Overexpression of Cinnamate 4-Hydroxylase Gene Enhances Biosynthesis of Decursinol Angelate in Angelica gigas Hairy Roots

Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.     

Optimal population of FoxP3+ T cells in tumors requires an antigen priming-dependent trafficking receptor switch

FoxP3⁺ T cells populate tumors and regulate anti-tumor immunity. The requirement for optimal population of FoxP3⁺ regulatory T cells in tumors remains unclear. We investigated the migration requirement and stability of tumor-associated FoxP3⁺ T cells. We found that only memory, but not naïve, FoxP3⁺ T cells are highly enriched in tumors. Almost all of the tumor-infiltrating FoxP3⁺ T cells express Helios, an antigen associated either with thymus-generated FoxP3⁺ T cells or activated T cells in the periphery. The tumor-infiltrating FoxP3⁺ T cells largely lack CD62L and CCR7, two trafficking receptors required for T cell migration into secondary lymphoid tissues. Instead, the tumor infiltrating FoxP3⁺ T cells highly express memory/tumor-associated CCR8 and CXCR4. Antigen priming is required for induction of this trafficking receptor phenotype in FoxP3⁺ T cells and only antigen primed, but not antigen-inexperienced naive, FoxP3⁺ T cells can efficiently migrate into tumors. While the migration of FoxP3⁺ T cells into tumors was a readily detectable event, generation of induced FoxP3⁺ T cells within tumors was unexpectedly inefficient. Genetic marking of current and ex-FoxP3⁺ T cells revealed that tumor-infiltrating FoxP3⁺ T cells are highly stable and do not readily convert back to FoxP3⁻ T cells. Taken together, our results indicate that population of tumors with thymus-generated FoxP3⁺ T cells requires an antigen priming-dependent trafficking receptor switch in lymphoid tissues.     

Salvianolic acid B prevents bone loss in prednisone-treated rats through stimulation of osteogenesis and bone marrow angiogenesis

Glucocorticoid (GC) induced osteoporosis (GIO) is caused by the long-term use of GC for treatment of autoimmune and inflammatory diseases. The GC related disruption of bone marrow microcirculation and increased adipogenesis contribute to GIO development. However, neither currently available anti-osteoporosis agent is completely addressed to microcirculation and bone marrow adipogenesis. Salvianolic acid B (Sal B) is a polyphenolic compound from a Chinese herbal medicine, Salvia miltiorrhiza Bunge. The aim of this study was to determine the effects of Sal B on osteoblast bone formation, angiogenesis and adipogenesis-associated GIO by performing marrow adipogenesis and microcirculation dilation and bone histomorphometry analyses. (1) In vivo study: Bone loss in GC treated rats was confirmed by significantly decreased BMD, bone strength, cancellous bone mass and architecture, osteoblast distribution, bone formation, marrow microvessel density and diameter along with down-regulation of marrow BMPs expression and increased adipogenesis. Daily treatment with Sal B (40 mg/kg/d) for 12 weeks in GC male rats prevented GC-induced cancellous bone loss and increased adipogenesis while increasing cancellous bone formation rate with improved local microcirculation by capillary dilation. Treatment with Sal B at a higher dose (80 mg/kg/d) not only prevented GC-induced osteopenia, but also increased cancellous bone mass and thickness, associated with increase of marrow BMPs expression, inhibited adipogenesis and further increased microvessel diameters. (2) In vitro study: In concentration from 10(-6) mol/L to 10(-7) mol/L, Sal B stimulated bone marrow stromal cell (MSC) differentiation to osteoblast and increased osteoblast activities, decreased GC associated adipogenic differentiation by down-regulation of PPARγ mRNA expression, increased Runx2 mRNA expression without osteoblast inducement, and, furthermore, Sal B decreased Dickkopf-1 and increased β-catenin mRNA expression with or without adipocyte inducement in MSC. We conclude that Sal B prevented bone loss in GC-treated rats through stimulation of osteogenesis, bone marrow angiogenesis and inhibition of adipogenesis.     

Complete mitochondrial genome of the seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae)

The complete mitochondrial genome (mitogenome) of the seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae), which is one of the best known insects capable of predation, is described with an emphasis on the noteworthy composition of the A+T-rich region. The C. septempunctata genome consists of 2 rRNAs, 22 tRNAs, 13 protein-coding genes, and 1 control region, designated as the A+T-rich region in insects. Along with an unusually long A+T-rich region (4469 bp), the 18,965-bp long C. septempunctata mitogenome was the largest in Coleoptera. The A+T-rich region is composed of a 2214-bp long non-repeat region composed of 80.17% A/T nucleotides and a 2256-bp long repeat region composed of 65.71% A/T nucleotides. The repeat region harbors 32 identical 70-bp long tandem repeats plus one 15-bp long incomplete first repeat. These repeat sequences may possibly have been caused by slipped-strand mispairing and unequal crossing-over events during DNA replication.     

