Antibacterial and hemolytic activity of the skin of the terrestrial salamander, Plethodon cinereus

As resistance increases against fungal antibiotics, antimicrobial peptides are receiving attention as possible replacements. The dermal glands of frogs secrete, among other things, antimicrobial peptides. As part of the innate immune system, stressors may affect the production of antimicrobial peptides by dermal glands. The dermal secretions of some salamanders have been examined for their toxic secretions, but little attention has been given to salamander antimicrobial peptides. This study examines the skin from the tail region for the production of antimicrobial peptides in the terrestrial salamander, Plethodon cinereus. Fractions of tail extracts were isolated using cation-exchange chromatography and reverse-phase HPLC. An HPLC fraction eluting at 15.75 min (HPLC run: 30 min, 30-80% acetonitrile/water gradient, Aquapore RP-300 C18 column) showed activity against Staphylococcus aureus but not against Escherichia coli. The antibacterial activity gradually increased over a 4-hr incubation time up to about 85% inhibition of bacterial growth. Lysis of guinea pig red blood cells also increased gradually over a 1-hr time period. J. Exp. Zool. 287:340-345, 2000.     

Evolutionary morphology of the caecilian urogenital system. IV. The cloaca

The gross and microscopic anatomy of male and female cloacae of caecilians (Amphibia: Apoda or Gymnophiona) is described and analyzed in terms of structure and function. The arrangement of musculature and cloacal accessory structures is species-specific in males. Contraction of certain cloacal and body wall musculature facilitates eversion of the male cloaca for use as an intromittent organ. The cloacae of females show less marked morphological differences from species to species, and are modified as receptors of male phallodea.     

Reverse hemolytic plaque assays: versatility in the study of secretion

The reverse hemolytic plaque assay has been used for several years to study hormone release from various endocrine cell types. The basic method utilizes a monolayer (consisting of indicator erythrocytes and the cells under study) that is fixed to the floor of an incubation chamber. Antibody directed against a peptide or protein is added to the chamber. Peptides released from the cells under study complex with the antibody and bind to protein-A on the surface of the indicator erythrocytes. The addition of complement causes the indicator cells to lyse, forming a "plaque" or zone of hemolysis surrounding the secreting cells. The size or rate of formation of these plaques can be used as indices to monitor peptide or protein release. In addition to this standard procedure, the plaque assay can be modified by using loose or unattached indicator cells and is termed the loose plaque assay (LPA). The LPA for a particular peptide can be used alone, sequentially with an assay directed toward another peptide, or repeatedly on the same cells to monitor release over time. In light of the fact that plaque assays do not compromise the function of living cells, it is possible to combine these plaque assays with other procedures such as immunocytochemistry, in situ hybridization, fluorescent microscopy, electrophysiology, and electron microscopy to explore other facets of the secretory process in conjunction with release. When taken together, the plaque assay has been quite useful in the study of endocrine cell secretion. Moreover, with the many adaptations possible, it should be particularly valuable in the future for the study of peptide release in other cell types such as neurons.     

Intracellular activity and secretion of various lysosomal glycosidases in cultured human skin fibroblasts

The intracellular activities of four lysosomal glycosidases (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) in human skin fibroblasts cultured in a medium with 0.1% serum increased in a greater degree than that in a medium with 10% serum. Only two glycosidases (alpha-L-fucosidase and beta-D-hexosaminidase) were secreted by fibroblasts in the culture medium. The extracellular activity of alpha-L-fucosidase and beta-D-hexosaminidase was equivalent to 80 and 25% of their intracellular activity in serum-sufficient fibroblasts and 40 and 15%--in serum-restricted fibroblasts. These results suggest that the observer phenomena are controlled by the levels of autophagy, endocytosis and membrane recycling.     

An insight into the skin glands,dermal scales and secretions of the caecilian amphibian Ichthyophis beddomei

The caecilian amphibians are richly endowed with cutaneous glands, which produce secretory materials that facilitate survival in the hostile subterranean environment. Although India has a fairly abundant distribution of caecilians, there are only very few studies on their skin and secretion. In this background, the skin of Ichthyophis beddomei from the Western Ghats of Kerala, India, was subjected to light and electron microscopic analyses. There are two types of dermal glands, mucous and granular. The mucous gland has a lumen, which is packed with a mucous. The mucous-producing cells are located around the lumen. In the granular gland, a lumen is absent; the bloated secretory cells, filling the gland, are densely packed with granules of different sizes which are elegantly revealed in TEM. There is a lining of myo-epithelial cells in the peripheral regions of the glands. Small flat disk-like dermal scales, dense with squamulae, are embedded in pockets in the dermis, distributed among the cutaneous glands. 1–4 scales of various sizes are present in each scale pocket. Scanning electron microscopic observation of the skin surface revealed numerous glandular openings. The skin gland secretions, exuded through the pores, contain fatty acids, alcohols, steroid, hydrocarbons, terpene, aldehyde and a few unknown compounds.     

