Silver staining of Drosophila polytene chromosomes and the effect of hyaluronidase and lysozyme pretreatment

The Ag-AS technique was used for staining the polytene chromosomes of D. melanogaster and D. lummei. Bands were stained dark reddish-brown, interbands light yellow. A toromere was heavily stained on the sixth chromosome of D. lummei. The staining intensity of nucleoli was lower than that of chromosomes. During a prolonged staining ectopic threads and the nonhomogeneous structure of nucleoli were revealed. Pretreatment with RNase caused slight changes in the silver staining pattern of chromosomes; pretreatment with DNase did not result in any visible changes, while after preincubation with proteolytic enzymes chromosome morphology was destroyed. Hyaluronidase and lysozyme removed the silver-reducing components from chromosomes without destroying the general chromosome structure. Each of these two enzymes acts specifically: hyaluronidase affects the morphology of chromosomes, but not nucleoli and bands at heat shock puffs, whereas the action of lysozyme is probably evenly distributed between chromosomes and nucleoli.     

Comparative study of proteolytic enzyme preparations of the actinomycete Streptomyces spheroides and its mutant

Preparations of proteolytic enzymes with high fibrinolytic and thrombolytic activities were isolated from the cultural broth filtrates of Streptomyces spheroides and its mutant by precipitation with ammonium sulfate, dialysis and freeze-drying. The fibrinolytic and thrombolytic activities of the enzymes produced by the mutant are 5.6 and 6 times higher, respectively, than those of the parent strain. Moreover, the enzymes of the mutant have an elevated stability in the alkaline pH region, the optimum of their activities is also shifted to the alkaline pH region, they are more resistant to the action of blood plasma inhibitors and more sensitive to the action of high temperatures. As was shown using isoelectric focusing in a sucrose density gradient, the preparations of both the parent and mutant strains contain several proteolytic enzymes (or their groups) with acid, neutral and alkaline isoelectric points.     

Inhibitor selectivity of a new class of oseltamivir analogs against viral neuraminidase over human neuraminidase enzymes

The viral neuraminidase enzyme is an established target for anti-influenza pharmaceuticals. However, viral neuraminidase inhibitors could have off-target effects due to interactions with native human neuraminidase enzymes. We report the activity of a series of known inhibitors of the influenza group-1 neuraminidase enzyme (N1 subtype) against recombinant forms of the human neuraminidase enzymes NEU3 and NEU4. These inhibitors were designed to take advantage of an additional enzyme pocket (known as the 150-cavity) near the catalytic site of certain viral neuraminidase subtypes (N1, N4 and N8). We find that these modified derivatives have minimal activity against the human enzymes, NEU3 and NEU4. Two compounds show moderate activity against NEU3, possibly due to alternative binding modes available to these structures. Our results reinforce that recognition of the glycerol side-chain is distinct between the viral and human NEU enzymes, and provide experimental support for improving the selectivity of viral neuraminidase inhibitors by exploiting the 150-cavity found in certain subtypes of viral neuraminidases.     

Effect of nitrosourea in small doses on the frequency of mutation in Salmonella typhimurium

The effect of N-nitroso-N-methylurea (NMU), N-nitroso-N,N'-dimethylurea (NDMU) and N-nitroso-N-ethylurea (NEU) at doses less than 100 mkg/ml on mutability of Salmonella typhimurium strains of Ames' system (G-46, TA-1950, TA-1535, TA-100, TA-1538) has been studied. NMU and NEU at doses of 5-10 mkg/ml have been found to increase the survival and decrease the number of reversions from auxotrophity in histidine to prototrophity. The effect of given doses of NMU and NEU on bacteria repair activity has been shown. The role of pk M101 plasmide in this process is being discussed. NDMU in contrast to NMU and NEU induces read frome shift mutations and exhibits high mutagenous activity at all doses examined.     

