The cytological localization of rat pancreas ribonuclease by means of fluorescent antibodies

Summary The occurrence of ribonuclease in murine and bovine pancreas has been studied with fluorescent antibodies. Results are identical in the two species. Staining occurred in the ergastoplasmic (basal) zone and in the nucleoli of the acinar cells. In the islets of Langerhans, none or only very faint nucleolar staining occurred. Extraction experiments indicate that ribonuclease might be present in at least two forms in the pancreas: one readily dissolvable and one which is more strongly bound to the cytoplasm.     

Effect of macrophages and antibodies on in vivo growth of Moloney sarcoma in the rat.

Brown Norway and Lewis rats were challenged with a Brown Norway Moloney sarcoma tumor, MST-1, admixed with nonimmune peritoneal exudate macrophages syngeneic to the host; or admixed with nonimmune peritoneal exudate macrophages and hyperimmune anti-MST-1 antibodies. In vivo growth of MST-1 in BN and Lewis rats was inhibited by admixing Brown Norway or Lewis macrophages, respectively, with BN anti-MST-1 antibodies. The inhibiting BN antibodies were of the IgG2 class, lacking IgG2a antibodies. Brown Norway anti-MST-1 of IgG2 class without macrophages did not affect growth of MST-1. Brown Norway and Lewis anti-MST-1 antibodies of IgG2a class enhanced tumor growth, whether admixed with macrophages or not. Anti-MST-1 antibodies of IgM and IgG1 classes did not influence tumor growth. Peritoneal exudate macrophages removed from Lewis donors 8 to 10 days after inoculation of MST-1 inhibited completely growth of the challenge tumor; macrophages of Brown Norway origin were inhibitory only when harvested from hyperimmune donors, that is, 40 or more days after inoculation of MST-1. Macrophages from hyperimmune donors were specifically cytotoxic to MST-1 and did not inhibit an unrelated syngeneic BN tumor of chemical origin.     

Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.     

Ultrastructural localization of epitopes recognized by monoclonal antibodies produced to rat Pneumocystis carinii.

The binding sites of five monoclonal antibodies (MAb) developed against rat Pneumocystis carinii were examined at the ultrastructural level by using a post-embedding labeling method. Although all five MAb reacted with the pellicle of P. carinii, they were divided into two groups by localization of binding sites. The MAb 168.2.1, 174.2.1, and 215.2.1 reacted mainly with the electron-dense outer layer, whereas MAb 227.1.1 and 228.1.1 labeled both the outer dense layer and the middle lucent layer. With in the first group of MAb, no significant differences were observed in the reactivity patterns seen with the different stages of P. carinii. In the second group, however, the intensity of labeling of the electron-dense layer was higher in the precyst, cyst, and ruptured cyst stages than in the trophozoite stage. These latter results indicate that there may be an increase in antigen accumulation during development from the trophozoite to the cyst stages, or that antigens may be modified the development.     

Immunocytochemical localization of microtubule-associated protein 1 in rat cerebellum using monoclonal antibodies

Immunohistochemical staining with monoclonal antibodies showed that microtubule-associated protein 1 (MAP1) has a restricted cellular distribution in the rat cerebellum. Anti-MAP1 staining was found only in neurons, where it was much stronger in dendrites than in axons. There were striking variations in the apparent concentration of MAP1 in different classes of neurons. Purkinje cells were the most strongly labeled, while granule cell neurons gave a faint, threshold-level reaction with the antibody. The reaction of Golgi neurons was intermediate between these two extremes. Equivalent results were obtained using two different methods of tissue preparation. Thus MAP1 appears to be a neuron-specific protein that is highly concentrated in dendrites and occurs at markedly different levels in different types of neurons. These observations provide further indications of heterogeneity among brain microtubules.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

In vivo localization of insulin binding to cells of the rat pancreas

The uptake of 125I-insulin by rat pancreas was studied in vivo. Following fixation and light microscope autoradiography, saturable uptake of 125I-insulin was quantitatively demonstrated on acinar and duct cells but not on blood vessels and islets of Langerhans. Electron microscopy revealed the localization of 125I-insulin to the basolateral cell membranes of acinar and duct cells.     

The use of region-specific antibodies in the characterization and localization of vasoactive intestinal polypeptide-like substances in the rat gastrointestinal tract

Antisera specific for different regions of porcine VIP have been used in radioimmunoassay and immunohistochemical studies of immunoreactive VIP in rat small and large intestine. Cation exchange chromatography of intestinal extracts separated two major and one minor peak of immunoreactivity. One major peak eluted in a similar position to natural porcine VIP and was read equally by NH₂-terminal-specific, and mid- and COOH-terminal-specific antisera. A second major peak, and the minor peak, eluted earlier than porcine VIP, and were read significantly less well with mid- and COOH-terminal antisera compared with NH₂-terminal-specific antisera. All forms of VIP occurred mainly in extracts of muscle layers of the gut, and no antiserum revealed more than trace amounts of immunoreactivity in mucosal extracts. In immunohistochemical studies all antisera demonstrated fluorescent nerve fibres in the enteric plexuses, circular smooth muscle and lamina propria; some antisera demonstrated nerve cell bodies predominantly in the submucous plexus. NH₂-terminal-specific antisera also demonstrated a sparse population of mucosal endocrine-like cells in the ileum and colon that were not seen with other antisera. It is concluded that VIPergic neurons of the rat gut contain a peptide closely resembling porcine VIP and at least two less basic variants with similar NH₂-terminal antigenic determinants. VIP-like peptides may also occur in endocrine cells, but since these peptides appearto fact that the majority of neuronal VIP in rat gut exists in a form that is both chromatographically and immunochemically distinct from porcine VIP, and may well possess different biological properties.