THE DIRECT ISOLATION OF THE MITOTIC APPARATUS

A method for isolating the mitotic apparatus from dividing sea urchin eggs without the use of ethyl alcohol or of detergents is described. In the present method, the eggs are dispersed directly in a medium containing 1 M (to 1.15 M) sucrose, 0.15 M dithiodiglycol, and 0.001 M Versene at pH 6, releasing the visibly intact mitotic apparatus. The method is designed for studies of enzyme activities, lipid components, and the variables affecting the stability of the apparatus.     

Increased Frequency of CD4 and CD8 Regulatory T Cells in Individuals under 15 Years with Multibacillary Leprosy

Background

Leprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4⁺/CD8⁺CD25^highFOXP3⁺ and CD4⁺/CD8⁺CD25^highFOXP3^high cells in leprosy patients and household contacts, in both cases under 15 years old.

Methodology/Principal Findings

PBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4⁺/CD8⁺ Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4⁺FOXP3⁺ (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4⁺FOXP3^high (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8⁺FOXP3⁺ (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8⁺FOXP3^high (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients.

Conclusions/Significance

We have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4⁺ and CD8⁺ Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.     

Water deficit modulates growth,morphology, and the essential oil profile in Lippia alba L. (Verbenaceae) grown in vitro

Lippia alba (Miller) N.E. Brown is an aromatic plant species of great economic importance due to the medicinal properties of its essential oils, which provide stress relief, respiratory and gastrointestinal disease control, and anti-inflammatory and natural sedative effects. The plant is also effective in biological control against various pathogens and in food preservation. Water deficit is the most critical abiotic factor limiting plant growth and morpho-physiological development, as well as production of secondary metabolism compounds. The objective of this work was to evaluate the effect of water deficit on growth, photosynthesis, essential oil profile, and the expression of genes related to the biosynthesis of these compounds in L. alba grown in vitro. Nodal segments were cultured on medium supplemented with 0, 1, 2, and 3% (w/v) polyethylene glycol for 45 days. Water stress had a negative effect on primary metabolism indicators, such as growth, leaf area, and photosynthetic rate; but a positive effect on amino acid and total protein content. Similarly, secondary metabolism exhibited an increase in linalool but a reduction in germacrene levels under water deficit. These findings provide a deeper understanding of how water deficit affects primary and secondary metabolism in L. alba, showing the potential of this medicinal species to adapt to soils with low water availability, while still being able to grow and synthesize essential oils.

     

Hormonal induction of ovulation and artificial insemination in suckled beef cows under nutritional stress

The objective was to develop a program for inducing estrus (followed by insemination) of suckled beef cows under nutritional stress (poor body condition). A total of 123 cows, from 60 to 75 days postpartum, were classified according to their body condition score (BCS; range from 1 to 5, in increments of 0.5) and allocated into two groups. On Day 0 (without regard to stage of the estrous cycle), cows (n = 59) in the hormone induction (HI) treatment group were given an intravaginal device (IVD) containing 250 mg of medroxiprogesterone acetate (MAP) and an i.m. injection of 2.5 mg estradiol benzoate (EB). On Day 6, these cows were given 500 IU eCG i.m. and calves were weaned for 96 h. The IVD were removed on Day 7. Cows detected in estrus by 45 h after IVD removal were inseminated 12 h after standing estrus; cows not in estrus by 45 h after IVD removal received an i.m. injection of 100 microg gonadorelin (GnRH) and were inseminated 16-18 h later. In the control group (C), cows (n = 64) only had their calves weaned at Day 6 (for 96 h), with estrus detection and AI from Days 6 to 11. Overall, the BCS ranged from 2.0 to 3.0. In the treatment group, estrus and pregnancy rates in cows with BCS 2.0 (20 and 30%, respectively) was lower (P < 0.05) than those with BCS 3.0 (50 and 66.6%, respectively), but did not differ (P > 0.05) from BCS 2.5 (23.3 and 47.6%). In C group, only 2 of 66 cows were detected in estrus and bred (neither was pregnant). In conclusion, the program for induction of ovulation using MAP, EB, eCG and GnRH increased the pregnancy rate in beef cows in poor body condition, enabling AI to be done in a 63-h interval.     

