A novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of MDCK cells in culture

Summary.  The sodium salts of 2-difluoromethyl-phenyl-α-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-α-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase. In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68. Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68. Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus. In non-infected MDCK cells no inhibition of cellular sialidase was observed. Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells. Even after 8 passages in its presence, no resistant strains were detected. Because of its high Ki (M) compared to the low Ki (M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged. Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain. Accepted February 3, 1997; Received November 12, 1996     

Kinetics of hematopoietic clones in reconstituted mice

In mice reconstituted with genetically marked bone marrow, hematopoiesis is shown to be effected, over a period of 14 months after transplantation, by numerous locally existing and short-lived cell clones that succeed one another as the potential of the clonogenic precursor cell is depleted. In each reconstituted mouse, several dozens of hematopoietic clones are functional in the bone marrow. Immortal self-maintaining stem cells with unlimited proliferative potential are not detected in this system. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 2, pp. 180–183, February, 1997     

Activity lysosomal enzymes in rat skin fibroblasts after treatment with progesterone and new gestagen ABMP

The effects of a new synthetic gestagen 17α-acetoxy-3β-butanoyloxy-6-methyl-pregna4,6-dien-20-on (ABMP) and reference drug progesterone on rat skin fibroblasts were evaluated by variations in lysosomal enzyme activity (cathepsin D and β-glucosidase). Our results suggest that ABMP exhibits lysosomotropic properties, which depended on its concentration and time of treatment. The direct effect of progesterone on lysosomal enzyme activity in skin fibroblasts was compared to the influence of systemic treatment with gestagens on skin lysosomes. The data indicate that local application of gestagen preparations holds much promise for the therapy of skin diseases accompanied by increased proliferation (e.g. psoriasis). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 1, pp. 50–52, January, 2008     

Chromosome and DNA analyses of rat 13762NF mammary adenocarcinoma cell lines and clones of different metastatic potentials

Chromosome morphologies revealed by Giemsa-banded karyotypes and chromosome numbers were compared between parental tumor-, lymph node- and lung metastasis-derived rat 13762NF mammary adenocarcinoma cell lines and clones having different spontaneous metastatic potentials. Although chromosome numbers in the cell lines and clones generally correlated with DNA content by flow cytometry, ploidy did not correlate with spontaneous metastatic potentials. Chromosone number and DNA content drifted during prolonged in vitro growth in each of the cell lines and clones. Common chromosome rearrangements were found, confirming a common origin for all the cell lines and clones, and the frequency and appearance of the individual marker chromosomes fluctuated during in vitro growth. Karyotypic analyses revealed that the markers coinciding with phenotypic drift in spontaneous metastatic potential and other biological properties of parental tumor-derived clones MTC and MTF7 and lung metastasis-derived clone MTLn3 involved chromosomes 3, 4, 5, 6, and 8. Clone MTC exhibited a shift in several markers and an increase in metastatic potential at passage T20, while clone MTF7 displayed a lesser spontaneous metastatic potential at high passage (T34) concomitant with an increase in the frequency of certain marker chromosomes. Lung metastasis-derived clone MTLn3 also exhibited a shift in some marker chromosomes, colonization preference and metastatic potential to lung and lymph nodes at high tissue culture passages. The changes in marker chromosomes during in vitro passage of clones MTC and MTLn3 suggested the presence of at least two cell subpopulations which could be responsible for the observed shift in spontaneous metastatic properties. Karyotypic features of the 13762NF cell lines and clones indicate that subtle cytogenetic changes, in contrast to gross chromosomal abnormalities, may be more important in determining metastatic phenotype.     

Characterization of the sialidase molecular defects in sialidosis patients suggests the structural organization of the lysosomal multienzyme complex

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation and hepatosplenomegaly (sialidosis type II). We have analyzed the genomic DNA from nine sialidosis patients of multiple ethnic origin in order to find mutations responsible for the enzyme deficiency. The activity of the identified variants was studied by transgenic expression. One patient had a frameshift mutation (G623delG deletion), which introduced a stop codon, truncating 113 amino acids. All others had missense mutations: G679G-->A (Gly227Arg), C893C-->T (Ala298Val), G203G-->T (Gly68Val), A544A-->G (Ser182Gly) C808C-->T (Leu270Phe) and G982G-->A (Gly328Ser). We have modeled the three-dimensional structure of sialidase based on the atomic coordinates of the homologous bacterial sialidases, located the positions of mutations and estimated their potential effect. This analysis showed that five mutations are clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We hypothesize that this region may be involved in the interface of sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in their high-molecular-weight complex required for the expression of sialidase activity in the lysosome.     

