U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3' end processing

The 3' cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors.

Alteration of ZnT5-mediated zinc import into the early secretory pathway affects the secretion of growth hormone from rat pituitary cells

BACKGROUND Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts. METHODS In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot). RESULTS Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway. CONCLUSION Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated.

Development of the First Fluorescence Screening Assay for the SLC39A2 Zinc Transporter.

Zinc is an essential micronutrient that is crucial for many vital cellular functions such as DNA and protein synthesis, metabolism, and intracellular signaling. Therefore, the intracellular zinc concentration is tightly regulated by zinc transporters and zinc-binding proteins. The members of the SCL39 transporter family transport zinc into the cytosol. The SLC39A2 (hZIP2) protein is highly expressed in prostate epithelial cells and was found to be involved in prostate cancer development. Thus far, there is no specific modulator available for the SLC39 transporters. The aim of this study was to develop a screening assay for compound screening targeting hZIP2. Employing the pIRES2-DsRed Express 2 bicistronic vector, we detected human ZIP2 expression at the plasma membrane in transiently transfected HEK293 cells. Using the FLIPR Tetra fluorescence plate reader, we demonstrated that ZIP2 transports Cd(2+) with an apparent Km value of 53.96 nM at an extracellular pH of 6.5. The cadmium influx via hZIP2 was inhibited by zinc in a competitive manner. We found that hZIP2 activity can be measured using cadmium in the range of 0.1 to 10 µM with our assay. In summary, for the first time we developed an assay for human ZIP2 that can be adapted to other zinc transporters.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

Purification and characterization of a specific antigen from Echinococcus multilocularis.

A polypeptide (Em2a) purified by affinity chromatography from the Echinococcus multilocularis metacestode showed a high degree of purity as assayed by SDS-PAGE and analytical isoelectrical focusing. A minor contamination with host albumin was revealed. Estimation of relative mol. mass gave a value of 54,000. The isoelectric point was found to be 4.8. Antigenic activity of the polypeptide was demonstrated by immunoprecipitation and western blotting. In these assays the protein was recognized only by homologous sera from patients infected with larval E. multilocularis. This antigen (Em2a) did not react in the ELISA with sera from patients infected with heterologous helminths; these sera were highly cross-reacting with antigen from E. granulosus hydatid fluid. Seventy-three (94%) from 78 investigated patients (alveolar echinococcosis) showed a seropositive reaction with the polypeptide Em2a.

Nickel(II)-, cobalt(II)-, copper(II)-, and zinc(II)-phthalate and 1-methylimidazole coordination compounds: synthesis, crystal structures and magnetic properties

Three new coordination polymers [M(Pht)(1-MeIm)2]n (where M=Cu (1), Zn (2), Co (3); Pht2−=dianion of o-phthalic acid; 1-MeIm=1-methylimidazole) and two compounds [M(1-MeIm)6](HPht)2 · 2H2O (M=Co (4), Ni (5)) have been synthesized and characterized by X-ray crystallography. The structures of 1–3 (2 is isostructural to 3) consist of [M(1-MeIm)2] building units connected by 1,6-bridging phthalate ions to form infinite chains. In complex 1, each copper(II) center adopts a square coordination mode of N2O2 type by two O atoms from different phthalate ions and two N atoms of 1-MeIm, whereas in 3 two independent metal atoms are tetrahedrally (N2O2) coordinated to a pair of Pht ligands and a pair of 1-MeIm molecules. There are only van der Waals interactions between the chains in 1, while the three-dimensional network in 3 is assembled by C–H⋯O contacts. In contrast to polymers 1–3 the structures of 4 and 5 (complexes are also isostructural) are made up of the [M(1-MeIm)6]2+ cation, two hydrogen phthalate anions (HPht−) and two H2O solvate molecules. The coordination around each metal(II) atom is octahedral with six nitrogen atoms of 1-MeIm. Extended hydrogen bonding networks embracing the solvate water molecules and a phthalate residue as well as the weak C–H⋯O interactions stabilize the three-dimensional structures. Magnetic studies clearly show that the magnetic ions do not interact with each other. Furthermore, in compound 4 we have another example of a highly anisotropic Co2+ ion with a rhombic g-tensor and large zero-field-splitting. The complexes were also characterized by IR and 1H NMR spectroscopy, thermogravimetric analysis, and all data are discussed in the terms of known structures.

Zinc supplementation reduced DNA breaks in Ethiopian women.

Assessment of zinc status remains a challenge largely because serum/plasma zinc may not accurately reflect an individual's zinc status. The comet assay, a sensitive method capable of detecting intracellular DNA strand breaks, may serve as a functional biomarker of zinc status. We hypothesized that effects of zinc supplementation on intracellular DNA damage could be assessed from samples collected in field studies in Ethiopia using the comet assay. Forty women, from villages where reported consumption of meat was less than once per month and phytate levels were high, received 20 mg zinc as zinc sulfate or placebo daily for 17 days in a randomized placebo-controlled trial. Plasma zinc concentrations were determined by inductively coupled plasma mass spectrometry. Cells from whole blood at the baseline and end point of the study were embedded in agarose, electrophoresed, and stained before being scored by an investigator blinded to the treatments. Although zinc supplementation did not significantly affect plasma zinc, mean (± SEM) comet tail moment measurement of supplemented women decreased from 39.7 ± 2.7 to 30.0 ± 1.8 (P< .005), indicating a decrease in DNA strand breaks in zinc-supplemented individuals. These findings demonstrated that the comet assay could be used as a functional assay to assess the effects of zinc supplementation on DNA integrity in samples collected in a field setting where food sources of bioavailable zinc are limited. Furthermore, the comet assay was sufficiently sensitive to detect changes in zinc status as a result of supplementation despite no significant changes in plasma zinc.

