Measurement of Horizontal and Vertical Movement of Ralstonia solanacearum in Soil

Two model systems were constructed to measure horizontal and vertical movement of bacteria in soil. These systems were applied to measuring movement of Ralstonia solanacearum (race 1, biovar 3), a causal agent of bacterial wilt of tomato, in andosol and sand at 28°C. The first system was used to measure horizontal movement of the bacteria in soil packed in a narrow horizontal frame. Suspension of the pathogen was applied to soil at one end of the frame, and bacterial number per gram of soil was measured over distance from the inoculation point after 4 days. Horizontal movement of R. solanacearum in supersaturated soil, but without flow, was possibly due to diffusion and the front advanced at 2.2 cm/day in andosol, and at 8.1 cm/day in sand. Using the same experimental system, but applying water inflow to one end of the frame only, the bacterium was detected at the front of water in andosol and sand. The front of the distribution advanced at 20.4 cm/h in andosol and 66.3 cm/h in sand. In the second experimental system, a cylinder of soil packed in a short tube was soaked with water, and soil at the top of the tube was inoculated with bacterial suspension. Immediately, soil cylinders were turned upward, and the bacterial number per gram of soil was measured along vertical distance from the inoculation point after 7 days. Using the system with andosol, the capillary water front rose to 32.5 cm over 7 days after inoculation, and R. solanacearum reached to 18.8 cm height. In sand, capillary water rose to 20.0 cm and the bacteria reached to 16.3 cm height.     

Isolation of variant carrot cell lines with altered pigmentation

Cultured carrot cells were treated with a known mutagenic compound, N-methyl-N′-nitro-N-nitroso-guanidine, and plated on a nutrient agar medium. Four variant cell lines whose pigmentation properties differed from stock calluses have been isolated. The contents of major carotenoid components, β-carotene and lycopene, of these cells were determined and compared with those of parent strains.     

Polymorphisms in the SOD2 and HLA-DRB1 genes are associated with nonfamilial idiopathic dilated cardiomyopathy in Japanese.

To reveal genetic risk factors of nonfamilial idiopathic cardiomyopathy (IDC) in Japanese, polymorphisms in the SOD2 and HLA-DRB1 genes were investigated in 86 patients and 380 healthy controls. There was a significant excess of homozygotes for the V allele [Val versus Ala (A allele), a polymorphism in the leader peptide of manganese superoxide dismutase at position 16] of the SOD2 gene in the patients compared with the controls (87.2% versus 74.7%, odds ratio = 2.30, p = 0.013, pc < 0.03), and a significant increase in the frequency of HLA-DRB1*1401 in the patients was confirmed (14.0% vs 4.5%, odds ratio = 3.46, p = 0.001, pc < 0.03). A two-locus analysis suggested that these two genetic markers (SOD2-VV genotype and DRB1*1401) may play a synergistic role in controlling the susceptibility to nonfamilial IDC. In addition, processing efficiency of Val-type SOD2 leader peptide in the presence of mitochondria was siginificantly lower than that of the Ala-type by 11 +/- 4%, suggesting that this lower processing efficiency was in part an underlying mechanism of the association between the SOD2-VV genotype and nonfamilial IDC.     

Differential effects of p63 mutants on transactivation of p53 and/or p63 responsive genes

p63, known to play a role in development, has more recently also been implicated in cancer progression. Mutations in p63 have been shown to be responsible for several human developmental diseases. Differential splicing of the p63 gene gives rise to p63 isoforms, which can act either as tumor suppressors or as oncogene. In this report, we studied the effects of naturally occurring TAp637 mutants on the regulation of p53/p63 and p63 specific target genes. We observed significant differences among p63 mutants to regulate the p53/p63 and p63 specific target genes. Additionally, we observed a differential effect of p63 mutants on wildtype-p63-mediated induction ofp53/p63 and p63 specific target genes. We also demonstrated that these mutants differentially regulate the binding of wildtype p63 to the promoter of target genes. Furthermore, the effects of these mutants on cell death and survival were consistent with their ability to regulate the downstream targets when compared to wildtype TAp63T. In summary, our data demonstrate that p63 mutants exhibit differential effects on p63 and p53/p63 specific target genes and on the induction of apoptosis, and provide further insight into the function of p63.     

