Role of epidermis-type lipoxygenases for skin barrier function and adipocyte differentiation

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.     

Brazilian green propolis on Helicobacter pylori infection. a pilot clinical study

Recent in vitro studies suggest that propolis and some of its phenolic components are able to inhibit Helicobacter pylori growth. To date, there are no clinical studies. AIMS: To evaluate the effect of Brazilian green propolis on H. pylori-infected individuals. PATIENTS AND METHODS: Eighteen (11 females, 7 males, mean age 47 years) participants were included. Before treatment, all participants were submitted to gastroscopy, and H. pylori infection was confirmed by histology, urease test, and (13)C-urea breath test (UBT). Participants with UBT showing a delta over baseline (DOB) value higher than 4 per thousand were considered positive for H. pylori infection. Twenty drops from an alcoholic preparation of Brazilian green propolis were administered three times a day for 7 days. Clinical evaluation and UBT were performed at 1-3 days and at 40 days after the end of therapy to evaluate H. pylori suppression or eradication, respectively. RESULTS: All participants took all medication and completed the study. Eighty-three percent of the subjects did not succeed in suppressing or eradicating H. pylori. Two participants reached partial suppression after treatment, but became positive again at UBT performed 40 days after treatment. Another participant presented negative at UBT 40 days after treatment, not confirmed by a second UBT performed 100 days after treatment. CONCLUSIONS: Brazilian green propolis used in popular dose showed minimal effect on H. pylori infection. Larger studies with longer duration, larger dose, and different frequency of administration of propolis extract should be undertaken to define its role on H. pylori therapy.     

Identification of transcription elements in the 5' intergenic region shared by LON and MDJ1 heat shock genes from the human pathogen Paracoccidioides brasiliensis. Evaluation of gene expression

The MDJ1/LON locus is conserved among pathogenic dimorphic fungi. We have mapped using DNase I footprinting and mobility shift assays three putative heat shock elements and one AP-1 binding domain (ARE) in the 5' intergenic region shared by PbMDJ1and PbLON (ML) from Paracoccidioides brasiliensis. The region bearing an ARE-like towards PbLON also has an opposite skn-1-like element. We studied genetically and pathogenically distinct isolates Pb18 and Pb3, where ML is polymorphic and the number of elements detected was higher. The functionality of the elements was suggested by the stimulatory response of both genes to heat shock and oxidative stress. Co-regulation occurred upon heat shock from 36 to 42 degrees C and, only in Pb3, also during mycelium to yeast transformation (26-36 degrees C). In Pb18, PbMDJ1 seemed to be preferentially expressed in yeast. Our study might help understand regulation of genes involved in fungal adaptation to the host.     

Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems

As the number of single-nucleotide polymorphism (SNP) screening and other mutation scanning studies have increased explosively, following the development of high-throughput instrumentation, it becomes even more important to have sufficient template DNA. The source of DNA is often limited, especially in epidemiological studies, which require many samples as well as enough DNA to perform numerous SNP screenings or mutation scannings. Therefore, the aim is to solve the problem of stock DNA limitation. This need has been an important reason for the development of whole genome amplification (WGA) methods. Several systems are based on Phi29 polymerase multiple displacement amplification (MDA) or on DNA fragmentation (OmniPlex). Using TaqMan SNP genotyping assays, we have tested four WGA systems -- AmpliQ Genomic Amplifier Kit, GenomiPhi, Repli-g, and GenomePlex -- on DNA extracted from Guthrie cards to evaluate the amplification bias, concordance- and call rates, cost efficiency, and flexibility. All systems successfully amplified picograms of DNA from Guthrie cards to micrograms of product without loss of heterozygosity and with minimal allelic bias. A modified AmpliQ set up was chosen for further evaluation. In all, 2,000 SNP genotyping results from amplified and nonamplified samples were compared and the concordance rates between the samples were 99.7%. The call rate using the TaqMan system was 99.8%. DNA extracted from Guthrie cards and amplified with one of the four evaluated WGA systems is applicable in epidemiological genetic screenings. System choice should be based on requirements for system flexibility, product yield, and use in subsequent analysis.     

