Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.     

Comparative analysis of GFP(UV)-beta1,3-N-acetylglucosaminyltransferase 2 production in two insect-cell-based expression systems

Active beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. beta3GnT2 was expressed as a secreted fusion protein with GFP(UV) with three different types of signal sequence to enhance the secretion of the fusion protein. In the stably transformed cells, the maximal beta3GnT2 activity differed between isolates, but their secretion efficiencies were similar. The difference between the maximal beta3GnT activities of the isolates studied was considered to be due to the presence of a copy number of the fusion gene, as determined on the basis of the results of Southern blot analysis. The beta3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a beta3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% on SDS-PAGE, in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, beta3GnT2.     

Molecular phylogeny of Vincetoxicum (Apocynaceae-Asclepiadoideae) based on the nucleotide sequences of cpDNA and nrDNA

Molecular phylogenetic analyses of Vincetoxicum and Tylophora (Apocynaceae-Asclepiadoideae) were conducted based on the nucleotide sequences of cpDNA (two intergenic spacers of trnL (UAA)-trnF (GAA) and psbA-trnH and three introns, i.e., atpF, trnG (UCC) and trnL (UAA)), and nrDNA (ITS and ETS regions). Our phylogenetic analysis revealed two monophyletic groups; one consisted of seven taxa of Tylophora and Vincetoxicum inamoenum, Vincetoxicum magnificum and Vincetoxicum macrophyllum (Clade I) and the other consisted of 17 accessions of Vincetoxicum (Clade II). The monophyly of the genus Vincetoxicum was not supported. Although many nucleotide substitutions were observed in Clade I, the genetic differentiation within Clade II was small. Low genetic diversification but considerable morphological divergence suggests that the species in Clade II had undergone rapid diversification. Although most species in Clade I have tiny flowers, those in Clade II have larger and more nectariferous ones. Thus, we hypothesized that the rapid morphological radiation in Clade II may have been due to the gaining of floral characters such as large flowers and large amounts of nectar corresponding to diverse pollinators.     

N-[18F]fluoroethyl-4-piperidyl acetate ([18F]FEtP4A): A PET tracer for imaging brain acetylcholinesterase in vivo

N-[(18)F]Fluoroethyl-4-piperidyl acetate ([(18)F]FEtP4A) was synthesized and evaluated as a PET tracer for imaging brain acetylcholinesterase (AchE) in vivo. [(18)F]FEtP4A was previously prepared by reacting 4-piperidyl acetate (P4A) with 2-[(18)F]fluoroethyl bromide ([(18)F]FEtBr) at 130 degrees C for 30 min in 37% radiochemical yield using an automated synthetic system. In this work, [(18)F]FEtP4A was synthesized by reacting P4A with 2-[(18)F]fluoroethyl iodide ([(18)F]FEtI) or 2-[(18)F]fluoroethyl triflate ([(18)F]FEtOTf in improved radiochemical yields, compared with [(18)F]FEtBr under the corresponding condition. Ex vivo autoradiogram of rat brain and PET summation image of monkey brain after iv injection of [(18)F]FEtP4A displayed a high radioactivity in the striatum, a region with the highest AchE activity in the brain. Moreover, the distribution pattern of (18)F radioactivity was consistent with that of AchE in the brain: striatum>frontal cortex>cerebellum. In the rat and monkey plasma, two radioactive metabolites were detected. However, their presence might not preclude the imaging studies for AchE in the brain, because they were too hydrophilic to pass the blood-brain barrier and to enter the brain. In the rat brain, only [(18)F]fluoroethyl-4-piperidinol ([(18)F]FEtP4OH) was detected at 30 min postinjection. The hydrolytic [(18)F]FEtP4OH displayed a slow washout and a long retention in the monkey brain until the PET experiment (120 min). Although [(18)F]FEtP4A is a potential PET tracer for imaging AchE in vivo, its lower hydrolytic rate and lower specificity for AchE than those of [(11)C]MP4A may limit its usefulness for the quantitative measurement for AchE in the primate brain.     

Oxidized LDL through LOX-1 modulates LDL-receptor expression in human coronary artery endothelial cells

Experimental studies have shown that oxidized low-density lipoprotein (ox-LDL) up-regulates its receptor LOX-1. Both ox-LDL and LOX-1 are expressed in atherosclerotic plaques. Native LDL concentrations are elevated in atherosclerosis, suggesting a reduction in LDL-receptors. We hypothesized that ox-LDL via LOX-1 could influence the expression of LDL-receptors. This study was designed to examine the interaction between ox-LDL, LOX-1, and LDL-receptors in human coronary artery endothelial cells (HCAECs). HCAECs were incubated with ox-LDL (10-80 microg/ml) for 3-24h. Ox-LDL decreased the expression of LDL-receptor in a concentration- and time-dependent fashion. The effects of ox-LDL were mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 microg/ml) prevented the effect of ox-LDL on LDL-receptor expression. The role of LOX-1 was further confirmed by the use of an antisense to LOX-1 mRNA, which also blocked the effect of ox-LDL in LDL-receptor expression. In other experiments, ox-LDL as expected induced superoxide anion generation; and pretreatment of HCAECs with the anti-oxidants trolox and alpha-tocopherol (each 10 microM) inhibited the formation of superoxide anions as well as the down-regulation of LDL-receptor in response to ox-LDL. These studies provide the first evidence that ox-LDL via LOX-1 modulates LDL-receptor expression in HCAECs. The generation of free radicals elicited by ox-LDL may be a key step in this process.     

