Effect of ascorbic acid preparations on certain indices of carbohydrate metabolism in tissues of animals with burns

The glucose content and glucokinase, amylase, hyaluronidase activities were determined in skin of guinea pigs after a burn. It is shown that in the wound focus the amylase and hyaluronidase activities increase, and the activity of glucokinase decreases, the content of glucose grows. Application of ascorbic acid and galascorbin as remedies induces a decrease in the intensity of hydrolytic processes and improves the glucose utilization, particularly with galascorbin introduction. This may evidence for the predominance of reparative processes in the burn area and improvement of their supply with energy.     

Furunculosis in cultured American eel Anguilla rostrata (Le Sueur)

Diseased American eels, Anguilla rostrata , were collected from an eel culture facility in South Carolina and examined. The disease was characterized by skin lesions located on the body. A bacterium identified as Aeromonas salmonicida was isolated from the lesions and shown to be the causative agent. The bacterium was also isolated from the kidney during later stages of the disease.     

Water content, body weight and acid mucopolysaccharides, hyaluronidase and beta-glucuronidase in response to aestivation in Australian desert frogs

This study investigates the effects of aestivation on body water content, body mass, acid mucopolysaccharide (AMPS) and some of its degrading enzymes in different tissues for some Australian desert frogs. The AMPS component of the liver, kidney, skin and cocoon alter during aestivation to help retain water, which is unchanged in most tissues of all frog species, and to protect the frogs from desiccation during extended periods of aestivation. Hepatic AMPS was unaltered in Cyclorana maini, C. platycephala and Neobatrachus sutor but increased significantly after 2 months of aestivation in C. australis. The level of AMPS in the kidney was elevated in all four frog species after 5 months of aestivation. Skin AMPS content in the skin of awake frogs decreases with aestivation period and increases in the cocoon. AMPS in the cocoon probably works as a cement between the cocoons' layers and its physical presence presumably contributes to preventing water flux. Changes in AMPS content in different tissues were accompanied by significant changes in both hyaluronidase and beta-glucuronidase activities, which play an important role in AMPS metabolism. Alcian blue staining of control and digested skin of C. australis and C. platycephala with testicular hyaluronidase indicated the presence of AMPS, concentrated in a thin layer (called ground substance, GS) located between stratum compactum and stratum spongiosum, and acid mucin concentrated in the mucous glands and in a 'tubular' structure which could be observed in the epidermal layer. Hyaluronidase digestion of the cocoon slightly changed the Alcian Blue colour, suggesting the presence of a large amount of acid mucin similar to that found in the skin mucous gland. The results of this study present data for the redistribution of AMPS, which may help in reducing water loss across the cocoon and reabsorption of water in the kidney during aestivation.     

Morphological and biochemical changes in carotenoid granules in the ventral skin during growth of the Japanese newt Cynops pyrrhogaster

The color of the ventral skin of the Japanese adult newt Cynops pyrrhogaster is red, whereas that of the small juvenile newts at metamorphosis is creamy. Xanthophores in the red skin have many ring carotenoid vesicles (rcv) and a few homogenous carotenoid granules (hcg), as reported earlier. To understand the reason for this change in coloration of the ventral skin of the newt, we carried out histological and biochemical studies to see whether the size and the number of carotenoid granules (hcg and rcv) in the xanthophores and also carotenoid content in the ventral skin change during the growth of this animal. By electron microscopic observation, only hcg were observed in the creamy skin of larvae at stage 59. The diameter of the hcg in the skin of the larvae was approximately 0.85 microm, but significantly decreased to 0.35 microm in the skin of the small juvenile newt. However, the number of the hcg/100 microm (2) of a xanthophore in the ventral skin was very low in the larva at stage 59, but increased in the small juvenile. The carotenoid content was very low in the creamy skin of small juveniles, but dramatically high in the red skin of the adult newts. In the red skin of the adult newt, many rcv (85%) and a few hcg (15%) were observed. However, the number of carotenoid granules (rcv and hcg)/100 microm(2) of a xanthophore in the red skin of adult newts was not different from that of hcg/100 microm (2) of a xanthophore in the creamy skin of small juveniles. The results, taken together, suggest that the increase in the size and the number of carotenoid granules and also carotenoid content in the ventral skin is very important for red body coloration during the growth of the Japanese newt Cynops pyrrhogaster.     

Effect of diethylstilbestrol on skin sterols of the male rat

Diethylstilbestrol (DES) was injected in doses ranging from 600 micro g to 0.4 micrograms/kg body weight into mature male rats over a 3 wk period. Profound effects on skin morphology and on sterol content of skin were noted. The sebaceous glands atrophied and the epidermis lost granularity. The concentrations of all skin sterols, with the exception of cholesterol, were reduced. At a dose level of DES of 4 micrograms/kg there was still a perceptible reduction in the concentration of Delta(7)-cholestenol. Incubation of skin fragments with acetate-2-(14)C for 2 hr demonstrated a reduced uptake of (14)C into the nonsaponifiable fraction of skin lipids at all dose levels studied. Preliminary thin-layer chromatography of the nonsaponifiable fraction revealed that the uptake of (14)C into cholesterol was only slightly decreased; uptake into cholesterol precursors was decreased somewhat more. The epidermis and dermis were separated by incubation of skin with elastase and hyaluronidase. The epidermis contained at least three times as much sterol per mg dry weight as did the dermis. Unesterified cholesterol was the major sterol present in both layers; the other sterols were present mainly as esters. DES injection resulted in no change in the free sterol content but markedly reduced the ester content of the epidermis and dermis.     

