Glycan-binding profile of DC-like cells

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-“vector” it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1–3Galβ (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.     

Epidemiological situation on tularemia in the regions of Stavropol Territory affected by flood

The data obtained in the analysis of the epidemiological situation in tularemia in the zone of inundation in the Stavropol Territory in 2002 are presented. The current systematic epidemiological surveillance, as well as the data of urgent epizootological and epidemiological survey in the zone of inundation permitted the objective prognostication of the situation in tularemia and formed the basis for the rational planning and realization of prophylactic measures.     

Epidemiological situation on anthrax in the regions of the Southern Federal District in connection with the flood in June 2002

Data on the epidemiological situation in anthrax in the regions of the Southern Federal District, situated in the zone of inundation, are presented. In 2002 no epidemic complications in anthrax were noted in the affected territories. The causes contributing to the appearance of anthrax cases among humans and animals are presented. Recommendations on the improvement of anti-epidemic measures are given.     

Downregulation of Rap1GAP in Human Tumor Cells Alters Cell/Matrix and Cell/Cell Adhesion

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, β-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.Mammalian Rap proteins Rap1a/b and Rap2a/b/c are members of the Ras superfamily of small GTPases. Rap proteins are active when bound to GTP and inactive when bound to GDP. Cellular Rap activity is regulated by the concerted action of guanine nucleotide exchange factors that activate Rap (RapGEFs) and Rap-specific GTPase-activating proteins (RapGAPs) that inactivate Rap (reviewed in reference 10). The Rap1GAP family is composed of several members, including Rap1GAP, Rap1GAPII, Spa-1/SIPA1, and E6TP1/SIPA1L1. Several lines of evidence suggest that RapGAPs function as tumor and/or invasion suppressors. Downregulation of E6TP1 by human papillomavirus protein E6 contributes to cervical cancer (20, 21), and Spa-1 deficiency in mice induces a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (26). The SPA1 gene was identified as a candidate for the metastasis efficiency modifier locus in mice (38). Although the relevance of this observation to humans is not yet clear, single-nucleotide polymorphisms in the SPA1 gene in human breast tumors have been associated with lymph node involvement and poor survival (15). Intriguingly, Spa-1 interacts with Brd4 (18) and Rrp-1b (13), the protein products of genes associated with patterns of extracellular matrix protein gene expression characteristic of metastatic tumors (14).The RAP1GAP gene maps to 1p35-36, a chromosomal region subject to copy number alterations in human tumors (36, 49). Rap1GAP protein levels are decreased in pancreatic adenocarcinomas (53), papillary thyroid carcinomas (37, 47, 57), and melanomas (56). Rap1GAP downregulation has been shown to arise as a consequence of proteasomal degradation (46), loss of heterozygosity (37, 53), and promoter methylation (56, 57). Mutations of unknown significance in RAP1GAP have been identified in breast cancer (42). Although downregulation of Rap1GAP is frequent in human tumors, the functional significance of decreased Rap1GAP expression is unknown. Up to now, studies assessing the role of Rap1GAP in tumor cells have relied exclusively on overexpression experiments. Overexpression of Rap1GAP in oropharyngeal squamous cell (54) and pancreatic (53) carcinoma lines impaired tumor formation in mouse xenograft models. In vitro, overexpression of Rap1GAP impaired tumor cell proliferation (34, 47, 53, 54, 56) and enhanced apoptosis (34, 53, 56). In some instances, overexpression of Rap1GAP inhibited tumor cell migration and invasion (3, 47, 53, 56), while in others, it enhanced invasion (34). While these studies provide insight into cellular processes that can be deregulated by overexpression, they do not assess the significance of depletion of endogenous Rap1GAP in human tumors.Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. The majority of CRC deaths arise as a consequence of distant metastases, most frequently to the liver. While the genetic basis of CRC is well understood (19, 48), less is known about the events that trigger the transition to metastatic disease. We report that Rap1GAP is highly expressed in normal colonic epithelium and that its expression is profoundly decreased in primary colorectal carcinomas. As one strategy to assess the significance of Rap1GAP depletion, the expression of Rap1GAP was silenced in human colon carcinoma cells. Silencing of Rap1GAP induced marked increases in Rap1 and Rap2 activity, the first evidence that Rap1GAP is an essential negative regulator of Rap GTPases in colon cancer. Rap1 regulates inside-out signaling through integrins (reviewed in references 8, 9, and 11) and is a target of outside-in signaling via cadherins (reviewed in reference 30). Downregulation of Rap1GAP induced profound alterations in cell/matrix and cell/cell adhesion. Suppressing Rap1GAP expression enhanced adhesion and spreading on collagen. Unexpectedly, based on the role of Rap1 in promoting cell/cell adhesion, silencing of Rap1GAP impaired cell/cell adhesion. These findings demonstrate a requirement for regulated Rap activity in the maintenance of epithelial cell structure and demonstrate a heretofore unappreciated role for Rap1GAP in the regulation of cell/cell adhesion. As the dissemination of tumor cells requires the weakening of cell/cell adhesion and an enhanced ability to adhere to collagen-rich interstitial matrices, our studies identify a potential mechanism through which loss of Rap1GAP contributes to tumor progression.     

