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Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O.
Get PDFNiemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O
Analogs of Cinnamic Acid Benzyl Amide As Nonclassical Inhibitors of Activated JAK2 Kinase.
Get PDFScaffold-based analogs of cinnamic acid benzyl amide (CABA) exhibit pleiotropic effects in cancer cells, and their exact molecular mechanism of action is under investigation. The present study is part of our systemic analysis of interactions of CABA analogs with their molecular targets. These compounds were shown to inhibit Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and JAK2/signal transducer and activator of transcription 5 (STAT5) signaling and thus are attractive scaffolds for anticancer drug design. To identify the potential mechanisms of action of this class of compounds, direct interactions of the selected CABA analogs with JAK2 kinase were examined. Inhibition of JAK2 enzymatic activity was assessed, and molecular modeling studies of selected compounds-(E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide (WP1065), (E)-2-cyano-N-[(S)-1-phenylbutyl]- 3-(3-bromopyridin-2-yl)acrylamide (WP1130), and (E)-2-cyano-N-[(S)-1,4-diphenylbutyl]-3-(3-bromopyridin-2-yl)acrylamide (WP1702)-in the JAK2 kinase domain were used to support interpretation of the experimental data. Our results indicated that the tested CABA analogs are nonclassical inhibitors of activated (phosphorylated) JAK2, although markedly weaker than clinically tested ATP-competitive JAK2 inhibitors. Relatively small structural changes in the studied compounds affected interactions with JAK2, and their mode of action ranged from allosteric-noncompetitive to bisubstrate-competitive. These results demonstrated that direct inhibition of JAK2 enzymatic activity by the WP1065 (half-maximal inhibitory concentration [IC50] = 14.8 µM), WP1130 (IC50 = 3.8 µM), and WP1702 (IC50 = 2.9 µM) potentially contributes, albeit minimally, to suppression of the JAK2/STAT signaling pathways in cancer cells and that additional specific structural modifications may amplify JAK2-inhibitory effects
Kultury starterowe do produkcji twarogów kwasowych – rola i oczekiwania.
Get PDFLactic acid bacteria are industrially important microbes used all over the world in a large variety of industrial food fermentations.
Cheese, cottage cheese, especially acid curd cheese, are characteristic dairy products for some Central and Eastern European countries. For
production of acid curd cheese, only two components are needed: milk and lactic acid bacteria starter cultures. Starter cultures determine
in large extent the technological process, the quality of the resulting quark and its shelf life. The composition of quark acid bacteria cultures
is constituted in majority by Lactococcus and Leuconostoc bacteria. Selection of strains and construction of cultures with balanced species
and stable composition for acid curd production is still very complex and presents a difficult challenge for producers of dairy cultures
Changes in digenean infection of the Antarctic fish Notothenia coriiceps in Admiralty Bay,King George Island, over three decades
Get PDFAbstract: The infection of black rockcod, Notothenia coriiceps, with digeneans in Admiralty
Bay (South Shetland Islands) within three months, from November 2007 to January 2008, is
compared with the infection in the same threemonths in 1978/79, based on the examination of
twenty fish collected in each month. Digenea found in 1978/1979 season were more numer−
ous, and more diverse. Only five digenean species, Macvicaria georgiana, Neolebouria
antarctica, Lepidapedon garrardi, Genolinea bowersi and Lecithaster macrocotyle, were re−
corded during both investigations,whereas three species, Neolepidapedon trematomi, Elytro−
phalloides oatesi and Gonocerca phycidis, only in 1978/79. M. georgiana was the dominant
species in 1978/79 and sub−dominant in 2007/08. Other digeneans were found in N. coriiceps
in 2007/08 invariably together with M. georgiana. G. bowersi was the sub−dominant species
in 1978/79 and the most common species in 2007/2008. Infections with Digenea belonging to
other species were much less intense. Of the three rare or common species in 1978/79, the
two, L. garrardi and L. macrocotyle, occurred in both seasons, whereas E. oatesi occurred
only in 1978/79. Three remaining species were sporadic or absent. The overall results there−
fore demonstrated that infections with almost all digenean species were less strong in 2007/08
than three decades earlier, in 1978/79. Only data on M. georgiana, G. bowersi and L. garrardi
were statistically significant (p <0.05). Data on the occurrence of 14 species of Digenea in N.
