Characterization and quantitation of fatty acids covalently bound to erythrocyte membrane proteins: anion transporter contains 1 mol of fatty acid thiol ester

Human erythrocyte membranes which had been thoroughly extracted with organic solvents contained 20 nmol of fatty acids/mg dry wt. The major fatty acids were palmitic and stearic with their monoethenoic derivatives as minor constituents. No other fatty acids were detected. When solvent-extracted membranes were digested with Pronase about 90% of the original content of fatty acids was retained in the insoluble residue. Fatty acids were linked to membrane proteins through alkali-labile bonds of which 30% were of a thiol ester and the remainder of an O-ester type. This conclusion is based on differential liberation of fatty acids by hydroxylamine at pH 7.0 and pH 11.0. Two extracts of membranes enriched in peripheral proteins (bands 1, 2, 5 and 2.1, 4.1, 4.2, 6) were prepared and extracted with organic solvents but each contained about six times less fatty acids than the parent solvent-extracted membranes. Glycophorin A contains little if any covalently bound fatty acids. Anion transporter (band 3) contains about 1 mol of thiol ester of fatty acid. This accounts for about half of the thiol ester-linked fatty acids in the parent solvent-extracted membranes. Most of the O-ester-linked fatty acids are linked to an undisclosed membrane protein.     

Interaction of ferric complexes with rat liver nuclei to catalyze NADH-and NADPH-Dependent production of oxygen radicals

The production of potent oxygen radicals by microsomal reaction systems has been well characterized. Relatively little attention has been paid to generation of oxygen radicals by liver nuclei, or to the interaction of nuclei with different ferric complexes to catalyze NADH- or NADPH-dependent production of reactive oxygen intermediates. Intact rat liver nuclei were capable of catalyzing an iron-dependent production of .OH as reflected by the oxidation of .OH scavenging agents such as 2-keto-4-thiomethylbutyrate, dimethyl sulfoxide, and t-butyl alcohol. Inhibition of .OH production by catalase implicates H2O2 as the precursor of .OH generated by the nuclei, whereas superoxide dismutase had only a partially inhibitory effect. The production of .OH with either cofactor was striking increased by addition of ferric-EDTA or ferric-diethylenetriamine-pentaacetic acid (DTPA) whereas ferric-ATP and ferric-citrate were not effective catalysts. All these ferric complexes were reduced by the nuclei in the presence of either NADPH or NADH. The pattern of iron chelate effectiveness in catalyzing lipid peroxidation by nuclei was opposite to that of .OH production; with either NADH or NADPH, nuclear lipid peroxidation was increased by the addition of ferric ammonium sulfate, ferric-ATP, or ferric-citrate, but not by ferric-EDTA or ferric-DTPA. NADPH-dependent nuclear lipid peroxidation was insensitive to catalase, superoxide dismutase, or .OH scavengers; the NADH-dependent reaction showed a partial sensitivity (30 to 40%) to these additions. The overall patterns of .OH production and lipid peroxidation by the nuclei are similar to those shown by microsomes, e.g., effect of ferric complexes, sensitivity to antioxidants; however, rates with the nuclei are less than 20% those of microsomes, which reflect the lower activities of NADPH- and NADH-cytochrome c reductase in the nuclei. The potential for nuclei to reduce ferric complexes and catalyze production of .OH-like species may play a role in the susceptibility of the genetic material to oxidative damage under certain conditions since such radicals would be produced site-directed and not exposed to cellular antioxidants.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

The immunogenic capacity of soluble "vaccinia" antigen (fractionated smallpox vaccine) administered simultaneously with inactivated typhoid vaccine and with purified and concentrated tetanus anatoxin

In this study we have investigated the immune humoral response in the associated vaccination with smallpox, tetanus and typhoid fractionated vaccine (trivaccine) administered in two series at 1 month interval, by dermojet, in a group of young people of 18-20 years old. The results were comparatively estimated with those obtained in two groups of young people of the same age (control group), separately immunized with two components of the tri-vaccine: fractionated smallpox vaccine and tetanus toxoid, following-up the humoral response to the two vaccine components. It was find that, at the end of the surveillance period, similar results were obtained for the testing group and control group, the antibody titers (in geometric mean) presenting very close values: 1/1,140,463 for the testing group, and 1/1,053,583 for the control group against vaccinia component, and 1/1,86,880,586 for the testing group and 1/79,900,431 for the control group, against the tetanus component. The results obtained entitle us to propose this vaccination scheme for the vaccination practice.     

