Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.     

Grass cover on forest clear-cut areas ameliorates some soil chemical properties

Clear-cut areas formed after forest decline due to acid deposition, pest attacks, or wind-breaks in temperate mountainous regions are often populated by grass (mainly Calamagrostis villosa). This study focused on the changes of soil chemical characteristics under the grass cover replacing the forest, focusing mainly on aluminium (Al) speciation. Clear-cut area due to strong acid deposition in the Jizera Mountains (Northern Bohemia) was studied. The soils under grass cover exhibit higher pH values and lower exchangeable Al content compared to adjacent surviving forest. Mobile Al species under the grass have larger proportion of non-toxic organic complexes. The content of exchangeable base cations is slightly higher under the grass. The positive effect of grass on soil chemistry was enhanced by liming. The temporary grass cover can therefore improve soil chemical quality for following reforestation. However, the differences are generally limited to surface organic horizons. Similar results were found also on a bark-beetle clear-cut area in the Bohemian Forest (Southern Bohemia) with smaller acid deposition; nevertheless, most differences were not significant there.     

(2-Benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid: an aldose reductase inhibitor and antioxidant of zwitterionic nature

Novel carboxymethylated pyridoindoles, characterized by antioxidant activity combined with the ability to inhibit aldose reductase, represent an example of a multitarget approach to the treatment of diabetic complications - severe diabetes-related health disorders of multifunctional nature. One of the novel carboxymethylated pyridoindoles, (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid (compound 1), was found to inhibit aldose reductase with the IC(50) value 18.2 ± 1.2 μM. Owing to aldose reductase pharmacophore requirements for an acidic proton, most aldose reductase inhibitors contain an acetic acid moiety, ionized at physiological pH, resulting in poor bioavailability of the drugs. The presence of a basicity center at the tertiary nitrogen of the carboxymethylated pyridoindoles, in addition to the acidic carboxylic function, predisposes these compounds to form double-charged zwitterionic species. The zwitterionic nature of compound 1 may remarkably affect its pH-lipophilicity profile allowing for increased membrane penetration in the pH region around its isoelectric point, which lies close to the physiological pH 7.4. In the first part of this study, the presence of zwitterionic species was experimentally proved by the concentration-dependent effect of sodium 1-hexanesulphonate on the distribution profile of compound 1. Then a series of experiments was performed in the cellular system of isolated erythrocytes in vitro. Isolated rat erythrocytes exposed to peroxyl radicals, generated in the solution by decomposition of the hydrophilic azoinitiator AAPH or intracellularly by decay of lipophilic t-BuOOH, underwent progressive hemolysis. The onset of the hemolysis was shifted from the starting zero point by the time interval assigned as a lag period. In the presence of compound 1 the lag period was significantly prolonged. Finally, under conditions of a short-term experiment in STZ-diabetic rats in vivo, increase in sorbitol levels in erythrocytes was recorded. Compound 1 administered in the dose 50mg/kg/day (i.g.) significantly decreased the sorbitol level in the erythrocytes. To conclude, the physico-chemical proof of the zwitterionic nature of compound 1 was established and the results obtained in isolated red blood cells indicated good cellular availability of the compound. In addition, in diabetic rats, sorbitol accumulation in red blood cells was significantly inhibited by compound 1 administered intra-gastrically, suggesting its ready uptake into the central compartment. The zwitterionic principle thus may have significant consequences for increased bioavailability of drugs bearing an acidic function.     

Impact of Cardiovascular Calcifications on the Detrimental Effect of Continued Smoking on Cardiovascular Risk in Male Lung Cancer Screening Participants

Background

Current smokers have an increased cardiovascular disease (CVD) risk compared to ex-smokers due to reversible as well as irreversible effects of smoking. We investigated if current smokers remain to have an increased CVD risk compared to ex-smokers in subjects with a long and intense smoking history. We in addition studied if the effect of smoking continuation on CVD risk is independent of or modified by the presence of cardiovascular calcifications.

Methods

The cohort used comprised a sample of 3559 male lung cancer screening trial participants. We conducted a case-cohort study using all CVD cases and a random sample of 10% (n = 341) from the baseline cohort (subcohort). A weighted Cox proportional hazards model was used to estimate the hazard ratios for current smoking status in relation to CVD events.

Results

During a median follow-up of 2.6 years (max. 3.7 years), 263 fatal and non-fatal cardiovascular events (cases) were identified. Age, packyears and cardiovascular calcification adjusted hazard ratio of current smokers compared to former smokers was 1.33 (95% confidence interval 1.00–1.77). In additional analyses that incorporated multiplicative interaction terms, neither coronary nor aortic calcifications modified the association between smoking status and cardiovascular risk (P = 0.08).

