Induction of rat muscle carbonic anhydrase by denervation demonstrated with immunofluorescence

Immunofluorescence microscopy of carbonic anhydrase III (CA III) was performed on sections of rat anterior tibialis (AT), extensor digitorum longus (EDL) and soleus after denervation. In contralateral control muscles, CAIII was located only in type I fibres whereas following the operation, CAIII was markedly induced in type II fibers of all the muscles, most strikingly in EDL.     

The size of the fibre populations in rabbit skeletal muscles as revealed by indirect immunofluorescence with anti-myosin sera.

Antibodies against myosin of the fast long. Dorsi and the slow soleus muscle of rabbits were induced in guinea pigs. With the aid of a new technique, the gel-electrophoresis-derived-enzyme-linked-immunosorbent assay (GEDELISA) it could be shown that they are directed against the heavy and the light chains of fast (M. long. dorsi) and slow (M. soleus) myosin. In the indirect immunofluorescence test each antiserum only stained one population of fibres in five different muscles tested. The single fibres were observed to react only with one of the two types of antisera. The following percentage of fibres showed a positive reaction with the anti-fast myosin serum: M. long. dorsi, 95%; M. psoa maior, 95%, M. psoa minor, 92%; M. tibialis ant., 90%; M. soleus, 15%.     

The size of the fibre populations in rabbit skeletal muscles as revealed by indirect immunofluorescence with anti-myosin sera

Summary Antibodies against myosin of the fast long. dorsi and the slow soleus muscle of rabbits were induced in guinea pigs. With the aid of a new technique, the gel-electrophoresis-derived-enzyme-linked-immunosorbent assay (GEDELISA) it could be shown that they are directed against the heavy and the light chains of fast (M. long. dorsi) and slow (M. soleus) myosin. In the indirect immunofluorescence test each antiserum only stained one population of fibres in five different muscles tested. The single fibres were observed to react only with one of the two types of antisera. The following percentage of fibres showed a positive reaction with the anti-fast myosin serum: M. long. dorsi, 95%; M. psoas maior, 95%, M. psoas minor, 92%; M. tibialis ant., 90%; M. soleus, 15%.Abbreviations AB antibodies - ETPase adenosintriphosphatase - Anti-LdM antiserum against LdM - Anti-SoM antiserum against SoM - BSA bovine serum albumin - CPf contaminating protein in LdM - CPs contaminating protein in SoM - EDTA ethylene diamine tetra-acetic acid - ELISA enzyme-linked immunosorbent assay - FITC fluoresceinisothiocyanate - FM last myosin showing 3LC in PAGE - GEDELISA gel electrophoresis-derived enzyme-linked immunosorbent assay - HC heavy chains of myosin - LC light chains of myosin - LdM myosin preparation of longissimus dorsi muscle - MCF microcomplement fixation - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline (140 mM NaCl, 20 mM potassium-phosphate, pH 7.4) - SDS sodiumdodecylsulfate - SM slow myosin, showing two LC in PAGE - SoM myosin preparation of soleus muscle     

Glycoprotein biosynthesis by human normal platelets

Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation.     

Aliesterase activity in normal and postheparin human blood sera.

An enzyme with characteristics typical of aliesterase has been found in human blood serum using a gas solid chromatographic assay technique. This conflicts with the findings of several authors that aliesterase is absent in the human blood. Another aliesterase is released into the blood stream after intravenous administration of heparin. Partial purification of the aliesterase in normal (preheparin) and postheparin sera was effected by column chromatography using CM- and DEAE-Sephadex. The preheparin aliesterase and postheparin aliesterase have different pH optima of 7.0 and 8.5 respectively. The preheparin aliesterase activity was very sensitive to sodium fluoride and insensitive to a negatively charged detergent, sodium lauryl sulfate, unlike the postheparin esterase which was highly sensitive to sodium lauryl sulfate and comparatively less sensitive to sodium fluoride.     

TMP-reactive autoantibodies in human SLE sera demonstrate thymine-dependent oligonucleotide specificity

Autoantibodies present in the sera of lupus patients and specific for single-stranded (ss) DNA were fractionated into subsets based upon their reactivity towards 5' nucleotide haptens. As evaluated by ELISA testing, antibodies retained by TMP-agarose bound to TMP-BSA and ssDNA but not to other nucleotide-BSA conjugates or to double-stranded (ds) DNA. Competition-inhibition studies further revealed that TMP-enriched oligo- and polynucleotides were the preferred antigens for these affinity purified antibodies. Similar assays with sequence- or size- defined oligonucleotides further implied that those oligonucleotides comprised entirely of TMP residues were most antigenic and that antigenicity increased with size (length). These results document the existence of a TMP-dependent oligonucleotide specificity among a diverse population of autoanti-ssDNA antibodies.     

