Anticancer effects of an oncolytic parvovirus combined with non-conventional therapeutics on pancreatic carcinoma cell lines.

Standard therapies such as surgery and chemotherapy offer only minimal improvement in pancreatic cancer. However, the viruses killing cancer cells and substances like some antibiotics and phytoalexins with anticancer potential may represent a candidate non-conventional mean of cancer treatment in the future. In this study, the effect of infection with oncolytic H-1 parvovirus (H-1PV) combined with antibiotic norfloxacin (NFX) or phytoalexin resveratrol on the survival of cell lines Panc-1 and BxPC3 derived from human pancreatic carcinoma was tested. Whereas H-1PV with NFX exerted a synergistic effect, H-1PV with resveratrol resulted in an additive effect only. All the effects were partial, but they were more pronounced in Panc-1 compared to BxPC3 cells.

Expression of cellular proteins Bcl-X(L), XIAP and Bax involved in apoptosis in cells infected with herpes simplex virus 1 and effect of pavine alkaloid (-)-thalimonine on virus-induced suppression of apoptosis.

The expression of three cellular proteins involved in the modulation of apoptosis, namely antiapoptotic Bcl-X(L) and XIAP and proapoptotic Bax, was investigated in cells infected with Herpes simplex virus 1 (HSV-1). To assess whether the regulation of apoptosis in virus-infected cells depends on strain specificity the wild-type (wt) strain Victoria and the mutant R-100 resistant to acyclovir (ACV) were used. In addition, the expression of Bcl-X(L), XIAP and Bax was studied in cells infected with HSV-1 and treated with pavine alkaloid (-)-thalimonine. Our previous work has demonstrated that (-)-thalimonine irreversibly inhibits the replication of wt HSV-1 in cultured cells. Our data showed that (-)-thalimonine down-regulates the expression of viral proteins U(L)17, VP11-12, VP22, VP24 and gamma1 34.5, and affects negatively the posttranslational processing of glycoproteins D (gD) and G (gG). As both gamma1 34.5 and glycoprotein D possess antiapoptotic activity, we investigated whether the antiviral effect of the alkaloid could also be due to its ability to suppress the antiapoptotic activity of the virus. Our results demonstrated that: (i) the virus induced overexpression of antiapoptotic proteins Bcl-X(L) and XIAP; (ii) (-)-thalimonine reduced their overexpression, and (iii) this effect was stronger with the acyclovir resistant mutant R-100 than with the wt virus. Taken together, these data suggest that: (i) the virus abolishes apoptosis by means of virus-induced up-regulation of cell-specific prosurvival proteins Bcl-X(L) and XIAP, and (ii) (-)-thalimonine, apart from affecting essential viral targets, inhibits the infectious progeny production via restoration of apoptosis during viral replication.

Immunocompetent cells in the pulp of human deciduous teeth.

This immunohistological study sought to determine how the distribution and density of various immunocompetent cells change in the pulp of human deciduous teeth during the process of physiological root resorption. Forty-three extracted deciduous teeth at various stages of resorption were subjected to immunoperoxidase staining with the use of antibodies directed to HLA-DR, CD68, factor XIIIa and lymphocyte subsets. In intact deciduous teeth (group 0), all types of cells examined, except for CD20+ B lymphocytes, were detected. In teeth in which resorption was less than 1/3 of the root length (group 1), all types of cells showed a statistically significant increase compared with group 0 (P<0.05; Mann-Whitney's U-test). HLA-DR+, CD68+, and factor XIIIa+ cells with a dendritic profile kept their distribution in the periphery of the pulp, and oval and round, newly recruited macrophages accumulated in the central portion of the pulp and near the resorption sites. In teeth where resorption was 1/2 to 2/3 (group 2), all the cell types increased further. Aggregations of HLA-DR+, CD68+, and factor XIIIa+ cells were frequently seen in the central portion of the pulp, and T and B lymphocytes occasionally formed some clusters. Comparisons with group 1 revealed that the density of these cells, except for CD20+ cells, showed significant increases (P<0.05; Mann-Whitney's U-test). These results provided evidence showing that immunocompetent cells of deciduous tooth pulp increase with the progress of physiological root resorption, suggesting that immunocompetency of deciduous teeth is altered by this process.

