Diffusion Tensor Imaging of Brain Abnormalities Induced by Prenatal Exposure to Radiation in Rodents

We assessed brain abnormalities in rats exposed prenatally to radiation (X-rays) using magnetic resonance imaging (MRI) and histological experiments. Pregnant rats were divided into 4 groups: the control group (n = 3) and 3 groups that were exposed to different radiation doses (0.5, 1.0, or 1.5 Gy; n = 3 each). Brain abnormalities were assessed in 32 neonatal male rats (8 per group). Ex vivo T₂-weighted imaging and diffusion tensor imaging (DTI) were performed using 11.7-T MRI. The expression of markers of myelin production (Kluver–Barrera staining, KB), nonpyramidal cells (calbindin-D28k staining, CaBP), and pyramidal cells (staining of the nonphosphorylated heavy-chain neurofilament SMI-32) were histologically evaluated. Decreased brain volume, increased ventricle volume, and thinner cortices were observed by MRI in irradiated rats. However, no abnormalities in the cortical 6-layered structure were observed via KB staining in radiation-exposed rats. The DTI color-coded map revealed a dose-dependent reduction in the anisotropic signal (vertical direction), which did not represent reduced numbers of pyramidal cells; rather, it indicated a signal reduction relative to the vertical direction because of low nerve cell density in the entire cortex. We conclude that DTI and histological experiments are useful tools for assessing cortical and hippocampal abnormalities after prenatal exposure to radiation in rats.     

Diabetic Osteopenia by Decreased β-Catenin Signaling Is Partly Induced by Epigenetic Derepression of sFRP-4 Gene

In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These in vitro data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression.     

Structural Insights into the Epimerization of β-1,4-Linked Oligosaccharides Catalyzed by Cellobiose 2-Epimerase,the Sole Enzyme Epimerizing Non-anomeric Hydroxyl Groups of Unmodified Sugars

Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of β1,4-mannobiose to 4-O-β-d-mannosyl-d-glucose in mannan metabolism. However, the detailed catalytic mechanism of CE is unclear due to the lack of structural data in complex with ligands. We determined the crystal structures of halothermophile Rhodothermus marinus CE (RmCE) in complex with substrates/products or intermediate analogs, and its apo form. The structures in complex with the substrates/products indicated that the residues in the β5-β6 loop as well as those in the inner six helices form the catalytic site. Trp-322 and Trp-385 interact with reducing and non-reducing end parts of these ligands, respectively, by stacking interactions. The architecture of the catalytic site also provided insights into the mechanism of reversible epimerization. His-259 abstracts the H2 proton of the d-mannose residue at the reducing end, and consistently forms the cis-enediol intermediate by facilitated depolarization of the 2-OH group mediated by hydrogen bonding interaction with His-200. His-390 subsequently donates the proton to the C2 atom of the intermediate to form a d-glucose residue. The reverse reaction is mediated by these three histidines with the inverse roles of acid/base catalysts. The conformation of cellobiitol demonstrated that the deprotonation/reprotonation step is coupled with rotation of the C2-C3 bond of the open form of the ligand. Moreover, it is postulated that His-390 is closely related to ring opening/closure by transferring a proton between the O5 and O1 atoms of the ligand.     

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.     

Metabolic Profiling of Alternative NAD Biosynthetic Routes in Mouse Tissues

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the “amidated” and “deamidated” routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes''rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme''s substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.     

Component manufacturing analysis

To date, numerous simplified Life Cycle Assessment methods and techniques have been developed to reduce complexities associated with practical application. However, these methods often identify critical elements according to subjective considerations. In this paper, we develop and apply a new type of Life Cycle Inventory method — Component Manufacturing Analysis (CMA) — that is easy to implement and less arbitrary. Application of CMA requires identification of all product components and their associated weights, which are then entered into a factory-type database. Because the factory database has a rigorous yet generic structure and because calculation is done automatically, the application of CMA tends to be less arbitrary and more complete than other simplified methods. Results of a case study on beverage vending machines show that the manufacturing stage is a significant phase in the whole life-cycle inventory of a product. We conclude that CMA shows promise for further development and future application.     

Mechanism of aluminium tolerance in Cyanidium caldarium

Cyanidium caldarium, an acidophilic, thermophilic red alga, specifically tolerates Al. The tolerance increases at lower culture temperatures. The intracellular Al concentration is kept at low levels, especially when the cells are cultured at lower temperatures. Lower Al incorporation accounts for the Al tolerance in this alga. Fe incorporation antagonizes the Al incorporation, implying that Fe transporters incorporate Al ions. Treatment with an uncoupler, carbonylcyanide m-chlorophenylhydrazone, increases the intracellular concentration of Al. These results support the hypothesis that Al ions taken up by the algal cells are exported by an energy-dependent mechanism.