Ani immunomodulator Hepon inhibits hepatitis C virus replication in human cell cultures in vitro]

Antiviral activity of immunomodulator "Hepon" was evaluated in human cells culture infected with hepatitis C virus. "Hepon" presence protected human cells SW-13 from cytopathogenic effect of hepatitis C virus. Maximum antiviral effect was demonstrated by "Hepon" at concentration 1 mcg/mL. Control antiviral agent reaferon (interferon alfa-2a) was more potent as vitality protecting agent in the case of SW-13 human cells culture. "Hepon" activity is based on changes of cytokins and interferons spectrum so this immunomodulator is expected to be effective against different viruses including herpes virus and encephalocarditis virus.     

Cyanogenic compounds as protecting agents for organisms

Biochemical and physiological arguments and several plant-predator relationships described in the literature are presented in which cyanogenesis plays a role as a protecting process. HCN arising from the cleavage of cyanogenics is regarded to be the most important agent, but also the cyanogenic itself, carbonyls and -cyanoalanine, which are products of degradation processes of cyanogenics, may possess protecting properties. Some examples show that these substances are also utilized by arthropods. This presents the opportunity to look at a coevolutionary system combined of snails, plants, moths and moth-parasites in which cyanogenesis obviously plays an interesting role.Lecture presented during the Tagung der Deutschen Botanischen Gesellschaft, Vienna, September 1984.     

The ultrastructural localization of the isozymes of aspartate aminotransferase in murine tissues

Two isozymes of aspartate aminotransferase have been demonstrated biochemically. One isozyme is found in the mitochondrial fraction of the cytoplasm, the other ("soluble") in the supernatant. Both isozymes can be demonstrated by the cytochemical technique of Lee and Torack, as reported in the preceding report. Aldehyde fixation rapidly inactivates both isozymes, especially the soluble one. Inactivation can be delayed by addition of ketoglutarate to the fixative. The ketoglutarate probably competes with the fixative for the active site of the enzyme, thus protecting that region of the molecule. This enables adequate tissue preservation with enough remaining enzymatic activity to be demonstrated by the precipitation of oxaloacetate as the lead salt from a medium containing α-ketoglutaric acid aspartic acid, and lead nitrate. Electron-opaque material was found not only in mitochondria but, as the result of substrate protection, on the plasma membranes of many cells including erythrocytes and bacteria, the limiting membrane of peroxisomes, and the transverse tubular system of striated muscle. Occasional centrioles, neurotubules, tubules in the tails of spermatozoa, the A-I band junction in myofibrils of striated muscle, and the ground substance between cisternae of endoplasmic reticulum in intestinal goblet cells also showed precipitate. In all cases, replacement of L-aspartic acid by D-aspartic acid in the medium resulted in unstained sections. The sensitivity of extramitochondrial sites to fixation, the need of ketoglutarate as an agent for protecting the enzymatic activity during the fixation process, and the known presence of only soluble isozyme in erythrocytes indicate that enzymatic activity at these sites can be attributed to the soluble isozyme. Localization of the soluble isozyme on the plasma membrane may be related to possible involvement in depolarization phenomena, amino acid transport, or synthesis of plasma membrane-bound mucopolysaccharides.     

Ultrastructural cytochemistry of non-specific esterase in murine peritoneal macrophages

Summary Non-specific esterase (NSE) activity was demonstrated in glutaraldehyde-fixed monolayers of murine peritoneal macrophages. Using 2-naphthylthiol acetate (NTA) as substrate and Fast Blue BB as coupling agent a strong osmiophilic reaction product was obtained. The reaction product was observed as electron-dense dots covering cisterns of the rough endoplasmic reticulum, Golgi saccules and vesicles, or as large aggregates in lysosomes. Using -naphthyl butyrate (ANB) as substrate and hexazotized pararosaniline as coupling agent the osmiophilic reaction product was observed extracellularly on the plasma membrane as an electron-dense continuous layer, whereas intracytoplasmic staining of lysosomes was rare. Substitution of the coupling agents in the respective media resulted in a slight reaction with the ANB medium whereas with the NTA medium reaction product was observed only in lysosomal structures. The substrate specificity of the different types of esterases was confirmed after isoelectric focusing on thin-layer polyacrylamide gels. The results indicate that in murine peritoneal macrophages different types on NSE are detected with NTA and ANB, having distinct ultrastructural localizations.     