Activity of lysosomal enzymes in the bile and serum of mice with intrahepatic cholestasis

Lysosomal enzyme activity in the bile and blood serum was compared in mice with experimental intrahepatic cholestasis induced by α-naphthyl isothiocyanate and Triton WR 1339. Triton WR 1339 increases the synthesis of cholesterol (fatty acid precursor) in liver cells. The development of intrahepatic cholestasis was confirmed by the increase in activities of alkaline phosphatase and γ-glutamyltransferase in blood serum. Administration of Triton WR 1339 in a dose of 100 mg/100 g was followed by a 10-fold increase in β-galactosidase activity (hepatocyte lysosomal enzyme) in the bile, but not in the serum of mice. β-Galactosidase activity significantly increased in the bile, but decreased in the serum of mice after treatment with α-naphthyl isothiocyanate in a dose of 200 mg/kg. Our results indicate that intrahepatic cholestasis is manifested in increased secretion of lysosomal glycosidases into the bile. Bile components can aggravate damage to liver cells by affecting the processes of hepatocyte apoptosis and necrosis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 496–499, May, 2008     

Functional activity of peritoneal macrophages from sensitive and resistant mouse strains during intravaginal infection with herpes simplex virus type 2 and mucosal vaccination

In the early period after intravaginal infection with herpes simplex virus type 2 (2 h), macrophages from sensitive DBA/2 mice were characterized by higher capacity to engulf the antigen, decreased function of the lysosomal apparatus, lower activity of cathepsin D, and reduced oxygen metabolism compared to cells from resistant BALB/c mice. Mucosal vaccination with herpes vaccine and hyaluronic acid promoted the increase in functional activity of macrophages and improved survival of sensitive mice (by 60%). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 2, pp. 196–200, February, 2008     

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation‐sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease. Hum Mutat 23:32–39, 2004. © 2003 Wiley‐Liss, Inc.     

Surface adhesins of lactobacilli loosely connected to the cell wall and eliminated into the environment during culturing in liquid nutrient media

Six lactobacillus species and 4 clones of one of them were studied in order to clear out the ratio between the adhesion capacities of concanavalin A-reactive glycoprotein adhesins on the surface of the bacterial cell and glycoprotein adhesins released into the broth during culturing in liquid nutrient media. The adhesive activity of cultures is largely determined by the strain rather than species appurtenance. Elimination of glycoprotein adhesins from the bacterial cell and their antagonistic activity towards Candida albicans were demonstrated in specific interactions of glycoprotein adhesins with immune serum and concanavalin A. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 557–561, November, 2006     

Intralysosomal accumulation of gadolinium and lysosomal damage during selective depression of liver macrophages in vivo

Kinetics of gadolinium accumulation was studied by inductively coupled plasma-emission spectroscopy after intravenous injection of this agent (7.5 mg/kg) to CBA mice. Gadolinium exhibits lysosomotropic properties (long-term selective accumulation in lysosomes in vivo). Gadolinium uptake by hepatic cells attained maximum 1 h after its intravenous injection and remained at this level during the next day. Accumulation of gadolinium in hepatocytic lysosomes disturbed their osmotic properties (as was seen from the increase in free acid phosphatase activity, which persisted for 19 days). Serum activities of β-D-galactosidase and β-D-glucuronidase also increased (24–72 h and day 19). Selective depression of liver macrophages (24–48 h) was accompanied by a decrease in serum chitotriosidase activity. We conclude that accumulation of gadolinium in lysosomes of liver macrophages leads to their damage and elimination of a certain population of macrophages (primarily large cells). Changes in activity of serum lysosomal enzymes also reflect repopulation of liver macrophages. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 10, pp. 369–372, October, 2006     

Clinical Significance of Levels of Molecular Biological Markers in Zones of Invasive Front-Line of Colorectal Cancer

