Comparative investigations of structure and properties of micro-arc wollastonite-calcium phosphate coatings on titanium and zirconium-niobium alloy.

Investigation results of micro-arc wollastonite-calcium phosphate (W-CaP) biocoatings on the pure titanium (Ti) and Zr-1wt.%Nb (Zr-1Nb) alloy were presented. The voltages of 150-300 V generate the micro-arc oxidation (MAO) process with the initial amplitude current of 150-550 A and 100-350 A for Ti and Zr-1Nb substrates, respectively. The identical dependencies of changes of the coating thickness, surface roughness and adhesion strength on the process voltage were revealed for the both substrates. The W-CaP coatings with the thickness of 10-11 µm were formed on Ti and Zr-1Nb under the low process voltage of 130-150 V. Elongated wollastonite particles with the size in the range of 40-100 µm were observed in such coatings. The structure of the coatings on Ti was presented by the X-ray amorphous and crystalline phases. The X-ray reflexes relating to the crystalline phases of Ti and wollastonite were observed only in XRD patterns of the coatings deposited under 130-200 V on Ti. While, the crystalline structure with phases of CaZr4(PO4)6, ß-ZrP2O7, ZrO2, and Zr was detected in the coatings on Zr-1Nb. FT-IRS, XRD, SEM, and TEM data confirmed that the increase of the process voltage to 300 V leads to the dissociation of the wollastonite. No toxic effect of specimens on a viability, morphology and motility of human adipose-derived multipotent mesenchymal stem cells was revealed in vitro.

Regulation of PTEN binding to MAGI-2 by two putative phosphorylation sites at threonine 382 and 383.

We have reported previously that the PTEN COOH-terminal 33 amino acids play a role in the maintenance of PTEN protein stability (Tolkacheva and Chan, Oncogene, 19: 680-689, 2000). By site-directed mutagenesis, we identified two threonine residues within this COOH-terminal region at codon 382 and 383 that may be targets for phosphorylation events. Interestingly, PTEN mutants rendered phosphorylation-incompetent at these two sites, T382A/T383A, and were found to have drastically reduced expression in cultured cells. The enhanced degradation of PTEN was most likely mediated by the proteosome-dependent pathway, we have evidence that PTEN was polyubiquitinated. More interestingly, the non-phosphorylated forms of PTEN displayed significantly greater binding affinity than the wild-type protein to a previously identified PTEN interacting partner, MAGI-2/ARIP1. On the basis of all these data, we propose that PTEN recruitment to the cell-cell junction may be regulated through the phosphorylation of its COOH terminus.

Inhibition of H-Ras transformation by the PTEN/MMAC1/TEP1 tumor suppressor gene.

The human PTEN/MMAC1/TEP1 (PTEN) tumor suppressor gene encodes a phosphatase with specificity towards the D3 phosphate of phosphatidylinositides. PTEN mutations have been reported in the endometrioid type of uterine tumors which are associated with frequent activations of the Ras oncogenes. In this study, we report the ability of PTEN to potently inhibit H-Ras induced morphological transformation and anchorage-independent growth in NIH3T3 cells. This novel activity of PTEN was correlated more with its ability to suppress the phosphatidylinositol 3-kinase (PI3-K)-dependent signaling cascade, but not the mitogen-activated protein kinase (MAPK) pathway. To define the minimal region in PTEN protein that is responsible for this anti-oncogenic activity, a panel of carboxyl-terminal truncation mutants was generated. While deletions of 4 and 33 amino acids do not have marked effects, removal of up to 68 amino acids drastically reduced the ability of PTEN to inhibit Ras transformation. The propensity of these mutants to suppress Ras transformation is correlated with their relative ability to dephosphorylate inositol (1,3,4,5)-tetrakisphosphate in vitro, and to suppress Akt kinase activity in cultured cells. In addition, we have evidence to suggest that the C-terminal region of PTEN contributes to the stability of the encoded gene product.

Differential roles of Akt, Rac, and Ral in R-Ras-mediated cellular transformation, adhesion, and survival.

Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.

[The empirical antibiotic therapy of patients with acute leukemias: the results of a multicenter study]. / Empiricheskaia antibioticheskaia terapiia u bol'nykh ostrymy leikozami: itogi mnogotsentrovogo issledovaniia.

AIM: To evaluate efficacy of ampicilline/sulbactame and fluconasole in the regimen of empirical antibiotic therapy in patients with acute leukemia. MATERIALS AND METHODS: The trial covered 14 hematological departments of Russia and 1 of Ukraine. Acute myeloid leukemia patients were included. 92 cases of fever in 56 patients with analysis of efficacy in 66 cases were considered. At the first stage of empirical antibiotic therapy, cefoperason (4 g/day) and gentamycin (240 mg/day) were administered. If no response was reached, ampicilline/sulbactam (7.5 g/day) was added. This was the second stage. If no response occurred for 5 days the three drugs were joined by fluconasol (400 mg followed by 200 mg). RESULTS: Fever of unclear genesis was cured in 82% (28 of 34), clinical infection--in 80% (20 of 25), microbiologically confirmed infection--in 4 of 7 cases. A complete response to the empirical antibiotic therapy was registered in 52 of 66 cases (79%). 7(10.5%) patients died of infectious complications. 7(10.5%) received other antibiotics.

