Lectin enhancement of the lipofection efficiency in human lung carcinoma cells.
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Poor transfection efficiency of human lung carcinoma cells by lipofection begs further development of more efficient gene delivery strategies. The purpose of this study was to determine whether lectins can improve the lipofection efficiency in lung carcinoma cells. A549, Calu3, and H292 cells grown to 90% confluence were transfected for 18 h with a plasmid DNA containing a beta-galactosidase reporter gene (pCMVlacZ) using lipofectin plus a lectin as the vector. Ten different lectins, which exhibit a wide range of carbohydrate-binding specificities, were examined for their abilities to enhance the efficiency of lipofection. The transfected cells were assessed for transfection efficiency by beta-galactosidase activity (units/microg protein) and % blue cells following X-Gal stain. Lipofectin supplemented with Griffonia simplicifolia-I (GS-I) yields largest enhancement of the lipofection efficiency in A549 and Calu3 cells (5.3- and 28-fold, respectively). Maackia amurensis gives the largest enhancement (6.5-fold) of lipofection efficiency in H292 cells. The transfection efficiency correlates with the amounts of DNA delivered to the nucleus. Binding of FITC-labeled GS-I and the enhancement of the lipofection efficiency by GS-I were inhibited by alpha-methyl-D-galactopyranoside, indicating an alpha-galactoside-mediated gene transfer to lung carcinoma cells. We conclude that lectin-facilitated lipofection is an efficient gene delivery strategy. Employment of cell type-specific lectins may allow for efficient cell type-specific gene targeting. (+info)
A spectroscopic assay for the analysis of carbohydrate transport reactions.
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A carbohydrate-transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or systems with high-affinity constants for sugars. (+info)
Interaction of the lacZ beta-galactosidase of Escherichia coli with some beta-D-galactopyranoside competitive inhibitors.
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1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (lacZ) beta-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg(2+) by Mn(2+) enhances both longitudinal and transverse relaxation of the methyl groups of the beta-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-beta-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn(2+) ion is located 8-9A (0.8-0.9nm) from the centroid of the trimethylammonium group and 9A (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (K(i) (o) and K(i) (+) respectively) for the isosteric ligands, beta-d-galactopyranosylbenzene and the beta-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=K(i) (+)/K(i) (o)) obtained with 2-(beta-d-galactopyranosyl)-naphthalene and the beta-d-galactopyranosylisoquinolinium ion at pH7 with Mg(2+)-enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg(2+)-enzyme can be described by Q=0.1(1+[H(+)]/4.17x10(-10))/1+[H(+)]/10(-8)). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg(2+)-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg(2+) as well as tyrosine-501. (+info)
Phosphorylation of HPr by the bifunctional HPr Kinase/P-ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion.
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We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion of methyl beta-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process. (+info)
DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex.
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The highly purified DNA Pol-alpha from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488-1492]. Binding of ConA and RCA to human recombinant DNA polymerase-alpha showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231-6237]. The catalytic polypeptide, DNA polymerase-alpha of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-alpha from embryonic chicken brain (ECB) contains an alpha-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-alpha reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567-573] by the treatment with methyl-alpha-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an alpha-galactose binding, 56kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-alpha as determined by immunostaining with the polymerase-alpha-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-alpha and a complete separation of polymerase complex and primase. (+info)
Molecular chaperone properties of serum amyloid P component.
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The selective binding of serum amyloid P component (SAP) to proteins in the pathological amyloid cross-beta fold suggests a possible chaperone role. Here we show that human SAP enhances the refolding yield of denatured lactate dehydrogenase and protects against enzyme inactivation during agitation of dilute solutions. These effects are independent of calcium ions and are not inhibited by compounds that block the amyloid recognition site on the B face of SAP, implicating the A face and/or the edges of the SAP pentamer. We discuss the possibility that the chaperone property of SAP, or its failure, may contribute to the pathogenesis of amyloidosis. (+info)
Evidence for binding protein-independent substrate translocation by the methylgalactoside transport system of Escherichia coli K12.
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Three genes, mgl A, B, and C, are required for active transport of substrate by the methylgalactose permease of E. coli K12. We report here that only two of these genes are required for substrate translocation, as seen by the ability or inability of isogenic mgl mutants (referred to as Tra+ and Tra minus, respectively) to grow on methyl-beta-D-galactopyranoside, supplied as sole carbon source. Individual mutants of both the Tra+ and Tra minus classes exhibited no detectable intracellular accumulation of methyl-beta-D-galactopyranoside; thus, the Tra+ phenotype cannot be explained by the mutants' levels of residual active transport. The phosphotransferase (Pts), the beta-galactoside (LacY), and the arabinose (Ara E and Ara F) transport systems are not required for substrate translocation by Tra+ cells. The Tra+ phenotype was identified with mutants defective in the mgl B, locus of the galactose-binding protein, by genetic complementation; the Tra minus phenotype was observed with both mgl A and mgl C mutants. The conclusion that the galactose-binding protein is not required for substrate translocation was supported by direct assays of the mgl mutants' binding protein activity. Mutants capable of translocation all showed reduced galactose-binding protein activity; mutants incapable of translocation exhibited binding protein activity equal to that of the mgl+ parent. (+info)
Regulation of the Escherichia coli methylgalactoside transport system by gene mglD.
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Constitutive activity of the methylgalactoside transport system of Escherichia coli K-12 is shown to result from mutation of a genetic locus distinct from the two previously described regulatory loci for this permease. Employing an autoradiographic procedure whereby constitutive and inducible cells can be differentiated, it is demonstrated that this locus, termed mglD, is 20% cotransducible with ptsF by bacteriophage P1. Selection for constitutive mutants among an inducible population yielded cells who mutations mapped in mglD. Cotransduction of mglD with mglB, minus C, and minus A, three genes required for activity of the methylgalactoside transport system, is 95, 88, and 81%, respectively. The results of recombination studies employing three and four factors indicate that the order of genes in this region is ptsF, mglD, B, C, A. (+info)