Enhancement of growth and cellulose accumulation by overexpression of xyloglucanase in poplar

Because the loosening of xyloglucan in the cell wall promotes plant growth (Takeda et al. (2002) Proc. Natl. Acad. Sci. USA 99, 9055-9060; Park et al. (2003) Plant J. 33, 1099-1106), we expressed Aspergillus xyloglucanase constitutively in Populus alba. The expression increased the length of stem even in the presence of sucrose. Increased stem growth was accompanied by a decrease in Young's elastic modulus in the growth zone but an increased elasticity in mature tissue. The increased internode length corresponded to an increase in cellulose content as well as specific gravity, showing that the removal of xyloglucan might cause an increase in cellulose density in the secondary xylem.     

Ceramide in lipid particles enhances heparan sulfate proteoglycan and low density lipoprotein receptor-related protein-mediated uptake by macrophages

Arterial wall sphingomyelinase (SMase) has been proposed to be involved in atherogenesis. SMase modification of lipoproteins has been shown to occur in atherosclerotic lesions and to facilitate their uptake by macrophages and foam cell formation. To investigate the mechanism of macrophage uptake enhanced by SMase, we prepared lipid emulsions containing sphingomyelin (SM) or ceramide (CER) as model particles of lipoproteins. SMase remarkably increased the uptake of SM-containing emulsions by J774 macrophages without apolipoproteins. The emulsion uptake was negatively correlated with the degree of particle aggregation by pretreatment with SMase, whereas the uptake of CER-containing emulsions was significantly larger than SM-containing emulsions, indicating that enhancement of uptake is due to the generation of CER molecules in particles but not to the aggregation by SMase. Heparan sulfate proteoglycans (HSPGs) and low density lipoprotein receptor-related protein (LRP) were crucial for CER-enhanced emulsion uptake, because heparin or lactoferrin inhibited the emulsion uptake. Confocal microscopy also showed that SMase promoted both binding and internalization of emulsions by J774 macrophages, which were almost abolished by lactoferrin. Apolipoprotein E further increased the uptake of CER-containing emulsions compared with SM-containing emulsions. These findings suggest the generation of CER in lipoproteins by SMase facilitates the macrophage uptake via HSPG and LRP pathways and plays a crucial role in foam cell formation. Thus, CER may act as an important atherogenic molecule.     

Activation of lignin peroxidase in organic media by reversed micelles

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.     

Biochemical and genetic characterization of mundticin KS,an antilisterial peptide produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells

Summary Activities of three lysosomal enzymes—acid RNase,N-acetyl-β-D-glucosaminidase and acid phosphatase—were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to five-fold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown, also increased as cultured approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH₄Cl, but the rate of turnover in the presence of NH₄Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops. This study was supported by Grant GM-21357 from the National Institutes of Health.     

Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart

Naked plasmid DNA (pDNA) and short interfering RNA (siRNA) duplexes were transduced into adult murine heart by means of sonoporation using the third-generation microbubble, BR14. Plasmid DNAs carrying luciferase, beta-galactosidase (beta-gal), or enhanced green fluorescent protein (EGFP) reporter genes were mixed with BR14 and injected percutaneously into the left ventricular (LV) cavity of C57BL/6 mice while exposed to transthoracic ultrasound at 1MHz for 60s. Sonoporation at an output intensity of 2.0W/cm(2) and a 50% pulse duty ratio resulted in the highest luciferase expression in the heart. Histological examinations revealed significant expression of the beta-gal and EGFP reporters in the subendocardial myocardium of LV. Intraventricular co-injection of siRNA-GFP and BR14 with concomitant ultrasonic exposure resulted in substantial reduction in EGFP expression in the coronary artery in EGFP transgenic mice. The present method may be applicable to gain-of-function and loss-of-function genetic engineering in vivo of adult murine heart.     

Systematic screening for genes specifically expressed in the anterior neuroectoderm during early Xenopus development

A cDNA library derived from the anterior neuroectoderm (ANE) of Xenopus late-gastrula embryos was systematically screened to isolate novel developmental regulatory genes involved in early brain development. We isolated 1,706 5 expressed sequence tags (ESTs), which were subdivided into 1,383 clusters and categorized into 19 classes based on predicted functions according to their similarities to other known genes. Of these, 757 clusters that were considered possible novel regulatory genes or unknown genes were subjected to expression pattern analysis using whole-mount in situ hybridization. Genes from 69 clusters (9%) were expressed in the ANE region. Based on their expression patterns and predicted amino acid sequences, 25 genes were selected for further analysis as novel Xenopus genes expressed broadly or region-specifically in the ANE. Eighteen genes were expressed in postulated patterning centers in the neuroectoderm, including the anterior (four genes) and lateral (nine genes) neural ridges, the midbrain-hindbrain boundary region (one gene) and the midline region of the neural plate (two genes), whereas 13 genes were expressed in the eye anlagen. Therefore, early regionalization of the neuroectoderm appears to occur mainly in those neural patterning centers and the eye anlagen. We determined the entire coding regions of p54nrb, Semaphorin 6D and a novel gene designated scribble-related protein 1 (SCRP1). Interestingly, Semaphorin 6D is expressed in the mesoderm with a dorsoventral gradient, as well as in the ectoderm at the gastrula stage, implying a new role for this protein in development other than in axon guidance.     

A phytochemical in the edible Tamogi-take mushroom (Pleurotus cornucopiae), D-mannitol, inhibits ACE activity and lowers the blood pressure of spontaneously hypertensive rats

D-Mannitol, one of the main phytochemicals of the edible Tamogi-take mushroom (Pleurotus cornucopiae), was found to inhibit an angiotensin I converting enzyme (ACE). The antihypertensive effect of D-mannitol and a hot water extract of Tamogi-take mushroom was demonstrated in spontaneously hypertensive rats (SHR) by oral administration.     

The orientation of nucleus, nucleus-associated body and protruding nucleolus in aggregating Dictyostelium discoideum

Dictyostelium discoideum growing or developing on cellulose dialysis membranes were fixed with acrolein vapour for electron microscopy. In interphase amoebae, nucleoli began to protrude from the nuclei. The percentage of cells with protruding nucleoli increased during aggregation by a value approximately twice as high in aggregation streams as in centers. Cells in pseudoplasmodia showed only a low percentage and protrusions disappeared at early culmination stage. The protrusions did not reappear when cells from dissociated pseudoplasmodia migrated toward cAMP. Thus the formation of the protrusions did not depend solely on chemotaxis; rather, it was specific to the aggregation stage. In aggregation streams, the nucleus was anterior in the cell, with the protrusion at its anterior periphery. In contrast, the nucleus associated body (NAB) was evident at the cell's mid-point. This orientation of nucleus and NAB in the aggregating slime mould amoeba is contrary to that seen in human neutrophils or cultured mouse 3T3 cells.     

Microdetermination of unbound tryptophan in plasma by a combination of ultrafiltration and high-performance liquid chromatography

Previous methods for measuring unbound plasma tryptophan are not completely satisfactory, and therefore we have developed an improved method for this purpose. Unbound tryptophan is separated from bound tryptophan by centrifugation through Amicon ultrafiltration membrane cones in a short period (within 2 min). The first fraction of filtrate is obtained in 30 s by centrifugation at 3000g at controlled pH and is assayed with a high-performance liquid chromatography system. The first filtrate has a higher tryptophan concentration than that obtained by prolonged centrifugation. We propose that the tryptophan concentration in the first filtrate is the value nearest that of the plasma, since a change of equilibrium between bound and unbound tryptophan during the separation procedure is quite small. The method is also simple and convenient for clinical application.