Formation of peptide bonds by carboxypeptidase c from orange leaves.

Carboxypeptidase c partially purified from orange leaves was studied as a catalyst for enzymatic peptide synthesis. Various N-protected ester- and nucleophile compounds were evaluated in order to determine the substrate specificity. For further characterization of the synthetic reaction, optimum pH and the influence of the N-terminal protecting group were studied. Kinetic investigations revealed considerable differences in Km and Vmax for the nucleophile when the N-terminal protecting group of the substrate was varied.     

Isolation and characterisation of DraI, a type II restriction endonuclease recognising a sequence containing only A:T basepairs, and inhibition of its activity by uv irradiation of substrate DNA

A type II restriction endonuclease, DraI, isolated from Deinococcus radiophilus ATCC 27603 recognises the palindromic hexanucleotide sequence (formula; see text) and cleaves it, as indicated by the arrows, to produce blunt-ended fragments. The yield of enzyme is 100 to 1000 times that of the only other known type II restriction endonuclease that recognises a sequence composed solely of A:T basepairs, the isoschizomer AhaIII (1). Ultraviolet irradiation of the DNA substrate at relatively low doses inhibits the activity of DraI by "protecting" the recognition sequence and this may be exploited to give control of partial digestion of DNA by DraI.     

Degradation of organophosphorous nerve agents by enzyme-polymer nanocomposites: efficient biocatalytic materials for personal protection and large-scale detoxification

The biocatalytic destruction of organophosphates has become an important focus area, as efficient "clean" technologies are sought for chemical weapons decommissioning, counteracting nerve agent attacks, and protecting against organophosphate pesticide poisoning. A novel method is advanced for immobilizing the broad-spectrum enzyme organophosphorous hydrolase (OPH) from Pseudomonas diminuta, based on the formation of nanocomposite protein-silicone polymers. The resulting materials are highly active, stable, and versatile biocatalysts for the liquid and gas phase detoxification of organophosphates, and can be fabricated as monoliths, sheets, thick films, granulates, or macroporous foams. This approach offers an efficient avenue to robust, high-performance biocatalytic OPH-containing polymers that outperform immobilized OPH catalysts reported to date. The method provides for the first time a route to biocatalytic materials that may be suitable for "active" protective wear, as well as bulk catalysts for the destruction of large volumes of organophosphates. The preparation of OPH-silicone biocomposites, their performances in the liquid and gas phase detoxification of paraoxon, dichlorvos, and diisopropyl fluorophosphate, and their features are discussed.     

4‐Fluorophenyl 3‐nitro‐2‐pyridinesulfenate as a practical protecting agent for amino acids

We report a new protecting agent ( 1 , Npys‐OPh(pF)) for 3‐nitro‐2‐pyridine (Npy) sulfenylation of amino, hydroxy, and thiol functional groups. Several Npys phenoxides were synthesized from Npys chloride (Npys‐Cl) and phenols in the presence of base in 1‐step reaction, and their ability for Npy‐sulfenylation was evaluated. As a result, 1 was selected as a new Npy‐sulfenylation agent with advantages including improved physicochemical stability, more controllable reactivity, and easier handling than the conventional protecting agent Npys‐Cl.     

Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk

The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and transient-state kinetic methods were applied to develop a catalytic pathway for substrate processing. Rapid quench flow techniques show that the phosphorylation of a substrate peptide, generated from a random library, occurs in two kinetic phases: a rapid, exponential "burst" phase followed by a slow, linear phase. The amplitude of the burst phase increases as a function of enzyme concentration, indicating that the biphasic kinetics are not the result of product inhibition. Analysis of the burst rate as a function of substrate concentration indicates that the phosphoryl transfer step is fast (k3 > or = 140 s(-1) and highly favorable (k3/k-3 > or = 6). The apparent dissociation rate constant for ADP (0.6 s(-1), measured using stopped-flow kinetic methods and a fluorescent trapping agent, mant-ATP, is close to kcat. Since the substrate dissociation constant is high, the release of phosphopeptide is not likely to limit turnover. These findings indicate that Csk rapidly delivers the gamma-phosphate of ATP to the substrate and rapidly releases the phosphoproduct. Overall rate limitation in the steady state is then attributed to the slow, net dissociation of ADP. Viscosometric studies suggest that this final event in the catalytic cycle is coupled with slow conformational changes.     

