Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

A sulfatase specific for glucuronic acid 2-sulfate residues in glycosaminoglycans

Although 2-O-sulfated L-iduronic acid (IdoA) residues have been known to occur in heparin, 2-O-sulfated D-glucuronic acid (GlcA) residues have been reported only recently (Bienkowski, M. J., and Conrad, H. E. (1985) J. Biol. Chem. 250, 356-365). Disaccharides prepared by cleavage of heparin and N-deacetylated chondroitin 6-sulfate with nitrous acid were used to demonstrate a new sulfatase that catalyzed the removal of the 2-O-sulfate substituents from GlcA but not IdoA residues. The deamination products were labeled by NaB3H4 reduction to give disaccharides from heparin and chondroitin sulfate which had reducing terminal 2,5-anhydro-D-mannitol ([3H]AManR) and 2,5-anhydro-D-talitol ([3H]ATalR) residues, respectively. IdoA(2-SO4)-[3H]AManR(6-SO4) from heparin and GlcA(2-SO4)-[3H]ATalR(6-SO4) from chondroitin sulfate were purified for use as substrates. GlcA(2-SO4)-[3H]AManR(6-SO4) was prepared by epimerization of IdoA(2-SO4)-[3H]AManR(6-SO4) with hydrazine at 100 degrees C. Lysosomal enzyme preparations from chick embryo chondrocytes and from two normal human fibroblast cell lines catalyzed the removal of the 2-O-SO4 substituent from the uronic acid residues of IdoA(2-SO4)-[3H]AManR(6-SO4), GlcA(2-SO4)-[3H] AManR(6-SO4), and GlcA(2-SO4)-[3H]ATalR(6-SO4). In contrast, a lysosomal enzyme preparation from a human fibroblast cell line deficient in idurono-2-sulfatase (Hunter's-syndrome), which had no activity on the IdoA(2-SO4)-[3H]AManR(6-SO4), converted GlcA(2-SO4)-[3H]AManR(6-SO4) to a mixture of GlcA-[3H] AManR(6-SO4) and [3H]AManR(6-SO4). This enzyme also converted GlcA(2-SO4)-[3H]ATalR(6-SO4) to a mixture of GlcA-[3H]ATalR(6-SO4) and [3H]ATalR(6-SO4). Digestion of both GlcA(2-SO4)-[3H]AManR(6-SO4) and GlcA(2-SO4)-[3H]ATalR(6-SO4) was inhibited by 35SO2-4 and was arrested at the monosulfated disaccharide stage by 1,4-saccharolactone. The glucurono-2-sulfatase exhibited a pH optimum of 4. The results indicate that there exists a separate sulfatase for the removal of sulfate substituents from C-2 of GlcA residues in glycosaminoglycans.     

Changes in Epstein-Barr virus antibody titers associated with aging

Antibody titers to the Epstein-Barr virus (EBV), early antigen (EA) IgG, and virus capsid antigen (VCA) IgG and IgA, were measured in 44 geriatric subjects to determine if the depression in cellular immunity known to be associated with aging affects the expression of latent EBV. Similar assays were performed on plasma obtained from a young adult (medical student) population as a control group. We found that 89% of the geriatric samples were positive for EA IgG, and 83% of the plasma obtained from medical students were positive for EA IgG. One hundred percent of the geriatric subjects were positive for VCA IgG, and 87% of the medical students were positive for VCA IgG. Seven percent of the medical student blood samples were positive for VCA IgA; in contrast, 36% of the blood samples obtained from the geriatrics subjects were positive. Significant differences were also found in the geometric mean titers (GMT) of antibodies to EBV antigens; the GMT to EBV EA and VCA were significantly higher in the geriatric group. The data suggest that there may be some loss of control over latent EBV by the cellular immune response in geriatric individuals.     

Hematologic and immunologic responses in common marmosets (Callithrix jacchus) infected with Plasmodium knowlesi and Epstein-Barr virus

The hematologic and immunologic responses to infection with either the Epstein-Barr virus alone or infection with Epstein-Barr virus and Plasmodium knowlesi were studied using common marmosets (Callithrix jacchus). The assays performed included complete blood cell counts, determinations of natural killer cell activity, and determinations of antibody titers to Epstein-Barr virus early antigen, virus capsid antigen and the nuclear antigen. While no animal showed signs of lymphoproliferative disease, it was found that animals infected with Epstein-Barr virus became positive for early antigen, virus capsid antigen and nuclear antigen at low levels. No difference in antibody titers between Epstein-Barr virus infected animals and co-infected animals was observed. An increase also was found in the number of leukocytes in all groups, and an increase in natural killer cells following infection with Epstein-Barr virus. Some depression in natural killer cells was observed in the co-infected animals when compared to Epstein-Barr virus infected animals.     

Pathology of larval Eustrongylides in the rabbit

Three fishermen from Maryland who swallowed live bait-minnows developed severe abdominal pain within 24 hr; 2 required abdominal surgery. Larvae of the nematode Eustrongylides sp. were found in the peritoneal cavity of both (Guerin et al., 1982). In the current study, the lesions produced by Eustrongylides larvae were investigated in New Zealand white rabbits. None of these exhibited any signs of clinical illness; however, postmortem examination within 24 hr of inoculation revealed that larvae had migrated through the walls of the esophagus and stomach and viable larvae were recovered from the pleural and peritoneal cavities as well as from gastric contents. Necropsies performed at different intervals of time postinoculation showed that the migrating larvae had produced multi-focal peritonitis and multiple granulomata in the liver.     

Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-1,3,5-hexatriene fluorescence

Using multifrequency phase and modulation fluorometry and a nonlinear least-squares analysis of lifetime data, we were able to determine the complex decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in synthetic phospholipid bilayers. Our results showed a monoexponential decay of DPH in the pure isotropic solvents studied, over a wide temperature range, and a double-exponential decay of DPH in phospholipids, both above and below the transition. During the transition, and in mixed-phase phospholipids, a three-component analysis was successfully accomplished, and the pre-exponential factors of the two main components have been shown to be quantitatively representative of the gel and liquid-crystalline phases of the bilayer. The fractional intensity of the shorter lifetime component depends on the modalities of the sample preparation. The factors affecting this component are discussed. From the DPH fluorescence lifetime and from the anisotropy data in L-alpha-dimyristoyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidyl choline mixtures, a phase diagram was independently constructed. Conclusions about the sensitivity and the partition of the probe between gel and the liquid-crystalline phases of the bilayer are derived. Lifetime experiments on DPH in a L-alpha-dilauroyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidylch oline mixture suggested a general method for the determination and quantitation of the two different phases in the bilayer.     

Electrorotation of lymphocytes—The influence of membrane events and nucleus

Electrorotation—the spin of cells in rotating high frequency electric fields—has been used to investigate properties of human peripheral blood lymphocytes. The rotation spectra of lymphocytes deviate from those of single shell spheres. The deviations are caused by the electrical properties of the nucleus in the cell interior.Electrorotation allows the distinction between successfully stimulated lymphocytes and unstimulated cells after application of concanavalin A. Notwithstanding the fact that only a proportion of the cells will be mitogenically stimulated we detected an enhanced cell membrane conductivity for the whole cell population immediately after the addition of mitogen.     

Living Vessel Elements in the Late Metaxylem of Sheathed Maize Roots

The two types of nodal roots of field-grown maize, sheathedand bare, were found to have such different water conductivitiesthat an investigation of the anatomy of their large metaxylemvessels was made. While the vessels of the bare roots were openfor scores of centimetres, those of the sheathed roots werefound to be not vessels but developing vessel elements, withcross walls at 1 mm intervals, and protoplasts. The cross wallsbetween the elements had several unique histochemical properties.Previous investigators have often failed to find the cross wallsbecause they are very easily dislodged during the usual methodsof tissue preparation. They are best identified by microdissectionof fresh xylem. The living elements persist in the late metaxylemup to 20 – 30 cm from the tip. As the roots become longerthan this both the cross walls and the soil sheaths disappearand there is a transition to a bare root with open vessels inthe proximal region. The soil sheath persists a little longerthan the cross walls. The two types are thus stages in a developmentalsequence through which all nodal roots pass. A fundamental differencebetween the two types is in their water status, since the estimatedconductive capacity of a bare root is about 100 times greaterthan that of a sheathed root. These observations point to theneed for a reassessment of the published work on transport ofions into the xylem of grass roots through a reinvestigationof the ‘maturity’ of their xylem vessels. Grass roots, dimorphic roots, ion secretion to xylem, soil sheaths, xylem vessels, xylem differentiation, water conduction, Zea mays L