EprS, an autotransporter serine protease, plays an important role in various pathogenic phenotypes of Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses an arsenal of both cell-associated (flagella, pili, alginate, etc.) and extracellular (exotoxin A, proteases, type III secretion effectors, etc.) virulence factors. Among them, secreted proteases that damage host tissues are considered to play an important role in the pathogenesis of P. aeruginosa infections. We previously reported that EprS, an autotransporter protease of P. aeruginosa, induces host inflammatory responses through protease-activated receptors. However, little is known about the role of EprS as a virulence factor of P. aeruginosa. In this study, to investigate whether EprS participates in the pathogenicity of P. aeruginosa, we characterized various pathogenic phenotypes of the wild-type PAO1 strain and its eprS-disrupted mutant. The growth assays demonstrated that the growth of the eprS mutant was somewhat lower than that of the wild-type strain in a minimal medium containing BSA as the sole carbon and nitrogen source. Thus, these results indicate that eprS would have a role in the growth of P. aeruginosa in the presence of limited nutrients, such as a medium containing proteinaceous materials as a sole nutrient source. Furthermore, disruption of eprS resulted in a decreased production of elastase, pigments, autoinducers and surfactants, and a reduction of swimming and swarming motilities. In addition, the eprS mutant exhibited a reduction in the ability to associate with A549 cells and an attenuation of virulence in leucopenic mice as compared with the wild-type strain. Collectively, these results suggest that EprS exerts pleiotropic effects on various pathogenic phenotypes of P. aeruginosa.

Protective Role of PEDF-Derived Synthetic Peptide Against Experimental Diabetic Nephropathy.

Pigment epithelium-derived factor (PEDF) is a glycoprotein with complex neuroprotective, anti-angiogenic, and anti-inflammatory properties, all of which could potentially be exploited as a therapeutic option for vascular complications in diabetes. We have previously shown that PEDF-derived synthetic peptide, P5-3 (FIFVLRD) has a comparable ability with full PEDF protein to inhibit rat corneal neovascularization induced by chemical cauterization. However, the effects of PEDF peptide on experimental diabetic nephropathy remain unknown. To address the issue, we modified P5-3 to stabilize and administered the modified peptide (d-Lys-d-Lys-d-Lys-Gln-d-Pro-P5-3-Cys-amide, 0.2 nmol/day) or vehicle to streptozotocin-induced diabetic rats (STZ-rats) intraperitoneally by an osmotic mini pump for 2 weeks. We further examined the effects of modified peptide on human proximal tubular cells. Renal PEDF expression was decreased in STZ-rats. Although the peptide administration did not affect blood glucose or blood pressure, it decreased urinary excretion levels of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker, and reduced plasminogen activator inhibitor-1 (PAI-1) gene expression, and suppressed glomerular expansion in the diabetic kidneys. High glucose or advanced glycation end products stimulated oxidative stress generation and PAI-1 gene expression in tubular cells, all of which were significantly suppressed by 10 nM modified P5-3 peptide. Our present study suggests that PEDF-derived synthetic modified peptide could protect against experimental diabetic nephropathy and inhibit tubular cell damage under diabetes-like conditions through its anti-oxidative properties. Supplementation of modified P5-3 peptide may be a novel therapeutic strategy for diabetic nephropathy.

Nitric oxide formation from hydroxylamine by myoglobin and hydrogen peroxide.

Hydroxylamine (HA), which is a natural product of mammalian cells, has been shown to possess vasodilatory properties in several model systems. In this study, HA and methyl-substituted hydroxylamines, N-methylhydroxylamine (NMHA) and N,N-dimethylhydroxylamine (NDMHA), have been tested for their ability to generate free diffusible nitric oxide (NO) in the presence of myoglobin (Mb) and hydrogen peroxide. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI), measured by electron spin resonance (ESR) spectroscopy, along with nitrite and nitrate production, was observed for HA but not for NMHA and NDMHA. ESR measurements at 77 K showed the formation of the ferrous nitrosyl myoglobin, Mb-NO, in the reaction mixtures containing Mb, H2O2 and HA. Our data also demonstrate that Mb-NO is an end product of the reaction pathway involving Mb, H2O2 and HA, rather than a reaction intermediate in the formation of NO. In summary, our results demonstrate a possible pathway of NO formation from HA, however, the significance of this mechanism for bioactivation of HA in vivo is unknown at the present time.

Biphenyl compounds are hydroxyl radical scavengers: their effective inhibition for UV-induced mutation in Salmonella typhimurium TA102.