Multiplex amplification test system for the identification and differentiation of Bacillus anthracis

The multiplex amplification test system for the identification of Bacillus anthracis with primers to plasmid cya (pX01), capC (pX02) genes and chromosomal sap gene were developed. The primers to sap gene were selected by the authors and, after being tested on 72 microbial strains of the genus Bacillus, proposed as more specific in comparison with the known primers to chromosomal locus Ba 813. The proposed test system permitted the simultaneous identification of B. anthracis of all plasmid variants, the evaluation of their potential virulence and the differentiation of B. anthracis nonplasmid strains from bacilli of the group Bacillus cereus.     

Use of DIR-PCR for elaboration of molecular markers of intraspecies bacterial groups as exemplified by Bacillus thuringiensis

A recently developed PCR-fingerprinting method, the so-called DIR (diverged inverted repeats)-PCR, was used for quick search for molecular markers of Bacillus thuringiensis subspecies carrying the cry1 genes. The analysis of the fingerprints obtained by this method made it possible to reveal PCR fragments characteristic of the subspecies that produce proteins toxic for insects of the order Lepidoptera. Cloning and sequencing of these fragments allowed systems of SCAR (sequence characterized amplified region) primers to be designed, which are specific to the above group of B. thuringiensis strains. Comparison of the specific fragments with sequences available in the GenBank database revealed their homology with the rpoC gene family and the adjacent spacer region, suggesting chromosomal localization of these markers. This increases the reliability of the designed system of SCAR primers, because plasmids may be lost or transferred by transformation between closely related strains. It was demonstrated that the DIR-PCR method allows markers to be developed that are linked to diagnostic genotypic and phenotypic characteristics of bacteria.     

Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.     

Activation of ERK by Ca2+ store depletion in rat liver epithelial cells

In rat liver epithelial (WB) cells,Ca²⁺ pool depletion induced by twoindependent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method,Ca²⁺ pool depletion bythapsigargin increased the activity of ERK, even when rise in cytosolicCa²⁺ was blocked with theCa²⁺ chelator BAPTA-AM. For thesecond method, addition of extracellular EGTA at a concentration shownto deplete intracellular Ca²⁺pools also increased ERK activity. In each instance, ERK activation, asmeasured by an immunocomplex kinase assay, was greatly reduced by thetyrosine kinase inhibitor genistein, suggesting thatCa²⁺ store depletion increased ERKactivity through a tyrosine kinase pathway. The intracellularCa²⁺-releasing agent thapsigarginincreased Fyn activity, which was unaffected by BAPTA-AM pretreatment,suggesting that Fyn activity was unaffected by increased cytosolic freeCa²⁺. Furthermore, depletion ofintracellular Ca²⁺ with EGTAcaused inactivation of protein phosphatase 2A and protein tyrosinephosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK wereaugmented in cells pretreated with BAPTA-AM, but ANG II-inducedexpression of the dual-specificity phosphatase mitogen-activatedprotein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment.Together these results indicate that ERK activity is regulated by thebalance of phosphorylation vs. dephosphorylation reactions in intactcells and that the amount of Ca²⁺stored in intracellular pools plays an important role in this regulation.