Polymorphic amyloid degeneration.
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Aged↗
Biomedical subjects
S Rajagopalan
Publications and source records attributed to S Rajagopalan.
Adriamycin-induced free radical formation in the perfused rat heart: implications for cardiotoxicity.
Adriamycin is an anthracycline drug with a wide spectrum of clinical antineoplastic activity. However, the usefulness of the drug is limited by its dose-dependent cardiotoxicity. Adriamycin-stimulated free radical formation has been suggested as one of the mechanisms for its cardiotoxic effects. In order to evaluate this underlying mechanism, we have perfused rat hearts with Adriamycin, using a modified Langendorf technique, and the free radicals formed were analyzed by electron spin resonance spectroscopy using spin-trapping techniques. Our studies show that Adriamycin stimulated the formation of .OH in the heart, and the maximum .OH was formed with 1 microM of the drug. The addition of superoxide dismutase (600 units/ml) inhibited the hydroxyl radical formation by 2- to 3-fold, while catalase (550 units/ml) abolished it completely, showing the intermediacy of superoxide and H2O2. Furthermore, ICRF-187, an iron chelator and a cytotoxic drug, was also an effective inhibitor of .OH formation in the rat heart. The heart rate was not significantly modified by all the above experiments. This study demonstrates that Adriamycin stimulates the formation of .OH in the isolated rat heart and suggests that this mechanism may be significant in Adriamycin-induced cardiotoxicity.
Animals↗
Retinal insulin receptors: localization using a polyclonal anti-insulin receptor antibody.
Although retinal insulin receptors have recently been described biochemically, the location of these receptors within the retina is unknown. The study presented here used a polyclonal anti-insulin receptor antibody (B10), immunofluorescence and immunoelectron microscopy to determine the location of insulin receptors in bovine, monkey and human retina. It was found that antibody immunofluorescence formed discrete bands localized predominantly to photoreceptor and neuronal cell bodies. In addition to the strong association with neuronal perikarya, a lower level of antibody binding was observed in photoreceptor outer segments. In human retina, some of the antibody immunofluorescence also had a pattern that suggested B10 binding to glial-like cells.
Animals↗
Ultrasonic velocity, attenuation, and nonlinearity constant in pure and Cd-doped KCl.
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Journal Article↗
The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses.
Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.
Arachidonic Acids↗
Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII.
The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.
Calcium↗
Essential iris atrophy. A clinical, immunohistologic, and electron microscopic study in an enucleated eye.
A 29-year-old woman had unilateral essential iris atrophy, corneal endothelial changes, and absolute glaucoma. The enucleated eye was examined by routine light microscopy. Separate portions of unfixed fresh frozen cornea were sectioned and reacted with monoclonal antibodies against keratins, vimentin, and inflammatory cell markers. Stains for filamentous actin (f-actin) were performed using the 7-nitro-benz-2-oxa-1,3-diazolylphallacidin (NBD phallacidin) probe. In addition, portions of cornea and iris were examined by scanning and transmission electron microscopy. Immunocytochemical stains with anti-keratin antibodies showed reactivity only in the corneal epithelium of the patient and normal control. Immunoreactivity with anti-vimentin antibodies was observed in corneal endothelium and keratocytes of the patient and control, but was negative in the epithelium. Staining for f-actin appeared more pronounced in the corneal endothelium of the patient. Scanning electron microscopy of the corneal endothelium showed irregularity in cell size and shape and filopodial processes characteristic of migrating cells. Transmission electron microscopy disclosed abnormalities of Descemet's membrane and endothelium with a posterior collagenous layer. The corneal endothelium displayed normal junctional complexes without desmosomal junctions or increased microvillus projections. Increased 10-nm cytoplasmic filaments were noted consistent with the expression of vimentin. Occasional chronic inflammatory cells perturbed the corneal endothelium and were detected within the endothelial layer. Alterations in the endothelium and Descemet's membrane suggest the acquired nature of this disease.
Actins↗
Meibomian gland changes in the rhino (hrrhhrrh) mouse.
The rhino mouse, a single gene recessive mutation, is characterized by abnormal epidermal differentiation and maturation leading to the loss of hair at 1 month of age as well as follicular and epidermal hyperkeratoses. We evaluated the lids and corneas of nine rhino mice and their normal litter mates at various ages from 3 months to 1 year. Tissue specimens were studied by light microscopy, scanning and transmission electron microscopy as well as immunoperoxidase using a polyclonal rabbit anti-keratin antibody. At 3 months of age there was a thickening and hyperkeratinization of the palpebral epidermis which extended into and included the meibomian gland central duct. Whereas in the skin, hyperkeratinization is followed by follicular hyperkeratosis and dermal cyst formation, in the meibomian gland, ductal hyperkeratinization appeared to lead to loss of well developed acini followed by atrophy of the gland at 1 year as confirmed by immunostaining for keratin proteins. Scanning electron microscopy revealed marked plugging of the meibomian gland orifice with keratinized cells or debris in contrast to the patent orifice of the normal lid. Ocular surface changes included the presence of a whitish exudate covering the surface of the eye and increased numbers of preexfoliative corneal epithelial cells. These findings suggest that the rhino mouse may represents the first naturally occurring disorder of the meibomian gland.