In our previous study, we found several hydroxylated biphenyl compounds have a great scavenging effect for hydroxyl radicals (.OH). In this study, to elucidate the relationship between generation of the .OH and photo-mutagenesis, six biphenyl compounds such as dehydrodieugenol, dehydrodivanillyl alcohol, dehydrodidihydroeugenol, dehydrodicreosol, magnolol and honokiol, respectively, were examined for their ability to inhibit UV-induced mutation in Salmonella typhimurium TA102. The relative mutagenic activities (RMA, %) indicated the mutation frequency of treated cells divided by the mutation frequency of control cells times 100%. The RMA (%) are as follows: 28 +/- 1, 31 +/- 1, 33 +/- 1, 41 +/- 2, 62 +/- 1, and 62 +/- 4 at concentrations of 5 micrograms per plate for dehydrodieugenol, dehydrodivanillyl alcohol, dehydrodidihydroeugenol, dehydrodicreosol, magnolol, and honokiol, respectively. These values indicate that low concentrations of these biphenyl compounds effectively suppress UV-induced mutagenesis. Also, these compounds acted as effective antimutagens in a dose-dependent manner (0.00005-5 micrograms per plate). These compounds are effective .OH scavengers. Consequently, the results obtained above suggest that these compounds could inhibit against UV-induced mutations by scavenging of .OH generated by UV irradiation. The results also suggest that .OH are associated with UV-induced mutation in Salmonella typhimurium TA102.

Apparent inhibition of superoxide dismutase activity in vitro by diesel exhaust particles.

The inhibitory effects of diesel exhaust particles (DEP) on superoxide dismutase (SOD) activity were examined in vitro because intratracheal administration of DEP to mice resulted in a suppression of the pulmonary enzyme activity (Sagai et al., Free Radic. Biol. Med. 14:37-47; 1993). Superoxide production, based on the reduction of cytochrome c, was suppressed considerably by the soluble fraction of mouse lung and by purified SOD from bovine erythrocytes, but the suppression was drastically diminished in the presence of methanol-extractable compounds of DEP. Inhibition of SOD by diethyldithiocarbamate was irreversible, but that by 1,2-naphthoquinone (1,2-NQ) and the methanol extract of DEP was removed by dialysis. Inhibition of superoxide mediated cytochrome c reduction by Tiron, a scavenging agent for superoxide, was blocked by the methanol extract and 1,2-NQ in a concentration-dependent manner. In contrast, addition of a large amount of SOD to the reaction mixture resulted in an almost complete disappearance of inhibitory action of not only 1,2-NQ but also the methanol extract. The existence of carbonyl compounds in the DEP was confirmed by thin-layer chromatography (TLC) with 2,4-dinitrophenylhydrazine reagent. Electron spin resonance (ESR) spectra of an incubation mixture of oxidized 1,2-dihydroxynaphthalene in the absence and presence of cytochrome c indicated a reaction between the semiquinone radical of 1,2-NQ and cytochrome c. These results indicate that the apparent reduction in SOD activity by DEP is due to the chemical reaction of superoxide with components like quinones, which reduce levels of superoxide.

Direct evidence for in vivo nitroxide free radical production from a new antiarrhythmic drug by EPR spectroscopy.

The new Class I anti-arrhythmic agent 2,2,5,5-tetramethyl-3-pyrroline-1-carboxamide derivative, is currently being evaluated in clinical trials in patients with a high risk for cardiac arrhythmias. In this study we show that this antiarrhythmic drug can be chemically converted to the nitroxide free radical analog. Further, using an in vivo Electron Paramagnetic Resonance (EPR) spectroscopy model by detecting free radicals in the distal portion of the tail of an anesthetized mouse, we demonstrate that the drug is oxidized to the corresponding nitroxide. In vitro studies using Chinese hamster V79 cells suggest that the oxidation products of the drug, namely, the hydroxylamine and the nitroxide protect against oxidative damage induced by hydrogen peroxide (H2O2). Taken together, our results suggest that, in addition to the antiarrhythmic effects of the parent drug, sufficient levels of nitroxides may accumulate from the parent drug in vivo to provide antioxidant defense to cardiac tissue that may be subject to ischemia and oxidation-driven injury.

Dimerumic acid as an antioxidant of the mold, Monascus anka.

We previously reported that the mold Monascus anka, traditionally used for fermentation of food, showed antioxidant and hepatoprotective actions against chemically induced liver injuries. In the present study, the antioxidant component of M. anka was isolated and identified. The antioxidant was elucidated to be dimerumic acid. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was significantly scavenged by the antioxidant whereas hydroxyl radical and superoxide anion were moderately scavenged. When the antioxidant (12 mg/kg) was given to mice prior to carbon tetrachloride (CCl(4), 20 microl/kg, ip) treatment, the CCl(4)-induced liver toxicity in mice seen in an elevation of serum aspartate aminotransferase and alanine aminotransferase activities was depressed, suggesting the hepatoprotective action of the antioxidant. The liver microsomal glutathione S-transferase activity, which is known to be activated by oxidative stress or active metabolites, was increased by CCl(4) treatment and the increase was also depressed by pretreatment with the mold antioxidant. Thus these data confirmed that the dimerumic acid isolated from M. anka is the potential antioxidant and protective against CCl(4)-induced liver injury.

