Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor

Incubation of several human tumor cell lines with human interferon-gamma (IFN-gamma) increased the specific binding of subsequently added 125I-labeled recombinant human tumor necrosis factor (TNF). A similar increase in TNF binding was seen in murine L929 cells after incubation with murine IFN-gamma, but not after incubation with human IFN-gamma. Increased TNF binding to cells incubated with IFN-gamma was due to an increase in the number of TNF receptors, with no demonstrable change in binding affinity. In one out of two human cell lines tested, IFN-alpha and IFN-beta also produced increased TNF binding, albeit with a lower efficacy than IFN-gamma. A maximal increase in TNF binding was seen after about 6 to 12 hr of incubation with IFN. Increased TNF binding due to enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity seen in some tumor cell lines after INF treatment. Modulation of TNF receptor expression by IFN may also influence other biological activities of TNF.     

Interferences and specificity of the 1-aminocyclopropane-1-carboxylic Acid assay with the hypochlorite reagent

1-Aminocyclopropane-1-carboxylic Acid (ACC), the immediate precursor of ethylene is routinely assayed by converting it into ethylene with NaOCl, and the ethylene liberated is then determined by gas chromatography (MCC Lizada, SF Yang 1979 Anal Biochem 100: 140-145). However, certain materials which may be present in crude plant extracts or in enzyme reaction mixtures interfere with this assay procedure. Mono, and di-alkyl amines cause poor yields of ethylene from ACC. Ethanol in the presence of NH₃ or amines but in the absence of ACC can produce ethylene under the assay procedure. The characteristics of these interfering reactions were studied and precautions to avoid these problems are suggested. Recovery of ACC during its extraction and purification from plant extracts were tested and are discussed.     

Insulin biosynthesis in the bullhead, Ictalurus nebulosus, and the effect of temperature

Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [(3)H]leucine into proteins of the principal islet. The tissue was incubated for 6-15h in Krebs-Ringer bicarbonate buffer with [(3)H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22 degrees C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor-product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12 degrees C, [(3)H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12 degrees C as was found after incubation at 22 degrees C. When the temperature was lowered from 22 degrees to 12 degrees C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22 degrees C. When the temperature was raised from 12 degrees to 22 degrees C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12 degrees C. No qualitative difference in the incorporation of [(3)H]leucine into proinsulin and its conversion into insulin at 12 degrees and 22 degrees C could be demonstrated between islet tissue from fish acclimated to less than 12 degrees C or to 22 degrees C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12 degrees C.     

The dye-exclusion test for cell viability: Persistence of differential staining following fixation

Summary A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differneces that are retained following aldehyde fixation. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada.     

An improved method of eliminating fungal contamination from monolayer cell cultures

A method is described for eliminating fungal and other forms of contamination from valuable monolayer cell cultures. The method employs the following sequence of operations: several changes of medium, trypsinization and removal of cells to coverglasses in appropriate tubes with medium plus amphotericin B (Fungizone) or other antibiotic, removal of coverglasses to new tubes with medium plus antibiotic, and removal in a few days to a new culture vessel without antibiotic. If microorganisms do not show up in 3–5 days, they have been eliminated. Greater success is achieved by using the same method with coverglass fragments in small culture wells.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine.     

Ethylene production by young Aranda orchid flowers and buds

A high rate of ethylene production was observed in buds and young flowers of Aranda orchid, which increased with bud growth, reaching a high value in half-opened flower. This was followed by a gradual decline but it increased again when the flowers showed sign of senescence. Aminooxylacetic acid (AOA) inhibited ethylene production and bud expansion of Aranda buds.     

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.