Animals↗
An immunohistopathologic study of trilateral retinoblastoma.
We examined retinal and midline pineal tumors from four patients with trilateral retinoblastoma who had antibodies against neural-associated antigens including neuron-specific enolase, photoreceptor cell proteins (S-antigen and rhodopsin), and glial fibrillary acidic protein. Expression of neuron-specific enolase was observed in all four patients. S-antigen immunoreactivity was present in three of four ocular tumors and two of four pineal tumors examined, whereas results of labeling with rhodopsin and glial fibrillary acidic protein were negative in each of the ocular and pineal tumors.
Brain Neoplasms↗
The temperature-dependent reaction between alpha 2-macroglobulin and streptokinase-plasmin(ogen) complex.
The reactions of alpha 2-macroglobulin (alpha 2M) with plasmin or streptokinase-plasmin(ogen) (SkPl) were studied as a function of temperature. alpha 2M and plasmin reacted relatively rapidly at all temperatures. The initial rates of reaction were identical at 24 and 37 degrees C. At 4 degrees C, the initial reaction rate was somewhat slower; however, the reaction was 90% complete in 2 min. The reaction of alpha 2M with SkPl was markedly temperature-dependent. Initial rates of reaction at 0 and 24 degrees C were only 3 and 40% of the rate of 37 degrees C, respectively. When these reactions occur, only the plasmin moiety is incorporated in the alpha 2M molecule. These data explain the inconsistencies in previous reports of SkPl interactions with alpha 2M, since experiments have been performed at different temperatures in various studies. In the present work, we present a model which interprets the data in terms of a possible high-energy transition state in the alpha 2M-SkPl reaction.
Fibrinolysin↗
Retinal insulin receptors. 1. Structural heterogeneity and functional characterization.
Neural cells of the bovine retina contain specific, high-affinity receptors for insulin. When solubilized and wheat-germ purified, these receptors exhibit a kinase activity that is capable of phosphorylating the receptor's beta-subunit (autophosphorylation) and a tyrosine-containing exogenous substrate, poly (Glu, Tyr) 4:1. Studies of the structure of retinal insulin receptors revealed the existence of two insulin receptor subpopulations. For these populations, the apparent molecular weights of the alpha-subunit were 120- and 133 kDa. This structural heterogeneity does not appear to be related to the presence of vascular contamination and stands in contrast to the brain and liver where a single alpha-subunit type was found (120 kDa for brain and 133 kDa for liver). In addition to being distinguishable by their molecular weights, the two populations of retinal insulin receptors could be distinguished in terms of (a) their solubility in Triton X-100, (b) glycosylation, and (c) recognition by anti-insulin receptor antibody. Despite these structural differences, the two populations of retinal insulin receptors appear to have similar insulin binding affinities.
Animals↗
Retinal insulin receptors. 2. Characterization and insulin-induced tyrosine kinase activity in bovine retinal rod outer segments.
Bovine retinal rod outer segments (ROS) possess specific, high-affinity receptors for insulin. These receptors exhibit an insulin-stimulatable tyrosine-specific activity that is capable of phosphorylating the receptor's own beta-subunit and exogenous substrate. ROS insulin receptors exhibit heterogeneity in the apparent molecular weight of the receptor's alpha-subunit. In this regard, insulin receptors from this single cell type resemble insulin receptors obtained from whole retina, but are unlike receptors from brain and liver.
Animals↗
Actin filament localization in developing and pathologic human corneas.
Actin is associated with motility, cell morphology, and cell-substrate adhesion. The molecular probe NBD phallacidin, which reacts with filamentous actin, was used to study the distribution of actin filaments in the corneal and conjunctival epithelium, stroma, and endothelium. Frozen sections of human fetal eyes from 8 weeks to 40 weeks of gestation were reacted with NBD phallacidin. Pathologic tissues included keratoplasty specimens from patients with hereditary posterior polymorphous corneal dystrophy (PPMD) and surgically excised tissues removed for treatment of epithelial down-growth. Normal human cornea was used as a control. Immunofluorescent staining disclosed actin filament distribution in corneal epithelium as early as 9-10 weeks of gestation. Staining increased with maturation until term. Adult human corneal epithelium showed more pronounced staining of the surface layers. Stromal staining was more extensive in earlier stages of gestation and decreased in later stages of gestation, after 20-21 weeks. In pathologic corneas with posterior polymorphous dystrophy, there was localization of actin, as well as keratin, in the abnormal epithelial-like layers lining the posterior cornea. In epithelial downgrowth, actin and keratin were demonstrated in multilayered squamous epithelium on the anterior iris surface. Actin appears to be involved in migration of corneal epithelial and endothelial cells.
