Monoclonal antibodies against chordin. Use in structural and immunohistochemical studies.

Eight MAbs have been developed against chordin and designated as At2-At9. It is shown that all antibodies are directed against identical, spatially overlapping or closely positioned epitopes of chordin. The chordin molecule has repetitive sites wherein epitopes for the eight MAbs are located. This site lies within a proteinase-resistant fragment of chordin, presumably a glycopeptide, of molecular mass between 2 and 10 kDa. Fluorescence staining of cryostat sections from stellate sturgeon with the use of At5 (indirect Coons' method) has revealed a positive reaction with notochord cells and sheath and with the spinal cord. No significant reaction with cartilage, muscle and kidney was detected.

Elongation factor-2 kinase: immunological evidence for the existence of tissue-specific isoforms.

eEF-2 kinase is a ubiquitous Ca2+/calmodulin-dependent protein kinase that is specific for protein synthesis elongation factor-2 (eEF-2). This study describes an improved procedure for the purification of eEF-2 kinase from rabbit reticulocyte lysate. The eEF-2 kinase preparation was used to raise polyclonal antibodies, which immunoprecipitated eEF-2 kinase protein and activity from rabbit reticulocyte lysate. The antibodies recognized a single 103 kDa band in extracts from several cell lines including NIH 3T3, PC12, C6 glioma, HeLa, and MCF-7 breast carcinoma. However, there was no immunoreactivity in extracts of rabbit or bovine liver or rabbit kidney despite the presence of abundant eEF-2 kinase activity in these tissues. Exposure of PC12 cells to nerve growth factor (NGF) resulted in rapid down-regulation of eEF-2 kinase activity and a decrease in immunoreactivity. After 24 h of incubation with NGF, the activity of the kinase recovered to 80% of initial values. In contrast, the immunoreactivity of eEF-2 kinase continued to decrease. These data suggest that tissue-specific isoforms of eEF-2 kinase may exist and that these isoforms may be regulated by growth factors.

Localization of apolipoprotein E in normal and atherosclerotic human aorta.

To elucidate the role of apolipoprotein E (apo E) in atherogenesis, we have investigated the localization of apo E in normal and atherosclerotic aortas as well as in other tissues of 32 post-mortem individuals. Using double immunofluorescence it has been found that normal intima of individuals older than 20 years and some adolescents contained immunoreactive material that reacted with poly- and monoclonal antibodies to apo E. A staining pattern of apo E differed from that of apolipoprotein B, the latter being seen in normal intima of each child older than 7 years. Apo E was present extracellularly in lipid streaks and atheromatous plaques, where its staining was particularly intensive around the necrotic zone of plaques. Some macrophages in the plaques of 4 aortas exhibited apo E-positive staining, while aortic endothelial and smooth muscle cells never contained apo E. Apo E-positive staining was not found in the majority of vessel cells, it was always, however, observed in other types of cells including hepatocytes. Kupffer cells, spleen macrophages and cerebral astrocytes. Our findings indicate that only some macrophages in human aorta may be responsible for the production of apo E that can participate in reverse cholesterol transport. At the same time, apo E accumulation in the aortic wall may promote the development of atherosclerosis.

Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases.

A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.

Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: very low density lipoprotein and bile acid production.

The interrelationship between very low density lipoprotein (VLDL) secretion and bile acid production was studied in primary culture of rabbit hepatocytes. Chylomicron remnants (CR) were added to the cultures to study their effect on VLDL secretion and bile acid production. After 24 hr preincubation of cells with CR (10-50 micrograms protein/mL), intercellular neutral lipid content was increased 1.5-4-fold in a dose-dependent manner. Neutral lipid accumulation was accompanied by a 70-90% reduction of [14C]acetate incorporation into cholesterol, while no stimulation of [14C]oleate incorporation into cholesteryl esters was observed. Incubation of cells with CR increased secretion of free cholesterol, triacylglycerol and apoproteins B and E in VLDL. Stimulation of VLDL cholesterol secretion was accompanied by a reduction of taurocholic acid synthesis. These data demonstrate the existence of an inverse relationship between secretion of VLDL cholesterol and bile acid production under conditions of effective uptake of triacylglycerol-rich CR by hepatocytes.

[Allelic exclusion in hybridomas. I. Isolation and properties of a hybrid clone producing two allelic variants of light chain immunoglobulins in the rat]. / Allel'noe iskliuchenie v gibridomakh. I. Poluchenie i svoistva gibridomnogo klona, produtsiruiushchego dva allel'nykh varianta legkikh tsepei immunoglobulinov krysy.