Affiliative relationships among Sorraia mares: influence of age, dominance, kinship and reproductive state

The influence of individual factors on dominance rank and the relationship between rank distance and patterns of aggression predicted by models of evolutionarily stable strategies (ESS) of animal conflict were investigated in a managed bachelor group of Sorraia horses, Equus caballus. The group was composed of four to six stallions 3- to 12-years-old during the study period. The dominance hierarchy was significantly linear and rank was not related to age, weight, height or aggressiveness. Frequency and intensity of agonistic interactions were low, but higher-ranking stallions did not receive lower aggressiveness than lower-ranking stallions. There was some evidence that dominance relationships were more contested among close-ranking stallions, as predicted. Agonistic-related interactions among close-ranking stallions served similar functions to those among distant-ranking stallions, but the latter interacted more frequently than expected for access to resting sites and/or resting partners. Therefore, we found some evidence that agonistic-related interactions among distant-ranking stallions play a larger role in providing access to valuable and defendable resources than those among close-ranking stallions. Nevertheless, the fact that space to escape from aggression was limited and breeding access was independent from dominance rank may have reduced the benefits relative to costs of aggression and therefore limited the occurrence of contests over dominance and resources.     

Dominance relationships and patterns of aggression in a bachelor group of Sorraia horses (Equus caballus)

The influence of individual factors on dominance rank and the relationship between rank distance and patterns of aggression predicted by models of evolutionarily stable strategies (ESS) of animal conflict were investigated in a managed bachelor group of Sorraia horses, Equus caballus. The group was composed of four to six stallions 3- to 12-years-old during the study period. The dominance hierarchy was significantly linear and rank was not related to age, weight, height or aggressiveness. Frequency and intensity of agonistic interactions were low, but higher-ranking stallions did not receive lower aggressiveness than lower-ranking stallions. There was some evidence that dominance relationships were more contested among close-ranking stallions, as predicted. Agonistic-related interactions among close-ranking stallions served similar functions to those among distant-ranking stallions, but the latter interacted more frequently than expected for access to resting sites and/or resting partners. Therefore, we found some evidence that agonistic-related interactions among distant-ranking stallions play a larger role in providing access to valuable and defendable resources than those among close-ranking stallions. Nevertheless, the fact that space to escape from aggression was limited and breeding access was independent from dominance rank may have reduced the benefits relative to costs of aggression and therefore limited the occurrence of contests over dominance and resources.     

Responses of chlorophyll fluorescence parameters of the facultative halophyte and C3-CAM intermediate species Mesembryanthemum crystallinum to salinity and high irradiance stress

Mesembryanthemum crystallinum L. (Aizoaceae) is a facultative annual halophyte and a C(3)-photosynthesis/crassulacean acid metabolism intermediate species currently used as a model plant in stress physiology. Both salinity and high light irradiance stress are known to induce CAM in this species. The present study was performed to provide a diagnosis of alterations at the photosystem II level during salinity and irradiance stress. Plants were subjected for up to 13 days to either 0.4M NaCl salinity or high irradiance of 1000 micromol m(-2)s(-1), as well as to both stress factors combined (LLSA=low light plus salt; HLCO=high light of 1000 micromol m(-2)s(-1), no salt; HLSA=high light plus salt). A control of LLCO=low light of 200 micromol m(-2)s(-1), no salt was used. Parameters of chlorophyll a fluorescence of photosystem II (PSII) were measured with a pulse amplitude modulated fluorometer. HLCO and LLSA conditions induced a weak degree of CAM with day/night changes of malate levels (Deltamalate) of approximately 12mM in the course of the experiment, while HLSA induced stronger CAM of Deltamalate approximately 20 mM. Effective quantum yield of PSII, DeltaF/F'(m), was only slightly affected by LLSA, somewhat reduced during the course of the experiment by HLCO and clearly reduced by HLSA. Potential quantum efficiency of PSII, F(v)/F(m), at predawn times was not affected by any of the conditions, always remaining at 0.8, showing that there was no acute photoinhibition. During the course of the days HL alone (HLCO) also did not elicit photoinhibition; salt alone (LLSA) caused acute photoinhibition which was amplified by the combination of the two stresses (HLSA). Non-photochemical, NPQ, quenching remained low (<0.5) under LLCO, LLSA and HLCO and increased during the course of the experiment under HLSA to 1-2. Maximum apparent photosynthetic electron transport rates, ETR(max), declined during the daily courses and were reduced by LLSA and to a similar extent by HLSA. It is concluded that M. crystallinum expresses effective stress tolerance mechanisms but photosynthetic capacity is reduced by the synergistic effects of salinity and light irradiance stress combined.     

Reconstruction of phylogenetic trees using the ant colony optimization paradigm

We developed a new approach for the reconstruction of phylogenetic trees using ant colony optimization metaheuristics. A tree is constructed using a fully connected graph and the problem is approached similarly to the well-known traveling salesman problem. This methodology was used to develop an algorithm for constructing a phylogenetic tree using a pheromone matrix. Two data sets were tested with the algorithm: complete mitochondrial genomes from mammals and DNA sequences of the p53 gene from several eutherians. This new methodology was found to be superior to other well-known softwares, at least for this data set. These results are very promising and suggest more efforts for further developments.