Characterization of human malignant melanoma cell lines. III. Membrane immunofluorescence reactivity with sera from patients with melanoma

Summary Seven well-characterized human malignant melanoma cell lines have been evaluated in terms of their reactivity in membrane immunofluorescence tests with sera from 48 patients with melanoma, 23 patients with other forms of cancer and 28 normal controls. There was a significantly greater degree of reactivity of melanoma sera (33.7%) than of sera of normal controls (22.2%) or of sera from patients with other forms of cancer (24.2%). The incidence of strong reactors among the melanoma patients was found to be inversely proportional to the extent of disease in the melanoma patients: Stage I, 54.5%, Stage III, 36.8% and Stage IV, 29.4%. Reactivity against non-melanoma cell lines was similar in the three subject groups and was unaffected by stage of disease in the melanoma patients. No single cell line showed preferential reactivity with melanoma sera. There was an increased overall incidence of reactivity of all three subject groups against non-pigmented cell lines.A-B-0 antigens and heterophile antigens were excluded as a cause of seropositivity. The antigen(s) was trypsin-sensitive and neuraminidase-resistant.These data suggest that long term cultures of human melanoma may contain melanoma-associated antigens which may be useful in the further study and search for melanoma-specific antigens.     

Keratin-like proteins in normal and neoplastic cells of human and rat mammary gland as revealed by immunofluorescence microscopy

Normal and neoplastic human breast tissue as well as lactating and nonlactating rat mammary glands and 7,12-dimethylbenz(alpha)-anthracene-induced mammary adenocarcinomas of rat, were examined by indirect immunofluorescence microscopy using guinea pig antibodies to human and bovine epidermal prekeratin and to cytokeratin polypeptide D from mouse hepatocytes. In normal mammary glands of both species, lactating rats included, the antibodies raised against human and bovine epidermal prekeratins strongly stained ductal and myoepithelial cells, whereas antibodies to hepatic cytokeratin D revealed, in addition, fibrillar staining in cells of the alveolus-like terminal lobular units and in milk secreting cells of the rat. The presence of some finely dispersed intermediate-sized filaments of the cytokeratin type in lactating alveolar cells of rat mammary gland was also demonstrated by electron microscopy. In human intraductal mammary carcinomas the antibodies to epidermal prekeratins showed staining in myoepithelial cells and intralumenal papillary protrusions of the tumor, whereas the antibodies to hepatic cytokeratin D presented an almost complementary pattern in that they showed strongest staining in the more basally located layers of tumor cells. Intraductal adenocarcinomas of rats showed strong staining with all keratin antibodies examined. In contrast to previous studies using exclusively antisera raised against epidermal prekeratin, out results show that all types of neoplastic and non-neoplastic epithelial cells of mammary gland of both species contain-at least some-filaments of the cytokeratin type identifiable by immunologic reaction, if antibodies are used that recognize a broad range of epidermal and nonepidermal cytokeratins. Consequently, such broad range antibodies to keratin-like proteins provide adequate tools to identify and characterize neoplastic and non-neoplastic epithelial cells and to eliminate false negative immunocytochemical findings in tumor diagnosis. In addition, our observation that in the same human carcinoma two cell types can be distinguished by their reaction with two different antibodies to cytokeratins from epidermis and liver, respectively, indicates that the cells of a given carcinoma can differ in their cytoskeletal composition, thus presenting further criteria for diagnostic differentiation.     

Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests

Introduction  

Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated.     

Plant DNA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.     

Plant DMA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.     

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A2, thromboxane A2-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholipase A2. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A2-like acitivity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A2. The results indicate that formation of thromboxane A2-like activity enhances aggregation in rat platelets, but that aggregation is not induced.     

Localization of neurophysin in the neurosecretory elements of the hypothalamus and neurohypophysis of the normal and osmotically stimulated guinea-pig as demonstrated by immunofluorescence histochemical techniques

Summary Antisera to porcine neurophysin-II and ovine neurophysin-III were used to localize neurophysin-like material in the hypothalamus and neurohypophysis of guinea-pigs with an immunofluorescence technique. Although the guinea-pig appears to have only one major neurophysin it was found to be localized in both of the bilateral magnocellular nuclei. Neurophysin-like material was also present in extreme rostral portions of the hypothalamus, in cells lying between the third ventricle and the supraoptic nucleus and in a cluster of cells dorsomedial to the fornix. Immunofluorescence was observed in neurosecretory fibres that followed pathways previously characterized with classical histological stains for neurosecretory material.The immunofluorescence in the hypothalamic elements of a normal guinea-pig was not greatly different from that in fluorescent structures present in an animal that had been severely dehydrated. In contrast, there was a marked depletion of neurophysin from the posterior pituitary gland of the dehydrated guinea-pig. The lack of graded fluorescence in the hypothalamus of the dehydrated animal is discussed.This work was financed by a grant from the New Zealand Medical Research Council and the Auckland Medical Research Foundation.