Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L.

Phototrophic growth of a Rubisco-deficient mesophilic purple nonsulfur bacterium harboring a Type III Rubisco from a hyperthermophilic archaeon.

The hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 harbors a structurally novel, Type III Rubisco (Rbc(Tk)). In terms of protein engineering of Rubiscos, the enzyme may provide an alternative target to the conventional Type I and Type II enzymes. With a future aim to improve the catalytic properties of Rbc(Tk), here we examined whether or not the enzyme could support growth of a mesophilic organism dependent on CO2 fixation.

A novel mouse model for MPO-ANCA-associated glomerulonephritis.

We established a novel model mouse for myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA)-associated glomerulonephritis with crescentic formation, which was induced by administering bovine serum albumin (BSA). Neutrophil infiltration into the renal glomeruli began at 8 weeks and crescent formation was observed from 10 weeks after the first BSA injection. Platelet and neutrophil counts significantly increased, and proteinuria was observed from 5 weeks.

Photoinactivation of ascorbate peroxidase in isolated tobacco chloroplasts: Galdieria partita APX maintains the electron flux through the water-water cycle in transplastomic tobacco plants.

We evaluated the H2O2-scavenging activity of the water-water cycle (WWC) in illuminated intact chloroplasts isolated from tobacco leaves. Illumination under conditions that limited photosynthesis [red light (>640 nm), 250 micromol photons m(-2) s(-1) in the absence of HCO3-] caused chloroplasts to take up O2 and accumulate H2O2. Concomitant with the O2 uptake, both ascorbate peroxidase (APX) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) lost their activities.

p53 protein transduction therapy: successful targeting and inhibition of the growth of the bladder cancer cells.

Introduction: Virus-mediated gene therapy for bladder cancer has some problems, such as efficiency of gene delivery and safety issues. We have reported that poly-arginine peptide (11R) has the ability to increase protein transduction in cells. Here, we show that p53 protein transduction using 11R is useful for targeting to bladder tumors and suppressing the growth of bladder cancer cells.

Cdk5-dependent regulation of glucose-stimulated insulin secretion.

Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications. Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K(+) (K(ATP)) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia.

Truncation and activation of calcineurin A by calpain I in Alzheimer disease brain.

A disturbance of calcium homeostasis is believed to play an important role in the neurodegeneration of the brains of Alzheimer disease (AD) patients, but the molecular pathways by which it contributes to the disease are not well understood. Here we studied the activation of two major Ca(2+)-regulated brain proteins, calpain and calcineurin, in AD brain. We found that calpain I is activated, which in turn cleaves and activates calcineurin in AD brain.

Effects of light intensity on cyclic electron flow around PSI and its relationship to non-photochemical quenching of Chl fluorescence in tobacco leaves.

We tested the hypothesis that plants grown under high light intensity (HL-plants) had a large activity of cyclic electron flow around PSI (CEF-PSI) compared with plants grown under low light (LL-plants). To evaluate the activity of CEF-PSI, the relationships between photosynthesis rate, quantum yields of both PSII and PSI, and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants which had been grown under different light intensities (150 and 1,100 micromol photons m(-2) s(-1), respectively) and with different amounts of nutrients supplied. HL-plants showed a larger value of non-photochemical quenching (NPQ) of Chl fluorescence at the limited activity of photosynthetic linear electron flow.

A new approach to inhibiting astrocytic IP3-induced intracellular calcium increase in an astrocyte-neuron co-culture system.

Astrocytes exhibit dynamic Ca2+ mobilization, such as Ca2+ wave and Ca2+ oscillation, via an inositol 1,4,5-triphosphate-induced Ca2+ release (IICR)-dependent mechanism. The physiological functions of astrocytic Ca2+ mobilization, however, are poorly understood. To investigate this issue, we created a plasmid encoding an enhanced green fluorescent protein-tagged inositol 1,4,5-triphosphate absorbent protein and expressed it in cultured astrocytes.

Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells.

Protein transduction therapy using poly-arginine can deliver the bioactive p53 protein into cancer cells and inhibits the proliferation of the cells. However, one disadvantage of such therapy is the short intracellular half-life of the delivered protein. Here, we generated mutant proteins in which multiple lysine residues in the C-terminal were substituted by arginines.

Impairment of hippocampal long-term depression and defective spatial learning and memory in p35 mice.

Cdk5 (cyclin-dependent kinase 5) activity is dependent upon association with one of two neuron-specific activators, p35 or p39. Genetic deletion of Cdk5 causes perinatal lethality with severe defects in corticogenesis and neuronal positioning. p35(-/-) mice are viable with milder histological abnormalities.

Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability.

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca(2+)/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation.

Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons.

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function.

Calpain inhibitors prevent neuronal cell death and ameliorate motor disturbances after compression-induced spinal cord injury in rats.

Traumatic spinal cord injury (SCI) results in widespread neuronal cell death. Recent studies have suggested that activated calpain mediates neuronal cell death in the central nervous system. We conducted a study to determine whether calpain mediates neuronal cell death in the motor neurons of the spinal cord after SCI, and whether postinjury administration of the calpain inhibitors N-acetyl- Leu-Leu-Met-CHO (ALLM) and calpain inhibitor III (CI III) (MDL28170) reduces the motor disturbances in rats with a model of SCI.

Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPM7/ChaK1.

TRPM7/ChaK1 is a unique channel/kinase that contains a TRPM channel domain with 6 transmembrane segments fused to a novel serine-threonine kinase domain at its C terminus. The goal of this study was to investigate a possible role of kinase activity and autophosphorylation in regulation of channel activity of TRPM7/ChaK1. Residues essential for kinase activity were identified by site-directed mutagenesis.

Regulation of synaptic vesicle recycling by calcineurin in different vesicle pools.

The synaptic vesicles keep recycling by the processes of endocytosis and exocytosis to maintain the normal synaptic transmission. The synaptic vesicles are classified as the readily releasable pool (RRP) and the reserve pool (RP). In the endocytosis process, calcineurin (CaN), a Ca2+/calmodulin-dependent protein phosphatase, has been shown to play important roles.

CO2 response of cyclic electron flow around PSI (CEF-PSI) in tobacco leaves--relative electron fluxes through PSI and PSII determine the magnitude of non-photochemical quenching (NPQ) of Chl fluorescence.

We hypothesized that cyclic electron flow around photosystem I (CEF-PSI) participates in the induction of non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence when the rate of photosynthetic linear electron flow (LEF) is electron-acceptor limited. To test this hypothesis, the relationships among photosynthesis rate, electron fluxes through both PSI and PSII [Je(PSI) and Je(PSII)] and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants at several light intensities and partial pressures of ambient CO2 (Ca). At low light intensities, decreasing Ca lowered the photosynthesis rate, but Je(PSI) and Je(PSII) remained constant.

The NH2 terminus of influenza virus hemagglutinin-2 subunit peptides enhances the antitumor potency of polyarginine-mediated p53 protein transduction.

Protein transduction therapy is a newly developing method that allows proteins, peptides, and biologically active compounds to penetrate across the plasma membrane by being fused with cell-penetrating peptides such as polyarginine. Polyarginine-fused p53 protein penetrates across the plasma membrane of cancer cells and inhibits the growth of the cells. However, the protein is often entrapped inside macropinosomes in the cytoplasm.

Enhancement of cyclic electron flow around PSI at high light and its contribution to the induction of non-photochemical quenching of chl fluorescence in intact leaves of tobacco plants.

Non-photochemical quenching (NPQ) of Chl fluorescence is a mechanism for dissipating excess photon energy and is dependent on the formation of a DeltapH across the thylakoid membranes. The role of cyclic electron flow around photosystem I (PSI) (CEF-PSI) in the formation of this DeltapH was elucidated by studying the relationships between O2-evolution rate [V(O2)], quantum yield of both PSII and PSI [Phi(PSII) and Phi(PSI)], and Chl fluorescence parameters measured simultaneously in intact leaves of tobacco plants in CO2-saturated air. Although increases in light intensity raised V(O2) and the relative electron fluxes through both PSII and PSI [Phi(PSII) x PFD and Phi(PSI) x PFD] only Phi(PSI) x PFD continued to increase after V(O2) and Phi(PSII) x PFD became light saturated.

2-methoxyestradiol enhances p53 protein transduction therapy-associated inhibition of the proliferation of oral cancer cells through the suppression of NFkappaB activity.

Protein transduction therapy using poly-arginine peptide can deliver the biologically active proteins. A previous study showed that 11 poly-arginine fused p53 protein (11R-p53) effectively penetrated across the plasma membrane and inhibited the proliferation of oral cancer cells. However, the intracellular half-life of the delivered protein was less than 36 h.

Prominent expression of spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1 in LH-producing cells, LbetaT2.

The LH-producing cell line, LbetaT2, and non LH-producing cell line, alphaT3-1 cells, established from a pituitary tumor, were employed for cDNA subtraction cloning to identify genes with expression unique to LH producing cells. Several cDNAs that code for known as well as for many unidentified clones were discovered. Most clones were the spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1, the abnormality of which causes neurodegeneration and loss of cerebellar Purkinje cells.

Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression.

Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2.

Exposure of mouse to high gravitation forces induces long-term potentiation in the hippocampus.

The central nervous system is highly plastic and has been shown to undergo both transient and chronic adaptive changes in response to environmental influences. The purpose of this study was to investigate the effect of hypergravic field on long-term potentiation (LTP) in the mouse hippocampus. Exposure of mice to 4G fields for 48 h had no effect on input-output coupling during extracellular stimulation of Schaffer collaterals and paired pulse facilitation, suggesting that the hypergravic exposure had no detrimental effect on basal neurotransmission in the hippocampus.

Photo-acceleration of protein release from endosome in the protein transduction system.

The protein transduction system has been employed for delivery of bioactive proteins into cells via an endocytotic mechanism. However, trapping of endocytosed proteins in the endosome may significantly attenuate biological actions in cells. The present investigation demonstrated that endosomal release of transduced protein could be artificially accelerated by exposure to fluorescent light.