Yeasts

Mol Cell Biol, 2005 Feb, 25(3), 1089 - 99
Nuclear Oncoprotein Prothymosin {alpha} Is a Partner of Keap1: Implications for Expression of Oxidative Stress-Protecting Genes; Karapetian RN et al.; Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2 . In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1 . Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin alpha . The in vivo and in vitro data indicated that the prothymosin alpha-Keap1 interaction is direct, highly specific, and functionally relevant . Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action . Prothymosin alpha was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1 . In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin alpha by using prothymosin alpha overproduction and mRNA interference approaches . Our data attribute to prothymosin alpha the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin alpha and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.

J Cell Biol, 2005 Jan 17, 168(2), 185 - 91
Cell growth-dependent coordination of lipid signaling and glycosylation is mediated by interactions between Sac1p and Dpm1p; Faulhammer F et al.; The integral membrane lipid phosphatase Sac1p regulates local pools of phosphatidylinositol-4-phosphate (PtdIns(4)P) at endoplasmic reticulum (ER) and Golgi membranes . PtdIns(4)P is important for Golgi trafficking, yet the significance of PtdIns(4)P for ER function is unknown . It also remains unknown how localization of Sac1p to distinct organellar membranes is mediated . Here, we show that a COOH-terminal region in yeast Sac1p is crucial for ER targeting by directly interacting with dolicholphosphate mannose synthase Dpm1p . The interaction with Dpm1p persists during exponential cell division but is rapidly abolished when cell growth slows because of nutrient limitation, causing translocation of Sac1p to Golgi membranes . Cell growth-dependent shuttling of Sac1p between the ER and the Golgi is important for reciprocal control of PtdIns(4)P levels at these organelles . The fraction of Sac1p resident at the ER is also required for efficient dolichol oligosaccharide biosynthesis . Thus, the lipid phosphatase Sac1p may be a key regulator, coordinating the secretory capacity of ER and Golgi membranes in response to growth conditions.

J Cell Sci . 2005 Jan 18; {Epub ahead of print}
TRIP6 is a RIP2-associated common signaling component of multiple NF-{kappa}B activation pathways; Li L et al.; Receptor-interacting protein 2 (RIP2) is a member of the RIP kinase family that has been shown to be crucially involved in inflammation, innate and adaptive immune responses . The physiological and pathological roles of RIP2 are mediated through its involvement in multiple NF-kappaB activation pathways, including those triggered by tumor necrosis factor (TNF), interleukin 1 (IL-1), Toll-like receptor 2 (TLR2), TLR3, TLR4 and Nod1 . In this report, we identified the LIM-domain-containing protein TRIP6 as a RIP2-interacting protein in yeast two-hybrid screens . In mammalian cells, TRIP6 interacts with RIP2 in a TNF- or IL-1-dependent manner . Overexpression of TRIP6 potentiates RIP2-mediated NF-kappaB activation in a dose-dependent manner . The LIM domains of TRIP6 are responsible for its interaction with RIP2 . TRIP6 also interacts with TRAF2, a protein that is crucially involved in TNF signaling, as well as the IL-1 receptor, TLR2 and Nod1 . Overexpression of TRIP6 potentiates NF-kappaB activation by TNF, IL-1, TLR2 or Nod1, whereas a dominant negative mutant or RNA-interference construct of TRIP6 inhibits NF-kappaB activation by TNF, IL-1, TLR2 or Nod1 . Moreover, TRIP6 also potentiates RIP2- and Nod1-mediated ERK activation . These data have established a physical and functional association between TRIP6 and RIP2, and suggest that RIP2's involvement in multiple NF-kappaB and ERK activation pathways is mediated through TRIP6.

J Biol Chem . 2005 Jan 18; {Epub ahead of print}
Amplification of telomeric arrays via rolling-circle mechanism; Nosek J et al.; Alternative (telomerase-independent) lengthening of telomeres mediated through homologous recombination is often accompanied by a generation of extrachromosomal telomeric circles (t-circles), whose role in direct promotion of recombinational telomere elongation has been recently demonstrated . Here we present evidence that t-circles in a natural telomerase-deficient system of mitochondria of the yeast Candida parapsilosis replicate independently of the linear chromosome via a rolling-circle mechanism . This is supported by an observation of (i) single-stranded DNA (ssDNA) consisting of concatemeric arrays of telomeric sequence, (ii) lasso-shaped molecules representing rolling-circle intermediates, and (iii) preferential incorporation of deoxyribonucleotides into telomeric fragments and t-circles . Analysis of naturally occurring variant t-circles revealed conserved motifs with potential function in driving the rolling-circle replication . This data indicates that extrachromosomal t-circles observed in a wide variety of organisms including yeasts, plants, Xenopus laevis, and certain human cell lines may represent independent replicons generating telomeric sequences and thus actively participating in telomere dynamics . Moreover, due to the promiscuous occurrence of t-circles across phyla, the results from yeast mitochondria have implications related to the primordial system of telomere maintenance providing a paradigm for evolution of telomeres in nuclei of early eukaryotes.

J Biol Chem . 2005 Jan 18; {Epub ahead of print}
Molecular characterization of the human Calpha -formylglycine generating enzyme; Preusser-Kunze A et al.; Calpha-Formylglycine (FGly) is the catalytic residue in the active site of sulfatases . In eukaryotes, it is generated in the endoplasmic reticulum by posttranslational modification of a conserved cysteine residue . The FGly generating enzyme (FGE), performing this modification, is an endoplasmic reticulum resident enzyme that upon overexpression is secreted . Recombinant FGE was purified from cells and secretions to homogeneity . Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE . Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity . FGE is a calcium binding protein containing an N-terminal (residues 86-168) and a C-terminal (residues 178-374) protease-resistant domain . The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh and the fifth to the sixth cysteine residue . The inner most cysteine pair is partially reduced . The first two cysteine residues are located in the sequence preceding the N-terminal protease resistant domain . They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers . The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocrosslinking of a substrate peptide to proline 182 . Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.

Gene, 2005 Jan 3, 344, 171 - 80
The OsLti6 genes encoding low-molecular-weight membrane proteins are differentially expressed in rice cultivars with contrasting sensitivity to low temperature; Morsy MR et al.; Rice (Oryza sativa L.) is sensitive to chilling particularly at early stages of seedling establishment . Two closely related genes (OsLti6a, OsLti6b), which are induced by low temperature during seedling emergence were isolated from a cold tolerant temperate japonica rice cultivar . These genes are closely related to the Arabidopsis rare cold-inducible (RCI2) and barley low-temperature-inducible (BLT101) genes . Based on direct biochemical and indirect physiological evidence and similarity with a conserved protein domain in the Cluster of Orthologous Groups (COG) database (e.g., yeast PMP3), the rice genes belong to a class of low-molecular-weight hydrophobic proteins involved in maintaining the integrity of the plasma membrane during cold, dehydration and salt stress conditions . Both genes exhibit a genotype-specific expression signature characterized by early and late stress-inducible expression in tolerant and intolerant genotypes, respectively . The differences in temporal expression profiles are consistent with cultivar differences in cold-induced membrane leakiness and seedling vigor . The presence of CRT/DRE promoter cis-elements is consistent with the synchronized expression of OsLti6 genes with the C-repeat binding factor/drought responsive element-binding protein (CBF/DREB) transcriptional activator . The present results indicate that the Oslti6 genes are part of a battery of cold stress defense-related genes regulated by a common switch.

Gene, 2005 Jan 3, 344C, 93 - 103 Epub 2004 Nov 26.
Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis; Morgante PG et al.; The xeroderma pigmentosum complementation group B (XPB) protein is involved in both DNA repair and transcription in human cells . It is a component of the transcription factor IIH (TFIIH) and is responsible for DNA helicase activity during nucleotide (nt) excision repair (NER) . Its high evolutionary conservation has allowed identification of homologous proteins in different organisms, including plants . In contrast to other organisms, Arabidopsis thaliana harbors a duplication of the XPB orthologue (AtXPB1 and AtXPB2), and the proteins encoded by the duplicated genes are very similar (95% amino acid identity) . Complementation assays in yeast rad25 mutant strains suggest the involvement of AtXPB2 in DNA repair, as already shown for AtXPB1, indicating that these proteins may be functionally redundant in the removal of DNA lesions in A . thaliana . Although both genes are expressed in a constitutive manner during the plant life cycle, Northern blot analyses suggest that light modulates the expression level of both XPB copies, and transcript levels increase during early stages of development . Considering the high similarity between AtXPB1 and AtXPB2 and that both of predicted proteins may act in DNA repair, it is possible that this duplication may confer more flexibility and resistance to DNA damaging agents in thale cress.

J Biochem Biophys Methods, 2005 Jan 31, 62(1), 1 - 12
Reconstitution of glycopeptide export in mixed detergent-solubilised and resealed microsomes depleted of lumenal components; Ali BR et al.; Export of macromolecules from the endoplasmic reticulum (ER) lumen into the cytosol is a major aspect of the quality control systems operating within the early secretory system . Glycopeptides are exported from the ER by an ATP- and GTP-dependent pathway, which shares many similarities to the protein export system . Significantly, for glycopeptides, there is no requirement for cytosolic factors, biochemically distinguishing the glycopeptide and protein paths and probably reflecting the lower conformational complexity of the former substrate . Genetic studies in yeast, and biochemical data from higher eukaryotes, indicate that glycopeptides utilise the Sec61 translocon . Here, we report a new system allowing access to lumenal ER components, facilitating assessment of their importance in glycopeptide retrotranslocation and potentially other processes . Saponin, in combination with CHAPS, but not saponin alone, facilitated removal of >95% of lumenal protein disulphide isomerase (PDI) and BiP . Upon resealing, these microsomes retained glycopeptide export competence . These data suggest that the majority of lumenal components of the ER are most likely nonessential for glycopeptide export . In addition, export competence was highly sensitive to the addition of external protease, indicating a role for protein factors with cytoplasmically exposed determinants.

BMC Cell Biol . 2005 Jan 18;6(1):3 {Epub ahead of print}
Hinderin, a five-domains protein including coiled-coil motifs that binds to SMC3; Patel CA et al.; BACKGROUND: The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase . The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions . RESULTS: In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait . This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins . Hinderin is ubiquitously expressed in human tissues . Orthologue forms of the protein are present in other vertebrates but not in lower organisms . A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3 . Hinderin/SMC3 complexes could be recovered in the immunoprecipitate from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo . On the contrary, Hinderin did not interact with SMC1 . In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin . CONCLUSIONS: Hinderin is a novel binding partner of SMC3 . Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.

Biochem J . 2005 Jan 19; {Epub ahead of print}
alphaII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts; Rotter B et al.; The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membrane is presumed to be involved in the stabilisation of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions . Spectrin tetramers made of alpha- and beta- subunits, are linked to actin microfilaments forming a network that binds a multitude of proteins . The most prevalent alpha-spectrin subunit in nonerythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, which host an SH3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains . Using yeast two-hybrid screening of kidney libraries, we identified two partners of the alpha9-alpha10 repeats: the potential tumour suppressor Tes, an actin binding protein mainly located at focal adhesions; and EVL, another actin-binding protein, equally recruited at focal adhesions . Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation . In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM domain of Tes and by the alpha10 repeat of alphaII-spectrin while EVL interacts with the SH3 domain located within alpha9 repeat . Morover we describe an in vitro interaction between Tes and EVL, and a colocalisation of these two proteins at focal adhesions . These interactions between alphaII-spectrin, Tes and EVL could indicate new functions for spectrin in actin dynamics and focal adhesions.

Bioconjug Chem, 2005 Jan-Feb, 16(1), 105 - 12
Synthesis and in Vitro Characterization of Organometallic Rhenium and Technetium Glucose Complexes against Glut 1 and Hexokinase; Schibli R et al.; A series of nine organometallic technetium-99m and rhenium complexes of glucose are presented and characterized in vitro regarding their potential as surrogates of {(18)F}-2-fluoro-desoxy glucose ({(18)F}-FDG) . The glucose derivatives are functionalized at positions C-1, C-2, C-3, and C-6 . Different spacer lengths and chelating systems have been introduced at these sites . For the (radio)labeling, the organometallic precursors {(99m)Tc(H(2)O)(3)(CO)(3)}(+) and {ReBr(3)(CO)(3)}(2)(-) respectively have been used . The resulting complexes have been characterized chemically and radiochemically . The formation of uniform products has been observed on the macroscopic (Re) and no-carrier-added level ((99m)Tc) . The Tc-99m complexes revealed good inertness against ligand exchange (Cys and His) and excellent stability in physiological buffered saline as well as in human plasma over a period of 24 h at 37 degrees C . The rhenium complexes have been tested for competitive inhibition of the (yeast) hexokinase . Only for C-2 derivatized glucose complexes with extended spacer functionalities K(i) values in the millimolar and sub-millimolar range have been observed . In silico molecular docking experiments supported these experimental findings . However, the competitive inhibitors are not recognized as a pseudosubstrate of hexokinase . The cellular uptake of all (99m)Tc-complexes into HT-29 colon carcinoma cells via Glut1 was generally low and unspecific independent of the position at the hexose ring, the chelating systems, or the overall charge of the corresponding metal complexes . The current results seem to preclude the use of these compounds as {(18)F}-FDG surrogates primarily due to the low cellular uptake via Glut1.

Cell Cycle . 2005 Jan 21;4(1) {Epub ahead of print}
A Case of Selfish Nucleolar Segregation; Strunnikov AV; Mitotic segregation of nucleolus in fission and budding yeast proceeds without disassembling its complex structure, creating challenging problems for transmission of nucleolus-organizing regions during nuclear division . The SMC complex called condensin, which plays a leading role in organizing mitotic structure of chromosomes in all eukaryotes, is essential for nucleolar segregation in budding yeast, where rDNA chromatin is the main target of mitotic condensin activity . Mitosis-specific condensin targeting to the nucleolus presents an attractive model to study mechanisms controlling condensin binding to specific chromatin domains . Recent reports suggest that the early-anaphase release of Cdc14 from the nucleolus (FEAR pathway) controls the proficiency of nucleolar segregation by promoting the mitotic condensin function in rDNA . This finding uncovers an essential function for the FEAR pathway and postulates the unique nucleolar self-regulatory mechanism, which evolved to recruit two essential enzymatic activities, Cdc14 phosphatase and condensin ATP-dependent supercoiling, for the specific task of segregating nucleoli without their disassembly.

Cell Cycle . 2005 Jan 13;4(1) {Epub ahead of print}
The Nucleolus: Playing by Different Rules?
Shaw P, Doonan J.
The nucleolus, although an integral part of all eukaryotic nuclei, plays by its own rules in many respects . Cytologically, it is the most prominent subnuclear domain; functionally, it is the site of transcription of 3 of the ribosomal RNAs from the tandemly repeated rDNA, and subsequently of ribosome biogenesis; biochemically, it possesses transcriptional and post-transcriptional machinery not shared with the rest of the nucleus . Making the huge number of ribosomes required by the cell represents an enormous investment of metabolic activity, and so nucleolar function can easily become a de facto limit to cell growth: nucleolar activity must also be actively regulated, but the detailed regulatory networks linking the nucleolus with cellular metabolism are still unclear . Several recent reports have now shown that segregation of the rDNA in yeast, along with telomeric DNA, is also controlled differently from the rest of the genome . This short review describes some of the features of the nucleolus and highlights recent progress in understanding this important, but enigmatic, nuclear structure.

Cell Cycle . 2005 Feb 3;4(2) {Epub ahead of print}
Revisiting the "Cdk-Centric" View of the Mammalian Cell Cycle; Malumbres M; Since the early genetic studies in yeast, regulation of the cell cycle has been associated to the sequential activation of several proline-directed serine-threonine protein kinases by cyclins . From yeast to humans, the activiy of these cyclin-dependent kinases (Cdks) have been thought to be essential for cell cycle regulation . Recent gene-targeted mouse models for different cyclins and Cdks have shown that members of these families show a certain level of redundancy and that specific complexes are not required for the mitotic cell cycle . However, the complexity of the Cdk-cyclin network and the promiscuity of their members makes it difficult to understand the relative contribution of these proteins to the mammalian cell division cycle . Compensatory roles by non-Cdk activities and Cdk-independent functions of cyclins are increasing the complexity of the current simplistic models . We still do not know whether at least one cyclin-dependent kinase activity is required for cell cycle progression in mammalian cells . Indeed, a relevant question for cancer therapy.

Cell Cycle . 2005 Feb 13;4(2) {Epub ahead of print}
The Second Subunit of the Replication Factor C complex (RFC40) and the Regulatory Subunit (RIalpha) of Protein Kinase A Form a Protein Complex Promoting Cell Survival; Gupte RS et al.; Replication Factor C (RFC) is required for the loading of Proliferating Cell Nuclear Antigen (PCNA) onto DNA during DNA replication, repair and recombination . RFC40, the second subunit of the RFC complex, and PCNA have been shown to be overexpressed in gestational trophoblastic diseases . Using RFC40 as the bait in a yeast two-hybrid screening, we have identified a novel interaction between RFC40 and the regulatory subunit (RIalpha) of cAMP-dependent Protein kinase A (PKA) . The interaction sites between these two proteins were investigated and mapped to the N-terminus of RIalpha and the C-terminus of RFC40 . Moreover, it was demonstrated that the C-subunit of PKA was not associated with the RFC40-RIalpha complex . Furthermore, RFC37, the third subunit of the RFC complex, competes with RIalpha and displaces it from the RFC40-RIalpha complex . Interestingly, downregulation of endogenous RIalpha by 8-chloro cAMP, in MCF7 breast cancer cells led to reduction in the amount of RFC40-RIalpha complex, together with decrease in cell survival.

Cell Cycle . 2005 Feb 7;4(2) {Epub ahead of print}
Cell Cycle-Dependent Regulation of Double-Strand Break Repair: A Role for the CDK; Aylon Y et al.; DNA Double-Strand Breaks (DSBs) are dangerous lesions that can lead to genomic instability and to cell death . Eukaryotic cells repair DSBs either by nonhomologous end joining (NHEJ) or by homologous recombination (HR) . Recent work has allowed to study the ability of yeast cells to repair a single, chromosomal DSB, at different stages of the cell cycle . Yeast cells repair the broken chromosome during the G(1) stage only by NHEJ, whereas HR is the mechanism of choice during the rest of the cell cycle . HR does not require duplicated chromatids or passage through S-phase . Control over the fate of the broken chromosome is exerted by Clb-CDK activity, which is required to carry out the first step of HR, ssDNA resection . Similar results in other organisms suggest that this control is a conserved feature in all eukaryotes.

Biochem J . 2005 Jan 18; {Epub ahead of print}
The trans-Golgi network GRIP-domain proteins form alpha-helical homodimers; Luke MR et al.; A recently described family of trans-Golgi network proteins, which all contain a GRIP domain targeting sequence, have been proposed to play a role in membrane transport . Based on the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled coil regions which have the potential to mediate protein-protein interactions . Four mammalian GRIP domain proteins have been identified which are targeted to the trans-Golgi network via their GRIP domains, namely p230, golgin-97, GCC88 and GCC185 . Here we have investigated the ability of the four mammalian GRIP domain proteins to interact . Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, crosslinking experiments and yeast two-hybrid interactions we have established that the GRIP proteins can self-associate to form homodimers exclusively . Two-hybrid analysis indicated that the N- and C- terminal fragments of GCC88 can interact with themselves but not with each other, suggesting GRIP domain proteins form parallel coiled coil dimers . Analysis of purified recombinant golgin-97 by circular dichoism spectroscopy indicated a 67% alpha-helical structure, consistent with a high content of coiled coil sequences . These data support a model for GRIP domain proteins as extended rod-like homodimeric molecules . The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently.

Nat Biotechnol . 2005 Jan 16; {Epub ahead of print}
Functional annotation and network reconstruction through cross-platform integration of microarray data; Zhou XJ et al.; The rapid accumulation of microarray data translates into a need for methods to effectively integrate data generated with different platforms . Here we introduce an approach, 2(nd)-order expression analysis, that addresses this challenge by first extracting expression patterns as meta-information from each data set (1(st)-order expression analysis) and then analyzing them across multiple data sets . Using yeast as a model system, we demonstrate two distinct advantages of our approach: we can identify genes of the same function yet without coexpression patterns and we can elucidate the cooperativities between transcription factors for regulatory network reconstruction by overcoming a key obstacle, namely the quantification of activities of transcription factors . Experiments reported in the literature and performed in our lab support a significant number of our predictions.