coriiceps from South Shetland Islands, South Orkney Islands, South Georgia, Argentine Is−
lands, Melchior Islands, Adelie Land and Heard Island are given
Bacteriophage T4 can produce progeny virions in extremely slowly growing Escherichia coli host: comparison of a mathematical model with the experimental data
Get PDFDevelopment of bacteriophage T4 depends on the physiological state of its host cell. Based on previous studies performed under laboratory conditions with different media determining various growth rates of Escherichia coli, a mathematical model was developed which suggested that phage T4 development cannot proceed efficiently in bacteria growing with a doubling time longer than 160 min. Contrary to this prediction, using a chemostat culture system allowing for culturing E. coli at different growth rates without changes in the medium composition, we found that T4 can yield progeny in host cells growing with a doubling time as long as 21 h. Our results indicate that the actual limiting growth rate of the host culture for the development of phage T4 is about 0.033 h(-1) , corresponding to the doubling time of about 21 h
The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation
No full textGenistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein- mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases
Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei.
Get PDFThe mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30 000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma.
Here, we present cloning and functional analysis of the erg20 gene from T. reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level
DiSCuS: an open platform for (not only) virtual screening results management.
No full textDiSCuS, a "Database System for Compound Selection", has been developed. The primary goal of DiSCuS is to aid researchers in the steps subsequent to generating high-throughput virtual screening (HTVS) results, such as selection of compounds for further study, purchase, or synthesis. To do so, DiSCuS provides (1) a storage facility for ligand-receptor complexes (generated with external programs), (2) a number of tools for validating these complexes, such as scoring functions, potential energy contributions, and med-chem features with ligand similarity estimates, and (3) powerful searching and filtering options with logical operators. DiSCuS supports multiple receptor targets for a single ligand, so it can be used either to evaluate different variants of an active site or for selectivity studies. DiSCuS documentation, installation instructions, and source code can be found at http://discus.ibb.waw.pl
Genome Sequence of the Probiotic Strain Lactobacillus rhamnosus (Formerly Lactobacillus casei) LOCK908.
Get PDFLactobacillus rhamnosus LOCK908, a patented probiotic strain (Polish patent no. 209987), was isolated from the feces of a healthy 6-year-old girl. Here, we present the complete genome sequence of LOCK908 and identify genes likely to be involved in the biosynthesis of exopolysaccharides (EPSs)
Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C
Get PDFMutations in the human mitochondrial ATP6 gene encoding ATP synthase subunit a/6 (referred to as Atp6p in yeast) are at the base of neurodegenerative disorders like Neurogenic Ataxia and Retinitis Pigmentosa (NARP), Leigh syndrome (LS), CharcoteMarieeTooth (CMT), and ataxia telangiectasia. In
previous studies, using the yeast Saccharomyces cerevisiae as a model we were able to better define how several of these mutations impact the ATP synthase. Here we report the construction of yeast models of two other ATP6 pathogenic mutations, T9185C and T9191C. The first one was reported as conferring a mild, sometimes reversible, CMT clinical phenotype; the second one has been described in a patient
presenting with severe LS. We found that an equivalent of the T9185C mutation partially impaired the functioning of yeast ATP synthase, with only a 30% deficit in mitochondrial ATP production. An equivalent of the mutation T9191C had much more severe effects, with a nearly complete block in yeast Atp6p assembly and an >95% drop in the rate of ATP synthesis. These findings provide a molecular basis for the
relative severities of the diseases induced by T9185C and T9191C