Molecular dynamics simulations of viral neuraminidase inhibitors with the human neuraminidase enzymes: Insights into isoenzyme selectivity

Inhibitors of viral neuraminidase enzymes have been previously developed as therapeutics. Humans can express multiple forms of neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) that share a similar active site and enzymatic mechanism with their viral counterparts. Using a panel of purified human neuraminidase enzymes, we tested the inhibitory activity of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir, oseltamivir, and peramivir against each of the human isoenzymes. We find that, with the exceptions of DANA and zanamivir, these compounds show generally poor activity against the human neuraminidase enzymes. To provide insight into the interactions of viral inhibitors with human neuraminidases, we conducted molecular dynamics simulations using homology models based on coordinates reported for NEU2. Simulations revealed that an organized water is displaced by zanamivir in binding to NEU2 and NEU3 and confirmed the critical importance of engaging the binding pocket of the C7–C9 glycerol sidechain. Our results suggest that compounds designed to target the human neuraminidases should provide more selective tools for interrogating these enzymes. Furthermore, they emphasize a need for additional structural data to enable structure-based drug design in these systems.     

The proteolytic enzymes as a highly sensitive criterion of effectiveness and safety of treatment

The effect of ozonated saline on proteolytic enzymes (trypsin, chymotrypsin, elastase, kallikrein, leucine aminopeptidase), inhibitors of proteolysis and lipid peroxidation (LPO) has been investigated. Injection of ozonated saline caused marked response of the proteolytic system. Low ozone doses did not cause activation of proteolytic enzymes, whereas high doses activated proteases, decreased the level of inhibitors of proteolysis (α₁-antitrypsin and α₂-macroglobulin) and stimulated accumulation of LPO products. Thus, analyses of proteolytic activity can be used as an indicator of effectiveness and safety of ozone therapy and other treatment programs.     

Effector controlling accumulation of N-acetylglucosaminidase during development of Dictyostelium discoideum

The accumulation of N-acetylglucosaminidase, an early developmentally regulated enzyme in Dictyostelium discoideum, is dependent upon the action of a heat-stable effector molecule secreted by the cells. Stimulation of enzyme accumulation is inhibited by cycloheximide and actinomycin, suggesting that it requires concomitant RNA and protein synthesis. The effector elutes from Sephadex columns as a molecule of 300 to 1000 daltons. It is stable to treatment with a variety of proteolytic enzymes and mild acid hydrolysis but can be inactivated by prolonged acid hydrolysis.     

The two sides of enzyme-substrate specificity: lessons from the aspartic proteinases

Like most proteolytic enzymes, the aspartic proteinases bind substrates and most inhibitors within an extended active site cleft. Bound ligands typically adopt a beta-strand conformation. Interactions with groups on both sides of the cleft determine the primary as well as secondary specificity of the enzymes. We have pursued the discovery of the sometimes subtle distinctions between members of the aspartic proteinase family by two routes. In the first case, we have constructed sets of oligopeptide substrates with systematic variation in each position to assess interactions at one position at a time. In the second type of experiment, we have altered residues of the enzymes in order to test theories of selectivity. The combination of the two approaches has provided a better understanding of the forces involved in determining specificity of enzyme action.     

Microorganisms from composting leaves: Ability to produce extracellular degradative enzymes

Mixed populations of bacteria, fungi, and actinomycetes in a leaf compost pile were examined over a 100-day test period for their ability to produce extracellular proteolytic, lipolytic, amylolytic, cellulolytic, pectolytic, and ureolytic enzymes and ability to utilize alkanes. Urea was added to the leaves to adjust the carbon to nitrogen ratio but was of little value in maintaining the proper ratio since it was degraded within the first few days. The degradative enzymes excreted by microorganisms was dependent on the temperature of the pile. In many cases organisms able to produce specific extracellular enzymes at medium temperatures were able to grow at high temperatures, but either did not excrete the specific enzymes or the enzymes were inactivated by the high temperature.     

New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa

Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.     

