Loss of Hda activity stimulates replication initiation from I-box, but not R4 mutant origins in Escherichia coli

Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA^ATP specific sites (I-boxes, τ-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC . Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA^ATP containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA^ATP specific I-boxes. However, competition between I-box mutant and wild-type origins , revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA^ATP/DnaA^ADP ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.     

Altered growth kinetics precede calcium-induced differentiation in mouse epidermal cells

Summary Primary cultures of newborn mouse epidermal cells proliferate rapidly and with a high growth fraction for several months when grown in medium with low calcium (0.02 to 0.1 mM). Addition of calcium to levels generally used in culture medium (1.2 mM) was followed by rapid changes in the pattern of proliferation. By using a combination of technics (a stathmokinetic method, autoradiography, [³H]thymidine incorporation into DNA, DNA flow cytometry) it was found that cell flux was blocked for 5 to 6 h, followed by a short rise in the mitotic rate at 10 h, and a gradual fall in all growth parameters until about 32 h after the calcium switch. There was no accumulation of cells in any particular cell cycle phase. The results indicate that the calcium switch is followed by a strong reduction in cell flux from G₁ whereas the majority of the cells that had left G₁ at the time of the switch completed one cell division before cessation of all proliferative activity. Both before and after the switch the primary epidermal cultures consisted of one diploid and one tetraploid G₁ DNA stemline that seemed to react in the same way to calcium. This work reported in this paper was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union Against Cancer (K. E.). The project was supported by funds partly provided by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, Bethesda, MD, under contract N01-C0-65341 (International Cancer Research Technology Transfer) and partly by the International Union Against Cancer (O.P.F.C.).     

Immunocytochemical evidence for a paracrine interaction between GIP and GLP-1-producing cells in canine small intestine

Glucagon-like-peptide 1 (GLP-1) released from the intestine is considered to be an important incretin. We have recently demonstrated that glucose-dependent insulinotropic peptide (GIP) stimulated GLP-1 secretion from canine ileal L cells in culture. To investigate further the interplay between GLP-1- and GIP-secreting cells, we set out to determine the exact location and abundance of both cell types throughout the canine intestine. Canine small intestine was subdivided into 15-20 segments and investigated by immunocytochemistry with computer-assisted imaging. The abundance of GIP-, GLP-1- and somatostatin-immunoreactive cells was determined. GIP-secreting K cells were equally distributed in duodenum and jejunum, with the GLP-1-secreting L cells concentrated in the jejunum (5% duodenum, 73% jejunum and 22% ileum). These results indicated that the middle section of the small intestine containing 69% of the K cells also contained 51% of the L cells. Double immunostaining confirmed this overlap and furthermore over 30% of the L cells in this region were found adjacent to K cells. These results suggest the existence of a paracrine interaction between the K and L cells and indicate the importance of the jejunum in the regulation of insulin release by enteric-derived incretins.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Field experiments on seed dispersal by wind in ten umbelliferous species (Apiaceae)

This report presents data from experiments on seed dispersal by wind for ten species of the family Apiaceae. Seed shadows were obtained in the field under natural conditions, using wind speeds between four and ten m/s. The flight of individual seeds was followed by eye, and seed shadows were acquired, with median distances varying from 0.7 to 3.1 m between species. Multiple regression models of wind speed and seed weight on dispersal distance were significant for six out of ten species; wind speed had significant effects in seven cases, but seed weight only once. A good correlation between mean terminal falling velocity of the seeds of a species and median dispersal distance, indicates the promising explanatory power that individual terminal velocity data might have on dispersal distance, together with wind speed and turbulence. The theory that seeds that seem to be adapted to wind dispersal travel much longer distances than seeds that have no adaptation was tested. Flattened and winged seeds were indeed found to be transported further by wind, but not much further. Moreover, the species with wind-adapted seeds were also taller, being an alternative explanation since their seeds experienced higher wind speeds at these greater heights. Furthermore, flattened and winged seeds were disseminated from ripe umbels at lower wind speeds in the laboratory. This means that the observed difference in dispersal distance would have been smaller when species specific thresholds for wind speed were incorporated in the field experiments. We argue therefore, that seed morphology is not always the best predictor in classifying species in groups with distinctly different dispersal ability.     

ABCG transporters export cutin precursors for the formation of the plant cuticle

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Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.