Interaction of the peptide antibiotic alamethicin with bilayer- and non-bilayer-forming lipids: influence of increasing alamethicin concentration on the lipids supramolecular structures.

Incorporation of the helical antimicrobial peptide alamethicin from aqueous phase into hydrated phases of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) was investigated within a range of peptide concentrations and temperatures by time-resolved synchrotron X-ray diffraction. It was found that alamethicin influences the organizations of the non-bilayer-forming (DOPE) and the bilayer-forming (DOPC) lipids in different ways. In DOPC, only the bilayer thickness was affected, while in DOPE new phases were induced. At low peptide concentrations (<1.10(-4) M), an inverted hexagonal (H(II)) phase was observed as with DOPE dispersions in pure buffer solution. A coexistence of two cubic structures was found at the critical peptide concentration for induction of new lipid/peptide phases. The first one Q224 (space group Pn3m) was identified within the entire temperature region studied (from 1 to 45 degrees C) and was found in coexistence with H(II)-phase domains. The second lipid/peptide cubic structure was present only at temperatures below 16 degrees C and its X-ray reflections were better fitted by a Q212 (P4(3)32) space group, rather than by the expected Q229 (Im3m) space group. At alamethicin concentrations of 1 mM and higher, a nonlamellar phase transition from a Q224 cubic phase into an H(II) phase was observed. Within the investigated range of peptide concentrations, lamellar structures of two different bilayer periods were established with the bilayer-forming lipid DOPC. They correspond to lipid domains of associated and nonassociated helical peptide. The obtained X-ray results suggest that the amphiphilic alamethicin molecules adsorb from the aqueous phase at the lipid head group/water interface of the DOPE and DOPC membranes. At sufficiently high (>1.10(-4) M) solution concentrations, the peptide is probably accommodated in the head group region of the lipids thus inducing structural features of mixed lipid/peptide phases.

Asymmetrical ion-channel model inferred from two-dimensional crystallization of a peptide antibiotic.

The structural organization of ion channels formed in lipid membranes by amphiphilic alpha-helical peptides is deduced by applying direct structural methods to different lipid/alamethicin systems. Alamethicin represents a hydrophobic alpha-helical peptide antibiotic forming voltage-gated ion channels in lipid membranes. Here the first direct evidence for the existence of large-scale two-dimensional crystalline domains of alamethicin helices, oriented parallel to the air/water interface, is presented using synchrotron x-ray diffraction, fluorescence microscopy, and surface pressure/area isotherms. Proofs are obtained that the antibiotic peptide injected into the aqueous phase under phospholipid monolayers penetrates these monolayers, phase separates, and forms domains within the lipid environment, keeping the same, parallel orientation of the alpha-helices with respect to the phospholipid/water interface. A new asymmetrical, "lipid-covered ring" model of the voltage-gated ion channel of alamethicin is inferred from the structural results presented, and the mechanism of ion-channel formation is discussed.

Morphine treatment affects the regulation of high mobility group I-type chromosomal phosphoproteins in C6 glioma cells.

High mobility group (HMG) I-type chromosomal phosphoproteins HMG I/Y and HMG I-C were investigated following morphine treatment of C6BU-1 glioma. Cells were labelled with [32P]-orthophosphoric acid. Electrophoretic profiles and autoradiograms of the control cells revealed the presence of HMG I and HMG I-C proteins. HMG Y was not detected. Northern blot analysis showed a single HMG I/Y transcript. Treatment with morphine lowered the [32P]-incorporation in HMG I and HMGI-C proteins and the level of the HMG I/Y transcript. However, it did not change the protein ratios on the Coomassie stained gels. These results suggest that morphine may trigger independent reaction pathways affecting either transcription regulation and/or postsynthetic phosphorylation of the preexisting HMG I-type proteins. In addition, opposing changes in the postsynthetic phosphorylation of HMG 14 and histones H1AB were also noticed.

