Role of lipopolysaccharide and outer membrane protein of Escherichia coli K-12 in the receptor activity for bacteriophage T4.

Lipopolysaccharide isolated from Escherichia coli K-12 did not inactivate phage T4, although the cell envelopes with 1% sodium deoxycholate resulted in the release of cytoplasmic membrane proteins, 70% of the lipopolysaccharide, and almost all of the phospholipid. The reconstitution of phage receptor activity was achieved from deoxycholate-soluble and -insoluble fractions by dialysis against a solution of magnesium chloride. Lipopolysaccharide was the only essential component in the deoxycholate-soluble fraction. PhageT4-resistant mutants YA21-6 and YA21-82, having defects in the deoxycholate-soluble and -insoluble fractions, respectively, were isolated. The deoxycholate-soluble fraction of YA21-6 possessed heptoseless lipopolysaccharide, and this defect was responsible for the phage resistance. The deoxycholate-insoluble fraction of YA21-82 lacked outer membrane protein O-8. The addition of O-8 to this fraction together with the wild-type lipopolysaccharide resulted in the appearance of the receptor activity. Furthermore, the reconstitution was successfully achieved with only O-8 and the wild-type lipopolysaccharide, indicating that O-8 was an essential component in the deoxycholate-insoluble fraction.     

Dynamic sweating response of man to infrared irradiation in various spectral regions

In an attempt to detect differences in the thermal effect of infrared irradiation of different wavelengths, transient sweating response to infrared irradiation in various spectral regions was examined. In Series 1, the ventral or dorsal surface of the nude subject was irradiated repetitively for a period of 4 min (2 min on, 2 min off) by each of three kinds of infrared heaters with main emissivity in near-infrared (NIR; 0.7–2.8 m), intermediate-infrared (MIR; 1.5–5.8 m), and far-infrared (FIR; 2.8–25 m) regions. The sweating response on a non-irradiated area tended to be the greatest with MIR, while the magnitude of the sweating response on the irradiated area showed no consistent differences among various wavelengths. The results infer that MIR stimulated cutaneous thomoreceptors most effectively, while its direct effect on local sweat gland activity was minimal. In Series 2, the effects of 9–12 min irradiations in more restricted ranges of wavelength were compared by the combination of the three kinds of heaters with filters (translucent to wavelength ranges of 1.3–2.7, 2.7–3.5, 3.6–8.0 m, respectively). The sweating response on a remote area was predominantly greater with the range of 2.7–3.5 m than with the other wavelength ranges, while the local effect on sweating was minimal with this range. The results of Series 2 reinforce those of Series 1, indicating that the degree of stimulation of cutaneous thermoreceptors and of direct thermal effect on sweat gland activity differ with spectral regions incident on the skin, thus affecting local and remote effects on the sweating response.     

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.     

Inhibition of actin filament depolymerization by the Dictyostelium 30,000-D actin-bundling protein

We have studied the effect of the Dictyostelium discoideum 30,000-D actin-bundling protein on the assembly and disassembly of pyrenyl-labeled actin in vitro. The results indicate that the protein is a potent inhibitor of the rate of actin depolymerization. The inhibition is rapid, dose dependent, and is observed at both ends of the filament. There is little effect of 30-kD protein on the initial rate of elongation from F-actin seeds or on the spontaneous nucleation of actin polymerization. We could detect little or no effect on the critical concentration. The novel feature of these results is that the filament ends are free for assembly but are significantly impaired in disassembly with little change in the critical concentration at steady state. The effects appear to be largely independent of the cross-linking of actin filaments by the 30-kD protein. Actin cross-linking proteins may not only cross-link actin filaments, but may also differentially protect filaments in cells from disassembly and promote the formation of localized filament arrays with enhanced stability.     

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.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Studies of the sites of intracellular degradation of apolipoprotein B in Hep G2 cells.

We previously reported that treatment of Hep G2 cells with oleate significantly increased apolipoprotein B (apoB) secretion by reducing early intracellular degradation of nascent apoB. In the current study, inhibitors of secretory protein transport (brefeldin A and monensin), cell fractionation studies, and protease protection assays were utilized to determine the location of apoB degradation and to better define the mechanism whereby oleate treatment reduces nascent apoB intracellular degradation. When cells were treated with brefeldin A, which blocks endoplasmic reticulum (ER) to Golgi protein transport, apoB degradation continued in control cells, suggesting that apoB is degraded in the ER. When oleate-treated cells were blocked with brefeldin A, oleate failed to protect apoB from intracellular degradation. The effects of brefeldin A were not due to effects on lipid synthesis as brefeldin A did not inhibit the synthesis of triglyceride, phospholipid, free cholesterol, or cholesteryl ester in control cells and did not prevent the increases in triglyceride (14-fold) and phospholipid (1.4-fold) synthesis seen in oleate-treated cells. Simultaneous treatment of cells with brefeldin A and nocodazole, which inhibits retrograde transport of proteins from Golgi to ER, added to the evidence for the ER as the site of apoB degradation. This conclusion received further support from experiments in which cells were treated with monensin, a Na+ ionophore which halts protein secretion at the level of the trans-Golgi network. Early degradation of nascent apoB (between 10 and 20 min of chase) was observed in monensin-treated cells, but then cellular apoB degradation ceased and apoB was stable during the remaining chase period. More apoB accumulated in the Golgi of cells that had been treated with oleate and monensin. These results suggest that ER degradation occurs in monensin-treated cells, but then stops as apoB is transferred to the Golgi. The results obtained in whole cells were confirmed in studies using isolated ER and Golgi, which indicated that ER contains a proteolytic activity which degrades apoB, in vitro, whereas Golgi does not. ApoB degradation in isolated ER was not reduced by pretreatment with oleate. Finally, protease protection assays carried out with isolated microsomes indicated that a majority of the apoB in both control or oleate-treated HepG2 cells was located on the cytosolic side of the membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues.

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.