On the development of a standard two-dimensional polypeptide map of the human X chromosome

Summary Two-dimensional gel electrophoresis analysis of a rodent-human somatic cell hybrid containing the X as the only human chromosome reveals three polypeptides that are absent in the parental cell line. The presence of these spots in human female fibroblasts indicates their human origin. The polypeptides have molecular weights and isoelectric points of 30,000/5.8, 37,000/5.4, and 57,000/4.7 and are designated as PFHG 1, PFHG 2, and PFHG 3. Comparison of their molecular characteristics with those of polypeptides assigned to the human X in two other investigations shows that some but not all polypeptides are similar. Factors are discussed that might interfere with the rapid development of a standardized polypeptide map of the human X.     

Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae

Isotope labeling of recombinant proteins is a prerequisite for application of nuclear magnetic resonance spectroscopy (NMR) for the characterization of the three-dimensional structures and dynamics of proteins. Overexpression of isotopically labeled proteins in bacterial or yeast host organisms has several drawbacks. In this work, we tested whether the recently described eukaryotic protein expression system based on the protozoa Leishmania tarentolae could be used for production of amino acid specific (15)N-labeled recombinant proteins. Using synthetic growth medium we were able to express in L. tarentolae and purify to homogeneity (15)N-valine labeled Enchanced Green Fluorescent Protein (EGFP) with the final yield of 5.7 mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identification of all 18 valine residues of the protein in the HSQC spectrum. Our results demonstrate the suitability of the L. tarentolae expression system for production of isotopically labeled proteins.     

Assessment of cytotoxicity, apoptosis and DNA damages in Colo320 colorectal cancer cells selected for oxaliplatin resistance

Despite the notable efficacy of oxaliplatin in the treatment of colorectal cancers, the metastatic tumours ultimately become resistant to the drug. This study investigated whether the oxaliplatin-resistant cells display different behaviour to this drug versus the sensitive cells and if this difference may be further exploited into the clinical treatments improvement. In order to establish a stable cell line resistant to oxaliplatin, a human colorectal cancer cell line (Colo320) was exposed to increasing doses of the drug up to the clinically relevant plasma concentration. Four cell groups with different levels of chemoresistance were subjected to additional doses of oxaliplatin, and their cytotoxicity, apoptosis and DNA damage production were assessed. Cells selected for resistance to oxaliplatin reacted differently to the application of additional doses of the drug, displaying lower toxicity and cellular death and fewer DNA cross-links formation, in accordance with the extent of the oxaliplatin pretreatments. As the cross-links formation by oxaliplatin being the main cause for cytotoxicity of this drug and a correlation between cytotoxicity and clinical outcome being shown repeatedly, we consider that the evaluation of oxaliplatin-induced cytotoxicity, apoptosis and DNA damage could be a valuable tool to assess the tumour cells sensitivity and thus to predict the response to chemotherapy.     

The use of non-dormant rods as planting material: A new approach to establishing willow for environmental applications

In this trial we tested a new planting technique for willows used in environmental applications such as green structures in urban settings. Three commercial willow clones were planted in spring 2008 at different periods as non-dormant woody rods. Comparing the main growth parameters at the end of the season, we found that planting date may affect plant establishment depending on the clone. Based on our results, the use of non-dormant willow material collected and planted in spring is possible in our environment with several different clones. Best results were obtained with Salix viminalis (Sv 5027), which can be planted with a delay of about one month without showing any significant decrease in biomass production or survival rate.     

A novel mutation in the putative DNA helicase XH2 is responsible for male-to-female sex reversal associated with an atypical form of the ATR-X syndrome.

We describe a pedigree presenting X-linked severe mental retardation associated with multiple congenital abnormalities and 46,XY gonadal dysgenesis, leading in one family member to female gender assignment. Female carriers are unaffected. The dysmorphic features are similar to those described in the alpha-thalassemia and mental retardation (ATR-X) syndrome, although there is no clinical evidence of alpha-thalassemia in this family. In addition, the family had other clinical features not previously observed in the ATR-X syndrome, including partial optic-nerve atrophy and partial ocular albinism. Mutations in a putative DNA helicase, termed XH2, have been reported to give rise to the ATR-X syndrome. We screened the XH2 gene for mutations in affected members of the family and identified a 4-bp deletion at an intron/exon boundary that removes an invariant 3' splice-acceptor site. The mutation cosegregates with the syndrome. The genomic deletion causes missplicing of the pre-mRNA, which results in the loss of 8 bp of coding sequence, thereby generating a frameshift and a downstream premature stop codon. Our finding increases the range of clinical features associated with mutations in the XH2 gene.     

Scalable heuristics for design of DNA probe arrays.

Design of DNA arrays for very large-scale immobilized polymer synthesis (VLSIPS) (Fodor et al., 1991) seeks to minimize effects of unintended illumination during mask exposure steps. Hannenhalli et al. (2002) formulate this requirement as the Border Minimization Problem and give an algorithm for placement of probes at array sites under the assumption that the array synthesis is synchronous; i.e., nucleotides are synthesized in a periodic sequence (ACGT)(k) and every probe grows by exactly one nucleotide with every group of four masks. Drawing on the analogy with VLSI placement, in this paper we describe and experimentally validate the engineering of several scalable, high-quality placement heuristics for both synchronous and asynchronous DNA array design. We give empirical results on both randomly generated and industry test cases confirming the scalability and improved solution quality enjoyed by our methods. In general, our techniques improve on state-of-the-art industrial results by over 4% and surpass academically published results by up to 35%. Finally, we give lower bounds that offer insights into the amount of available further improvements.     

Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis

Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 +/- 0.5 g (means +/- SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system.