Control of AMP deaminase 1binding to myosin heavy chain

AMP deaminase (AMPD) plays a central role in preserving theadenylate energy charge in myocytes following exercise and in producingintermediates for the citric acid cycle in muscle. Prior studies havedemonstrated that AMPD1 binds to myosin heavy chain (MHC)in vitro; binding to the myofibril varies with the state of musclecontraction in vivo, and binding of AMPD1 to MHC is required foractivation of this enzyme in myocytes. The present study has identifiedthree domains in AMPD1 that influence binding of this enzyme to MHCusing a cotransfection model that permits assessment of mutationsintroduced into the AMPD1 peptide. One domain that encompasses residues178-333 of this 727-amino acid peptide is essential for binding ofAMPD1 to MHC. This region of AMPD1 shares sequence similarity withseveral regions of titin, another MHC binding protein. Two additionaldomains regulate binding of this peptide to MHC in response tointracellular and extracellular signals. A nucleotide binding site,which is located at residues 660-674, controls binding of AMPD1 toMHC in response to changes in intracellular ATP concentration. Deletionanalyses demonstrate that the amino-terminal 65 residues of AMPD1 playa critical role in modulating the sensitivity to ATP-induced inhibitionof MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8-12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP.These findings are discussed in the context of the various rolesproposed for AMPD in energy production in the myocyte.

     

Identification of novel mRNAs expressed in oligodendrocytes

To identify new proteins, which are expressed in oligodendrocytes and which may have a functional role in myelination, a rat oligodendrocyte cDNA library was screened using differential and subtractive screening techniques. Ten clones that have elevated levels of expression in brain were isolated. Two of these clones were characterized further and one clone, pC26.H2, was found to be closely related to mouse stearoyl-CoA desaturase 2 (SCD2), which catalyzes the synthesis of unsaturated fatty acid. From Northern blot and in situ hybridization studies, SCD2 mRNA was expressed primarily in brain with lower levels found in lung and spleen. In brain sections, SCD2 mRNA was found primarily in oligodendrocytes, although mRNA was detected at a low level in neurons, in particular in Purkinje cells in the cerebellum. Northern blot analysis of the other clone, p973.HB, indicated that it was expressed more selectively in brain. In mixed glial cultures oligodendrocytes were the only cells that expressed this mRNA, whereas in brain, neurons expressed this mRNA at a higher level than in oligodendrocytes. This clone is being actively pursued because of its unique expression exclusively in oligodendrocytes and neurons.Special issue dedicated to Dr. Marjorie B. Lees.     

Quantitative footprinting analysis

This review outlines the steps for obtaining relative constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA sites. Slopes of plots outside the drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netrospin to a 139-base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netrospin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.     

Construction of YAC Contigs at Human Chromosome 11q22.3-q23.1 Region Covering the Ataxia Telangiectasia Locus

Ataxia telangiectasia (AT) is an autosomal recessive diseaseof unknown etiology associated with cerebellar ataxia, telangiectasia,immune dysfunction, higher cancer risk, genomic instabilityand hypersensitivity to ionizing radiation. The major AT loci,AT-A and AT-C, are shown to be closely linked at chromosome11q22–q23. The most recent genetic linkage mapping andlinkage disequilibrium analysis have localized the major ATloci to a sequence of approximately 850 kb between the markersD11S1819 and D11S1818. The isolation of yeast artificial chromosomesspanning the AT region is an essential step to identify thegene or genes responsible for the mutation(s). We isolated atotal of 20 YAC clones from three independent YAC libraries,using sequence tagged sites mapped in the AT region as primersfor PCR-based YAC screening. The PCR assay for the presenceor absence of 16 different DNA markers allowed us to constructand to order four YAC contigs at the AT region. One of the contigswhich consists of the 10 YAC clones, covers about 2 Mb of DNAat the boundary between Giemsa-positive band 11q22.3 and Giemsa-negativeband 11q23.1 and includes the entire region of the major ATlocus between D11S1819 and D11S1818. Thus, the YAC contigs willfacilitate the positional cloning approach for searching transcribedsequences from the defined genomic region.     

Increased level of serum vascular endothelial growth factor by long-term exposure to hypergravity.

