Differential biological effects of 1,25-dihydroxyVitamin D3 on melanoma cell lines in vitro

1,25-DihydroxyVitamin D(3) and analogs have been shown to inhibit proliferation and to induce differentiation in different cell types, including human melanocytes. However, various tumor cell lines that fail to respond to the antiproliferative effects of Vitamin D analogs have also been reported. Using real-time PCR (LightCycler), we have compared mRNA expression of Vitamin D receptor (VDR), Vitamin D-25-hydroxylase (25-OHase), 25-hydroxyVitamin D-1alpha-hydroxylase (1alpha-OHase), and 1,25-dihydroxyVitamin D-24-hydroxylase (24-OHase) in a melanoma cell line that responds to antiproliferative effects of Vitamin D (MeWo) with a non-responsive melanoma cell line (SkMel5). Additionally, modulation of cell proliferation by calpain inhibitors, as well as regulation of mRNA expression of VDR, 1alpha-OHase, and 24-OHase genes by Vitamin D analogs were assessed in melanoma cell lines in vitro using a WST-1 based colorimetric assay and real-time PCR, respectively. RNA for VDR, 25-OHase, 1alpha-OHase, and 24-OHase was detected in melanoma cell lines. In contrast to SkMel5 cells, treatment of MeWo cells with calcitriol resulted in a dose-dependent increase in mRNA for VDR and 24-OHase as well as in a suppression of cell proliferation (up to approximately 50%). Our findings demonstrate that local synthesis or metabolism of Vitamin D metabolites may be of importance for growth regulation of MM and melanoma cell lines. Additionally, metastasizing MM represents a promising target for palliative treatment with new Vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of calcitriol synthesis/metabolism in these tumors.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Formin homology 2 domains occur in multiple contexts in angiosperms

Background  

Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions.     

Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage

Angiotensin II contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One possibility that has been advanced is that angiotensin II activates NADPH oxidase, generating free radicals that trigger apoptosis. In apparent support of this notion, it was found that angiotensin II-mediated apoptosis in the cardiomyocyte is blocked by the NADPH oxidase inhibitor diphenylene iodonium. However, three lines of evidence suggest that peroxynitrite, rather than superoxide, is responsible for angiotensin II-mediated DNA damage and apoptosis. First, the inducible nitric oxide inhibitor aminoguanidine prevents angiotensin II-induced DNA damage and apoptosis. Second, based on ligation-mediated PCR, the pattern of angiotensin II-induced DNA damage resembles peroxynitritemediated damage rather than damage caused by either superoxide or nitric oxide. Third, angiotensin II activates p53 through the phosphorylation of Ser15 and Ser20, residues that are commonly phosphorylated in response to DNA damage. It is proposed that angiotensin II promotes the oxidation of DNA, which in turn activates p53 to mediate apoptosis.     

Conformational changes,plasma membrane penetration,and infection by human rhinovirus type 2: role of receptors and low pH

Human rhinovirus type 2 (HRV2) is internalized by members of the low-density lipoprotein (LDL) receptor (LDLR) family. It then progresses into late endosomes, where it undergoes conversion from D- to C-antigenicity at pH < 5.6. Upon uncoating, the viral RNA is transferred into the cytoplasm across the endsosomal membrane. However, C-antigenic particles fail to attach to LDLR; this raised the question of whether the virus remains attached to the receptors and is carried to late compartments or rather falls off at the higher pH in early endosomes. We therefore determined the pH dependence of virus-receptor dissociation and virus conversion to C-antigen under conditions preventing endocytosis. (35)S-HRV2 was attached to HeLa cells at 4 degrees C and incubated in buffers of pH 7.4 to 5.0; levels of native virus and C-antigenic particles remaining cell associated or having been released into the medium were determined by immunoprecipitation. At pH 6.0, HRV2 was readily released from plasma membrane receptors in its native form, whereas at pH < or = 5.4, it was entirely converted to C-antigen, which, however, only dissociated from the surface upon prolonged incubation. The antigenic conversion occurred at the same pH regardless of whether HRV2 was free in solution or bound to its receptors. These data suggest that, in vivo, the virus is no longer bound to its receptors when the antigenic conversion and uncoating occur in more acidic late endosomes. When virus was bound to HeLa cells at 4 degrees C, converted into C-antigen by exposure to pH 5.3, and subsequently warmed to 34 degrees C in the presence of bafilomycin (to prevent endosomal uncoating), viral de novo synthesis was detected. This study demonstrates for the first time that a nonenveloped virus such as HRV2 can infect from the plasma membrane when artificially exposed to low pH. This implies that the viral RNA can gain access to the cytoplasm from the plasma membrane.     

Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda

Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible.