Residue R120 is essential for the quaternary structure and functional integrity of human alphaB-crystallin

The missense mutation Arg-120 to Gly (R120G) in the human alphaBeta-crystallin sequence has been reported to be associated with autosomal dominant myopathy, cardiomyopathy, and cataract. Previous studies of the mutant showed a significant ability to aggregate in cultured cells and an increased oligomeric size coupled to an important loss of the chaperone-like activity in vitro. The aim of this study was to further analyze the role of the R120 residue in the structural and functional properties of alphaBeta-crystallin. The following mutants were generated, Arg-120 to Gly (R120G), Cys (R120C), Lys (R120K), and Asp (R120D). In cellulo, after expression in two cultured cell lines, NIH-3T3 and Cos-7, the capacity of the wild-type and mutant crystallins to aggregate was evaluated and the protein location was determined by immunofluorescence. In vitro, the wild-type and mutant crystallins were expressed in Escherichia coli cells, purified by size exclusion chromatography, and characterized using dynamic light scattering, electron microscopy, and chaperone-like activity assays. Aggregate sizes in cellulo and in vitro were analyzed. The whole of the data showed that the preservation of an Arg residue at position 120 of alphaBeta-crystallin is critical for the structural and functional integrity of the protein and that each mutation results in specific changes in both structural and functional characteristics.     

Biochemical and biophysical characterization of inhibitor binding to caspase-3 reveals induced asymmetry

Activation of the caspase family of cysteine proteases results in the deregulation of cellular homeostasis and apoptosis. This deregulation is a key factor in the development of Alzheimer's disease, Parkinson's disease, and cancer. Thus, the caspases are important drug targets for the therapeutic intervention of a number of pathological states involving inflammation and apoptosis. In this article, we report the results of inhibition kinetics and binding studies utilizing fluorescence spectroscopy and isothermal titration calorimetry to characterize the mechanism of interaction of caspase-3 with three different classes of inhibitors: peptidomimetics, isatins, and pyrimidoindolones. The peptidomimetics and pyrimidoindolones bind to both active sites of the caspase-3 homodimer with equal affinity and favorable enthalpic and entropic binding contributions. Enzyme activity is abolished when both active sites are occupied with the above inhibitors. In contrast, the isatins bind to caspase-3 with significant heat release (-12 kcal/mol) and negative entropy. In addition, enzyme activity is abolished upon isatin binding to one active site of the homodimer resulting in half-site reactivity. Our studies provide important mechanistic insight into inhibitor interactions with caspase-3 and a way to characterize inhibitor interactions that may not be readily apparent from the crystal structure.     

Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas)

An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H(2)O(2) at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 10(3)-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72).     

No signature of Y chromosomal resemblance between possible descendants of the Cimbri in Denmark and Northern Italy

Two European populations are believed to be related to the ancient Germanic tribe Cimbri: one living in Northern Italy, the other living in Jutland, Denmark. The people called Cimbri are documented in the ancient Roman historical record. Arriving from the far north their movements can be tracked from successive battles with the Romans. The Cimbri finally entered Italy from the northeast and were defeated at Vercellae (present day Vercelli) in 101 BC by Gaius Marius and his professional legions. Classical sources from the first centuries AD relate the homeland of the Cimbri to the coasts around the Elb estuary (northern Germany) or specifically towards the north (Himmerland in northern Jutland). In the alpine parts of Veneto, northeast of the historical battlefield, local traditions dating back to late medieval time, identify a local population as Cimbri living in Terra dei Cimbri. They are considered the descendents of the Germanic combatants that fled the battlefield at Vercelli. As the defeated Cimbri that possibly fled to the mountains of Northern Italy most likely would have been male (warriors), the present study investigated the possible Y chromosomal diversity of the two present populations using microsatellite markers and single nucleotide polymorphisms. While Cimbri from Himmerland resembled their geographical neighbors from Denmark for the Y-chromosome markers, Cimbri from Italy were significantly differentiated both from Cimbri from Himmerland and from Danes. Therefore, we were not able to show any biological relationship for uniparentally transmitted markers.     

Incubation of Environmental Samples in a Diffusion Chamber Increases the Diversity of Recovered Isolates

The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their natural environment. An inoculum is sandwiched between semipermeable (0.03-μm-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and Acidobacteria. Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.     

Side population cells in anaplastic thyroid cancer and normal thyroid

Comparison of studies of cells derived from normal and pathological tissues of the same organ can be fraught with difficulties, particular with cancer where a number of different diseases are considered cancer within the same tissue. In the thyroid, there are 4 main types of cancer, three of which arise from follicular epithelial cells; papillary and follicular which are classified as differentiated, and anaplastic which is classified as undifferentiated.One assay that can be utilised for isolation of cancer stem cells is the side population (SP) assay. However, SP studies have been limited in part due to lack of optimal isolation strategies and in the case of anaplastic thyroid cancer (ATC) are further compounded by lack of access to ATC tumors. We have used thyroid cell lines to determine the optimal conditions to isolate viable SP cells. We then compared SP cells and NSP cells (bulk tumour cells without the SP) of a normal thyroid cell line N-thy ori-3-1 and an anaplastic thyroid cancer cell line SW1736 and showed that both SP cell populations displayed higher levels of stem cell characteristics than the NSP. When we compared SP cells of the N-thy ori-3-1 and the SW1736, the SW1736 SP had a higher colony forming potential, expressed higher levels of stem cell markers and CXCR4 and where more migratory and invasive, invasiveness increasing in response to CXCL12.This is the first report showing functional differences between ATC SP and normal thyroid SP and could lead to the identification of new therapeutic targets to treat ATC.     

FXR Regulates Intestinal Cancer Stem Cell Proliferation

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