The evaluation of cytotoxic activity of planar pentadentate ligand 2',2"'-(2,6-pyridindiyldiethylidyne) dioxamohydrazide dihydrate (H2l x 2H2O) and its metal coordination complexes; pitfalls in the use of the MTT-assay.

In this study we have investigated, for the first time to our knowledge, the antineoplastic activity of a planar pentadentate ligand (H2L.2H2O = 2', 2'''-(2,6-pyridindiyldiethylidyne)dioxamohydrazide dihydrate) and some of its metal coordination complexes [Cu(L)(H2O)].H2O, [Cu(HL)(H2O)]Cl04, [Co(L)(H20)2] 6H20, [Co(H2L)(H2O)(MeOH)](ClO4)2 and [Fe(L)(H2O)2]ClO4-3H2O, as well as of inorganic salts CuCl2 2H20, CoCl2 6H2O and FeCl3.6H2O of corresponding metal ions. The antiproliferative activity of these compounds was examined in a human melanoma cell line FemX with exposure time of 48 hours by performing two cytotoxicity tests: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sulforhodamine B (SRB) assay. Among these substances, the ligand H2L.2H2O expressed the greatest antineoplastic activity IC50 = 45.40 microM, while the IC50 of others could not be determined by SRB assay in the examined range of concentrations due to their low activity. FeCl3.6H2O showed stimulatory activity. We have found remarkable discrepancies between the results obtained by MTT assay and SRB assay that influence IC50 value as well as other measures of cytotoxicity, which led to the conlusion of uncertainty of using the MTT assay in evaluation of antineoplastic activity of organometalic complexes and inorganic metal salts.

Study on protolytic equilibria of lorazepam and oxazepam by UV and NMR spectroscopy.

Protolytic equilibria in homogeneous and heterogeneous systems of lorazepam and oxazepam, which are sparingly soluble ampholytes from the class of 1,4-benzodiazepines, were studied at 25 degrees C and ionic strength of 0.1 M. Acidity constants and equilibrium constants in a heterogeneous system were determined. On the basis of the analysis of the corresponding 13C- and 1H-NMR spectra, deprotonation site in the molecules of the investigated compounds was predicted. Finally, the correlation between chemical shifts in the 1H-NMR spectra and the acidity of the amide proton of 1,4-benzodiazepines was established.

Protection of broiler breeders by an inactivated combined water-in-oil-in-water viral vaccine.

A four-component vaccine, prepared by combining the single vaccines, contains subunits of Newcastle disease and infectious bronchitis viruses, as well as whole inactivated infectious bursal disease and egg drop syndrome viruses. The vaccine is prepared in the form of a low-viscosity water-in-oil-in-water emulsion with low mineral oil content. Heavy breeders were vaccinated at the age of 20 weeks by intramuscular administration of 0.5 ml vaccine/bird in an experiment carried out under field conditions, involving 5000 female and 450 male parents. The birds had previously been vaccinated with live vaccines according to an obligatory field vaccination programme. Vaccination with the WOWE vaccine near the point of lay elicited serological responses protecting both the parents and their progeny. Each of the antigens administered in the four-component vaccine was as effective as the respective single component vaccine. The mortality, recorded during the 31-week experimental period, was 6.2%. Mortality and morbidity were not triggered by viruses against which vaccination was carried out. Egg production was not affected by the vaccination and was 170.2 eggs per hen during the 28-week production period.

Tween 80-solubilized Newcastle disease virus prepared as a water-in-oil-in-water vaccine.

The Newcastle disease virus (NDV) was solubilized with 10% w/v Tween 80 and inactivated with 0.05% v/v formalin. The average molecular mass of the released antigenic subunits was 307 kD. The tested vaccine was prepared in the form of a water-in-oil-in-water emulsion (WOWE) vaccine. The oil-to-aqueous ratio was 1:2. The solubilized NDV was administered alone or built in a tetravalent WOWE vaccine. A dose of the monovalent vaccine containing an equivalent of 44.7 microliters of detergent-treated NDV-allantoic fluid (NDV-AF) was sufficient for the complete protection of the commercially available chickens vaccinated at the age of 5 wk and challenged 7 wk later. The anti-NDV-free chickens, vaccinated at 4 wk of age and challenged 2 wk postvaccination, were 100% and 73% protected by a vaccinal dose containing 178.6 and 89.3 microliters of detergent-treated NDV-AF, respectively. Commercially available light pullets, primary vaccinated with live lentogenic NDV vaccine, generated a protective level of NDV antibodies after revaccination with WOWE vaccine containing 89.3 microliters of detergent treated NDV-AF. Laying hens were revaccinated under field conditions at the beginning of the laying cycle by the tetravalent vaccine. A vaccinal dose/bird containing 11.2 microliters of detergent-treated NDV-AF elicited a long-lasting high level of NDV neutralizing antibodies.