Expression of RI-1a and RI-1b allelic genes controlling the production of rat lg kappa L-chains by hybridoma cells in vitro was studied. By fusing mouse myeloma cells with RI-1a/RI-1b heterozygous rat splenocytes the unique cloned hybrid cell line secreting both allelic variants has been established. This line may have appeared because cell hybridization made it possible to fix the rare case of correct rearrangement of the kappa chain gene segments on both homologous chromosomes.

[Structural study of the chromatin of the myxomycete Physarum polycephalum in the mitotic cycle]. / Izuchenie struktury khromatina miksomitseta Physarum polycephalum v mitoticheskom tsikle.

The chromatin structure of Physarum polycephalum was studied with electron microscope at different phases of its mitotic cycle. At the S-phase and during mitosis, the chromatin has a nucleosomal structure. At the early G2-phase the chromatin structure changes, long regions of non-beaded structure being found in the chromatin fibers. At the late G2-phase, the major part of chromatin loses its globular organization, with chromatin fibres without a pronounced subunit structure prevailing in the preparations. Biochemical data show that the amount of chromatin resistant to staphylococcal nuclease varies during the mitotic cycle. The amount of nuclease-resistant chromatin is equal to 80% at the S-phase, to decrease up to 50-60% by the early G2-phase. Successive changes of chromatin structure at different levels of its transcriptional activity are found. Lability of nucleosomes is shown to increase with the increase in the transcriptional activity of chromatin, thus leading presumably to the chromatin structural alterations during the mitotic cycle.

[Immuno-morphologic detection of specific APO-B antigenic determinants in normal intima and atherosclerotic plaques in the human aorta]. / Immunomorfologicheskoe vyiavlenie otdel'nykh antigennykh determinant apoB v normal'noi intime i ateroskleroticheskikh bliashkakh aorty cheloveka.

Various apo-B antigenic determinants exposures in the normal aortic intima and atherosclerotic plaques were examined by immunofluorescence using 5 clones from monoclonal antibodies (BAb) to apo-B. As a rule, 4 clones were found to react with apo-B in the normal aortic intima, whereas the clone 12G10 failed to respond to it. All the 5 determinants of apo-B were exposed in the fibrous tissue of most atherosclerotic plaques. However, there were atherosclerotic plaques, in whose fibrous tissue only did 1 to 3 clones positively react with apo-B. The similar expression of apo-B antigenic determinants was observed in the atheronecrotic zone of fibrous plaques. Apo-B did not respond to apo-B MAb at all in some atherosclerotic plaques present in the zone of atheronecrosis. Whether it is possible to modify low-density lipoproteins just in the arterial wall, especially in atherosclerotic plaques and whether this process is significant in the pathogenesis of atherosclerosis are discussed in the paper.

[The localization of apoprotein E in the normal human aortic wall and in atherosclerosis]. / Lokalizatsiia apoproteina E v stenke aorty cheloveka v norme i pri ateroskleroze.

The authors studied the distribution of apoprotein E (apoE) in normal and atherosclerotic human aortic wall. Double immunofluorescent technique and a set of mono- and polyclonal antibodies were used in the study. Apo E was found in normal intima of every aorta taken from people over 20 years of age and in vessels of some adolescents. The protein was localized extracellularly and was noted in some portion of macrophages but not in the endothelial and smooth muscle cells of human aorta. The accumulation of apo E increased in lipid strips and was particularly high in acellular zone of the atherosclerotic plaque. This effect may be due to the retention of apo E by changed sulfated glycosaminoglycans of aortic connective tissue. The accumulation of apo E in the vessel wall may have an important role in the pathogenesis of atherosclerosis.

[Obtaining hybridomas producing monoclonal antibodies with different specificities against Mycobacterium tuberculosis of the human and bovine types]. / Poluchenie gibridom, produtsiruiushchikh monoklonal'nye antitela s razlichnoi spetsifichnost'iu k mikobakteriiam tuberkuleza chelovecheskogo i bych'ego vidov.

