Balanced homodyning for apertureless near-field optical imaging.

A quadrature optical detection technique, based on polarized balanced-homodyne interferometry, has been developed for specific application to apertureless near-field scanning optical microscopy (ANSOM). With such technique, multiplicative background interference, inficiating quantitative optical imaging in standard homodyne-based ANSOM, can be suppressed. Periodic modulation of interferometric optical phase, typically employed in heterodyne-based ANSOMs even to such purpose, is not needed in the present configuration. Homodyne detection also facilitates detection of harmonic components of the ANSOM optical signal at the probe/sample distance modulation frequency, necessary for near-field discrimination and suppression of artifacts. Furthermore, since amplitude signal is not affected by phase fluctuations generated in the optical path of the interferometer, an optical fiber could be included in one interferometer arm, to couple the ANSOM head to the detection system, obtaining improved versatility of the instrument. A demonstration of the interferometer performance is given by a test confocal optical scan of a mirror surface. This technique, as applied to near-field microscopy, is anticipated to provide absolute values of optical contrast not depending on background interference and topography artifacts.

Confocal microscopy of electro-optic materials: effect of aberrations on the axial response in ac mode

Confocal scanning optical microscopy can be used to investigate the structure of electro-optic materials. Application of an ac electric field allows one to measure sensitively small changes in the reflection of light from a sample surface, and those changes can be related to the electro-optic properties. We observe the axial dependence of the ac light intensity to be a linear combination of the dc component and its axial derivative. Our analysis shows that astigmatic aberrations and the azimuthal dependence of the optical index in anisotropic materials can explain this behavior.

[A retrospective clinico-morphologic study of insulinoma]. / Retrospektivnoe kliniko-morfologicheskoe issledovanie insulinom.

Surgical material from 51 patients operated on for insulinoma is studied. Three histological variants of insulinoma are distinguished: adenoma-like, insular-ductal and carcinoid-like. The structure of Langerhans islands outside the tumour is described and the diagnostic criteria of insuloma-microadenoma are presented. Multiple nests of the endocrine cells are regarded either as a manifestation of the multifocal island pathology or as a marker of non-diagnosed insuloma. There is a need of distinguishing between the terms related to the nosology and histogenesis of endocrine pancreatic tumours.

[Neuronal activity of microganglia of the vena cava orifice]. / Neironal'naia aktivnost' mikrogangliev ust'ia polykh ven.

Spontaneous activity of ganglionic neurons was studied in vitro in subserous plexus of the isolated preparations of inferior vena cava segments attached to the ventricle. Presence of the active neurons along with the "silent" those was shown. Mechanosensitive neurons with a single response to mechanical stimulation constituted a separate group. The neurons under study belong to the metasympathetic part of the nervous system and are supposed to affect the functional state regulation of the intracardiac sinus node.

[The effect of muscular loading on carbohydrate and fat metabolism in the current year's brood of Salmo irideus trout]. / Vliianie myshechnoi nagruzki na uglevodnyi i zhirovoi obmen u segoletok foreli Salmo irideus

In trout fingerlings with a mean weight 1.1 g and body length 4.3 cm, kept in a water stream at the velocity of 0.2 m/sec for 1-3 hours, the content of glycogen in muscles, liver and brain decreases whereas the content of unsaturated fatty acids and glucose in the blood as well as the level of lactate in muscles increase. After 5-hour swimming of the fingerlings carbohydrate metabolism and the content of unsaturated fatty acids return to the initial levels; the content of fat in the liver significantly decreases. Presumably trout fingerlings exhibit high capacity for adaptation to the given muscular activity and do not show fatigue.

Geldanamycin induces ErbB-2 degradation by proteolytic fragmentation.

Exposure of carcinoma cell lines to the antibiotic geldanamycin induces the degradation of ErbB-2, a co-receptor tyrosine kinase that is frequently overexpressed in certain tumors. Using ErbB-2 mutants expressed as chimeric receptors or green fluorescent protein fusion proteins, we report that the kinase domain of ErbB-2 is essential for geldanamycin-induced degradation. The kinase domain of the related epidermal growth factor receptor was not sensitive to this drug. The data further indicate mechanistic aspects of ErbB-2 degradation by geldanamycin. The data show that exposure to the drug induces at least one cleavage within the cytoplasmic domain of ErbB-2 producing a 135-kDa fragment and a 23-kDa fragment. The latter represents the carboxyl-terminal domain of ErbB-2, whereas the former represents the ectodomain and part of the cytoplasmic domain. Degradation of the carboxyl-terminal fragment is prevented by proteasome inhibitors, whereas degradation of the membrane-anchored 135-kDa ErbB-2 fragment is blocked by inhibitors of the endocytosis-dependent degradation pathway. Confocal microscopy studies confirm a geldanamycin-induced localization of ErbB-2 on intracellular vesicles.

