Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.     

Monoclonal antibodies to epitopes on different regions of the 200 00 dalton neurofilament protein : Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.     

Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.     

Role of supernatant protein factor and anionic phospholipid in squalene uptake and conversion by microsomes

Supernatant protein factor (SPF), a cytosolic protein (Mr = 47,000) stimulates microsomal squalene epoxidase activity 4- to 10-fold in the presence of anionic phospholipid such as phosphatidylglycerol (PG) (Saat, Y., and Bloch, K. (1976) J. Biol. Chem. 251, 5155-5160). This effect has been ascribed to substrate translocation from inactive to active pools within the membrane of the endoplasmic reticulum (Friedlander, E. J., Caras, I. W., Lin, L. F. H., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). Here we show that SPF and PG also stimulate squalene uptake per se by microsomes as well as stimulate squalene epoxidase. Microsomes preloaded with substrate in the presence of SPF and PG show full epoxidase activity. They do not require further addition of these factors during enzyme assay. Addition of SPF and PG to assay mixtures containing microsomes preloaded with substrate in the presence of SPF and PG did not further increase epoxidase activity. We also show that PG tightly binds to microsomes. This binding of PG is essential for the response of microsomal epoxidase to SPF. Solubilized microsomal enzymes have been reconstituted and show high epoxidase activity. In this system, SPF and PG do not stimulate the conversion of squalene into products.     

Alzheimer''s paired helical filaments share epitopes with neurofilament side arms.

A panel of monoclonal antibodies to neurofilaments have been investigated with regard to the location of their respective epitopes on neurofilament polypeptides and their ability to label the neurofibrillary tangles and paired helical filaments (PHF) which are characteristic of Alzheimer's disease. All of the neurofilament monoclonal antibodies that label tangles and PHF are directed against epitopes in the side arm domains of the two larger neurofilament polypeptides, NF-H and NF-M, and do not recognise the alpha-helical rod domains of these proteins. Immuno-electron microscopy demonstrates that the neurofilament antibodies label the constituent PHF per se and do not simply stain neurofilaments that might be admixed with PHF. These neurofilament epitopes are differentially retained by PHF, following isolation. Thus, antibody labelling of PHF is not simply due to the presence of normal neurofilament polypeptides. We propose that in tangle-bearing neurons, neurofilaments are degraded by proteases and that it is fragments of the side arms which contribute to the composition of PHF.     

Convenient,laboratory procedure for producing solid d-arabino-hexos-2-ulose (d-glucosone)

The production of solid d-arabino-hexos-2-ulose (d-glucosone) from d-glucose by use of an enzyme, pyranose-2-oxidase (EC 1.1.3.10), is described. The enzyme is extracted from the mycelia of Polyporus obtusus, partially purified, and then immobilized on activated CH-Sepharose 4B. The enzymic conversion of d-glucose into d-glucosone is simple and convenient, and provides a product free from residual d-glucose. Lyophilization of the filtered reaction-solution yields the product, solid d-glucosone. Assay methods have been developed for monitoring the enzymic reaction and evaluating the purity of the final product.     

Studies on the Indochina I/CDC strain of Plasmodium falciparum in Colombian and Bolivian Aotus monkeys and different anophelines

The Indochina I/CDC strain of Plasmodium falciparum was isolated from a physician returning to the United States after working in the refugee camps along the Thailand-Kampuchean border. The strain was established in splenectomized Aotus monkeys from Colombia after being grown in vitro for 50 days. During the first three passages in Colombian monkeys, the parasites were not infective to Bolivian Aotus monkeys. After six intervening passages in Saimiri sciureus monkeys, the parasites produced high parasitemias in both Colombian and Bolivian Aotus, but gametocytes were no longer produced. Mosquito infections were obtained only during the first three passages in the Colombian monkeys. The most susceptible mosquito was Anopheles freeborni, followed by An. dirus, An. stephensi, An. maculatus, An. culicifacies, and, rarely, An. gambiae. Sporozoites were found in the salivary glands of the An. freeborni, An. dirus, An. stephensi, and An. maculatus.     

Studies of the Sal I strain of Plasmodium vivax in the squirrel monkey (Saimiri sciureus)

The Sal I strain of Plasmodium vivax was successfully adapted to three phenotypes of the squirrel monkey, Saimiri sciureus. Through five linear blood passages, parasitemias in excess of 200,000/mm3 blood were attained; Bolivian phenotype Saimiri appear to develop higher peak parasitemias. Sporozoites of the Sal I strain inoculated intravenously produced patent parasitemias in all five squirrel monkeys challenged, with prepatent periods ranging from 21 to 38 days. Anopheles freeborni and An. gambiae were the most susceptible of eight anopheline species fed on infected squirrel monkeys. As a model for in vivo studies of P. vivax the Sal I strain in Saimiri has great potential.     

The proteolytic digestion of ox neurofilaments with trypsin and alpha-chymotrypsin.

Brief digestion of ox neurofilaments with trypsin liberates fragments that are soluble and have molecular weights ranging from 164 000 to 97 000. Peptide fingerprinting indicates that these regions, termed the tryptic head-regions, arise from the 205 000- and 158 000-mol.wt. components of the triplet. The remains of the parent polypeptides sediment with normal filaments and have been termed tail-regions. Digestion of neurofilaments with chymotrypsin also liberates soluble fragments (chymotryptic head-regions) but these have mol.wts. 171 000 and 119 000, though they too originate from the higher-molecular-weight triplet polypeptides. Tryptic and chymotryptic head-regions have extensive homology, and a low (less than or equal to 20%) helix content. Electron microscopy shows that chymotryptic digestion rapidly reduces the length of filaments, probably because this enzyme preferentially attacks the 72 000-mol.wt. polypeptide. In contrast, brief digestion with trypsin does not reduce filament length even though more than 90% of the two higher-molecular-weight components have been cleaved. These results indicate that the backbone of native filaments is formed from the 72 000-mol.wt. polypeptide together with the tail-regions from the 205 000- and 158 000-mol.wt. polypeptides. The corresponding head-regions of these components, which can represent nearly 75% of each molecule, are not necessary for preserving the backbone of native neurofilaments and are therefore good candidates for being the side arms that connect these filaments in nerve cells.