Hydroxyl radical formation by UV-irradiated epidermal cells.

To elucidate the mechanism of sunlight-induced skin damage, guinea pigs were exposed to UV light (280-320 nm, UV B, 4 J/cm2) and a homogenate of the epidermis was examined by means of the thiobarbituric acid (TBA) test. Three hours after the exposure, TBA-malondialdehyde adducts had increased while glutathione reductase activity had decreased, indicating lipid peroxidation. To detect the initial species, spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to a suspension of illuminated epidermal cells (0.5 J/cm2). An ESR signal obtained only with irradiation comprised a 1:2:2:1 quartet [a(N)= a(beta H) = 1.49 mT] attributable to a spin adduct of hydroxyl radicals. These results suggest that sunlight exposure of skin may lead to hydroxyl radical generation and simultaneous lipid peroxidation.

Pigmented lesions in actinically damaged skin. Histopathologic comparison of biopsy and excisional specimens.

BACKGROUND AND DESIGN: A consecutive sample of 46 cases was collected for comparative histologic evaluation. Results of incisional biopsies of cutaneous pigmented lesions interpreted as lentigo maligna, melanoma in situ, or invasive melanoma, and those suggestive, but not diagnostic, of melanoma were collected. Those lesions that were on actinically damaged skin and in which biopsy was followed by complete excision within 6 months were included. Incisional biopsies that removed greater than 50% of the surface area of the lesion were excluded. RESULTS: Of the excisional specimens, 40% demonstrated histopathologic features more pronounced than those in the biopsy specimens. Areas of invasive melanoma not detected in the biopsy specimens were observed in 20% of the excisional specimens. Accurate diagnosis based on small biopsy specimens was not always possible because of the absence of a classic lentigo maligna histologic pattern in many cases. The most frequent deviation from the pattern was the presence of lentiginous epidermal hyperplasia within these lesions. CONCLUSIONS: These results suggest that limited sampling may be inadequate for an accurate diagnosis of pigmented melanocytic lesions on actinically damaged skin. Areas chosen for biopsy may not contain the most advanced areas histologically and may fail to detect foci of invasive melanoma elsewhere within the lesion.

Mucinous eccrine nevus.

We report a solitary lesion with histologic characteristics of an organoid nevus occurring on the lower extremity of a forty-seven-year-old woman. This lesion is thought to represent a variant of eccrine nevus composed of an intradermal proliferation of hyperplastic eccrine sweat glands with associated mucinous change.

Basal cell carcinoma and melanocytic nevus in the same lesion.

We report a case of basal cell carcinoma and intradermal melanocytic nevus in the same lesion. A review of the literature indicates that the simultaneous occurrence of two or more neoplasms in one specimen is a relatively infrequent finding. The pathogenesis of simultaneously occurring neoplasms remains the subject of speculation. Based on currently available evidence, we believe that the coexistence of lesions in this particular patient is most likely coincidental.

Lobular capillary hemangioma (pyogenic granuloma) with satellitosis.

We describe a 43-year-old white man who rapidly developed multiple, extensive angiomatous lesions on the temple and scalp after excision of a solitary lobular capillary hemangioma. This is a well-recognized but rare event. Our case differs from previously reported examples in terms of the age of the patient, the location and extent of the lesions, the histologic features in the form of small foci of angiosarcoma-like infiltration, and possibly with respect to the response to therapeutic intervention. Because of the alarming clinical picture produced by multiple lobular capillary hemangiomas, in addition to the occurrence of disturbing histologic features, the benign and self-limited nature of this disease must be emphasized.

Effective inhibition of hydroxyl radicals by hydroxylated biphenyl compounds.

In aqueous media, approximate rate constants for the reactions between hydroxyl radicals (.OH) and biphenyl compounds such as dehydrodieugenol, magnolol, honokiol, dehydrodidihydroeugenol, dehydrodivanillyl alcohol, and dehydrodicreosol were estimated by competition reactions for .OH between these biphenyls and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). By measuring the decrease in the height of the EPR signals of the .OH spin adduct, rate constants in the order of 10(9) to 10(10) M were measured.

Small-diameter invasive melanomas: clinical and pathologic characteristics.