Labelling of breast carcinoma, thyroid carcinoma and melanoma with manno- and galacto-specific lectins from marine invertebrates.

Three marine invertebrate FITC-labelled lectins, CNL, GCL, and GSL, isolated respectively, from the sponges Chondrilla nucula, Geodia cydonium, and the hexacoral Gerardia savaglia, were used as potential diagnostic tools for different breast tumors. The lectins vary in their carbohydrate binding properties: GSL is D-mannose specific, GCL and CNL D-galactose specific. GSL labels most investigated types of malignant tissues distinctively, while the results with CNL and GCL are less consistent. The well known D-mannose specific lectin, concanavalin A, also binds to tumor tissues, but with much lower intensity than GSL.

Application of the immune complex for immune protection against viral disease.

An immune complex (IC), composed of antigenic subunits of the Newcastle disease virus (NDV) and specific polyclonal allogeneic antibodies, was used to protect chickens against NDV. Antibodies in the IC were chicken immunoglobulin G. The antibody:antigen ratio in IC was 2.03. The IC was prepared at equivalence by direct mixing of NDV-infected allantoic fluid, treated with Triton X-100, and chicken anti-NDV serum. In order to bind NDV antigenic subunits to specific antibodies, previous isolation and purification of antigen is not required. Chickens were immunized with 1 mg IC, containing 0.3788 mg of viral antigens. The IC, prepared in the form of an oil-emulsion, was administered intramuscularly. The IC generated high levels of anti-NDV antibodies and successfully protected chickens against live virus challenge. Therefore, the IC could be recommended as a safe and environmentally convenient vaccine.

Suppression of the modulatory effects of the antileukemic and anti-human immunodeficiency virus compound avarol on gene expression by tryptophan.

The amino acid L-tryptophan is known to be a modulator of many processes of cell metabolism. In this contribution we show that L-tryptophan interferes with some biological effects of the antileukemic and anti-human immunodeficiency virus agent avarol, possibly by different mechanisms. Avarol has been shown to be able to modulate posttranscriptional events of mRNA synthesis, resulting in an increase of the base-sequence complexities of the nonabundant and rare mRNA classes. Here it is demonstrated that this change in mRNA abundancy distribution is accompanied by an increase in the level of some specific, low abundant mRNAs (ras and c-myc). Addition of L-tryptophan was found to abolish avarol-caused gene relaxation in L1210 mouse leukemia cells. In addition, L-tryptophan suppressed the induction of gamma-interferon mRNA production in human peripheral blood lymphocytes. At the level of DNA, L-tryptophan inhibited the production of strand breaks by cytotoxic avarol concentrations in Friend erythroleukemia cells in vitro. Moreover, it competed with avarol for binding to the nuclear envelope binding site; this effect was not shown by other amino acids.

Avarol-induced DNA strand breakage in vitro and in Friend erythroleukemia cells.

The hydroquinone-containing cytostatic compound avarol inhibits predominantly growth of those cell lines which have a low level of superoxide dismutase. The substrate of this enzyme, the superoxide anion, was found to be formed during the in vitro oxidation reaction of avarol to its semiquinone radical in the presence of oxygen. Under the same incubation conditions plasmid DNA (pBR322) was converted from the fully supercoiled circular form mainly to the nicked circular form, indicating that the compound causes primarily single-strand breaks. Using Friend erythroleukemia cells (FLC) it was found that avarol induces a dose-dependent DNA damage; the maximum number of DNA strand breaks was observed at 5 h after addition of the compound to the cells. Removal of avarol resulted in a rapid DNA rejoining with biphasic repair kinetics [first half-time, 8 min (90% of the breaks) and a second half-time, 40 min (10% of the breaks)]. When the degree of avarol-induced DNA damage in FLC was compared with the drug-caused inhibition of cell growth a close correlation was established. Avarol displayed no effect on dimethyl sulfoxide-induced erythrodifferentiation of FLC as determined by the benzidine reaction and by dot blot hybridization experiments. From incubation studies of FLC with [3H]avarol no hint was obtained for the formation of an adduct between DNA and the compound. The subcellular distribution of [3H]avarol was studied in liver cells after i.v. application of the compound. The predominant amount of the compound was present in the cytosolic fraction; little avarol was associated with plasma membranes, nuclei, and mitochondria. Using (a) oxidative phosphorylation and (b) oxygen uptake as parameters for mitochondria function, no effect of the compound on the activity of this organelle was determined. These results suggest that avarol forms superoxide anions (and in consequence possibly also hydroxyl radicals) especially in those cells which have low levels of superoxide dismutase. Moreover, evidence is provided that the active oxygen species cause DNA damage resulting in the observed cytotoxic effect.