Synthesis and biological evaluations of oleanolic acid indole derivatives as hyaluronidase inhibitors with enhanced skin permeability

Oleanolic acid (OA) is a natural cosmeceutical compound with various skin beneficial activities including inhibitory effect on hyaluronidase but the anti-hyaluronidase activity and mechanisms of action of its synthetic analogues remain unclear. Herein, a series of OA derivatives were synthesised and evaluated for their inhibitory effects on hyaluronidase. Compared to OA, an induction of fluorinated (6c) and chlorinated (6g) indole moieties led to enhanced anti-hyaluronidase activity (IC₅₀ = 80.3 vs. 9.97 and 9.57 µg/mL, respectively). Furthermore, spectroscopic and computational studies revealed that 6c and 6g can bind to hyaluronidase protein and alter its secondary structure leading to reduced enzyme activity. In addition, OA indole derivatives showed feasible skin permeability in a slightly acidic environment (pH = 6.5) and 6c exerted skin protective effect by reducing cellular reactive oxygen species in human skin keratinocytes. Findings from the current study support that OA indole derivatives are potential cosmeceuticals with anti-hyaluronidase activity.     

Vascular permeability-increasing activity possessed byTreponema phagedenis (Reiter strain)

Treponema phagedenis possessed the ability to induce an increase in vascular permeability (blueing) in guinea pig skin, which was exerted not only by cell-free preparations but also directly by intact cells. Cell-free preparations induced maximum blueing 0 h after sample injection, while whole cells did so after 1 h. Log dose-response curves for cell-free preparations were linear within the range of blue spots with diameters of 10–24 mm, with slopes ( ) of 8.5–8.7. The vascular permeability-increasing activity was not ascribable to hyaluronidase and probably to -galactosidase; this organism did not produce any detectable hyaluronidase activity.     

Dietary protein as a potent regulator of the hyaluronan synthase gene in rat skin

The status of hyaluronan, the major glycosaminoglycan in the skin, is regulated by many factors such as cytokines and glucocorticoids. To examine whether and how protein malnutrition affects the status of skin hyaluronan, the hyaluronan content and mRNA levels of hyaluronan synthases (has) were analyzed in the skin of rats fed on a protein-free diet or on a 12% gluten diet. When these malnourishing diets had been given for 1 week, the hyaluronan content was significantly reduced as compared with that in rats fed on a 12% casein diet. Substantial falls in the mRNA levels of rhas2 and rhas3 were also observed. The reduction of mRNAs was already evident on the second day of treatment with the malnourishing diets. These results suggest that protein malnutrition has a primary impact on the gene expression of rhass, which leads to the reduction of hyaluronan content and to disfunction of the skin.     

A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms

Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis.     

Effect of hyaluronidase on the quantum content and binomial p and n parameters of neuromuscular transmission in frogs

In the frog dermo-sternal muscle hyaluronidase (0.01--0.1%) caused e.p.p. amplitude and quantum content of transmission to fall when acting in a solution containing 1 mM Ca2+ and 4 mM Mg2+. The change in the quantum content was related to a decrease in binomial parameter n, the probability of mediator p release increased in the first moments of the enzyme action. With the prolongation of hyaluronidase action, negative values of p appeared. Hyaluronidase caused an increase in e.p.p. amplitude and in quantum content of transmission when acting in a solution containing 8 mM Ca2+ and d-tubocurarine. It was suggested that hyaluronidase modifies the normal calcium exchange between the bulk solution and the unstirred layer near nerve terminal membrane thus affecting the transmission.     

Hyaluronan affects extravascular water in lungs of unanesthetized rabbits

We have determined whether changes in lung hyaluronan content affect extravascular water in lungs of unanesthetized rabbits. Three groups of experiments were performed. In group 1 (n = 12), no infusions were given; in group 2, nine pairs of rabbits received either intravenous hyaluronidase (750 U.kg-1.min-1) or an equivalent volume of saline; in group 3, nine pairs of rabbits received either hyaluronidase or saline, followed by intravenous saline infusion amounting to 24% of body weight. At the end of each experiment, one lung was analyzed for extravascular lung water by the wet-dry method. Except for group 3, in all animals the other lung was analyzed for hyaluronan content by a method that involved hydrolyzing lung hyaluronan with fungal hyaluronidase to release reducing N-acetyl glucosamine groups, which were quantified. In group 1, lung hyaluronan, which varied from 50 to 159 micrograms/g dry wt (mean 106 +/- 35 micrograms/g dry wt), significantly correlated with variation in extravascular lung water (mean 4.2 +/- 0.3 g/g dry wt). In group 2 rabbits given hyaluronidase, lung hyaluronan was 40% lower and extravascular lung water was 14.6% lower than in paired controls (P less than 0.01). In group 3, volume expansion did not affect lung water, except after hyaluronidase when lung water was 47% higher than paired controls. We conclude that in the lung the content of hyaluronan is one of the determinants of extravascular water content.     

