Nuclear behaviour during ascus formation in Saccharomyces rosei (Guilliermond) Lodder et Kreger-van Rij

Stained cells of Saccharomyces rosei prepared from 4 to 10-day-old cultures were studied under the light microscope. Mitotic and meiotic divisions involving a ring-like structure as well as preceding and subsequent stages were observed. Cells presenting supernumerary mitoses in a varying number were frequent. These mitoses, having terminated their multiplication activity, suspended the process shortly before its conclusion and, in a development which was identical at all, assumed a curious arrangement forming a mitoses-ring. Meiosis-buds were detected. These especial buds, where karyogamy and meiosis took place, resulted from the development of the mitoses-ring, whose mitoses upon resuming their activity moved toward the cell wall giving rise to the appearance of these appendices. Each one of these buds received the corresponding pair of daughter nuclei, diploidization occurring subsequently. Meiosis was usually processed in a single bud (effective-meiosis-bud) and all four meiotic nuclei migrated to the mother cell, and gave rise to a tetra-nucleate spore or binucleate spores if two were formed.Other modalities of sporulation were observed. These may result either from the association of two cells, in which one assumed the function of meiosis-bud (false-meiosis-bud), or from a cell association in which this function was performed for several linearly arranged cells forming a protuberance.Conjugation between mother cell and an attached bud, or between independent cells, was not observed.     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C₁₈ column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.     

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.     

P-glycoprotein induction: an antidotal pathway for paraquat-induced lung toxicity

The widespread use of the nonselective contact herbicide paraquat (PQ) has been the cause of thousands of deaths from both accidental and voluntary ingestion. The main target organ for PQ toxicity is the lung. No antidote or effective treatment to decrease PQ accumulation in the lung or to disrupt its toxicity has yet been developed. The present study describes a procedure that leads to a remarkable decrease in PQ accumulation in the lung, together with an increase in its fecal excretion and a subsequent decrease in several biochemical and histopathological biomarkers of toxicity. The administration of dexamethasone (100 mg/kg ip) to Wistar rats, 2 h after PQ intoxication (25 mg/kg ip), decreased the lung PQ accumulation to about 40% of the group exposed to only PQ and led to an improvement in tissue healing in just 24 h as a result of the induction of de novo synthesis of P-glycoprotein (P-gp). The involvement of P-gp in these effects was confirmed by Western blot analysis and by the use of a competitive inhibitor of this transporter, verapamil (10 mg/kg ip), which, given 1 h before dexamethasone, blocked its protective effects, causing instead an increase in lung PQ concentration and an aggravation of toxicity. In conclusion, the induction of P-gp, leading to a decrease in lung levels of PQ and the consequent prevention of toxicity, seems to be a new and promising treatment for PQ poisonings that should be further clinically tested.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Secretory structures at syconia and flowers of Ficus enormis (Moraceae): A specialization at ostiolar bracts and the first report of inflorescence colleters

The fig (Ficus L.) infructescence, called syconium, is a receptacle with an apical opening, the ostiole, closed by bracts. The ostiolar bracts produce an exudate, which is rather conspicuous in some species. It has not been histochemically analyzed yet, and the structures responsible for its production are still unknown. Some wild growing species of Ficus from Brazil produce high amounts of this ostiolar exudate. Ficus enormis (Mart. ex Miq.) Miq. grows as trees or shrubs in the Atlantic rainforest. Our goal was to identify the secretory structures present in the inflorescence and, to characterize histochemically the ostiolar tissues and exudates. Syconia samples of F. enormis were processed and stained according to the usual techniques in plant anatomy. The morphological analysis revealed different types of bracts, one type specialized in secretion, another showing transitional characteristics between secretory and non-secreting bracts, and a third one being non-secreting. They are designated as secretory ostiolar bracts, transitional bracts and wall bracts. The floral bracteoles, digital-shaped colleters present in the ostiole, at the syconium axis and at the flower receptacle, were also analyzed. All have similar structure, like finger-shaped secretory trichomes. The colleters present among ostiolar bracts may contribute to production and composition of the ostiole exudate.     

Beta2 Adrenergic Receptor (ADRβ2) Haplotype Pair (2/4) Is Associated with Severe Asthma

Background

β2 adrenergic receptor (ADRβ2) polymorphisms including ADRβ2+46G>A have been reported to cause adverse outcomes in mild asthmatics. The extent to which ADRβ2 polymorphisms and in particular their haplotypes contribute to severe asthma is unknown.

Objective

To determine the association of ADRβ2 polymorphisms and haplotypes with asthma severity.

Methods

Caucasians (n = 2979) were genotyped for 11 ADRβ2 polymorphisms. The cohort (mean age 39.6, 60% female) included 2296 non-asthmatics, 386 mild asthmatics, 172 moderate asthmatics and 125 severe asthmatics. Haplotype frequency and haplotype pair for each subject was determined using the PHASE algorithm.

Results

The three asthmatic cohorts were comparable in age and gender but were distinguishable from each other in terms of symptoms, spirometry, medication use and health care utilisation (p <0.001). None of the polymorphisms showed a genotypic or allelic association with asthma diagnosis or severity. Nine haplotypes were identified and no association was found with asthma diagnosis or severity per se. Haplotype pair 2/4 was associated with asthma severity (Trend Test, OR 1.42, p = 0.0008) but not with asthma per se. Prevalence of haplotype pair 2/2 appeared to decrease with asthma severity (Trend Test, OR 0.78, p = 0.067). Two new haplotypes were identified, occurring exclusively in asthmatics at a frequency of ≥ 1%. In addition, a positive association between carriage of ADRβ2 +523*C and increased risk of atopy was discovered.

Conclusions

ADRβ2 haplotype pair 2/4 is associated with severe asthma and is consistent with findings of poor bronchodilator response in mild asthmatics who are also haplotype 2/4.