Antagonistic interaction between oxygenation-linked lactate and CO2 binding to human hemoglobin

Oxygen binding to hemoglobin (Hb) depends on allosteric effectors (CO(2), lactate and protons) that may increase drastically in concentration during exercise. The effectors share common binding sites on the Hb molecules, predicting mutual interaction in their effects on Hb (de)oxygenation. We analysed the effects of lactate and CO(2), separately and in combination, on O(2) binding of purified human Hb at 37 degrees C and physiological pH and chloride values. We demonstrate pH-dependent, inhibitory interactions between lactate binding and CO(2) binding (carbamate formation); at pH 7.4, physiological CO(2) tension ( approximately 43 mm Hg) reduced lactate binding more markedly ( approximately 75%), than lactate (50 mM) inhibited carbamate formation ( approximately 25%). In contrast to previous studies on blood and Hb solutions, we moreover find that added lactate neither 'reverses' oxylabile carbamate formation (resulting in lower carbamate levels in deoxyHb than in oxyHb) nor exerts greater allosteric effects on Hb-O(2) affinity than equal increases in chloride ion concentrations.     

Characterization of a Polycyclic Aromatic Hydrocarbon-Degrading Microbial Consortium from a Petrochemical Sludge Landfarming Site

Anthracene, phenanthrene, and pyrene are polycyclic aromatic hydrocarbon (PAHs) that display both mutagenic and carcinogenic properties. They are recalcitrant to microbial degradation in soil and water due to their complex molecular structure and low solubility in water. This study presents the characterization of an efficient PAH (anthracene, phenanthrene, and pyrene)-degrading microbial consortium, isolated from a petrochemical sludge landfarming site. Soil samples collected at the landfarming area were used as inoculum in Warburg flasks containing soil spiked with 250 mg kg^-1 of anthracene. The soil sample with the highest production of CO₂-C in 176 days was used in liquid mineral medium for further enrichment of anthracene degraders. The microbial consortium degraded 48%, 67%, and 22% of the anthracene, phenanthrene, and pyrene in the mineral medium, respectively, after 30 days of incubation. Six bacteria, identified by 16S rRNA sequencing as Mycobacterium fortuitum, Bacillus cereus, Microbacterium sp., Gordonia polyisoprenivorans, two Microbacteriaceae bacteria, and a fungus identified as Fusarium oxysporum were isolated from the enrichment culture. The consortium and its monoculture isolates utilized a variety of hydrocarbons including PAHs (pyrene, anthracene, phenanthrene, and naftalene), monoaromatics hydrocarbons (benzene, ethylbenzene, toluene, and xylene), aliphatic hydrocarbons (1-decene, 1-octene, and hexane), hydrocarbon mixtures (gasoline and diesel oil), intermediary metabolites of PAHs degradation (catechol, gentisic acid, salicylic acid, and dihydroxybenzoic acid) and ethanol for growth. Biosurfactant production by the isolates was assessed by an emulsification index and reduction of the surface tension in the mineral medium. Significant emulsification was observed with the isolates, indicating production of high-molecular-weigh surfactants. The high PAH degradation rates, the wide spectrum of hydrocarbons utilization, and emulsification capacities of the microbial consortium and its member microbes indicate that they can be used for biotreatment and bioaugumentation of soils contaminated with PAHs.     

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.     

Extracellular proteolytic activity and molecular analysis of Microsporum canis strains isolated from symptomatic and asymptomatic cats

Microsporum canis is the main zoophylic dermatophyte in dogs and cats, and it is also an important zoonotic agent. The literature showed that cats are asymptomatic carriers of M. canis. This is apparently due to host resistance and/or the presence of strains with lower virulence. This study was aimed to evaluate the keratinolytic, elastinolytic and collagenolytic activities of M. canis strains and their relationship with symptomatic and asymptomatic cats. In addition, these strains were analysed by RFLP. The strains isolated from cats with clinical dermatophytosis had higher keratinase and elastase activity than those isolated from asymptomatic animals (p minus than 0.05). There were not differences in RFLP patterns based on Hind III digestion.     

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.