Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.     

Increasing the Catalytic Performance of a Whole Cell Biocatalyst Harboring a Cytochrome P450cam System by Stabilization of an Electron Transfer Component

Catalytic activity of a recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system from Pseudomonas putida coupled with enzymatic co-factor regeneration was investigated. About 0.7 μmol camphor was hydroxylated per mg dry cells at 4°C in 50 mM Tris/HCl buffer (pH 7.4) when utilizing a stable putidaredoxin (Pdx) mutant, C73S/C85S-Pdx (Cys73Ser, Cys85Ser double mutant), instead of wild-type Pdx, which was about two-fold improvement in the substrate conversion. Ten-micromole camphor was completely hydroxylated at 20°C in 6 h by 15 mg dry cell weight of whole cell biocatalyst including C73S/C85S-Pdx. Thus, modulation of protein-protein interaction in multicomponent enzymatic catalysis in whole cells is important.     

New Assay for Rhizobitoxine Based on Inhibition of 1-Aminocyclopropane-1-Carboxylate Synthase

Rhizobitoxine is synthesized by the legume symbiont Bradyrhizobium elkanii and the plant pathogen Burkholderia andropogonis. Rhizobitoxine competitively inhibited 1-aminocyclopropane-1-carboxylate (ACC) synthase bLE-ACS2 from the tomato, a key enzyme in the pathway of ethylene biosynthesis. Based on this inhibition of ACC synthase, we have developed a new assay for rhizobitoxine.     

Stromal over-reduction by high-light stress as measured by decreases in P700 oxidation by far-red light and its physiological relevance

The oxidation level of P700 induced by far-red light (DeltaA(FR)) in briefly dark-treated leaves of some sun plants decreased during the daytime and recovered at night. The dark recovery of decreased DeltaA(FR) proceeded slowly, with a half-time of about 5 h. We propose that stromal over-reduction induced by sunlight was the direct cause of the depression of DeltaA(FR). The depression of DeltaA(FR) found during the daytime was reproduced by controlled illumination with saturating light of fully dark-treated leaves. Simultaneous measurement of P700 redox and chlorophyll fluorescence showed that the depression of DeltaA(FR) was associated with dark reduction of the plastoquinone pool, which represented cyclic electron transport activity. The decrease of DeltaA(FR) in the light-stressed chloroplasts was partly reversed by treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of electron transport at the cytochrome b6/f complex, and the subsequent addition of methyl viologen, an efficient electron acceptor from photosystem I (PSI), stimulated further recovery, showing that both cyclic electron flow around PSI and the charge recombination within PSI were responsible for the light-induced depression of DeltaA(FR). The dark level of blue-green fluorescence, an indicator of NAD(P)H concentration, from intact chloroplasts was increased by high-light stress, suggesting that NADPH accumulated in stroma as a result of the high-light treatment. Possible effects on photosynthetic activity of over-reduction and its physiological relevance are discussed.     

Natural recombinant between equine herpesviruses 1 and 4 in the ICP4 gene

Equine herpesvirus 1 (EHV-1) is a pathogen causing rhinopneumonia in young horses, abortion in mares, and myeloencephalitis in adult horses. Two types, EHV-1 P and EHV-1 B, have recently been dominant among 16 electropherotypes. EHV-1 P and EHV-1 B viruses were compared by long and accurate polymerase chain reaction (LA-PCR) and restriction fragment length polymorphism (RFLP) analysis. Differences in restriction sites were found to be focused in ORF64, which encodes the infected cell protein 4 (ICP4), and downstream of the ICP4 gene. The 3 ' -end and downstream of ICP4 gene of EHV-1 B were found to be replaced by the corresponding region of EHV-4, indicating that EHV-1 B is a naturally occurring recombinant virus between progenitors of EHV-1 P and EHV-4. This is the first report showing a natural interspecies recombinant in alphaherpesviruses.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

ICOS costimulates invariant NKT cell activation

It has been reported that costimulatory molecules, CD80/86-CD28 and CD154-CD40, critically contribute to activation of CD1d-restricted invariant NKT (iNKT) cells. Here we have demonstrated that ICOS, a new member of the CD28 family, plays a substantial role in iNKT cell activation. iNKT cells constitutively expressed ICOS as well as CD28 independently, and ICOS expression was further up-regulated 2-3 days after alpha-galactosylceramide (alpha-GalCer) treatment. Blockade of ICOS-mediated costimulation by administration of anti-ICOS ligand (B7RP-1) mAb or by ICOS gene knockout substantially inhibited alpha-GalCer-induced IFN-gamma and IL-4 production, cytotoxic activity, and anti-metastatic effect. Moreover, blockade of both B7RP-1-ICOS and CD80/86-CD28 interactions mostly abolished the alpha-GalCer-induced immune responses. These findings indicate that iNKT cell activation is regulated by CD28 and IOCS independently.     

Zinc-induced sodium-dependent vitamin C transporter 2 expression: potent roles in osteoblast differentiation

Zinc is an essential trace element that increases osteoblast numbers and bone formation. However, the mechanisms involved in the Zn-induced differentiation of osteoblasts are poorly understood. We examined the roles of L-ascorbic acid (AA) and its transporter, sodium-dependent vitamin C transporter (SVCT) 2, in the Zn-induced expression of osteoblastic differentiation markers. Zinc time- and dose-dependently induced SVCT2 mRNA expression in the absence or presence of AA. Western blotting and kinetic assays showed that Zn increased functional SVCT2 protein levels and AA transport. In the presence of AA, 50 microM Zn enhanced mRNA expression of the osteoblastic differentiation markers alkaline phosphatase, alpha(1)(I) procollagen, osteopontin (OPN), and osteocalcin (OCN) by 3.9-, 3.8-, 3.3-, and 3.5-fold, respectively; in the absence of AA, the Zn-induced increase was 2.8-, 2.5-, 1.3-, and 1.1-fold, respectively. These findings suggest that AA and SVCT2 mediate Zn-induced OPN and OCN expression and partly regulate Zn-induced osteoblastic differentiation.     

Possible involvement of leaf gibberellins in the clock-controlled expression of XSP30, a gene encoding a xylem sap lectin, in cucumber roots

Root-produced organic compounds in xylem sap, such as hormones and amino acids, are known to be important in plant development. Recently, biochemical approaches have revealed the identities of several xylem sap proteins, but the biological functions and the regulation of the production of these proteins are not fully understood. XYLEM SAP PROTEIN 30 kD (XSP30), which is specifically expressed in the roots of cucumber (Cucumis sativus), encodes a lectin and is hypothesized as affecting the development of above-ground organs. In this report, we demonstrate that XSP30 gene expression and the level of XSP30 protein fluctuate in a diurnal rhythm in cucumber roots. The rhythmic gene expression continues for at least two or three cycles, even under continuous light or dark conditions, demonstrating that the expression of this gene is controlled by a circadian clock. Removal of mature leaves or treatment of shoots with uniconazole-P, an inhibitor of gibberellic acid (GA) biosynthesis, dampens the amplitude of the rhythmic expression; the application of GA negates these effects. These results suggest that light signals perceived by above-ground organs, as well as GA that is produced, possibly, in mature leaves, are important for the rhythmic expression of XSP30 in roots. This is the first demonstration of the regulation of the expression of a clock-controlled gene by GA.