Continuous measurement of changes in intracellular calcium concentration in mouse splenic T cells attached to a glass substrate

Mitogen- and isoproterenol-induced changes of [Ca²⁺]_i in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak® (3.5 µg/cm²). The cells were then loaded with fluorescent dye (2 µg/ml of fura2-AM or fluo3-AM) and changes in [Ca²⁺]_i in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca²⁺]_i demonstrated that concanavalin A (Con A, 2 or 5 µg/ml) caused an increase in [Ca²⁺]_i that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05–5 µg/ml) had an EC₅₀ of 0.3 µg/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca²⁺]_i. In single cell studies, the increase in [Ca²⁺]_i in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together, these data demonstrate that Cell Tak® can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells.     

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.     

Structures of wild-type and mutant signal sequences of Escherichia coli ribose binding protein.

The structure of a chemically synthesized 25-residue-long functional signal peptide of Escherichia coli ribose binding protein was compared with that of a nonfunctional mutant-signal peptide using circular dichroism and two-dimensional 1H NMR in solvents mimicking the amphiphilic environments. The functional peptide forms an 18-residue-long alpha-helix starting from the NH2-terminal region and reaching to the hydrophobic stretch in a solvent consisting of 10% dimethylsulfoxide, 40% water, and 50% trifluoroethanol (v/v). The nonfunctional mutant peptide, which contains a Pro at position 9 instead of a Leu in the wild-type peptide, does not have any secondary structure in that solvent but forms a 12-residue-long alpha-helix within the hydrophobic stretch in water/trifluoroethanol (50:50, v/v) solvent. It seems that the Pro-9 residue in the nonfunctional peptide disturbs the helix propagation from the hydrophobic stretch to the NH2-terminal region. Because both of these peptides have stable helices within the hydrophobic stretch, it may be concluded that the additional 2 turns of the alpha-helix in the NH2-terminal region of the wild-type signal peptide is important for its function.     

Production of panose from maltose by intact cells of Aureobasidium pullulans

Panose, a major component of isomalto-oligosaccharides, was selectively produced from maltose using transglucosylation reaction catalyzed by intact cells of Aureobasidium pullulans. When 50 %(w/v) maltose was used as a substrate, the maximum concentration of panose accumulated in the final reaction mixture was about 50 %(w/w) after 120 hr reaction at 55 °C.     

Expression of the Lactobacillus casei lactate dehydrogenase gene in Bacillus subtilis

Summary A gene for allosteric lactate dehydrogenase (LDH) of Lactobacillus casei ATCC393 was transferred into Bacillus subtilis. The LDH was produced in a growth-associated type, and comprised up to 40 % of the total cellular protein. The maximum specific activity in the transformant was 208 U/mg protein which was approximately 16 times higher than in L. casei or in the previously constructed Escherichia coli transformant.     

Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens

Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl^-1 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm^-2 cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of Sweet Gem were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter--glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl^-1 BA and 200 M -hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl^-1 BA 250 mgl^-1 carbenicillin, and 100 mgl^-1 kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl^-1 BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.     

Production of high fructo-oligosaccharide syrup with two enzyme system of fructosyltransferase and glucose oxidase

Summary A novel two enzyme system of fructosyltransferase and glucose oxidase to enhance the content of the net fructo—oligosaccharide (FOS) fractions in the industrial production of FOS syrup from sucrose was devised. The net FOS content in the commercial FOS syrup has been limited only to 55–60 % due to the accumulation of glucose which acts as a feedback inhibitor of the fructosyltransferase. By supplementing glucose oxidase to the conventional FOS reaction system, we could convert the glucose to gluconic acid readily separable from neutral sugars by simple ion exchange operation in the next step. The simultaneous removal of glucose was proved effective in proceeding the reaction by fructosyltransferase further by relieving the product inhibition caused by glucose. By this way, we could raise the net FOS content as high as 90 %.     

A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.     

Population differentiation inSpartina patens: Water potential components and bulk modulus of elasticity

Pressure-volume technique was utilized to evaluate salinity response among three populations ofSpartina patens (Ait.) Muhl. from Louisiana Gulf coast marshes. Plants were subjected to salinities of 85 and 425 mol m^?3 for 77 d in a greenhouse. Ψ_w and Ψ_π decreased in all populations in response to increases in salinity. There were 32% decrease in Ψ_sat, 42% decrease in Ψ_tlp in response to salinity changes from 85 to 425 mol m^?3 in the Ferblanc population. Similarly, there were 35% and 41% decrease in Ψ_sat in the Clovelly and Lake Tambour populations, respectively. All populations showed the ability to adapt to the increased salinity as was evidenced by osmotic adjustment. However, the Lake Tambour population appeared to have superior ability to adapt to high salinity through having a significantly lower osmotic potential at saturation (Ψ_sat), osmotic potential at turgor loss point (Ψ_tlp), and maximum turgor potential (Ψ_P(max)) compared to other populations. Ferblanc and Clovelly populations revealed the ability to adapt to saline environments to a lesser extent as compared to the Lake Tambour population. Results indicate that there is a potential for selection of superior strains ofSpartina patens for use in marsh restoration projects aiming at prevention of wetland loss in certain coastal areas.     

Interleukin-4 plus tumor necrosis factor α augments the antigenicity of melanoma cells

Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor α (TNF) or interferon γ (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3⁺CD4⁺ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.