Genetics . 2005 Jan 16; {Epub ahead of print}
SepBCTF4 is required for the formation of DNA damage-induced UvsCRAD51 foci in Aspergillus nidulans; Gygax S et al.; SepB is an essential, conserved protein required for chromosomal DNA metabolism in Aspergillus nidulans . Homologues of SepB include yeast Ctf4p and human hAND-1 . Molecular and bioinformatic characterization of these proteins suggests that they act as molecular scaffolds . Furthermore, recent observations implicate the yeast family members in lagging strand replication and the establishment of sister chromatid cohesion . Here, we demonstrate that SepB functions in the A . nidulans DNA damage response . In particular, analysis of double mutants reveals that SepB is a member of the UvsC(RAD51) epistasis group . In accord with this prediction, we show that UvsC(RAD51) forms DNA damage-induced nuclear foci in a manner that requires SepB function . We also provide evidence that implicates SepB in sister chromatid cohesion, thereby suggesting that cohesion may play a role in regulating the localization and/or assembly of UvsC(RAD51) complexes.

Genetics . 2005 Jan 16; {Epub ahead of print}
Rapid subfunctionalization accompanied by prolonged and substantial neofunctionalization in duplicate gene evolution; He X et al.; Gene duplication is the primary source of new genes . Duplicate genes that are stably preserved in genomes usually have divergent functions . The general rules governing the functional divergence, however, are not well understood and are controversial . The neofunctionalization (NF) hypothesis asserts that after duplication one daughter gene retains the ancestral function while the other acquires new functions . In contrast, the subfunctionalization (SF) hypothesis argues that duplicate genes experience degenerate mutations that reduce their joint levels and patterns of activity to that of the single ancestral gene . We here show that neither NF nor SF alone adequately explains the genome-wide patterns of yeast protein interaction and human gene expression for duplicate genes . Instead, our analysis reveals rapid SF, accompanied by prolonged and substantial NF in a large proportion of duplicate genes, suggesting a new model termed sub-neo-functionalization (SNF) . Our results demonstrate that enormous numbers of new functions have originated via gene duplication.

J Biol Chem . 2005 Jan 14; {Epub ahead of print}
R1, a novel repressor of the human monoamine oxidase A; Chen K et al.; Monoamine oxidase catalyzes the oxidative deamination of a number of neurotransmitters . Deficiency in monoamine oxidase A results in aggressive behavior in both human and mice . Studies on the regulation of monoamine oxidase A gene expression have shown that Sp1 family is important for monoamine oxidase A expression . In order to search for novel transcription factors, the sequence of three Sp1 sites in monoamine oxidase A core promoter was used in the yeast one-hybrid system to screen a human cDNA library . A novel repressor, R1 (RAM2), has been cloned . The R1 cDNA encodes a protein with 454 amino acids and an open reading frame at the 5' end . Transfection of R1 in a human neuroblastoma cell line, SK-N-BE(2)-C, inhibited the monoamine oxidase A promoter and enzymatic activity . The degree of inhibition of monoamine oxidase A by R1 correlated with the level of R1 protein expression . R1 was also found to repress monoamine oxidase A promoter activity within a natural chromatin environment . Gel-shift assay indicated that the endogenous R1 protein in SK-N-BE(2)-C cells interacted with R1 binding sequence . R1 also bound directly to the natural monoamine oxidase A promoter in vivo as shown by chromatin immunoprecipitation assay . Immunocytochemical analysis showed that R1 was expressed in both cytosol and nucleus which suggested a role for R1 in transcriptional regulation . Northern blot analysis revealed the presence of endogenous R1 mRNA in human brain and peripheral tissues . Taken together, this study shows that R1 is a novel repressor which inhibits monoamine oxidase A gene expression.

J Cell Sci, 2005 Jan 15, 118(Pt 2), 283 - 90
The developmental cell biology of Trypanosoma brucei; Matthews KR; Trypanosoma brucei provides an excellent system for studies of many aspects of cell biology, including cell structure and morphology, organelle positioning, cell division and protein trafficking . However, the trypanosome has a complex life cycle in which it must adapt either to the mammalian bloodstream or to different compartments within the tsetse fly . These differentiation events require stage-specific changes to basic cell biological processes and reflect responses to environmental stimuli and programmed differentiation events that must occur within a single cell . The organization of cell structure is fundamental to the trypanosome throughout its life cycle . Modulations of the overall cell morphology and positioning of the specialized mitochondrial genome, flagellum and associated basal body provide the classical descriptions of the different life cycle stages of the parasite . The dependency relationships that govern these morphological changes are now beginning to be understood and their molecular basis identified . The overall picture emerging is of a highly organized cell in which the rules established for cell division and morphogenesis in organisms such as yeast and mammalian cells do not necessarily apply . Therefore, understanding the developmental cell biology of the African trypanosome is providing insight into both fundamentally conserved and fundamentally different aspects of the organization of the eukaryotic cell.

J Biol Chem . 2005 Jan 14; {Epub ahead of print}
The c-myc DNA unwinding element binding protein modulates the assembly of DNA replication complexes in vitro; Casper JM et al.; The presence of DNA unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein binding sites, or both . We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE B, with a predicted mass of 23.4 kDa . Based on homology to yeast proteins DUE-B was previously classified as an amino acyl tRNA synthetase, however the human protein is approximately 60 amino acids longer than its orthologues in yeast or worms, and is primarily nuclear . In vivo, chromatin bound DUE-B localizes to the c myc DUE region . DUE-B levels are constant over the cell cycle although the protein is preferentially phosphorylated in cells arrested early in S phase . Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G1 to S phase, and induced cell death . DUE-B extracted from HeLa cells or expressed from baculovirus migrates as a dimer during gel filtration, and co-purifies with ATPase activity . In contrast to endogenous DUE-B, baculovirus expressed DUE-B efficiently forms high molecular weight complexes in Xenopus egg or HeLa extracts . In Xenopus extracts baculovirus expressed DUE B inhibits chromatin replication and RPA loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication . Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.

J Biol Chem . 2005 Jan 14; {Epub ahead of print}
SIRT3, a mitochondrial Sirtuin deacetylase, regulates mitochondrial function and thermogenesis in Brown adipocytes; Shi T et al.; SIRT3 is one of the seven mammalian sirtuin homologues of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and C.elegans . Since adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat . We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane . Caloric restriction activates SIRT3 expression in both white and brown adipose . Additionally, cold exposure up regulates SIRT3 expression in brown fat, while elevated climate temperature reduces the expression . Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1a, UCP1, and a series of mitochondria related genes . Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action . Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression . This inhibitive effect can be abolished by the co-expression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action . In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly . Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species (ROS) production, while increasing cellular respiration . Finally, SIRT3, along with genes related to mitochondrial function, is down regulated in the brown adipose tissue of several genetically obese mice . In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.

J Biol Chem . 2005 Jan 14; {Epub ahead of print}
Probing the mechanism of the Archaeoglobus fulgidus inositol-1-phosphate-synthase; Neelon K et al.; Myo-inositol-1-phosphate synthase (mIPS) catalyzes the conversion of glucose-6-phosphate (G-6-P) to inositol-1-phosphate . In the sulfate reducing archaeon Archaeoglobus fulgidus it is a metal dependent thermozyme that catalyzes the first step in the biosynthetic pathway of the unusual osmolyte di-myo-1,1'-phosphate . Several site-specific mutants of the archaeal mIPS were prepared and characterized to probe the details of the catalytic mechanism that was suggested by the recently solved crystal structure and by the comparison to the yeast mIPS . Six charged residues in the active site (Asp225, Lys274, Lys278, Lys306, Asp332, and Lys367), and two non-charged residues (Asn255 and Leu257) have been changed to alanine . The charged residues are located at the active site, and, were proposed to play binding and/or direct catalytic roles while non-charged residues are likely to be involved in proper binding of the substrate . Kinetic studies showed that only N255A retains any measurable activity, while two other mutants, K306A and D332A, can carry out the initial oxidation of G-6-P and reduction of NAD+ to NADH . The rest of the mutant enzymes show major changes in binding of G-6-P (monitored by the 31P linewidth of inorganic phosphate when G-6-P is added in the presence of EDTA) or NAD+ (detected via changes in the protein intrinsic fluorescence) . Characterization of these mutants provides new twists on the catalytic mechanism previously proposed for this enzyme.

Nucleic Acids Res, 2005 Jan 13, 33(1), 347 - 55 Print 2005.
RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans; Tops BB et al.; In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline . One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner . The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease . Here, we show that the MUT-7 protein resides in complexes of approximately 250 kDa in the nucleus and in the cytosol . In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size . This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production . Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex . This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing . Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo . Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.

Biol Chem, 2004 Dec, 385(12), 1203 - 8
Nuclear fibroblast growth factor-2 interacts specifically with splicing factor SF3a66; Gringel S et al.; Fibroblast growth factor 2 (FGF-2) has a dual role as a classical extracellular signaling protein and as an intracellular factor . Isoforms of FGF-2, resulting from alternatively used start codons on one mRNA species, locate differentially to nuclear compartments . In this study we aimed to analyze functions of intracellular FGF-2 by identification of interacting proteins . We identified the 66-kDa subunit of splicing factor 3a (SF3a66) as a binding partner in a yeast two-hybrid screen and confirmed this interaction by pull-down assays . The splicing factor interacted with the 18-kDa (FGF-2(18)) and with the 23-kDa (FGF-2(23)) isoforms, indicating an interaction with a domain common to both isoforms . Moreover, FGF-2 interacted with the C-terminus of SF3a66, a sequence that has not previously been assigned a functional role . In a functional neurite outgrowth assay, SF3a66 enhanced neurite lengths similar to FGF-2(18) . We have previously identified the spliceosomal assembly factor survival of motoneuron (SMN) protein as a protein interacting specifically with the FGF-2(23) isoform {Claus et al., J . Biol . Chem . 278 (2003), 479-485} . The identification of two FGF-2 interacting proteins from the same biochemical pathway suggests a novel intranuclear role of FGF-2.

Clin Chim Acta, 2005 Feb, 352(1-2), 217 - 24
Effects of Xuezhikang, an extract of cholestin, on lipid profile and C-reactive protein: a short-term time course study in patients with stable angina; Li JJ et al.; BACKGROUND: Reduction of cholesterol and inflammation can be achieved by administration of a statin . Xuezhikang, an extract of cholestin, available from Chinese red yeast rice, could effectively modify the lipid profile . However, limited information is available regarding rapid effects of Xuezhikang on plasma C-reactive protein (CRP) and the lipid profile in patients with stable angina . We evaluated the short-term time course effects of lipid profile and CRP by Xuezhikang in patients with stable angina . METHODS: Forty-eight consecutive patients with stable angina were randomly assigned to 1200 or 2400 mg/day of Xuezhikang . Blood samples were drawn at days 0, 1, 7 and 14 for lipid profile and CRP levels in all patients, and hepatic enzymes were also evaluated at days 0 and 14 . RESULTS: Both doses of Xuezhikang induced significant reductions in median CRP levels and in mean CRP levels at day 1 (13.0% with 1200 and 16.6% with 2400 mg/day; 14.7% with 1200 and 18.4% with 2400 mg/day), and at day 7 (18.3% with 1200 and 20.2% with 2400 mg/day; 18.5% with 1200 and 22.6% with 2400 mg/day) as well as at day 14 (28.6% with 1200 and 30.4% with 2400 mg/day; 21.7% with 1200 and 24.8% with 2400 mg/day) compared with baseline without a dose-dependent effect but a time-dependent manner . In addition, no changes were found at days 1 and 7 regarding lipid profile . However, both doses of Xuezhikang induced significant reductions in total cholesterol (TC, 13% and 22%), and low-density lipoprotein (LDL) cholesterol (23% and 32%) compared with baseline at day 14 . The higher dose of Xuezhikang (2400 mg/day) resulted in significantly greater reductions in TC and LDL cholesterol compared with 1200 mg/day group (p<0.05, p<0.01, respectively) . A less significant reduction was observed in triglycerides (TG) level (13% and 23%) compared with TC and LDL cholesterol . There was no significant difference in mean high-density lipoprotein (HDL) cholesterol levels compared with baseline in both groups . CONCLUSIONS: Xuezhikang resulted in rapid reduction of CRP within 24 h and lipid profile within 2 weeks, which may be clinically important for patients with coronary artery disease.

Biochem Biophys Res Commun, 2005 Feb 25, 327(4), 1052 - 7
Biosynthesis of epoxyeicosatrienoic acids varies between polymorphic CYP2C enzymes; Lundblad MS et al.; Arachidonic acid is oxidized by cytochromes P450 2C (CYP2C) to epoxyeicosatrienoic acids (EETs), possessing vasoactive properties, with 11,12-EET as the endothelium derived hyperpolarization factor . Genetic variants of CYP2C enzymes have altered drug metabolizing capacity . Our primary aim was to determine whether EET biosynthesis differed in human liver microsomes with known CYP2C genotypes . Human liver microsomes (n=25) of different CYP2C genotypes or yeast-expressed CYP2C enzymes were used . Analysis of metabolites was performed by liquid chromatography/mass spectrometry . Samples genotyped as CYP2C8*3/*3/CYP2C9*2/*2 exhibited a 34% (p<0.05) decreased EET biosynthesis, compared to other CYP2C8/CYP2C9 haplotypes . Inhibition experiments suggested CYP2C8 and CYP2C9 to be the predominant catalysts of EETs . We found no differences between the three recombinantly expressed CYP2C9 variants, but CYP2C8.1 had lower K(m) than these isoforms . In conclusion, there are genetic differences in the CYP2C-dependent oxidation of arachidonic acid to vasoactive metabolites, of which the relevance to cardiovascular pathophysiology is still unclear.

Cell, 2005 Jan 14, 120(1), 73 - 84
A Series of Ubiquitin Binding Factors Connects CDC48/p97 to Substrate Multiubiquitylation and Proteasomal Targeting; Richly H et al.; Protein degradation in eukaryotes usually requires multiubiquitylation and subsequent delivery of the tagged substrates to the proteasome . Recent studies suggest the involvement of the AAA ATPase CDC48, its cofactors, and other ubiquitin binding factors in protein degradation, but how these proteins work together is unclear . Here we show that these factors cooperate sequentially through protein-protein interactions and thereby escort ubiquitin-protein conjugates to the proteasome . Central to this pathway is the chaperone CDC48/p97, which coordinates substrate recruitment, E4-catalyzed multiubiquitin chain assembly, and proteasomal targeting . Concomitantly, CDC48 prevents the formation of excessive multiubiquitin chain sizes that are surplus to requirements for degradation . In yeast, this escort pathway guides a transcription factor from its activation in the cytosol to its final degradation and also mediates proteolysis at the endoplasmic reticulum by the ERAD pathway.

Exp Cell Res, 2005 Feb 15, 303(2), 457 - 470
Identification of MAGI-3 as a transforming growth factor-alpha tail binding protein; Franklin JL et al.; The cytoplasmic domain of the transforming growth factor-alpha precursor (proTGFalpha) contains a C-terminal PSD-95/SAP90, Discs Large, and Zona Occludens-1 (PDZ) recognition motif (TVV) . By yeast two-hybrid screening of a mouse embryo library, we have found that a third member of a family of PDZ-containing proteins, membrane associated guanylate kinase inverted-3 (MAGI-3), binds to TGFalpha's TVV . MAGI-3 is widely expressed in multiple mouse tissues, including brain . Immunolocalization showed that MAGI-3 and TGFalpha were colocalized in neurons in the cortex and dentate gyrus, as well as in ependymal cells and some astrocytes . In vitro, proTGFalpha bound the PDZ-1 domain of MAGI-3 and MAGI-2, but not MAGI-1 . MAGI-3 and the 17-kDa cell surface form of proTGFalpha interact transiently in MDCK cells stably transfected with both MAGI-3 and human proTGFalpha cDNAs . MAGI-3 and wild-type proTGFalpha colocalize at the cell surface . In contrast, MAGI-3 forms a stable complex with membrane-fixed TGFalpha early in the secretory pathway and interacts with immature and cell surface forms of membrane-fixed TGFalpha . Overexpression of MAGI-3 resulted in increased levels of TGFalpha in the basolateral medium of polarized MDCK cells, suggesting that MAGI-3 has a role in efficient trafficking of TGFalpha to the cell surface in polarized epithelial cells.

Exp Cell Res, 2005 Feb 15, 303(2), 432 - 46 Epub 2004 Nov 13.
Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining; Ashley N et al.; Human mitochondria DNA (mtDNA) is arranged within the mitochondria into discrete DNA-protein complexes, termed nucleoids . The size of the human mitochondrial genome is less than that of yeast and is more difficult to visualise by fluorescent DNA stains such as DAPI and Hoescht . We have developed a simple yet effective method to visualise mtDNA in situ within living cells using the fluorescent stain PicoGreen . Quantitative analysis shows that PicoGreen can be used to estimate the degree of mtDNA depletion within living cells . We have used this approach to study the arrangement and fluorescence of nucleoids in cells depleted of mtDNA by treatment with the anti-viral nucleoside analogue, 2',3'-dideoxycytidine . We also studied the distribution of mtDNA in fibroblasts cultured from patients with mitochondrial disease . Combining PicoGreen staining with histochemical and immunocytochemical approaches enabled us to examine the effects of mtDNA depletion on mtDNA-related components at the level of single cells . This method is able to detect an intermediate degree of mtDNA depletion in living cells, and can be used to detect mtDNA free cells (rho(0) cells) in culture even at very low numbers . We have also adapted the technique to efficiently sort rho(0) cells from populations of normal cells by fluorescent-assisted cell sorting (FACS), without the need for selection of respiratory competence . This should be useful for the construction of new trans-mitochondrial 'cybrid' cell lines.

Exp Cell Res, 2005 Feb 15, 303(2), 343 - 359 Epub 2004 Nov 2.
dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats; Baladron V et al.; The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development . The molecular mechanisms by which dlk regulates cell differentiation remain unknown . By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner . Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity . Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk . The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression . On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression . These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.

Biochem Pharmacol, 2005 Feb 1, 69(3), 525 - 529 Epub 2004 Dec 10.
Identification and functional analysis of two rare allelic variants of the thiopurine S-methyltransferase gene, TPMT*16 and TPMT*19; Hamdan-Khalil R et al.; Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs . TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy . During routine genotyping of patients with Crohn's disease, one novel missense mutation, 365A>C (TPMT*19, Lys(122)Thr), and a recently described missense mutation, 488G>A (TPMT*16, Arg(163)His), were identified in a Caucasian and a Moroccan patient, respectively . Using a heterologous yeast expression system, kinetic parameters (K(m) and V(max)) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme . The Lys(122)Thr exchange did not significantly decrease the intrinsic clearance value (V(max)/K(m)) of the variant enzyme . In contrast, the Arg(163)His substitution significantly decreased the intrinsic clearance value by three-fold . The Arg(163) is located in a highly conserved region of the human TPMT protein and, as such, the Arg(163)His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data . Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys(122)Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg(163)His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data . The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity . These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy.

Biochem J . 2005 Jan 17; {Epub ahead of print}
Lsb5p interacts with actin regulators Sla1p and Las17p, ubiquitin and Arf3p to couple actin dynamics to membrane trafficking processes; Costa R et al.; The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified . Sla1p is a well characterised yeast protein that binds both activators of actin dynamics, Las17p and Pan1p, and to cargo proteins such as the pheromone receptor Ste2p . Previously we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localises to the plasma membrane . Lsb5p has a similar structure to the GGA family of proteins with an N-terminal VHS domain and a GAT domain . It does not however contain either a gamma adaptin ear, or a clathrin binding motif . Here we have further defined its interaction site with both Sla1p and with Las17p, two regulators of actin dynamics . The site of interaction with Sla1p involves Sla1 HD1 domain which also was previously shown to interact with the pheromone receptor Ste2p . We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin . Finally, we demonstrate a requirement for Arf3p expression in order to localise Lsb5p to the correct cortical location in cells . Taken together our data provide further evidence for the role of Lsb5p in membrane trafficking events at the plasma membrane and also demonstrate for the first time an interaction of Arf3 with the endocytic machinery in yeast.