Phenolic and iridoid glycosides from Strychnos axillaris

Five phenolic glycosides 1-5 and an iridoid glucoside 6 were isolated, together with 22 known compounds, from the dried barks and woods of Strychnosaxillaris. Their structures were determined by application of spectroscopic (NMR, MS) and chemical methodologies.     

Synthesis,SAR, and X-ray structure of tricyclic compounds as potent FBPase inhibitors

With the aim of discovering a novel class of fructose-1,6-bisphosphatase (FBPase) inhibitors, a series of compounds based on tricyclic scaffolds was synthesized. Extensive SAR studies led to the finding of 8l with an IC₅₀ value of 0.013 μM against human FBPase. An X-ray crystallographic study revealed that 8l bound at AMP binding sites of human liver FBPase with hydrogen bonding interactions similar to AMP.     

MG53 Regulates Membrane Budding and Exocytosis in Muscle Cells

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.When myoblasts exit the cell cycle during myogenesis, dramatic changes in membrane organization occur as myoblast fusion allows the formation of multinucleated muscle fibers. In addition to cell fusion events, differentiation of myotubes involves establishment of specialized membrane structures (1, 2). The transverse tubular invagination of sarcolemmal membrane and the intracellular membrane network known as the sarcoplasmic reticulum are two highly organized membrane architectures in cardiac and skeletal muscle. Establishment of these intricate membrane compartments requires extensive remodeling of the immature myoblast membranes. Dynamic membrane remodeling also contributes to many physiologic processes in mature muscle, including Ca²⁺ signaling, trafficking of glucose transporter (GLUT4), and other membrane internalization events involving caveolae structures (3-6). Although defects in membrane integrity have been linked to various forms of muscular dystrophy (7, 8), the molecular machinery regulating these specific membrane recycling and remodeling events in striated muscle is not well defined.The large tripartite motif (TRIM)⁵ family of proteins is involved in numerous cellular functions in a wide variety of cell types. Members of this protein family contain signature motifs that include a RING finger, a zinc binding moiety (B-box), and a coiled coil structure (RBCC), which invariably comprise the amino-terminal domain of TRIM family members (9). The carboxyl-terminal sequence of TRIM proteins is variable; in some cases a subfamily of TRIM proteins contains a SPRY domain, a sequence first observed in the ryanodine receptor Ca²⁺ channel in the sarcoplasmic reticulum membrane of excitable cells (10). Extensive studies have revealed that protein-protein interactions in the cytosol mediate the defined functions of TRIM proteins. For example, the ubiquitin E3 ligase enzymatic activity of several TRIM family members requires the B-box motif (11, 12). Recent studies have also indicated a role for TRIM proteins in defense against events involving membrane penetration, such as protection against infection by various viruses, including human immunodeficiency virus (13-15). Although most of the studies concentrate on the cytosolic action of TRIM, limited reports have investigated the role of TRIM proteins in membrane signaling or recycling.We have previously established an immunoproteomics approach that allows definition of novel components involved in myogenesis, Ca²⁺ signaling, and maintenance of membrane integrity in striated muscle (16). Using this approach, we have shown that junctophilin is a structural protein that establishes functional communication between sarcoplasmic reticulum and transverse tubule membranes at triad and dyad junctions in striated muscle (17-19). Further studies identified mitsugumin 29, a synaptophysin-related protein that is essential for biogenesis of triad membrane structures and Ca²⁺ signaling in skeletal muscle (20, 21). Screening of this immunoproteomics library led to the recent identification of MG53, a muscle-specific TRIM family protein (22). Domain homology analysis revealed that MG53 contains the prototypical RBCC motifs plus a SPRY domain at the carboxyl terminus. Genetic knock-out and functional studies reveal that MG53 nucleates the assembly of the sarcolemmal membrane repair machinery to restore cellular integrity following acute damage to the muscle fiber (22).Here we present evidence illustrating that MG53, in contrast to other known TRIM proteins, can localize to intracellular vesicles and the sarcolemmal membrane. A functional interaction between MG53 and caveolin-3, another muscle-specific protein, plays an essential role in regulating the dynamic process of membrane budding and exocytosis in skeletal muscle.