Simple method for separating the chlamydoconidia of Candida albicans from its mycelium

Different physical and chemical methods were used to detach the chlamydoconidia of Candida albicans from its mycelium. The action of concentrated H₂SO₄ acid for a 4-min period on cultures lysed both the mycelium and the outer but not the inner wall layer of the chlamydoconidia.The sulfuric acid procedure is recommended as the best method to obtain mycelium free chlamydoconidia because of its simplicity, rapidity and low cost.     

Activation of β- and γ-carbonic anhydrases from pathogenic bacteria with tripeptides

Six tripeptides incorporating acidic amino acid residues were prepared for investigation as activators of β- and γ-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacteria Vibrio cholerae, Mycobacterium tuberculosis, and Burkholderia pseudomallei. The primary amino acid residues that are involved in the catalytic mechanisms of these CA classes are poorly understood, although glutamic acid residues near the active site appear to be involved. The tripeptides that contain Glu or Asp residues can effectively activate VchCAβ and VchCAγ (enzymes from V. cholerae), Rv3273 CA (mtCA3, a β-CA from M. tuberculosis) and BpsCAγ (γ-CA from B. pseudomallei) at 0.21–18.1?µM levels. The position of the acidic residues in the peptide sequences can significantly affect bioactivity. For three of the enzymes, tripeptides were identified that are more effective activators than both l-Glu and l-Asp. The tripeptides are also relatively selective because they do not activate prototypical α-CAs (human carbonic anhydrases I and II). Because the role of CA activators in the pathogenicity and life cycles of these infectious bacteria are poorly understood, this study provides new molecular probes to explore such processes.     

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen‐induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia‐dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin‐4 (AQP4) is involved in the hypoxia‐dependent VEGF upregulation in the retina of a mouse model of oxygen‐induced retinopathy (OIR). The expression levels of VEGF, the hypoxia‐inducible factor‐1α (HIF‐1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF‐1 binding site (HBS) in the VEGF gene promoter, the binding of HIF‐1α to the HBS, the retinal vascularization and function have been determined in the retina of wild‐type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF‐1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF‐1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF‐1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF‐1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF‐induced retinal damage in response to hypoxia.     

Aquaporin‐1 inhibition reduces metastatic formation in a mouse model of melanoma

Aquaporin‐1 (AQP1) is a proangiogenic water channel protein promoting endothelial cell migration. We previously reported that AQP1 silencing by RNA interference reduces angiogenesis‐dependent primary tumour growth in a mouse model of melanoma. In this study, we tested the hypothesis that AQP1 inhibition also affects animal survival and lung nodule formation. Melanoma was induced by injecting B16F10 cells into the back of C57BL6J mice. Intratumoural injection of AQP1 siRNA and CTRL siRNA was performed 10 days after tumour cell implantation. Lung nodule formation was analysed after the death of the mice. Western blot was used to quantify HIF‐1α, caspase‐3 (CASP3) and metalloproteinase‐2 (MMP2) protein levels. We found that AQP1 knock‐down (KD) strongly inhibited metastatic lung nodule formation. Moreover, AQP1 siRNA‐treated mice showed a twofold survival advantage compared to mice receiving CTRL siRNAs. The reduced AQP1‐dependent tumour angiogenesis caused a hypoxic condition, evaluated by HIF‐1α significant increase, in turn causing an increased level of apoptosis in AQP1 KD tumours, assessed by CASP3 quantification and DNA fragmentation. Importantly, a decreased level of MMP2 after AQP1 KD indicated a decreased activity against extracellular matrix associated with reduced vascularization and metastatic formation. In conclusion, these findings highlight an additional role for AQP1 as an important determinant of tumour dissemination by facilitating tumour cell extravasation and metastatic formation. This study adds knowledge on the role played by AQP1 in tumour biology and supports the view of AQP1 as a potential drug target for cancer therapy.     

Activation of CaMKKβ/AMPKα pathway by 2‐AG in human platelets

The objective of this study was to determine whether AMPK is activated by 2‐arachidonoylglycerol (2‐AG) and participates to the cytoskeleton control in human platelets. We found that 2‐AG stimulates the AMPKα activation through a Ca²⁺/Calmodulin‐dependent pathway as the specific inhibition of the CaMKKβ by STO‐609 inhibits the AMPKα phosphorylation/activation. Moreover, the CaMKKβ/AMPKα pathway activated by 2‐AG is involved in the phosphorylation of cofilin, vasodilator stimulated phosphoprotein (VASP), and myosin light chain (MLCs). These proteins participate to actin cytoskeletal remodelling during aggregation. We found that the phosphorylation/activation inhibition of these proteins is associated with a significant reduction in actin polymerization, aggregation, ATP, and α‐granule secretion. Finally, AMPKα activation, Cofilin, VASP, and MLCs phosphorylation are significantly reduced by SR141716, the specific inhibitor of type 1 cannabinoid (CB1) receptor, suggesting that the CB1 receptor is involved in the 2‐AG effect. In conclusion, we have shown that the CaMKKβ/AMPKα pathway is activated by 2‐AG in human platelets and controls the phosphorylation of key proteins involved in actin polymerization and aggregation.     