Yi Chuan Xue Bao, 2004 Nov, 31(11), 1321 - 6
{Progress on cis-acting regulatory elements in nonsense-mediated mRNA decay}; Huang Z et al.; In eukaryotic cells, nonsense-mediated mRNA decay (NMD) is an effective mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons ( PTC) and protects cells from the potentially deleterious effects of truncated proteins . Some cis-acting regulatory elements have been reported involving in NMD . They are PTC presence, PTC recongnation by downstream elements that termed as downstream sequence element (DSE) in yeast and primarily exon-exon junction ( EEJ) in mammalian cells, stabilizer sequence (STE) inactivation the NMD, and other sequences correlated with NMD such as extended poly (A) in 3' UTRs,upstream open reading frame (uORF) located in 5' UTR and programmed -1 ribosomal frameshift (-1 PRF) . Progress on cis-acting regulatory elements in nonsense-mediated mRNA decay was reviewed in this paper.

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz, 2005 Jan, 48(1), 29 - 35
{Background concentrations of molds in house dust . Determination of mold concentrations in dwellings without known mold infestations in three parts of Germany.}; Trautmann C et al.; Mold concentrations of house dust samples from dwellings reportedly free from mold infestations were analysed to obtain background values . Samples from carpet floors were taken from 80 dwellings in three parts of Germany in winter and summer . Samples were analysed with the cultivation method (using suspension) . This resulted in the detection of 35 mold species or genera . Concentrations of the genera Alternaria, Cladosporium, Fusarium and yeast increased in summer . In contrast concentrations of Aspergillus and Penicillium in the summer samples were nearly equal to those in the winter samples . The majority of the various molds were only found in a number of samples too small for a reliable comparison of the winter and summer findings to be made . The results were compared with findings of other authors . The authors propose the concentrations of the 95th percentile of each species (when representing over 10,000 KBE/g of dust) as background values, while a threefold higher value is regarded as an indication of mold presence . An exception should be made for the concentrations of molds which increase sharply in the outdoor air in summer . Because of the difficulty in estimating the outdoor influence, no evaluation proposals can be given for these species for the summer season.

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz, 2005 Jan, 48(1), 12 - 20
{Background concentrations of molds in air . Determination of mold concentrations in dwellings without known mold infestations in three parts of Germany.}; Trautmann C et al.; The mold concentration of indoor air and outdoor air in three parts of Germany was investigated in both winter and summer . In total, air samples from 80 dwellings, reportedly free from mold infestations, were analysed with both the cultivation method and the total spore count method . With the cultivation method, 40 mold species and genera were differentiated, while with the total spore count method, 11 spore types were distinguished . The concentration of the genera Alternaria, Cladosporium, Fusarium and yeast increased in summertime . In contrast, the concentrations of Aspergillus and Penicillium measured in summer were nearly equal to those measured in winter . The majority of the various molds were only found in a small number of samples, too small for a reliable comparison of the winter and summer findings . The 95th percentile of the indoor mold concentrations is suggested as the upper limit of the background concentration . The results are discussed comparing the assessment proposals of various authors, and a new assessment proposal is described.

J Biol Inorg Chem, 2005 Jan, 10(1), 25 - 32 Epub 2004 Nov 18.
Effect of complex formation between Zn(2+) ions and the anticancer drug mithramycin upon enzymatic activity of zinc(II)-dependent alcohol dehydrogenase; Das S et al.; Mithramycin (MTR), a member of the aureolic group of anticancer antibiotics, is a drug that supposedly acts via the inhibition of transcription . It binds to bivalent cations such as Mg(2+) and Zn(2+) . In this paper, we report the association of MTR with Zn(2+), a biologically important bivalent cation whose coordination property leads to its important role as a cofactor in different enzymes and nucleosomal DNA-binding proteins . First, we have characterized the complex formation between MTR and Zn(2+) using spectroscopic methods . In the second part, we have examined the effect of the association of Zn(2+) with MTR on the enzymatic activity of a typical zinc(II)-dependent alcohol dehydrogenase (ADH) from yeast . Our data show that MTR forms a single complex with Zn(2+) in a mole ratio of 2:1 in terms of MTR:Zn(2+) . We also observe a negative effect for the preincubation of ADH with MTR upon the enzymatic activity . These results indicate that MTR induced structural changes in the enzyme as a sequel to its complex formation with Zn(2+) present in the enzyme, thereby leading to a loss of enzymatic activity.

Cancer Gene Ther . 2005 Jan 14; {Epub ahead of print}
Development of a hypoxia-inducible cytosine deaminase expression vector for gene-directed prodrug cancer therapy; Wang D et al.; One important feature of human solid tumors is the presence of a hypoxic microenvironment . Under hypoxia, genes that contain a hypoxia-response element (HRE) can be activated by the binding of hypoxia-inducible factor-1 . To reach the goal of selectively killing tumor cells in a hypoxic microenvironment using a gene therapy approach, we developed a cytosine deaminase (CD) gene construct (pH9YCD2) that contains an HRE gene enhancer . CD is an enzyme that catalyzes the conversion of noncytotoxic 5-fluorocytosine (5-FC) to the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU) . Yeast CD was cloned into an SV40 promoter-based mammalian expression vector, and an HRE enhancer was inserted in front of the promoter . Human glioblastoma U-87 MG cells were transfected with pH9YCD2 . Western blots revealed that CD was strongly expressed under hypoxic conditions (0.3-1% O(2)), whereas only minor CD expression was seen under normoxic conditions . To confirm that the expressed CD enzyme retains catalytic activity, we performed a 5-FC/5-FU-conversion assay in which 5-FC was incubated with the lysates of pH9YCD2-transfected cells . The percentage of conversion from 5-FC to 5-FU was 63% under hypoxia versus 13% under normoxia . In vitro, cell viability and colony-forming efficiency assays demonstrated that the gene construct was able to significantly kill glioblastoma cells in a hypoxia-dependent manner . In addition, 5-FC treatment of hypoxic pH9YCD2-transfected cells produced a marked bystander effect, which could be a distinct advantage for gene therapy . If this construct exhibits antitumor efficacy in vivo, it may have promise as an antitumor agent in humans.Cancer Gene Therapy advance online publication, 14 January 2005; doi:10.1038/sj.cgt.7700748.

Mol Cells, 2004 Dec 31, 18(3), 369 - 73
Activity Staining of Glutathione Peroxidase after Two-dimensional Gel Electrophoresis; Kho CW et al.; We have developed a method for rapid activity staining of proteins with glutathione peroxidase (GPx) activity after 2-D gel electrophoresis . After separating proteins extracted from yeast, or mouse red blood cells, by two-dimensional gel electrophoresis, SDS was removed and the gel was submerged in a Tris-HCl buffer containing glutathione and hydrogen peroxide, followed by incubation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phenazine methosulfate (PMS) . After this proteins with GPx activity appeared as clear zones on a purple background . This relatively simple activity staining method could be useful for rapid screening of proteins with GPx activity in cell extracts.

Ann N Y Acad Sci, 2004 Dec, 1028, 38 - 55
Volume-sensitive organic osmolyte/anion channels in cancer: novel approaches to studying channel modulation employing proteomics technologies; Davies AR et al.; Key elements of tumor development include proliferation, migration, invasiveness, and angiogenesis . Activation of the volume-sensitive organic osmolyte/anion channel (VSOAC) has been suggested to play a role in all of these processes . VSOACs may therefore represent an important therapeutic target in the etiology of cancer . However, pharmacological inhibitors of VSOAC are nonselective and of low potency, highlighting the importance of identifying novel regulators of the channel . The use of electrophysiological methods coupled with techniques such as pull-down assays, yeast 2-hybrid, and functional protein arrays have already proved valuable in studying protein-protein interactions in a variety of systems . Some of these methods have been used to identify small molecules that modulate the function of other types of ion channels . Given that several proteins have already been identified as putative modulators of VSOACs, proteomics technologies may prove useful in elucidating the molecular identity of VSOACs and helpful in identifying novel modulators of channel function . In this paper, we review the involvement of VSOACs in tumor development processes and its regulation by pharmacological agents and cellular proteins . Proteomic approaches to study protein-protein interactions and how such approaches may be used to study VSOACs are also discussed . We speculate on how modulation of protein-protein interactions may result in the identification of a novel class of compounds for modulating VSOACs.

Mol Endocrinol . 2005 Jan 14; {Epub ahead of print}
The elongation factor ELL is a selective coregulator for steroid receptor functions; Pascual-LE Tallec L et al.; The dynamic and coordinated recruitment of coregulators by steroid receptors is critical for specific gene transcriptional activation . To identify new cofactors of the human mineralocorticoid receptor (hMR), its highly specific N-terminal domain was used as bait in a yeast two-hybrid approach . We isolated ELL (Eleven-nineteen Lysine rich Leukemia), a RNA polymerase II elongation factor which, when fused to MLL (Mixed Lineage Leukemia) contributes to the pathogenesis of acute leukemia . Specific interaction between hMR and ELL was confirmed by GST pull-down and coimmunoprecipitation experiments . Transient transfections demonstrated that ELL increased receptor transcriptional potency and hormonal efficacy, indicating that ELL behaves as a bona fide MR coactivator . Of major interest, ELL differentially modulates steroid receptor responses, with striking opposite effects on hMR and glucocorticoid receptor-mediated transactivation, without affecting that of androgen and progesterone receptors . Furthermore, the MLL-ELL fusion protein as well as several ELL truncated mutants lost their ability to potentiate MR transcriptional activities, suggesting that both the elongation domain and the ELL Associated Factor 1 (EAF1) interaction domains are required for ELL to fulfill its selector activity on steroid receptors . This study is the first direct demonstration of a functional interaction between a nuclear receptor and an elongation factor . These results provide further evidence that the selectivity of the mineralo vs . glucocorticoid signaling pathways also occurs at the transcriptional complex level and may have major pathophysiological implications most notably in leukemogenesis and corticosteroid-induced apoptosis . These findings allow us to propose the concept of "transcriptional selector" for ELL on steroid receptor transcriptional functions.

J Physiol . 2005 Jan 13; {Epub ahead of print}
Role of the N-terminal negative charges of actin in force generation and cross-bridge kinetics in reconstituted bovine cardiac muscle fibres; Lu X et al.; Mutant yeast actins were used to determine the role of actin's N-terminal nega nottive charges in force generation . The thin filament was selectively removed from bovine cardiac skinned muscle fibres by gelsolin, and the actin filament was reconsti nottut noted from purified G-actin . In this reconstitution, yeast wild type actin (2Ac: two N-terminal negative charges), yeast mutant actins (3Ac and 4Ac), and rabbit skeletal muscle actin (MAc) were used . The effects of phos notphate, ATP, and ADP on force development were studied at 25 degrees C . With MAc, iso notmet notric tension was 77% of the initial tension owing to the lack of a regula nottory sys nottem . With 2Ac, isometric tension was 10% of the initial tension; with 3Ac, isometric tension was 23%; and with 4Ac, isometric tension was 44% . Stiffness followed a similar pattern (2Ac < 3Ac < 4Ac < MAc) . A similar trend was observed during rigor induction and relaxation . Sinusoidal analysis was performed to obtain the kinetic constants of the cross-bridge cycle . The results showed that the variability of the kinetic constants was </=2.5-fold among the 2Ac, 4Ac and MAc muscle models . When the cross-bridge distribution was examin noted, there was no signifi notcant re notapportionment among these three models examined . These results indi notcate that force support noted by each cross-bridge is modi notfied by the N-terminal neg notative charges of actin, presumably via the acto notmyosin interface . We conclude that two N-terminal negative charges are not adequ notate, three negative charges are intermediate, and four negative charges are necessary to generate force.

J Biol Chem . 2005 Jan 13; {Epub ahead of print}
Identification and mutational analysis of amino acid residues involved in dipyridamole interactions with human and caenorhabditis elegans equilibrative nucleoside transporters; Visser F et al.; The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology . Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile 429 in TM 11 of CeENT1 and Met 33 in TM 1 of hENT1 . Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole . To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high-affinity dipyridamole binding (i.e., a Met at the TM 1 and/or an Ile at the TM 11 positions) . The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wildtype . Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that an Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that L442 of hENT1 was involved in permeant selectivity . Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed . This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2 and CeENT1 are important for dipyridamole interactions and nucleoside transport.

Comp Biochem Physiol B Biochem Mol Biol, 2005 Feb, 140(2), 299 - 307
Fatty acid compositions of Collembola: unusually high proportions of C(20) polyunsaturated fatty acids in a terrestrial invertebrate; Chamberlain PM et al.; To examine the C(20) polyunsaturated fatty acid (PUFA) compositions of Collembola, we raised five species of Collembola on yeast diets, and then quantified body mass, neutral lipid fatty acid (NLFA) and phospholipid fatty acid (PLFA) compositions . PLFA content was always less than 5% of dry weight, but NLFA content varied from 5.9% to 29.6% of dry weight, depending upon species . Combined C(20) PUFA proportions of up to 9.2% and 48% were observed in the NLFA and PLFA fractions, respectively, resulting in total C(20) PUFA proportions of up to 19.4% of the total fatty acid compositions of Collembola . C(20) PUFAs were also detected in Collembola specimens from a deciduous woodland at proportions up to 29.7% of the total fatty acid composition . Terrestrial invertebrates generally contain <4% and <22% C(20) PUFAs in PLFAs and NLFAs, respectively; therefore, these results demonstrate that Collembola often possess the highest proportions of C(20) PUFAs yet observed in terrestrial invertebrates . The biochemical reasons for such high C(20) PUFA proportions, which were biosynthesised by the Collembola since these components were absent from the yeast diets, remain unclear . The distinctive fatty acid compositions of Collembola may be useful in soil food web studies utilising fatty acids as biomarkers of trophic behaviour.

Biochem Soc Symp, 2005, (72), 31 - 8
Lipids, Rafts and Traffic: Chapter 3 - Phosphoinositides and membrane traffic at the trans-Golgi network; Choudhury RR et al.; Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes . Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P . This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane . Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear . Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved.

Proteomics . 2005 Jan 13; {Epub ahead of print}
Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme; Yi EC et al.; Quantitative protein profiling using the isotope-coded affinity tag (ICATtrade mark) method and tandem mass spectrometry (MS) enables the pair-wise comparison of protein expression levels in biological samples . A new version of the ICAT reagent with an acid-cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes (13)C substitution for (12)C in the heavy-ICAT reagent rather than (2)H (for (1)H) as in the original reagent (Gygi, S . P., Rist, B., Gerber, S . A., Frantisek, T., Gelb, M . H., Aebersold, R., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags . Nat . Biotechnol . 1999, 17, 994-999), was investigated . We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification . This was achieved by following a single survey (precursor) ion scan and serial collisioninduced dissociations (CIDs) of four different precursor ions observed in the prior survey scan . This strategy is common to many high-performance liquid chromatography-electrospray ionization (HPLC-ESI)-MS shotgun proteomic strategies, but heretofore not to ICAT experiments . This advance is possible because the new ICAT reagent uses (13)C as the "heavy" element rather than (2)H, thus, eliminating the slight delay in retention time of ICAT-labeled "light" peptides on a C18-based HPLC separation that occurs with (2)H and (1)H . Analyses using this new scheme of an ICAT-labeled trypsin-digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification . complement.

Cell Mol Biol Lett, 2004, 9(4A), 739 - 53
Molecular characterisation of the sand protein family: a study based on comparative genomics, structural bioinformatics and phylogeny; Cottage A et al.; The activities of vertebrate lysosomes are critical to many essential cellular processes . The yeast vacuole is analogous to the mammalian lysosome and is used as a tool to gain insights into vesicle mediated vacuolar/lysosome transport . The protein SAND, which does not contain a SAND domain (PFAM accession number PF01342), has recently been shown to function at the tethering/docking stage of vacuole fusion as a critical component of the vacuole SNARE complex . In this publication we have identified SAND in diverse eukaryotes, from single celled organisms such as the yeasts to complex multi-cellular chordates such as mammals . We have demonstrated subfamily divisions in the SAND proteins and show that in vertebrates, a duplication event gave rise to two SAND sequences . This duplication appears to have occurred during early vertebrate evolution and conceivably with the evolution of lysosomes . Using bioinformatics we predict a secondary structure, solvent accessibility profile and protein fold for the SAND proteins and determine conserved sequence motifs, present in all SAND proteins and those that are specific to subsets . A comprehensive evaluation of yeast and human functional studies in conjunction with our in silico analysis has identified potential roles for some of these motifs.

Nature . 2005 Jan 12; {Epub ahead of print}
Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation; Pray-Grant MG et al.; The specific post-translational modifications to histones influence many nuclear processes including gene regulation, DNA repair and replication . Recent studies have identified effector proteins that recognize patterns of histone modification and transduce their function in downstream processes . For example, histone acetyltransferases (HATs) have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription . Yeast SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous and conserved multi-subunit HAT complexes, which preferentially acetylate histones H3 and H2B and deubiquitinate histone H2B . Here we identify the chromatin remodelling protein Chd1 (chromo-ATPase/helicase-DNA binding domain 1) as a component of SAGA and SLIK . Our findings indicate that one of the two chromodomains of Chd1 specifically interacts with the methylated lysine 4 mark on histone H3 that is associated with transcriptional activity . Furthermore, the SLIK complex shows enhanced acetylation of a methylated substrate and this activity is dependent upon a functional methyl-binding chromodomain, both in vitro and in vivo . Our study identifies the first chromodomain that recognizes methylated histone H3 (Lys 4) and possibly identifies a larger subfamily of chromodomain proteins with similar recognition properties.

Am J Physiol Cell Physiol . 2005 Jan 12; {Epub ahead of print}
CD63 Interacts with the Carboxy-Terminus of the Colonic H+,K+-ATPase to Increase Plasma Membrane Localization and Rb+-Uptake; Codina J et al.; The carboxy-terminus of the colonic H(+),K(+)-ATPase is required for stable assembly with the beta-subunit, translocation to the plasma membrane and efficient function of the transporter . To identify protein-protein interactions involved in the localization and function of HKalpha2, we selected 84 amino acids in the carboxy-terminus of the alpha-subunit of mouse colonic H(+),K(+)-ATPase (CT-HKalpha2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library . The longest identified clone was CD63 . To characterize interaction of CT-HKalpha2 with CD63, recombinant CT-HKalpha2 and CD63 were synthesized in vitro, incubated, and complexes immunoprecipitated . CT-HKalpha2 protein (but not CT-HKalpha1) co-precipitated with CD63, confirming stable assembly of HKalpha2 with CD63 . In HEK-293 transfected with HKalpha2 plus beta1-Na(+),K(+)-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKalpha2/NKbeta1 and (86)Rb(+)-uptake . These studies demonstrate that CD63 participates in the regulation of the abundance of the HKalpha2/NKbeta1 complex in the cell membrane.

Mol Biol Cell . 2005 Jan 12; {Epub ahead of print}
Cyclical Regulation of the Exocyst and Cell Polarity Determinants for Polarized Cell Growth; Zajac A et al.; Monitoring Editor: Keith Mostov Polarized exocytosis is important for morphogenesis and cell growth . The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis . In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion . To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants . We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p . The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable . However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins . We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization . The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled . Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site . Reversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway . We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex . We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.

J Biol Chem . 2005 Jan 12; {Epub ahead of print}
Stability of the topoisomerase II closed clamp conformation may influence DNA stimulated ATP hydrolysis; Vaughn J et al.; Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for t he enzyme reaction . We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II a that has phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme . This substitution reduces the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme . A similar, but stronger effect was seen when the homologous mutation (Tyr28AEPhe) was introduced in yeast TOP2 . The ATPase activities of both hTOP2a (Tyr50AEPhe) and yTOP2 (Tyr28AEPhe) were resistant to both bisdioxopiperazines, and also to ATPase inhibitor sodium orthovanadate . Vanadate, like bisdioxopiperazines, traps the enzyme in a salt-stable closed conformation termed the closed clamp that can be detected in the presence of circular DNA substrates . Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP . Similarly, ADP trapped wild type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes . Our results demonstrate that bisdioxopiperazine resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme, and that this difference in stability may lead to a loss of DNA stimulated ATPase . We suggest that the DNA stimulated ATPase of topoisomerase II is intimately connected with steps occurring while the N-terminal domain of the enzyme is dimerized.