cDNA microarray analysis of altered gene expression in Ara-C-treated leukemia cells

The acute lymphoblastic leukemia cell line CCRF-CEM is sensitive to Ara-C and undergoes apoptosis. In contrast, the chronic myelogenous leukemia (CML) cell line K562 is highly resistant to Ara-C, which causes the cells to differentiate into erythrocytes before undergoing apoptosis. We used cDNA microarrays to monitor the alterations in gene expression in these two cell lines under conditions leading to apoptosis or differentiation. Ara-C-treated CCRF-CEM cells were characterized by a cluster of down-regulated chaperone genes, whereas Ara-C-treated K562 cells were characterized by a cluster of up-regulated hemoglobin genes. In K562 cells, Ara-C treatment induced significant down-regulation of the asparagine synthetase gene, which is involved in resistance to L-asparaginase. Sequential treatment with Ara-C and L-asparaginase had a synergistic effect on the inhibition of K562 cell growth, and combination therapy with these two anticancer agents may prove effective in the treatment of CML, which cannot be cured by either drug alone.     

Enhanced chemiluminescence of 6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one by attachment of a cyclomaltooligosaccharide (cyclodextrin). Attachment of cyclomaltononaose (delta-cyclodextrin)

2-Methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one (MCLA) is an oxygen-induced chemiluminescent compound. It has been shown that the chemiluminescence can be enhanced by forming a cyclomaltooligosaccharide (cyclodextrin)-bound MCLA, and therefore, in continuation of the survey of the types of cyclodextrins, in this study, MCLA was attached to the secondary hydroxyl face of delta-cyclodextrin, which consists of nine D-glucose units. Although the oxygen-induced chemiluminescence efficiency of delta-cyclodextrin-bound MCLA in a pH 8.0 aqueous phosphate buffer was 12 times greater than that of MCLA, the efficiency was markedly lower than that of gamma-cyclodextrin-bound MCLA, which exhibited the highest chemiluminescence efficiency in the previous investigation. Although fluorescence efficiency and light-emitter formation efficiency for delta-cyclodextrin-bound MCLA were similar to those for gamma-cyclodextrin-bound MCLA, singlet-excited state formation efficiency for delta-cyclodextrin-bound MCLA was lower than that for gamma-cyclodextrin-bound MCLA. This study distinctly indicated the optimum cyclodextrin for construction of greatly luminescent cyclodextrin-bound MCLA is gamma-cyclodextrin.     

Gene silencing in chick embryos with a vector-based small interfering RNA system

In this paper, the use of vector-based RNA interference (RNAi) to specifically interfere with gene expression in chick embryos is reported. In ovo electroporation was carried out to transfer a small interfering RNA (siRNA) expression vector into chick embryos. En2 was chosen for the target gene because the family gene, En1, is expressed in a similar pattern. Four sets of 19-mer sequences were designed with the En2 open reading frame region connected to a sequence of short hairpin RNA (shRNA), which exerts siRNA effects after being transcribed, and inserted into pSilencer U6-1.0 vector. En2 and En1 expression were suppressed by the siRNA whose sequence completely matched En2 and En1. Suppression occurred when the siRNA sequence differed by up to two nucleotides from the target sequence. The sequence that differed by four nucleotides from the target gene did not show siRNA effects. One set that completely matched the En2 target did not show siRNA effects, which may be due to location of the siRNA in the target gene. Thus, multiple sets of shRNA must be prepared if we are to consider. This system will greatly contribute to the analysis of function of genes of interest, because the target gene can be silenced in a locally and temporally desired manner.     

Reduction of phosphodiesterase 3B gene expression in peroxisome proliferator-activated receptor gamma (+/-) mice independent of adipocyte size

Phosphodiesterase 3B (PDE3B) gene expression is generally reduced in large adipocytes of obese, insulin-resistant mice. This reduced gene expression is restored by peroxisome proliferator-activated receptor (PPAR) gamma ligands accompanied by a reduced fat cell size. To determine whether PDE3B gene expression is regulated by PPAR gamma itself, we analyzed lean PPAR gamma (+/-) mice with adipocyte size comparable to control PPAR gamma (+/+) mice. In adipocytes of PPAR gamma (+/-) mice, PDE3B mRNA and protein were both reduced to 63% of wild-type levels. Basal PDE activity tended to be decreased to 70% of wild-type levels, and, similarly, insulin-induced PDE activity was significantly decreased to 70%. Thus, PPAR gamma is required for PDE3B gene expression independent of adipocyte size.     

Type III Cu mutants of Myrothecium verrucaria bilirubin oxidase

Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.