Plasmid pAP20 controlling the hemolytic activity of E. coli

The authors investigated pAP20 plasmid identified in E. coli cells isolated from man. According to the evidence obtained pAP20 plasmid determines the synthesis of alpha-hemolysin, being an F-like plasmid of the drd type. Having medium molecular size, the plasmid belongs to the inc FIV group and is partly incompatible with pAP38 plasmid which is a reference plasmid of the inc FVII group.     

Science of hemolytic activity of some radiation-resistant micrococci in food.

Micrococci resistant to 1 Mrad of gamma radiation were isolated from irradiated chicken. Three isolates were hemolytic on blood agar plates and were selected for further study. Two other radiation-resistant micrococci, Micrococcus radiodurans and Micrococcus radiophilus, were included in the study because there is only a very limited amount of information regarding hemolytic activity of these organisms and their potential role of public health importance. Tests to determine hemolytic patterns, hemolytic activity of extracellular substances, leukocytic activity, presence of enzymes commonly associated with pathogenicity (coagulase, deoxyribonuclease, phosphatase), and pathogenicity for laboratory animals all suggested that the organisms would not be of public health significance.     

On the hemolytic activity of geophilic dermatophytes

10 species each ofK. ajelloi, M. gypseum, M. cookei andT. terrestre were tested for hemolytic activity on media contamining horse, calf or sheep blood. Hemolysis was regularly produced by all species except those ofM. cookei.The test for hemolysis may thus be a complimentary physiological test for the differentiation of the related speciesK. ajelloi andM. cookei.

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Zusammenfassung Zehn Arten vonK. ajelloi, M. gypseum, M. cookei undT. terrestre sind für hämolytische Aktivität an Kulturmedia mit Pferd-, Kalb-, und Schaafblut untersucht worden. Hämolyse war regelmäßig durch alle Arten mit der Ausnahme vonM. cookei hervorgerufen. Der Hämolyse-test mag so als ein ergänzender, physiologischer Test für die Unterscheidung der verwandten Arten vonK. ajelloi undM. cookei verwendet werden.

     

Increased secretion of latent elastase activity following contact between human skin fibroblasts and elastin derived peptides.

Elastin-derived peptides were previously shown to influence human skin fibroblasts (HSF) chemotaxis and proliferation (Ghuysen et al., 1992). We report here that culturing HSF on κ-elastin (κE) but not onto fibronectin (FN) enhanced the secretion of latent elastinolytic activity. The proteinase involved was identified as the 72 kDa gelatinase A. Moreover, HSF-κE as well as HSF-FN interactions modulated the secretions of Il₁ induced expressions of elastinolytic activities.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

Anti-Legionella activity of staphylococcal hemolytic peptides

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic.     

Comparative effects of sugars on the hemolytic activity of Schistosoma mansoni and other hemolytic agents

1. The rate of red blood cell lysis by the hemolytic agent in adult worm homogenates of Schistosoma mansoni is slowed in the presence of added sugars (50 mM). 2. Trisaccharides were the most effective in slowing and reducing lysis. Disaccharides were more effective than monosaccharides. 3. The addition of sodium, potassium or lithium chloride salts (25 mM) stimulated hemolysis by the S. mansoni agent. 4. Hemolysins with known mechanisms were tested to determine the effects of added sugars (50 mM) or salts (25 mM). 5. The S. mansoni hemolytic agent responds to the addition of sugars and salts in a manner similar to small membrane pore formers.     

Synthesis and secretion of apolipoprotein A-I by chick skin.

Chick skin slices were incubated with [35S]methionine and labeled apoA-I was immunoprecipitated from incubation medium and tissue homogenate. ApoA-I accounted for approximately 13 and 2.5% of radioactive medium and cell proteins, respectively. After ultracentrifugation of the medium, 55% of labeled apoA-I was found as a constituent of lipoproteins (d less than 1.210 g/ml) and 45% in a lipid-poor form (1.210-1.260 g/ml). To ascertain whether this large proportion of lipid-poor apoA-I was due to a dissociation of this peptide from medium lipoproteins during ultracentrifugation, labeled incubation medium was applied to an anti-chick apoA-I immunoaffinity column. The material bound to the column was analyzed by nondenaturing polyacrylamide gradient gel electrophoresis and found to contain three subpopulations of lipoproteins with a particle size of 12, 11, and 9 nm, respectively. The radioactivity of these subpopulations accounted for 82% of total radioactive medium apoA-I. ApoA-I was localized by immunohistochemistry in the viable cells of the epidermis and in the stratum corneum. Rat skin slices were found to synthesize and secrete apoE but no apoA-I. ApoA-I and apoE secreted by chick and rat skin, respectively, may play a role in the secretion of lipids from the differentiating keratinocytes and thus contribute to the formation of the hydrophobic barrier of the skin.