A homogeneous cell-based assay to identify N-linked carbohydrate processing inhibitors

Malignant transformation is accompanied by altered cell surface glycosylation. N-Linked oligosaccharides carrying beta1-6GlcNAc branches are associated with tumor invasion and metastasis. Therefore, compounds that can enter cells and block biosynthesis of beta1-6GlcNAc-branched glycans without overt cytotoxicity are potential anticancer agents. We have developed a homogeneous cell-based assay for detection of such compounds. The method enables identification of agents that block beta1-6GlcNAc-branched glycan expression after incubation for 16-20 h with MDAY-D2 tumor cells, thereby protecting the cells from the subsequent addition of leukoagglutinin, a cytotoxic plant lectin. We observed that MDAY-D2 cell number is directly proportional to the level of endogenous alkaline phosphatase activity measured spectrophotometrically in cultures after the addition of substrate. The alkaline phosphatase assay was capable of detecting as few as 1,500 cells. The assay was readily adapted for high-throughput screening as reagent costs are low and no cell harvesting and washing steps are required. Under high-throughput operating conditions, the coefficient of variation for controls was found to be 4.2%. The results suggest that measurement of alkaline phosphatase in this cell assay format may be adapted for wider applications in high-throughput screenings for compounds that relieve cells from other growth inhibitors.     

Studies on pituitary follitropin. III. Functional role of the amino groups in subunit and receptor interactions of the ovine hormone.

The effectiveness of Glc, mannose, 2-deoxyglucose, fructose, galactose, arabinose, and N-acetylglucosamine at protecting rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) from inactivation by chymotrypsin, glutaraldehyde, heat, and Ellman's reagent have been compared. The relative effectiveness at protecting against these inactivating agents decreases in the order Glc > mannose > 2-deoxyglucose > fructose, galactose, and arabinose, the last three providing no significant protection at all. The nonphosphorylatable substrate analog, N-acetylglucosamine, provides substantial protection against heat inactivation, but is ineffective against inactivation by the other agents. Similar inactivation studies were conducted using several hexose 6-phosphates. Glc-6-P and 1,5-anhydroglucitol-6-P provided substantial protection while 2-deoxyglucose-6-P, fructose-6-P, mannose-6-P, and galactose-6-P were all relatively ineffective at protecting hexokinase activity. The protective effect of these ligands is taken as an indication of ligand-induced conformational changes in brain hexokinase. The results are interpreted in terms of, and considered to support, a recently proposed model (J. E. Wilson, 1978, Arch. Biochem. Biophys.185, 88–99) in which the suitability of a carbohydrate as a substrate depends directly on its ability to induce specific conformational changes prerequisite for subsequent catalytic events while the inhibitory effectiveness of a hexose 6-phosphate is likewise related to its ability to evoke appropriate conformational change in the enzyme. Synergistic interactions between hexose and hexose-6-P binding sites, first reported for Glc and Glc-6-P by Ellison et al. (1975, J. Biol. Chem.250, 1864–1871), have been confirmed. Thus, although fructose and galactose were themselves quite ineffective at providing protection against inactivation of hexokinase by chymotrypsin or glutaraldehyde, they greatly increased the protection afforded by suboptimal (with respect to degree of protection in the absence of added hexose) levels of Glc-6-P. Conversely, the 6-phosphates of fructose, galactose, mannose, and 2-deoxyglucose, which were themselves ineffective at protecting the enzyme activity, markedly enhanced the protection provided by suboptimal levels of Glc or mannose. Based on the relationship between this enhancement of Glc-dependent protection and the hexose-6-P concentration, the dissociation constants for the complexes of hexokinase with 2-deoxyglucose-6-P and mannose-6-P were estimated to be ?0.5 mm.     

Hexafluoroacetone as protecting and activating reagent: Site-selective functionalization of iminodiacetic acid

Summary. Hexafluoroacetone was applied as a bidentate protecting and activating agent for the syntheses of RGD-peptide mimetics starting from iminodiacetic acid in solution and on solid phase.     

Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity

The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (a_w, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.     

Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity

The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (a_w, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.     

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein.     

