Interactions of Mitochondrial Presequence Peptides with the Mitochondrial Outer Membrane Preprotein Translocase TOM

TOM protein-conducting channels serve as the main entry sites into mitochondria for virtually all mitochondrial proteins. When incorporated into lipid bilayers, they form large, relatively nonspecific ion channels that are blocked by peptides derived from mitochondrial precursor proteins. Using single-channel electrical recordings, we analyzed the interactions of mitochondrial presequence peptides with single TOM pores. The largest conductance state of the translocon represents the likely protein-conducting conformation of the channel. The frequency (but not the duration) of the polypeptide-induced blockage is strongly modulated by the substrate concentration. Structural differences between substrates are reflected in characteristic blockage frequencies and duration of blockage. To our knowledge, this study provides first quantitative data regarding the kinetics of polypeptide interaction with the mitochondrial TOM machinery.     

Functional Cooperation of the Proapoptotic Bcl2 Family Proteins Bmf and Bim In Vivo

Bcl2-modifying factor (Bmf) is a member of the BH3-only group of proapoptotic proteins. To test the role of Bmf in vivo, we constructed mice with a series of mutated Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation by the c-Jun NH₂-terminal kinase (JNK) on Ser⁷⁴, or mimic Bmf phosphorylation on Ser⁷⁴. We report that the loss of Bmf causes defects in uterovaginal development, including an imperforate vagina and hydrometrocolpos. We also show that the phosphorylation of Bmf on Ser⁷⁴ can contribute to a moderate increase in levels of Bmf activity. Studies of compound mutants with the related gene Bim demonstrated that Bim and Bmf exhibit partially redundant functions in vivo. Thus, developmental ablation of interdigital webbing on mouse paws and normal lymphocyte homeostasis require the cooperative activity of Bim and Bmf.Bmf is a proapoptotic BH3-only member of the Bcl2-related protein family that is implicated in cell death caused by anoikis (23, 26, 27), arsenic trioxide (19), histone deacetylase inhibitors (33, 34), transforming growth factor β (24), and tumor necrosis factor alpha (8). Mice with a loss of Bmf expression exhibit B-cell hyperplasia and increased sensitivity to γ-radiation-induced B-cell lymphoma (14). These observations indicate that Bmf represents an important mediator of cell death signaling pathways.The structure of Bmf includes a BH3 domain that is essential for apoptosis induction. In addition, Bmf contains a sequence motif that is required for interactions with dynein light chain 2 (DLC2), a component of the myosin V motor complex (23). The interaction of Bmf with DLC2 is required for the recruitment of Bmf to the cytoskeleton. The release of Bmf from complexes sequestered on the cytoskeleton may contribute to anoikis (23). Interestingly, this regulatory mechanism is shared by the related proapoptotic BH3-only protein Bim, which interacts via a similar sequence motif with dynein light chain 1 (DLC1), a component of the dynein motor complex (22).The similarities between Bmf and Bim include the presence of a conserved phosphorylation site (Bmf Ser⁷⁴ and Bim Thr¹¹²) that is a substrate for the c-Jun NH₂-terminal kinase (JNK) (15). Data from biochemical studies indicate that the JNK-mediated phosphorylation of Bmf and Bim may increase apoptotic activity (15). Indeed, mice with a germ line point mutation in the Bim gene (Thr¹¹² replaced with Ala) exhibit decreased apoptosis (10). These studies indicate that Bmf and Bim may mediate, in part, proapoptotic signaling by JNK (3, 30).The purpose of this study was to examine the role of Bmf using mouse models with germ line defects in the Bmf gene, including mice with Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation, or mimic Bmf phosphorylation. We examined the effects of these mutations in mice with both wild-type and mutant alleles of the related gene Bim. The results of our analysis demonstrate that Bmf and Bim exhibit partially redundant functions, that phosphorylation on Ser⁷⁴ is not essential for Bmf activity, and that phosphorylation on Ser⁷⁴ can contribute to increased levels of Bmf activity in vivo.     

