Epigenetic inactivation of the miR-124-1 in haematological malignancies

miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2'-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (p<0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.     

An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.     

Iron metabolism in diabetes-induced Alzheimer’s disease: a focus on insulin resistance in the brain

Alzheimer’s disease (AD) is characterized by an excessive accumulation of toxic amyloid beta (Aβ) plaques and memory dysfunction. The onset of AD is influenced by age, genetic background, and impaired glucose metabolism in the brain. Several studies have demonstrated that diabetes involving insulin resistance and glucose tolerance could lead to AD, ultimately resulting in cognitive dysfunction. Even though the relationship between diabetes and AD was indicated by significant evidences, the critical mechanisms and metabolic alterations in diabetes induced AD are not clear until now. Recently, iron metabolism has been shown to play multiple roles in the central nervous system (CNS). Iron deficiency and overload are associated with neurodegenerative diseases. Iron binds to Aβ and subsequently regulates Aβ toxicity in the CNS. In addition, previous studies have shown that iron is involved in the aggravation of insulin resistance. Considering these effects of iron metabolism in CNS, we expect that iron metabolism may play crucial roles in diabetic AD brain. Thus, we review the recent evidence regarding the relationship between diabetes-induced AD and iron metabolism.     

Temperature range of thermodynamic stability for the native state of reversible two-state proteins

The difference between the heat (T(G)) and the cold (T(G)') denaturation temperatures defines the temperature range (T(Range)) over which the native state of a reversible two-state protein is thermodynamically stable. We have performed a correlation analysis for thermodynamic parameters in a selected data set of structurally nonhomologous single-domain reversible two-state proteins. We find that the temperature range is negatively correlated with the protein size and with the heat capacity change (DeltaC(p)) but is positively correlated with the maximal protein stability [DeltaG(T(S))]. The correlation between the temperature range and maximal protein stability becomes highly significant upon normalization of the maximal protein stability with protein size. The melting temperature (T(G)) also shows a negative correlation with protein size. Consistently, T(G) and T(G)' show opposite correlations with DeltaC(p), indicating a dependence of the T(Range) on the curvature of the protein stability curve. Substitution of proteins in our data set with their homologues and arbitrary addition or removal of a protein in the data set do not affect the outcome of our analysis. Simulations of the thermodynamic data further indicate that T(Range) is more sensitive to variations in curvature than to the slope of the protein stability curve. The hydrophobic effect in single domains is the principal reason for these observations. Our results imply that larger proteins may be stable over narrower temperature ranges and that smaller proteins may have higher melting temperatures, suggesting why protein structures often differentiate into multiple substructures with different hydrophobic cores. Our results have interesting implications for protein thermostability.     

Gramicidin A channel as a test ground for molecular dynamics force fields

We use the well-known structural and functional properties of the gramicidin A channel to test the appropriateness of force fields commonly used in molecular dynamics (MD) simulations of ion channels. For this purpose, the high-resolution structure of the gramicidin A dimer is embedded in a dimyristoylphosphatidylcholine bilayer, and the potential of mean force of a K(+) ion is calculated along the channel axis using the umbrella sampling method. Calculations are performed using two of the most common force fields in MD simulations: CHARMM and GROMACS. Both force fields lead to large central barriers for K(+) ion permeation, that are substantially higher than those deduced from the physiological data by inverse methods. In long MD simulations lasting over 60 ns, several ions are observed to enter the binding site but none of them crossed the channel despite the presence of a large driving field. The present results, taken together with many earlier studies, highlights the shortcomings of the standard force fields used in MD simulations of ion channels and calls for construction of more appropriate force fields for this purpose.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Quinic acid derivatives from Saussurea triangulata attenuates glutamate-induced neurotoxicity in primary cultured rat cortical cells

The leaves of Saussurea triangulata (Compositae) have been eaten with rice as a wrapping vegetable for preventing neuro-aging. However, the components responsible for the neuroprotective effects of S. triangulata still remain unidentified. In the process of investigating the neuroprotective activity of S. triangulata, we found that a methanol extract of S. triangulata exhibited significant protection against glutamate-induced toxicity in primary cultured rat cortical cells. Three quinic acid derivatives were isolated from the n-BuOH fraction of S. triangulata. Among these three quinic acid derivatives, methyl 5-caffeoylquinic acid (3) exhibited significant neuroprotective activities against glutamate-induced toxicity exhibiting cell viability of about 50%, at concentrations ranging from 0.1 microM to 10 microM. Therefore, the neuroprotective effect of S. triangulata might be due to the inhibition of glutamate-induced toxicity by the quinic acid derivatives from S. triangulata.