Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.     

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

Phylogenetic diversity of stress signalling pathways in fungi

Background  

Microbes must sense environmental stresses, transduce these signals and mount protective responses to survive in hostile environments. In this study we have tested the hypothesis that fungal stress signalling pathways have evolved rapidly in a niche-specific fashion that is independent of phylogeny. To test this hypothesis we have compared the conservation of stress signalling molecules in diverse fungal species with their stress resistance. These fungi, which include ascomycetes, basidiomycetes and microsporidia, occupy highly divergent niches from saline environments to plant or mammalian hosts.     

Differential binding of lectins IL-2 and CSL to candida albicans and cancer cells

The demonstration that interleukin 2 (IL-2) is a lectin specific for oligomannosides allows to understand a new function for this cytokine: as a bifunctional molecule when bound to its receptor ss, IL-2 associates the latter which the CD3/TCR complex, interacting with oligosaccharides of CD3 through its carbohydrate-recognition domain (Zanetta et al. , 1996, Biochem. J., 318, 49-53). This induces the tyrosine phosphorylation of the IL-2R beta by ++p56(lck) , the first step of the IL-2-dependent signaling. Since this specific association is disrupted in vitro by oligomannosides with five and six mannose residues, we made the hypothesis that pathogenic cells or microorganisms could bind IL-2, consequently disturbing the IL-2- dependent response. This study shows that the pathogenic yeast Candida albicans (in contrast with nonpathogenic yeasts) binds high amounts of IL-2 as did cancer cells. In contrast with cancer cells, yeasts do not bind the Man6GlcNAc2-specific lectin CSL, an endogenous "amplifier of activation signals" (Zanetta et al. , 1995, Biochem. J., 311, 629-636).      

Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes

In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. We demonstrate that B-Raf is activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocked the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. We show that Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. Our results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor, Son of sevenless (SOS), nor the cAMP-responsive Rap-1 exchange factor, Epac, participate in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP.     

The p53/p21Cip1/ Waf1 pathway mediates the effects of SPARC on melanoma cell cycle progression

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, belongs to the family of matricellular proteins that modulate cell-matrix interactions and cellular functions. SPARC is highly expressed in melanoma, and we reported that SPARC promotes epithelial/mesenchymal-like changes and cell migration. Here, we used siRNA and conditional shRNA to investigate the contribution of tumor-derived SPARC to melanoma cell growth in vitro and in vivo. We found that depletion of SPARC induces G2/M cell cycle arrest and tumor growth inhibition with activation of p53 and induction of p21(Cip1/Waf1) acting as a checkpoint, preventing efficient mitotic progression. In addition, we demonstrate that reduced mesenchymal features and the invasive potential of SPARC-silenced cells are independent of p21(Cip1/Waf1) induction and cell cycle arrest. Importantly, overexpression of SPARC reduces p53 protein levels and leads to an increase in cell number during exponential growth. Our findings indicate that in addition to its well-known function as a mediator of melanoma cell migration and tumor-host interactions, SPARC regulates, in a cell-autonomous manner, cell cycle progression and proliferation through the p53/p21(Cip1/Waf1) pathway.