Mycopathologia, 2004 Oct, 158(3), 271 - 4
Epidemiology of onychomycosis and paronychia in the area of ANCONA (ITALY) over a period of 5 years; Simonetti O et al.; We retrospectively evaluated the epidemiology of onychomycosis and/or paronychia in 172 patients attending the Clinic of Dermatology and Venereology over a 5 year period . Although yeast isolates, belonging to the Candida species, represented the most frequent etiologic agents of these infections, an increasing prevalence of fungal infections due to emerging fungal pathogens (EFP) was noted throughout this time period . In particular, EFP as causative agents of these infections increased from 0 to 28.4% from 1998 to 2002.

J Cosmet Sci, 2004, 55 Suppl, S207 - 14
Enhanced delivery of an anti-dandruff active in a shampoo vehicle; Georgalas A; Formulating a delivery vehicle to enhance activity can potentially give higher activity or allow for adjustment to a lesser percent of active . A study was conducted in which the anti-dandruff active, Octopirox(R), INCI Piroctone Olamine {1-Hydroxy-4-methyl-6-(2,4,4-trimethylpentyl)-2(1H)-pyridinone}, was incorporated into a simple shampoo base at two levels as well as in the same base with an added amphiphilic surfactant blend (Biobase (R) SMC) at the lower level . A group of thirty (30) male subjects with moderate to severe dandruff were divided into three groups each of which evaluated one of three products for four weeks . Methods of evaluation included gravimetric determination of actual dandruff flakes, fluorescent staining of suspect yeast populations, blind evaluation by trained clinical personnel and panelist self assessment . The study demonstrated that the Octopirox(R) at 0.2% active delivered in the amphiphile blend was superior to the same level in the simple shampoo base and equivalent in activity to a much higher level (0.5%) in the base only . A proposed mechanism postulates the formation of liposome-like association structures that solubilize and entrap the Octopirox(R) and deposit is substantively to the scalp for enhanced longer lasting activity.

J Anim Sci, 2005 Feb, 83(2), 422 - 9
Effect of the chemical form of supranutritional selenium on selenium load and selenoprotein activities in virgin, pregnant, and lactating rats; Taylor JB et al.; Virgin, pregnant, and lactating rats were used to assess the influence of selenomethionine and selenocystine, fed at four to seven times the daily Se requirement (supranutritional), on Se load and selenoprotein activities . Female Sprague Dawley rats (n = 48; age = 13 wk), reared on a low-Se torula yeast diet, were assigned to one of three reproductive states (n = 16 per reproductive state) to occur simultaneously: virgin, pregnant, and lactating . Once reproductive state was achieved, rats were fed (ad libitum) either l-selenomethionine (n = 24) or L-selenocystine (n = 24) diets providing 2.0 microg Se/g of diet (as-fed basis) for 18 d, and then killed . Lactating rats consuming selenomethionine had the greatest Se concentration in the brain, with pregnant rats being intermediate, and virgin rats having the least (P < 0.02) . When selenocystine was fed, the concentration of Se in the brain was greater (P = 0.008) in lactating rats, but not different (P = 0.34) between pregnant and virgin rats . Selenium concentrations in the heart, liver, lung, muscle, spleen, plasma, placenta, uterus, and fetus were greatest (P < 0.001) in rats consuming selenomethionine . Brain, kidney, and liver thioredoxin reductase, and brain, erythrocyte, kidney, and liver glutathione peroxidase activities did not differ (P = 0.13 to P = 0.85) between Se treatments . Lactating rats exhibited the greatest (P < 0.006) Se concentration in the heart, lung, muscle, plasma, and spleen compared with pregnant and virgin rats . Thioredoxin reductase was greatest (P < 0.004) in the brain of pregnant rats, greatest (P < 0.004) in the liver of lactating rats, and greater (P < 0.03) in the kidney of lactating and pregnant vs . virgin rats . Regardless of reproductive state, supranutritional Se (2.0 microg/g of diet) fed as selenocystine resulted in less Se load, and when fed as selenomethionine, was equally available for thioredoxin reductase synthesis as the Se in selenocystine . Independent of dietary Se chemical form, thioredoxin reductase activity was responsive to reproductive state.

J Biol Chem . 2005 Jan 11; {Epub ahead of print}
Delta-interacting protein A, a new inhibitory partner of C/EBPbeta implicated in adipocyte differentiation; Bezy O et al.; CCAAT/enhancer-binding protein beta (C/EBPbeta) is expressed early during the adipocyte differentiation program and plays an important role in this process . In an attempt to identify novel proteins that interact with C/EBPbeta, we performed a yeast two-hybrid screen with a preadipocyte cDNA library, and identified a new coregulator, delta-interacting protein A (DIPA) . DIPA mRNA is expressed during adipocyte differentiation of clonal cell lines . DIPA interacts with C/EBPbeta and delta proteins in intact cells and inhibits their transcriptional activity but not that of C/EBPalpha . Stable overexpression of DIPA in preadipocytes partially inhibits adipocyte differentiation while its gene silencing enhances this process . DIPA and C/EBPbeta colocalize in the nucleus, and overexpression of DIPA in preadipocytes results in a partial inhibition of the mitotic clonal expansion, which is critical for differentiation . Thus, DIPA is a novel partner of C/EBPbeta that down-regulates early events of adipogenesis.

J Biol Chem . 2005 Jan 11; {Epub ahead of print}
Grif-1 and OGT interacting protein, OIP106, members of a novel gene family of coiled-coil domain proteins: Association in vivo and in vitro with kinesin; Brickley K et al.; -Aminobutyric AcidA Receptor Interacting Factor, GRIF-1, is a 913 amino acid protein proposed to function as a GABAA receptor ss2 subunit-interacting, trafficking protein . GRIF-1 shares ~ 44% amino acid sequence identity with O-N-acetylglucosamine transferase interacting protein 106, OIP106 . Both proteins contain predicted coiled-coil domains and probably constitute a novel gene family . The Drosophila orthologue of this family of proteins may be Milton . Milton shares ~ 44% amino acid homology with GRIF-1 . Milton is proposed to function in kinesin-mediated transport of mitochondria to nerve terminals . We report here that GRIF-1 and OIP106 also associate with kinesin and mitochondria . Following expression in human embryonic kidney 293 cells, both GRIF-1 and OIP106 were shown by co-immunoprecipitation to be specifically associated with an endogenous kinesin heavy chain species, Mr = 115 kDa, and exogenous KIF5C . Association of GRIF-1 with kinesin was also evident in native brain and heart tissue . In brain, anti-GRIF-18-633 antibodies specifically co-immunoprecipitated two kinesin immunoreactive species with Mrs 118 and 115 kDa and in heart, one kinesin immunoreactive species, Mr 115 kDa, was immunoprecipitated . Further studies revealed that GRIF-1 was predominantly associated with KIF5A in brain and with KIF5B in both heart and in HEK 293 cells . Yeast two-hybrid interaction assays and immunoprecipitations showed that GRIF-1 associated directly with KIF5C with the GRIF-1/KIF5C interaction domain localized to GRIF-1 (124-283) . These results further support a role for GRIF-1 and OIP106 in protein and/or organelle transport in excitable cells in a manner analogous to Glutamate-Receptor-Interacting-Protein 1, GRIP1, in the motor-dependent transport of AMPA glutamate excitatory neurotransmitter receptors to dendrites.

J Biol Chem . 2005 Jan 11; {Epub ahead of print}
The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells; Ward DM et al.; We examined the function of LIP5 in mammalian cells, as the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation . LIP5 is predominantly a cytosolic protein . Depletion of LIP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles . Depletion of LIP5 by siRNA also decreases HIV-1 budding by 70% . We identify CHMP5 as a LIP5 binding protein and show that CHMP5 is primarily cytosolic . Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5 . Surprisingly, CHMP5 depletion results in an increase in release of infectious HIV-1 particles . Overexpression of CHMP5 with a large carboxyl terminal epitope affects the distribution of both early and late endocytic compartments whereas overexpression of LIP5 does not alter the endocytic pathway . Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting whereas only LIP5 is required for HIV release.

J Biol Chem . 2005 Jan 11; {Epub ahead of print}
BIG1 is a binding partner of myosin IXB and regulates its Rho gap activity; Saeki N et al.; Myosin IXb, a member of the myosin superfamily, is a molecular motor that possesses a GTPase activating protein (GAP) for Rho . Through the yeast two-hybrid screen using the tail domain of myosin IXb as bait we found BIG1, a guanine nucleotide exchange factor (GEF) for Arf1, as a potential binding partner for myosin IXb . The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in NRK cells . Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other . Various truncation mutants of the myosin IXb tail domain were produced and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain . Interestingly, the GAP activity of myosin IXb was significantly inhibited by addition of BIG1 with IC(50) of 0.06 muM . The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with the concentration similar to the inhibition of the GAP activity . Likewise, RhoA inhibited the BIG1 binding of myosin IXb . These results suggest that BIG1 and RhoA compete each other for the binding to myosin IXb, thus resulting the inhibition of the GAP activity by BIG1 . The present study identified BIG1, the ArfGEF, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb . The findings raise a concept that the myosin transports the signaling molecule as a cargo that functions as a regulator for the myosin molecule.

Eukaryot Cell, 2005 Jan, 4(1), 166 - 77
Mcl1p Is a Polymerase {alpha} Replication Accessory Factor Important for S-Phase DNA Damage Survival; Williams DR et al.; Mcl1p is an essential fission yeast chromatin-binding protein that belongs to a family of highly conserved eukaryotic proteins important for sister chromatid cohesion . The essential function is believed to result from its role as a Pol1p (polymerase alpha) accessory protein, a conclusion based primarily on analogy to Ctf4p's interaction with Pol1p . In this study, we show that Mcl1p also binds to Pol1p with high affinity for the N terminus of Pol1p during S phase and DNA damage . Characterization of an inducible allele of mcl1(+), (nmt41)mcl1-MH, shows that altered expression levels of Mcl1p lead to sensitivity to DNA-damaging agents and synthetic lethality with the replication checkpoint mutations rad3Delta, rqh1Delta, and hsk1-1312 . Further, we find that the overexpression of the S-phase checkpoint kinase, Cds1, or the loss of Hsk1 kinase activity can disrupt Mcl1p's interaction with chromatin and Pol1p during replication arrest with hydroxyurea . We take these data to mean that Mcl1p is a dynamic component of the polymerase alpha complex during replication and is important for the replication stress response in fission yeast.

Am J Physiol Cell Physiol, 2005 Feb, 288(2), C223 - 39
Evolutionary origins of eukaryotic sodium/proton exchangers; Brett CL et al.; More than 200 genes annotated as Na(+)/H(+) hydrogen exchangers (NHEs) currently reside in bioinformation databases such as GenBank and Pfam . We performed detailed phylogenetic analyses of these NHEs in an effort to better understand their specific functions and physiological roles . This analysis initially required examining the entire monovalent cation proton antiporter (CPA) superfamily that includes the CPA1, CPA2, and NaT-DC families of transporters, each of which has a unique set of bacterial ancestors . We have concluded that there are nine human NHE (or SLC9A) paralogs as well as two previously unknown human CPA2 genes, which we have named HsNHA1 and HsNHA2 . The eukaryotic NHE family is composed of five phylogenetically distinct clades that differ in subcellular location, drug sensitivity, cation selectivity, and sequence length . The major subgroups are plasma membrane (recycling and resident) and intracellular (endosomal/TGN, NHE8-like, and plant vacuolar) . HsNHE1, the first cloned eukaryotic NHE gene, belongs to the resident plasma membrane clade . The latter is the most recent to emerge, being found exclusively in vertebrates . In contrast, the intracellular clades are ubiquitously distributed and are likely precursors to the plasma membrane NHE . Yeast endosomal ScNHX1 was the first intracellular NHE to be described and is closely related to HsNHE6, HsNHE7, and HsNHE9 in humans . Our results link the appearance of NHE on the plasma membrane of animal cells to the use of the Na(+)/K(+)-ATPase to generate the membrane potential . These novel observations have allowed us to use comparative biology to predict physiological roles for the nine human NHE paralogs and to propose appropriate model organisms in which to study the unique properties of each NHE subclass.

Proc Natl Acad Sci U S A, 2005 Jan 18, 102(3), 892 - 7 Epub 2005 Jan 10.
A potent small molecule inhibits polyglutamine aggregation in Huntington's disease neurons and suppresses neurodegeneration in vivo; Zhang X et al.; Polyglutamine (polyQ) disorders, including Huntington's disease (HD), are caused by expansion of polyQ-encoding repeats within otherwise unrelated gene products . In polyQ diseases, the pathology and death of affected neurons are associated with the accumulation of mutant proteins in insoluble aggregates . Several studies implicate polyQ-dependent aggregation as a cause of neurodegeneration in HD, suggesting that inhibition of neuronal polyQ aggregation may be therapeutic in HD patients . We have used a yeast-based high-throughput screening assay to identify small-molecule inhibitors of polyQ aggregation . We validated the effects of four hit compounds in mammalian cell-based models of HD, optimized compound structures for potency, and then tested them in vitro in cultured brain slices from HD transgenic mice . These efforts identified a potent compound (IC(50) = 10 nM) with long-term inhibitory effects on polyQ aggregation in HD neurons . Testing of this compound in a Drosophila HD model showed that it suppresses neurodegeneration in vivo, strongly suggesting an essential role for polyQ aggregation in HD pathology . The aggregation inhibitors identified in this screen represent four primary chemical scaffolds and are strong lead compounds for the development of therapeutics for human polyQ diseases.

J Cell Biol, 2005 Jan 17, 168(2), 201 - 7 Epub 2005 Jan 10.
The offloading model for dynein function: differential function of motor subunits; Lee WL et al.; During mitosis in budding yeast, dynein moves the mitotic spindle into the mother-bud neck . We have proposed an offloading model to explain how dynein works . Dynein is targeted to the dynamic plus end of a cytoplasmic microtubule, offloads to the cortex, becomes anchored and activated, and then pulls on the microtubule . Here, we perform functional studies of dynein intermediate chain (IC) and light intermediate chain (LIC) . IC/Pac11 and LIC/Dyn3 are both essential for dynein function, similar to the heavy chain (HC/Dyn1) . IC and LIC are targeted to the distal plus ends of dynamic cytoplasmic microtubules, as is HC, and their targeting depends on HC . Targeting of HC to the plus end depends on IC, but not LIC . IC also localizes as stationary dots at the cell cortex, the presumed result of offloading in our model, as does HC, but not LIC . Localization of HC to cortical dots depends on both IC and LIC . Thus, the IC and LIC accessory chains have different but essential roles in dynein function, providing new insight into the offloading model.

FEBS Lett, 2005 Jan 17, 579(2), 441 - 8
Oligomerization of the diaphanous-related formin FHOD1 requires a coiled-coil motif critical for its cytoskeletal and transcriptional activities; Madrid R et al.; The diaphanous-related formin homology 2 domain containing protein 1 (FHOD1) interacts with the Rac GTPase and activates the Rho-ROCK cascade leading to the formation of actin stress fibers . Here, we report the detection of homotypic interactions of FHOD1 in the yeast two-hybrid system, by co-immunoprecipitation and co-localization in mammalian cells . A predicted coiled-coil motif C-terminal to the core FH2 domain, but not the core FH2 domain itself, was critical for self-association of FHOD1 . Deletion of both the coiled-coil motif and the core FH2 domain abrogated formation of actin stress fibers and activation of transcription of the serum response element by FHOD1 . In contrast, these motifs were dispensable for the physical and functional interaction of FHOD1 with Rac1 . Together, these results indicate that oligomerization of FHOD1 via the coiled-coil motif is a critical parameter for its biological activities.

Genome Biol . 2005;6(1):R3 . Epub 2004 Dec 22.
Genome-scale approaches for discovering novel nonconventional splicing substrates of the Ire1 nuclease; Niwa M et al.; BACKGROUND: The unfolded protein response (UPR) allows intracellular feedback regulation that adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need . The signal from the ER lumen is transmitted by the ER-transmembrane kinase Ire1, which upon activation displays a site-specific endoribonuclease activity . Endonucleolytic cleavage of the intron from the HAC1 mRNA (encoding a UPR-specific transcription factor) is the first step in a nonconventional mRNA splicing pathway; the released exons are then joined by tRNA ligase . Because only the spliced mRNA is translated, splicing is the key regulatory step of the UPR . RESULTS: We developed methods to search for additional mRNA substrates of Ire1p in three independent lines of genome-wide analysis . These methods exploited the well characterized enzymology and genetics of the UPR and the yeast genome sequence in conjunction with microarray-based detection . Each method successfully identified HAC1 mRNA as a substrate according to three criteria: HAC1 mRNA is selectively cleaved in vitro by Ire1; the HAC1 mRNA sequence contains two predicted Ire1 cleavage sites; and HAC1 mRNA is selectively degraded in tRNA ligase mutant cells . CONCLUSION: Within the limits of detection, no other mRNA satisfies any of these criteria, suggesting that a unique nonconventional mRNA-processing mechanism has evolved solely for carrying out signal transduction between the ER and the nucleus . The approach described here, which combines biochemical and genetic 'fractionation' of mRNA with a novel application of cDNA microarrays, is generally applicable to the study of pathways in which RNA metabolism and alternative splicing have a regulatory role.

Langmuir, 2005 Jan 18, 21(2), 705 - 9
Encapsulated living cells on microstructured surfaces; Krol S et al.; The immobilization of cells in defined arrays (cell patterning) is a key step towards cell-based biosensors or other cell-based devices . While cell patterning is usually achieved by modifying the surface on which only the cells should adhere and leaving the cells unmodified, we present here a different approach in which cells are first coated with polyelectrolytes and subsequently immobilized on patterned surfaces . By coating, the cells are protected and their interactions with the substrate are modified such that patterning is simplified . We used microcontact printing of polyelectrolytes to structure surfaces such that regions of opposite charges and the same charge as the cell coating were present and found that we can thus achieve patterning of the coated yeast cells . In accordance with prior work, we find that coating does not kill the cells and coated GFP-expressing cells still function after immobilization, which we checked by fluorescence microscopy.

Oncogene, 2005 Jan 10, 24(2), 277 - 86
Regulation of cell cycle checkpoints by polo-like kinases; Xie S et al.; Protein kinases play a pivotal role in execution of cell division . Polo and Polo-like kinases have emerged as major regulators for various cell cycle checkpoints . Early genetic studies have demonstrated that CDC5, a budding yeast counterpart of vertebrate Plks, is essential for successful mitotic progression . Mammalian Plks localize primarily to the centrosome during interphase and the mitotic apparatus during mitosis . Many key cell cycle regulators such as p53, Cdc25C, cyclin B, components of the anaphase-promoting complex, and mitotic motor proteins are directly targeted by Plks . Although the exact mechanism of action of these protein kinases in vivo remains to be elucidated, Plks are important mediators for various cell cycle checkpoints that monitor centrosome duplication, DNA replication, formation of bipolar mitotic spindle, segregation of chromosomes, and mitotic exit, thus protecting cells against genetic instability during cell division.

Nucleic Acids Res . 2005 Jan 07;33(1):e2.
Detection and discovery of RNA modifications using microarrays; Hiley SL et al.; Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes . We show that at least five types of RNA modification (dihydrouridine, m1G, m2(2)G, m1A and m6(2)A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites . Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex . complex.

J Biol Chem . 2005 Jan 7; {Epub ahead of print}
Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5; Yang WH et al.; The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity) . Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities, and preferentially mediate one of the two 17,20-lyase reactions . In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary . CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that Xenopus CYP17's high lyase activity might be due to a lesser dependence on b(5) . The kinetics of XeCYP17 expressed in yeast microsomes were therefore examined in the absence and presence Xenopus or human b(5) . Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5) . However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5) . Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17 . Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of HuCYP17's lyase activity, possibly serving as an interacting domain with the enzyme . CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.