We have previously demonstrated that short-term exposure to hypergravity at 2G for 4 h induces expression of cyclooxygenase-2 (COX-2) in the mouse heart. Moreover, expression of vascular endothelial growth factor (VEGF) is also induced in the heart in a COX-2-dependent manner. Here, we demonstrate that long-term exposure of mice to 2G for 24 h resulted in a significant increase of serum VEGF level, although expression of COX-2 and VEGF in the heart decreased to the 1G-control level. Moreover, increase of serum VEGF was not suppressed by treatment with COX-2 inhibitor, indicating that VEGF was induced in a COX-2-independent manner. These results suggest that gravitational force contributes to maintenance of the serum VEGF level.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

PROTON GRADIENT REGULATION 5 contributes to ferredoxin-dependent cyclic phosphorylation in ruptured chloroplasts

Antimycin A-sensitive cyclic electron flow (CEF) was discovered as cyclic phosphorylation by Arnon et al. (1954). Because of its sensitivity to antimycin A, PROTON GRADIENT REGULATION 5 (PGR5)/PGR5-like Photosynthetic Phenotype 1 (PGRL1)-dependent CEF has been considered identical to the CEF of Arnon et al. However, this conclusion still needs additional supportive evidence, mainly because of the absence of definitive methods of evaluating CEF activity. In this study, we revisited the classical method of monitoring cyclic phosphorylation in ruptured chloroplasts to characterize two Arabidopsis mutants: pgr5, which is defective in antimycin A-sensitive CEF, and chlororespiratory reduction 2-1 (crr2-1), which is defective in chloroplast NDH-dependent CEF. We observed a significant reduction in CEF-dependent pmf formation and consequently ATP synthesis in the pgr5 mutant, although LEF-dependent pmf formation and ATP synthesis were not impaired at photosynthetic photon flux densities below 130?μmol?m^?2?s^?1. In contrast, the contribution of chloroplast NDH complex to pmf formation and ATP synthesis was not significant. Antimycin A partially inhibited CEF-dependent pmf formation, although there may be further inhibition sites. Unlike in the observation in leaves, the proton conductivity of ATP synthase, monitored as g_H⁺, was not enhanced in ruptured chloroplasts of the pgr5 mutant.     

Synthesis of 1,5-Anhydro-d-fructose derivatives and evaluation of their inflammasome inhibitors

Synthesis of several 1,5-Anhydro-d-fructose (1,5-AF) derivatives to evaluate inhibitory activities of the inflammasome was carried out. Recently, 1,5-AF reported to suppress the inflammasome, although with only low activity. We focused on the hydration of 2-keto form of 1,5-AF and speculated that this hydration was the cause of low activity. Therefore, we synthesized some 1,5-AF derivatives that would not be able to form the dimer conformation and can be expected to have high activity against inflammasome, and then evaluated their inhibitory activities with respect to the NLRP3 inflammasome by using mouse bone marrow-derived macrophages and human THP-1 cells. As a result, some synthesized 2-keto form compounds had much higher inhibitory activities with respect to the NLRP3 inflammasome than did 1,5-AF.     

Unconventional Myosin ID is Involved in Remyelination After Cuprizone-Induced Demyelination

Myelin, which is a multilamellar structure that sheathes the axon, is essential for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes (OLs), which wrap their plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we reported that myosin ID (Myo1d) is distributed in rat CNS myelin and is especially enriched in the outer and inner cytoplasm-containing loops. Further, small interfering RNA (siRNA) treatment highlighted the involvement of Myo1d in the formation and maintenance of myelin in cultured OLs. Myo1d is one of the unconventional myosins, which may contribute to membrane dynamics, either in the wrapping process or transport of myelin membrane proteins during myelination. However, the function of Myo1d in myelin formation in vivo remains unclear. In the current study, to clarify the function of Myo1d in vivo, we surgically injected siRNA in the corpus callosum of a cuprizone-treated demyelination mouse model via stereotaxy. Knockdown of Myo1d expression in vivo decreased the intensities of myelin basic protein and myelin proteolipid protein immunofluorescence staining. However, neural/glial antigen 2-positive signals and adenomatous polyposis coli (APC/CC1)-positive cell numbers were unchanged by siRNA treatment. Furthermore, Myo1d knockdown treatment increased pro-inflammatory microglia and astrocytes during remyelination. In contrast, anti-inflammatory microglia were decreased. The percentage of caspase 3-positive cells in total CC1-positive OLs were also increased by Myo1d knockdown. These results indicated that Myo1d plays an important role during the regeneration process after demyelination.