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111.
The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture. 相似文献
112.
Final steps in the synthesis of platelet activating factor (PAF) occur via two enzymatic reactions: the acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine by a specific acetyltransferase or the transfer of the phosphocholine base group from CDP-choline to 1-alkyl-2-acetyl-sn-glycerol by a dithiothreitol (DTT)-insensitive cholinephosphotransferase. Our studies demonstrate that rat kidney inner medulla microsomes synthesize PAF primarily via the DTT-insensitive cholinephosphotransferase since the specific activity of this enzyme is greater than 100-fold higher than the acetyltransferase. The two cholinephosphotransferases that catalyze the biosynthesis of phosphatidylcholine and PAF have similar Mg2+ or Mn2+ requirements and are inhibited by Ca2+. Also topographic experiments indicated that both activities are located on the cytoplasmic face of microsomal vesicles. PAF synthesis was slightly stimulated by 10 mM DTT, whereas the enzymatic synthesis of phosphatidylcholine was inhibited greater than 95% under the same conditions. The concept of two separate enzymes for PAF and phosphatidylcholine synthesis is further substantiated by the differences in the two microsomal cholinephosphotransferase activities with respect to pH optima, substrate specificities, and their sensitivities to temperature, deoxycholate, or ethanol. Study of the substrate specificities of the DTT-insensitive cholinephosphotransferase showed that the enzyme prefers a lipid substrate with 16:0 or 18:1 sn-1-alkyl chains. Short chain esters at the sn-2 position (acetate or propionate) are utilized by the DTT-insensitive cholinephosphotransferase, but analogs with acetamide or methoxy substituents at the sn-2 position are not substrates. Also, CDP-choline is the preferred water-soluble substrate when compared to CDP-ethanolamine. Utilization of endogenous neutral lipids as a substrate by the DTT-insensitive cholinephosphotransferase demonstrated that sufficient levels of alkylacetylglycerols are normally present in rat kidney microsomes to permit the synthesis of physiological quantities of PAF. These data suggest the renal DTT-insensitive cholinephosphotransferase could be a potentially important enzyme in the regulation of systemic blood pressure. 相似文献
113.
Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed. 相似文献
114.
B cell growth-promoting activity of recombinant human interleukin 4 总被引:23,自引:0,他引:23
T Defrance B Vanbervliet J P Aubry Y Takebe N Arai A Miyajima T Yokota F Lee K Arai J E de Vries 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(4):1135-1141
Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells. 相似文献
115.
Equilibrium binding parameters of an autoimmune monoclonal antibody specific for double-stranded DNA 总被引:2,自引:0,他引:2
Two techniques have been developed to estimate binding parameters for Jel 241 under equilibrium conditions. Jel 241 is an autoimmune monoclonal antibody derived from an NZB/NZW mouse which binds to double-stranded DNA. Thermal denaturation profiles of poly[d(AT)] were measured in the presence of increasing concentrations of IgG Jel 241. From these data it was estimated that the IgG occludes 12 base-pairs on duplex DNA, and the binding to double-stranded DNA was at least four orders of magnitude greater than to single-stranded DNA. In addition, intrinsic association constants (K(O)) were measured by a gel filtration technique for the interaction of both Fab and IgG Jel 241 to native calf thymus DNA. K(O) for the IgG was only 60-fold greater than for the Fab fragment for which K(O) was 4.4 X 10(4) M-1 at an NaCl concentration of 150 mM. Also, K(O) for the Fab increased dramatically with decreasing ionic strength, suggesting that there are four phosphates involved in the interaction. These techniques should be applicable to most autoimmune antibodies which bind to nucleic acid polymers. 相似文献
116.