A procedure for isolation of hybridomes producing monoclonal antibodies (McAB) to tubercle bacilli is described. Specificity of the McABs was studied with the solid phase radioimmune and immunoenzyme tests. Supernatant of tubercle bacilli destroyed with ultrasound was used as antigens. The McABs did not practically react with antigens of the tubercle bacilli atypical forms. Five ascitic monoclonal hybridomes were isolated. Four of them produced antibodies with selective specificity to antigens of bovine tubercle bacilli (M. bovis-8 and BCG) and one produced antibodies to antigens of human tubercle bacilli (H37Rv).

[The plasmoimmunosorption of low-density lipoproteins in the treatment of patients with hereditary hypercholesterolemia]. / Plazmoimmunosorbtsiia lipoproteidov nizkoi plotnosti v lechenii bol'nykh s nasledstvennoi giperkholesterinemiei.

The authors describe the experience gained with the use of apheresis of low density lipoproteins with the aid of the Soviet immunosorption columns with polyclonal and monoclonal antibodies in patients with hereditary hypercholesterolemia resistant to the diet and hypolipidemic drug therapy. High specificity of apheresis of low density lipoproteins and high efficacy of the use of the sorption columns to correct hypercholesterolemia have been demonstrated.

Monoclonal antibody to human lung angiotensin-converting enzyme.

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunized with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by enzyme-linked immunosorbent assay and that of soluble ACE by an immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not affect ACE activity. We believe this antibody will be useful not only for immunoassay and immunopurification of ACE, but also as a tool for the investigation of the tissue distribution of the enzyme as well as for the study of the structure and mechanism of action of ACE.

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies.

The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9 B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9 B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9 B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.

[Transformation of human corneal endothelial cells by micro- injection of oncogenes]. / Transformatsiia kletok rogovichnogo éndoteliia cheloveka mikroin''ektsirovannymi onkogenami.

Human corneal endothelial cells (HCEC) were transfected with some cloned oncogenes. The direct microinjection of either early region (E1) genes of monkey (SA7) and human (Ad5) adenoviruses or Ha-ras oncogen in conjunction with the Ad5 Ela-gene into embryonic HCEC nuclei was shown to result in immortalization of these cells. 3 independent immortalized HCEC lines were established in their growth and morphological properties were studied. These properties were very similar to those of primary HCEC, but unlike primary HCEC the immortalized cells didn't need the endothelial cell growth factor.

[Immunocytochemical analysis of apoprotein E distribution in human tissues]. / Immunotsitokhimicheskii analiz raspredeleniia apoproteina E v tkaniakh cheloveka.

Localization of apoprotein E (apo E) has been studied in different human tissues. For this aim the immunoperoxidase method and peroxidase-antiperoxidase complex, polyclonal and monoclonal antibodies, to apo E have been used. In every human tissue analysed apo E-containing cells have been revealed. To the latter the following cell types belong: hepatocytes and hepatic sinusoidal cells, macrophages of the spleen, lymph nodes and pulmonary tissues, glial cells and cells in all layers of the adrenals, skin keratinocytes, cells of the glomerular capsule and convoluted tubules of the kidney, spermatocytes and smooth muscle cells of the testis. Besides, apo E is revealed in endothelium of some vessels. As demonstrate the results obtained, apo E is found practically in all human tissues. A suggestion is made that besides its participation in reverse cholesterol transport, this protein performs a number of additional functions, such as regulation of local hormonal homeostasis of an organ.

Lung is the target organ for a monoclonal antibody to angiotensin-converting enzyme.

125I-labeled mouse monoclonal antibody (MoAb) to human angiotensin-converting enzyme (ACE), termed 9B9 and cross-reacting with rat and monkey ACE, when injected into the circulation, accumulates in the lung in up to 10 to 20 greater concentrations than in other organs and blood. That 111In-labeled MoAb 9B9 also accumulates in the lungs of both rats and monkeys very selectively, was clearly revealed by gamma-scintigraphy. Unlike polyclonal anti-ACE antibodies that induce an immunodependent lethal reaction when administered intravenously, MoAb 9B9 was well tolerated by rats even at very high doses (up to 300 mg/kg/body weight). At the same time, the administration of this antibody (which does not inhibit the catalytic activity of ACE) resulted in both a 3-fold decrease of the lung ACE activity and an increase in the activity of serum ACE. The highly organ-specific, nondamaging accumulation of the MoAb 9B9 makes it a promising vector for targeted drug delivery to the lung, for modeling of lung pathology, and for gamma-scintigraphic visualization of the lung vascular bed. We also suggest that MoAb 9B9 accumulation in the lung may serve as a highly sensitive marker of lung vessel damage upon various lung pathology.