BACKGROUND: Most melanomas when first diagnosed clinically are larger than common benign melanocytic nevi ( > 6 mm). Smaller melanomas, however, may occasionally occur. Yet, very little is known about them. OBJECTIVE: To determine the incidence and delineate the clinical and histologic characteristics of small-diameter invasive melanomas. METHODS: Slides of all melanomas diagnosed between 1984-1993 were reviewed and measured. All invasive lesions less than 6 mm were further scrutinized clinically and morphologically. RESULTS: Among 357 invasive melanomas, 16 (4.4%) were less than 6 mm. Most lesions lacked some of the histologic features usually seen in large melanomas. One half of them were over 0.75 mm deep. Patients with small melanomas were on average 15 years younger than those with large lesions. In females, sites on the upper extremities predominate. In females, small melanomas had clinical signs of melanoma, while in males they resembled nevi. CONCLUSION: Melanomas smaller than 6 mm are not rare. Clinicians and pathologists must learn their features in order to diagnose them properly.

Essential oil phenyl propanoids. Useful as .OH scavengers?

In order to search for radical scavengers which could be used as raw materials for cosmetics, phenyl propanoids (eugenol, isoeugenol, dehydrodieugenol, dehydrodieugenol B and coniferyl aldehyde) were examined for their hydroxyl radical (.OH) scavenging ability. A Fenton system was used to produce .OH. In order to see scavenging by these phenyl propanoids, competition reactions between a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and these phenyl propanoids for .OH were studied. The relative yield of the spin adduct of .OH (DMPO-OH) was measured by electron spin resonance spectroscopy. The approximate rate constants of the reactions between these phenyl propanoids and .OH estimated by measuring the reduced height of the ESR signals of DMPO-OH were found to be at least in the order of 10(9) M-1 s-1 (diffusion-controlled). Also, using the TBA tests, the reactions between .OH and several compounds reactive with .OH were investigated in the presence of the phenyl propanoids and it was found that the phenyl propanoids compete with such reactive compounds for .OH. These results indicate that these phenyl propanoids can be used as antioxidants for skin damage perhaps caused by .OH generated by UV-light.

Stimulation by nitroxides of catalase-like activity of hemeproteins. Kinetics and mechanism.

The ability of stable nitroxide radicals to detoxify hypervalent heme proteins such as ferrylmyoglobin (MbFeIV) produced in the reaction of metmyoglobin (MbFeIII) and H2O2 was evaluated by monitoring O2 evolution, H2O2 depletion, and redox changes of the heme prosthetic group. The rate of H2O2 depletion and O2 evolution catalyzed by MbFeIII was enhanced by stable nitroxides such as 4-OH-2,2,6,6-tetramethyl-piperidinoxyl (TPL) in a catalytic fashion. The reduction of MbFeIV to MbFeIII was the rate-limiting step. Excess TPL over MbFeIII enhanced catalase-like activity more than 4-fold. During dismutation of H2O2, [TPL] and [MbFeIV] remained constant. NADH caused: (a) inhibition of H2O2 decay; (b) progressive reduction of TPL to its respective hydroxylamine TPL-H; and (c) arrest/inhibition of oxygen evolution or elicit consumption of O2. Following depletion of NADH the evolution of O2 resumed, and the initial concentration of TPL was restored. Kinetic analysis showed that two distinct forms of MbFeIV might be involved in the process. In summary, by shuttling between two oxidation states, namely nitroxide and oxoammonium cation, stable nitroxides enhance the catalase mimic activity of MbFeIII, thus facilitating H2O2 dismutation accompanied by O2 evolution and providing protection against hypervalent heme proteins.

Fibronectin and the extracellular matrix in the perforating disorders of the skin.

Despite detailed microscopic descriptions and clinical observation, little is known regarding the pathogenesis of the perforating disorders of skin, which have traditionally been subdivided into numerous microscopic entities associated with various clinical settings. An increasing body of evidence now suggests that the perforating disorders of skin are akin, and may constitute an expanded single pathologic entity. Each of the classic perforating disorders of skin, including elastosis perforans serpiginosa, perforating folliculitis, reactive perforating collagenosis, Kyrle's disease, and perforating disorder of uremia, have been shown to extrude collagen, elastin, and related extracellular matrix components through the epidermis. Considering a shared pathogenic mechanism among these entities, we explored the possible role of the extracellular matrix, in particular fibronectin, in perforating disorders of skin. Using immunohistochemical and serum determinations of extracellular matrix constituents, including fibronectin, collagen type IV, laminin, and tenascin, we showed consistent serum elevation and/or deposition of fibronectin, in each case, without a commensurate increase in laminin, collagen type IV, and tenascin. We propose that elevated serum and tissue concentrations of fibronectin may be responsible for inciting, in a physiologically aberrant manner, increased epithelial migration and proliferation culminating in perforation.