The role of expression of markers (β-catenin, matrix metalloproteinase 9, collagen IV, and laminin) in rimary colorectal adenocarcinomas and their metastases in the liver and lymph nodes of patients with colorectal cancer was studied. High level of matrix metalloproteinase 9 expression in zones of invasive growth of colorectal cancer was associated with high accumulation of β-catenin in cancer cell nuclei in the peripheral zones of 30% studied tumors. The presence of nuclear β-catenin and high content of matrix metalloproteinase 9 in the tumor were associated with abnormal accumulation of laminin in the cytoplasm and with the absence of basal membranes containing collagen IV. These changes were characteristic of colorectal cancer with high invasive metastatic potential. It was found that β-catenin, matrix metalloproteinase 9, laminin, and collagen IV were important markers for prediction of the clinical course of colorectal cancer. The expression of proteins associated with risk of metastases in the liver was coordinated and most pronounced in zone of invasive front-line of tumors. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 11, pp. 552–555, November, 2008     

Identification,detection and transmission of a new vitivirus from <Emphasis Type="Italic">Mentha</Emphasis>

Summary Mentha × gracilis ‘Variegata’ is an ornamental clone with a phenotype caused by virus infection. Several clones were ordered from mail-order nurseries in an attempt to identify a virus consistently associated with symptoms. One of these clones did not exhibit typical ‘Variegata’ symptoms, and steps were taken to identify any agents causing the ‘off-type’ symptoms. One of the viruses identified in the atypical ‘Variegata’ clone is a previously unknown virus, a member of the family Flexiviridae. Sequence and phylogenetic analysis indicate that the virus, designated as mint virus-2, is related to members of the species Grapevine virus A, Grapevine virus B and Heracleum latent virus, placing it in the genus Vitivirus. A detection protocol for the virus has been developed, and the mint aphid (Ovatus crataegarius) was able to transmit the virus in the presence of a helper virus but not from single infected plants.     

Antitumor and antimetastatic effects of betulonic acid amides in mice with transplantable Lewis carcinoma

We studied the effects of four synthetic amides of betulonic acid containing amino acid fragments (d,l-α-alanine, β-alanine, and their methyl esters) on the rate of growth and metastatic dissemination of transplantable Lewis lung carcinoma in C57Bl/6 mice. The test compounds were administered intragastrically in a single dose of 500 mg/kg. 3-[-oxo-20(29)-lupen-28-oilamino]-propionic acid suppressed primary tumor growth (by 26%) and decreased the number of lung metastases (by more than 2 times). Antitumor and antimetastatic activity of triterpenoids decreased after methylation of the amino acid fragment in betulonic acid. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 78–81, July, 2006     

Expression of ganglioside GM3 and H-2 antigens in clones with different metastatic and growth potentials isolated from Lewis lung carcinoma (3LL) cell line

In view of the evidence that cell expression of gangliosides in several tumors is positively involved in the metastatic phenotype, Lewis lung carcinoma (3LL) cell line, expressing GM3 as the major ganglioside, was analysed for the cell surface expression of GM3. An indirect immunofluorescence assay, using a M2590 monoclonal antibody recognizing GM3, was used for this purpose. Since the parental 3LL cells consist of heterogenous subpopulations differing in the degrees of GM3 expression, we have developed clones of this cell line with different degrees of metastatic potentials by using anin vitro non-selective procedure in order to investigate whether the expression of GM3 is associated with metastatic potential. The degree of cell surface expression of GM3 among the clones correlated well with their total cellular content of this ganglioside. However, we were unable to confirm the report of increased level of GM3 in high metastatic 3LL clones, nor did a decreased level correlate with weak metastatic ability. In our recent work, an inhibitor of glucosylceramide synthase,d-threo-l-phenyl-2-decanoylamino-3-morpholino-l-propanol (DPDMP), was found to decrease the levels of all cellular glucosphingolipids and cause the accumulation of the precursors of glucosylceramide. The present study does not, however, rule out the possible involvement of this lipid family in metastatic dissemination, since treatment of 3LL cells with D-PDMP resulted in significant inhibition of their experimental metastatic potential. Clones expressing very low GM3 grew slowly in culture dishes, suggesting that GM3 may have a regulatory role in cell proliferation. The low metastatic clones expressed high levels of H-2K^b antigen, while the expression of the same antigen on the high metastatic clones was relatively low, confirming the previous observation of this tumor system. Moreover, a clone showing the lowest tumorigenic potency revealed both a high cell surface expression of H-2K^b and a high H-2K^b/H-2D^b ratio.     

