Анти-адгезивная стратегия в разработке комплексных противодифтерийных вакцин как перспективная мера снижения циркуляции Corynebacterium diphtheriae среди населения

A study is devoted to the development of new bacterial diphtheria vaccines aimed at prevention adhesion of microbial cells to host’s pharyngeal epithelial cells and thus limit colonization of mucous membranes, bacteria carrier state formation and promoted to C. diphtheriaе circulation decrease among the population. As a base of the candidate-vaccine we suggest the diphtheria bacterial antigenic preparation obtained by use of electro-magnetic radiation of ultrahigh frequency from a culture of C. diphtheriaе, var. gravis, tox +.Исследование посвящено разработке новых бактериальных дифтерийных вакцин, направленных на предупреждение колонизации слизистых оболочек, формирование бактерионосительства и способствующих снижению циркуляции C. diphtheriaе среди населения. В качестве основы кандидат-вакцины предлагается дифтерийный бактериальный антигенный препарат, полученный с помощью электро-магнитного излучения сверхвысокой частоты из культуры C. diphtheriaе, var. gravis, tox +

Origin of an Alternative Genetic Code in the Extremely Small and GC–Rich Genome of a Bacterial Symbiont

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop→Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an α-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC–biased base composition (58.4%), and a coding reassignment of UGA Stop→Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment

Original Russian Text Originally published in Biochemistry (Moscow) On Line Papers in Press, as Manuscript BM04 310

Trypsin like proteinases play an important role in digestion in most insects studied [1 4]. They are also involved in processing of insecticide toxins of Bacillus thuringiensis, and this underlines protease dependent resistance of these species to the toxins In this paper, we describe a relatively simple method for purification of the main trypsin like enzyme of the posterior part of the midgut of yellow mealworm (Tenebrio molitor) larvae (TmT1) and characterize the proteinase of this stored product pest. MATERIALS AND METHODS Yellow mealworm (Tenebrio molitor) larvae were reared on a mixture of wheat flour, bran, and brewer's yeast and transferred to milled oat flakes (Raisio, Finland) 1 1.5 weeks prior to dissection. Proteinase purification. Isolated larvae midguts were separated into anterior and posterior parts. The collected posterior parts were homogenized in 0.14 M NaCl and centrifuged at 15,000g for 10 min. The supernatant dia lyzed against 20 mM K,Na phosphate buffer, pH 6.9, containing 0.02% NaN 3 , was centrifuged again and sub jected to batch chromatography on DEAE Sephadex A 50 using the same buffer. The filtrate containing the Vol. 70, No. 3, 2005, pp. 300 305. Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 370 377. Original Russian Text Copyright © 2005 by Tsybina, Dunaevsky, Belozersky, Zhuzhikov, Oppert, Elpidina. Originally published in Biochemistry (Moscow) On Line Papers in Press, as Manuscript BM04 310, February 20, 2005 ACCELERATED PUBLICATION 0006 2979/05/7003 0300 ©2005 Pleiades Publishing, Inc. Abbreviations: Bz) benzoyl; FPLC) fast performance liquid chromatography; For) formyl; PMSF) phenylmethylsulfonyl fluoride; pNA) p nitroanilide; TLCK) N α p tosyl L lysine chloromethyl ketone; TmT1) trypsin like proteinase from T. molitor larvae; E 64) L trans epoxysuccinyl L leucine amido(4 guanidine)butane; Z) N benzoyloxycarbonyl. * To whom correspondence should be addressed. Abstract-A new trypsin like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion exchange chromatography on DEAE Sephadex A 50 and gel filtration on Superdex 75. The isolated enzyme had molec ular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and K m value of 0.04 mM (for hydrolysis of Bz Arg pNA). According to inhibitor analysis the enzyme is a trypsin like serine proteinase stable within the pH range of 5.0 9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N terminal amino acid sequence, IVGGSSI SISSVPXQIXLQY, shares 50 72% identity with other insect trypsin like proteinases, and 44 50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest. Digestive Proteinases of Yellow Mealworm (Tenebrio molitor

Anti-adhesive Strategy in Development of Combined Antidiphtheritic Vaccine as a Perspective Preventive Measure of Corynebacteria diphtheriae Circulation Among the Population