Caspase-dependent cleavage of ErbB-2 by geldanamycin and staurosporin.

The geldanamycin-induced degradation of ErbB-2 produces a 23-kDa carboxyl-terminal fragment, which has been isolated and subjected to amino-terminal microsequencing. The obtained sequence indicates that the amino terminus of this fragment corresponds to Gly-1126 of ErbB-2. Analysis of the residues immediately before Gly-1126 suggests that cleavage may involve caspase activity. Site-directed mutagenesis of Asp-1125 in ErbB-2 prevents geldanamycin-provoked formation of the 23-kDa fragment, consistent with the requirement of this residue for caspase-dependent cleavage in known substrates. Also, the addition of the pan-caspase inhibitor Z-VAD-FMK blocks formation of the 23-kDa ErbB-2 fragment in cells exposed to geldanamycin. Interestingly, staurosporin and curcumin are also shown to provoke the degradation of ErbB-2 with formation of the 23-kDa carboxyl-terminal fragment. The generation of this fragment by staurosporin or curcumin is likewise blocked by caspase inhibition. Caspase inhibition does not prevent accelerated degradation of the 185-kDa native ErbB-2 in geldanamycin-treated cells but does significantly prevent staurosporin-stimulated metabolic loss of ErbB-2.

Preference for IgG mAb binding insulin in solution or on surfaces is related to immunoglobulin variable region structures.

Studies on insulin autoantibodies often show a lack of correlation between enzyme-linked immunosorbent assays (ELISAs) and fluid phase radioimmunoassays (RIAs). Similarly, a set of IgG anti-insulin monoclonal antibodies (mAb) from BALB/c mice are found to differ in their binding in ELISAs and RIAs. To understand the structural basis for differential insulin binding, soluble and complexed biotinylated insulin is used to confirm binding properties independent of insulin-coated plastic and radioiodination. The binding properties of intact mAb are also present in Fab fragments, indicating ligand preference is not related to valence or to the Fc portion of Ig. Analysis of binding to soluble or bound ligand in relationship to antibody variable (V) region structures indicates that differential binding in the two assays is a property of heavy chain variable region structure. Studies also show that limited amino acid replacements arising during maturation of the immune response may change the binding preference for an individual mAb. These findings indicate that differences in detection of insulin binding in solid phase and fluid phase are not artefactual but reflect intrinsic structural features of immunoglobulin interaction with insulin.

Alanine scanning mutants of rat proinsulin I show functional diversity of anti-insulin monoclonal antibodies.

In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a "molten globule" may contribute to the diversity and autoreactivity of the anti-insulin repertoire.

[Ultramicromethod of erythrocyte immunoadsorption in screening for somatic antigens in acute myocardial infarct]. / Ul'tramikrometod éritroimmunoadsorbtsii v skrininge somaticheskikh antigenov pri ostrom infarkte miokarda.

Test-systems for the identification of somatic myoglobin antigens and fibrin-fibrinogen degradation products in the sera of patients with acute myocardial infarction have been developed and tested. A significant correlation between erythrocyte immunoadsorption technique and solid-phase immunoenzyme assay was observed, the former technique being simpler and express.

[Conformational changes of the platelet membrane glycoprotein IIb-IIIa complex stimulated by a monoclonal antibody to the N-terminal segment of glycoprotein IIIa]. / Konformatsionnye izmeneniia kompleksa glikoproteidov IIb-IIIa membrany trombotsitov, stimulirovannye monoklonal'nym antitelom k N-kontsevomu uchastku glikoproteida IIIa.