Increased Efficacy of Zinc Complexes With Picolinic and Aspartic Acids Against Herpes Simplex Virus (HSV) Infection When Combined With the Pavine Alkaloid (-)-Thalimonine.

Complexes of zinc with picolinic and aspartic acids inhibit key steps of HSV-1 replication affecting different virus-specific targets. As was recently demonstrated by us, the pavine alkaloid (-)-thalimonine irreversibly inhibits HSV-1 infection in cultured cells. The aim of the present study was the evaluation of the combined effect of zinc complexes and (-)-thalimonine on uninfected and HSV-1 infected cells. The data obtained have shown that zinc complexes and the alkaloid exert decreased cytotoxicity (antagonistic effect) and significantly increased anti-HSV-1 activity (synergistic effect) when applied in dual chess-board combinations as compared to the individual effects of compounds tested. These combinations are also effective against the infection caused by a resistant to acyclovir (ACV) HSV-1 mutant and the effect has been recognised as synergistic.

Catecholamine concentration in rat liver after high level transection of the spinal cord.

The high level transection of the spinal cord (C-7) provokes a sustained increase of rat liver catecholamines: biphasic increase in norepinephrine level 1 hour and 24 hour after the operation and 7-fold increase of dopamine content 4 hour after the chordotomy. In contrast to cervical transection, sham operation causes only an initial catecholamine increase, the maximum being at the first hour after the surgery. Our experimental data indicate a possible participation of cervical spinal pathways in regulation of liver catecholamine content. It is also shown that bilateral adrenalectomy augments liver norepinephrine concentration in spinal rats as compared to the non-adrenalectomized ones. The results presented here indicate that cervical chordotomy affects the functioning of the sympatho-adrenal system, thus provoking specific changes in liver catecholamine content. The potential effect of such changes on a liver metabolic system (tyrosine aminotransferase induction) is discussed.

Drug-Cyclodextrin Association Constants Determined by Surface Tension and Surface Pressure Measurements.

The complexation reaction between the amphiphilic peptide antibiotic polymyxin B and naturally occurring cyclodextrins, used as potential drug carriers, was quantitatively evaluated from surface tension measurements at various drug concentrations. The association constant, Ka, of polymyxin B:beta-cyclodextrin inclusion complex formation of 1:1 stoichiometry was determined from the change in the drug interfacial activity upon the addition of beta-cyclodextrin at the excess solution concentration (10(-3) M). The obtained Ka value is discussed in terms of molecular matching of the host cyclodextrin cavity and the guest drug molecule. Copyright 1999 Academic Press.

Drug-Cyclodextrin Association Constants Determined by Surface Tension and Surface Pressure Measurements.

The compression of water-insoluble drug monolayers spread on the aqueous subphase containing cyclodextrins (CD) led to a shift of surface pressure (pi)-area (A) isotherms toward smaller molecular areas with respect to the pi-A isotherms on the pure water subphase. The displacement of the compression isotherm obtained for the retinol spread on the beta-CD containing aqueous subphase was used to quantify the depletion process and to determine the drug-CD association constants. The proposed method appeared to be sensitive enough to account for extremely low amounts of sequestered drug molecules. The obtained values of the association constants Ka ranged from about 1.4.10(-2) to 36 m2/mol. The magnitudes of these constants are discussed in terms of drug bioavailability and of the stoichiometry of retinol-beta-cyclodextrin inclusion complex which was shown to have a 1:1 correspondence. Copyright 1999 Academic Press.

Morphine-induced alterations on DNA-binding proteins in developing rat brain.

