Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae

The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.     

Analysis of the essential DNA region for OsEBP-89 promoter in response to methyl jasmonic acid

In rice, the characterization of OsEBP-89 is inducible by various stress-or hormone-stimuli, including ethylene, abscisic acid (ABA), jasmonate acid (JA), drought and cold. Here, we report the investigation of essential DNA region within OsEBP-89 promoter for methyl jasmonic acid (MeJA) induction. PLACE analysis indicates that this promoter sequence contains multiple potential elements in response to various stimuli. First, we fused this promoter with GUS gene and analyzed its expression under MeJA treatment through Agrobacterium infiltration mediating transient expression in tobacco leaves. Our results revealed that this chimeric gene could be inducible by MeJA in tobacco leaves. To further determine the crucial sequences responsible for MeJA induction, we generated a series of deletion promoters which were fused with GUS reporter gene respectively. The results of transient expression of GUS gene driven by these mutant promoters show that the essential region for MeJA induction is positioned in the region between −1200 and −800 in OsEBP-89 promoter containing a G-box (−1127), which is distinct from the essential region containing ERE (−562) for ACC induction. In all, our finding is helpful in understanding the molecular mechanism of OsEBP-89 expression under different stimuli. OsEBP-89, essential DNA region, methyl jasmonic acid, transient assay, promoter, tobacco leaves Contributed equally to this work Supported by the National Basic Research Program of China (Grant No. 2006CB101700) and the National Natural Science Foundation of China (Grant Nos. 30671135, 30525034 and 30730060)     

Design,synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of 89Zr-labeled antibodies

Two compact and symmetrical bifunctional tetrahydroxamate chelators, 1 and 2, were synthesized and evaluated for labeling antibodies with ⁸⁹Zr for imaging with positron emission tomography. Using 2,2′-iminodiacetamide as the backbone, four hydroxamate-containing moieties coupled to the diacetamide nitrogen were used for ⁸⁹Zr labeling, while a pendant connected to the amino group provided an isothiocyanate group for coupling to the antibody. Both 1- and 2-conjugated Trastuzumab were labeled with ⁸⁹Zr efficiently (>90% radiolabeling yield), and their ⁸⁹Zr-labeled products maintained comparable immunoreactivity to Trastuzumab. Compared to ⁸⁹Zr-labeled deferoxamine-conjugated Trastuzumab, ⁸⁹Zr-1- and ⁸⁹Zr-2-Trastuzumab showed faster demetalation in mouse plasma, and also displayed higher bone uptake in mice. Despite suboptimal stability of ⁸⁹Zr complexes of 1 and 2, our design strategy led to tetrahydroxamate chelators for efficient ⁸⁹Zr labeling, and could be potentially modified to provide novel chelators with improved stability.     

89Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 (⁸⁹Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on⁸⁹Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to ⁸⁹Zr-mAb3481 administration. Subsequently, ex vivo ⁸⁹Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). ⁸⁹Zr-mAb3481 BT474 tumor uptake was remarkably high at SUV_mean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg ⁸⁹Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect ⁸⁹Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. ⁸⁹Zr-mAb3481 PET accurately reflected HER3 tumor status. ⁸⁹Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.     

OsBP-5与OsEBP-89两蛋白之间相互作用区域的确定

OsBP-5(MYC类转录因子)与OsEBP-89(AP2/EREBP类转录因子)两个蛋白之间可以相互作用,并能协同调控水稻waxy基因的表达.将OsBP-5与OsEBP-89 cDNA的不同限制性片段分别克隆到酵母双杂交系统的载体上,利用酵母双杂交的方法确定了OsEBP-89中与OsBP-5相互作用的区域位于AP2/EREBP保守域的RAYD元件内;OsBP-5中与OsEBP-89相互作用的区域则有两个区段,它们分别位于Pro68与Val171之间和Leu284与Gly335之间,而不是位于HLH保守域内.酵母系统中的实验还表明,OsEBP-89的3′端部分氨基酸在一定程度上防碍了它与OsBP-5蛋白的相互作用.     

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process. It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.     

Similarity Study on Peptide ?-turn Conformation Mimetics

The ability of a series of structures to mimic the geometric and electronic properties of an ideal –turn has been studied. Initially, an exhaustive conformational analysis was carried out using the molecular dynamics technique at high temperature followed by minimization. Additionally, each minimum was optimized with the semi–ab initio molecular orbital method SAM1. Then, the unique minima found have been superimposed with ideal –turns, classic and inverse, using the SEAL program which takes into account steric and electronic parameters for the superpositions and finally, three molecular similarity indices were determined for each superposition. These indices consider the general steric and electronic characteristics of the structures, as well as, the position of the carbon atoms that correspond to the Cⁱ and Cⁱ⁺² in the peptide chain.     

Development of the caput epididymidis studied by expressed proteins (a glutamate transporter,a lipocalin and β-galactosidase) in the c-<Emphasis Type="Italic">ros</Emphasis> knockout and wild-type mice with prepubertally ligated efferent ducts

Expression of a glutamate transporter (EAAC1), a lipocalin (MEP17) and -galactosidase (-Gal) in histological sections was used to monitor post-natal development of the murine epididymis. Three epithelia in the adult caput of wild-type mice were distinguished: I, the initial segment; II, the proximal caput; and III, the distal caput. The regions in which epithelia I, II and III were situated were called regions I, II and III, respectively. Regions I, II and III developed from a precursor epithelium present on day 14; from day 16, a presumptive region I epithelium was evident and, by day 21, epithelia characteristic of future regions II and III appeared. The relationship between the c-ros gene and the initial segment was studied by investigating the development of the caput epididymidis in transgenic homozygous c-ros knockout (–/–) mice that lack the initial segment, heterozygous (+/–) males and wild-type males in which the efferent ducts had been ligated prepubertally so that the initial segment failed to develop. In mice with prepubertally ligated efferent ducts, regions II and III developed normally but region I was missing in the adult and expression of c-ros was partially decreased. In (–/–) mice, the precursor epithelium was present, differentiation of epithelium II was delayed until day 32 and epithelium I never developed. Thus, caput region I develops before c-ros expression, high testosterone secretion and differentiation of regions II and III but not if the organ is deprived of the oncogene c-ros or testicular exocrine secretions. The caput of the knockout male lacks solely the initial segment so that the efferent ducts are in continuity with the post-initial segment, proximal caput region. The ligand for c-ros may be present in testicular fluid and both ligand and receptor may be necessary for differentiation of epithelia I and II.This work was funded by the Deutsche Forschungsgemeinschaft (the male gamete: production, maturation, function, FOR 197/3-1)