[Use of meropenem in patients with neutropenia]. / Primenenie meropenema u bol'nykh s neitropeniei.

Meropenem was used in the treatment of 14 infectious complications in 11 patients including 8 with acute myeloid leukemia due to the cytostatic therapy, 1 with chronic myeloid leukemia, 1 with aplastic anemia and 1 with acute intermittent porphyria. At the moment of the meropenem use critical neutropenia (less than 500 granulocytes per 1 ml) in 11 cases (79 per cent) was stated. The drug was administered as intravenous infusions in a dose of 1 g every 8 hours for 4 to 41 days (the median of 11 days). 9 out of the 14 infectious complications were cured (the body temperature normalized and all the inflammation foci were eliminated), among them 6 out of 8 pyocyanic sepsis. Eradication of gram-negative bacteria was observed in 8 out of 10 cultures of the biological materials. No toxic complications or electrolytic disorders due to the drug use were recorded. The drug tolerance was good.

Identification and characterization of R-ras3: a novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution.

Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins.

Cooperative transformation of NIH3T3 cells by G alpha12 and Rac1.

The heterotrimeric G-protein, G alpha12, together with the closely-related G alpha13, are members of the G12 class of alpha-subunits important in mediating the signaling from seven transmembrane domain-spanning receptors. Recent evidence implicating both G alpha12 and G alpha13 in the activation of signaling pathways involving members of the RHO gene family led us to examine the role of Rac1, RhoA and Cdc42Hs in the transforming properties of G alpha12. Asparagine 17 (Asn 17) dominant inhibitory mutants of Rac1, and to a lesser extent RhoA, block focus forming ability of the GTPase-deficient mutant of G alpha12 (G alpha12 Leu 229) in NIH3T3 cells. In turn, wild-type G alpha12 cooperates well with Rac1 Val 12 but not with RhoA Leu 63 mutant in transforming NIH3T3 cells. Interestingly, the morphology of foci induced by G alpha12 and RhoA mutants are strikingly similar and is distinct from those displayed by Rac1 Val 12 mutant. The fact that G alpha12's ability to induce mitogenesis in NIH3T3 cells is not significantly perturbed by C3 ribosyltransferase suggested that RhoA does not play a major role in G alpha12-induced mitogenic events. Activated mutant of Rac1 has previously been demonstrated to stimulate the activity of the stress-induced c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs). Transient co-transfection of Rac1 Val 12 mutant with the wild-type G alpha12 in COS7 cells leads to the further activation of an exogenously expressed hemagglutinin(HA)-tagged JNK. Furthermore, the cooperation between G alpha12 and Rac1 in cellular transformation is correlated with their ability to stimulate transcription from c-fos serum response element (SRE).

Cloning of a Cryptococcus neoformans gene, GPA1, encoding a G-protein alpha-subunit homolog.

We have isolated a gene, GPA1, from Cryptococcus neoformans by the PCR technique. DNA sequencing of the GPA1 clone suggested that it encodes a protein homologous to the G-protein alpha-subunit family. Comparison of the deduced amino acid sequence of the GPA1-encoded protein revealed that it is about 45% identical to several mammalian Gi alpha subunits and 48% identical to the G alpha protein Gpa2 from Saccharomyces cerevisiae. G alpha proteins are known to be involved in mating of other yeasts, such as S. cerevisiae and Schizosaccharomyces pombe. Southern analysis demonstrated that GPA1 is present in a single copy within the Cryptococcus genome. Isolation of the cDNA for GPA1 confirmed that the gene contains six introns within the coding region. The GPA1 transcript was identified by Northern (RNA) analysis as a 1.6-kb RNA present in exponentially growing cells of both the alpha and a mating types. Moreover, the abundance of this transcript increased in cells shifted to starvation medium. Coincubation of alpha and a cells on starvation medium is required for mating of cryptococcal cells. Thus, our results are consistent with the involvement of C. neoformans GPA1 in mating.

[Bacteriotherapy of intestinal dysbacteriosis in patients with acute leukemia]. / Bakterioterapiia disbakterioza kishechnika u bol'nykh ostrymi leikozami.

The results have been presented of correcting intestinal microflora in 127 patients with acute leukemia by preparations containing Bacillus bifidus and acidophilus: dried and milk bifidum-bacterin, biofructolact, acidophilic milk.