Endogenous opioids and opiate drugs inhibit nervous system maturation through both direct and indirect mechanisms. Recently much attention has been directed toward changes in the postreceptor events and it has been speculated that the regulation of gene expression may be involved in the development of drug tolerance and dependence. We investigated the changes in the levels of in vitro RNA synthesis in developing rat brain after continuous block of opioid receptors. Repeated naloxone treatment induced increased levels (27-48%) of RNA synthesis during the early postnatal period. Using mobility gel shift assay the presence of octamer binding proteins (Oct-1) and the replication differentiation transcription factor CTF/NF1 in the developing rat brain were studied both after single or repeated morphine and naloxone treatment. Decreased Oct-1 binding activity in brain protein extracts 1 h after morphine application was registered, while opioid antagonist naloxone exerted an opposite effect on this octamer protein following single drug treatment. Repeated administration of morphine or naloxone decreased markedly the DNA-binding affinity of Oct-1. The binding activity of CTF/NF1 changes differently showed higher levels assessed 30-120 min after morphine administration. The opposite trend of the changes in opiate drug and opioid antagonist animals suggests opioid receptor-mediated regulation of Oct-1 and CTF/NF1 transcription factors.

HMG-2 protein in developing rat brain cells.

1. The distribution of HMG-2 protein was followed in unfractionated rat brain cells at different stages of development. Its amount gradually decreased and reached the lowest level in the terminally differentiated and non-proliferating cells. 2. In isolated oligodendrocyte nuclei the changes in the content of HMG-2 followed the same pattern of distribution which corresponded to their stage of development and proliferative activity, while in the terminally differentiated and non-proliferating cortical neurons a substantial amount of HMG-2 protein was present up to the twenty-eighth postnatal day. 3. In the presence of anti-HMG-2 antibodies the DNA synthetic activity of oligodendrocyte nuclei in vitro was significantly decreased. The treatment with antibodies affected mainly the DNA replicative activity of the nuclei, while their DNA repair activity remained unchanged.

[Changes in the hemoglobin A2 level of patients with ischemic heart disease]. / Promeni v sudurzhanieto na khemoglobin A2 pri bolni ot iskhemichna bolest na surtseto.

40 patients with ischemic heart disease were studied. In 60% of them a higher content of HbA2 was found. These data for higher frequency of HbA2 among the patients with ischemic heart disease do not correspond with the average incidence of the genetically determined anomaly A2-beta-thalassemia among the Bulgarian population. The negative data for increased methemoglobin, the shift of the oxygen dissociation curve to the right toward increased oxygen release from the hemoglobin molecule, the normalization of HbA2 after several days, the lack of anomalous fraction in the analyses lead to the conclusion that HbA2 plays a compensatory role in patients with ischemic heart disease and its dynamic changes could be used as a diagnostic test for ischemia.

[Effect of Furacillin antiseptic tablets on dental plaque]. / Vliianie na antiseptichnite tabletki Furacillin vurkhu zubnata plaka.

According to the modern concepts the ideal oral hygiene is decisive for the prophylaxis of caries and periodontal diseasese. However, its realization is not easy since dental plaque rapidly and ceaslessly cumulates upon all dental surfaces and is difficult to remove by the ordinary means for oral hygiene (tooth brush and tooth paste). Lately hope is centered on the antiseptics, being the base of various preparations as solutions for mouth wash for inhibition of dental plaque. The action of Furacillin tablets on the dental plaque was studied in 10 volunteers with sanated dentition, with on diseases of gingiva and periodontium. The probands sucked one tablet every three hours (6 tablets daily) in the course of 5 days with interrupted oral-hygienic cares. Dentalplaque was controlled by the oral higienic index of the whole dentition daily, after staining with 1 per cent alkaline fuxin. Probands, left on an ahygienic regimen for 5 days served as a control. The results from the study show an essential inhibitory effect of the tablets on trial upon the dental plaque and the possibilities to use the tablets for some patient groups with restrictions as regards the maintenance of oral hygiene via the ordinary means.

Distribution and metabolic behaviour of "tightly bound" acid-soluble non-histone chromosomal proteins in developing rat brain cells.

"Tightly bound" acid-soluble non-histone chromosomal proteins of rat brain were studied, and limited but detectable tissue specificity of their pattern was demonstrated. This class of proteins showed specific distribution in rat brain cells at different stages of development. The most prominent differences were observed between non-differentiated and terminally differentiated cells. In non-differentiated rat brain cells the acid-soluble non-histone protein fraction contained both metabolically labile and metabolically stable proteins, while in fully developed cells the main portion of acid-soluble proteins showed metabolic stability.