Transferrin conjugates in the anticancer therapy

To enhance the therapeutic efficiacy of anticancer drugs and reducing its systemic side-effects carriers are used. Transferrin is one of the very promising protein which can be used to transport drugs, DNA and ions into the cancer cells. Because of the fact that neoplastic cells have increased number of transferrin receptors, the transferrin can deliver the drugs directly to the neoplastic cells without injury of normal cells.     

Synthesis and antibacterial activity of desosamine-modified macrolide derivatives

Structural factors behind erm macrolide resistance were studied through synthesis of new macrolide derivates possessing truncated desosamine sugar moieties and subsequent determination of their antibacterial activity. Synthesized compounds with 2'-deoxy and 3'-desmethyl desosamine rings demonstrated decreased antibacterial activity on the native Staphylococcus aureus strain and were inactive against constitutively resistance S. aureus. The obtained results indicate that steric repulsion between the dimethylated A2058 and desosamine ring cannot be considered as a primary reason for erm-resistance.     

Genes and structure of selected cytokines involved in pathogenesis of psoriasis

Psoriasis is a common skin disease involving 1-4% of human population worldwide, of strong genetic background. The following cytokines are directly involved in psoriasis: TNF, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-18, IL-19, IL-20, IL-23 whereas IL-4, IL-10, IL-12 as well as IL-11, IL-17 and IFN-gamma are rather indirectly engaged. This work is a review of some genetic factors and structure of selected cytokines and receptors and their genes location.     

Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (～60 kDa) is processed into active BMP10 (～14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.     

