Does yeast shmooing mean a commitment to apoptosis?

Treatment of yeast Saccharomyces cerevisiae with alpha-pheromone has been reported to lead to massive apoptosis of cells finding no conjugation partner [Severin FF, Hyman AA. Pheromone induces programmed cell death in S. cerevisiae. Curr Biol 2002;12:R233-5]. We report here that this effect is not common in yeast. Using different yeast strains, we demonstrate that identical treatment results in a low mortality even after prolonged treatment with the pheromone. These findings are followed by a general discussion of the biological relevance of apoptosis in yeast.     

Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily

Proteins belonging to PD-(D/E)XK phosphodiesterases constitute a functionally diverse superfamily with representatives involved in replication, restriction, DNA repair and tRNA–intron splicing. Their malfunction in humans triggers severe diseases, such as Fanconi anemia and Xeroderma pigmentosum. To date there have been several attempts to identify and classify new PD-(D/E)KK phosphodiesterases using remote homology detection methods. Such efforts are complicated, because the superfamily exhibits extreme sequence and structural divergence. Using advanced homology detection methods supported with superfamily-wide domain architecture and horizontal gene transfer analyses, we provide a comprehensive reclassification of proteins containing a PD-(D/E)XK domain. The PD-(D/E)XK phosphodiesterases span over 21 900 proteins, which can be classified into 121 groups of various families. Eleven of them, including DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII and Replic_Relax, are newly assigned to the PD-(D/E)XK superfamily. Some groups of PD-(D/E)XK proteins are present in all domains of life, whereas others occur within small numbers of organisms. We observed multiple horizontal gene transfers even between human pathogenic bacteria or from Prokaryota to Eukaryota. Uncommon domain arrangements greatly elaborate the PD-(D/E)XK world. These include domain architectures suggesting regulatory roles in Eukaryotes, like stress sensing and cell-cycle regulation. Our results may inspire further experimental studies aimed at identification of exact biological functions, specific substrates and molecular mechanisms of reactions performed by these highly diverse proteins.     

Neuroprotection from Tissue Inhibitor of Metalloproteinase-1 and its nanoparticles

Matrix metalloproteinases (MMPs) are family of zinc dependent endopeptidases, which cleave extracellular matrix proteins, and play an important role in tissue remodelling in physiological and pathological processes. There is enhanced expression of MMPs, in particular MMP-9, during numerous pathological conditions, including epilepsy and ischemic stroke. Therefore, inhibition of MMP-9 is considered as a potential therapeutic target. Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) is a 28 kDa endogenous inhibitor of MMP-9. In this study we examined recombinant mouse TIMP-1 for its in-vitro neuroprotective effects, against Kainic Acid (KA) induced excitotoxicity in organotypic hippocampal slice culture (OHC) model. We also studied, sustained release effects of TIMP-1 in OHC by using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). TIMP-1 and TIMP-1 PLGA NPs were added to the slice cultures at different time points, i.e., 30 min before treatment with KA and 6 h after KA treatment. Propidium iodide staining was used to reveal cell toxicity in the cultures. In addition, neurotoxicity was assessed using standard lactate dehydrogenase (LDH) release assay. Gelatinolytic activity in conditioned cultured medium of OHC was accessed by a fluorescent substrate assay. Briefly, our result show that TIMP-1 provided significant level of neuroprotection, especially when given before 30 min of KA and released from the NPs. Since gelatinolytic activity assay showed a decrease in MMP-9 activity, it can be suggested that this neuroprotection might be mediated by the gelatinase inhibition.     

Carnitine Deficiency in OCTN2?/? Newborn Mice Leads to a Severe Gut and Immune Phenotype with Widespread Atrophy,Apoptosis and a Pro-Inflammatory Response

We have investigated the gross, microscopic and molecular effects of carnitine deficiency in the neonatal gut using a mouse model with a loss-of-function mutation in the OCTN2 (SLC22A5) carnitine transporter. The tissue carnitine content of neonatal homozygous (OCTN2^−/−) mouse small intestine was markedly reduced; the intestine displayed signs of stunted villous growth, early signs of inflammation, lymphocytic and macrophage infiltration and villous structure breakdown. Mitochondrial β-oxidation was active throughout the GI tract in wild type newborn mice as seen by expression of 6 key enzymes involved in β-oxidation of fatty acids and genes for these 6 enzymes were up-regulated in OCTN2^−/− mice. There was increased apoptosis in gut samples from OCTN2^−/− mice. OCTN2^−/− mice developed a severe immune phenotype, where the thymus, spleen and lymph nodes became atrophied secondary to increased apoptosis. Carnitine deficiency led to increased expression of CD45-B220⁺ lymphocytes with increased production of basal and anti-CD3-stimulated pro-inflammatory cytokines in immune cells. Real-time PCR array analysis in OCTN2^−/− mouse gut epithelium demonstrated down-regulation of TGF-β/BMP pathway genes. We conclude that carnitine plays a major role in neonatal OCTN2^−/− mouse gut development and differentiation, and that severe carnitine deficiency leads to increased apoptosis of enterocytes, villous atrophy, inflammation and gut injury.     