The influence of ethylene on in vitro rooting of GF 677 (Prunus persica × Prunus amygdalus) hybrid peach rootstock

Summary Treatments differing from each other for the type of tube closure (i.e., cotton plug for free gas exchange, airtight rubber cap, and rubber cap with ethysorb) and/or rooting culture medium (i.e., enriched or not by 25 to 100 μM acetylsalicylic acid) were compared for their effects on gaseous composition of the culture atmosphere and microcutting rooting of the GF 677 (Prunus persica × Prunus amygdalus) hybrid. Rubber capping, which leads to rapid ethylene accumulation inside tubes, strongly reduced rooting time and in some cases enhanced final rooting percentage over that of cotton plugs. Ethysorb almost completely absorbed ethylene produced by shoots, which showed lower rooting percentages within 9 d than microcuttings cultured in the absence of ethysorb. In contrast, no significant difference in rooting was found between the two treatments after 14 d. Carbon dioxide concentration was similar in all treatments within 5 to 9 d and seemed to be ineffective for rooting. The influence of acetylsalicylic acid on rooting was unclear. Root number and length were not significantly influenced by the treatments. These results demonstrate that the use of airtight closures, leading to rapid ethylene accumulation, can reduce time of rooting expression for GF 677 microcuttings. However, free gas exchange towards the end of the rooting period (from Day 9 to Day 14) is advisable to prevent leaf yellowing. No significant difference in plantlet survival and growth after transfer ex vitro was found among treatments.     

Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.

We evaluated the IGF1 system in cholangiocytes of primay biliary cirrhosis (PBC) patients and investigated the relationships with apoptosis. Biopsies of PBC patients (n=32) and normal subjects (n=5) were investigated by immunohistochemistry for expression in cholangiocytes of IGF1, IGF1-R, pAKT, terminal deoxynucleotide transferase end labeling (TUNEL), Bax (proapoptotic protein), and Bcl2 (antiapoptotic protein). Whereas normal cholangiocytes were almost negative, cholangiocytes of PBC patients showed strong IHC staining for IGF1, IGF1-R, and pAKT, which increases from stage I to stage IV, where >70% of cholangiocytes were positive. Bax/Bcl2 ratio reached the highest value (4.6) in PBC stage III when apoptosis is maximal (24% TUNEL positivity), whereas it declines in stage IV (1.4) when only 7.8% cholangiocytes were TUNEL positive. In PBC stages III and IV, expression of IGF1, IGF1-R, and pAKT in cholangiocytes was directly correlated with the antiapoptotic Bcl2 and inversely correlated with proapoptotic Bax, Bax/Bcl2 ratio, and TUNEL positivity. In conclusion, cholangiocytes of PBC patients showed a marked increase in IGF1, IGF1-R, and pAKT expression involving most cholangiocytes surviving in the terminal ductopenic stage. This was associated and correlated with a balance of pro- and antiapoptotic proteins favoring survival rather than apoptosis, suggesting a major role of IGF1 system in promoting cholangiocyte survival.     

Evolution of Hypervariable Region 1 of Hepatitis C Virus in Primary Infection

The hypervariable region 1 (HVR-1) of the putative envelope encoding E2 region of hepatitis C virus (HCV) RNA was analyzed in sequential samples from three patients with acute type C hepatitis infected from different sources to address (i) the dynamics of intrahost HCV variability during the primary infection and (ii) the role of host selective pressure in driving viral genetic evolution. HVR-1 sequences from 20 clones per each point in time were analyzed after amplification, cloning, and purification of plasmid DNA from single colonies of transformed cells. The intrasample evolutionary analysis (nonsynonymous mutations per nonsynonymous site [K_a], synonymous mutations per synonymous site [K_s], K_a/K_s ratio, and genetic distances [gd]) documented low gd in early samples (ranging from 2.11 to 7.79%) and a further decrease after seroconversion (from 0 to 4.80%), suggesting that primary HCV infection is an oligoclonal event, and found different levels and dynamics of host pressure in the three cases. The intersample analysis (pairwise comparisons of intrapatient sequences; rK_a, rK_s, rK_a/rK_s ratio, and gd) confirmed the individual features of HCV genetic evolution in the three subjects and pointed to the relative contribution of either neutral evolution or selective forces in driving viral variability, documenting that adaptation of HCV for persistence in vivo follows different routes, probably representing the molecular counterpart of the viral fitness for individual environments.