DYF-1 Is Required for Assembly of the Axoneme in Tetrahymena thermophila

In most cilia, the axoneme can be subdivided into three segments: proximal (the transition zone), middle (with outer doublet microtubules), and distal (with singlet extensions of outer doublet microtubules). How the functionally distinct segments of the axoneme are assembled and maintained is not well understood. DYF-1 is a highly conserved ciliary protein containing tetratricopeptide repeats. In Caenorhabditis elegans, DYF-1 is specifically needed for assembly of the distal segment (G. Ou, O. E. Blacque, J. J. Snow, M. R. Leroux, and J. M. Scholey. Nature. 436:583-587, 2005). We show that Tetrahymena cells lacking an ortholog of DYF-1, Dyf1p, can assemble only extremely short axoneme remnants that have structural defects of diverse natures, including the absence of central pair and outer doublet microtubules and incomplete or absent B tubules on the outer microtubules. Thus, in Tetrahymena, DYF-1 is needed for either assembly or stability of the entire axoneme. Our observations support the conserved function for DYF-1 in axoneme assembly or stability but also show that the consequences of loss of DYF-1 for axoneme segments are organism specific.Cilia are microtubule-rich cellular extensions that arise from basal bodies near the surfaces of most eukaryotic cell types. Defective cilia cause a wide variety of diseases, including polycystic kidney disease, primary ciliary dyskinesia, and retinal degeneration (3). A typical motile cilium has a microtubule-based framework, the axoneme, which contains nine outer (mostly doublet) microtubules and two central (singlet) microtubules. In most cilia, the axoneme can be subdivided into three segments: proximal (transition zone), middle (containing outer doublet microtubules), and distal (containing singlet extensions of peripheral microtubules). The outer doublet microtubules of the middle segment have a complete tubule A made of 13 protofilaments and an incomplete tubule B made of 11 protofilaments that is fused to the wall of the A tubule (36, 57). The outer microtubules in the distal segment lack the B tubule (32, 49). The distal segment also lacks dynein arms and radial spokes, and its microtubules are terminated by caps that are associated with the plasma membranes at the tips of cilia (11, 50). The distal segments are characterized by a high level of microtubule turnover, which could play a role in the regulation of the length of cilia (31).The mechanisms that establish the segmental subdivision of the axoneme are not well understood. Studies of Caenorhabditis elegans indicate that the distal segment is assembled using a mechanism that differs from the one utilized in the middle and proximal segments (54). In most cell types, ciliogenesis is dependent on the intraflagellar transport (IFT) pathway, a bidirectional motility of protein aggregates, known as IFT particles, that occurs along outer microtubules (10, 28, 29, 42). IFT particles are believed to provide platforms for transport of axonemal precursors (23, 44). The anterograde component of IFT that delivers cargo from the cell body to the tips of cilia is carried out by kinesin-2 motors (28, 63), whereas the cytoplasmic dynein DHC1b is responsible for the retrograde IFT (41, 43, 53). Importantly, in the well-studied amphid cilia of C. elegans, two distinct kinesin-2 complexes are involved in the anterograde IFT and differ in movement velocity: the “slow” heterotrimeric kinesin-II and the “fast” homodimeric OSM-3 kinesin (54). While kinesin-II and OSM-3 work redundantly to assemble the middle segment, OSM-3 alone functions in the assembly of the distal segment (39, 56).In C. elegans, DYF-1 is specifically required for assembly of the distal segment (39). In the DYF-1 mutant, the rate of IFT in the remaining middle segment is reduced to the level of the slow kinesin-II, suggesting that the Osm3 complex is nonfunctional and that kinesin-II functions alone in the middle segment. Thus, DYF-1 could either activate OSM-3 kinesin or dock OSM-3 to IFT particles (14, 39).However, a recent study of zebrafish has led to a different model for DYF-1 function. Zebrafish embryos that are homozygous for a loss of function of fleer, an ortholog of DYF-1, have shortened olfactory and pronephric cilia and ultrastructural defects in the axonemes. In the middle segment, the fleer axonemes have B tubules that are disconnected from the A tubule, indicating that DYF-1 functions in the middle segment and could play a role in the stability of doublet microtubules (40). Earlier, a similar mutant phenotype was reported in Tetrahymena for a mutation in the C-terminal tail domain of β-tubulin, at the glutamic acid residues that are used by posttranslational polymodifications (glycylation and glutamylation) (47). Glycylation (46) and glutamylation (12) are conserved polymeric posttranslational modifications that affect tubulin and are highly enriched on microtubules of axonemes and centrioles (reviewed in reference 20). Other studies have indicated that tubulin glutamylation contributes to the assembly and stability of axonemes and centrioles (4, 8). The fleer mutant zebrafish cilia have reduced levels of glutamylated tubulin (40). Pathak and colleagues proposed that the primary role of DYF-1/fleer is to serve as an IFT cargo adapter for a tubulin glutamic acid ligase (25) and that the effects of lack of function of DYF-1/fleer could be caused by deficiency in tubulin glutamylation in the axoneme (40). As an alternative hypothesis, the same authors proposed that DYF-1 is a structural component that stabilizes the doublet microtubules in the axoneme (40).Here, we evaluate the significance of a DYF-1 ortholog, Dyf1p, in Tetrahymena thermophila. Unexpectedly, we found that Tetrahymena cells lacking Dyf1p either fail to assemble an axoneme or can assemble an axoneme remnant. While our observations revealed major differences in the significance of DYF-1 for segmental differentiation in diverse models, it is clear that DYF-1 is a conserved and critical component that is required for assembly of the axoneme.     

Combination of vasostatin and cyclophosphamide in the therapy of murine melanoma tumors

Growth of tumors is strongly dependent upon supply of nutrients and oxygen by de novo formed blood vessels. Inhibiting angiogenesis suppresses growth of primary tumors as well and affects development of metastases. We demonstrate that recombinant MBP/vasostatin fusion protein inhibits proliferation of endothelial cells in vitro. The therapeutic usefulness of such intratumorally delivered recombinant protein was then assessed by investigating its ability to inhibit growth of experimental murine melanomas. In the model of B16-F10 melanoma the MBP/vasostatin construct significantly delayed tumor growth and prolonged survival of treated mice. A combination therapy involving MBP/vasostatin construct and cyclophosphamide was even more effective and led to further inhibition of the tumor growth and extended survival. We show that such combination might be useful in the clinical setting, especially to treat tumors which have already formed microvessel networks.     

TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants

Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni’s correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease.