The slow wound-response of<Emphasis Type="Italic"> γVPE</Emphasis> is regulated by endogenous salicylic acid in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Vacuolar processing enzyme (VPE) is a cysteine protease responsible for the maturation of various vacuolar proteins in higher plants. The Arabidopsis thaliana (L.) Heynh. VPE gene, encoding a VPE homologue, is slowly up-regulated in both local and systemic leaves in response to wounding. To clarify the activation mechanism of VPE, we examined the accumulation of VPE mRNA after hormone treatments or after wounding in wild-type and various mutant plants of Arabidopsis. Both ethylene and jasmonic acid (JA) are known as signal molecules that activate the wound-responsive genes. However, treatment with exogenous JA had little effect on the VPE response, although JA activated the vegetative storage protein (VSP) gene, a typical wound-responsive gene. Wounding activated VPE even in two ethylene-insensitive plants (etr1-1 and ein2-1). Thus, the wound-induced expression of VPE was independent of ethylene and JA. We found that the wound-induced expression of VPE was reduced in two SA-deficient plants (pad4-1 and NahG), while the wound-induced expression of VSP increased in these mutants. Appreciable accumulation of SA was not observed in either the local or systemic leaves after wounding. These results suggest that endogenous SA enhances the wound-induced expression of VPE and attenuates the wound-induced expression of VSP, although SA is not a wound-signal that directly activates these genes.Abbreviations ABA abscisic acid - GST glutathione S-transferase - INA 2,6-dichloroisonicotinic acid - JA jasmonic acid - MeJA methyl jasmonate - PR pathogenesis-related - RBCS Rubisco small subunit - SA salicylic acid - VPE vacuolar processing enzyme - VSP vegetative storage protein     

Hip1-related mutant mice grow and develop normally but have accelerated spinal abnormalities and dwarfism in the absence of HIP1

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.     

Structural and functional characterization of ovotransferrin produced by Pichia pastoris

Ovotransferrin is an egg white protein with complex disulfide and bilobal structures, which is derived from the same gene as chicken serum transferrin. We demonstrate here the structural and functional characteristics of bilobal ovotransferrin, produced at a high level using Pichia pastoris expression system. The recombinant protein was secreted into the medium, and the secretion signal peptide was processed correctly. The secretion level was almost 100 mg/l culture and the yield after purification by two-step anion exchange chromatography was 57 mg/l. The CD spectrum and fluorescence spectra indicate the correct folding of the recombinant protein. The analyses for the Fe3+ binding ability by urea-PAGE and visible absorption spectrum revealed that two Fe3+ sites exist in a recombinant ovotransferrin molecule as in the egg white protein. Endoglycosidases, such as endo-beta-N-acetylglucosaminidase H (Endo-H), peptide:N-glycosidase F (PNGaseF), and endo-beta-N-acetylglucosaminidase from Mucor hiemalis, showed differential activities for the native Fe3+-loaded, native Fe3+-free, and denatured forms of recombinant ovotransferrin; only the first enzyme displayed the cleavage ability for all the ovotransferrin forms. The results from the enzyme specificity and from the molecular weight difference for the intact and deglycosylated proteins were consistent with the view that recombinant ovotransferrin have one N-linked carbohydrate chain which mainly consists of two GlcNac and 10 mannoses.     

Crystal structure of archaeal ribonuclease P protein Ph1771p from Pyrococcus horikoshii OT3: an archaeal homolog of eukaryotic ribonuclease P protein Rpp29

Ribonuclease P (RNase P) is the endonuclease responsible for the removal of 5' leader sequences from tRNA precursors. The crystal structure of an archaeal RNase P protein, Ph1771p (residues 36-127) from hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined at 2.0 A resolution by X-ray crystallography. The structure is composed of four helices (alpha1-alpha4) and a six-stranded antiparallel beta-sheet (beta1-beta6) with a protruding beta-strand (beta7) at the C-terminal region. The strand beta7 forms an antiparallel beta-sheet by interacting with strand beta4 in a symmetry-related molecule, suggesting that strands beta4 and beta7 could be involved in protein-protein interactions with other RNase P proteins. Structural comparison showed that the beta-barrel structure of Ph1771p has a topological resemblance to those of Staphylococcus aureus translational regulator Hfq and Haloarcula marismortui ribosomal protein L21E, suggesting that these RNA binding proteins have a common ancestor and then diverged to specifically bind to their cognate RNAs. The structure analysis as well as structural comparison suggested two possible RNA binding sites in Ph1771p, one being a concave surface formed by terminal alpha-helices (alpha1-alpha4) and beta-strand beta6, where positively charged residues are clustered. A second possible RNA binding site is at a loop region connecting strands beta2 and beta3, where conserved hydrophilic residues are exposed to the solvent and interact specifically with sulfate ion. These two potential sites for RNA binding are located in close proximity. The crystal structure of Ph1771p provides insight into the structure and function relationships of archaeal and eukaryotic RNase P.     

Molecular Mechanism in Activation of Glutathione System by Ropinirole,a Selective Dopamine D2 Agonist

We have previously reported that ropinirole, a non-ergot dopamine agonist, has neuroprotective effects against 6-hydroxydopamine in mice based on in vivo antioxidant properties such as the glutathione (GSH)-activating effect. In the present study, we determined that the effects of ropinirole on the level of expression of GSH-related enzyme mRNA, these enzymes were shown to regulate GSH contents in the brain. This study focused on the mechanism of GSH enhancement by ropinirole. Striatal GSH contents were significantly increased by 7-day daily administration of ropinirole. Furthermore, the expression levels of -glutamylcysteine synthetase (-GCS), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) mRNA increased following daily injections of ropinirole for 7 days. In addition, ropinirole treatment for 7 days suppressed auto-oxidation in mouse striatal homogenates, in contrast to the vehicle treatment. In conclusion, ropinirole was able to suppress auto-oxidation, most probably by increasing GSH levels due to an increase of GSH synthesis. In addition, it is likely that auto-oxidation was also suppressed by the activation of GSH-regulating enzymes such as GPx, GR, and GST in the mouse striatum. Thus, our results indicate that the GSH-activating effect of ropinirole may render this dopamine agonist beneficial as a neuroprotective drug.     

Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment

A coupled cell-free translation/glycosylation system, prepared from Spodoptera frugiperda insect cells, was established and optimized for protein production and glycosylation efficiency. Both translation and glycosylation were stimulated by addition of Mg2+, K+, ATP, GTP, creatine kinase and creatine phosphate, suggesting that glycoprotein productivity is largely determined by translation efficiency. However, high concentrations of creatine phosphate significantly inhibited translation. Spermidine stimulated both translation and glycosylation, but glycosylation required higher concentrations of spermidine than translation. Furthermore, extracts prepared at a nitrogen pressure of 10 kg/cm2 with the Mini-Bomb cell disruption chamber had the highest glycoprotein productivity; and extracts prepared at the higher nitrogen pressure of 15 kg/cm2 retained glycosylation ability. While extracts prepared with the Potter-Elvehjem homogenizer could mediate translation, no glycosylation was achieved. This indicated that the posttranslational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 microg of glycosylated gp120/ml of reaction mixture.     

Characterization of the γc chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID)

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.     

Functional reconstitution and characterization of the Arabidopsis Mg(2+) transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.     

Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic F(o)F(1)-ATP synthase expressed in Escherichia coli

F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.     

Hyperproliferation of mitotically active germ cells due to defective anti-Müllerian hormone signaling mediates sex reversal in medaka

The function of AMH (Anti-Müllerian hormone), a phylogenetically ancient member of the TGFβ family of proteins, in lower vertebrates is largely unknown. Previously, we have shown that the gene encoding the type II anti-Müllerian hormone receptor, amhrII, is responsible for excessive germ cell proliferation and male-to-female sex reversal in the medaka hotei mutant. In this study, functional analyses in cultured cells and of other amhrII mutant alleles indicate that lack of AMH signaling causes the hotei phenotype. BrdU incorporation experiments identified the existence of both quiescent and mitotically active germ cells among the self-renewing, type I population of germ cells in the developing gonad. AMH signaling acts in supporting cells to promote the proliferation of mitotically active germ cells but does not trigger quiescent germ cells to proliferate in the developing gonad. Furthermore, we show that the male-to-female sex reversal phenotype in hotei mutants is not a direct consequence of AMH signaling in supporting cells, but is instead mediated by germ cells. Our data demonstrate that interfollicular AMH signaling regulates proliferation at a specific stage of germ cell development, and that this regulation is crucial for the proper manifestation of gonadal sex directed by sex determination genes.