Bone marrow angiogenesis and angiogenic factors in multiple myeloma treated with novel agents

Introduction. An increased bone marrow (BM) angiogenesis is associated with poor outcome in multiple myeloma (MM). Objective. Angiogenesis study in MM treated with novel antimyeloma agents: thalidomide, lenalidomide, bortezomib, and with dexamethasone. Patients and methods. Forty-four patients with MM (14 newly diagnosed, 30 refractory/relapsed) were treated with novel agents at our institution. A BM biopsy was obtained before the initiation of therapy in 19. Angiogenesis was assessed by microvessel density (MVD) estimation in BM biopsies stained with the monoclonal anti-CD34 antibody, and by serum levels of angiogenic factors (VEGF, bFGF, and HGF) and cytokines (IL-6 and TNF-α). Results. A positive correlation was found between BM plasma cell involvement and MVD estimation (p = 0.01). However, MVD was not significantly correlated with either disease phase (p = 0.065) or response to therapy (p = 0.79). Neither baseline serum levels of angiogenic cytokines correlated to response to treatment. No significant correlation was found between BM MVD and serum levels of angiogenic cytokines. Serum levels of angiogenic cytokines before and after therapy showed a significant increase of bFGF (p = 0.008). Conclusion. There is no relationship between MVD estimation and baseline serum levels of angiogenic cytokines, neither between each of them and response to therapy.     

Propofol lipidic infusion promotes resistance to antifungals by reducing drug input into the fungal cell

Background  

The administration of non-antifungal drugs during patient hospitalization might be responsible for discrepancies between in vitro and in vivo susceptibility to antifungals. Propofol is often administered to intensive care units as a sedative.     

Non-neuronal cells involved in the degeneration and regeneration of the fish retina

In the present work we show that during the degenerative process occurring after the cryo-elimination of the tench peripheral growing zone many non-neuronal cell types in addition to the resident microglial cells, appear within the affected areas. Some of them are normally found in the retina, such as the retinal pigmented epithelium cells and others originate from extra-retinal tissues. We identified these as granular leukocytes and macrophages. The microglial cells and macrophages, those resident in the sub-retinal space, and the invasive ones, act as phagocytes. The analysis of the injured retina following lesion shows that the invasive macrophages, arising from the scleral extra-retinal tissues, penetrate the neural retina, and migrate from the scleral to the vitreal portion. In contrast those coming from the vitreal extra-retinal tissues migrate in the opposite direction. Moreover, the retinal pigmented epithelium cells present remarkable modifications in their morphology and distribution and enter the neural retina, where they disrupt the surrounding tissue. We have also observed that this cryo-lesion causes an inflammation mediated by a type of granular leukocyte, denominated heterophils which penetrate the neural retina and probably come from the blood supply. Our results suggest that, during the first days after the lesion, the participation of diverse non-neuronal cells removing cell debris from the damaged zone should create a favourable environment allowing the regeneration of the neural retina.     

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.     

Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla

Hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla. These seedling-derived explants were incubated on a modified MS medium (SP medium), supplemented with 2.0 M TDZ. After 1 month, the calluses obtained were transferred to SP medium containing different concentrations of BA and NAA or zeatin and NAA. Shoots were induced from these calluses at a high frequency. Shoot elongation was then stimulated on SP medium supplemented with BA, NAA and GA₃ for 20 to 30 days. For rooting, 50 mm long shoots were cultivated on root induction medium containing IBA (2.5 M) for different periods and then transferred to the same medium but without auxin, for 30 days. Plantlets were then successfully transplanted to greenhouse conditions.     

Synthesis of 3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles,displaying combined 5-HT uptake inhibiting and alpha(2)-adrenoceptor antagonistic activities: a novel series of potential antidepressants

The synthesis of a series of novel 3-piperazinylmethyl-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles as novel dual 5-HT reuptake inhibitors and alpha(2)-adrenoceptor antagonists is described. Their affinity at the three different human alpha(2)-adrenoceptor subtypes and the 5-HT transporter site is reported. The in vivo activity of the compounds was measured in two different assays: (1). inhibition of pCA-induced excitation, which evaluates the ability to block the central 5-HT transporter, and (2). inhibition of xylazine-induced loss of righting, which evaluates the ability to block central alpha(2)-adrenoceptors.     

