Gene/Protein Disease Symptom Drug Enzyme Compound
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Enzymes, which degrade elements of the extracellular environment, were studied for their actions upon stereocilia and their cross-linkages by scanning electron microscopy. Chondroitinase, hyaluronidase and keratanase, which attack carbohydrate moieties of the extracellular matrix, had little effect upon hair bundles. Collagenase and plasmin (fibrinolysin) also had only marginal effects. Elastase produced dramatic effects upon hair bundles. Both lateral and tip links were degraded resulting in separation and splaying of stereocilia. Many stereocilia showed no marked loss of rigidity, although some were bent or kinked. In general, inner hair cells were the most susceptible to elastase followed by row 3, row 2, row 1 of the outer hair cells. The proteolytic enzyme trypsin did not noticeably disrupt the hair bundles. Protease caused loss of rigidity and fracture of stereocilia resulting in considerable collapse of hair bundles. Crosslinkages between stereocilia were less noticeably degraded. These results indicate that both lateral and tip links of stereocilia comprise a proteinaceous moiety which could be elastin or some chemically related structure.
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PMID:Action of elastase, collagenase and other enzymes upon linkages between stereocilia in the guinea-pig cochlea. 216 89

Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens contained round granules (luminal diameter 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl2 indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC. Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the 35SO4 labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/dermatan sulphates comprised about one fifth of the total activity incorporated. The distributions of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.
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PMID:Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis. 244 72

To assess myocardial lymphatics during the evolution of myocardial infarction we performed lymphangiographic studies thirty and three hundred sixty minutes after occlusion of the left anterior descending coronary artery in 92 dogs. A morphometric index was employed on a coded basis to assess the lymphangiograms. Well before myocardial necrosis was evident, at thirty minutes, a striking reduction was evident in lymphatic filling in the ischemic zone: similar changes were seen three hundred sixty minutes after occlusion. Heparin in doses that rendered blood incoagulable did not prevent the lymphatic occlusion or collapse, but they were prevented by two agents that act as cardiac lymphagogues, hyaluronidase and CLS 2210. Lymph flow from the heart was assessed in another 23 dogs. Lymph flow fell sharply after coronary artery occlusion in placebo-treated dogs but was well maintained in dogs treated with hyaluronidase and with CLS 2210. The reduction in cardiac lymphatic filling and lymph flow occurred too early to be a consequence of myocardial necrosis. To the extent that reduced lymphatic drainage allows the local accumulation of potentially toxic products, it could contribute to the local damage. Treatment with the lymphagogues not only maintained lymphatic patency but also reduced evidence of myocardial damage evident on examination by light and electron microscopy. These studies provide an alternative to commonly held concepts on how hyaluronidase reduces myocardial infarction after coronary artery occlusion and support the concept that lymphatic occlusion or collapse plays a role in myocardial infarction.
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PMID:Early disappearance of lymphatics draining ischemic myocardium in the dog. 381 24

To allow a more valid comparison between our previous ultrastructural data and the immunolocalization of type IX and other minor collagen species in cryosectioned cartilage, we examined both normal and testicular hyaluronidase-digested canine tibial cartilage by electron microscopy. Removal of matrix proteoglycans caused the pericellular capsule to collapse against the cell surface, suggesting that its normal anatomical position is mediated by pericellular matrix hydration. Detailed examination of the pericellular capsule and pericellular channel revealed fine, faintly banded fibrils and an amorphous component somewhat similar in structure to basement membrane collagens. Matrix vesicles and the electron-dense material of the interterritorial matrix were only partially digested by hyaluronidase. We propose that the pericellular capsule is composed of a "felt-like" network of minor collagen species which act synergistically to maintain both the composition of the pericellular matrix and the integrity of the chondrocyte/pericellular matrix complex during compressive loading.
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PMID:Morphology of the pericellular capsule in articular cartilage revealed by hyaluronidase digestion. 405 39

In this report, the effects of hyaluronidase on the absorption of intramuscular succinylcholine chloride in goats are discussed. Twenty-five mature, mixed-strain dairy goats were immobilized with 0.33 mg/kg of the drug. An additional 25 goats were given corresponding doses to which hyaluronidase (150 NF units per vial of succinylcholine) had been added. The latent period (the time from injection to collapse) is reduced by 25% in goats given the drug-enzyme mixture. It is concluded that hyaluronidase increases the rate of absorption of succinylcholine chloride. However, it produces no significant difference in the duration of paralysis.
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PMID:The effects of hyaluronidase on the immobilization of goats by intramuscular succinylcholine chloride. 423 90

The structural roles of cardiac jelly components were examined in the early developing chick embryonic heart. Cardiac jelly matrix components were enzymically removed in situ by injecting nanogram quantities of enzymes directly into the cardiac jelly. Injection of ovine testicular hyaluronidase caused shrinkage and the heart became flaccid, but overall heart shape did not change. These responses were the result of enzymatic removal of glycosaminoglycan sugar moieties and were not due to lumenal collapse. Although purified collagenase did not cause any noticeable change, enzymes with non-specific proteolytic activity induced marked cardiac shape changes. In such hearts the dorsal mesocardium opened completely, and the myocardium as well as splanchnic mesoderm of foregut detached from their substrate and the entire heart region swelled. Consequently the shape of the heart was altered completely. The results suggested that in the normal condition the myocardial envelope was under an internal pressure due to the presence of glycosaminoglycans in the cardiac jelly space, and that some matrical non-collagenous protein components were essential to control the internal pressure. Therefore it is suggested that the internal pressure of cardiac jelly may be the direct driving force for the looping process and protein components of cardiac jelly may be important in directing the force for the morphogenetic process.
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PMID:An experimental study of the relation of cardiac jelly to the shape of the early chick embryonic heart. 627 43

Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b-HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan-aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.
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PMID:The dynamic structure of the pericellular matrix on living cells. 827 5

Synovial hydraulic resistance is vital for the retention of intra-articular fluid, and originates within the matrix of biopolymers in the intercellular gaps. Specific digestion of hyaluronan resulted in a increase in synovial hydraulic permeability from 0.478+/-0.24 microl min(-1) cm H(2)O(-1) in control tissue to 4.561+/-0.40 microl min(-1) cm H(2)O(-1) (mean+/-S.D., n=6 rabbits, P<0.001 t test). To investigate whether hyaluronidase also altered the interstitial ultrastructure, morphometry of hyaluronidase treated synovium was carried out. The most striking novel finding was that hyaluronidase treatment reduced extrafibrillar volume fraction within the synovial collagen bundles from 50.5+/-11.1% to 36.8+/-15.5% (mean+/-S.D., n=6 rabbits, P<0.001, two-way anova). This was accompanied by a reduction in interfibrillar centre to centre spacing from 101+/-11 (control) to 84+/-6 nm (mean+/-S.D.; n=6 rabbits, P<0.001) in enzyme-treated bundles. Individual fibrils showed a small but highly significant reduction in cross-sectional diameter from 76.9+/-6.3 to 72.5+/-6.3 nm (mean+/-S.E.; P<0.001) after hyaluronidase treatment. The findings indicate that hyaluronan chains have a major organisational role within the collagen bundle itself. The trans-synovial pathway comprises bundles and substantial areas of intervening, bundle-free matrix, and it is possible that bundle collapse contributes to a rise in overall permeability by increasing the inter-bundle space.
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PMID:Evidence for a role of hyaluronan in the spacing of fibrils within collagen bundles in rabbit synovium. 1209 Sep 31

EXPERIMENTS DESIGNED TO CHARACTERIZE AN UNIDENTIFIED TRANSMISSIBLE AGENT BROUGHT FORTH THE FOLLOWING FINDINGS: The cytopathology consisted of the formation of intranuclear globules, collapse of the involved nuclei, and the extrusion of nuclear materials. The relatively dormant primary human amnion cells were less susceptible than the rapidly growing cell lines. Similarly, the slowly multiplying ribose variants were less susceptible than their corresponding parent cell lines. Interferon-like activity was released from infected cells. Infectivity was readily demonstrated following storage at 0-4 degrees C for at least 8 months or at 37 degrees C for at least 2 weeks. Freeze-thawing, however, markedly reduced or completely destroyed its infectivity. Infectivity was destroyed completely by ether and chloroform; partially by desoxycholate, and not affected by trypsin, papain, RNAse, DNAse, hyaluronidase, lysozyme, lecithinase, or pancreatic lipase. The rate of inactivation by 0.025 per cent formalin was much slower than that of vaccinia and herpes viruses. Its synthesis was suppressed by 5-fluorodeoxyuridine. This suppression was not reversed by thymidine and/or uracil. Heat-stable neutralizing antibody could not be demonstrated in 379 human and animal serums, in human gamma globulins, or in serums from animals "immunized" with this agent. Heat-labile inhibitors (lipoprotein-like) capable of inhibiting the infectivity of this agent were demonstrated in 154 of the 157 serums tested. Experimental evidence indicated the non-identity of this ubiquitous inhibitor and the properdin system. The non-infectious complex between this agent and the ubiquitous serum inhibitor may be dissociated (hence, become infectious) by simple dilution. Repeated attempts to reisolate a similar agent have not been successful. We have hypothesized that this agent is a virus consisting of DNA wrapped in a surface coat rich in lipid, and suggest that this virus be referred to tentatively as a lipovirus.
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PMID:The biological, immunological, and physicochemical characterization of a transmissible agent capable of inducing DNA and thymine degradation in cultured human cells. 1387 2

A crude filtrate from a culture of Clostridium perfringens, type A, was fractionated by a heavy metal-alcohol technique, and some degree of concentration of biologically active factors was achieved. Both the crude material and the fractions obtained were characterized in terms of their alpha toxin, theta toxin, hyaluronidase, and collagenase activities. The filtrate and fractions were tested for their effect on the peripheral circulation of the rat, using the epinephrine threshold technique. The crude material and several fractions caused a sharp increase in epinephrine sensitivity of the capillary bed of the meso-appendix of the rat; fraction R3B1 giving an 86-fold increase in sensitivity. This reaction could be inhibited by specific antitoxic serums but not by normal serum. The "circulation factor" was shown to be heat-labile and appears to be independent of either the alpha or theta toxins. Bilateral nephrectomy greatly reduced, but did not abolish, the effect of the toxin, while the threshold response to epinephrine was not materially changed following bilateral adrenalectomy. The crude filtrate and several fractions were shown to inhibit the phagocytosis of heat-killed B. anthracis to the extent of 40 to 50 per cent. Fraction R3C2 was devoid of all biological properties studied here, except phagocytosis inhibition, suggesting that the factor responsible for this activity is distinct from the "classical" toxins and the "circulation factor." Moreover, a 1:5000 dilution of an antiserum prepared against this fraction would completely neutralize the phagocytosis inhibition factor but failed to inhibit any of the other toxic activities. Since cardiovascular collapse and absence of phagocytosis are two significant clinical findings in gas gangrene, the possible roles of the "circulation" and "phagocytosis inhibition" factors in the pathogenesis of this disease are discussed.
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PMID:The relationship of toxic fractions of a filtrate of Clostridium perfringens type A to the pathogenesis of clostridial myonecrosis. 1436 82


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