A study of the ability of Bordetella pertussis toxin to induce the formation of B-suppressors

Mouse spleen cells not adhering to the plastic surface and B-cells isolated from them were treated with B. pertussis toxin in vitro, washed and injected into recipients (allogeneic, syngeneic, intact or lethally irradiated) whose immune response to sheep red blood cells was then evaluated by Jerne's method. Treatment with B. pertussis toxin was shown to induce the development of immunosuppressive activity in intact spleen cells and in B-cells, to abolish the activity of memory B-cells and to enhance the suppressor activity of autoimmune mice. Supernatants obtained after autoimmune mice. Supernatants obtained after the 18- to 24-hour cultivation of spleen cells, previously treated with B. pertussis toxin for 60 minutes, suppressed the reaction of blast transformation of spleen cells to Con A and lipopolysaccharide and induced the appearance of immunosuppressive activity in intact spleen cells. The suppressing effect of the cells studied in this investigation may be linked with the ability of B. pertussis cells to stimulate the synthesis of cAMP, prostaglandins E and/or suppressor factors.     

Lymphocytes with antigen receptors in drug allergy

The data on the possibility of using the rosette-formation tests for the diagnosis of drug allergy are presented. Tests based on changes in the levels of activated T- and B-lymphocytes after their incubation with allergenic drugs have proved to be low informative. The test found to be highly informative is the antigen-specific rosette-formation test based on the detection of lymphocytes, capable of binding allogeneic erythrocytes loaded with antibiotics causing allergy in patients, in the peripheral blood. This test may be of importance not only in diagnosis, but also for prognosis, as it permits the detection of sensitization to a drug before the clinical manifestations of allergy.     

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.     

Paraendocervical route for metronidazole administration in complex treatment of chronic Trichomonas endocervicitis]

The data on paraendocervical administration of metronidazole in the treatment of patients with relapsing Trichomonas endocervicitis are presented. Metronidazole was administered as 0.5% solution in a dose of 0.04 g once a day for 8-10 days in complex traditional therapy including oral use of metronidazole and immunocorrigating and local treatment. It was shown that paraendocervical administration of the protistocidal agent provided earlier regression of the urogenital symptoms and 2.8 times lower frequency of the relapses.     

Structural effects of naturally occurring human blood group B galactosyltransferase mutations adjacent to the DXD motif

Human blood group A and B antigens are produced by two closely related glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) utilizes UDP-GalNAc to extend H antigen acceptors (Fuc alpha(1-2)Gal beta-OR) producing A antigens, whereas a galactosyltransferase (GTB) utilizes UDP-Gal as a donor to extend H structures producing B antigens. GTA and GTB have a characteristic (211)DVD(213) motif that coordinates to a Mn(2+) ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the (211)DVD(213) motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the B(el) variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to A(weak)B phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in k(cat) compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn(2+) was disrupted by Arg-214 causing displacement of the metal by a water molecule. Kinetic characterizations of the M214T and M214V mutants revealed they both had GTA and GTB activity consistent with the serology. The crystal structure of the M214T mutant showed no change in DVD coordination to Mn(2+). Instead a critical residue, Met-266, which is responsible for determining donor specificity, had adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity.     

Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5

The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.     

Thiols as biomarkers of heavy metal tolerance in the aquatic macrophytes of Middle Urals,Russia

Aquatic macrophytes, viz. Sagittaria sagittifolia L., Lemna gibba L., Elodea canadensis Michx., Batrachium trichophyllum (Chaix.) Bosch., Ceratophyllum demersum L. and Potamogeton sp. (P. perfoliatus L., P. alpinus Balb., P. crispus L., P. berchtoldii Fieber, P. friesii Rupr., P. pectinatus L.) were collected from 11 sites for determining their metal accumulation and thiols content. Cu²⁺, Ni²⁺, Mn²⁺, Zn²⁺, and Fe³⁺ exceeded maximum permissible concentrations in chosen sites. Significant transfer of metals from water to leaves is observed in the order of Ni²⁺ < Cu²⁺ < Zn²⁺ < Fe³⁺ < Mn²⁺. The maximum variation of bioconcentration factor was noticed for manganese. The accumulation of heavy metals in leaves was correlated with non-protein and protein thiols, confirming their important role in metal tolerance. The largest contribution was provided by Cu²⁺ (on the average r = 0.88, p < 0.05), which obviously can be explained as an important role of these ions in thiols synthesis. Increased synthesis of thiols in the leaves allows the usage of SH-containing compounds as biomarkers of metal tolerance. Considering accumulation of metals and tolerance, B. trichophyllum, C. demersum and L. gibba are the most suitable species for phytoremediation of highly multimetal contamination, while E. canadensis and some species of Potamageton are suitable for moderately metal-polluted sites.     

In vitro activity of a new glycopeptide antibiotic eremomycin in relation to obligate anaerobic Gram-positive bacteria]

Antibacterial activity of eremomycin, a novel glycopeptide antibiotic, against obligate anaerobic Gram-positive++ bacteria was studied. Eremomycin was shown to inhibit the growth of obligate anaerobic Gram-positive++ cocci and bacteria belonging to Clostridium in rather low concentrations and within narrow ranges of the MIC which was indicative of the antibiotic undoubted advantages. The antibacterial activity of eremomycin was 2 times as high as that of vancomycin and 8 times as high as that of ristomycin with respect to Gram-positive++ anaerobic cocci. Pathogenic strains of Clostridium spp. were 2 to 4 times more sensitive to eremomycin than to vancomycin. A significant property of the novel glycopeptide antibiotic was shown to be its capacity for inhibiting the growth of Gram-positive++ aerobic and obligate anaerobic cocci within the same concentration ranges which might be of importance in monotherapy of mixed aerobic and anaerobic infections.