Genetic relationships of Moscow and foreign strains of Haemophilus influenzae, serotype B

46 Haemophilus influenzae strains of serotype b, isolated on the territory of Moscow and compared with foreign strains, were characterized by the method of multilocus sequencing-typing. Among the strains circulating in Moscow 10 variants of sequence-types were observed; of these, only one variant (CT-6) had been described earlier. The analysis of the data revealed that the strains under study were distributed in two clonal complexes; the overwhelming majority of the strains belonged to sequence-type 6. The distribution of Moscow strains in clonal complexes repeated the clonal organization detected in foreign strains. The conclusion was made that lower morbidity rate in Hib meningitis in Moscow (in comparison with that in the countries of Europe and America before the introduction of prophylactic vaccination) was not due to the peculiar genetic features of the circulating strains.     

Cycloimide bacteriochlorin p derivatives: photodynamic properties and cellular and tissue distribution

Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure-activity relationships. The studied CIBCs absorb light within a tissue transparency window (780-830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8-13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54-0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy.     

PHOG-BLAST – a new generation tool for fast similarity search of protein families

Background  

The need to compare protein profiles frequently arises in various protein research areas: comparison of protein families, domain searches, resolution of orthology and paralogy. The existing fast algorithms can only compare a protein sequence with a protein sequence and a profile with a sequence. Algorithms to compare profiles use dynamic programming and complex scoring functions.     

Repair in the nuclear matrix of DNA damaged by benz(a)pyrene

The confluent culture of hamster embryo cells was incubated with benzo(a)pyrene for 24 hours. Then the medium was replaced by maximal lacking both the serum and benzo(a)pyrene. The process of DNA repair was observed in four nuclear fractions according to two indexes: the disappearance of metabolites of benzo(a)pyrene covalently bound to DNA and the incorporation of 3H-thymidine to DNA in the period from I min to 72 hours. Hydroxyurea at the concentration of 5 mM was added 2-19 hours before 3H-thymidine. The highest concentration of benzo(a)pyrene metabolites was found in the DNA of nuclear matrix fraction throughout all the experiment. The initial concentration of 3H-thymidine right after its addition into the cell culture medium was the highest in DNA of nuclear matrix fraction and the lowest in DNA fraction soluble in the buffer with low ionic strength. Later on, the concentration of 3H-thymidine was decreased in matrix-bound fractions and increased in other fractions up to the total DNA level. The results suggest that the repair process requires joining of benzo(a)pyrene damaged DNA region to the nuclear matrix with the following reverse transition into the fraction where the fragment was initially located.     

SST versus EST in gene recognition

The expressed sequence tag (EST) data provide a powerful tool for identification of transcribed DNA sequences. However, as EST are relatively short, many exons are poorly covered by EST, thus reducing the utility of EST data. Recently, signature sequence tag (SST) fingerprints were proposed as an alternative to EST fingerprints. Given a fingerprint set of probes, SST of a clone is a subset of probes from the fingerprint set that hybridize with the clone. We demonstrate that besides being a powerful technique for screening cDNA libraries, SST technology provides for very accurate gene predictions. Even with a small fingerprint set (600-800 probes), SST-based gene recognition outperforms many conventional and EST-based methods. The increase in the size of the fingerprint set to 1500 probes provides almost perfect gene recognition. Even more importantly, SST-based gene predictions miss very few exons and, therefore, provide an opportunity to bypass the cDNA sequencing step on the way from finished genomic sequence to mutation detection in gene-hunting projects. Because SST data can be obtained in a highly parallel and inexpensive way, SST technology has a potential of complementing EST technology for gene hunting.     

Targeted intracellular site-specific drug delivery: photosensitizer targeting to melanoma cell nuclei

A number of drugs are regarded as possessing local activity because their effects take place at an extremely short distance from their location site in the cell. The response of different cellular compartments to these effects is different. Such substances as photosensitizers (PSs), which are used in photodynamic cancer therapy, should be targeted to the cell compartments where their effect is the most pronounced. This study describes the construction and properties of the chimeric modular recombinant transporters (MRTs) expressed in Escherichia coli and used for PS targeting. These constructs include (1) the alpha-melanocyte-stimulating hormone as a ligand module, which is internalized by the target cells (mouse melanoma); (2) the optimized SV40 large T-antigen nuclear localization signal; (3) the hemoglobin-like protein from E. coli as a carrier module; (4) the endosomolytic module, the translocation domain of the diphtheria toxin. These MRTs were used for PS targeting to the mouse melanoma cell nuclei, the most PS-damaged intracellular compartment, which resulted in a PS photocytotoxic effect increase of several orders of magnitude. In our opinion, MRTs, which target locally active drugs into the desired cell compartment and thereby enhance the drug response, represent a new generation of the pharmacological agents.