Characterization of dermatan sulfate in mucopolysaccharidosis VI Evidence for the absence of hyaluronidase-like enzymes in human skin fibroblasts

Dermatan sulfate-chondroitin sulfate copolymers with a high content of dermatan sulfate are stored in cultured human skin fibroblasts from patients affected with mucopolysaccharidosis VI (Maroteaux-Lamy disease). Characterization of the storage material provided evidence that hyaluronidase-like enzymes are not present in these fibroblasts. This is based on the following observations: (i) dermatan sulfate chains stored intracellularly show no reduction of molecular size as compared with intact chains isolated from the extracellular space; (ii) the stored dermatan sulfate chains lack reducible end groups generated by endoglycosidases; (iii) homogenates of human skin fibroblasts do not degrade hyaluronate and (iv) the stored dermatan sulfate chains are degraded by testes hyaluronidase.     

Quantitative determination of hyaluronidase in tissue sections

Summary A method for the determination of hyaluronidase in histological sections is described. This method is based on incubation of tissue sections in a medium containing hyaluronic acid as substrate. Depolymerisation of the substrate during incubation as well as the total nitrogen content are measured in the same section. The comparison of these two values gives information concerning the hyaluronidase activity. This assay was tested in experiments with rat testis. It was also found that our procedure is sensitive enough for the estimation of hyaluronidase activity in sections from kidney. From these experiments with kidney sections it can be concluded that: 1. The pH optimum for renal hyaluronidase (3.5) differs from that in the testis (4.7). 2. In the renal cortex more hyaluronidase activity was detected than in the medulla.Department of Transplantology.Department of Histology and Embryology.     

Quantitation of hyaluronic acid in tissues by ion-pair reverse-phase high-performance liquid chromatography of oligosaccharide cleavage products

A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

Canine submandibular-gland hyaluronidase. Identification and subcellular distribution

1. Submandibular glands from four species of mammal have been shown to contain a hyaluronidase active at acid pH; glands from dog and cat had a much higher content of this enzyme than has been found in other sources. 2. Product formation from hyaluronate after 24hr. incubation was almost the same as with testicular hyaluronidase, indicating that the enzyme is an endo-poly-β-hexosaminidase. 3. When submandibular-gland homogenates were fractionated by the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed, except cytochrome c oxidase, were found to occur partly in the soluble fraction and partly in the particulate fractions. Among the particular fractions, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome c oxidase, in the microsomal fraction for alkaline phosphatase and in the light-mitochondrial fraction for acid phosphatase, β-N-acetylhexosaminidase and acid-active hyaluronidase. 4. Release of the enzyme activity from the sedimentable fractions occurred in 0·1% Triton X-100 or after high-speed homogenization. 5. Stimulation of dogs by pilocarpine was found to decrease the hyaluronidase content of the submandibular gland by 5% and to cause the occurrence of a corresponding amount of acid-active hyaluronidase in the submandibular saliva. 6. The results are discussed in relation to the subcellular localization of hyaluronidase.     

Role of the glycosaminoglycan microenvironment of hyaluronidase in regulation of its endoglycosidase activity

The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the 1-3 and 1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking—(1e-4e) and (1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.     

Rubidium and zinc fluctuations in selected tissues during the development of the BW7756 murine hepatoma

In separate studies, radioisotopes ⁶⁵Zn and ⁸⁶Rb were used to monitor trace element fluctuations from normal in C57L/J mice throughout the progression of a murine hepatoma. Amounts too small to upset normal levels were injected directly into the blood stream. After an equilibration period, the whole mouse and various resected organs and tissues were counted. Compared to normal levels, rubidium in diseased mice was lower in kidney and blood, and elevated in skin and muscle. Diseased mice showed depressed levels of zinc in skin and muscle. Large fluctuations during different stages of tumor growth were observed for various other tissues and organs of diseased mice.     

Feline Histoplasmosis in Brazil: Clinical and Laboratory Aspects and a Comparative Approach of Published Reports

The present study described clinical and epidemiological aspects of three cases of feline histoplasmosis and compared them to previously described cases. A detailed mycological identification and antifungal susceptibility profile of each isolate are presented. Secondarily, a serological survey for anti-Histoplasma antibodies was performed with domestic and wild cats. Diseased animals presented nodular to ulcerated skin lesions and respiratory disorders as main clinical signs. H. capsulatum var. capsulatum was isolated and the strains showed to be susceptible to antifungal drugs. Considering that feline histoplasmosis is uncommonly observed in veterinary clinics, diagnosis, and clinical management in endemic areas should be improved.