Biosynthesis of endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase

In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC50 of about 6 microM. This activation was dependent on NADPH and Ca2+. The EC50 for Ca2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor.     

Recognition and use of the unusual X-DNA as a primer-template by Klenow DNA polymerase enzyme

Based on CD spectra, 2-amino-2'-deoxyadenosine-containing synthetic alternating DNA, poly(amino2dA-dt) undergoes a conformational transition from a B-form to a non-Z zig-zag form of DNA, called X, even under conditions where enzymes can work. Kinetic parameters of the E. coli Klenow DNA polymerase enzyme-catalyzed copying of both the B- and X-forms of poly(amino2dA-dT) have been determined. Binding affinity of X-DNA to the enzyme proved to be even higher than that of the B-DNA; primer-chain extension of X-poly(amino2dA-dT) was however hindered as compared to its B-form. This differential utilization of X-DNA versus B-DNA by a DNA polymerase is an in vitro enzymatic evidence of an unusual DNA conformation.     

The biological activity of interferon alpha is influenced by two distinct regions in the protein

With the aim to assign differences in activity between murine interferon-alpha 1 and -alpha 4 to specific amino acids, we have constructed hybrid genes and analysed the antiviral properties of the corresponding hybrid proteins. The hybrid genes were constructed by means of homologous recombination between the alpha 1 and alpha 4 genes in Escherichia coli. Hybrids in which the N-terminal part is derived from alpha 1 show that two regions have a major effect on the activity: amino acid 10-20 and 55-67. When comparing hybrids with N-terminal alpha 4 sequences, transitions in activity are found in the same regions. Interestingly, the curves for the two sets of hybrids are exactly each others mirror image.     

Transformation of erythrocytes and their ultrastructure with respect to aging

Transformation and ultrastructure of erythrocytes have been investigated in 58 white non-inbred rats on the 1st, 2nd, 3d days after birth and at the age of 1.5, 3, 8, 20 months. The transformation index of the erythrocytes drops by the 8th month of life and further it rises in old animals. In newborn rats echino-poikiloid forms predominate; they are mainly presented as reticulocytes with remnants of nuclei, mitochondria and other organells. During first three days of life cells of the erythroid line actively free themselves from the nucleus by means of its pyknosis and chromatinolysis. In newborn rats erythrocytes have folds of plasmolemma of linear and spotted form on their surface; their number sharply decreases in mature animals. The least changes in form and ultrastructure of erythrocytes are noticed in 3-8-month-old animals.     

Chromatin matrix activity in hepatocyte nuclei after disturbance of the hepatic vagal innervation

The level of matrix activity of extranucleolar and nucleolar chromatin in hepatocytes of white noninbred rats has been studied in dynamics after disturbance of the vagal innervation. In order to estimate the labelling intensity of nuclear structures, the histoautoradiographic method is used. After bilateral subdiaphragmal vagotomy at early stages (1-2 weeks) the level of the nucleoplasm and nucleolus increases essentially, and by the 30th day a tendency to its normalization is noted. A conclusion is made that the peripheral nervous system influences the intensity of metabolic processes in the nucleus.     

Vimentin-positive epithelial cells in aggregated lymphoid nodules (Peyer's patches) in rabbits

Peculiarities of cytoskeleton in membranous cells and disposition of the latter in the cupola epithelium in aggregated lymphoid nodules++ (ALN) have been studied in the ileum of 5 rabbits. The material has been fixed in liquid nitrogen and in the mixture of paraformaldehyde and glutar aldehyde. Methods of immunomorphology, high resolving light and transmissive electron microscopy have been used. Monoclonal antibodies to vimentin are selectively bind with a specific population of the ALN cupola epithelial cells. These cells are regularly arranged in the epithelium of the cupola lateral part and they are absent in the epithelium of the intestinal crypts, villi and apex of the cupolas. In the lateral epithelium of the cupolas surface, nearer to their base vimentin-positive++ epitheliocytes make contacts with single interepitheliocytic lymphocytes, and nearer to the apex they surround compact groups of the interepitheliocytic lymphocytes. The vimentin-positive++ epitheliocytic cells are identified as M-cells.