降解剂对森林凋落物可溶性有机碳含量的影响及凋落物降解模式比较研究

森林凋落物是森林生态系统中可溶性有机碳(DOC)的主要来源,在生态系统碳循环过程中起重要作用。以帽儿山地区胡桃楸、兴安落叶松以及胡桃楸-兴安落叶松人工林凋落物为研究对象,通过液体发酵培养将纤维素高效降解真菌Peniophora intranata与Sarocladium strictum制成单一(A菌剂、B菌剂)及混合菌剂(C菌剂),测定野外条件下经降解剂处理的凋落物基质在不同时期的DOC含量,分析降解剂对不同类型森林凋落物DOC动态变化的影响,并比较各凋落物基质降解模式异同。结果表明：(1)凋落物基质DOC含量在降解剂处理后均随降解时间增加呈下降趋势,且下降幅度大小表现为混合基质>胡桃楸基质>落叶松基质;在最初的1个月,各凋落物基质DOC含量均显著高于其他降解时期DOC含量。(2)经混合菌剂处理后的胡桃楸基质DOC含量相较于2种单一菌剂处理后的胡桃楸基质DOC含量低,而经混合菌剂处理后的兴安落叶松基质与胡桃楸-兴安落叶松基质的DOC含量未表现出相同的显著性。(3)经3种菌剂处理后的胡桃楸基质降解模式均相同,经B菌剂与C菌剂处理的落叶松基质降解模式相同,经A菌剂与C菌剂处理...     

Potential dermatological application on Asian plants

Skin diseases are among some of the most common types of health problems faced in Malaysia, as reported by the World Health Organization (WHO). Correspondingly, research into the use of medicinal plants for skin disease treatment has become important. Through the ages, medicinal plants have been used widely to treat a variety of skin diseases. The demand for plant-based medicines is growing, as they are generally considered to be safer and less harmful than conventional allopathic drugs. This article reviews the potential of Asian plants to be epidermal protecting agents. There are eleven remarkable Asian plants that are known for their skin barrier protecting agent properties. Important studies have shown that natural products offer a rich potential source of epidermal protecting agents. Nevertheless, further surveys and clinical evidence are needed to establish the potential of identified species in contributing to the treatment of skin disease, especially atopic eczema.     

Induction of glucose 6-phosphatase in cultured hepatoma cells by dexamethasone

The synthetic glucocorticoid, dexamethasone, induced high levels of glucose 6-phosphatase in a line of cultured hepatoma cells (2S FAZA). This conflicts with current theory that glucocorticoids induce a microsomal translocase, specific for glucose 6-phosphate, but not the hydrolase itself. The earlier conclusion was based on experiments with rat livers in vivo in which cortisone was the inducing agent. In the present study, 5 X 10(-6) M dexamethasone induced a tenfold increase in glucose 6-phosphatase activity in "intact" as well as disrupted microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. This induction was blocked by cycloheximide (50 micrograms/ml). The broad pH maxima between 5.5 and 7.0 were similar for both "intact" and disrupted microsomes.     

An Inducible System for the Hydrolysis and Transport of β-Glucosides in Yeast : I. Characteristics of the β-glucosidase activity of intact and of lysed cells

A strain of bakers'' yeast was isolated which could utilize cellobiose and other β-D-glucosides quantitatively as carbon and energy sources for growth. Cellobiose-grown cells contained a largely cryptic enzyme active against the chromogenic substrate p-nitrophenyl-β-D-glucoside. The patent (intact cell) activity of such cells was inhibited by azide and, competitively, by cellobiose; neither agent inhibited the β-glucosidase activity of lysed cells or of extracts. The enzyme induced by growth in cellobiose medium had no affinity for cellobiose as either substrate or inhibitor; its substrate specificity classifies it as an aryl-β-glucosidase. It was concluded that growth in cellobiose also induced the formation of a stereospecific and energy-dependent system whose function determined the rate at which intact cells could hydrolyze substrates of the intracellular β-glucosidase.     

Rapid Synthesis of Oligodeoxyribonucleotides VIII.1 An Improved Procedure for Solid-Phase Oligodeoxyribonucleotide Synthesis Using a Controlled Pore Glass Support and Phosphotriester Chemistry

Abstract

An improved method for solid phase oligodeoxyribonucleotide synthesis is described that uses phosphotriester chemistry, controlled pore glass as support, a new and more stable linkage agent and a better protecting group combination.