Detection of extracellular protease in Mucor species

The fungi are characterized by their abilities to produce and secrete enzymes to the external environment. The species of genus Mucor are a group of fungal microbes with important biotechnological potential, which are responsible for production of industrial enzymes. This work evaluated the ability of protease production in twelve species of genus Mucor. The strains were kept for 120 h under the incubation temperature of 28 degrees C on a shaker at 120 rpm. The detection of proteolytic activity was evaluated in all species, the higher activity was detected in Mucor racemosus Fres. f. chibinensis (Neophytova) Schipper.     

Estrogen upregulates renal angiotensin II AT1 and AT2 receptors in the rat

We studied renal AT1 and AT2 receptors in male, female, ovariectomized and ovariectomized-estrogen-treated Wistar-Hanover and Wistar-Kyoto rats. AT1 receptors and AT1A receptor mRNA predominated, with no significant differences between males and females. AT2 receptor expression was restricted in female rats to the capsule, the transition zone between outer and inner medulla, the endothelium lining the papilla, and arcuate arteries and veins. There were no AT2 receptors in male rats, while male mice express substantial numbers of estrogen-dependent AT2 receptors. Arcuate arteries and veins expressed AT1B mRNA in males and females, and AT2 mRNA in females only. AT1 receptor and AT2 receptor expression were estrogen-dependent, with increases in AT1 and AT2 receptor expression after estrogen treatment in ovariectomized rats. Estrogen treatment increased prostaglandin E2 (PGE2) and cGMP concentrations in the renal medulla, and eNOS expression in cortical arteries. In rodents, expression of renal Angiotensin II receptor types is estrogen-dependent, with significant species, strain and area differences. Our results support an important role for AT2 receptors in the regulation of renal function and in the protective effects of estrogen in the kidney.     

Identification of a tomatinase in the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin alpha-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by alpha-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of alpha-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.     

Quality of screening for diabetic retinopathy in the Rijeka region of Croatia

The aim of this study was to compare the quality of screening for diabetic retinopathy in cities of Rijeka and Zagreb, Croatia. Review of a random sample of 500 diabetic patient records and prospective ophthalmologic survey of 466 randomly selected diabetic patients in a secondary level diabetologic service in Rijeka (coastal region of Croatia). The main outcome measures were proportion of diabetic patient records with notes on ophthalmologic examination; rate of diabetic patients involved with screening for diabetic retinopathy; comparison with rates in Zagreb (Croatian capital). A total of 67% patients visited the ophthalmologist at least once after diagnosed with diabetes, and notes on ophthalmologic examination were found in only 28% patient records. Fifty percent of patients underwent an ophthalmologic examination within two years. Only one third of patients diagnosed with DM in last two years visited the ophthalmologist within this time, and 14% of patients older than 50 years never visited the ophthalmologist. Model of screening for diabetic retinopathy in Croatia works better in Zagreb than in Rijeka region, and needs certain improvements. The authors suggested modern methods of screening, the incorporation of the mechanisms of quality control, the obligatory reporting of newly diagnosed diabetic patients to the national registry, and the direct referral from diabetologist to ophthalmologist.     

Restoration of normal vascular relaxation mechanisms in cerebral arteries by chromosomal substitution in consomic SS.13BN rats

This study sought to identify the mechanisms of vascular relaxation that are rescued in middle cerebral arteries (MCA) of SS.13BN consomic rats by substituting chromosome 13 containing the renin gene from Brown Norway (BN) rats into the Dahl salt-sensitive (SS) genetic background. Isolated MCA from SS rats exhibited an indomethacin-sensitive constriction in response to acetylcholine (ACh) and hypoxia. ACh-induced dilation was NO dependent and hypoxic dilations were cyclooxygenase (COX) dependent in BN and SS.13BN rats. In SS rats, hypoxic dilation was restored by indomethacin and abolished by inhibiting cytochrome P-450 epoxygenases, suggesting a role for epoxyeicosatrienoic acids. MCA from SS and SS.13BN rats constricted and MCA from BN rats dilated in response to the stable prostacyclin analog iloprost. MCA from SS.13BN and BN rats (but not SS rats) dilated in response to the prostaglandin E2 receptor agonist butaprost. Hypoxia increased prostacyclin release in cerebral arteries from all the strains, whereas thromboxane A2 production was reduced in BN rat vessels only. These data suggest that SS rats may be less sensitive to vasodilator prostaglandins and that normalization of renin-angiotensin system regulation causes a switch from production of COX-derived vasoconstrictor metabolites (in SS rats) toward NO-dependent relaxation in response to ACh- and prostaglandin-dependent dilation in response to hypoxia in SS.13(BN) rats.     

