulase-negative staphylococci (CNS) isolates consecutively collected from blood culture in patients admitted in a 600-bed teaching hospital was evaluated. The isolates were identified to the species level by conventional methods and the MicroScan Positive Combo Panel type 6 system, and their susceptibility to vancomycin, teicoplanin, and oxacillin were tested by agar dilution, disk diffusion, and MicroScan-WalkAway system. The species distribution was as follows: Staphylococcus epidermidis 120 (50.2%), S. hominis 29 (12.1%), S. haemolyticus 24 (10.0%), S. cohnii 14 (5.9%), and isolates from other CNS species 52 (21.8%). The percentage of resistance to oxacillin was 74.5% by agar dilution. The highest percentages of oxacillin resistance were found among S. haemolyticus (95.8%) and S. epidermidis (80.8%). Teicoplanin resistance (MIC > or = 32 micrograms/mL) was detected in five S. haemolyticus isolates, whereas intermediate resistance (MIC = 16 micrograms/mL) was detected in nine strains. These isolates with reduced susceptibility to teicoplanin were resistant to oxacillin, but remained susceptible to vancomycin (MIC < or = 4 micrograms/mL). Two isolates, one S. haemolyticus and one S. epidermidis, showed a vancomycin MIC of 8 micrograms/mL, and both MicroScan and disk diffusion methods classified these isolates as susceptible. Our results showed that glycopeptide resistance is emerging among CNS isolates in our institution and the disk diffusion method may not detect isolates with decreased susceptibility to these antimicrobial agents.

An improved nonisotopic test to screen a large series of new inhibitor molecules of poly(ADP-ribose) polymerase activity for therapeutic applications.

A reliable ELISA for screening large numbers of poly(ADP-ribose) polymerase (PARP) inhibitors is described. The test is based upon the drop in PARP activity estimated by the decrease in poly(ADP-ribose) synthesis in the presence of inhibitor. This ELISA is easy to perform, rapid, and specific. It is extremely sensitive because a clear inhibition of the total reaction could be visualized with molecules used in the nanomolar range. The assay uses no radioactivity, and automation is possible with robots for large-scale investigations. This test is of great interest for the screening of chemical libraries and the discovery of new inhibitors (and possibly activators) of PARP. Such molecules have important applications in all abnormal situations involving DNA damage and oxidative stress, such as cancer, autoimmunity, diabetes, myocardial dysfunctions, certain infections, aging, and radiation/chemical exposure.

Recovery of cellulase by HPMC-salt precipitation: analysis by statistical experimental design.

Production of industrial enzymes including cellulases requires minimum cost with the downstream processing. The objective of this work was to analyze the precipitation of cellulases by ammonium sulfate in the presence of hydroxypropyl(methylcellulose) as a co-precipitant through the use of statistical experimental design. The model generated with the experimental results showed that high protein recovery can be achieved at high levels of temperature, aging times, and rate of salt-solution addition, and at a low mixing level. The results also allowed the observation that activity recovery was improved at high levels of temperature, rate of salt addition and mixing level, and a low level of aging time.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain.

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 10(9) infected cells in three liters of culture.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for largescale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive biorector consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 10(9) infected cells in three liters of culture.

Production of monoclonal antibodies for the identification of the eggs of Taenia solium.

Two antibodies, one monoclonal and one polyclonal, were produced and used to identify Taenia solium eggs, using the enzyme-linked immuno-electrotransfer blot technique (EITB). Different life-stages of Taenia solium and T. saginata, including eggs from gravid proglottids recovered, post-treatment, from patients infected with the tapeworms, and eggs of Diphyllobothrium pacificum and Hymenolepis nana from other patients were tested with these antibodies. The monoclonal antibody only recognized the eggs and immature proglottids of T. solium. The polyclonal antibody, however, not only reacted with the eggs, cysticerci and immature proglottids of T. solium but also with the eggs and immature proglottids of T. saginata. The sensitivity and specificity of the EITB were both 100% using the monoclonal antibody but only 78% and 60%, respectively, using the polyclonal antibody. Diagnostic bands for T. solium eggs corresponded to proteins of 22.5 kDa using the polyclonal antibody and 22.5-37 kDa using the monoclonal antibody. Species-specific fluorescence was obtained with an anti-T. solium monoclonal antibody which bound to egg-derived oncospheres of T. solium but not to those of T. saginata.

Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase.

In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described. Here, we have used mutagenesis with hydroxylamine to generate a random library of PARP mutants. The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F) is described. We show that the kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged. The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme. This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage.

Poly(ADP-ribose) polymerase: structure-function relationship.

Dissection of the human poly(ADP-ribose) polymerase (PARP) molecule in terms of its structure-function relationship has proved to be an essential step towards understanding the biological role of poly(ADP-ribosylation) as a cellular response to DNA damage in eukaryotes. Current approaches aimed at elucidating the implication of this multifunctional enzyme in the maintenance of the genomic integrity will be presented.

Crystallization and X-ray crystallographic analysis of recombinant chicken poly(ADP-ribose) polymerase catalytic domain produced in Sf9 insect cells.

Poly (ADP-ribose) polymerase (PARP) participates in the immediate response in mammalian cells exposed to DNA-damaging agents. Recombinant baculovirus harboring the cDNA of the chicken PARP catalytic domain (40 kDa) have been used to infect Spodoptera frugiperda (Sf9) insect cells. The recombinant polypeptide (30 mg per 1 x 10(9) cells) was purified to homogeneity by 3-aminobenzamide affinity chromatography. The enzymatic properties of the recombinant domain were similar to those of the native fragment. Crystals of the purified recombinant catalytic domain were grown by vapor diffusion. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 59.2 A, b = 65.0 A, c = 96.9 A. They are suitable for X-ray analysis and diffract to 2.0 A.

Evaluation of column flotation in the downstream processing of fermentation products: recovery of a genetically engineered alpha-amylase.

Flotation is a simple, inexpensive, and versatile unit operation with a largely unexplored potential in biotechnology. There is a general lack of research concerning biotechnological applications in this area, especially in the recovery of fermentation products. Moreover, the few reports in the literature do not consider the modern concept of column flotation as practiced in the mineral industry. We report herein the application of column flotation for the recovery of a Bacillus stearothermophilus alpha-amylase expressed in Escherichia coli by the use of a food-grade polymer, (hydroxypropyl)methylcellulose (HPMC), and ammonium sulfate. First, the enzyme was removed from the liquid phase by partition to a salted-out HPMC phase. The enzyme-containing polymer flocs were then floated from the liquid. Recovery of active enzyme was as high as 90%, with throughput as high as 94 m3/(h.m2). The floatability of the enzyme from a periplasmic extract was higher than extracellular enzyme in the broth due to the presence of depressors of molecular weight lower than 10,000 in the broth.

Recovery of Clostridium thermosulfurogenes produced beta-amylase by (hydroxypropyl)methylcellulose partition.

A procedure for recovering Clostridium thermosulfurogenes produced beta-amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropyl)methylcellulose. The beta-amylase-containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. The process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous-phase partitions, such as poly(ethylene glycol)/dextran, the current approach appeared to be biospecific.