Yi Chuan, 2003 Sep, 25(5), 573 - 6
{Cloning and Characterization of Hsp70a cDNA Fragment of Dunaliella salina.}; Jiang GZ et al.; The present study is to obtain heat shock protein 70a cDNA fragment from Dunaliella salina.Two pairs of degenerate primers were designed according to conserved motifs of DIDLGTT,DQGNRTTP,PAYFNDS and ATKDAG of the homologous amino acid sequences and used to amplify hsp70a cDNA fragment from heat-shock-treated Dunaliella salina by nest PCR technique.The resulting PCR products were inserted into T-vector then transformed into JM109.Ten colonies were selected to determine their sequences.Homologous analysis of the deduced amino acid sequences were performed by BLAST and subsequently compared with GenBank data.Three of ten nucleotide sequences were obtained,of which there was 372 bp coding 126 amino acids.The sequences shared high homology with hsp70a,with identity 96% to Chlamydomonas reinhardtii,94% to Petunia,93% to Pisumsativum,92% to tomato,92% to human,90% to Drosophila and 89% to yeast respectively.It can be concluded that the cloned sequence is putatively hsp70a cDNA fragment from Dunaliella salina.

Yi Chuan, 2003 Jan, 25(1), 30 - 6
{RNA Responsible for Conferring a DNase I Sensitive Structure on Albumin Gene in Assembled Chromatin.}; Lv ZJ et al.; Although the set of genes is virtually the same in all tissues,differential gene expression is appeared in cells of different kinds.Differentiation and ageing are associated with regulation of gene expression that is a fundamental mechanism in eukaryotic development and survival.The sensitivity to DNase I of actively transcribed genes seems to be a general phenomenon.The purpose of the study is to test whether RNAs obtained from different organs or cells can enhance susceptibility of albumin gene to DNase I digestion in BALB/c mouse brain chromatin assembled.RNAs extracted from rat liver,lung,kidney,brain,tRNA from yeast and synthesized RNAs (23nt completed with mouse alb gene) were added to a system of chromatin reconstitution that was achieved by dialysis from high ionic strength solution.Assembled chromatin was digested with DNase I (12.5mug/mL) at 20 degrees for 1 min,then PCR assay was used to detect the level of albumin gene digested.PCR products (1200 bp) were run on a 6% polyacylamide gel and analyzed by silver stain assay.RNAs from different organs and synthesized RNAs all increased the sensitivity of albumin gene to DNase I attack in mouse assembled chromatin.The effect was more obvious in liver and lung RNAs than in kidney and brain ones.tRNA from yeast did not enhance the sensitivity of albumin gene to DNase I digestion.RNA increased albumin gene sensitivity to DNase I in a dose-dependent manner.We report here for the first time that RNAs can enhance susceptibility of albumin gene to DNase I digestion.The effect is associated with RNA sources or sequences.It is generally agreed that the formation of gene sensitivity to DNase I ,by unfolding of a tightly packed chromatin fiber,is the first step in gene activation,then RNAs that recognize complementary DNA sequences may be the specific factors that affect DNA supercoiling and determine the sensitivity of gene to DNase I digestion.Here we describes "RNA Population Gene Activating Model" that gives a logical interpretation of events leading to expression of specific genes during normal development and differentiation,in the same time,explains ageing and oncogenesis.Gene expression in eukaryotic cells requires two level regulations.The first may be controlled by RNAs that locate complementary regions within the genomes and make these regions loosened potentially,and the second is mainly involved in sequence specific and nonspecific proteins by which genomic regions bound by RNAs are unfolded.In eukaryotic cells,RNA fragments cleaved from all transcripts mix together to form "RNA populations" in which the majority is intron RNA.Every type of RNA fragments and its homologous sequences act as a group to form certain concentration in which repetitive sequences are more effective.If it is considered that there are many groups of RNA fragments in a particular cell,then different groups of RNA fragments are presented in dissimilar cell types of differentiation.Between DNA replication and nucleosome formation,RNA fragments in nuclear liquid will compete with DNA for binding to complement regions,then the chromatin regions bound to RNA can not be wrapped to form typical nucleosomes.After DNA doubles and is divided into 2 cells,these regions containing atypical nucleosomes become loose by function of non-histone.Transcriptionally active regions of chromatin are loose conformation but loosened regions are not always transcriptionally active.In every division,cells surfer in the described procedure that genes express RNAs,then RNAs recognize and imprint DNA.There are different RNA populations in different cells so that they imprint different genes,which is the primary mechanism by which same genes have expression distinctness.Since loosened genes are similar to bacterial operator system,factors in environment around cells play roles in inducing different gene expression to form different RNA population,which is the primary reason of cell differentiation.RNA population produced by certain impressions in genome can not imprint to form the same ones,otherwise immortal cells will be emerged,so that this program also controls ageing and oncogenesis.

Mol Genet Metab, 2005 Jan, 84(1), 32 - 8
Functional characterization of the K257R and G319E-hGALE alleles found in patients with ostensibly peripheral epimerase deficiency galactosemia; Wasilenko J et al.; Epimerase deficiency galactosemia is an autosomal recessive condition resulting from the impairment of UDP-galactose 4'-epimerase (hGALE) . Although a small number of clinically severe patients have been reported who exhibit "generalized" GALE deficiency, the vast majority exhibit an apparently benign "peripheral" form of the disorder in which enzyme impairment is restricted to the circulating red and white blood cells . Previously, preliminary data were reported suggesting that GALE deficiency is 10-fold more common among African-Americans than among non-African-Americans, and that two missense mutations, K257R and G319E, are found in at least some of these patients . We report here functional studies of these alleles involving expression of the substituted human enzymes in a null-background strain of yeast . Although under normal assay conditions both substituted proteins demonstrate enzyme activities indistinguishable from the wild-type, one (G319E) demonstrates mild impairment under conditions of substrate limitation . No impairments are evident under conditions of cofactor (NAD) limitation . These results are consistent with the apparently benign status of peripheral epimerase deficiency galactosemia, but leave open the question of why patients with these substitutions demonstrate GALE deficiency in their red blood cells . While the possibility remains that K257R and G319E may cause tissue-specific impairments not recapitulated in vitro or in yeast, an equally if not more plausible explanation suggested by interspecies sequence alignments is that both substitutions may be polymorphisms that exist in linkage disequilibrium with other, as yet unidentified causal mutations.

Biomacromolecules, 2005 Jan 10, 6(1), 174 - 179
Separation Technique for Messenger RNAs by Use of Schizophyllan/Poly(A) Tail Complexation(1); Kimura T et al.; Schizophyllan (SPG) is one of the water soluble beta-1,3-glucans and has a peculiar molecular recognition capability, namely, the single stranded SPG (s-SPG) can form a stoichiometric complex with certain polynucleotides such as poly(C) and poly(A), although it cannot bind poly(G) and poly(dC) at all . In this paper, we prepared an s-SPG-appended column and made an attempt to separate polynucleotides on the bases of this molecular recognition capability . The s-SPG-appended column trapped only such RNAs that could form the complex with s-SPG but eluted other RNAs which did not form the complex . Encouraged by the results in the model system, we extended the s-SPG-appended column into separation of native messenger RNAs (mRNAs) from a RNA mixture (total RNA) obtained from yeast . Since eukaryotic mRNAs have a poly(A) tail with 150-300 bases, we supposed that the tails would be trapped by the s-SPG-appended column . The results indicate that mRNAs were separated from total RNA in good yield and with high purity . It should be emphasized that this is the first device to separate natural mRNAs without using a dA/dT Watson-Crick-type interaction.

J Biol Chem . 2005 Jan 6; {Epub ahead of print}
PRAM-1 potentiates arsenic trioxide-induced JNK activation; Denis FM et al.; PML-RARalpha target gene encoding an Adaptor Molecule -1 (PRAM-1) is involved in a signaling pathway induced by retinoic acid in acute promyelocytic leukemia (APL) cells . To better understand the function of PRAM-1, we have undertaken the identification of its partners through a yeast two-hybrid screen . Here, we show that the proline-rich domain of PRAM-1 interacted with the Src-homology (SH3) domain of hematopoietic progenitor kinase 1 (HPK-1)-interacting protein of 55 kDa (HIP-55, also called SH3P7 and Abp1) known to stimulate the activity of HPK-1 and c-Jun N-terminal kinase (JNK) . Overexpression of PRAM-1 in the NB4 APL cell line increased arsenic trioxide-induced JNK activation through a caspase 3-like dependent activity . Dissociation of the SH3 domain from the rest of the HIP-55 protein was observed in the NB4 APL cell line treated with arsenic trioxide due to specific cleavage by caspase 3-like enzymes . The cleavage of HIP55 correlated with the induction of PRAM-1 mRNA and protein expression . Taken together, our results suggest that the caspase-3 cleaved SH3 domain of HIP-55 is likely involved in PRAM-1-mediated JNK activation upon arsenic trioxide-induced differentiation of NB4 cells.

EMBO J . 2005 Jan 06; {Epub ahead of print}
Lipid-mediated, reversible misfolding of a sterol-sensing domain protein; Shearer AG et al.; Cellular quality control requires recognition of common features of misfolding, and so is not typically associated with the specific targeting of individual proteins . However, physiologically regulated degradation of yeast HMG-CoA reductase (Hmg2p) occurs by the HRD endoplasmic reticulum quality control pathway, implying that Hmg2p undergoes a regulated transition to a quality control substrate in response to a sterol pathway molecule . Using in vitro structural assays, we now show that the pathway derivative farnesol causes Hmg2p to undergo a change to a less folded structure . The effect is reversible, biologically relevant by numerous criteria, highly specific for farnesol structure, and requires an intact Hmg2p sterol-sensing domain . This represents a distinct lipid-sensing function for this highly conserved motif that suggests novel approaches to cholesterol management . More generally, our observation of reversible small-molecule-mediated misfolding may herald numerous examples of regulated quality control to be discovered in biology or applied in the clinic.

Nature . 2005 Jan 05; {Epub ahead of print}
Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain; Otomo T et al.; The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament . Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin . Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus . Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation . Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain . Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.

Mol Biol Cell . 2005 Jan 5; {Epub ahead of print}
The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ABC-Transporter Ste6 in the Endocytic Pathway; Schmitz C et al.; Monitoring Editor: David Drubin Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes . Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) familiy, the Ubp1 protein . We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that appear to be independently expressed from the same gene . The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose-density-gradient centrifugation and by immunofluorescence microscopy . Overexpression of the soluble Ubp1 variant stabilizes the ABC-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination . Ste6 stabilization was not the result of a general increase in deubiquitination activity, since overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation (ERAD) substrate CPY(*) and most importantly on Ste6 ubiquitination itself . Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover . This suggests that the Ubp1 target is a component of the protein transport machinery . On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment.

Mol Biol Cell . 2005 Jan 5; {Epub ahead of print}
EBAG9 Adds a New Layer of Control on Large Dense-Core Vesicle Exocytosis via Interaction with Snapin; Ruder C et al.; Monitoring Editor: Benjamin Glick Regulated exocytosis is subject to several modulatory steps that include phosphorylation events and transient protein-protein interactions . The EBAG9 gene product was recently identified as a modulator of tumor-associated O-linked glycan expression in nonneuronal cells, however this molecule is expressed physiologically in essentially all mammalian tissues . Particular interest gained this molecule because in some human tumor entities high expression levels correlated with clinical prognosis . To gain insight into the cellular function of EBAG9, we scored for interaction partners employing the yeast two-hybrid system . Here, we demonstrate that EBAG9 interacts with Snapin, which is likely to be a modulator of Synaptotagmin-associated regulated exocytosis . Strengthening of this interaction inhibited regulated secretion of neuropeptide Y from PC12 cells, whereas evoked neurotransmitter release from hippocampal neurons remained unaltered . Mechanistically, EBAG9 decreased phosphorylation of Snapin, subsequently association of Snapin with SNAP25 and SNAP23 was diminished . We suggest that the occurrence of SNAP23, Snapin and EBAG9 also in nonneuronal cells might extend the modulatory role of EBAG9 to a broad range of secretory cells . The conjunction between EBAG9 and Snapin adds an additional layer of control on the exocytosis process, in addition mechanistic evidence is provided that inhibition of phosphorylation has a regulatory function in exocytosis.

J Clin Microbiol, 2005 Jan, 43(1), 101 - 11
Multigene phylogenetic analysis of pathogenic candida species in the Kazachstania (Arxiozyma) telluris complex and description of their ascosporic states as Kazachstania bovina sp . nov., K . heterogenica sp . nov., K . pintolopesii sp . nov., and K . slooffiae sp . nov; Kurtzman CP et al.; A yeast causing widespread infection of laboratory mice was identified from 26S rRNA gene sequences as Candida pintolopesii . To determine the relationship of C . pintolopesii with other members of the Kazachstania (Arxiozyma) telluris species complex, nucleotide sequences from domains 1 and 2 of the 26S rRNA gene, the mitochondrial small-subunit rRNA gene, and the RNA polymerase II gene were phylogenetically analyzed . That analysis resolved the 48 strains examined into five closely related species: K . telluris, Candida bovina, C . pintolopesii, Candida slooffiae, and a previously unknown species . One or more strains of each of the last four species formed an ascosporic state much like that of K . telluris . To place these ascosporogenous strains taxonomically, it is proposed that they be assigned to the teleomorphic genus Kazachstania as K . bovina (type strain NRRL Y-7283, CBS 9732, from the nasal passage of a pigeon), K . heterogenica (type strain NRRL Y-27499, CBS 2675, from rodent feces), K . pintolopesii (type strain NRRL Y-27500, CBS 2985, from the peritoneal fluid of a dead guinea pig), and K . slooffiae (type strain NRRL YB-4349, CBS 9733, from the cecum of a horse) . On the basis of multigene sequence analyses, K . heterogenica appears to be a hybrid of K . pintolopesii and a presently unknown species . With the exception of K . bovina, the phylogenetically defined species show a moderate degree of host specificity.

Zhonghua Bing Li Xue Za Zhi, 2004 Dec, 33(6), 536 - 40
{Infection of penicillium Marneffei.}; Lu ZH et al.; OBJECTIVES: To elucidate the etiology, pathohistology, clinical characteristic and differentiatial diagnosis, reduce missed diagnosis and improve the early detection and treatment of Penicillium Marneffei infection, by means of this case report and literature review . METHODS: A patient hospitalized Penicillium Marneffei infection were presented here, together with 27 cases in the literature, among which 10 patients had complications of AIDS and 5 with other diseases . RESULTS: Penicillium Marneffei is a temperature-sensitive, two-phase fungus, which can infect healthy and immunocompromised subjects . The common symptoms are lymphadenopathy and infection of the lung . The infection may be local or diffuse, involving the intestinal tract, soft tissue, bone, liver, spleen and bone marrow etc . The lesion can be classified into the granuloma type, suppurative type and anergy/necrosis type histologically . The yeast-like fungus were mainly found in the cytoplasm of macrophages, which were demonstrated by PAS and Giemsa staining . The wine red color developed on the culture confirms the diagnosis . CONCLUSIONS: The diagnosis of Penicillium Marneffei infection should be considered when tuberculosis is suspected but not confirmed, and if the patient has a history of having lived or traveled in Southeast Asia, is anemic or resistant to anti-tuberculosis treatment . The major differentiatial diagnosis is histoplasmosis . Early administration of anti-fungus drugs is essential for recovery.

FEBS J, 2005 Jan, 272(1), 37 - 46
The Rab5 effector Rabaptin-5 and its isoform Rabaptin-5delta differ in their ability to interact with the small GTPase Rab4; Korobko E et al.; Rabaptin-5 is an effector for the small GTPase Rab5, a regulator of the early steps in endocytosis . In addition, Rabaptin-5 interacts with the small GTPase Rab4 that has been implicated in recycling from early endosomes to the cell surface . Recently we have identified a ubiquitous transcript encoding the Rabaptin-5 isoform, Rabaptin-5delta . To evaluate the interaction properties of Rabaptin-5delta with the small GTPases Rab4 and Rab5, we have applied protein interaction assays using the yeast two-hybrid system and a glutathione S-transferase pull-down assay . We found that unlike Rabaptin-5, that interacts with both GTPases in GTP-bound conformations, Rabaptin-5delta interacts only with GTP-bound Rab5, and does not interact with Rab4, presumably due to a disrupted Rab4 binding site . Immunofluorescence microscopy analysis carried out to address the localization of Rabaptin-5delta relative to GTP-bound Rab4 and Rab5 in BHK-21 cells supported these data . Our data suggests that while Rabaptin-5 was proposed to act as a molecular linker between Rab5 and Rab4, to coordinate endocytic and recycling traffic, Rabaptin-5delta is involved only in the Rab5-driven events.

Traffic, 2005 Feb, 6(2), 75 - 82
Sorting nexins - unifying trends and new perspectives; Carlton J et al.; The sorting nexins (SNXs) are a family of PX domain-containing proteins found in yeast and mammalian cells that have been proposed to regulate intracellular trafficking . Mammalian SNXs have been suggested to function variously in pro-degradative sorting, internalization, endosomal recycling, or simply in endosomal sorting . In yeast, the defining function for these proteins is a regulation of cargo retrieval . Here we examine recent data on the SNX family of proteins and attempt to draw out unifying themes between the work performed in yeast and mammalian systems.

Biochem J . 2005 Jan 6; {Epub ahead of print}
Truncated human serum albumin retains general anesthetic binding activity; Liu R et al.; The multiple binding sites for anesthetics in human serum albumin (HSA) make solution studies difficult to interpret . Here, we expressed HSA domain III (wtHSAd3), a peptide with two known anesthetic binding sites in a yeast expression system . We also expressed a site-directed mutant of domain 3 (Y411Wd3) . The stability and secondary structure of the constructed fragments were determined by hydrogen-tritium (HX) and circular dichroism (CD) spectroscopy . The binding of two general anesthetics, halothane and propofol, to wtHSAd3 and Y411Wd3 was determined using isothermal titration calorimetry (ITC), HX, and intrinsic tryptophan fluorescence quenching . Although the expressed fragments are less stable than intact wtHSA as indicated by both CD and HX, they retain the secondary structure and anesthetic-binding characteristics of intact HSA molecule, but with fewer binding sites . Y411Wd3 had decreased affinity for propofol but not for halothane, consistent with steric hindrance . Retention of structural features and anesthetic binding properties with fewer binding sites in this truncated protein provide feasibility for using scaled-down models of otherwise intractable systems to gain an understanding of anesthetic binding requirements and binding/stability relationships.

Pest Manag Sci . 2005 Jan 4; {Epub ahead of print}
p-Hydroxyphenylpyruvate dioxygenase inhibitor-resistant plants; Matringe M et al.; The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisic acid, the aromatic precursor of plastoquinone and vitamin E . HPPD is the specific target of several herbicide families: isoxazoles, triketones and pyroxazoles . Its inhibition results in the depletion of the plant plastoquinone and vitamin E pools, leading to bleaching symptoms . These herbicides are very potent for the selective pre- and in some cases post-emergence control of a wide range of broadleaf and grass weeds in maize and rice . Their herbicidal potential raised interest in the development of highly resistant transgenic crops . This goal was first achieved by over-expression of a bacterial HPPD in crop plants, and an increased level of resistance was obtained by using a mutant enzyme . A second strategy based on bypassing HPPD in the production of homogentisate was then developed . Recently, a third strategy of resistance based on the increase of p-hydroxyphenylpyruvate substrate flux has been developed . This was achieved by the introduction of the yeast prephenate dehydrogenase gene (PDH) into transgenic plants already overexpressing HPPD . In addition to a high level of herbicide resistance, a massive accumulation of vitamin E, mainly tocotrienols, was observed in leaves of the transgenic HPPD-PDH plants . Copyright (c) 2004 Society of Chemical Industry.

Microbiology, 2005 Jan, 151(Pt 1), 291 - 9
Melanization of Penicillium marneffei in vitro and in vivo; Youngchim S et al.; Melanins are found universally in nature and are implicated in the pathogenesis of several important human fungal pathogens . This study investigated whether the conidia and the yeast cells of the thermally dimorphic fungal pathogen Penicillium marneffei produce melanin or melanin-like compounds in vitro and during infection . Treatment of conidia with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles that were similar in size and shape to the conidia . A melanin-binding monoclonal antibody (mAb) labelled pigmented conidia, yeast cells and the isolated particles as determined by immunofluorescence microscopy . Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical compound, consistent with their identification as melanins . Skin tissue from penicilliosis marneffei patients contained yeast cells that were labelled by melanin-binding mAb . Additionally, sera from P . marneffei-infected mice developed a significant antibody response (both IgG and IgM) against melanin . Phenoloxidase activity capable of synthesizing melanin from l-DOPA was detected in cytoplasmic yeast cell extracts . These findings indicate that P . marneffei conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that the yeast cells can synthesize pigment in vivo . Accordingly this pigment may play some role in the virulence of P . marneffei.

