The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.     

p130Cas Over-Expression Impairs Mammary Branching Morphogenesis in Response to Estrogen and EGF

p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the in vivo consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.     

Involvement of the Tubular ClC-Type Exchanger ClC-5 in Glomeruli of Human Proteinuric Nephropathies

Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndrome. Histopathological findings in renal biopsies from severely proteinuric patients support the likelihood of protein endocytosis by podocytes. ClC-5 is involved in the endocytosis of albumin in the proximal tubule.

Aim

To investigate whether ClC-5 is expressed in the glomerular compartment and whether it has a role in proteinuric nephropathies. ClC-5 expression was studied using Real-time PCR in manually- and laser-microdissected biopsies from patients with type 2 diabetes (n 37) and IgA nephropathy (n 10); in biopsies of membranous glomerulopathy (MG) (n 14) immunohistochemistry for ClC-5 (with morphometric analysis) and for WT1 was done. Controls: cortical tissue (n 23) obtained from unaffected parts of tumor-related nephrectomy specimens.

Results

ClC-5 was expressed at glomerular level in all biopsies. Glomerular ClC-5 levels were significantly higher in diabetic nephropaty and MG at both mRNA and protein level (p<0.002; p<0.01). ClC-5 and WT1 double-staining analysis in MG showed that ClC-5 was localized in the podocytes. ClC-5 ultrastructural immunolocalization was demonstrated in podocytes foot processes. Our study is the first to demonstrate that ClC-5 is expressed in human podocytes. The ClC-5 overexpression found in biopsies of proteinuric patients suggests that proteinuria may play a part in its expression and that podocytes are likely to have a key role in albumin handling in proteinuric states.     

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

Cardiac cellular coupling and the spread of early instabilities in intracellular Ca2+

Recent experimental and modeling studies demonstrate the fine spatial scale, complex nature, and independent contribution of Ca²⁺ dynamics as a proarrhythmic factor in the heart. The mechanism of progression of cell-level Ca²⁺ instabilities, known as alternans, to tissue-level arrhythmias is not well understood. Because gap junction coupling dictates cardiac syncytial properties, we set out to elucidate its role in the spatiotemporal evolution of Ca²⁺ instabilities. We experimentally perturbed cellular coupling in cardiac syncytium in vitro. Coupling was quantified by fluorescence recovery after photobleaching, and related to function, including subtle fine-scale Ca²⁺ alternans, captured by optical mapping. Conduction velocity and threshold for alternans monotonically increased with coupling. Lower coupling enhanced Ca²⁺ alternans amplitude, but the spatial spread of early (<2 Hz) alternation was the greatest under intermediate (not low) coupling. This nonmonotonic relationship was closely matched by the percent of samples exhibiting large-scale alternans at higher pacing rates. Computer modeling corroborated these experimental findings for strong but not weak electromechanical (voltage-Ca²⁺) coupling, and offered mechanistic insight. In conclusion, using experimental and modeling approaches, we reveal a general mechanism for the spatial spread of subtle cellular Ca²⁺ alternans that relies on a combination of gap-junctional and voltage-Ca²⁺ coupling.     

Haemopexin affects iron distribution and ferritin expression in mouse brain

Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme‐mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer’s disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron‐loaded oligodendrocytes in the basal ganglia and thalamus of Hx‐null mice compared to wild‐type controls. Interestingly, there was no increase in H‐ and L‐ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer’s disease and Parkinson’s disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin‐positive cells was observed in the cerebral cortex of Hx‐null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu–Zn superoxide dismutase‐1 expression were higher in the brain of Hx‐null mice than in that of wild‐type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron‐related neurodegenerative diseases.     

Essential role of diastolic oscillatory potentials in adrenergic control of guinea pig sino-atrial node discharge

Background  

The diastolic oscillatory after-potential V_os and pre-potential ThV_os play an essential role in the pacemaker mechanism of sino-atrial node (SAN). The aim of this study was to investigate whether these oscillatory potentials are also involved in adrenergic control of SAN discharge.     

AFLP genotyping of Candida metapsilosis clinical isolates: Evidence for recombination

In a collection of 395 independent clinical isolates classified as Candida parapsilosis on a biochemical profile basis, 20 Candida metapsilosis strains were identified by molecular tests with an isolation frequency of 5%. Isolates were screened for their susceptibility to conventionally used antifungals and for virulence determinants, such as biofilm formation and protease production. Molecular characterization of C. metapsilosis independent isolates by amplified fragment length polymorphism (AFLP) revealed a high percentage of polymorphic bands. Statistical analysis of the pairwise genetic distances and bootstrapping revealed that recombination occurs and significantly contributes to C. metapsilosis genetic population variability. No association between specific AFLP markers and drug resistance or other phenotypes was observed.     

Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

Background  

A wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.