Selective Transport of Zinc, Manganese, Nickel, Cobalt and Cadmium in the Root System and Transfer to the Leaves in Young Wheat Plants

• Background and Aims The uptake, translocation and redistribution of the heavy metals zinc, manganese, nickel, cobalt and cadmium are relevant for plant nutrition as well as for the quality of harvested plant products. The long-distance transport of these heavy metals within the root system and the release to the shoot in young wheat (Triticum aestivum ‘Arina’) plants were investigated. • Methods After the application of 65Zn, 54Mn, 63Ni, 57Co and 109Cd for 24 h to one seminal root (the other seminal roots being excised) of 54-h-old wheat seedlings, the labelled plants were incubated for several days in hydroponic culture on a medium without radionuclides. • Key Results The content of 65Zn decreased quickly in the labelled part of the root. After the transfer of 65Zn from the roots to the shoot, a further redistribution in the phloem from older to younger leaves was observed. In contrast to 65Zn, 109Cd was released more slowly from the roots to the leaves and was subsequently redistributed in the phloem to the youngest leaves only at trace levels. The content of 63Ni decreased quickly in the labelled part of the root, moving to the newly formed parts of the root system and also accumulating transiently in the expanding leaves. The 54Mn content decreased quickly in the labelled part of the root and increased simultaneously in leaf 1. A strong retention in the labelled part of the root was observed after supplying 57Co. • Conclusions The dynamics of redistribution of 65Zn, 54Mn, 63Ni, 57Co and 109Cd differed considerably. The rapid redistribution of 63Ni from older to younger leaves throughout the experiment indicated a high mobility in the phloem, while 54Mn was mobile only in the xylem and 57Co was retained in the labelled root without being loaded into the xylem.

Redistribution of Nickel, Cobalt, Manganese, Zinc, and Cadmium via the Phloem in Young and Maturing Wheat

The phloem mobility of heavy metals is relevant to the redistribution of micronutrients and pollutants and, ultimately, to the quality of harvested plant parts. The relative mobility in wheat may vary considerably between different cations. In the study reported here, radio-labeled nickel (Ni), cobalt (Co), manganese (Mn), zinc (Zn) and cadmium (Cd) were introduced into either intact young winter wheat (Triticum aestivum L. cv. Arina) via a leaf flap, or detached maturing shoots via the cut stem. Elements fed into the lamina of the second leaf of 21-day-old plants were translocated to the younger (expanding) leaves and to the roots but not or only in trace amounts to the first (already fully expanded) leaf. The 63Ni and 65Zn were exported more rapidly compared with the other heavy metals. Most of 54Mn was retained in the originally labeled leaf. The peduncle of some maturing shoots was steam-girdled below the ear to distinguish between xylem and phloem transport. This phloem interruption reduced the content of 63Ni in the ear to about 25%. Intermediate effects were observed for 65Zn, 57Co, and 109Cd. Total 54Mn accumulation in the ear was hardly affected by steam-girdling, indicating a transport of this element within the xylem to the ear. These results suggest that the relative phloem mobility of Ni and Zn in young wheat plants and in maturing wheat shoots is higher than the mobility of Co and Cd, whereas the mobility of Mn is very low.

Stereoselective virtual screening of the ZINC database using atom pair 3D-fingerprints

Background Tools to explore large compound databases in search for analogs of query molecules provide a strategically important support in drug discovery to help identify available analogs of any given reference or hit compound by ligand based virtual screening (LBVS). We recently showed that large databases can be formatted for very fast searching with various 2D-fingerprints using the city-block distance as similarity measure, in particular a 2D-atom pair fingerprint (APfp) and the related category extended atom pair fingerprint (Xfp) which efficiently encode molecular shape and pharmacophores, but do not perceive stereochemistry. Here we investigated related 3D-atom pair fingerprints to enable rapid stereoselective searches in the ZINC database (23.2 million 3D structures). Results Molecular fingerprints counting atom pairs at increasing through-space distance intervals were designed using either all atoms (16-bit 3DAPfp) or different atom categories (80-bit 3DXfp). These 3D-fingerprints retrieved molecular shape and pharmacophore analogs (defined by OpenEye ROCS scoring functions) of 110,000 compounds from the Cambridge Structural Database with equal or better accuracy than the 2D-fingerprints APfp and Xfp, and showed comparable performance in recovering actives from decoys in the DUD database. LBVS by 3DXfp or 3DAPfp similarity was stereoselective and gave very different analogs when starting from different diastereomers of the same chiral drug. Results were also different from LBVS with the parent 2D-fingerprints Xfp or APfp. 3D- and 2D-fingerprints also gave very different results in LBVS of folded molecules where through-space distances between atom pairs are much shorter than topological distances. Conclusions 3DAPfp and 3DXfp are suitable for stereoselective searches for shape and pharmacophore analogs of query molecules in large databases. Web-browsers for searching ZINC by 3DAPfp and 3DXfp similarity are accessible at www.gdb.unibe.ch webcite and should provide useful assistance to drug discovery projects.

A fast protocol for purification of translating mRNAs

We present a protocol that enables easy extraction of translating mRNAs by purification of tagged ribosomes and associated mRNAs. By studying three different examples of stress influenced genes, we demonstrate the effectiveness and compare our rapid method to an older protocol (Halbeisen et al., 2009).

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^