A study is devoted to the development of new bacterial diphtheria vaccines aimed at prevention adhesion of microbial cells to host’s pharyngeal epithelial cells and thus limit colonization of mucous membranes, bacteria carrier state formation and promoted to C. diphtheriaе circulation decrease among the population. As a base of the candidate-vaccine we suggest the diphtheria bacterial antigenic preparation obtained by use of electro-magnetic radiation of ultrahigh frequency from a culture of C. diphtheriaе, var. gravis, tox +

METHODS OF CONTROL DIPHTHERIA VACCINE SAFETY

Vaccination success depends not only on the timely coverage of threatened contingents, but also on the quality of vaccines. Every day, the requirements for security guarantees vaccines and their use guarantees of security increases. For the fast, reliable and independent scientific assessment of vaccine safety issues, WHO in 1999 created the Global Advisory Committee on Vaccine Safety. To enhance the capacity of pharmaceutical supervision in relation to vaccines in 2012 it was developed the Global Vaccine Safety Initiative. The main directions of the Global Vaccine Safety programs are considered in this review. It’s noted more strict requirements of Ukrainian pharmaceutical industry to produce public immunization drugs regulated Supplements to the State Pharmacopoeia of Ukraine, in comparison with other countries. This review considered diphtheria vaccine safety monitoring in the process of production according to the recommendations of the World Health Organization (WHO), described a subcutaneous method for determining the specific toxicity of the combined purified toxoid, characterized an intracutaneous method of determining of the presence of diphtheria toxin in each sample of the combined purified toxoid, that additionally used by some manufacturers. The definition of diphtheria toxin in dilutions of purified toxoid is presented. This review considered diphtheria vaccine safety monitoring in the process of production according to the recommendations of the World Health Organization (WHO), described a subcutaneous method for determining the specific toxicity of the combined purified toxoid, characterized an intracutaneous method of determining of the presence of diphtheria toxin in each sample of the combined purified toxoid, that additionally used by some manufacturers. The definition of diphtheria toxin in dilutions of purified toxoid is presented. As methods for determination of diphtheria toxin must be able to detect even a small amount of toxin it's examined the WHO's proposal to use an intradermal method on guinea pigs and tests on cell cultures. Attention is drawn to the fact that the determination of specific toxicity in cell culture can be carried out at presence of the approval of this method of a national control authority and sensitivity rates no less than in experiments on guinea pigs. The determining of specific toxicity of ready vaccine by subcutaneous method is described. The publication gave a test for elevated toxicity of the final product by intraperitoneal infection of mice and guinea pigs. It’s cited the WHO recommendations aimed at removing the possibility of recovery of the refined toxin toxicity. Checking vaccines toxicity, pyrogenicity, sterility, allergenicity, teratogenicity, mutagenicity and immunogenicity mainly performed on laboratory animals. The review examined the unreliability of animal experiments and the need to find alternative methods for determining the toxicity without their use particularly in light of the “3R”concept. Methods for determining diphtherial toxin using cell cultures is considered, namely, colony overlay test (COT), tests using a monolayer of HeLa cell culture, a culture of Vero cells (kidney cells of african green monkeys ), a culture of CHO cells (cells of Chinese hamster ovary), which are based on the toxin cytopathic effect on sensitive cell culture. Their advantages and disadvantages are listed. An alternative method for the quantitative detection of C. diphtheriae toxin using the polystyrene plate coated with monoclonal antibody to the part of the diphtheria toxin which defines its binding to the cell, is described. It’s regarded the cytotoxic effect of diphtheria toxin on cells of the immune system of mice and guinea pigs: splenocytes, adhesive phagocytes i B- lymphocytes

Purification, Characterization and Partial Amino Acid Sequencing of Two New Aspartic Proteinases from Fresh Flowers of Cynara cardunculus L.

Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two-step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da where as the chains of cardosin B migrated as bands of 34000 Da and 14000 Da. The partial amino acid sequences of the two cardosins revealed that they are similar but not identical, and that they differ horn the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although the cardosins show some degree of similarity to each other, we could detect no immunological cross-reactivity between them. Both cardosins were active at low pH and were inhibited by pepstatin, with Ki values of 3 nM for cardosin A and 1 nM for cardosin B, indicating that they belong to the class of aspartic proteinases. Significant differences between the two enzymes were also found for the Kcat/Km values for the hydrolysis of two chromophoric synthetic peptides. The active-site ionization constants, pKe1 and pKe2, for cardosin A are 2.5±0.2 and 5.3±20.2, whereas for cardosin R they are 3.73±10.09 and 6.7±50.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which have probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed