Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.     

Induction of ornithine decarboxylase in rat liver by phorbol ester is mediated by prostanoids from Kupffer cells

Administration of phorbol 12-myristate 13-acetate (PMA) to rats in vivo resulted in the induction of ornithine decarboxylase activity in the liver which could be blocked by preinjection of indomethacin, a cyclooxygenase inhibitor. In vitro administration of PMA to primary cultures of rat parenchymal cells did not lead to an induction of ornithine decarboxylase activity. It was investigated to what extent non-parenchymal liver cells could play an intermediary role in the expression of the PMA effect on ornithine decarboxylase activity in parenchymal liver cells. Addition of conditioned medium from PMA-activated Kupffer cells to cultured parenchymal cells led to the induction of ornithine decarboxylase activity in parenchymal cells. This effect was not observed with conditioned medium from untreated Kupffer cells or from Kupffer cells treated with PMA plus indomethacin. Conditioned media from PMA-treated or untreated endothelial liver cells were ineffective in the induction of ornithine decarboxylase activity in parenchymal liver cells. Prostaglandin D2, the main eicosanoid produced by Kupffer cells, was able to stimulate the synthesis of ornithine decarboxylase in parenchymal liver cells (up to 40-fold) in a dose-dependent way. Prostaglandin (PG) D2 appeared to be a more potent inducer of ornithine decarboxylase activity in parenchymal cells than PGE1 and PGE2. It is concluded that intercellular communication inside the liver mediated by prostaglandins derived from activated Kupffer cells may form a mechanism to induce synthesis of specific proteins in parenchymal cells.     

Biosynthesis of gastric mucus glycoprotein of the rat

We have studied the biosynthesis of rat gastric mucin in stomach segments using an antiserum against rat gastric mucin specific for peptide epitopes. Pulse-chase experiments were performed with [35S]methionine, [3H]galactose, and [35S]sulfate to label mucin precursors in different stages of biosynthesis, which were analyzed after immunoprecipitation. The earliest mucin precursor that could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a 300-kDa protein. The occurrence of N-linked "high-mannose" oligosaccharides on this protein was shown by susceptibility to degradation by endo-beta-N-acetylglucosaminidase H. This precursor could be labeled with [35S]methionine and not with [3H]galactose or [35S]sulfate. The 300-kDa precursor was converted into mature mucin after extensive glycosylation and sulfation. The mature mucin but not the 300-kDa precursor was in part secreted into the medium. Specific inhibition of sulfation with sodium chlorate had no effect on rate and amount of mucin secretion. In addition, we show that two core proteins are expressed in rats, slightly varying in Mr among individual animals.     

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.     

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.     

Genetic and morphological divergence among sympatric canids

Numerous studies have suggested that the extent of character divergence observed between two sympatric species reflects the intensity of competition for resources or space. However, the influence of time on divergence is often overlooked. We examined the relationship between time and character divergence in two groups of congeneric, sympatric canids on two continents: South American foxes and African jackals. Character divergence was assessed from measurements of body mass and dental and cranial shape. Divergence time was estimated from data on mitochondrial DNA restriction site polymorphisms. Our findings indicate that African jackals are morphologically similar despite having diverged more than 2 million years ago. By contrast, South American foxes differ substantially in both size and morphology after only 250,000 years of evolution. Thus, the lack of character divergence among the African jackals cannot be explained as a result of very recent common ancestry.     

Mammomonogamus laryngeus (Railliet, 1899) infections in cattle from Sri Lanka

During one year 1249 male cattle were examined for Mammomonogamus laryngeus infections in the slaughterhouse at Kandy, Sri Lanka. The overall prevalence was 40% with only light monthly variations (34 to 52%). The infection rate was highest (47%) in 2 to 2.5 year old animals. In infected animals an average of 6.4 parasite pairs was found with higher numbers in older animals. The majority of worms were located on the posterior side of the epiglottis. Lesions observed were mucosal plugs at the site where the parasites were attached to the mucosa and moderate to severe erosions and ulcers in other zones.     

Suppression of human lymphocyte chemotaxis and transendothelial migration by anti-LFA-1 antibody

The role of lymphocyte function-associated antigen 1 (LFA-1) in human T cell chemotaxis was investigated by using mAb specific to the beta-chain (TS1/18) (CD18) and alpha-chain (TS1/22) (CD11a) of LFA-1. T cell chemotaxis in response to IL-2 and to lymphocyte chemotactic factor (LCF) was markedly suppressed by the addition of TS1/18. TS1/22 was a less effective inhibitor than TS1/18 with only LCF stimulated responses showing significant inhibition when compared in seven different T cell preparations. Neither TS1/18 nor TS1/22 antibody inhibited random T cell migration. Control mAb to CD4 T cells failed to inhibit T cell random migration or chemotaxis. Additional studies to evaluate the adherence and migration of T cells through IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration through HUVEC, but failed to inhibit adherence of T cells to these cells. These studies indicate that LFA-1 plays a role in the migration of T cells through HUVEC and in the in vitro chemotactic response of T lymphocytes to IL-2 and LCF.     

Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies

A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.     

Superinduction of the murine B cell Fc epsilon RII by T helper cell clones. Role of IL-4

Th cell clones are known to induce an IL-4 dependent polyclonal IgE synthesis. Because IL-4 can induce the expression of the low affinity FcR for IgE (Fc epsilon RII) the ability of Th cell clones to induce Fc epsilon RII on purified splenic B cells was analyzed. It was found that a TH2 clone could cause a 50- to 100-fold superinduction of Fc epsilon RII after 2 days in culture; after 3 days, the Fc epsilon RII levels had almost returned to base line. The superinduction was inhibited by an anti-IL-4 antibody, 11B11, indicating its dependence on IL-4. A TH1 clone could cause a modest (four fold) induction of Fc epsilon RII, and this induction was not influenced by 11B11. A similar Fc epsilon RII induction was seen when using the supernatant from activated TH1 cells. The component(s) causing this relatively low level Fc epsilon RII induction is not known; a variety of known lymphokines were tested, and only IL-4 demonstrated any capacity for Fc epsilon RII induction on LPS-activated B cells. Addition of rIL-4 at concentrations of 400 U/ml or greater to the TH1 culture was sufficient to cause a Fc epsilon RII superinduction similar to that seen with the TH2 clone, while 40 U/ml was not. In order to determine a potential role for the Fc epsilon RII or its soluble fragment on the IgE synthesis mediated by TH2, a monoclonal anti-Fc epsilon RII, B3B4, was added to the culture. The addition of B3B4 did not have an influence on IgE levels in this system.