J C Lee M J Dimartino B J Votta N Hanna 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(10):3268-3274
Adjuvant-induced arthritis (AA) in rats is associated with a number of immunologic abnormalities which include a marked decrease in spleen cell mitogenic responses. In this study we investigated the altered production of interleukins in arthritic rats and evaluated the effects of auranofin treatment on disease progression and aberrant interleukin production. The capacity of the AA rat spleen cells to produce interleukin (IL) 2 and IL-3 was found to decrease during the development of the arthritic lesion, with maximum suppression occurring 16 to 17 days after adjuvant injection. In contrast, the production of IL-1 by splenic adherent cells from arthritic rats was markedly increased. Prophylactic treatment of AA rats with auranofin resulted in a slight reduction in paw edema, a complete normalization of the depressed IL-2 production, and a reduction of the elevated IL-1 production, but had no effect on the depressed IL-3 production. In contrast, auranofin administered to normal rats, in the same dosing regimen, did not affect interleukin production. Therapeutic administration of auranofin to AA rats with established disease resulted in normalization of IL-1 production without affecting the suppressed IL-2 and IL-3 levels. In contrast, while indomethacin treatment effectively decreased paw edema, it did not appreciably affect the systemic aberrant interleukin production. Taken together, these results suggest that disease-associated abnormalities in interleukin production may be mediated by different mechanisms with differential sensitivity to the effects of the disease-modifying drug auranofin. Furthermore, defining the relationship between drug-mediated normalization of aberrant immune parameters and clinical improvement will provide a basis for the elucidation of the mechanism of action of disease-modifying antiarthritic drugs as well as for assessment of clinical efficacy of drug treatment. 相似文献
117.
Two platelet-activating factor (PAF) analogs containing a methyl group at C2 of the glycerol moiety were synthesized, and some of their biochemical properties were investigated. 1-O-Hexadecyl-2-C,O-dimethyl-rac-glycero-3-phosphocholine (2-methyl-2-methoxy PAF) was prepared in a synthetic scheme beginning with the etherification of 2-methylpropen-1-ol. A reaction sequence involving hydroxylation, tritylation, alkylation, and detritylation afforded 1-O-hexadecyl-2-C,O-dimethyl-rac-glycerol, which was converted into the phosphocholine. A 2-lyso derivative of this PAF analog (2-methyl-lyso PAF) was synthesized from 1-O-hexadecyl-2-C-methyl-3-O-trityl-rac-glycerol. Benzylation followed by detritylation gave 1-O-hexadecyl-2-C-methyl-2-O-benzyl-rac-glycerol, which was converted into the phosphocholine compound. Hydrogenolysis afforded 1-O-hexadecyl-2-C-methyl-rac-glycero-3-phospholine (2-methyl-lyso PAF). The 2-methyl-lyso PAF analog served as a substrate for the acetyl-CoA-dependent acetyltransferase that acetylates 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine. However, 2-methyl-lyso PAF did not have a significant effect on the activities of a CoA-independent transacylase or of the acetylhydrolase that inactivates PAF, and thus does not appear to be a substrate or an inhibitor, respectively, for these enzymes. In addition, this analog exhibited only one-half of the antitumor activity of rac-1-O-alkyl-2-methoxy-rac-glycero-3-phosphocholine in human leukemic (HL-60) cells, and elicited no hypotensive response in rats and no platelet-activating activity.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
118.
All 503 fine needle aspirations (FNAs) of the breast performed over a three-year period at the Medical Center Hospital of Vermont were analyzed. There were 93 aspirates diagnosed as "positive," all of which were from patients eventually shown to have cancer. However, there were 38 patients with primary carcinoma in which the FNA was not diagnosed as positive, for a diagnostic failure rate of 31.4%. In order to determine the possible effect of technique as practiced by an experienced aspirator in diminishing such diagnostic failures, we compared 190 aspirates obtained by a single individual with 193 aspirates obtained by 15 individuals in the same community. For the single experienced aspirator, the technical failure rate was 9.8% whereas in the group with many aspirators it was 45.9%. This finding confirms that, although fine needle aspiration of the breast is considered easy to perform, skill on the part of the aspirator is important for satisfactory results. 相似文献
119.
Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM). 相似文献
120.
Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity 总被引:2,自引:0,他引:2
A R Clarke C J Smith K W Hart H M Wilks W N Chia T V Lee J J Birktoft L J Banaszak D A Barstow T Atkinson 《Biochemical and biophysical research communications》1987,148(1):15-23
Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate. 相似文献