Actin Cytoskeleton↗
Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.
We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.
Animals↗
Specificity of alpha 2-macroglobulin covalent cross-linking for the active domain of proteinases.
The reaction of alpha 2-macroglobulin (alpha 2M) with the two-chain enzyme plasma kallikrein results in covalent bond formation between the catalytic subunit and the inhibitor. We have recently published a model of alpha 2M which suggests that this phenomenon may be a general mechanism when multisubunit proteinases are inactivated by alpha 2M. In order to test this hypothesis, we studied the reactions of factor Xa, plasmin, streptokinase-plasmin and alpha-thrombin with alpha 2M. In the case of factor Xa the catalytic heavy chain demonstrated greater than 99% covalent incorporation while over 97% of the light chain failed to crosslink to the inhibitor. Preferential binding of the catalytic light chains of plasmin (70% covalent incorporation) and plasmin in complex with streptokinase (79% covalent incorporation) was also observed. Finally, 82% covalent incorporation of the catalytic heavy chain of alpha-thrombin was found. These studies demonstrate that in the case of multisubunit proteinases, the chain containing the active site demonstrates preferential binding as predicted by the model supporting placement of the site of covalent binding close to the "bait region" of alpha 2M.
Binding Sites↗
Characterization of murine peritoneal macrophage receptors for fibrin(ogen) degradation products.
The binding of human fibrinogen degradation fragments D1, E, X, and Y, as well as fibrin fragment D1 dimer, to mouse peritoneal macrophages was examined. A Scatchard plot of fragment D1 binding was biphasic, suggesting two classes of receptors. Fragments D1, D1 dimer, X, and Y in low concentrations bound to macrophages with high affinity (Kd = 23 to 73 X 10(-11) mol/L). Fragment E bound specifically but at a much lower level than the other fragments. Fragment D1 was able to compete for the binding of radiolabeled fragments X and Y but not radiolabeled fragment E. These studies indicate that fragments D and E are recognized by separate receptor systems but that all of the fibrinogen degradation products that contain the D domain are recognized by the same receptor system.
Animals↗
A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.
A series of new, covalent polyethylene glycol (PEG)-streptokinase adducts were synthesized and characterized. PEGs with average molecular weights of 2,000, 4,000, and 5,000 were activated with carbonyldiimidazole and coupled to the protein under standardized reaction conditions. Steady-state kinetic analysis demonstrated comparable Km values for the activation of plasminogen by streptokinase, PEG-2-streptokinase, and PEG-4-streptokinase. The kcat values were somewhat decreased when PEG-2 or PEG-4 was coupled to the streptokinase. Activation by the PEG-5 adduct did not follow Michaelis-Menten kinetics under the conditions employed in this study. Plasmin activity obtained by incubating streptokinase derivatives with plasminogen also was studied as a function of time with each of the PEG-streptokinase derivatives. By this assay, incubations containing PEG-5-streptokinase and unmodified streptokinase demonstrated comparable activity while reaction mixtures containing PEG-2-streptokinase and PEG-4-streptokinase were slightly more active. Streptokinase incubated with plasminogen at a 1:1 molar ratio was extensively degraded after 30 min whereas PEG-2-streptokinase was resistant to plasmin cleavage. The derivatized proteins were radioiodinated and incubated in plastic microtiter plates that were coated with an immunoglobulin fraction containing antibodies to streptokinase. Binding of the PEG-streptokinase adducts was decreased by greater than 95% compared with unmodified streptokinase. Plasminogen activator complexes were formed by reacting the streptokinases with human plasminogen in vitro and the clearance studied in mice. Radioiodinated plasmin in complex with the PEG-streptokinase adducts cleared at a slower rate than did plasmin complexed with unmodified streptokinase. Catabolism of the protease still occurred by a mechanism that involved reaction with alpha 2-macroglobulin as has been described for nonderivatized streptokinase-plasminogen complex (Gonias, S. L., M. Einarsson, and S. V. Pizzo, 1982, J. Clin. Invest., 70:412-423). When more extensive derivatization procedures were utilized, PEG-2-streptokinase preparations were obtained that further prolonged the clearance of complexed 125I-plasmin; however, these adducts did not uniformly retain comparable activity. It is suggested that PEG-streptokinase complexes with greatly reduced antigenicity may be useful in the treatment of thrombotic disorders.
Animals↗
Hinf family: a novel repeated DNA family of the human genome.
The isolation of a mutant adenovirus carrying an insertion of cellular DNA has led to the identification of a new family of human repetitive sequences, which are found tandemly arranged in the genome. The sequence of the viral insert resembles that of eukaryotic transposable elements.
Adenoviruses, Human↗