What do we know about the yeast strains from the Brazilian fuel ethanol industry?

The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a large-scale bioprocess that delivers an advanced biofuel at competitive prices and low environmental impact. Two to three fed-batch fermentations per day, with acid treatment of the yeast cream between consecutive cycles, during 6–8 months of uninterrupted production in a nonaseptic environment are some of the features that make the Brazilian process quite peculiar. Along the past decades, some wild Saccharomyces cerevisiae strains were isolated, identified, characterized, and eventually, reintroduced into the process, enabling us to build up knowledge on these organisms. This information, combined with physiological studies in the laboratory and, more recently, genome sequencing data, has allowed us to start clarifying why and how these strains behave differently from the better known laboratory, wine, beer, and baker's strains. All these issues are covered in this minireview, which also presents a brief discussion on future directions in the field and on the perspectives of introducing genetically modified strains in this industrial process.     

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A(1) (PLA(1)) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA(1). Addition of this recombinant PLA(1) significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA(1); 2) PLA(1) generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.     

Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.     

New evidence for a role for thioredoxin h in germination and seedling development

Thioredoxin of the h-type — earlier linked to the reduction of wheat (Triticum durum Desf. cv. Monroe) endosperm proteins — was converted from an oxidized to a partially reduced state during germination and seedling development. While the abundance of thioredoxin progressively decreased during this period, the availability of reducing equivalents, defined as the product of the relative abundance of thioredoxin and the percent reduction, increased. The amount of the enzyme catalyzing the reduction of thioredoxin h (NADP-thioredoxin reductase) remained constant. The activities of enzymes generating the NADPH needed for the reduction of thioredoxin (glucose 6-phosphate and 6-phosphogluconate dehydrogenases) increased. The level of thioredoxin h in the endosperm appeared to be controlled by the embryo via hormones. Gibberellic acid enhanced the disappearance of thioredoxin, whereas abscisic acid showed the opposite effect. Moreover, uniconazole, an inhibitor of gibberellic acid synthesis, slowed seedling growth and inhibited the disappearance of thioredoxin in a manner reversible by gibberellic acid. The results are consistent with a role for thioredoxin h in initiating the mobilization of nitrogen and carbon needed for germination and seedling development.Abbreviations ABA cis-abscisic acid - DTT dithiothreitol - GA gibberellin - GA₃ gibberellic acid - mBBr monobromobimane - NTR NADP-thioredoxin reductase This work was supported by National Science Foundation grant MCB-9316496. We thank Dr. Yuji Kamiya for advice and the Sumitomo Chemical Co. for a generous sample of uniconazole.     

Association between birth weight in preterm neonates and the BclI polymorphism of the glucocorticoid receptor gene

Endogenous and exogenous glucocorticoids influence fetal growth and development, and maternal administration of synthetic glucocorticoids may decrease the risk of perinatal morbidity including lung disease in preterm neonates. Because polymorphisms of the glucocorticoid receptor gene are known to influence the sensitivity to glucocorticoids, in the present study we examined whether any associations could exist among the BclI, N363S and ER22/23EK polymorphisms of the glucocorticoid receptor gene and gestational age, birth weight and/or perinatal morbidity of 125 preterm neonates born at 28-35 weeks' gestation with (n=57) or without maternal dexamethasone treatment (n=68). The prevalence of the three polymorphisms in the whole group of preterm infants was similar to that reported in healthy adult Hungarian population. However, we found that the BclI polymorphism significantly associated with higher birth weight adjusted for the gestational age (p=0.004, ANOVA analysis). None of the three polymorphisms showed an association with perinatal morbidities, including necrotizing enterocolitis, intraventricular hemorrhagia, patent ductus arteriosus, respiratory distress syndrome, bronchopulmonary dysplasia and sepsis in the two groups of preterm neonates with and without maternal dexamethasone treatment. These results suggest that the BclI polymorphism of the glucocorticoid receptor gene may have an impact on gestational age-adjusted birth weight, but it does not influence perinatal morbidities of preterm neonates.     

Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo

The proline-rich designer antibacterial peptide dimer A3-APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic-resistant Gram-negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro, exhibiting a half-life of approximately 100 min as documented by reversed-phase chromatography. Indeed, after a 30-min incubation period in undiluted mouse serum ex vivo, mass spectrometry failed to identify any degradation product. The peptide was still a major peak in full blood ex vivo, however, with degradation products present corresponding to amino-terminal cleavage. When injected into mice intravenously, very little, if any unmodified peptide could be detected after 30 min. Nevertheless, the major early metabolite, a full single-chain fragment, was detectable until 90 min, and this fragment exhibited equal or slightly better activity in the broth microdilution antimicrobial assay against a panel of resistant Enterobactericeae strains. The Chex1-Arg20 metabolite, when administered three times at 20 mg/kg to mice infected with a sublethal dose (over LD(50)) of an extended spectrum beta-lactamase-producing Escherichia coli strain, completely sterilized the mouse blood, similar to imipenem added at a higher dose. The longer and presumably more immunogenic prodrug A3-APO, injected subcutaneously twice over a 3-wk period, did not induce any antibody production, indicating the suitability of this peptide or its active metabolite for clinical development.     

Sphingosylphosphorylcholine as a novel calmodulin inhibitor

S1P (sphingosine 1-phosphate) and SPC (sphingosylphosphorylcholine) have been recently recognized as important mediators of cell signalling, regulating basic cellular processes such as growth,differentiation, apoptosis, motility and Ca2+ homoeostasis.Interestingly, they can also act as first and second messengers. Although their activation of cell-surface G-protein-coupled receptors has been studied extensively, not much is known about heir intracellular mechanism of action, and their target proteins are yet to be identified. We hypothesized that these sphingolipids might bind to CaM (calmodulin), the ubiquitous intracellular Ca2+sensor. Binding assays utilizing intrinsic tyrosine fluorescence of the protein, dansyl-labelled CaM and surface plasmon resonance revealed that SPC binds to both apo- and Ca2+-saturated CaM selectively, when compared with the related lysophospholipid mediators S1P, LPA (lysophosphatidic acid) and LPC (lysophosphatidylcholine). Experiments carried out with the model CaM-binding domain melittin showed that SPC dissociates the CaM-target peptide complex, suggesting an inhibitory role. The functional effect of the interaction was examined on two target enzymes, phosphodiesterase and calcineurin, and SPC inhibited the Ca2+/CaM-dependent activity of both. Thus we propose that CaM might be an intracellular receptor for SPC, and raise the possibility of a novel endogenous regulation of CaM.     

Immunohistoblot analysis on whole human hemispheres from normal and Alzheimer diseased brains

We demonstrate the feasibility and usefulness of the histoblot immunostaining of cryosections of whole hemispheres of healthy and Alzheimer diseased (AD) human brains by localizing a neuron-specific marker, the anti-neuronal nuclei (NeuN) antigen. As expected, cortical NeuN-immunopositive regions were generally thinner and lighter in the AD brains than in the controls. The advantages of using whole hemisphere histoblots: (1) they provide a low-resolution overview/outline of the antigen distribution in a large surface area, (2) large, thick, and/or unfixed tissue sections from post-mortem samples (perhaps of inferior tissue quality) can be compared, and (3) subsequent immunohistochemistry can be performed on the tissue sections used for the histoblots.     

Ablation of internalization signals in the carboxyl-terminal tail of the cystic fibrosis transmembrane conductance regulator enhances cell surface expression

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999) J. Biol. Chem. 274, 3602-3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr(1424)-X-X-Ile(1427) where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.