Development of a highly sensitive, field operable biosensor for serological studies of Ebola virus in central Africa.

We describe herein a newly developed optical immunosensor for detection of antibodies directed against antigens of the Ebola virus strains Zaire and Sudan. We employed a photo immobilization methodology based on a photoactivatable electrogenerated poly(pyrrole-benzophenone) film deposited upon an indium tin oxide (ITO) modified conductive surface fiber-optic. It was then linked to a biological receptor, Ebola virus antigen in this case, on the fiber tip through a light driven reaction. The photochemically modified optical fibers were tested as an immunosensor for detection of antibodies against Ebola virus, in animal and human sera, by use of a coupled chemiluminescent reaction. The immunosensor was tested for sensitivity, specificity, and compared to standard chemiluminescent ELISA under the same conditions. The analyte, anti-Ebola IgG, was detected at a low titer of 1:960,000 and 1:1,000,000 for subtypes Zaire and Sudan, respectively. While the same serum tested by ELISA was one order (24 times) less sensitive.

Filamentous phage displaying the extracellular domain of the hLH/CG receptor bind hCG specifically.

We have expressed the extracellular binding domain of the human LH/CG receptor as a fusion with the cpIII filamentous phage coat protein. These fusion phage are able to specifically bind ELISA plates coated with hCG. Preincubation of the phage with hCG before exposure to the coated plates inhibited binding of phage, whereas human FSH had no effect. Furthermore, addition of anti-hLH/CG receptor antisera inhibited binding of phage to the plates. We have also tested the effect of monoclonal antibodies to hCG on phage binding. Monoclonal antibodies that can bind hCG when it is bound to native receptor do not inhibit phage binding. These include B105 and A109 Alternatively, B107 and B109 have an inhibitory effect on phage binding. They cannot bind hCG when it is bound to receptor and are likely directed against epitopes at or near the receptor binding interface. Therefore, these fusion phage exhibit the same binding specificity as the wild-type receptor and likely display the extracellular domain of the hLH/CG receptor on their surface in the native conformation. Phage display is a potentially useful tool for studying the hLH/CG receptor structure and in screening for synthetic compounds that compete with hormone for receptor binding.

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.

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.

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.

Flow sorting of hybrid hybridomas using the DNA stain Hoechst 33342.

A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described for the selection of hybrid hybridomas producing bispecific monoclonal antibodies. Parental hybridoma cells were first labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC); heterofluorescent cells were recovered after fusion. After a period of growth in culture, the cells were then stained with the DNA-specific dye bis-benzimidazole Hoechst 33342 and sorted on the basis of their DNA content. The staining conditions (10 micrograms/ml of Hoechst 33342, 90 min incubation time at 37 degrees C) were found to be optimal for obtaining a well resolved DNA histogram with minimal effect on the growth properties of cells from different mouse hybridoma lines. Employing this method we have isolated hybrid hybridomas synthesizing bispecific monoclonal antibodies reacting with human low density lipoprotein and alkaline phosphatase from calf intestine.

Three-dimensional structures of gonadotropins.

Most secreted proteins are modified post-translationally with the addition of carbohydrate. It has been difficult to use crystallography to solve the structures of these proteins due to the inherent heterogeneity of the carbohydrate. The structure of the chemically deglycosylated form (hydrogen fluoride treated) of human chorionic gonadotropin (hCG) has been solved through crystallographic techniques. Unfortunately this form of hCG is not biologically active, and exhibits immunochemical differences from native hormone. In addition, subunit interactions appear altered after chemical deglycosylation as indicated by the increased thermal stability of the HF-treated hormone. The Asn 52 glycan on the alpha-subunit of hCG has been identified as being required for biological activity, it is, therefore, of physiological importance to determine the structure of the hormone with its carbohydrate intact. Also, it has not been possible to obtain crystals of the individual glycosylated subunits of hCG. Therefore an alternative method to solve the structure of the biologically active form of the hormone in solution as well as its separated subunits is necessary. Structural information utilizing NMR techniques can be obtained from native hCG subunits in solution if they can be uniformly labeled with 13C and 15N isotopes. We have developed a universal nonradioactive isotope, labeling medium enriched in 13C and 15N which can be used to express uniformly labeled hCG from Chinese hamster ovary cells suitable for solving the structure of the individual subunits and ultimately that of the native, biologically active hormone. The isotopically labeled recombinant hCG and its purified subunits are essentially identical to urinary hCG on comparison by biochemical, immunochemical, biological activity and the ability of the isolated subunits to recombine to form a biologically active dimer. Mass spectrometric analysis and preliminary structural NMR data indicate that the labeling is uniform and there is greater than 90% incorporation, sufficient for complete structural determination studies. This labeled growth medium represents a technological advance which will enable the rapid solution of the structures of the other glycoprotein hormones, as well as other glycoproteins which have proven unsuitable for crystallographic study.

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.

Melatonin, environmental light, and breast cancer.

Although many factors have been suggested as causes for breast cancer, the increased incidence of the disease seen in women working in night shifts led to the hypothesis that the suppression of melatonin by light or melatonin deficiency plays a major role in cancer development. Studies on the 7,12-dimethylbenz[a]anthracene and N-methyl-N-nitrosourea experimental models of human breast cancer indicate that melatonin is effective in reducing cancer development. In vitro studies in MCF-7 human breast cancer cell line have shown that melatonin exerts its anticarcinogenic actions through a variety of mechanisms, and that it is most effective in estrogen receptor (ER) alpha-positive breast cancer cells. Melatonin suppresses ER gene, modulates several estrogen dependent regulatory proteins and pro-oncogenes, inhibits cell proliferation, and impairs the metastatic capacity of MCF-7 human breast cancer cells. The anticarcinogenic action on MCF-7 cells has been demonstrated at the physiological concentrations of melatonin attained at night, suggesting thereby that melatonin acts like an endogenous antiestrogen. Melatonin also decreases the formation of estrogens from androgens via aromatase inhibition. Circulating melatonin levels are abnormally low in ER-positive breast cancer patients thereby supporting the melatonin hypothesis for breast cancer in shift working women. It has been postulated that enhanced endogenous melatonin secretion is responsible for the beneficial effects of meditation as a form of psychosocial intervention that helps breast cancer patients.