Down-regulation of model yeast proteins by ubiquitin-dependent proteolysis

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a multitude of proteins. I will attempt to bring together our recent data on the down-regulation of two yeast model proteins, the galactose transporter Gal2 and fructose-1,6-bisphosphatase, by ubiquitin-dependent proteolysis triggered by the addition of easily fermentable carbon sources.     

The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a wide variety of proteins. Two of the best characterized functions of protein ubiquitination are to mark proteins for degradation by cytosolic proteasome and to promote the internalization of certain plasma membrane proteins via the endocytotic pathway, followed by their degradation in the vacuole. Recent studies of membrane proteins both in yeast and mammalian cells suggest that the role of ubiquitin may extend beyond its function as an internalization signal in that it also may be required for modification of some component(s) of the endocytotic machinery, and for cargo protein sorting at the late endosome and the Golgi apparatus level. In this review, I will attempt to bring together what is currently known about the role of ubiquitination in controlling protein trafficking between the yeast plasma membrane, the trans-Golgi network, and the vacuole/lysosome.     

The serologic screening for celiac disease in the general population (blood donors) and in some high-risk groups of adults (patients with autoimmune diseases,osteoporosis and infertility) in the Czech republic

The prevalence of celiac disease (CD) was determined in healthy blood donors and in high-risk groups of adults (a total of 1835 adults—randomly selected 1312 healthy blood donors, 102 patients with primary osteoporosis, 58 patients with autoimmune diseases and 365 infertile women). It was calculated on the basis of a two-step serologic screening method—in the first step IgA and IgG antigliadin antibodies (AGA) and IgA anti-γ-glutamyltransferase (‘transglutaminase’) antibodies (ATG) were estimated, in the second step sera positive for IgA AGA and/or IgA ATG were examined for antiendomysial IgA (AEA) antibodies. Immunoenzymic assay (ELISA) was used for determining of AGA and ATG antibodies; immunoflurescence method, performed on human umbilical cord tissue, was used for assaying of AEA antibodies. Total serum IgA level in only IgG AGA positive subjects was measured by routine turbidimetric method. 0.45% of healthy blood donors, 0.98% of osteoporotic patients, 2.7% of patients suffering from autoimmune disease and 1.13% of women with infertility considered as immunologically mediated were found to be positive in both steps of serologic screening (AGA and/or ATG and antiendomysium positive). The presumed high prevalence of seropositivity for CD in apparently healthy Czech adult population was confirmed. In the high-risk groups, the prevalence of seropositivity for CD was approximately 2–4 times higher than in healthy blood donors. The real prevalence of CD in the tested groups, however, can be estimated after performing small intestinal biopsy in the seropositive patients.     

Presence of fetal cells in maternal circulation after delivery

Fetal cells can still be detected in maternal blood 8 month postpartum.     

Preventive effect of several antioxidants after oxidative stress on rat brain homogenates

Brain homogenate was used as a model system to study antioxidant properties of several natural and synthetic antioxidants under oxidative stress. Oxidative stress was induced by Fe/ascorbate system and lipid peroxidation as well as protein modification were studied. Thiobarbituric acid reactive substances (TBARS) were used as a marker of lipid peroxidation. The preventive effect concerning lipid peroxidation decreased in the order: buthylated hydroxytoluene (BHT) (3.5), stobadine (ST) (35), serotonin (54), trolox (98), U 74389G (160), melatonin (3100), (the numbers in the brackets represent IC50 in micromol/l). Methylprednisolone had no effect, and spin traps interfered with TBARS determination. Concerning creatine kinase (CK) activity as a selected marker of oxidative modification of proteins, the preventive effect of antioxidants (30 micromol/l) decreased in the order: BHT (30), trolox (75), stobadine (ST) (77), alpha-phenyl-N-tert-buthylnitrone (PBN) (87), sodium salt of N-tert-buthyl-C-(phenyl-2-sulfone) nitrone (SPBN) (90), (the numbers in the brackets represent the loss of CK activity in percentages, when 100% was the loss of CK activity in the absence of any antioxidant). The nonglucocorticoid steroid U 74389G, methylprednisolone and serotonin had no preventive effects, while melatonin had antioxidant effect only in a higher concentration (1 mmol/l).     

Enhancement of Isoflavone Synthase Activity by Co-expression of P450 Reductase from Rice

Plant cytochrome P450s interact with a flavoprotein, NADPH-cytochrome P450 reductase (CPR), to transfer electrons from NADPH. The gene for rice P450 reductase (RCPR) was cloned and expressed in Saccaromyces cerevisiae, where the specific activity of the expressed RPCR was 0.91 U/mg protein. When isoflavone synthase gene (IFS) from red clover, used as a model system of plant cytochrome P450, was co-expressed with RCPR in yeast, the production of genistein from naringein increased about 4.3-fold, indicating that the RCPR efficiently interacts with cytochrome P450 to transfer electrons from NADPH.     

Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei

The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.