Study of channel-forming properties of latrotoxin in liposomes

Interaction of latrotoxin with phospholipid vesicles and bilayer lipid membranes is shown to proceed differently. Latrotoxin when interacting with liposomes is sorbed on the membrane surface forming no ionic channels in this case. Only latrotoxin fragments obtained due to the toxin hydrolysis by pronase or trypsin are able to form channels. These fragments being inserted into liposomes are coupled strongly with the membrane and are not subjected to the further splitting by proteinases. The electrophoretic spectrum of peptides bound with liposomes is presented by protein components with a molecular weight of 116, 100, 92, 67, 52 and 45 kDa, while zone typical of latrotoxin is absent in this spectrum. The method of small-angle X-rays scattering has shown that tryptic fragments of latrotoxin penetrate into the phospholipid bilayer of liposomes.     

Applications of microorganisms to geotechnical engineering for bioclogging and biocementation of soil in situ

Microbial Geotechnology is a new branch of geotechnical engineering that deals with the applications of microbiological methods to geological materials used in engineering. The aim of these applications is to improve the mechanical properties of soil so that it will be more suitable for construction or environmental purposes. Two notable applications, bioclogging and biocementation, have been explored. Bioclogging is the production of pore-filling materials through microbial means so that the porosity and hydraulic conductivity of soil can be reduced. Biocementation is the generation of particle-binding materials through microbial processes in situ so that the shear strength of soil can be increased. The most suitable microorganisms for soil bioclogging or biocementation are facultative anaerobic and microaerophilic bacteria, although anaerobic fermenting bacteria, anaerobic respiring bacteria, and obligate aerobic bacteria may also be suitable to be used in geotechnical engineering. The majority of the studies on Microbial Geotechnology at present are at the laboratory stage. Due to the complexity, the applications of Microbial Geotechnology would require an integration of microbiology, ecology, geochemistry, and geotechnical engineering knowledge.     

Mathematical modeling of the reproduction of the hepatitis C virus replicon in cell culture. Simulation of the action of potential therapeutics

Nowadays, search for efficient pharmaceuticals against hepatitis C virus (HCV) is an urgent task. In addition to conventional medicines, such as interferon and ribavirin, new specific drugs are being developed. Recently, it has been shown that a peptidomimetic substance, competitive inhibitor of viral NS3 protease, efficiently suppresses replication of the viral RNA replicon in Huh-7 cells. Computer simulation of the operation of the gene network comprising major processes of the viral RNA in the cell provides grounds for analysis of the HCV life cycle and search for key targets for efficient attack with drugs. The gene network of viral RNA replication in Huh-7 cells in the presence of a highly specific and efficient viral NS3 protease inhibitor has been reconstructed by analysis of reported experimental results and application of the GenNet technology. A mathematical model describing the operation of this network has been developed. The kinetics of the decrease in the level of viral RNA in the presence of the inhibitor predicted on the basis of this model is close to experimental results.

     

Bioaugmentation and enhanced formation of microbial granules used in aerobic wastewater treatment

Microbial aggregates of an aerobic granular sludge can be used for the treatment of industrial or municipal wastewater, but their formation from a microbial activated sludge requires several weeks. Therefore, the aim of this research was the selection of microbial cultures to shorten the granule-forming period from several weeks to a few days. An enrichment culture with the ability to accelerate granulation was obtained by repeating the selection and batch cultivation of fast-settling microbial aggregates isolated from the aerobic granular sludge. Bacterial cultures of Klebsiella pneumoniae strain B and Pseudomonas veronii strain F, with self-aggregation indexes of 65 and 51%, respectively, and a coaggregation index of 58%, were isolated from the enrichment culture. A mixture of these strains with the activated sludge was used as an inoculum in an experimental sequencing batch reactor to start up an aerobic granulation process. Aerobic granules with a mean diameter of 446±76 μm were formed in an experiment after 8 days of cultivation, but microbial granules were absent in controls. Considering biosafety issues, K. pneumoniae strain B was excluded from further studies, but P. veronii strain F was selected for larger-scale testing.Stephen Tiong-Lee Tay Passed away on 27 July 2005.     

Screening for caspase-3 inhibitors: effect of a reducing agent on identified hit chemotypes

When studying cysteinyl proteases in general and caspases in particular, it is generally accepted that a reaction buffer must contain a reducing agent to prevent essential cysteinyl groups from spontaneous oxidation. Dithiothreitol (DTT) and beta-mercaptoethanol (beta-MCE) are 2 of the most broadly used reducing agents. While screening a library of small molecules against caspase-3, the authors have found that the nature of the reducing agent used, DTT or beta-MCE, dramatically affects screening results and leads to identification of nonoverlapping hits. Screening in DTT-containing buffer revealed few novel classes of small molecules that selectively and reversibly inhibit caspase-3 but failed to identify isatin sulfonamides recently found to be potent and selective caspase-3 inhibitors (false negatives). On the other hand, screening in the presence of beta-MCE failed to identify a series of hit compounds, 1,3-dioxo-2,3-dichloro-1H-pyrrolo[3,4-c]quinolines, discovered with DTT, whereas isatin sulphonamides in these conditions exhibited strong caspase-3 inhibition. In this work, the authors show that thiol-containing reducing agents can affect catalytic activity of caspase-3 and modify its thermostability in a redox-potential-independent manner. The authors speculate that the differential structural modifications of caspase-3 seen with different reducing agents represent structurally different caspase-3 conformations and are responsible for its differential sensitivity to small molecules of different chemotypes. Hence, selection of the reducing agent may dramatically affect the quality of high-throughput screening campaigns.     