Microbiology, 2005 Jan, 151(Pt 1), 35 - 43
Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization; Kappler U et al.; Metallosphaera sedula is a thermoacidophilic Crenarchaeon which is capable of leaching metals from sulfidic ores . The authors have investigated the presence and expression of genes encoding respiratory complexes in this organism when grown heterotrophically or chemolithotrophically on either sulfur or pyrite . The presence of three gene clusters, encoding two terminal oxidase complexes, the quinol oxidase SoxABCD and the SoxM oxidase supercomplex, and a gene cluster encoding a high-potential cytochrome b and components of a bc(1) complex analogue (cbsBA-soxL2N gene cluster) was established . Expression studies showed that the soxM gene was expressed to high levels during heterotrophic growth of M . sedula on yeast extract, while the soxABCD mRNA was most abundant in cells grown on sulfur . Reduced-minus-oxidized difference spectra of cell membranes showed cytochrome-related peaks that correspond to published spectra of Sulfolobus-type terminal oxidase complexes . In pyrite-grown cells, expression levels of the two monitored oxidase gene clusters were reduced by a factor of 10-12 relative to maximal expression levels, although spectra of membranes clearly contained oxidase-associated haems, suggesting the presence of additional gene clusters encoding terminal oxidases in M . sedula . Pyrite- and sulfur-grown cells contained high levels of the cbsA transcript, which encodes a membrane-bound cytochrome b with a possible role in iron oxidation or chemolithotrophy . The cbsA gene is not co-transcribed with the soxL2N genes, and therefore does not appear to be an integral part of this bc(1) complex analogue . The data show for the first time the differential expression of the Sulfolobus-type terminal oxidase gene clusters in a Crenarchaeon in response to changing growth modes.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
SIRT1 deacetylation and repression of P300 involves lysine residues 1020/1024 within the cell-cycle regulatory domain 1; Bouras T et al.; The SIR2 family of nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases modulates diverse biological functions in different species, including longevity, apoptosis, cell-cycle exit and cellular differentiation . SIRT1, the closest mammalian orthologue of the yeast SIR2 gene, represses several transcription factors, including p53, NFB and forkhead proteins . The p300 protein serves as a rate-limiting transcriptional cointegrator of diverse transcription factors to either activate or repress transcription through modular subdomains . Herein, SIRT1 physically interacted with and repressed p300 transactivation, requiring the NAD-dependent deacetylase activity of SIRT1 . SIRT1 repression involved the CRD1 transcriptional repression domain of p300 . Two lysine residues within the CRD1 domain (K1020/1024) were required for SIRT1 repression and served as substrates for SIRT1 deacetylation . These residues also serve as acceptor lysines for modification by the ubiquitin-like SUMO protein . The SUMO-specific protease SSP3 relieved SIRT1 repression of p300 . SSP3 antagonism of SIRT1 required the SUMO-deconjugating function of SSP3 . Thus, p300 serves as a deacetylase substrate for SIRT1 through a conserved SUMO consensus motif . Since p300 is a limiting transcriptional cofactor, deacetylation and repression of p300 by SIRT1 may serve an important integration point during metabolism and cellular differentiation.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
RING finger E3 ligase Nrdp1/FLRF regulates parkin stability and activity; Zhong L et al.; Parkin is an ubiquitin E3 ligase . It has been suggested that loss-of-function in parkin causes accumulation and aggregation of its substrates, leading to death of dopaminergic neurons in Parkinson's disease . Using the yeast two-hybrid screen, we isolated a RING finger protein that interacted with the N-terminus of parkin in a Drosophila cDNA library . Interaction between human parkin and the mammalian RING finger protein homologue Nrdp1/FLRF, an E3 ligase that ubiquitinates ErbB3 and ErbB4, was validated by in vitro binding assay, co-immunoprecipitation and immunofluorescence co-localization . Significantly, pulse-chase experiments showed that co-transfection of Nrdp1 and parkin reduced parkin's half-life from 5 hours to 2.5 hours . Consistent with these findings, we further observed that degradation of CDCrel-1, a parkin substrate, was facilitated by overexpression of parkin protein . However, co-transfection of Nrdp1 with parkin reversed parkin's effects on CDCrel-1 degradation . We conclude that Nrdp1 is a parkin modifier that accelerates degradation of parkin, resulting in reduction of parkin activity.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
Transmembrane topology of the protein palmitoyl transferase Akr1p; Politis EG et al.; The two recently identified protein acyl transferases (PATs), Akr1p and Erf2p/Erf4p, point toward the DHHC protein family as a likely PAT family . The DHHC protein family, defined by the novel, zinc finger-like, DHHC cysteine-rich domain (DHHC-CRD), is a diverse collection of polytopic membrane proteins extending through all eukaryotes . To define the PAT domains that are oriented to the cytoplasm and are thus available to effect the cytoplasmically-limited palmitoyl modification, we have determined the transmembrane topology of the yeast PAT Akr1p . Portions of the yeast protein invertase (Suc2p) were inserted in-frame at ten different hydrophilic sites within the Akr1 polypeptide . Three of the Akr1-Suc2-Akr1 insertion proteins were found to be extensively glycosylated, indicating that the invertase segment inserted at these Akr1p sites is luminally-oriented . The remaining seven insertion proteins were not glycosylated, consistent with a cytoplasmic orientation for these sites . The results support a model in which the Akr1 polypeptide crosses the bilayer six times with the bulk of its hydrophilic domains disposed towards the cytoplasm . Cytoplasmic domains include both the relatively large, ankyrin repeat-containing N-terminal domain and the DHHC-CRD, which maps to a cytosolic loop segment . Functionality of the different Akr1-Suc2-Akr1 proteins also was examined . Insertions at only four of the ten sites were found to disrupt Akr1p function . Interestingly, these four sites all map cytoplasmically, suggesting key roles for these cytoplasmic domains in Akr1 PAT function . Finally, extrapolating from the Akr1p topology, topology models are proposed for other DHHC protein family members.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
The mitochondrial type II peroxiredoxin F is essential for redox homeostasis and root growth of Arabidopsis thaliana under stress; Finkemeier I et al.; Peroxiredoxins (Prx) have recently moved into the focus of plant and animal research in the context of development, adaptation and disease, as they function both in antioxidant defence by reducing a broad range of toxic peroxides and in redox signaling relating to the adjustment of cell redox and antioxidant metabolism . At-PrxII F is one of six type II Prx identified in the genome of Arabidopsis thaliana and the only Prx that is targeted to the plant mitochondrion . Therefore it might be assumed to have functions similar to the human 2-Cys Prx (PRDX3) and type II Prx (PRDX5) and yeast 1-Cys Prx that likewise have mitochondrial localisation . This paper presents a characterization of PrxII F at the level of subcellular distribution, activity and reductive regeneration by mitochondrial thioredoxin and glutaredoxin . Employing T-DNA-insertion mutants of A . thaliana lacking expression of AtprxII F (KO-AtPrxII F), it is shown that under optimal environmental conditions the absence of PrxII F is almost fully compensated for, possibly by increases in activity of mitochondrial ascorbate peroxidase and glutathione-dependent peroxidase . However, a stronger inhibition of root growth in KO-AtPrxII F seedlings as compared to wild type is observed under stress conditions induced by CdCl2 as well as after administration of salicylhydroxamic acid, an inhibitior of cyanide-insensitive respiration . Simultaneously, major changes in the abundance of both nuclear- and mitochondria-encoded transcripts were observed . These results assign a principal role to PrxII F in antioxidant defence and possibly redox signaling in plants cells.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
GPI7 is the second partner of PIG-F and involved in modification of glycosylphosphatidylinositol; Shishioh N et al.; Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI) . GPI is synthesized from phosphatidylinositol by stepwise reactions and attached en bloc to nascent proteins . In mammalian cells, the major GPI species transferred to proteins is termed H7 . By attachment of an additional ethanolaminephosphate (EtNP) to the second mannose, H7 can be converted to H8, which acts as a minor type of protein-linked GPI and also exists as a free GPI on the cell surface . Yeast GPI7 is involved in the transfer of EtNP to the second mannose, but the corresponding mammalian enzyme has not yet been clarified . Here, we report that the human homolog of Gpi7p (hGPI7) forms a protein complex with PIG-F and is involved in the H7-to-H8 conversion . We knocked down hGPI7 by RNA interference and found that H7 was accumulated with little production of H8 . Immunoprecipitation experiments revealed that hGPI7 was associated with and stabilized by PIG-F, which is known to bind to and stabilize PIG-O, a protein homologous to hGPI7 . PIG-O is a transferase that adds EtNP to the third mannose, rendering GPI capable of attaching to proteins . We further found that the overexpression of hGPI7 decreased the level of PIG-O and transfer of EtNP to the third mannose . Finally, we propose a mechanism for the regulation of GPI biosynthesis through competition between the two independent enzymes, PIG-O and hGPI7, for the common stabilizer, PIG-F.

Mol Cell Biol, 2005 Jan, 25(2), 779 - 88
BRCTx Is a Novel, Highly Conserved RAD18-Interacting Protein; Adams DJ et al.; The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control . Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif . Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis . Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile . BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus . Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents . MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable . Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells . We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.

Mol Cell Biol, 2005 Jan, 25(2), 652 - 60
The Histone Chaperone Anti-Silencing Function 1 Is a Global Regulator of Transcription Independent of Passage through S Phase; Zabaronick SR et al.; We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1) in global transcriptional regulation in budding yeast . Deletion of ASF1 or CAF-1 components led to global transcriptional misregulation, both activation and repression, of genes scattered throughout the 16 yeast chromosomes . To investigate direct effects on gene regulation, we developed an approach to destabilize Asf1p that results in its rapid degradation within minutes of transcriptional repression . Upon degradation of Asf1p, rapid global changes in gene expression occur without the requirement for passage through S phase or de novo protein synthesis . In particular, we demonstrate that the previously reported influence of Asf1p on histone gene expression is not a direct effect of loss of Asf1p . These data indicate that the histone chaperones CAF-1 and Asf1p regulate the gene expression of a broad array of genes in yeast and, in the case of Asf1p, this is likely to be due to a direct role in chromatin modulation during transcriptional regulation.

Mol Cell Biol, 2005 Jan, 25(2), 621 - 36
Mutations in the RNA Polymerase III Subunit Rpc11p That Decrease RNA 3' Cleavage Activity Increase 3'-Terminal Oligo(U) Length and La-Dependent tRNA Processing; Huang Y et al.; Termination by RNA polymerase III (Pol III) produces RNAs whose 3' oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3' exonucleases and promotes their maturation . Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function . The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3' cleavage and to be important for termination . We used a La-dependent opal suppressor, tRNA(Ser)UGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression . Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3' cleavage activity and produce pre-tRNA(Ser)UGAM transcripts with elongated 3'-oligo(U) tracts that are better substrates for La . A substantial fraction of pre-tRNA(Ser)UGAM contains too few 3' Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants . The data indicate that Rpc11p limits RNA 3'-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.

Mol Cell Biol, 2005 Jan, 25(2), 612 - 20
The parafibromin tumor suppressor protein is part of a human paf1 complex; Rozenblatt-Rosen O et al.; Parafibromin, the product of the HRPT2 (hyperparathyroidism-jaw tumor syndrome 2) tumor suppressor gene, is the human homologue of yeast Cdc73, part of the yeast RNA polymerase II/Paf1 complex known to be important for histone modification and connections to posttranscriptional events . By purifying cellular parafibromin and characterizing its associated proteins, we have identified a human counterpart to the yeast Paf1 complex including homologs of Leo1, Paf1, and Ctr9 . Like the yeast complex, the parafibromin complex associates with the nonphosphorylated and Ser2 and Ser5 phosphorylated forms of the RNA polymerase II large subunit . Immunofluorescence experiments show that parafibromin is a nuclear protein . In addition, cotransfection data suggest that parafibromin can interact with a histone methyltransferase complex that methylates histone H3 on lysine 4 . Some mutant forms of parafibromin lack association with hPaf1 complex members and with the histone methyltransferase complex, suggesting that disruption of these complexes may correlate with the oncogenic process.

J Cell Biol, 2005 Jan 3, 168(1), 67 - 77
Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation; Cheng Z et al.; The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel . Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway . Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes . Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore . In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.

Biochem J . 2005 Jan 5; {Epub ahead of print}
Interactions between Cdc42 and the scaffold protein Scd2: requirement of SH3 domains for GTPase binding; Wheatley E et al.; The multi-domain protein Scd2 acts as a scaffold upon which the small GTPase Cdc42, its nucleotide exchange factor Scd1 and the p21 activated kinase Shk1 assemble to regulate cell polarity and the mating response in fission yeast . In the present study we show by isothermal titration calorimetry that Scd2 binds two molecules of active, GTP-bound Cdc42 simultaneously, but independently of one another . The two binding sites have significantly different affinities, 21 nM and 3 muM, suggesting that they play distinct roles in the Shk1 signalling network . Each of the Cdc42 binding sites includes one of the SH3 domains of Scd2 . Our data indicate that complex formation does not occur in a conventional manner via the conserved SH3 domain ligand-binding surface . Neither of the isolated SH3 domains is sufficient to interact with the GTPase and they both require adjacent regions to either stabilise their conformations or contribute to the formation of the Cdc42-binding surface . Furthermore, we show that there is no evidence for an intramolecular PX-SH3 domain interaction, which could interfere with SH3 domain function . This work suggests that SH3 domains might directly contribute to signalling through small GTPases and thereby adds another aspect to the diverse nature of SH3 domains as protein interaction modules.

J Mol Med . 2005 Jan 4; {Epub ahead of print}
The telomerase cycle: normal and pathological aspects; Brunori M et al.; Telomeres are nucleoprotein complexes that cap the end of eukaryotic chromosomes and are essential for their function and stability . Telomerase, a reverse transcriptase that extends the single-stranded G-rich 3' protruding ends of chromosomes, stabilizes telomere length in germ line cells and regenerative tissues as well as in tumor cells . In the absence of telomerase telomeres shorten with cell division, a process able to trigger cell growth arrest . When telomerase is present in the cell, its activity is tightly regulated at its site of action by factors specifically bound to the telomeric DNA . Recent data indicate that telomeres reorganize during the cell cycle . This review summarizes our current knowledge on how telomeres are dynamically organized and remodeled during cell cycle and stress response, pointing out the conservation and the difference between yeast and human . We then focus on the cellular consequences of telomere modifications in normal and cancer cells . This leads to a discussion of the different roles, seemingly contradictory, of telomeres and telomerase during the initiation and the progression of a cancer.

Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 349 - 54 Epub 2005 Jan 03.
A-kinase-interacting protein localizes protein kinase A in the nucleus; Sastri M et al.; The genetic variability and covalent modifications associated with the amino terminus of the protein kinase A (PKA) catalytic (C) subunit suggest that it may contribute to protein-protein interactions and/or localization . By using a yeast two-hybrid screen, we identified a PKA-interacting protein (AKIP1) that binds to the amino terminus (residues 1-39) of the C subunit of PKA . The interaction was localized to the A helix (residues 14-39) of the C subunit and to the carboxyl terminus of AKIP1 . AKIP1 thus defines the amino-terminal A helix of PKA as a protein interaction motif . In normal breast (Hs 578 Bst) and HeLa cells, AKIP1 is present in the nucleus as speckles . A nuclear localization signal (Arg-14 and Arg-15) was identified . Upon stimulation with forskolin, HeLa cells expressing AKIP1 accumulated higher levels of the endogenous C subunit in the nucleus . Deletion of the carboxyl terminus of AKIP1 or overexpression of residues 1-39 of the C subunit abolished nuclear localization of the activated endogenous C subunit . Thus, AKIP1 describes a PKA-interacting protein that can contribute to localization by a mechanism that is distinct from A-kinase anchoring proteins that interact with the regulatory subunits.

Arch Pediatr Adolesc Med, 2005 Jan, 159(1), 64 - 7
Pityrosporum folliculitis: diagnosis and management in 6 female adolescents with acne vulgaris; Ayers K et al.; BACKGROUND: Pityrosporum folliculitis is a common inflammatory skin disorder that may mimic acne vulgaris . Some adolescents with recalcitrant follicular pustules or papules may have acne and Pityrosporum folliculitis simultaneously . Clinical response is dependent on treating both conditions . OBJECTIVES: To demonstrate the similarity in clinical manifestation between acne vulgaris and Pityrosporum folliculitis, the benefit of potassium hydroxide preparation, and the benefit of appropriate antifungal therapy . PATIENTS: We describe 6 female adolescents with concurrent Pityrosporum folliculitis infection and acne vulgaris . INTERVENTION: A potassium hydroxide examination was performed on all 6 patients from the exudate of follicular pustules exhibiting spores consistent with yeast . All patients were treated with oral antifungals, and 5 of the 6 patients were also treated with topical antifungals . RESULTS: Six of 6 patients improved with antifungal treatment . All patients also required some ongoing therapy for their acne . CONCLUSIONS: These patients demonstrate that follicular papulopustular inflammation of the face, back, and chest may be due to a combination of acne vulgaris and Pityrosporum folliculitis, a common yet less frequently identified disorder . Symptoms often wax and wane depending on the patient's activities, time of the year, current treatment regimens, and other factors . Pityrosporum folliculitis will often worsen with traditional acne therapy and dramatically respond to antifungal therapy.

FASEB J, 2005 Jan, 19(1), 115 - 7 Epub 2004 Nov 01.
Implication of the MAGI-1b/PTEN signalosome in stabilization of adherens junctions and suppression of invasiveness; Kotelevets L et al.; We recently established the critical role of the lipid phosphatase activity of the PTEN tumor suppressor in stabilizing cell-cell contacts and suppressing invasiveness . To delineate the effector systems involved, we investigated the interaction of PTEN with E-cadherin junctional complexes in kidney and colonic epithelial cell lines . PTEN and the p85 regulatory subunit of phosphatidylinositol 3-OH kinase (PI3K) co-immunoprecipitated with E-cadherin and catenins . By using a yeast two-hybrid assay, we demonstrated that PTEN interacted indirectly with beta-catenin by binding the scaffolding protein MAGI-1b . This model was corroborated in various ways in mammalian cells . Ectopic expression of MAGI-1b potentiated the interaction of PTEN with junctional complexes, promoted E-cadherin-dependent cell-cell aggregation, and reverted the Src-induced invasiveness of kidney MDCKts-src cells . In this model, MAGI-1b slightly decreased the activity of AKT, a downstream effector of PI3K . By using dominant-negative and constitutively active AKT expression vectors, we demonstrated that this kinase was included in the pathways involved in Src-induced destabilization of junctional complexes and was necessary and sufficient to trigger invasiveness . We propose that the recruitment of PTEN at adherens junctions by MAGI-1b and the local down-regulation of phosphatidylinositol-3,4,5-trisphosphate pools and downstream effector systems at the site of cell-cell contacts are focal points for restraining both disruption of junctional complexes and induction of tumor cell invasion.

Virology, 2005 Jan 20, 331(2), 316 - 24
The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus; Ren T et al.; We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17 . TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor . We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro . Secondly, we have performed gel shift experiments demonstrating that TIP does not bind to DNA in a sequence-specific manner . The subcellular localization of TIP was also investigated by transiently expressing green fluorescence protein (GFP)-tagged TIP in Nicotiana benthamiana plant cells, which showed that GFP-tagged TIP localizes primarily to nuclei . Significantly, co-expression of TCVCP and GFP-TIP prevented the nuclear localization of TIP . Together, these results suggest that TIP might be a transcription factor involved in regulating the defense response of Arabidopsis to TCV and that its normal role is compromised by interaction with the invading viral CP.

