Inhibition of hydroxyproline synthesis by palladium ions.

Palladium ions, administered as PdSO4, markedly affect the incorporation of L-[3,4-3H2] proline into non-dialyzable fractions in 10-day chick embryo cartilage explants with a 55-65% reduction in the concentration range 0.06-0.6 mM. Under these conditions the synthesis of [3H]hydroxyproline was nearly completely inhibited. Experiments with prolyl hydroxylase (EC 1.14.11.2) indicated a strong irreversible inhibition of the enzyme with a competition between Fe2+ and Pd2+. The Ki for the inhibition was 0.02 mM. Pd2+-treated enzyme remained inactive after extensive dialysis. These studies suggest that Pd2+ may inhibit collagen synthesis by replacing Fe2+ in the active site of prolyl hydroxylase and forming strong complexes with the enzyme. These studies also point to a potential mechanism of Pd2+ toxicity.     

Interactions between anesthetics and lipid mixtures. Normal alcohols.

The effects of normal alcohols up to 1-dodecanol on phase transitions in phosphatidylcholines and phosphatidylethanolamines have been studied using chlorophyll a as fluorescent probe. With the phosphatidylcholines, alcohols up to octanol cause a lowering of the transition temperature, and a broadening of the transition, whereas for dipalmitoylphosphatidylethanolamine, only a lowering of the transition is observed. The lowering of the phase transition temperature in dipalmitoylphosphatidylcholine by butanol and hexanol is close to that expected for ideal behavior, but the behavior of the longer chain alcohols becomes less ideal. The effects of these alcohols on mixtures of lipids have been studied, and they illustrate the care necessary if these plots of temperatures of onset and completion of gel phase formation are to be called "phase diagrams". The effect of 1 -octanol on mixtures of lipids is to increase the proportion of lipid present in the lipid-crystalline state. In contrast, 1-decanol causes an increase in the phase transition temperature for dimyristoylphosphatidylcholine, although it lowers the transition temperature for dipalmitoylphosphatidylcholine, and 1 -dodecanol raises the transition temperature for both of these phosphatidylcholines, although it lowers that for dipalmitoylphosphatidylethanolamine. Dodecanol appears to behave in these lipid bilayer membranes as a lipid with a phase transition temperature of ca. 55 degrees C. Anesthesia is discussed as a phenomenon of liquidus extension: alcohols up to 1 -octanol increase the proportion of lipid in the liquidus state and result in anesthesia, whereas the longer alcohols do not, and result in catalepsy.     

Enzymic and immunochemical properties of lysozyme. XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site.

Previous studies from this laboratory on the immunochemistry of specific chemical derivatives of native lysozyme and of the two disulfide peptide 62-68 (Cys 64-Cys 80) 74-97 (Cys 76-Cys 94) (i.e. (SS)2-peptide), have established an antigenic reactive site to comprise the spatially contiguous surface residues: Trp 72, Lys 97, Lys 96, Asn 93, Thr 89 and Asp 87. In the present work, the identity of the site was verified by an entirely different and novel approach. The aforementioned amino acids were linked directly into a single linear peptide with an intervening spacer where appropriate and substituting phenylalanine for tryptophan (i.e. Phe-Gly-Lys-Asn-Thr-Asp). This peptide (which does not exist in native lysozyme but simulates a surface region of the protein) possessed a remarkable inhibitory activity towards the reaction of lysozyme with its antisera. The immunochemical reactivity of the peptide was equal to the maximum expected reactivity of the site (i.e. a third of the total antigenic reactivity of lysozyme). These findings define quite conclusively and accurately the reactive site which is clearly composed of spatially adjacent residues that are distant in sequence reacting as if in direct linear linkage. The unequivocal establishment of this concept indicates that antigenic sites need not always be composed of residues in direct peptide linkage in the sequence. The nature of the site may depend on the protein. This unorthodox attack at the problem provides a novel and powerful approach for final delineation of the antigenic reactive sites (and perhaps other types of binding sites) in native proteins, following the completion of accurate narrowing down by chemical methods.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine preferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

Application of step-wise discriminant analysis and Bayesian classification procedure in determining prognosis of acute myocardial infarction.

A retrospective study was carried out to assess the feasibility of computer-assisted prognostication by discriminant analysis and the Bayesian classification procedure based on clinical information collected on patients with acute myocardial infarction. The overall accuracy was 94.2% in predicting hospital death but the prediction of late death after discharge was less accurate. It was found that not all of the 44 variables used for analysis were necessary to reach the same level of predictive accuracy--16 to 20 variables would result in almost the identical prediction. The Bayesian classification procedure was applied to estimate probabilities of individual patients belonging to the different prognostic categories.     