High-metastatic clones selectedin vitro from a recent spontaneous BALB/c mammary adenocarcinoma cell line

The metastatic TS/A line has been recently derived from a spontaneous BALB/c mammary tumor. When TS/A cells were cultured in 0·33 per cent agar, two morphologically distinct types of colonies were observed from which two sets of clones were obtained. E clones were derived from small, transparent colonies, whereas F clones were from large, thick, actively growing colonies.All the clones were tumorigenic in syngeneic BALB/c females. However, E clones showed higher ability than F clones to metastasize spontaneously to the lung. Comparison between E and F clones shows that the high level of spontaneous metastasization to the lung is associated with epithelial-likein vitro growth pattern, spontaneous dome formation and growth pattern in 0·33 per cent agar cultures. The ability to give rise to lung colonies following intravenous inoculation is not a predictive parameter for the spontaneous metastatic potential.     

Cytotoxic activity of lymphocytes isolated from mouse liver involved into tumor process

We studied cytotoxic activity and immunophenotype of mononuclear cells isolated from the liver of mice after implantation of ovarian cancer cells into the liver parenchyma. The isolated cells exhibited higher natural killer cell activity and possessed higher cytotoxic potential against autologous tumor cells compared to spleen lymphocytes. The ratio of CD3⁺ lymphocytes and natural killer cells was high in the liver with tumor metastases. Lymphoid cells were practically absent in the liver of intact animals. Natural killer cells and T cells from liver tumor tissue play an important role in antitumor immunity and can be used for local and regional adjuvant immunotherapy of liver metastases. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 1, pp. 76–79, January, 2006     

Growth in an organ microenvironment as a selective process in metastasis

The purpose of these studies was to determine whether the ability of tumor cells to grow in an organ parenchyma selects for cells with enhanced potential to metastasize to this organ. B16-F1 melanoma cells (with low metastatic potential) were culturedin vitro on fragments of mouse lung or kidney suspended in medium supplemented with only 1 per cent fetal bovine serum. Seven days later, the organs were enzymatically dissociated and tumor cells recovered and expanded in monolayer culture. Tumor cells were then harvested and seeded onto fresh organ fragments. This sequence was repeated six times. The cells designated as B16 Lung-6 and B16 Kidney-6 were then injected intravenously into CS7BL/6 mice and the number of experimental metastases counted after 21 days. B16 Lung-6 and, to a lesser degree, B16 Kidney-6 cells produced significantly more lung tumor colonies than B16-F1 cells. Some factor(s) in the organ environment did appear, therefore, to select out cells with greater metastatic potential from the low metastatic B16-F1. Forty-five clones of the B16-F1 melanoma, isolated by limiting dilution, were screened for their ability to grow on expiants of mouse lung in a low-serum medium. Four clones exhibiting least growth and four clones exhibiting most growth, as assessed by examining histological sections of the lung explants, were injected intravenously into syngeneic mice. The eight clonal populations differed in experimental metastatic potential, but this behavior did not correlate with the ability of cells to grow in lung expiantsin vitro.The data suggest that selecting for cells with enhanced ability to grow in an organ is a necessary but not sufficient condition for isolating cells with high metastatic potential.     

Study of Magnetic Field of the Brain in Parkinson’s Disease

The magnetic field of the brain in parkinsonism was studied. Magnetic encephalography data were analyzed by a comprehensive spectral method. Sources of magnetic activity of the brain were simulated by punctate current dipoles. Based on the results of classification, we detected the pattern of high magnetic activity; this opens new vistas for the diagnosis and treatment of Parkinson’s disease. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 3, pp. 351–354, March, 2009     

Neuroprotective effect of ultra-low doses of antibodies against S100 protein in neuroblastoma culture during oxygen and glucose deprivation

Antibodies against S100 protein applied in high and ultra-high dilutions possess neuroprotective activity and maintain survival of neuroblastoma C-1300 cells under conditions of oxygen and glucose deprivation. The examined antibody preparations stimulated differentiation in neuroblastoma culture thereby demonstrating pronounced neurotrophic activity. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 260–263, September, 2007