Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.     

Synthesis and biological evaluation of prodrugs of 2-fluoro-2-deoxyribose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate

We report in this Letter the synthesis of prodrugs of 2-fluoro-2-deoxyarabinose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate. We demonstrate the difficulty of realising a phosphorylation step on the anomeric position of 2-deoxyribose, and we discover that introduction of fluorine atoms on the 2 position of 2-deoxyribose enables the phosphorylation step: in fact, the stability of the prodrugs increases with the degree of 2-fluorination. Stability studies of produgs of 2-fluoro-2-deoxyribose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate in acidic and neutral conditions were conducted to confirm our observation. Biological evaluation of prodrugs of 2,2-difluoro-2-deoxyribose-1-phosphate for antiviral and cytotoxic activity is reported.     

A convenient synthesis of lubeluzole and its enantiomer: Evaluation as chemosensitizing agents on human ovarian adenocarcinoma and lung carcinoma cells

Lubeluzole, a neuroprotective anti-ischemic drug, and its enantiomer were prepared following a convenient procedure based on hydrolytic kinetic resolution. The ee values were >99% and 96%, respectively, as assessed by HPLC analysis. The chemosensitizing effects of both enantiomers were evaluated in combination with either doxorubicin (human ovarian adenocarcinoma A2780 cells) or paclitaxel (human lung carcinoma A549 cells) by the MTT assay. At the lowest concentrations used, lubeluzole showed an overall and remarkable tendency to synergize with both anticancer drugs. In ovarian cancer cells a clear prevalence of antagonistic effect was observed for the R-enantiomer. The synergistic effects of lubeluzole for both drugs were observed over a wide concentration window (0.005–5 μM), the lowest limit being at least 40 times lower than human plasma concentrations previously reported as causing serious side effects.     

Maximising adaptive potential in translocated populations of clonal saltmarsh plants: a case study on Wilsonia backhousei, Convolvulaceae

We investigated the implications of clonality for translocation of Wilsonia backhousei, a threatened, outbreeding, saltmarsh plant with tidally-dispersed fruit. Eight microsatellite loci were used to characterise samples from three estuaries in New South Wales, Australia, and to determine the size and distribution of genetically distinct individuals (genets). Within-population diversity was compared to the presence or absence of seed production using the t test. Ordinal logistic regression was used to investigate the relative influence on seed yield of soil characteristics (soil moisture, salinity, pH) and the number of clonal lineages within a 5 and 10 m radius. Principal coordinate analysis, analysis of molecular variance and Bayesian analysis were used to investigate the extent of gene flow within and among the three estuaries. We found individual genets could cover extensive areas (up to 225 m²) and apparently large populations could consist of only a few individuals. Populations that failed to produce seed had significantly less genetic diversity than populations that produced seed (P = 0.001). Seed yield showed a significant positive response to both increasing soil moisture content (P = 0.003) and increasing genetic diversity in a 5 m radius (P = 0.003). Gene flow was found to occur chiefly within estuaries though occasional longer-distance gene transfer was evident. To maximise adaptive potential in translocated populations of W. backhousei, we recommend sourcing propagules from multiple populations and planting representatives of the different populations in close proximity to facilitate sexual reproduction. These findings are likely to be applicable to other outbreeding clonal saltmarsh plants with tidally-dispersed fruit or seed.     

Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic

Of the 200+ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a non-endemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in non-O1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.     

Inhibition of melanoma development in the Nras(Q61K)::Ink4a−/− mouse model by the small molecule BI‐69A11

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15–20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI‐69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon‐ and cell death‐related genes that were associated with responsiveness of melanoma cell lines to BI‐69A11. Strikingly, the administration of BI‐69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras^(Q61K)::Ink4a^?/?). Biweekly administration of BI‐69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI‐69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI‐69A11‐resistant Nras^(Q61K)::Ink4a^?/? tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI‐69A11 for clinical assessment.     

Automated,High Accuracy Classification of Parkinsonian Disorders: A Pattern Recognition Approach

Progressive supranuclear palsy (PSP), multiple system atrophy (MSA) and idiopathic Parkinson’s disease (IPD) can be clinically indistinguishable, especially in the early stages, despite distinct patterns of molecular pathology. Structural neuroimaging holds promise for providing objective biomarkers for discriminating these diseases at the single subject level but all studies to date have reported incomplete separation of disease groups. In this study, we employed multi-class pattern recognition to assess the value of anatomical patterns derived from a widely available structural neuroimaging sequence for automated classification of these disorders. To achieve this, 17 patients with PSP, 14 with IPD and 19 with MSA were scanned using structural MRI along with 19 healthy controls (HCs). An advanced probabilistic pattern recognition approach was employed to evaluate the diagnostic value of several pre-defined anatomical patterns for discriminating the disorders, including: (i) a subcortical motor network; (ii) each of its component regions and (iii) the whole brain. All disease groups could be discriminated simultaneously with high accuracy using the subcortical motor network. The region providing the most accurate predictions overall was the midbrain/brainstem, which discriminated all disease groups from one another and from HCs. The subcortical network also produced more accurate predictions than the whole brain and all of its constituent regions. PSP was accurately predicted from the midbrain/brainstem, cerebellum and all basal ganglia compartments; MSA from the midbrain/brainstem and cerebellum and IPD from the midbrain/brainstem only. This study demonstrates that automated analysis of structural MRI can accurately predict diagnosis in individual patients with Parkinsonian disorders, and identifies distinct patterns of regional atrophy particularly useful for this process.     

Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

Background

Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.

Methods

RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.

Results

Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.

Conclusions

Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.