Monoclonal antibodies to alpha-chain regions of human fibrinogen that participate in polymer formation

Monoclonal antibodies have been generated against a cross-link-containing derivative of alpha polymer (alpha XLCNBr), isolated following CNBr digestion of fibrin [Sobel, J. H., Ehrlich, P. H., Birken, S., Saffran, A. J., & Canfield, R. E. (1983) Biochemistry (preceding paper in this issue)]. One cloned cell line (F-102) was chosen for characterization based on its apparent specificity for the A alpha-chain region A alpha 518-584 (CNBr X). A second line (F-103) was selected because of its anti-A alpha 241-476 (CNBr VIII) properties. These two regions of the A alpha chain have previously been implicated as major contributors to the cross-linking process that leads to alpha-polymer formation. Radioimmunoassays have been developed, employing the immunoglobulins produced by clones F-102 and F-103. These assays have been applied, in conjunction with high-performance liquid chromatography purified tryptic and chymotryptic derivatives of CNBr VIII and CNBr X, to localize the respective determinants involved in antibody binding. In each case, virtually full immunoreactivity was exhibited by both the CNBr fragment and a single tryptic or chymotryptic peptide originating from it. These findings indicate that sequence-specific, rather than conformational, determinants were operative in the generation of antibodies F-102 and F-103. The epitope recognized by F-102 was localized to the region of A alpha 540-554, while the F-103 binding site resided within A alpha 259-276. When these radioimmunoassays were applied to study the relative immunoreactivity exhibited by a variety of fibrinogen derivatives, the results obtained support earlier suggestions that the COOH-terminal portion of the A alpha chain contains regions of random conformation.     

Quantification of choline and ethanolamine phospholipids in rabbit myocardium

To facilitate evaluation of the influence of myocardial phospholipid metabolites on the development of electrophysiologic abnormalities induced by ischemia, a method for the quantification of choline and ethanolamine phospholipids suitable for accurate and reproducible analysis of small amounts of myocardium was developed. The procedure combines chloroform and methanol extraction of phospholipids after tissue homogenization with subsequent separation by sequential thin-layer and high-performance liquid chromatography. Phosphorus in purified lipid classes was determined with the correction for recovery based on 14C-labeled internal standards.     

Changes in the distribution of a spectrin-like protein during development of the preimplantation mouse embryo

The mouse blastocyst expresses a 240,000-mol-wt polypeptide that cross-reacts with antibody to avian erythrocyte alpha-spectrin. Immunofluorescence localization showed striking changes in the distribution of the putative embryonic spectrin during preimplantation and early postimplantation development. There was no detectable spectrin in either the unfertilized or fertilized egg. The first positive reaction was observed in the early 2-cell stage when a bright band of fluorescence delimited the region of cell-cell contact. The blastomeres subsequently developed continuous cortical layers of spectrin and this distribution was maintained throughout the cleavage stages. A significant reduction in fluorescence intensity occurred before implantation in the apical region of the mural trophoblast and the trophoblast outgrowths developed linear arrays of spectrin spots that were oriented in the direction of spreading. In contrast to the alterations that take place in the periphery of the embryo, spectrin was consistently present in the cortical cytoplasm underlying regions of contact between the blastomeres and between cells of the inner cell mass. The results suggest a possible role for spectrin in cell-cell interactions during early development.     

A fixation procedure suitable for autoradiography of endogenous long-chain acyl carnitine

A tissue processing procedure was evaluated for fixation of endogenous long-chain acyl carnitine (LCA) to facilitate autoradiographic subcellular localization of this amphiphile. Suspensions of neonatal rat myocytes labeled with exogenous 14C-palmitoyl carnitine retained 85.2% of the radiolabel after tissue processing. Autoradiography demonstrated no significant translocation of radiolabeled LCA from myocytes to unlabeled sheep erythrocytes mixed in equal proportions and processed together. To evaluate endogenous LCA fixation, cultured myocytes were incubated for 3 days with 3H-carnitine. Radioactivity was distributed in LCA, short-chain acyl carnitine, and free carnitine pools in proportion to the physiological concentrations of the metabolites traced. Before tissue processing, LCA contained 4.5% of total radioactivity. After tissue processing, labeled water-soluble components were lost and 88% of the retained radioactivity was in the LCA pool. The enrichment of endogenous LCA radioactivity was attributable to the selective extraction of endogenous short-chain and free carnitine. Nearly 75% of endogenous LCA was preserved. In contrast, 99.5% of both endogenous short-chain and free carnitine were extracted. Thus, endogenous LCA can be selectively preserved, permitting quantitative subcellular localization of this amphiphile with ultrastructural autoradiography.     

