Purification and characterization of yeast mitochondrial initiation factor 2

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.     

Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.     

Modulation of lipid synthesis,apolipoprotein biogenesis,and lipoprotein assembly by butyrate

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [14C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P < 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [14C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [14C]oleic acid as a precursor, butyrate significantly (P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [35S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations.     

Peripheral benzodiazepine receptor ligand-melphalan conjugates for potential selective drug delivery to brain tumors

To gain insight into the strategy to target PBR ligand-drug conjugates to brain tumors, novel N-imidazopyridinacetyl-melphalan conjugates and the corresponding ethyl esters have been prepared and evaluated for their cytotoxicity in melphalan-sensitive human (SF126, SF188) and rat (RG-2) glioma cell lines. These conjugates exhibited PBR binding affinity with IC(50) values ranging from 57 and 2614 nM. By a computational approach it can be predicted that these conjugates possess significant brain penetration. The stability of the conjugates in 0.05 M phosphate buffer at pH 7.4 and, in some cases, in dilute human serum solution was determined. All the ethyl ester derivatives were stable in 0.05 M phosphate buffer at pH 7.4 and their half-lives exceeded 28 h. Conversely, under the same conditions, the corresponding acids were found to undergo a fast cleavage within a few minutes. HPLC-MS analysis of the mixture from degradation in buffer and physiological medium of the representative cases allowed the identification of their main degradation products. A plausible degradation pathway accounting for the available experimental data is presented.     

Platinum toxicity and gene expression in Xenopus embryos: analysis by FETAX and differential display

Since the level of platinum in the environment is destined to increase, because of its use in vehicle catalytic converters, the toxicity of platinum needs further investigation. In this study, the frog embryo teratogenesis assay-Xenopus (FETAX) was used to compare the embryotoxicity and teratogenicity of two common platinum species, (NH4)2PtCl4 and (NH4)2PtCl6. The uptake rates of the two platinum species were studied, and also their effects on the expression of genes encoding metallothionein and heat-shock protein 70, which are known to be induced by several stress factors. In addition, the differential display technique was used to search for genes that were specifically induced by platinum. A gene for the type I collagen alpha-chain and a novel gene were identified.     

Expression of GM3 microdomains on the surfaces of murine fibroblasts correlates with inhibition of cell proliferation

The expression and surface distribution of monosialoganglioside GM3 on the plasma membranes of NIH3T3 fibroblasts cultured at semiconfluence were analyzed by immunofluorescence as well as by immunogold electron microscopy on thin sections and surface replicas. The GM3 expression was highly variable from cell to cell and the distribution of the ganglioside on the positive cells appeared punctate. Quantitative immunogold electron microscopy showed the existence of well-defined GM3 clusters of different sizes scattered all over the cell surfaces. Double immunofluorescence analysis of 5-bromo-2’-deoxyuridine incorporation to identify proliferating cells and of GM3 expression indicated that most of the GM3-positive cells appear unable to synthesize DNA and demonstrated a growth-dependent expression of GM3. Accepted: 16 November 1999     

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56^lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56^lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56^lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56^lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56^lck, we analyzed this association in U937, a CD4+and p56^lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56^lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.     

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56Ick in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56Ick complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56Ick were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56Ick complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56Ick, we analyzed this association in U937, a CD4 + and p56Ick negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56Ick. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.