Bronchial reactivity to histamine before and after sodium cromoglycate in bronchial asthma.

Out of 19 patients with extrinsic bronchial asthma challenged with 123 mug histamine acid phosphate by intravenous infusion only 13 responded with a fall in FEV1 of over 10% (mean 16%). Seventeen of these patients were given histamine 2 mg/ml by aerosol, and all responded with a mean decrease in FEV1 of 37.8%. When challenged with allergen extract by aerosol the mean decrease in FEV1 was 37.5%. After 40 mg sodium cromoglycate 15 of the 17 patients showed significant protection against allergen challenge with a mean decrease in FEV1 of only 23.6%. Inhalation of 40 mg sodium cromoglycate, however, failed to protect against histamine given by either the intravenous or aerosol route. Histamine given intravenously to asthmatic patients produces less of a bronchial response than when given by aerosol, even though the intravenous route produces many more systemic symptoms, such as flushing and throbbing headache. The protection of sodium cromoglycate against an allergen inhalation challenge is not due to histamine antagonsim.     

Cytotoxic activity of lymphocytes. VI. Heterogeneity of cytotoxins in supernatants of mitogen-activated lymphocytes.

Two different lymphotoxins synthesized by human blood lymphocytes stimulated with phytohemagglutin (PHA) are described. The two toxins are called alpha-LT and beta-LT relative to their elution order on gel chromatography. Their m.w. are 75,000 and 45,000 daltons, respectively. Both toxins appear as early as 7 hr after the addition of PHA, with the amount of beta-toxin exceeding that of alpha-LT initially. Both toxins are differentiated from a third toxin (adherent cell toxin, ACT) made by plastic-adherent cells without requiring mitogen-stimulation. Depletion of macrophages or B cells does not affect the synthesis of either lymphotoxin. Monospecific antisera were elicited to alpha-LT. Antisera elicited to beta-LT also neutralized alpha-LT but to a significantly lesser degree. Alpha-LT appears to be the lymphotoxin described by most other workers. Beta-LT is unstable at 37 degrees C which may explain why the low m.w. lymphotoxin has not been described previously.     

Suppression of reaginic antibody formation. III. Relationship between immunogenecity and tolerogenicity of hapten-carrier conjugates.

From the study of the effect of epitope density on the immunogenicity of haptenated ovalbumin (DNP-OA) it was concluded that the lightly haptenated conjugate, DNP0-5-OA, induced, on the one hand, only low titers of anti-DNP hemagglutinating antibody and no reaginic antibodies to the hapten and, on the other, high reaginic and high hemagglutinating antibody responses to the carrier. The conjugate with a slightly higher degree of haptenation, i.e., DNP2.3-OA, induced both reaginic and hemagglutinating antibodies to both the hapten and the carrier. By contrast, the heavily haptenated conjugate, DNP20-OA, elicited reaginic and hemagglutinating antibodies only against the hapten but not against the carrier. Specific suppression of anti-hapten reaginic antibody formation had been achieved by treatment of mice with a tolerogen consisting of the hapten (DNP) conjugated covalently to isologous gamma globulins (MgammaG). The epitope density of the DNPx-MgammaG conjugates was shown to play a dominant role in determining whether or not the conjugate was tolerogenic. Thus, lightly haptenated conjugates (DNP0.5-MgammaG, DNP1.3-MgammaG or DNP1.9-MgammaG) were not tolerogenic, moderately haptenated conjugates (DNP4.2-MgammaG, DNP8-MgammaG, and DNP 14-MgammaG) were tolerogenic, and heavily haptenated conjugates (DNP32-MgammaG and DNP53-MgammaG) were immunogenic, being capable of priming the recipients for the DNP hapten. Further evidence for the nonimmunogenicity of DNP 8-MgammaG conjugate was inferred from its rate of clearance in tolerized and normal mice. Thus, the half-life of 125I-labeled DNP8-MgammaG in circulation was not significantly different for normal and tolerized mice; it was 3.7 and 3.5 days, respectively, which is within the range of data reported for clearance of normal MgammaG. These results suggest that DNP8-MgammaG was catabolized at a rate similar to that of nonconjugated, isologous MgammaG. Moreover, there was no significant difference in the localization of DNP8-MgammaG in identical difference in the localization of DNP8-MgammaG in identical organs (spleen, thymus, kidney, and liver) of normal and tolerized mice. All the multivalent DNPx-MgammaG conjugates were shown to be able to elicit passive cutaneous anaphylaxis (PCA) reaction on i.v. challenge of rats which had been pre-sensitized i.d. with anti-DNP reaginic antibodies.