Exposure of neonates to ochratoxin A: first biomonitoring results in human milk (colostrum) from Chile

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OTα) were determined in milk and blood from nine lactating women who provided samples soon after delivery at a hospital in southern Chile. The analytical method applied liquid–liquid extraction with chloroform, and in the case of blood, an extra purification with solid phase extraction prior to HPLC analysis with fluorescence detection. OTA was detected in all human milk samples, with an average concentration of 106?±?45 ng/L (range 44–184 ng/L). Levels of OTα were 40?±?30 ng/L (LOQ 40 ng/L), but increased considerably upon enzymatic hydrolysis with ß-glucuronidase/sulfatase (up to 840?±?256 ng/L) in human milk. By contrast, there was no evidence for conjugates of OTA. The data on OTA in breast milk and levels reported in blood from women in Chile are indicative of an efficient lactational transfer of the mycotoxin. Infant exposure to OTA was estimated by considering their daily OTA intake with human milk at early stages of nursing. For the majority of milk samples, the calculated OTA intake of infants exceeded the tolerable daily intake (TDI) of 5 ng/kg body weight (bw)/day proposed by the Nordic Expert Group, and infant exposure approached the provisional tolerable doses of 14–16 ng/kg bw/day suggested by the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) and by EFSA for adults. The present study documents and confirms the presence of OTA in human milk at levels where the TDI can be exceeded. These results point out the need to continue food and biological monitoring and to develop strategies, e.g. dietary recommendations to pregnant and lactating women, aimed to reduce OTA exposure in early periods of life.     

Histochemistry and storage of organic compounds during basidiosporogenesis in the ectomycorrhizal fungus <Emphasis Type="Italic">Pisolithus microcarpus</Emphasis>

Knowledge on the distribution and storage of different organic compounds during basidiosporogenesis in P. microcarpus is paramount to a better understanding of basidiospore recalcitrance to germination. The objective of this work was to detect the presence and distribution of phenolics, reducing sugars, starch, glycogen, total polysaccharides, RNA, and proteins during P. microcarpus basidiosporogenesis. Starch and reducing sugars were not detected in the fungal basidiocarps, while other polysaccharides predominated in the extracellular matrix at the base of the basidiocarp containing unconsolidated peridioles. Phenolics were also detected in this region. Glycogen was present inside the hyphae, basidia, and basidiospores and constitutes an important storage compound in the fungal basidiocarps. In mature basidiospores, RNA accumulation occurred at discrete locations in the cytoplasmatic periphery, while polysaccharides and proteins were shown to predominate in the cell wall. The presence of glycogen, RNA, and proteins inside the basidiospores strongly indicates provision for future germination and suggests that other factors may also influence basidiospore recalcitrance.     

Temperature tolerance and energetics: a dynamic energy budget-based comparison of North Atlantic marine species

Temperature tolerance and sensitivity were examined for some North Atlantic marine species and linked to their energetics in terms of species-specific parameters described by dynamic energy budget (DEB) theory. There was a general lack of basic information on temperature tolerance and sensitivity for many species. Available data indicated that the ranges in tolerable temperatures were positively related to optimal growth temperatures. However, no clear relationships with temperature sensitivity were established and no clear differences between pelagic and demersal species were observed. The analysis was complicated by the fact that for pelagic species, experimental data were completely absent and even for well-studied species, information was incomplete and sometimes contradictory. Nevertheless, differences in life-history strategies were clearly reflected in parameter differences between related species. Two approaches were used in the estimation of DEB parameters: one based on the assumption that reserve hardly contributes to physical volume; the other does not make this assumption, but relies on body-size scaling relationships, using parameter values of a generalized animal as pseudo-data. Temperature tolerance and sensitivity seemed to be linked with the energetics of a species. In terms of growth, relatively high temperature optima, sensitivity and/or tolerance were related to lower relative assimilation rates as well as lower maintenance costs. Making the step from limited observations to underlying mechanisms is complicated and extrapolations should be carefully interpreted. Special attention should be devoted to the estimation of parameters using body-size scaling relationships predicted by the DEB theory.     