Verotoxin-producing Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) isolated from healthy cattle in Spain

AIMS: To determine the prevalence and characteristics of verotoxigenic Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) in healthy cattle. METHODS AND RESULTS: Faecal samples from 412 healthy cattle were screened for the presence of VTEC, EPEC and NTEC. Four isolates from each sample were studied. VTEC, EPEC and NTEC were isolated in 8.7%, 8.2% and 9.9% of the animals, respectively. VTEC and NTEC were isolated more frequently from calves and heifers than from adults. Seventy (4.2%), 69 (4.2%) and 74 (4.5%) of the 1648 E. coli isolates were VTEC, EPEC and NTEC, respectively. Seventeen (24.3%) of the VTEC strains were eae-positive. Thirty-six (51.4%) of VTEC strains belonged to E. coli serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. The serogroups most prevalent among the EPEC strains were O10, O26, O71, O145 and O156. CONCLUSIONS: Healthy cattle are a reservoir of VTEC, EPEC and NTEC. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most of the VTEC strains were eae-negative, a high percentage of VTEC strains belonged to serogroups associated with severe disease in humans.     

Response to thalidomide in multiple myeloma: impact of angiogenic factors

Thalidomide has antiangiogenic and immunomodulatory effects, mediated by several cytokines such as vascular endothelial growth factor (VEGF), fibroblastic growth factor (FGF-2), hepatocyte growth factor (HGF), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). Although extramedullary plasmacytomas (EMP) have a high vascularization, the response of these patients to thalidomide is controversial. Thirty-eight patients with refractory/relapsed MM were treated with thalidomide. Eleven patients had EMP when therapy was initiated. Serum specimens were obtained in patients before treatment was started and at the time of maximum response in responding patients or at thalidomide discontinuation in non-responders. Serum levels of VEGF, HGF and FGF-2 were determined in 18 patients whereas IL-6 and TNF-alpha were measured in 19 patients. Sixteen of the 38 patients (42%) responded to thalidomide. The response rate was significantly higher in patients without EMP (59% vs 0%, p = 0.0006 ). VEGF serum levels were significantly higher in responding patients. In contrast, baseline serum levels of HGF were significantly lower in responders. Neither VEGF nor HGF serum levels showed correlation with the presence of EMP. Baseline TNF-alpha serum levels were significantly lower in responding patients and in those without EMP. The serum levels of FGF-2 and IL-6 did not correlate with response to treatment or presence of EMP.     

Extracellular conserved cysteine forms an intersubunit disulphide bridge in the KCNK5 (TASK-2) K+ channel without having an essential effect upon activity

The functional channel unit of K(+) channels with two pore regions in tandem is thought to be a homodimer and it has been suggested that this dimeric structure occurs by interaction of an extracellular domain, the self-interacting domain. Interaction and functional assembly have been studied in some detail for KCNK1. It is proposed that a disulphide bond between highly conserved C69 residues of the self-interacting domain is formed which is essential for channel activity. We mutated C51, the equivalent residue in the pH-dependent KCNK5, to study its effect on channel function. Western analysis of proteins from cells expressing epitope-tagged KCNK5 and KCNK5-C51S was consistent with reduction-sensitive self-association of monomers dependent upon the presence of C51. Patch-clamp analysis of heterologously expressed KCNK5-C51S, however, revealed it was functional and indistinguishable in rectification properties and pH dependence from the non-mutated channel. The same result was found with KCNK5-C115S. It is concluded that the proposed disulphide bond between cysteine 51 residues of KCNK5 subunits does occur and preserves a dimeric structure in the detergent solubilized complex. Functional assays, on the other hand, suggest that such a disulphide bridge is not essential for correct functional expression.     

A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.