Association between FGFR1OP2/wit3.0 Polymorphisms and Residual Ridge Resorption of Mandible in Korean Population

Background

A previous study on the genetic association between single nucleotide polymorphisms in FGFR1OP2/wit3.0 and the long term atrophy of edentulous mandible hypothesized that the excessive jawbone atrophy after dental extraction may be associated with abnormal oral mucosa contraction induced by the FGFR1OP2/wit 3.0 gene. It was reported that the minor allele of rs840869 or rs859024 in FGFR1OP2/wit3.0 was associated with the excessive atrophy of edentulous mandible. The present study represents an attempt to replicate the results of this previous study and to examine the genetic association between polymorphisms in FGFR1OP2 and residual ridge resorption of mandible in a Korean population.

Methodology/Principal Findings

134 subjects (70.46±9.02 years) with partially or completely edentulous mandible were recruited. The mandibular bone height was measured following the protocol of the American College of Prosthodontists (ACP). From 24 subjects, seven variants in FGFR1OP2 were discovered and four of them were novel. Selected SNPs that are not in high LD at r2 threshold of 0.8 were genotyped for the remaining population. There was no frequency of the minor allele of SNP rs859024 in Korean population. SNP rs840869 was not associated with residual ridge resorption (p = 0.479). The bone height of the subject with the ss518063493 minor allele (8.52 mm) was shorter than that of those subjects with major alleles (18.96±5.33 mm, p = 0.053).

Conclusions/Significance

The patient with minor allele of ss518063493 may be associated with excessive atrophy of edentulous mandible whereas the patients with that of rs840869 are not associated in Korean population. The result from this study may assist in developing a novel genetic diagnostic test and be useful in identifying Koreans susceptible to developing excessive jawbone atrophy after dental extraction.     

Valproic acid attenuates blood-spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury

In a retinal ischemic ex vivo model, we have reported protective effects of somatostatin (SRIF) receptor 2 (sst(2) ). As an ischemic condition not only causes cell death but also induces a vascular response, we asked whether vascular endothelial growth factor (VEGF) is altered in this model and whether its expression, release or localization are affected by sst(2) activation. Ex vivo retinas of wild-type (WT) and sst(1) KO mice (which over-express sst(2) ) were incubated in ischemic conditions with SRIF, octreotide (OCT) or a VEGF trap. Ischemia in WT retinas caused increase of VEGF release and decrease of VEGF mRNA. Both effects were counteracted by SRIF or OCT. VEGF immunoreactivity was in retinal neurons and scarcely in vessels. Ischemia caused a significant shift of VEGF immunoreactivity from neurons to vessels. The increase of vascular VEGF was reduced in sst(1) KO retinas and in WT retinas treated with SRIF or OCT. VEGF trap also limited this increase, demonstrating that vascular VEGF was of extracellular origin. Together, the data show a VEGF response to ischemia, in which VEGF released by damaged neurons reaches the retinal capillaries. The activation of sst(2) protects neurons from ischemic damage, thereby limiting VEGF release and the VEGF response.     

In vitro stability and in vivo anti-inflammatory efficacy of synthetic jasmonates

A chlorinated methyl jasmonate analog (J7) was elaborated as an in vitro anti-inflammatory lead. However, its in vitro efficacy profile was not reproduced in a subsequent in vivo evaluation, presumably due to its rapid enzymatic hydrolysis in a biological system. In an attempt to improve the metabolic stability of the lead J7 by replacement of its labile methyl ester with reasonable ester groups, several analogs resistant to enzymatic hydrolysis were synthesized. In vivo evaluation of the stability-improved analogs showed that these compounds displayed higher efficacy than the lead J7, suggesting that these new jasmonate analogs may serve as potential anti-inflammatory leads.     

Effect of trichostatin A on fertilization and embryo development during extended culture of mouse oocyte

We performed this study to investigate the effect of histone deacetylase inhibition during extended culture of in vitro matured mouse oocytes. In vitro matured mouse (BDF1) oocytes were cultured in vitro for 6, 12, and 24 h, respectively, and then inseminated. During in vitro culture for 6 and 12 h, two doses of trichostatin A (TSA), a histone deacetylase inhibitor, were added (100 nM and 500 nM) to the culture medium and the oocytes were then inseminated. During the 24-h in vitro culture, two doses of TSA were added (100 nM and 500 nM) to the medium and the oocytes were activated with 10 mM SrCl2. After the 6-h culture, the fertilization rate was similar to that of the control group, but the blastocyst formation rate was significantly decreased. After the 12-h culture, both the fertilization and blastocyst formation rates were significantly decreased. After the 24-h culture, total fertilization failure occurred. In the oocytes cultured for 6 and 12 h, the fertilization and blastocyst formation rates did not differ between the TSA-supplemented and control groups. Although extended culture of the mouse oocytes significantly affected their fertilization and embryo development, TSA supplementation did not overcome their decreased developmental potential.