The immunopathology of experimental allergic encephalomyelitis (EAE). III. Differential in situ expression of strain 13 Ia on endothelial and inflammatory cells of (strain 2 x strain 13)F1 guinea pigs with EAE

This study was undertaken to determine if there is differential expression of the relevant strain-specific Ia antigen on endothelial and parenchymal inflammatory cells in the central nervous systems (CNS) of guinea pigs (GP) with acute experimental allergic encephalomyelitis (EAE). Adult inbred GP were sensitized with GP spinal cord homogenate and complete Freund's adjuvant. Strain 13 GP and (2 x 13)F1 hybrids developed clinical disease within 2 to 3 wk after sensitization, whereas strain 2 GP did not, although all sensitized GP had CNS mononuclear inflammatory infiltrates. By using monoclonal antibodies to strain-specific and framework Ia epitopes with an immunoperoxidase technique, the distribution and amount of the strain 2 and strain 13 Ia were analyzed. Equivalent strain 2 and strain 13 Ia expression was found in normal tissues from F1 animals. Strain 2 and strain 13 GP sensitized for EAE had increased strain-specific Ia staining of CNS vessels and inflammatory cells over controls. However, F1 GP with EAE had markedly increased strain 13, but not strain 2, Ia on CNS parenchymal vessels and mononuclear inflammatory cells (p less than 0.001, for both). These results suggest for the first time that specific major histocompatibility complex gene products are selectively expressed on endothelial and inflammatory cells in situ in immune reactions in the target organ of individuals of heterogeneous immunogenetic composition.     

Aortic accumulation and plasma clearance of beta-VLDL and HDL: effects of diet-induced hypercholesterolemia in rabbits

To assess the role of beta-VLDL in diet-induced atherogenesis, the in vivo metabolism and aortic accumulation of 125I-labeled beta-VLDL were investigated in cholesterol-fed rabbits and chow-fed controls. 125I-labeled HDL and 125I-labeled albumin were studied for comparison. The fractional catabolic rate of 125I-labeled beta-VLDL was reduced in cholesterol-fed rabbits (0.011 vs 0.139 hr-1), but due to the high endogenous pool, the total beta-VLDL flux was very high (13.1 vs less than 1.1 mg/kg per 24 hr). These results suggest that elevated levels of beta-VLDL during cholesterol feeding were due to an enhanced rate of synthesis, a finding confirmed in hypercholesterolemic rabbits subjected to plasmapheresis. Following acute reduction of plasma cholesterol by plasmapheresis, the quantitative increases in beta-VLDL cholesterol concentrations (210 to 364 mg/dl) over the subsequent 24 hr were in agreement with the rise calculated from the plasma clearance kinetics of 125I-labeled beta-VLDL (378 mg/dl per 24 hr). Aortic accumulation of beta-VLDL in hypercholesterolemic rabbits was increased greater than 15-fold over controls. Accumulation was predominantly in the intimal atheromatous lesions. The fractional catabolic rate of 125I-labeled HDL was increased during cholesterol feeding (0.037 vs 0.021 hr-1). A decreased rate of synthesis appeared to be responsible for the markedly depleted plasma HDL. HDL accumulation within the aorta was attenuated greater than 9-fold in cholesterol-fed rabbits compared to those fed normal chow. Plasma kinetics and aortic accumulation of 125I-labeled albumin were similar in hypercholesterolemic and control rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylation of type II and type I collagen genes in differentiated and dedifferentiated chondrocytes

