Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Serum Albumin Washing Specifically Enhances Arachidonate Incorporation into Synaptosomal Phosphatidylinositols

Arachidonate incorporation into synaptosomal phospholipids was shown to be affected by factors including the procedure for preparation of the membrane fractions and preincubation of synaptosomes prior to assay of incorporation of arachidonate into both phosphatidylcholine (PC) and phosphatidylinositol (PI). However, the inhibition toward incorporation into PIs, but not PCs, was fully reversed when the membranes were washed with bovine serum albumin. A twofold increase in arachidonate incorporation into PIs was also observed when freshly prepared synaptosomes were washed with serum albumin immediately before assay of incorporation activity. The inhibitory action is thought to be due to an increase in polyunsaturated fatty acids and/or their oxidation products which may then elicit a special effect on the acyltransferase responsible for transferring arachidonate into phosphatidylinositols. The differences in fatty acid uptake and response to serum albumin also suggest the presence of different acyltransferase for acyl transfer to PIs and PCs.     

In vivo interactions of acrylonitrile with macromolecules in rats

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.     

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.     

The mode of action of cytotoxic and antitumor 1-nitroacridines. I. The 1-nitroacridines do not exert their cytotoxic effects by physicochemical binding with DNA

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.     

Transport properties of rigid, symmetrical oligomeric structures composed of prolate, ellipsoidal subunits

We have calculated translational and rotational diffusion coefficients and intrinsic viscosities of oligomeric structures composed of n identical subunits having a prolate ellipsoidal shape with axial ratio p. Results are presented for p = 1-6 for a variety of structures with n = 1-6. We compare our results with those obtained by a different modeling procedure, proposed by other workers, in which the monomeric subunit is represented as a string of touching, colinear spheres. If n and an estimate of p are known, the structure of the oligomer can be. in most cases, unambiguously determined by comparison of the experimental oligomer-to-monomer ratios of a given property with the numerical results of this work. As examples of the applicability of our results, we examine the relationship between structure and properties for neurophysin. bovine serum albumin, hemoglobin and phycocyanin.     

Nematospiroides dubius: Mechanisms of host immunity: II. Skin testing and immunosuppression of orally or subcutaneously sensitized mice

The present study was designed to acquire further understanding of the differences in the immune response of mice orally (OS) or subcutaneously (SS) sensitized to Nematospiroides dubius. Two immunosuppressive agents and skin tests were utilized in this regard.Rabbit antimouse thymus serum (RAMTS) and cyproheptadine (antihistamine-antiserotonin) were similarly effective in suppressing the immune response of subcutaneously sensitized mice. When compared to normal rabbit serum and SS control (sensitized, untreated) animals, observations of the intestinal tunica muscularis in the immunosuppressed SS groups revealed granulomatous lesions in which fewer eosinophils enveloped the sequestered parasite. Cyproheptadine was more successful than the other treatments in interrupting the immune expulsion of N. dubius from orally sensitized mice, but this was only at a borderline significance level.The size and intensity of the active cutaneous anaphylactic skin tests in OS mice injected with an adult or larval antigen, was greater than the response elicited in SS mice or uninfected control mice injected with the same preparations. Similarly, the reaction in subcutaneously sensitized mice exceeded that observed in the noninfected controls.     

Trypanosoma congolense. I. Clinical observations of experimentally infected cattle

The course of disease was studied in 8 cattle infected with Trypanosoma congolense. Although the onset of patency was dependent on the numbers of infecting organisms, the duration of the infection was not. High fevers were present on the day of or the day after initial patency. Succeeding peaks of parasitemia, and a progressive weight loss of over 30% occurred. A decrease in packed cell volume (PCV) beginning the first week after infection was observed. Early in the course of the developing anemia, many polychromatophilic erythrocytes and occasional normoblasts were found in the blood. A leucopenia persisted for the duration of the disease. Total serum protein concentrations fell sharply during the first 5 weeks of infection, then gradually increased to low normal levels. Serum albumin levels followed a similar pattern for the first 5 weeks, and remained at a relatively low level. Although gamma globulin levels also declined during the first 5 weeks, their levels gradually surpassed those of preinfection samples. No marked changes in serum glucose were noted. A mild elevation of serum urea nitrogen values occurred early during infection, but subsided. The animals dying early after infection developed elevated total bilirubin levels.     

Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth,galleria mellonella

The levels of an 81K storage protein in the waxmoth, Galleria mellonella, were monitored during the course of development using rocket immunoelectrophoresis. During the fifth and sixth larval stadia, 81K protein levels increased during feeding and growth but sharply declined at each larval molt. During the fifth and sixth stadia hemolymph levels of the 81K protein increased to about 1 and 2.5 mg/ml, respectively, with no discernible differences between levels in males and females. Neither the fat body nor the remainder of the carcass contained the 81K protein, indicating that the accumulation of this protein during the intermolt period was exclusively in the hemolymph and redistribution of the 81K protein into other tissues does not occur at the final two larval molts. During the seventh (final) larval stadium the absolute quantities of the 81K protein increased from 23 μg per insect to over 1,600 μg in females and to 300 μg in males. The hemolymph concentration of the 81K protein reached 28 mg/ml in females and 6 mg/ml in males with only low levels found in the remaining tissues. Shortly after pupal apolysis, marked by eyespot retraction, the fat body in both sexes rapidly and quantitatively sequestered the 81K protein from the hemolymph. The 81K protein in the hemolymph of both males and females rapidly dropped to nearly zero concentration by pupation. The 81K storage protein remained localized in the fat body cells after uptake occurred, even though the fat body cells disaggregate and reaggregate during metamorphosis. During pharate adult development the 81K storage protein disappeared from the fat body without entering the hemolymph. At adult eclosion 81K was virtually absent from the tissues of both males and females.