During platelet activation, the glycoprotein (GP) complex IIb-IIIa (alpha 11b beta 3-integrin) changes its conformation, resulting in binding of adhesive proteins of RGD containing an amino acid sequence as well as in expression of new ligand-induced binding sites (LIBS) on the GPIIb-IIIa molecule. Like its F(ab)-fragments, the monoclonal antibody CRC54, whose epitope is located in the N-terminal part of the GPIIIa molecule, binds to no more than 10% of GPIIb-IIIa on the resting platelet surface. However, the binding of CRC54 increases considerably during activation of platelets by thrombin, platelet adhesion on plastic, GPIIb-IIIa interaction with RGDS-peptide as well as during dissociation of the complex in the presence of EDTA. These finding suggest that CRC54 is specifically directed against the LIBS epitope on the GPIIIa molecule. This epitope differs from those of other known conformation-dependent antibodies against GPIIb-IIIa (LIBS1, LIBS6, PMI-1, pl55 and p180), since those antibodies did not block the CRC54 binding to GPIIb-IIIa on the surface of adhering platelets. Unlike whole platelets, the binding of GPIIb-IIIa from lysates of platelets treated with Triton X-100 with immobilized CRC54 did not depend on the presence of the RGDS peptide. Under these conditions another anti-LIBS-antibody, p180 specifically directed against GPIIb, preserved its ability to discriminate the RGDS-occupied and resting conformations of GPIIb-IIIA. CRC54 and its F(ab) fragments induced platelet aggregation in both platelet-enriched plasma and in suspensions of washed platelets. CRC54 also stimulated the binding to platelets of GPIIb-IIIa ligand fibrinogen, labelled with 125I as well as adhesion of 51Cr-labelled platelets to immobilized ligands-fibrinogen and fibronectin. The CRC54-dependent aggregation was fully blocked by RGDS-peptide and antibody CRC64 inhibiting the GPIIb-IIIa binding to the ligands. However, the platelet activation inhibitor, prostaglandin EI, and the mixture of metabolic inhibitors, deoxyglucose-sodium azide, only party inhibited the CRC54-dependent aggregation. Incubation of platelets with CRC54 induced the binding to platelets of the anti-GPIIb LIBS antibody p180 and of the anti-GPIIb-IIIa activation-dependent antibody p155. The binding of GPIIb-IIIa from lysates of CRC54-treated platelets with immobilized p180 and p155 was also several times as high as that of GPIIb-IIIa from control platelet lysates. The data obtained indicate that the GPIIb-IIIa transition to the active state and its interaction with ligands induces conformational changes in the N-terminal part of GPIIIa and that the CRC54 binding to the N-terminal part of GPIIIa stimulates conformational changes in GPIIb-IIIa, complex interaction with ligands and platelet aggregation.

[Probucol and alpha-tocopherol stimulate the synthesis of bile acids in cultured rabbit hepatocytes]. / Probukol i alpha-tokoferol stimuliruiut sintez zhelchnykh kislot v kul'tiviruemykh gepatotsitakh krolika.

Primary cultures of rabbit hepatocytes were used to examine the effect of probucol and alpha-tocopherol on cholesterol and bile acid metabolism. After 24-hour preincubation of cells with 100 microM probucol, the cholesterol and cholesteryl ester content increased--by 30% and 50%, respectively. This was accompanied by decreasing incorporation of [14C]acetate into cholesterol (down to 25-35%). At the same time, alpha-tocopherol had no effect on cholesterol accumulation in hepatocytes, while cholesterol synthesis was stimulated by 30-50%. Addition of 100 microM probucol or alpha-tocopherol to a culture medium containing 10% fetal calf serum and [14C]cholesterol caused a significant (30-40%) stimulation of bile acid synthesis. Stimulation by probucol was dependent on the presence of exogenous plasma or HDL2 cholesterol, while alpha-tocopherol enhanced this process in a cholesterol-free medium. Stimulation (40%) of bile acid secretion by probucol in the presence of physiological concentrations of apo E-free HDL2 was found. Study of receptor-mediated uptake of HDL2 revealed that: (i) probucol stimulated apo E secretion; (ii) HDL2 isolated from a medium of probucol-treated cells contained 2-3 times more apo E than the particles preincubated with cells without the drug; (iii) apo E-enriched HDL2 particles were incorporated into cultured human skin fibroblasts 1.5-3.0 times more effectively than apo E depleted HDL2; (iv) monoclonal antibody against the LDL-receptor binding domain of apo E effectively (by 40%) inhibited the apo E-enriched HDL2 uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of soluble P-selectin by platelets and endothelial cells.

The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for approximately 10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A2 analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up approximately 10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.