Asparagine peptide lyases: a seventh catalytic type of proteolytic enzymes

The terms "proteolytic enzyme" and "peptidase" have been treated as synonymous, and all proteolytic enzymes have been considered to be hydrolases (EC 3.4). However, the recent discovery of proteins that cleave themselves at asparagine residues indicates that not all peptide bond cleavage occurs by hydrolysis. These self-cleaving proteins include the Tsh protein precursor of Escherichia coli, in which the large C-terminal propeptide acts as an autotransporter; certain viral coat proteins; and proteins containing inteins. Proteolysis is the action of an amidine lyase (EC 4.3.2). These proteolytic enzymes are also the first in which the nucleophile is an asparagine, defining the seventh proteolytic catalytic type and the first to be discovered since 2004. We have assembled ten families based on sequence similarity in which cleavage is thought to be catalyzed by an asparagine.     

Molecular analysis of homocysteic acid-induced neuronal stress

Hyperhomocysteinemia is a risk factor for vascular and neuronal lesions often observed with concomitant high levels of homocysteic acid. In contrast to homocysteine, homocysteic acid induces calcium influx into neurons, with characteristics of an excitotoxic glutamatergic agonist at elevated concentrations. On the molecular level this is correlated to fast modifications of proteins (phosphorylation and proteolysis). Within the homocysteic acid induced molecular signature we focused in more detail on phosphorylation of two proteins implicated as risk factors in schizophrenia and neurodegeneration: Dihydropyrimidinase related protein and 14-3-3 protein isoforms. Among the identified proteins there are known chaperones and oxidative metabolism enzymes, but a few are new in context of neuronal stress: Lasp-1, a vitamin D associated factor and an expressed sequence with features of a Rho GDP dissociation inhibitor. Moreover, we detect a specific proteolytic processing of heat shock protein 70 and proteindisulfide isomerase, which is abolished by vitamins (folic acid, vitamin B12, and vitamin B6), which also decrease elevated intracellular calcium levels induced by homocysteic acid.     

The protease problem in Neurospora. Variable stability of enzymes in aromatic amino acid metabolism.

A proteolytic activity isolated from Neurospora crassa is shown to be responsible for the variable stability observed in vitro for enzymes involved in aromatic amino acid metabolism. For example, the activity of kynurenine formamidase was insensitive to the action of this protease preparation over a 24-h period of incubation at 25 °C, whereas chorismate synthase, anthranilate synthase, kynureninase, and the five activities of the arom multienzyme system were inactivated during this time. Anthranilate synthase and two of the arom system activities (dehydroquinate synthase and shikimate kinase) were inactivated by the protease preparation within 2 h. Phenylmethanesulfonylfluoride and a specific proteolytic inhibitor from N. crassa prevented inactivation of these enzymes. Spontaneous loss of activity at 25 °C of purified samples of anthranilate synthase, dehydroquinate synthase and shikimate kinase was also prevented by the inhibitors. A method for purifying the inhibitor from N. crassa is described, and its use as a reagent in the analysis of proteolytic action is demonstrated.     

Expression of a novel human sialidase encoded by the NEU2 gene

Sialidases (E.C.3.2.1.18) belong to a group of glycohydrolytic enzymes, widely distributed in nature, which remove sialic acid residues from glycoproteins and glycolipids. All of the sialidase so far characterized at the molecular level share an Asp block, repeated three to five times in the primary structure, and an F/YRIP sequence motif which is part of the active site. Using a sequence homology-based approach, we previously identified a human gene, named NEU2, mapping to chromosome 2q37. NEU2 encoded protein is a polypeptide of 380 amino acids with two Asp block consensuses and the YRIP sequence in the amino terminal part of the primary structure. Here we demonstrate that NEU2 encodes a functional sialidase. NEU2 was expressed in COS7 cells, giving rise to a dramatic increase in the sialidase activity measured in cell extracts with the artificial substrate 4-MU-NANA. Using a rabbit polyclonal antiserum, on Western blots a protein band with a molecular weight of about 42 kDa was detectable, and its cytosolic localization was demonstrated with cell fractionation experiments. These results were confirmed using immunohistochemical techniques. NEU2 expression in E.coli cells allowed purification of the recombinant protein. As already observed in the enzyme expressed in COS7 cells, NEU2 pH optimum corresponds to 5.6 and the polypeptide showed a K(m)for 4-MU-NANA of 0.07 mM. In addition, based on the detectable similarities between the NEU2 amino acid sequence and bacterial sialidases, a prediction of the three-dimensional structure of the enzyme was carried out using a protein homology modeling approach.     

