Establishment of a variety of human bone marrow stromal cell lines by the recombinant SV40-adenovirus vector.

Various human bone marrow stromal cell lines were established from the adherent cell populations by introduction of the recombinant SV40-adenovirus vector with an infection or electric poration procedure. As compared with DNA transfection, the vector introduction was able to immortalize the cells with more than 100 times higher efficiency. Morphological and cytochemical analyses indicated that various cloned cell lines with different properties were isolated by the vector introduction. All the established cell lines expressed SV40 large T antigen. These results provided the evidence indicating that the recombinant SV40-adenovirus vector was a useful tool to establish a variety of cell lines with different biological activities from human bone marrow stroma.     

Preferred ambient temperature for old and young men in summer and winter

To investigate the effects of age on thermal sensitivity, preferred ambient temperature (T _pref) was compared between old (71–76 years) and young (21–30 years) groups, each consisting of six male subjects in summer and winter. The air temperature (T _a) was set at either 20° C or 40° C at commencement. The subject was directed to adjust theT _a for 45 min by manipulating a remote control switch to the level at which he felt most comfortable. In the older group, theT _pref was significantly lower in trials starting at 20° C than that starting at 40° C in summer. The fluctuation ofT _pref (temperature difference between maximum and minimumT _a during the last 10 min) was significantly wider in the older group in both summer and winter. Repetition of the same experiment on each subject showed a poorer reproducibility ofT _pref in the older group than in the younger group in summer. Tympanic and esophageal temperatures of the older group kept falling throughout the trial starting at 20° C in summer. These results suggest that thermal sensitivity is decreased with advancing age and that thermal perception in the elderly, especially to cold, is less sensitive in summer.     

A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion-exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross-reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemically visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent M(r) of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent M(r) of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50-kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor-stimulated astroblasts, but not neuronal cells.     

Activation of immunoglobulin control elements in trasgenic mice

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EkCAT, contains the Ig heavy chain enhancer (E) and the light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, EkCAT, CAT is under the control of the promoter alone. E and relative activity were assessed by CAT assay. In EkCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In EkCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon- (IFN-) increased CAT expression to varying extents in cells derived from EkCAT mice but not in spleen cells prepared from EkCAT mice. Thus, the presence of E, in addition to the promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EkCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN- caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.This work has been submitted in partial fulfillment of the requirements for the doctoral degree from the George Washington University.     

Construction of shuttle vectors derived fromAcetobacter xylinum for cellulose-producing bacteriumAcetobacter xylinum

Summary Shuttle vector pUF106 was constructed by ligation ofAcetobacter xylinum plasmid pFF6 toEscherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. pUF106 transformed cellulose-producingA. xylinum ATCC10245 as well asE. coli JM109.     

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.     

Phospholipid monolayers at the triolein-saline interface: production of microemulsion particles and conversion of monolayers to bilayers

Interfacial tensions of phospholipid monolayer at the triolein (TO)-saline interface were measured. The adsorption isotherms and the interfacial pressure-molecular area curves were evaluated on the basis of the measurements. Phosphatidylcholine (PC) forms a highly condensed monolayer, with a large lateral attractive interaction; phosphatidylethanolamine (PE) and phosphatidylserine (PS) form expanded monolayers with smaller lateral interaction energies. At the lowest interfacial tension (the highest interfacial pressure), the mole fractions of PC, PE, and PS in the monolayers are estimated as 0.95, 0.73, and 0.88, respectively. Therefore, PC forms the most stable monolayer at the interface. These results are consistent with the finding that the stable TO particles in aqueous solution were produced by using PC as an emulsifier, and PE and PS did not stabilize the particles. The phase diagram of TO and PC mixtures in saline obtained from theoretical considerations predicts the equilibrium conversion of the monolayers on TO particles to bilayers. This process may be closely related to the transformations of very low density lipoproteins and chylomicrons to high-density lipoproteins in plasma. The particle sizes of the emulsion are calculated theoretically as a function of PC mole fraction in the TO-PC mixture and compared with the experimental values obtained from quasi-elastic light scattering (QLS) measurements.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.