Fuzzy oil drop model to interpret the structure of antifreeze proteins and their mutants

Mutations in proteins introduce structural changes and influence biological activity: the specific effects depend on the location of the mutation. The simple method proposed in the present paper is based on a two-step model of in silico protein folding. The structure of the first intermediate is assumed to be determined solely by backbone conformation. The structure of the second one is assumed to be determined by the presence of a hydrophobic center. The comparable structural analysis of the set of mutants is performed to identify the mutant-induced structural changes. The changes of the hydrophobic core organization measured by the divergence entropy allows quantitative comparison estimating the relative structural changes upon mutation. The set of antifreeze proteins, which appeared to represent the hydrophobic core structure accordant with “fuzzy oil drop” model was selected for analysis.     

Understanding chlorophylls: central magnesium ion and phytyl as structural determinants

Phytol, a C20 alcohol esterifying the C-17(3) propionate, and Mg2+ ion chelated in the central cavity, are conservative structural constituents of chlorophylls. To evaluate their intramolecular structural effects we prepared a series of metal- and phytyl-free derivatives of bacteriochlorophyll a and applied them as model chlorophylls. A detailed spectroscopic study on the model pigments reveals meaningful differences in the spectral characteristics of the phytylated and non-phytylated pigments. Their analysis in terms of solvatochromism and axial coordination shows how the central Mg and phytyl residue shape the properties of the pigment. Surprisingly, the presence/absence of the central Mg has no effect on the solvatochromism of (bacterio)chlorophyll pi-electron system and the hydrophobicity of phytyl does not interfere with the first solvation shell of the chromophore. However, both residues significantly influence the conformation of the pigment macrocycle and the removal of either residue increases the macrocycle flexibility. The chelation of Mg has a flattening effect on the macrocycle whereas bulky phytyl residue seems to control the conformation of the chromophore via steric interactions with ring V and its substituents. The analysis of spectroscopic properties of bacteriochlorophyllide (free acid) shows that esterification of the C-17(3) propionate is necessary in chlorophylls because the carboxyl group may act as a strong chelator of the central Mg. These observations imply that the truncated chlorophylls used in theoretical studies are not adequate as models of native chromophores, especially when fine effects are to be modeled.     

Allosteric inhibition of the nonMyristoylated c-Abl tyrosine kinase by phosphopeptides derived from Abi1/Hssh3bp1

Here we report c-Abl kinase inhibition mediated by a phosphotyrosine located in trans in the c-Abl substrate, Abi1. The mechanism, which is pertinent to the nonmyristoylated c-Abl kinase, involves high affinity concurrent binding of the phosphotyrosine pY213 to the Abl SH2 domain and binding of a proximal PXXP motif to the Abl SH3 domain. Abi1 regulation of c-Abl in vivo appears to play a critical role, as demonstrated by inhibition of pY412 phosphorylation of the nonmyristoylated Abl by coexpression of Abi1. Pervanadate-induced c-Abl kinase activity was also reduced upon expression of the wild type Abi1 but not by expression of the Y213 to F213 mutant Abi1 in LNCaP cells, which are naturally deficient in the regulatory pY213. Our findings suggest a novel mechanism by which Abl kinase is regulated in cells.     

Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10-30% lower than that of UGPase-2, depending on direction of the reaction, whereas its K(m) values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03-0.14 mM vs. 0.07-0.36 mM, respectively). Both proteins were "true" UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.     

Synthesis of orthogonally protected vicinal diamines with amino acid-based skeleton

Summary A convenient route to amino acid-based orthogonally protected 1,2-diamines starting from materials readily available for a peptide chemist is presented. The key step of the procedure is the Mitsunobu reaction ofN-protected aminoalcohol, obtained by the reduction of commercially available Z- or Boc-protected amino acid, with imidodicarbonate or sulfonylcarbamate related to standard amino-protecting groups used in peptide chemistry yielding triprotected vicinal diamines.     

Effects of hydrogen, oxygen, and ammonia low-pressure glow plasma on granular starches

Cassava, corn, high amylose corn, potato, rice Indica, rice Japonica, sweet potato, waxy corn, and wheat starches were exposed to low-pressure ammonia, hydrogen, and oxygen plasma. In every case, depolymerization of the starch polysaccharides was noted. The extent of the depolymerization depended on the nature of the starch as well as the type of plasma applied. Among three fractions of polysaccharides distinguished by their molecular weight average, the fraction of the highest molecular weight suffered the most efficient depolymerization. The relative depolymerization for the middle- and low-molecular fractions of polysaccharides was found to be starch and plasma specific. The chemical character of the plasma had very little influence on the starch polysaccharides. Only subtle oxidation effects were observed in oxygen plasma. Low-pressure glow plasma treatment appeared to be a convenient tool for a waste-less dextrinization of starch. Manipulation of the plasma variety and the time of exposure resulted in a wide spectrum of dextrins of various molecular weights and paste-forming properties.