Gamma-irradiated chymotrypsin-like proteins. II. Connection between inactivation and structural changes.

The radiation yeilds of unfolding (Gconf) determined by the method of tryptophan fluorescence coincide with the radiation yields of proteolytic inactivation (Gin) for chymotrypsin-like (CT-like) enzymes on irradiation in air, both in solution and in the dry state with futher dissolution at pH7. It can be supposed that the unfolding is the main process determining the proteolytic gamma-inactivation of CT-like enzymes. It was also shown that the transition of chymotrypsin and trypsin gamma-irradiated at acid pH to neutral pH is an additional action, leading to unfolding of part of the molecules.     

Increased ATP-dependent proteolytic activity in lon-deficient Escherichia coli strains lacking the DnaK protein.

Extracts made from Escherichia coli null dnaK strains contained elevated levels of ATP-dependent proteolytic activity compared with levels in extracts made from dnaK+ strains. This ATP-dependent proteolytic activity was not due to Lon, Clp, or Alp-associated protease. Comparison of the levels of ATP-dependent proteolytic activity present in lon rpoH dnaK mutants and in lon rpoH dnaK+ mutants showed that the level of ATP-dependent proteolytic activity was elevated in the lon rpoH dnaK mutant strain. These findings suggest that DnaK negatively regulates a new ATP-dependent proteolytic activity, independently of sigma 32. Other results indicate that an ATP-dependent proteolytic activity was increased in a lon alp strain after heat shock. It is not yet known whether the same protease is associated with the increased ATP-dependent proteolytic activity in the dnaK mutants and in the heat-shocked lon alph strain.     

MHC molecules protect T cell epitopes against proteolytic destruction.

There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures that some of the Ag is salvaged? Using a cell-free system we demonstrate that an Ag fragment, once bound to a MHC class II molecule, is effectively protected against proteolytic destruction by cathepsin B and pronase E. The bound fragment, however, can be modified by aminopeptidase N. We suggest that MHC class II molecules play an important regulatory role in the physiologic processing of Ag.     

Correlation of the virulence of Bacillus anthracis with expression of signs, coded for by chromosomal genes

Clonal analysis of Bacillus anthracis strains showed heterogeneity of the majority of populations by the proteolytic, hemolytic, and pigment-adsorbing activities. Phenotypes isolated within each population were subdivided into 4 subpopulations. The appearance of sub-populations of virulent and vaccine strains (B. anthracis Sterne, Zenkowskii) was due to the ability of clones adsorbing Congo red and possessing high activities of proteolytic and hemolytic enzymes to dissociate with the formation of phenotypes characterized by different expression of proteolysis, hemolysis, pigment adsorption, pigment production, and sporulation signs. A characteristic feature of vaccine strains of B. anthracis CTIl, Wright, and Pasteur is a relative homogeneity of their populations consisting mainly of cells with low activities of proteolytic, hemolytic, and pigment-adsorbing enzymes. Study of the heterogeneity of di-, mono-, and plasmid-free derivatives of B. anthracis showed that the process of phenotype formation did not depend on the plasmids proper. Evaluation of the virulence of clones isolated from highly virulent B. anthracis strain 81/1 showed that the processes associated with loss of sporulation capacity notably decrease the virulence for laboratory animals.     

Effect of Bacillus mucilaginosus biomass on the exocrine function of the pancreas in piglets]

The effect of mucous bacilli on the excretory activity of pancreas in pigs has been investigated. The use of biomass at doses of 0.1, 0.2 and 0.3 g/kg stimulates to a different extent the pancreatic exocrine function in pigs: the pancreatic juice volume increases, on the average, by 17-20% and the secretion of amylolytic, proteolytic and lipolytic enzymes of the pancreatic juice becomes 1.6-2.2, 1.5-1.8 and 1.4-1.7 times higher, respectively. The increased functional activity of pancreas indicates the high reserve secretory potential of the pancreatic secretory apparatus. When brought into action, this apparatus favours more complete segregation and absorption of food nutrients.