The methyl-sensitive restriction endonucleases HpaII and HhaI as well as the methyl-insensitive enzyme MspI were used to examine the methylation status of the pro-alpha 1(II) collagen gene of cartilage. Five different cell types with varying abilities to express type II collagen were studied. Chick embryo chondrocytes express type II collagen, while 5-bromodeoxyuridine-treated chondrocytes, retinoic acid-treated chondrocytes, chick embryo fibroblasts, and erythrocytes do not synthesize type II collagen. Both cDNA and genomic probes for the pro-alpha 1(II) collagen gene were used, covering the complete 3' end of the gene and its flanking sequences. The pro-alpha 1(II) collagen DNA was undermethylated in chondrocytes, compared to either fibroblasts or erythrocytes. However, the methylation of the 5-bromodeoxyuridine-treated and retinoic acid-treated chondrocytes was identical to that of control chondrocytes. The methylation pattern of two regions of the gene of the pro-alpha 2(I) collagen chain was identical in all cell types tested, whether or not the gene was expressed. Our results indicate that genes for these collagen chains differ in their methylation pattern. The type II collagen gene shows reduced methylation in expressing cartilage, but does not acquire an increase in methylation in "dedifferentiated" chondrocytes. The changes in DNA methylation that occur during cell differentiation do not appear to be sufficient to explain gene activation and deactivation.     

The immunopathology of experimental allergic encephalomyelitis. I. Quantitative analysis of inflammatory cells in situ

Acute experimental allergic encephalomyelitis (EAE) is a T cell-mediated, neurologic disease that is under immunogenetic control. We systematically analyzed the quantity and distribution of T cells, B cells, and macrophages in the central nervous system (CNS) of susceptible and resistant guinea (GP) with a panel of seven monoclonal antibodies by using the avidin-biotin complex (ABC) immunoperoxidase technique and alpha-naphthyl-butyrate esterase (ANBE) staining. Adult EAE-susceptible strain 13 GP immunized with isogeneic spinal cord homogenate (SC) or with myelin basic protein (MBP) developed clinical signs (paralysis, weight loss, etc.) in 2 to 3 wk. T cells were present in all CNS inflammatory foci and comprised 44% of the perivascular mononuclear cells. T cells diffusely infiltrated the neuropil away from inflammatory cell aggregates. These T cells were judged to be extravascular by the lack of an associated identifiable vessel in counter-stained sections, and by their persistence following exhaustive perfusion of the brains. In routine sections, mononuclear cells could be detected only in perivascular aggregates. IgM+ B cells comprised 9% of the perivascular infiltrates and did not diffusely infiltrate the parenchyma. ANBE+ macrophages comprised the remaining 47% of the identified perivascular cells. SC- and MBP-immunized GP showed equivalent numbers of inflammatory foci, T cells, and macrophages, but SC-immunized GP had more IgM+ cells in the meninges and choroid plexus (p less than 0.001, p less than 0.02, respectively). Virtually all cells in perivascular locations were Ia+. Ia+ mononuclear cells were also present in the neuropil. EAE-resistant strain 2 GP immunized with SC developed no clinical signs. These GP had fewer perivascular foci than strain 13 GP but, when present, the cellular composition, including the density of diffuse parenchymal T cell infiltrates, was indistinguishable. Significantly fewer parenchymal mononuclear cells in the strain 2 GP, however, displayed Ia, both in perivascular and diffuse infiltrates (p less than 0.001). We conclude that T cell migration into the CNS parenchyma is a characteristic feature of acute EAE in the GP, but that T cells can occur in this pattern without clinical signs of disease. The two features that distinguish susceptible and resistant strains were the frequency of perivascular infiltrates and the expression of Ia on parenchymal mononuclear cells, which probably reflects their enhanced immunologic activation in situ.     

In vivo adherence of Pseudomonas aeruginosa to rat bladder epithelium

To date, the study of bacterial adherence to uroepithelial cells has utilized considerable variation in methodology, resulting in highly divergent conclusions. In an attempt to standardize methodology, a modified method is described to more accurately measure the in vivo bacterial adherence to rat bladder uroepithelium utilizing Pseudomonas species labeled with 2-[3H]adenine. Two isolates of P. aeruginosa were selected for more intensive study; one showed consistently good adherence; the second strain always adhered poorly, thus providing a negative control. Maximum adherence was detected after 60 min of incubation. Neither of the two Pseudomonas isolates when examined by transmission electron microscopy showed evidence of pili formation. The morphological features of Pseudomonas adherence to bladder mucosa as studied by scanning electron microscopy are described.