Microexudates from Cells Grown in Tissue Culture

Cellular substrata of known molecular structure and measurable dimensions can be constructed as transferred films from Langmuir troughs or as adsorbed films. In addition, large molecules in culture media form measurable adsorbates. With the techniques of ellipsometry and surface chemistry it is possible to characterize and measure (within ± 3A) as a function of several parameters a microexudate of molecular dimensions deposited when tissue cultured cells contact certain substrata. The selective attraction of substratum and cell for microexudate has been determined, and the time course of deposition in Eagle's medium is characterized by a rapid initial accretion of material. During this period, microexudate can diffuse several cell diameters and cannot be detected in the culture medium. In Eagle's medium the cells cannot be detached from glass surfaces by versene or trypsin unless the surface of cell or substratum is coated with certain molecules. Trypsin becomes adsorbed to cell surfaces, continues to be enzymatically active on the surface, and digests protein components of microexudate and substratum. Microexudate appears to be a complex mosaic of molecules (including protein) synthesized within or on the surfaces of cells and secreted by cells or transferred from their surfaces to specific substrata. It is proposed that this mosaic plays, on the molecular level, a significant role in cell-to-cell interactions, cell locomotion and adhesion, and the selective application and spreading of cells on various surfaces.     

Production of the Milk Agent in Cultures of Mouse Mammary Carcinoma

Thin sections of tissue cultures grown from tumors of the RIII high-breast-cancer strain mice were studied in the electron microscope. These tissues contain an abundance of particles whose morphology is consistent with biophysical measurement of the milk agent. These particles, found only extracellularly in our cultures, are formed at the cell membrane. The process of formation, as reconstructed from sections, appears to include a thickening and protrusion of the cell membrane which then evolves gradually into a dense sphere and separates from the cell in much the same manner as does influenza virus. The contents of the newly formed body are later rearranged to form a nucleoid within a membranous sac.     

Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells

1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.     

Fluoride metabolism in Acacia georginae Gidyea

1. The metabolism of fluoride in seedlings and small plants of Acacia georginae has been studied with the idea of finding the conditions under which the plant makes fluoroacetate in the laboratory. 2. Individual seedlings vary in the extent to which they take up fluoride and convert it into a form other than inorganic which is here called `organic' fluoride, F(org.). The differences between the toxicity of A. georginae Gidyea trees may therefore be genetic in origin. 3. The uptake of fluoride from solutions 0·525–1·05mm (10–20p.p.m.) was not large. In 1–4 days it reached 8 p.p.m. in the aerial parts and 16 p.p.m. in the roots. Unlike the distribution of the halogen in grass, total fluoride was greater than inorganic fluoride. It was almost a rule that more `organic' fluoride was present in the roots than in the aerial parts. 4. With higher concentrations of fluoride 10·5–15·75mm (200–300p.p.m.) much larger amounts of fluoride were taken up, especially by the roots, and much more apparent organic fluoride was formed. 5. pH had a large influence upon the intake, this being lowest at an initial pH8·4 and highest at pH4·0. The pH outside this range was not investigated.     

Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23.

Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cell line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cells. These initial observations are now extended through an expanded study of EBNA- and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative B-lymphoma cell line BL41 and the EBV-positive Burkitt's lymphoma cell line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2- and LMP1-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-activity relationships in the in vitro modulation of rat hepatic microsomal androst-4-ene-3,17-dione hydroxylase activities by derivatives of 5 alpha- and 5 beta-androstane

The relationships between structure and inhibitory potency toward microsomal cytochrome P-450 (P-450)-mediated androst-4-ene-3,17-dione hydroxylase activities were investigated in rat liver with a series of 5 alpha- and 5 beta-androstane derivatives. 5 beta-Reduced steroids (containing a cis-A/B ring junction) were more potent inhibitors than the 5 alpha-reduced epimers (containing a trans-A/B ring junction) except in the case of the 17 beta-hydroxy-substituted derivatives. The most effective inhibitor was 5 beta-androstane-3 beta-ol which exhibited I50 values of 7 and 27 microM against androstenedione 16 alpha- and 6 beta-hydroxylase activities, which are catalysed by P-450 IIC11 and IIIA2, respectively. In general, these two pathways of steroid hydroxylation were more susceptible to inhibition than the 7 alpha- and 16 beta-hydroxylase pathways. The 7 alpha-hydroxylase enzyme (P-450 IIA1) was only inhibited by 5 beta-reduced steroids that contained an oxygenated function at C17. All of the test compounds elicited type I spectral binding interactions with P-450 in oxidised microsomes. The most effective steroid inhibitors generally exhibited the greatest capacity to interact with P-450. Additional studies with one of the more potent compounds, 5 beta-androstane-3 beta-ol-17-one, revealed that the inhibition kinetics were competitive and that preincubation of the inhibitor with NADPH-supplemented microsomes prior to substrate (androstenedione) addition decreased the extent of inhibition observed. These findings are consistent with the assertion that the inhibition of hepatic steroid hydroxylases by 5 beta-androstanes involves an effective competitive interaction with the steroid substrate at the P-450 active site. Since the relative overproduction of 5 beta-reduced metabolites of certain androgens has been reported in clinical conditions, such as androgen insensitivity, it now appears important to investigate the hepatic drug oxidation capacity of patients with hormonal abnormalities.     

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.     

Differential Binding Properties of the Peripheral-Type Benzodiazepine Ligands [3H]PK 11195 and [3H]Ro 5-4864 in Trout and Mouse Brain Membranes

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.