Irreversible global DNA hypomethylation as a key step in hepatocarcinogenesis induced by dietary methyl deficiency

Dietary methyl group deprivation is now well recognized as a model of hepatocarcinogenesis in rodents. In the present study, we examined the effects of feeding a methyl-deficient diet followed by a methyl-adequate diet on the extent of methylation of liver DNA and on the formation and evolution of altered hepatic foci. Male F344 rats were fed a methyl-deficient diet for 9, 18, 24, and 36 weeks, followed by re-feeding a methyl-adequate diet for a total of 54 weeks. Similar to previous findings, the methyl-deficient diet resulted in decreased levels of S-adenosylmethionine (SAM), SAM/SAH ratios, and global DNA hypomethylation. Feeding the methyl-adequate diet restored the liver SAM levels and SAM/SAH ratios to control levels in all experimental groups. In contrast, re-feeding the complete diet restored DNA methylation to normal level only in the group that had been fed the methyl-deficient diet for 9 weeks; in animals exposed to methyl deprivation longer, the methyl-adequate diet failed to reverse the hypomethylation of DNA. Liver tissue of rats exposed to methyl deficiency for 9, 18, 24, or 36 weeks was characterized by the persistent presence of placental isoform of glutathione-S-transferase (GSTpi)-positive lesions despite re-feeding the methyl-adequate diet. The persistence of altered hepatic foci in liver after withdrawal of methyl-deficient diet serves as an indication of the carcinogenic potential of a methyl-deficient diet. Substitution of the methyl-deficient diet with complete diet failed to prevent the expansion of initiated foci and restore DNA methylation in animals exposed to deficiency for 18, 24, or 36 weeks. The association between DNA hypomethylation and expansion of foci suggests that stable DNA hypomethylation is a promoting factor for clonal expansion of initiated cells. These results provide an experimental evidence and a mechanistic basis by which epigenetic alterations may contribute to the initiation and promotion steps of carcinogenesis.     

Repressing the expression of the SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid-insensitive phenotype

The classic role of SUCROSE NONFERMENTING-1 (Snf1)-like kinases in eukaryotes is to adapt metabolism to environmental conditions such as nutrition, energy, and stress. During pea (Pisum sativum) seed maturation, developmental programs of growing embryos are adjusted to changing physiological and metabolic conditions. To understand regulation of the switch from cell proliferation to differentiation, SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE (SnRK1) was antisense repressed in pea seeds. Transgenic seeds show maturation defects, reduced conversion of sucrose into storage products, lower globulin content, frequently altered cotyledon surface, shape, and symmetry, as well as occasional precocious germination. Gene expression analysis of embryos using macroarrays of 5,548 seed-specific genes revealed 183 differentially expressed genes in two clusters, either delayed down-regulated or delayed up-regulated during transition. Delayed down-regulated genes are related to mitotic activity, gibberellic acid/brassinosteroid synthesis, stress response, and Ca2+ signal transduction. This specifies a developmentally younger status and conditional stress. Higher gene expression related to respiration/gluconeogenesis/fermentation is consistent with a role of SnRK1 in repressing energy-consuming processes in maturing cotyledons under low oxygen/energy availability. Delayed up-regulated genes are mainly related to storage protein synthesis and stress tolerance. Most of the phenotype resembles abscisic acid (ABA) insensitivity and may be explained by reduced Abi-3 expression. This may cause a reduction in ABA functions and/or a disconnection between metabolic and ABA signals, suggesting that SnRK1 is a mediator of ABA functions during pea seed maturation. SnRK1 repression also impairs gene expression associated with differentiation, independent from ABA functions, like regulation and signaling of developmental events, chromatin reorganization, cell wall synthesis, biosynthetic activity of plastids, and regulated proteolysis.     

Water soluble (η-arene) ruthenium(II) complexes incorporating marine derived bioligand: Synthesis, spectral and structural studies

A series of water soluble compounds of general formula [{(η⁶-arene)Ru(HMP)Cl}], [η⁶-arene = η⁶-cymene (1), η⁶-HMB (2), η⁶-C₆H₆ (3); HMP = 5-hydroxy-2-(hydroxymethyl)-4-pyrone] have been prepared by the reaction of [{(η⁶-arene) RuCl₂}₂] with HMP. The complexes 1 and 2 react with NaN₃ to give in excellent yield tetra-azido complexes [{(η⁶-arene)Ru(μN₃)N₃}₂] (arene = cymene 4, HMB = 5) but similar reaction of complex 3 with NaN₃ yielded di-azdo complex [{(η⁶-C₆H₆)Ru(μN₃)Cl}₂] (6). Reaction of [{(η⁶-arene)Ru(μN₃)Cl}₂] with HMP in the presence of NaOMe resulted in the formation of azido complex [{(η⁶-arene)Ru(HMP)N₃}]. Mono and dinuclear complexes [{(η⁶-arene)Ru(HMP)(L₁)}]⁺ and [{(η⁶-arene)Ru(HMP)}₂(μL₂)]²⁺ were also prepared by the reaction of complexes 1 and 2 with the appropriate ligand, L₁ or L₂ in the presence of AgBF₄ (L₁ = PyCN, DMAP; L₂ = 4,4′-bipy, pyrazine). The complexes are characterized on the basis of spectroscopic data and molecular structures of three representative compounds have been determined by single crystal X-ray diffraction study.     

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.