Conclusions

Current smokers have an increased CVD risk compared to former smokers even in subjects with a long and intense smoking history. Smoking exerts its hazardous effects on CVD risk by pathways partly independent of cardiovascular calcifications.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Salivary levels of tumor necrosis factor-alpha in oral lichen planus

OBJECTIVE: Oral lichen planus (OLP) is chronic inflammatory disease of the oral mucosa, presenting in various clinical forms. The etiology of OLP is still unknown but mounting evidence points to the immunologic basis of this disorder. AIM: Our study was undertaken to quantify the salivary levels of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) in the reticular and the erosive/atrophic forms of OLP, compared with age-matched healthy control volunteers. SUBJECTS AND METHODS: Whole saliva from 40 patients with active lesions of OLP, as well as from 20 healthy persons, was investigated for the presence of TNF-alpha by enzyme immunoassay. RESULTS: Salivary TNF-alpha levels were significantly increased in patients with OLP in comparison with healthy subjects. The presence of TNF-alpha showed positive correlation to clinical forms of OLP, being significantly higher in the erosive/atrophic type than in the reticular type of disease. CONCLUSION: Saliva provides an ideal medium for the detection of pro-inflammatory markers of the oral cavity. In patients with OLP, TNF-alpha levels in saliva are elevated, correlating with the severity of illness. Salivary TNF-alpha analysis may be a useful diagnostic tool and a potential prognostic marker in OLP.     

Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach

Recent advances in cytokinin analysis have made it possible to measure the content of 22 cytokinin metabolites in the tissue of developing tobacco seedlings. Individual types of cytokinins in plants are interconverted to their respective forms by several enzymatic activities (5'-AMP-isopentenyltransferase, adenosine nucleosidase, 5'-nucleotidase, adenosine phosphorylase, adenosine kinase, trans-hydroxylase, zeatin reductase, beta-glucosidase, O-glucosyl transferase, N-glucosyl transferase, cytokinin oxidase). This paper reports modelling and measuring of the dynamics of endogenous cytokinins in tobacco plants grown on media supplemented with isopentenyl adenine (IP), zeatin (Z) and dihydrozeatin riboside (DHZR). Differences in phenotypes generated by the three cytokinins are shown and discussed, and the assumption that substrate concentration drives enzyme kinetics underpinned the construction of a simple mathematical model of cytokinin metabolism in developing seedlings. The model was tested on data obtained from liquid chromatography/tandem mass spectrometry cytokinin measurements on tobacco seedlings grown on Murashige and Skoog agar nutrient medium, and on plants grown in the presence of IP, Z and DHZR. A close match was found between measured and simulated data, especially after a series of iterative parameter searches, in which the parameters were set to obtain the best fit with one of the data sets.     

Craniofacial anthropometric analysis in Down's syndrome patients

Past investigations of Down's syndrome (DS) have indicated that there are marked abnormalities in the craniofacial morphology. The aim of this study was to establish the craniofacial anthropometric variables which discriminate DS group from healthy population and also to observe the changes occurring with growth. Using noninvasive method of craniofacial anthropometry, craniofacial pattern profile (CFPP) analysis (from twenty-five anthropometric measurements per person) was performed in 104 DS individuals and 365 healthy controls, aged seven to fifty-seven and divided into four age ranges. Z-scores were calculated for each variable and the variations in the craniofacial region have been identified by multivariate discriminative analysis. The results showed that three variables (head length (g-op), head circumference (OFC) and outer canthal distance (ex-ex) were responsible for 85.68% variability (p < 0.001). The analysis of z-scores showed that the majority of variables were in subnormal (under -2 SD) and normal range (from -2SD to +2SD), but none of them was in the supernormal range (over the +2SD). Some craniofacial characteristics are age-related. On the basis of craniofacial anthropometric traits it was possible to separate even 91.35% of DS patients from the healthy population. It could be concluded that these findings demonstrate the usefulness of application of CFPP in defining abnormal craniofacial dimensions in DS individuals.     

Epidemiology of dermatomycosis in the eastern Croatia--today and yesterday

The aim of our investigation was to compare the distribution of dermatomycosis species in Eastern Croatia between two different periods: first period from 1997-2001 year, and second period from 1986-88 year. The outpatients from Department of Dermatovenerology University Hospital "Osijek" with confirmed diagnosis infection. Tinea, were selected on the basis o age, gender, localization and dermatomycosis species. During the first period (1997-2001) among 75,691 outpatients Tinea infection was confirmed in 558 (0.73%), while in the second period among 47,832 outpatients there were 126 (0.26%) cases with Tinea, what showed significant increase of fungal infections among population this region. According the age and gender in both periods predominant population were under of the age 16(40.14%: 41.26%), and female population was predominant (58.60% and 57.14%) in comparison to males (41.39% and 42.85%). The most frequent localization of lesions in period I were cutis glabrae (47.31%), palms and soles (31.36%), capitis (17.38%) and unguis (9.31%) and isolated species were as followed: Trichophyton (39.06%), Microsporum (31.72%) and Candida (28.13%) species. In period II the most frequent localization were palms and soles (40.47%), cutis glabrae (36.50%), capitis (12.69%) and unguis (10.31%). The isolated species in this period were: Trichophyton (80.15%), Candida (12.69%) and Microsporum (4.76%) species. From the data collected during two different periods we can observe 1) increase of fungal infection generally in our region; 2) significant changes in causative species (increase of Microsporum and Candida species infection, but Trichophyton spp still remain the first causative agent); and 3) changes in the localization of lesions.