Identification of chalcones as in vivo liver monofunctional phase II enzymes inducers

Cancer preventive agents (CPA) are drugs able to suppress the carcinogen metabolic activation or block the formation of ultimate carcinogens. CPA could act through various molecular mechanisms, for example by interfering with the action of procarcinogen. This could be attained by increasing the phase II enzymes levels of quinone reductase (QR) and glutathione S-transferase (GST). New flavonoids, especially chalcones, have been identified as in vivo monofunctional phase II enzymes inducers. Oral administration of chalcone, 4, and both p-methoxy-substituted chalcones, 6 and 14, increased hepatic QR activity with concomitant decrease in CYP1A1 activity, a member of the most important group of phase I enzymes cytochrome P450. Among them, 4 also increased GST activity. While p-bromo-substituted chalcone 8 was the best inducer of QR it decreased hepatic GST expression and cytochrome P450, being the most effective decreasing cytochrome P450-expression. Thienyl-chalcone 20 being the bioisostere of chalcone 4 did not display the same in vivo profile in the phase I level modification. As chalcone 4 its bioisostere, chalcone 20, displayed low DNA strand breakage and absence of mutagenicity. Also, in our preliminary in vivo tumourigenesis/chemopreventive and acute-toxicity studies, chalcones 4, 6 and 8 showed the best behaviours as CPA justifying additional studies that are ongoing.     

Features of Saccharomyces cerevisiae as a culture starter for the production of the distilled sugar cane beverage,cachaça in Brazil

Aims: To evaluate the dominance and persistence of strains of Saccharomyces cerevisiae during the process of sugar cane fermentation for the production of cachaça and to analyse the microbial compounds produced in each fermentative process. Methods and Results: Three S. cerevisiae strains were evaluated during seven consecutive 24‐h fermentation batches using recycled inocula. The UFLA CA 116 strain had the largest population of viable organisms, and the maximum population was achieved in the fourth batch after 96 h of fermentation. The UFLA CA 1162 and UFLA CA 1183 strains grew more slowly, and the maximum population was reached in the seventh batch. Molecular characterization of isolated yeast cells using PFGE (pulse field gel electrophoresis) revealed that more than 86% of the isolates corresponded to the initially inoculated yeast strain. The concentration of aldehydes, esters, methanol, alcohol and volatile acids in the final‐aged beverages were within the legal limits. Conclusions: Cachaça produced by select yeast strains exhibits analytical differences. UFLA CA 1162 and UFLA CA 116 S. cerevisiae isolates can be considered the ideal strains for the artisanal production of cachaça in Brazil. Significance and Impact of the Study: The use of select yeast strains can improve the quality and productivity of cachaça production. Our findings are important for the appropriate monitoring of yeast during sugar cane fermentation. In addition, we demonstrate that UFLA CA 116 and UFLA CA 1162, the ideal yeast strains for cachaça production, are maintained at a high population density. The persistence of these yeast strains in the fermentation of sugar cane juice promotes environmental conditions that prevent or decrease bacterial contamination. Thus, the use of select yeast strains for the production of cachaça is a viable economic alternative to standardize the production of this beverage.     

Cytogenetic characterization of Partamona cupira (Hymenoptera, Apidae) by fluorochromes

Four colonies of the stingless bee Partamona cupira (Hymenoptera: Apidae) were cytogenetically analyzed using conventional staining and the fluorochromes CMA(3) e DAPI. The females have 2n = 34 chromosomes (2K = 32 Mˉ+2 Aˉ). Some females, however, presented an additional large B acrocentric chromosome, to a total of 2n = 35. Chromosome B and the chromosomal pairs 2, 9 and 10 showed CMA (3) (+) bands, indicating an excess of CG base-pairs. A clear association was verified between the P. helleri B chromosome SCAR marker and the presence of a B chromosome in P. cupira. The data obtained suggests that B chromosomes in P. helleri and P. cupira share a common origin.     

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo‐adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C‐terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co‐localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light‐induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two‐dimensional gel electrophoresis discriminated, for the first time, the 58‐kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC‐MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.