Phospholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipid production

This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries.     

Prolonged blockade of VEGF receptors does not damage retinal photoreceptors or ganglion cells

It has recently been reported that relatively short‐term inhibition of vascular endothelial growth factor (VEGF) signaling can cause photoreceptor cell death, a potentially clinically important finding since VEGF blockade has become an important modality of treatment of ocular neovascularization and macular edema. However, in a set of studies in which we achieved extended and complete blockage of VEGF‐induced vascular leakage through retinal expression of a VEGF binding protein, we did not observe any toxicity to retinal neurons. To follow‐up on these apparently discrepant findings, we designed a set of experiments with the kinase inhibitor SU4312, which blocks phosphorylation of VEGF receptors, to look directly for evidence of VEGF inhibition‐related retinal toxicity. Using transgenic mice with sustained expression of VEGF in photoreceptors, we determined that periocular injection of 3 µg of SU4312 every 5 days markedly suppressed subretinal neovascularization, indicating effective blockade of VEGF signaling. Wild‐type mice given periocular injections of 5 µg of SU4312 every 5 days for up to 12 weeks showed normal scotopic and photopic electroretinograms (ERGs), no TUNEL stained cells in the retina, and no reduction in outer nuclear layer thickness. Incubation of cultured ganglion cells or retinal cultures containing photoreceptors with high doses of SU4312 did not reduce cell viability. These data suggest that blocking VEGF signaling in the retina for up to 12 weeks does not damage photoreceptors nor alter ERG function and should reassure patients who are receiving frequent injections of VEGF antagonists for choroidal and retinal vascular diseases. J. Cell. Physiol. 224:262–272, 2010 © 2010 Wiley‐Liss, Inc.     

A GC polymorphism associated with serum 25-hydroxyvitamin D level is a risk factor for hip fracture in Japanese patients with rheumatoid arthritis: 10-year follow-up of the Institute of Rheumatology,Rheumatoid Arthritis cohort study

Introduction

Vitamin D deficiency has been reported to be common in patients with rheumatoid arthritis (RA) who have a higher prevalence of osteoporosis and hip fracture than healthy individuals. Genetic variants affecting serum 25-hydroxyvitamin D (25(OH)D) concentration, an indicator of vitamin D status, were recently identified by genome-wide association studies of Caucasian populations. The purpose of this study was to validate the association and to test whether the serum 25(OH)D-linked genetic variants were associated with the occurrence of hip fracture in Japanese RA patients.

Methods

DNA samples of 1,957 Japanese RA patients were obtained from the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort DNA collection. First, five single nucleotide polymorphisms (SNPs) that were reported to be associated with serum 25(OH)D concentration by genome-wide association studies were genotyped. The SNPs that showed a significant association with serum 25(OH)D level in the cross-sectional study were used in the longitudinal analysis of hip fracture risk. The genetic risk for hip fracture was determined by a multivariate Cox proportional hazards model in 1,957 patients with a maximum follow-up of 10 years (median, 8 years).

Results

Multivariate linear regression analyses showed that rs2282679 in GC (the gene encoding group-specific component (vitamin D binding protein)) locus was significantly associated with lower serum 25(OH)D concentration (P = 8.1 × 10^-5). A Cox proportional hazards model indicated that rs2282679 in GC was significantly associated with the occurrence of hip fracture in a recessive model (hazard ratio (95% confidence interval) = 2.52 (1.05-6.05), P = 0.039).

Conclusions

A two-staged analysis demonstrated that rs2282679 in GC was associated with serum 25(OH)D concentration and could be a risk factor for hip fracture in Japanese RA patients.     

Regulation of lipid metabolism by palmitoleate and eicosapentaenoic acid (EPA) in mice fed a high-fat diet

We investigated whether oral administration of palmitoleate ameliorates disorders of lipid metabolism to clarify the effects of one of the components of fish oil. Lipid levels in the liver and plasma were significantly decreased by palmitoleate and by EPA administration. These results suggest that palmitoleate, in addition to EPA, plays a role in the regulation of lipid metabolism by fish oil.     

Direct interaction between DNA methyltransferase DIM-2 and HP1 is required for DNA methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genomic stability in mammals, plants, and fungi. Genetics studies of Neurospora crassa have revealed that a DNA methyltransferase (DIM-2), a histone H3K9 methyltransferase (DIM-5), and heterochromatin protein 1 (HP1) are required for DNA methylation. We explored the interrelationships of these components of the methylation machinery. A yeast two-hybrid screen revealed that HP1 interacts with DIM-2. We confirmed the interaction in vivo and demonstrated that it involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1. Both regions are essential for proper DNA methylation. We also determined that DIM-2 and HP1 form a stable complex independently of the trimethylation of histone H3K9, although the association of DIM-2 with its substrate sequences depends on trimethyl-H3K9. The DIM-2/HP1 complex does not include DIM-5. We conclude that DNA methylation in Neurospora is largely or exclusively the result of a unidirectional pathway in which DIM-5 methylates histone H3K9 and then the DIM-2/HP1 complex recognizes the resulting trimethyl-H3K9 mark via the chromo domain of HP1.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Molecular characterization of a novel beta1,3-galactosyltransferase for capsular polysaccharide synthesis by Streptococcus agalactiae type Ib

A group B streptococcus, Streptococcus agalactiae type Ib, produces a high-molecular-weight polysaccharide consisting of the following pentasaccharide repeating unit: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. The type-specific capsular polysaccharide (CP) synthesis (cps) genes of this strain were cloned and analyzed. A cloned 10-kb DNA fragment contained cpsIbE to L and neu (neuraminic acid synthesis gene) B. Comparison of the gene products with those of S. agalactiae type Ia, which has a similar but distinct CP, showed that the translation products of cpsIa and cpsIb genes exhibited very high homology except for those of cpsJ and K. In the type Ia strain, cpsIaJ encodes beta1,4-galactosyltransferase, which catalyzes the transfer of galactose as the fourth monosaccharide of the sugar repeating unit. In the type Ib CP, this galactose forms a beta1,3-linkage to GlcNAc. The low homology between the type Ia and Ib CpsJs seems to reflect this difference. By enzymatic activity measurement, the cpsIbJ product was found to display beta1,3-galactosyltransferase activity. Furthermore, hydrophobic cluster analysis clarified the similarities and differences of the structures in N-terminal regions, including the DXD motif, between the galactosyltransferases.     

Ex vivo intraoperative angiography for rectus abdominis musculocutaneous free flaps

In this study, the vascular architecture of rectus abdominis free flaps nourished by deep inferior epigastric vessels was investigated using an ex vivo intraoperative angiogram. Oblique rectus abdominis free flaps were elevated and isolated from the donor site. In 11 patients, the vascular architecture of these flaps was analyzed before the flap was thinned. Radiographic study identified an average of 2.1 large deep inferior epigastric arterial perforators in each flap. In nine of the 11 flaps, the axial artery was visible. In four flaps, the axial artery originated from the perforator of the lateral branch of the deep inferior epigastric artery; in five others, it originated from the medial branch. In each flap, the angle of the axial perforator from its anterior rectus sheath in the vertical plane was measured; its mean was 50.6 degrees. All flaps survived, although three showed partial necrosis in the distal portions. In two of these three flaps, the axial artery was not visible in the angiograms, and the third revealed a one-sided distribution of axial flap arteries. Using ex vivo intraoperative angiography, the architecture of the individual flap, its axial perforator, and its connecting axial flap vessel could be investigated. This information can help the surgeon safely thin and separate the flap.