Identification of homologues to the pathogenicity factor Pat-1, a putative serine protease of Clavibacter michiganensis subsp. michiganensis

Hybridization of Clavibacter michiganensis subsp. michiganensis total DNA against the pathogenicity gene pat-1 indicated the presence of pat-1 homologous nucleotide sequences on the chromosome and on plasmid pCM2. Isolation of the corresponding DNA fragments and nucleotide sequence determination showed that there are three pat-1 homologous genes: chpA (chromosome) and phpA and phpB (plasmid pCM2). The gene products share common characteristics, i.e. a signal sequence for Sec-dependent secretion, a serine protease motif, and six cysteine residues at conserved positions. Gene chpA located on the chromosome is a pseudogene since it contains a translational stop codon after 97 of 280 amino acids. In contrast to pat-1, cloning of the plasmid encoded homologs phpA and phpB into the avirulent plasmid free Cmm strain CMM100 did not result in a virulent phenotype. So far no proteolytic activity could be demonstrated for Pat-1, however, site specific mutagenesis of pat-1 showed that the serine residue in the motif GDSGG is required for the virulent phenotype of pat-1 and thus Pat-1 could be a functional protease.     

Manifestations of inflammatory arthritis are critically dependent on LFA-1

Leukocyte infiltration of synovial fluid and tissues is the hallmark of inflammatory arthritis. Selectins and beta2 integrins have been implicated in the multistep process of leukocyte adhesion to vascular endothelium. However, previous work has revealed disparate requirements for leukocyte recruitments to specific anatomic locales. Moreover, the mechanisms regulating recruitment of leukocytes to the joint in inflammatory arthritis models are not fully understood. We hypothesized that beta2 integrins, expressed on leukocytes, might play a pathogenic role in synovial inflammation. Using mice deficient in all beta2 integrins (CD18 null mice), we demonstrate that expression of these heterodimeric adhesion molecules is critical for arthritis induction in the K/B x N serum transfer model. Using null-allele mice and blocking mAbs, we demonstrate specifically that CD11a/CD18 (LFA-1) is absolutely required for the development of arthritis in this model. Blocking mAbs further revealed an ongoing requirement for LFA-1 I-domain adhesive function in disease perpetuation. These findings suggest that the LFA-1 I-domain forms an attractive target for treatment of human inflammatory arthritis.     

Hom s 4, an IgE-reactive autoantigen belonging to a new subfamily of calcium-binding proteins, can induce Th cell type 1-mediated autoreactivity

Skin inflammation in atopic dermatitis starts with Th2 and IgE-mediated responses against exogenous allergens and, for unknown reasons, resembles features of a Th1-driven reaction in the chronic stages. We report the characterization of a human protein, Hom s 4, recognized by IgE autoantibodies from atopic dermatitis patients. The complete Hom s 4 cDNA codes for a 54-kDa basic protein containing two typical calcium-binding domains separated by an unusually long alpha-helical domain. Therefore, Hom s 4 and homologous proteins found by sequence comparison in mice, fruit flies, and nematodes constitute a novel subfamily of calcium-binding proteins. Using Hom s 4-specific Abs, it is demonstrated that the protein is strongly expressed within epidermal keratinocytes and dermal endothelial cells. Purified Hom s 4 showed IgE cross-reactivity with exogenous calcium-binding allergens from plants and fish but, in contrast to the exogenous allergens, induced only weak histamine release from patient basophils. However, the analysis of Hom s 4-specific cytokine and humoral immune responses indicated that Hom s 4 strongly induces Th1 responses which are accompanied by the release of IFN-gamma, a cytokine implicated in epithelial cell damage. Hom s 4-induced IFN-gamma production was found in normal individuals, in patients with chronic inflammatory skin diseases and in Th2-prone atopic persons, suggesting that Hom s 4 represents a protein with an intrinsic property to induce Th1-mediated autoreactivity. It may thus contribute to chronic skin inflammation in atopic as well as in nonatopic persons.     

Hepatocyte growth factor promotes lymphatic vessel formation and function

The lymphatic vascular system plays a pivotal role in mediating tissue fluid homeostasis and cancer metastasis, but the molecular mechanisms that regulate its formation and function remain poorly characterized. A comparative analysis of the gene expression of purified lymphatic endothelial cells (LEC) versus blood vascular endothelial cells (BVEC) revealed that LEC express significantly higher levels of hepatocyte growth factor receptor (HGF-R). Whereas little or no HGF-R expression was detected by lymphatic vessels of normal tissues, HGF-R was strongly expressed by regenerating lymphatic endothelium during tissue repair and by activated lymphatic vessels in inflamed skin. Treatment of cultured LEC with HGF promoted LEC proliferation, migration and tube formation. HGF-induced proliferation of LEC did not require vascular endothelial growth factor receptor-3 activation, and HGF-induced cell migration was partially mediated via integrin alpha-9. Transgenic or subcutaneous delivery of HGF promoted lymphatic vessel formation in mice, whereas systemic blockade of HGF-R inhibited lymphatic function. These results identify HGF as a novel, potent lymphangiogenesis factor, and also indicate that HGF-R might serve as a new target for inhibiting pathological lymphangiogenesis.