RNAi-Mediated Silencing of the Flavanone 3-Hydroxylase Gene and Its Effect on Flavonoid Biosynthesis in Strawberry Fruit

Anthocyanin biosynthesis requires the activities of several enzymes in vivo. Flavanone 3-hydroxylase (F3H) converts flavanone into dihydroflavanol at an early step in the anthocyanin biosynthesis pathway. In this study we constructed an RNAi gene-silencing vector that encodes a hairpin F3H RNA. Agrobacterium strain GV3101 harboring the F3H RNAi vector was injected into strawberry fruits which were still attached to the plants 14 days after pollination. The phenotype was observed 10 days postinjection, and fruits were tested by RT-PCR and northern blot assays. The results showed that the F3H gene was downregulated by approximately 70 % in the agroinfiltrated fruits compared with the control. HPLC–MS analysis showed that anthocyanin content was greatly reduced, flavonol was also decreased, and the levels of p-coumaroyl glucoside and p-coumaroyl-1-acetate were markedly increased. We conclude that the precursors were shunted to the phenylpropanoid pathway, and that F3H is one of the key enzymes required for the biosynthesis of flavonoids in strawberry fruit. According to our results, reducing gene function via RNA interference is a rapid, simple, and effective way to identify gene function in strawberry fruit.     

Intestinal Parasites among Wild Rodents in Northern Gangwon-do,Korea

To determine geographical patterns of natural parasite infections among wild rodents, a total of 46 wild rodents from 3 different localities in northern Gangwon-do (Province), Korea were examined for intestinal parasite infections. Along with nematodes such as hookworms and Syphacia spp., Plagiorchis muris (2 specimens) (Trematoda) were collected from striped field mice, Apodemus agrarius. In a Korean wood mouse, Apodemus peninsulae, the overall nematode infections were similar to A. agrarius, but an adult worm of Echinostoma hortense (Trematoda) was collected. In addition, 2 species of cestodes, i.e., Hymenolepis nana and Hymenolepis diminuta, were collected from A. agrarius. Through this survey, A. agrarius and A. peninsule were confirmed as the natural definite hosts for zoonotic intestinal helminths, i.e., P. muris, E. hortense, H. nana, and H. diminuta, in northern Gangwon-do, Korea. Considering increased leisure activities around these areas, seasonal and further comprehensive surveys on wild rodents seem to be needed to prevent zoonotic parasite infections.     

Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.     

Mesenchymal stem cells contribute to endogenous FVIII:c production

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative‐RT‐PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord‐derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis is SOD1‐immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S‐nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up‐regulated, which generated high levels of nitric oxide (NO) and subsequently induced S‐nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH‐SY5Y cells with NO donor S‐nitrosocysteine triggered a dose‐dependent formation of S‐nitrosylated PDI. When mutant SOD1 was over‐expressed in SH‐SY5Y cells, the iNOS expression was up‐regulated, and NO generation was consequently increased. Furthermore, both S‐nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1^G93A. Blocking NO generation with the NOS inhibitor N‐nitro‐l ‐arginine attenuated the S‐nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO‐mediated S‐nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

Highly enantioselective oxidation of phenyl methyl sulfide and its derivatives into optically pure (S)-sulfoxides with Rhodococcus sp. CCZU10-1 in an n-octane–water biphasic system

Enantiopure sulfoxides can be prepared via the asymmetric oxidation of sulfides using sulfide monooxygenases. The n-octane–water biphasic system was chosen for the bio-oxidation of a water-insoluble phenyl methyl sulfide (PMS) by Rhodococcus sp. CCZU10-1. In this n-octane–water system, the optimum reaction conditions were obtained. (S)-phenyl methyl sulfoxide ((S)-PMSO) with >99.9 % enantiomeric excess formed at 55.3 mM in the n-octane–water biphasic system. Using fed-batch method, a total of 118 mM (S)-PMSO accumulated in 1-L reaction mixture after the 7th feed, and no (R)-PMSO and sulfone were detected. Moreover, Rhodococcus sp. CCZU10-1 displayed fairly good activity and enantioselectivity toward other sulfides. In conclusion, Rhodococcus sp. CCZU10-1 is a promising biocatalyst for synthesizing highly optically active sulfoxides.