Biochem Biophys Res Commun, 2005 Feb 11, 327(2), 557 - 64
Comparative analysis of the signaling capabilities of the insulin receptor-related receptor; Klammt J et al.; Although insulin receptor (InsR) and type I insulin-like growth factor receptor (IGF-IR) elicit different physiological effects in their target tissues, their signaling capabilities are similar to a large extent . In the present work, we investigated the potential of the third member of the family, insulin receptor-related receptor (IRR), to associate with known interaction partners of the InsR and the IGF-I receptor in a yeast two-hybrid assay . Using the intracellular part of the IRR we found no association with any of the tested signaling molecules . Phosphotyrosine detection revealed a lack in the constitutive activation of the IRR described for analogous constructs of the two other members of the family . Replacement of the kinase domain of the IGF-IR or its C-terminal lobe alone into the IRR caused a complete restoration of the tyrosine phosphorylation of the IRR . The reestablishment of autophosphorylation was paralleled by restoration of interaction with a specific range of signaling molecules.

Genomics Proteomics Bioinformatics, 2003 Aug, 1(3), 193 - 7
The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity; Liu G et al.; In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter . Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast . To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls . The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

Biosci Rep, 2004 Apr, 24(2), 117 - 26
Bioenergetics of Yarrowia lipolytica cells grown at alkaline conditions; Zvyagilskaya RA et al.; Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined . Cells grown under such severe conditions were found to preserve high respiratory activity . The oxidative phosphorylation system dominated in the energy budget of the cell . A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions . The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4-3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used.

Mol Cell Proteomics . 2004 Dec 31; {Epub ahead of print}
A genome wide screening approach for membrane-targeted proteins; Jaaro H et al.; Membrane-associated proteins are critical for intra- and intercellular communication . Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue . Here we describe a modification of the yeast Ras Recruitment System (RRS) to this end, and designate the modified approach the Ras Membrane Trap (RMT) . A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome . 112 gene products were identified in the screen, of which 79 lack assignable homologues in available databases . Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested . Thus, genome annotation using currently available tools is likely to under-predict representation of membrane-associated gene products . The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes . Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks, and indeed from representatives of all major invertebrate phyla.

Biochim Biophys Acta, 2005 Jan 11, 1681(2-3), 59 - 73 Epub 2004 Nov 23.
Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes; Mohrmann L et al.; By regulating the structure of chromatin, ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in the maintenance, transmission and expression of the eukaryotic genome . Although all known chromatin-remodeling complexes contain an ATPase as a central motor subunit, a number of distinct classes have been recognized . Recent studies have emphasized a more extensive functional diversification among closely related chromatin remodeling complexes than previously anticipated . Here, we discuss recent insights in the functional differences between two evolutionary conserved subclasses of SWI/SNF-related chromatin remodeling factors . One subfamily comprises yeast SWI/SNF, fly BAP and mammalian BAF, whereas the other subfamily includes yeast RSC, fly PBAP and mammalian PBAF . We review the subunit composition, conserved protein modules and biological functions of each of these subclasses of SWI/SNF remodelers . In particular, we will focus on the roles of specific subunits in developmental gene control and human diseases . Recent findings suggest that functional diversification among SWI/SNF complexes allows the eukaryotic cell to fine-tune and integrate the execution of diverse biological programs involving the expression, maintenance and duplication of its genome.

Biophys J . 2004 Dec 30; {Epub ahead of print}
Mathematical modeling of the eukaryotic heat shock response: Dynamics of the hsp70 promoter; Rieger TR et al.; The heat shock response in humans and other eukaryotes is a highly conserved genetic network that coordinates the cellular response to protein damage and is essential for adaptation and survival of the stressed cell . It involves an immediate and transient activation of heat shock transcription factor-1 (HSF1) which results in the elevated expression of genes encoding proteins important for protein homeostasis including molecular chaperones and components of the protein degradative machinery . We have developed a mathematical model of the critical steps in the regulation of HSF1 activity to understand how chronic exposure to a stress signal is converted into specific molecular events for activation and feedback regulated attenuation of HSF1 . The model is utilized to identify the most sensitive steps in HSF1 activation and to evaluate how these steps affect the expression of molecular chaperones . This analysis allows the formulation of hypotheses about the differences between the heat shock responses in yeast and humans and generates a model with predictive abilities relevant to diseases associated with the accumulation of damaged and aggregated proteins including cancer and neurodegenerative diseases.

Biochem Biophys Res Commun, 1976 Dec 20, 73(4), 1101 - 8
Characterization of a mannan-like oligosaccharide from Mycobacterium smegmatis; Maitra SK et al.; A nitrogen-free neutral mannooligosaccharide, similar in structure to the polysaccharide component of yeast mannoproteins, has been isolated from Mycobacterium smegmatis ATCC-356 . It has a molecular weight of 3200 and is terminated at the reducing end by mannose . nuclear magentic resonance spectroscopy, methylation analysis, selective enzymic degradation and acetolysis indicates that the molecule consists of an alpha1 --> 6-linked backbone to which single mannose units are attached in alpha1 --> 2 linkage as sidechains.

Mol Reprod Dev . 2004 Dec 29;70(3):301-307 {Epub ahead of print}
Identification and characterization of a novel testicular germ cell-specific gene Ggnbp1; Zhou Y et al.; A novel gene Ggnbp1 was identified during yeast two-hybrid screening of gametogenetin protein 1 (GGN1)-interacting proteins . Ggnbp1 gene was found in mouse, rat, and human genomes but not in sequenced yeast, worms, fly, or fish genomes . Northern blotting analysis revealed that the gene was specifically expressed in the testis but not expressed in the other tissues . In situ hybridization showed that it was testicular germ cell-specific and was specifically expressed in later primary spermatocytes, meiotic cells, and early round spermatids . Western blotting analysis detected a protein of expected size in and only in the testis . By making membrane and cytosolic fractions of germ cells, we were able to show that GGNBP1 associated with the membrane . The identification and characterization of a novel germ cell-specific gene Ggnbp1 is the first step toward the defining of the functions of Ggnbp1 in spermatogenesis . Mol . Reprod . Dev . 70: 301-307, 2005 . (c) 2005 Wiley-Liss, Inc.

J Biochem (Tokyo), 2004 Oct, 136(4), 421 - 5
Establishment of Stable hFis1 Knockdown Cells with an siRNA Expression Vector; Arai R et al.; Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p . Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1 . We established stable transformants with an hFis1 siRNA expression vector . In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed . In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1 . These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.

Mol Endocrinol . 2004 Dec 29; {Epub ahead of print}
p62, a TFIIH subunit directly interacts with thyroid hormone receptor and enhances T3-mediated transcription; Liu Y et al.; Currently little is known about the direct interactions of general transcription factors and nuclear hormone receptors . To investigate the potential role of the general transcription factor, TFIIH, in T3-mediated transcriptional activation, we examined thyroid hormone receptor (TR) interaction with individual TFIIH subunits in a yeast-two hybrid system . Among the nine subunits of TFIIH studied, only p62 subunit interacted with TRbeta in a ligand-dependent manner . GST pull-down and in vivo co-immunoprecipitation studies also demonstrated direct TR/p62 interaction . Using chromatin immunoprecipitation (ChIP) assays, we showed that TFIIH subunits were co-recruited on or near an endogenous thyroid hormone response element (TRE) upon T3 addition . Co-transfection studies with TSA201 cells showed that p62 increased T3-mediated transcription, which could be further enhanced when p62 and another TFIIH subunit, p44, were co-transfected simultaneously . Taken together, these data suggest that TRs can interact directly with a subunit of TFIIH and may provide an alternative pathway for nuclear receptor communication with the general transcription machinery that circumvents co-activators.

Genes Dev, 2005 Jan 15, 19(2), 234 - 41 Epub 2004 Dec 29.
Regulation of the Neurospora circadian clock by an RNA helicase; Cheng P et al.; The eukaryotic circadian oscillators consist of autoregulatory negative-feedback loops . FRQ, WC-1, and WC-2 are three known components of the negative-feedback loop of the Neurospora circadian oscillator . FRQ represses its own transcription by interacting with the WC-1/WC-2 complex and inhibiting WC's role in transcriptional activation . Here we show that all FRQ associates with FRH, an essential DEAD box-containing RNA helicase in Neurospora . The budding yeast homolog of FRH, Dob1p/Mtr4p, is a cofactor of exosome, an important regulator of RNA metabolism in eukaryotes . Down-regulation of FRH by inducible expression of a hairpin RNA leads to low levels of FRQ but high levels of frq RNA and the abolishment of circadian rhythmicities . FRH is associated with the WC complex and this interaction is maintained in a frq null strain . Disruption of the FRQ-FRH complex by deleting a domain in FRQ eliminates the FRQ-WC interaction, suggesting that FRH mediates the interaction between FRQ and the WC complex . These data demonstrate that FRH is an essential component in the circadian negative-feedback loop and reveal an unexpected role of an RNA helicase in regulating gene transcription.

J Cell Biol, 2005 Jan 3, 168(1), 79 - 88 Epub 2004 Dec 28.
Specialized membrane-localized chaperones prevent aggregation of polytopic proteins in the ER; Kota J et al.; The integral endoplasmic reticulum (ER) membrane protein Shr3p is required for proper plasma membrane localization of amino acid permeases (AAPs) in yeast . In the absence of Shr3p AAPs are uniquely retained in the ER with each of their twelve membrane-spanning segments correctly inserted in the membrane . Here, we show that the membrane domain of Shr3p specifically prevents AAPs from aggregating, and thus, plays a critical role in assisting AAPs to fold and correctly attain tertiary structures required for ER exit . Also, we show that the integral ER proteins, Gsf2p, Pho86p, and Chs7p, function similarly to Shr3p . In cells individually lacking one of these components only their cognate substrates, hexose transporters, phosphate transporters, and chitin synthase-III, respectively, aggregate and consequently fail to exit the ER membrane . These findings indicate that polytopic membrane proteins depend on specialized membrane-localized chaperones to prevent inappropriate interactions between membrane-spanning segments as they insert and fold in the lipid bilayer of the ER membrane.

J Biol Chem . 2004 Dec 28; {Epub ahead of print}
Src phosphorylates Ezrin at tyrosine 477 and induces a phosphospecific association between Ezrin and a Kelch-repeat protein family member; Heiska L et al.; Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells . Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking . The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion . We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells . We show that ezrin is phosphorylated in 293 human embryonic kidney cells in a Src-family dependent way . In SYF cells lacking Src, Yes and Fyn, ezrin was not tyrosine phosphorylated, but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation . Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Y477 as the primary target residue . We generated a pY477 phosphospecific antibody, which confirmed that Y477 becomes phosphorylated in cells in a Src-dependent manner . Y477 phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Y477 phosphorylation is not related to the intramolecular regulation of ezrin . A modified yeast two-hybrid screen, in which ezrin bait was phosphorylated by Src, identified a novel interaction with a kelch-repeat protein family member, Kelch repeat and BTB (POZ) domain containing 2 (KBTBD2) . The Src-dependency of the interaction was further verified by affinity precipitation assays . Identification of a functional interplay with Src opens novel avenues for further characterization of ezrin's biological activities.

Br Poult Sci, 2004 Oct, 45(5), 677 - 83
Selenium supplementation affects broiler growth performance, meat yield and feather coverage; Choct M et al.; (1) Selenium (Se) is an essential part of numerous selenoproteins, most of which are involved in the antioxidant system of the body . It is also required by poultry for the maintenance of optimal health and meat quality . This paper reports data from a study examining the effect of dietary source and concentration of selenium on broiler performance and meat quality . (2) Increased dietary selenium content markedly reduced feed conversion ratio (FCR) as a result of significantly lower feed intakes of birds while maintaining the same weight gains . (3) Selenium supplementation increased feathering, with organic selenium (selenised yeast) being superior to inorganic selenium (sodium selenite) . (4) Birds receiving organic selenium in their diets had improved eviscerated weight, breast yield and reduced drip loss . (5) There were significant concentration x source interactions on yields of breasts and marylands (thigh plus drumstick), with elevated levels of organic selenium increasing the yields, whereas the opposite was true for the inorganic selenium.

Zhonghua Xue Ye Xue Za Zhi, 1997 Jun, 18(6), 308 - 10
{Detection of bcr gene rearrangement in chronic myeloid leukemia by fluorescence in situ hybridization}; Liu M et al.; OBJECTIVE: To detect bcr gene rearrangement in chronic myeloid leukemia (CML) . METHODS: Bcr rearrangement was detected by fluorescence in situ hybridization (FISH), using 765E3, a yeast artificial chromosome (YAC)-derived probe flanking the breakpoint within bcr gene . RESULTS: Nine patients with CML were examined, bcr gene rearrangement was revealed in 5 cases in chronic phase, 2 in blastic phase, and one after interferon-alpha therapy . The karyotype of 1 case after autologous bone marrow transplantation (ABMT) was chimera with normal and bcr gene rearrangement chromosomes . CONCLUSION: YAC765E3 is a useful probe for detecting bcr gene rearrangement . FISH technique is likely an important tool for monitoring of treatment and revealing minimal residual disease in CML.

Curr Biol, 2004 Dec 29, 14(24), 2264 - 70
Loss of hPot1 function leads to telomere instability and a cut-like phenotype; Veldman T et al.; The human telomere binding protein hPot1 binds to the most distal single-stranded extension of telomeric DNA in vitro, and probably in vivo, as well as associating with the double-stranded telomeric DNA binding proteins TRF1 and TRF2 through the bridging proteins PTOP (also known as PIP1 or TINT1) and TIN2 . Disrupting either the DNA binding activity of hPot1 or its association with PTOP results in elongated telomeres, suggesting a role for hPot1 in telomere length regulation . However, mutations to POT1 and Cdc13p, the fission and budding yeast genes encoding the structural orthologs of this protein, leads to telomere instability and cell death . Thus, it is possible that the hPot1 protein may also serve to cap and protect telomeres in humans . Indeed, we now find that knocking down the expression of hPot1 in human cells causes apoptosis or senescence, as well as an increase in telomere associations and anaphase bridges, telltale signs of telomere instability . In addition, knockdown cells also displayed chromatin bridges between interphase cells, reminiscent of the cut phenotype that was first described in fission yeast and in which cytokinesis progresses despite a failure of chromatid separation . However, unlike the yeast cut phenotypes, we suggest that the cut-like phenotype observed in hPot1 knockdown cells is a consequence of the fusion of chromosome ends and that this fusion impedes proper chromosomal segregation . We conclude that hPot1 protects chromosome ends from illegitimate recombination, catastrophic chromosome instability, and abnormal chromosome segregation.

Cell, 2004 Dec 29, 119(7), 969 - 79
TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1; Martin DE et al.; The regulation of ribosome biogenesis in response to environmental conditions is a key aspect of cell growth control . Ribosomal protein (RP) genes are regulated by the nutrient-sensitive, conserved target of rapamycin (TOR) signaling pathway . TOR controls the subcellular localization of protein kinase A (PKA) and the PKA-regulated kinase YAK1 . However, the target transcription factor(s) of the TOR-PKA pathway are unknown . We show that regulation of RP gene transcription via TOR and PKA in yeast involves the Forkhead-like transcription factor FHL1 and the two cofactors IFH1 (a coactivator) and CRF1 (a corepressor) . TOR, via PKA, negatively regulates YAK1 and maintains CRF1 in the cytoplasm . Upon TOR inactivation, activated YAK1 phosphorylates and activates CRF1 . Phosphorylated CRF1 accumulates in the nucleus and competes with IFH1 for binding to FHL1 at RP gene promoters, and thereby inhibits transcription of RP genes . Thus, we describe a signaling mechanism linking an environmental sensor to ribosome biogenesis.

Cell, 2004 Dec 29, 119(7), 955 - 67
Sir-mediated repression can occur independently of chromosomal and subnuclear contexts; Gartenberg MR et al.; Epigenetic mechanisms silence the HM mating-type loci in budding yeast . These loci are tightly linked to telomeres, which are also repressed and held together in clusters at the nuclear periphery, much like mammalian heterochromatin . Yeast telomere anchoring can occur in the absence of silent chromatin through the DNA end binding factor Ku . Here we examine whether silent chromatin binds the nuclear periphery independently of telomeres and whether silencing persists in the absence of anchorage . HMR was excised from the chromosome by inducible site-specific recombination and tracked by real-time fluorescence microscopy . Silent rings associate with the nuclear envelope, while nonsilent rings move freely throughout the nucleus . Silent chromatin anchorage requires the action of either Ku or Esc1 . In the absence of both proteins, rings move throughout the nucleoplasm yet remain silent . Thus, transcriptional repression can be sustained without perinuclear anchoring.

Cell, 2004 Dec 29, 119(7), 911 - 4
A ribonucleolytic rat torpedoes RNA polymerase II; Luo W et al.; Three recent papers reveal a fascinating link between pol II termination and ribonucleolytic decay of the nascent transcript by a 5'-3' exonuclease (yeast Rat1 and human Xrn2) . The exonuclease travels with pol II and gains access to the nascent RNA after endonucleolytic cleavage at the poly(A) site or at a second cotranscriptional cleavage site (CoTC) . It is then thought to track in a 5'-3' direction like a guided torpedo that ultimately helps dissociate the RNA polymerase elongation complex.

Cell, 2004 Dec 29, 119(7), 901 - 2
Ribosome biogenesis: giant steps for a giant problem; Powers T; In this issue of Cell, the Hall group describes how the rapamycin-sensitive TOR signaling network controls ribosomal protein (RP) gene expression via the Forkhead-like transcription factor FHL1 in budding yeast (Martin et al., 2004) . These findings represent a crucial advance in our understanding of the mechanism by which TOR regulates ribosome biogenesis in response to environmental cues.

DNA Seq, 2004 Aug, 15(4), 303 - 5
Cloning and analysis of human Apg16L; Zheng H et al.; Autophagy is an intracellular bulk degradation system, which delivers cytoplasmic components to the lysosome/vacuole . In yeast and mammalian cells, the Apg12-Apg5 conjugate, together with Apg16, form a multimeric complex, which plays an essential role in autopihageosome formation . By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a putative protein with 607 amino acid residues, which shows 90% identity and 93% similarity to mouse Apg16L . This protein, designated human Apg16L, contains a coiled-coil domain and a motif with seven WD repeats, which are also shared by mouse Apg16L . Database searching revealed that Apg16L is mapped to chromosome 2q37.1 and there exist at least four splice variants.

Mass Spectrom Rev . 2004 Dec 23; {Epub ahead of print}
Defining mitogen-activated protein kinase pathways with mass spectrometry-based approaches; Powell DW et al.; I.Introduction000 II.Mitogen-Activated Protein Kinase Pathways000A . ERK1/2 Pathway000B . JNK Pathway000C . p38 MAPK Pathway000D . ERK5 Pathway000 III.Specificity and Crosstalk in MAPK Pathways000A . Organization of MAPK Pathways Into Networks000B . Docking Sites000C . Scaffold Proteins000 1 . ERK Scaffolds000 2 . JNK and p38 Scaffolds000 IV.Role of MAPKs in Neutrophil Functions000 V.MAPK Substrates000 VI.Proteomic Methods for Identification of Phosphoproteins000A . Gel Shift of pI000B . Phosphopeptide Enrichment000C . Phosphoprotein Staining000D . Precursor-Ion Scanning000E . Neutral-Loss Scanning000 VII.Proteomic Methods for Identification of MAPK-Signaling Components in Neutrophils000A . Protein Extraction000B . Kinase-Specific Phosphorylation000C . Protein Separation000D . Mass Spectrometry000E . Bioinformatic Identification of MAPKAPK2 Substrates000F . Substrate Confirmation and Functional Role of Phosphorylation000G . Phosphopeptide Enrichment000VIII.Conclusions000References000Mitogen-activated protein kinases are a group of ubiquitously expressed kinase pathways that have been conserved from yeast through humans . They control a large number of critical cell functions . Identification of targets of those kinases is necessary to define signal transduction pathways that lead to cell responses . The application of a number of mass spectrometry-based techniques to the identification of phosphoproteins is reviewed . A new proteomic approach is described for the identification of the downstream targets of specific kinases that combines phosphorylation of cell lysates in in vitro kinase reactions by active recombinant kinase with protein separation by two-dimensional (2D) gel electrophoresis or SDS-PAGE and phosphoprotein identification by MALDI-TOF mass spectrometry or by phosphopeptide enrichment and tandem mass spectrometry . The results suggested that a combination of multiple approaches will be required to fully identify phosphoproteomes . Published 2004 Wiley Periodicals, Inc.

Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2598 - 606
Molecular Characterization of the Cytoplasmic Interacting Protein of the Receptor Kinase IRK Expressed in the Inflorescence and Root Apices of Arabidopsis; Hattan J et al.; Meristem maintenance and differentiation is regulated by intercellular communication through receptor-like kinases (RLKs) in plants, but the underlying molecular mechanisms of RLK signaling remain largely unknown . A cytoplasmic interactor for inflorescence and root apices receptor-like kinase (IRK), which is a typical meristematic RLK with leucine-rich repeats in Arabidopsis, was identified using a yeast two-hybrid assay and named IRK-interacting protein (IRKI) . IRKI is a novel but highly conserved protein found in higher plants . The interaction between IRK and IRKI was confirmed by an in vitro pull-down assay and supported by their simultaneous expression in actively dividing cells in meristems . In the root tip, IRKI expression and localization visualized by green fluorescence protein (GFP) were observed in the quiescent center, initial cells, and immature stele cells . IRKI expression was expanded by exogenous auxin treatment and repressed by inhibitor treatment of polar auxin transport.

Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2581 - 7
Identification of Three Clones Which Commonly Interact with the Kinase Domains of Highly Homologous Two Receptor-Like Kinases, RLK902 and RKL1; Tarutani Y et al.; We have previously reported the characterization of highly homologous two leucine-rich repeat (LRR)-receptor-like kinase (RLK) genes, RLK902 and RKL1, which showed 75% identity at the amino acid sequence level . To investigate the RLK902 and RKL1 mediated signal transduction pathways, we performed yeast two-hybrid screening using the kinase domains of RLK902 and RKL1 as baits . Three clones, Y-1, 2 and 3, were found to interact commonly with the kinase domain of RLK902 and RKL1 and not to interact with the kinase domain of BRI1, a member of LRR-RLKs . This result suggests that RLK902 and RKL1 may have common biochemical functions, especially in their downstream signal transduction . Furthermore, the detail analysis of their responsiveness to various conditions suggests their involvement in such stress conditions as mechanical wounding, treatment with salicylic acid, and pathogen infection.

Plant Physiol, 2005 Jan, 137(1), 83 - 93 Epub 2004 Dec 23.
Arabidopsis AtCUL3a and AtCUL3b Form Complexes with Members of the BTB/POZ-MATH Protein Family; Weber H et al.; The ubiquitin proteasome pathway in plants has been shown to be important for many developmental processes . The E3 ubiquitin-protein ligases facilitate transfer of the ubiquitin moiety to substrate proteins . Many E3 ligases contain cullin proteins as core subunits . Here, we show that Arabidopsis (Arabidopsis thaliana) AtCUL3 proteins interact in yeast two-hybrid and in vitro pull-down assays with proteins containing a BTB/POZ (broad complex, tramtrack, bric-a-brac/pox virus and zinc finger) motif . By changing specific amino acid residues within the proteins, critical parts of the cullin and BTB/POZ proteins are defined that are required for these kinds of interactions . In addition, we show that AtCUL3 proteins assemble with the RING-finger protein AtRBX1 and are targets for the RUB-conjugation pathway . The analysis of AtCUL3a and AtCUL3b expression as well as several BTB/POZ-MATH genes indicates that these genes are expressed in all parts of the plant . The results presented here provide strong evidence that AtCUL3a and AtCUL3b can assemble in Arabidopsis with BTB/POZ-MATH and AtRBX1 proteins to form functional E3 ligases.

Mol Endocrinol . 2004 Dec 23; {Epub ahead of print}
Novel association of Vav2 and Nek3 modulates signaling through the human prolactin receptor; Miller SL et al.; Prolactin receptor activation contributes to the progression and motility of human breast cancer . This event activates multimeric signaling pathways, including the activation of the Vav family of guanine nucleotide exchange factors . To detect novel proteins interacting with Vav, yeast two-hybrid analysis was performed and demonstrated an interaction between the serine/threonine NIMA-related family kinase p56(Nek3) and Vav1 . The prolactin-dependent interaction of Nek3 with Vav1 and Vav2, was confirmed by co-immunoprecipitation analysis . Prolactin stimulation of T47D cells induced Nek3 kinase activity, and the interaction of Vav2/Nek3 with the prolactin receptor . Increased Nek3 levels upregulated Vav2 serine and tyrosine phosphorylation, while knock-down of Nek3 resulted in a reduction of Vav2 phosphorylation . Activation of GTPase Rac-1 in CHO transfectants, required both Nek3 and Vav2, and was inhibited by the over-expression of a kinase inactivating Nek3 mutant . However, overexpression of either Nek3 or kinase-inactive Nek3 had no effect on Vav2-potentiated Stat5-mediated gene expression . Overexpression of kinase inactive Nek3 in T47D cells led to a 50% increase in apoptosis vs . controls . These data suggest that the prolactin-mediated activation of Nek3 contributes differentially to Vav2 signaling pathways involving Rac1 and Stat5 and implicates Nek3 during prolactin-mediated actions in breast cancer.

J Biol Chem . 2004 Dec 23; {Epub ahead of print}
TTK/hMps1 participates in the regulation of DNA damage checkpoint response by phosphorylating CHK2 on Thr68; Wei JH et al.; CHK2 (hCds1) plays important roles in DNA damage-induced cell cycle checkpoint by phosphorylating several important targets, such as Cdc25 and p53 . To obtain a better understanding of the CHK2 signaling pathway, we have carried out yeast two hybrid screen to search for potential CHK2-interacting proteins . Here, we report the identification of the mitotic checkpoint kinase, TTK/hMps1, as a novel CHK2-interacting protein . TTK/hMps1 directly phosphorylates CHK2 on Thr68 in vitro . Expression of a TTK kinase-dead mutant, TTKD647A, interferes with the G2/M arrest induced by either ionizing radiation or UV . Interestingly, induction of CHK2 Thr68 phosphorylation and of several downstream events, such as cyclin B1 accumulation and Cdc2 Tyr15 phosphorylation, are also affected . Furthermore, ablation of TTK expression using siRNA results not only in reduced CHK2 Thr68 phosphorylation, but also in impaired growth arrest . Our results are consistent with a model in which TTK functions upstream of CHK2 in the response to DNA damage, and suggest possible crosstalk between the spindle assembly checkpoint and the DNA damage checkpoint.

Dev Biol, 2005 Jan 15, 277(2), 457 - 71
Range of HOX/TALE superclass associations and protein domain requirements for HOXA13:MEIS interaction; Williams TM et al.; AbdB-like HOX proteins form DNA-binding complexes with the TALE superclass proteins MEIS1A and MEIS1B, and trimeric complexes have been identified in nuclear extracts that include a second TALE protein, PBX . Thus, soluble DNA-independent protein-protein complexes exist in mammals . The extent of HOX/TALE superclass interactions, protein structural requirements, and sites of in vivo cooperative interaction have not been fully explored . We show that Hoxa13 and Hoxd13 expression does not overlap with that of Meis1-3 in the developing limb; however, coexpression occurs in the developing male and female reproductive tracts (FRTs) . We demonstrate that both HOXA13 and HOXD13 associate with MEIS1B in mammalian and yeast cells, and that HOXA13 can interact with all MEIS proteins but not more diverged TALE superclass members . In addition, the C-terminal domains (CTDs) of MEIS1A (18 amino acids) and MEIS1B (93 amino acids) are necessary for HOXA13 interaction; for MEIS1B, this domain was also sufficient . We also show by yeast two-hybrid assay that MEIS proteins can interact with anterior HOX proteins, but for some, additional N-terminal MEIS sequences are required for interaction . Using deletion mutants of HOXA13 and HOXD13, we provide evidence for multiple HOX peptide domains interacting with MEIS proteins . These data suggest that HOX:MEIS interactions may extend to non-AbdB-like HOX proteins in solution and that differences may exist in the MEIS peptide domains utilized by different HOX groups . Finally, the capability of multiple HOX domains to interact with MEIS C-terminal sequences implies greater complexity of the HOX:MEIS protein-protein interactions and a larger role for variation of HOX amino-terminal sequences in specificity of function.

Biochem J . 2004 Dec 23; {Epub ahead of print}
Identification and assessment the role of a nominal phospholipid binding region of oxysterol binding protein related protein ORP1S in the regulation of vesicular transport; Fairn GD et al.; Oxysterol binding protein related proteins (ORPs) constitute an enigmatic family of intracellular lipid receptors related through a shared lipid binding domain . Emerging evidence suggests that ORPs conjoin lipid metabolism with membrane transport . Current data imply that the yeast ORP, Kes1p, is a negative regulator of Golgi derived vesicular transport mediated by the essential phosphatidylinositol/phosphatidylcholine transfer protein Sec14p . Inactivation of Kes1p function allows for restoration of growth and vesicular transport in cells lacking Sec14p function, and Kes1p function in this regard can be complemented by human ORP1S . Recent studies have determined that Kes1p and ORP1S both bind phospholipids as ligands . To explore the function of distinct linear segments of ORP1S in phospholipid binding and vesicular transport regulation we generated a series of 15 open reading frames coding for diagnostic regions within ORP1S . Purified versions of these ORP1S deletion proteins were characterized in vitro and allowed for the identification of a nominal phospholipid binding region . The in vitro analysis was interpreted in the context of in vivo growth and vesicle transport assays for analytic members of the ORP1S deletion set . The results determined that phospholipid binding domain per se was insufficient for ORP1S inhibition of vesicular transport and that CPY and invertase transport from the Golgi may be regulated differentially by specific regions of ORP1S/Kes1p.

Dalton Trans, 2005 Jan 21, (2), 256 - 67 Epub 2004 Dec 09.
From model compounds to protein binding: syntheses, characterizations and fluorescence studies of {Ru(II)(bipy)(terpy)L}(2+) complexes (bipy = 2,2{prime or minute}-bipyridine; terpy = 2,2{prime or minute}:6{prime or minute},2{double prime}-terpyridine; L = imidazole, pyrazole and derivatives, cytochrome c); Yang XJ et al.; Compounds {Ru(II)(bipy)(terpy)L}(PF(6))(2) with bipy = 2,2{prime or minute}-bipyridine, terpy = 2,2{prime or minute}:6{prime or minute},2{double prime}-terpyridine, L = H(2)O, imidazole (imi), 4-methylimidazole, 2-methylimidazole, benzimidazole, 4,5-diphenylimidazole, indazole, pyrazole, 3-methylpyrazole have been synthesized and characterized by (1)H NMR, ESI-MS and UV/Vis (in CH(3)CN and H(2)O) . For L = H(2)O, imidazole, 4,5-diphenylimidazole and indazole the X-ray structures of the complexes have been determined with the crystal packing featuring only few intermolecular C-H{small pi} or {small pi}-{small pi} interactions due to the separating action of the PF(6)-anions . Complexes with L = imidazole and 4-methylimidazole exhibit a fluorescence emission with a maximum at 662 and 667 nm, respectively ({small lambda}(exc)= 475 nm, solvent CH(3)CN or H(2)O) . The substitution of the aqua ligand in {Ru(bipy)(terpy)(H(2)O)}(2+) in aqueous solution by imidazole to give {Ru(bipy)(terpy)(imi)}(2+) is fastest at a pH of 8.5 (as followed by the increase in emission intensity) . Coupling of the {Ru(bipy)(terpy)}(2+) fragment to cytochrome c(Yeast iso-1) starting from the Ru-aqua complex was successful at 35 {degree}C and pH 7.0 after 5 d under argon in the dark . The {Ru(bipy)(terpy)(cyt c)}-product was characterized by UV/Vis, emission and mass spectrometry . The location where the {Ru(bipy)(terpy)} complex was coupled to the protein was identified as His44 (corresponding to His39 in other numbering schemes) using digestion of the Ru-coupled protein by trypsin and analysis of the tryptic peptides by HPLC-high resolution MS.

Mol Biol Cell . 2004 Dec 22; {Epub ahead of print}
Opposing Roles for Actin in Cdc42p Polarization; Irazoqui JE et al.; Monitoring Editor: Anthony Bretscher In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells . Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton . Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells . We provide evidence that dispersal is due to endocytosis associated with cortical actin patches, and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity . Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis . In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.

Mol Biol Cell . 2004 Dec 22; {Epub ahead of print}
Identification and Functional Characterization of Ankyrin-Repeat Family Protein, ANKRA, as a Protein Interacting with BKCa Channel; Lim HH et al.; Monitoring Editor: Guido Guidotti Ankyrin-repeat family A protein (ANKRA) was originally cloned in mouse as an interacting protein to megalin, a member of low-density lipoprotein receptor superfamily . Here we report that the isolation of rat ANKRA as a new binding partner for the alpha-subunit of rat large-conductance Ca(2+)-activated K(+) channel (rSlo) . We mapped the binding region of each protein using yeast two-hybrid and in vitro binding assays . ANKRA expressed together with rSlo channels were colocalized near the plasma membrane and coimmunoprecipitated in transfected cells . We also showed that BKCa channel in rat cerebral cortex coprecipitated with rANKRA and colocalized in cultured rat hippocampal neuron . While the coexpression of ANKRA did not affect the surface expression of rSlo, the gating kinetics of rSlo channel was significantly altered and the effects were highly dependent on the intracellular calcium . These results indicate that ANKRA could modulate the excitability of neurons by binding directly to endogenous BKCa channel and altering its gating kinetics in a calcium dependent manner.

Mol Biol Cell . 2004 Dec 22; {Epub ahead of print}
The FHA-domain Protein Cep170 Interacts with Plk1 and Serves as a Marker for Mature Centrioles; Guarguaglini G et al.; Monitoring Editor: Trisha Davis We report the characterization of Cep170, a forkhead-associated (FHA) domain protein of previously unknown function . Cep170 was identified in a yeast two-hybrid screen for interactors of Polo-like kinase 1 (Plk1) . In human cells, Cep170 is constantly expressed throughout the cell cycle but phosphorylated during mitosis . It interacts with Plk1 in vivo and can be phosphorylated by Plk1 in vitro, suggesting that it is a physiological substrate of this kinase . Both overexpression and siRNA-mediated depletion studies suggest a role for Cep170 in MT organization and cell morphology . Cep170 associates with centrosomes during interphase and with spindle microtubules during mitosis . As shown by immunoelectron-microscopy, Cep170 associates with subdistal appendages, typical of the mature mother centriole . Thus, anti-Cep170 antibodies stain only one centriole during G1, S and early G2, but two centrioles during late G2 phase of the cell cycle . We show that Cep170 labeling can be used to discriminate bonafide centriole overduplication from centriole amplification that results from aborted cell division.

Mol Biol Evol . 2004 Dec 22; {Epub ahead of print}
Comparative Genomics of Centrality and Essentiality in Three Eukaryotic Protein-Interaction Networks; Hahn MW et al.; Most proteins do not evolve in isolation, but as components of complex genetic networks . Therefore, a protein's position in a network may indicate how central it is to cellular function, and hence how constrained it is evolutionarily . In order to look for an effect of position on evolutionary rate, we examined the protein-protein interaction networks in three eukaryotes: yeast, worm, and fly . We find that the three networks have remarkably similar structure, such that the number of interactors per protein and the centrality of proteins in the networks have similar distributions . Proteins that have a more central position in all three networks, regardless of the number of direct interactors, evolve more slowly and are more likely to be essential for survival . Our results thus are consistent with a classic proposal of Fisher's that pleiotropy constrains evolution.

Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 152 - 7 Epub 2004 Dec 22.
RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production; Sugiyama T et al.; In fission yeast, factors involved in the RNA interference (RNAi) pathway including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for heterochromatin assembly at centromeric repeats and the silent mating-type region . Previously, we have shown that RNA-induced initiation of transcriptional gene silencing (RITS) complex containing the Argonaute protein and small interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism to couple siRNA generation with heterochromatin formation . Here, we investigate the role of RNA-dependent RNA polymerase (Rdp1) and its polymerase activity in the assembly of heterochromatin . We find that Rdp1, similar to RITS, localizes to all known heterochromatic loci, and its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins . We show that a point mutation within the catalytic domain of Rdp1 abolished its RNA-dependent RNA polymerase activity and resulted in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering . Moreover, the RITS complex in the rdp1 mutant does not contain siRNAs, and is delocalized from centromeres . These results not only implicate Rdp1 as an essential component of a self-enforcing RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase activity in siRNA production necessary for heterochromatin formation.

J Biol Chem . 2004 Dec 22; {Epub ahead of print}
Friedreich's ataxia: no changes in mitochondrial labile iron in human lymphoblasts and fibroblasts . A decrease in antioxidative capacity?
Sturm B, Bistrich U, Schranzhofer M, Sarsero JP, Rauen U, Scheiber-Mojdehkar B, de Groot H, Ioannou P, Petrat F.
Friedreich s ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein . Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage . Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-{(1,10-phenanthrolin-5-yl)aminocarbonyl}benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells . No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels . Uptake studies with 55(r)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls . However, sensitivity to H2{sub}O2{sub} was significantly increased in patient cells and H2{sub}O2{sub} toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2 -dipyridyl, but not by the mitochondrion-selective chelator RPA . Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells . These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA...

Arch Virol . 2004 Dec 21; {Epub ahead of print}
Human hepatitis B virus X protein promotes cell proliferation and inhibits cell apoptosis through interacting with a serine protease Hepsin; Zhang JL et al.; The X protein of human hepatitis B virus (HBV) acts as an indirect transcriptional transactivator to regulate the expression of many viral and cellular genes as well as playing a critical role in the development of hepatocellular carcinoma . While the biological importance of HBx has been well established, the cellular and molecular bases of its function remain largely undefined . In this study, we isolated a new HBV field strain from a patient with chronic viral infection . The X protein encoded by this virus was used as a bait protein for screening a human liver cDNA library using a yeast two-hybrid system . Several cell proteins were identified as new HBx interacting partners, including a transmembrane serine protease, Hepsin . Direct interaction between HBx and Hepsin proteins was confirmed by in vitro and in vivo co-immunoprecipitation assays . HBx also co-localized with Hepsin in human cells as determined by confocal immunofluorescence microscopy . The interaction between HBx and Hepsin protein appeared to play a role in both promoting cell proliferation and blocking apoptosis in human liver tumor cell and normal liver cell lines . In addition, the complex of HBx and Hepsin promoted the expression of HBeAg in Hep G2.2.1.5 cells indicating that the association of these two proteins stimulated viral replication.

RNA . 2004 Dec 21; {Epub ahead of print}
3D models of yeast RNase P/MRP proteins Rpp1p and Pop3p; Dlakic M; Sensitive profile searches and fold recognition were used to predict the structures of two yeast RNase P/MRP proteins . Rpp1p, which is one of the subunits common to eukaryotes and archaea, is predicted to adopt the seven-stranded TIM-barrel fold found in PHP phosphoesterases . Pop3p, initially thought to be one of the RNase P/MRP subunits unique to yeast, has been assigned the L7Ae/L30e fold . This RNA-binding fold is also present in human RNase P subunit Rpp38, raising the possibility that Pop3p and Rpp38 are functional homologs.

J Virol, 2005 Jan, 79(2), 910 - 7
Covalent modification of human immunodeficiency virus type 1 p6 by SUMO-1; Gurer C et al.; The p6 domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein mediates virion budding from infected cells via protein-protein contacts with the class E vacuolar protein sorting factors, Tsg101 and AIP1/ALIX . Interaction with Tsg101 is strengthened by covalent attachment of monovalent ubiquitin to HIV-1 p6 . To identify additional host factors that bind to HIV-1 p6, a human cDNA library was screened in the yeast two-hybrid system . HIV-1 p6 was found to interact with small ubiquitin-like modifier 1 (SUMO-1) as well as the E2 SUMO-1 transfer enzyme, Ubc9 . Interaction with p6 was also detected with Daxx, a cellular protein to which SUMO-1 is sometimes covalently attached . SUMO-1 was incorporated into HIV-1 virions where it was protected within the virion membrane from digestion by exogenous protease . Of the two lysine residues in p6, lysine 27 uniquely served as a site of covalent SUMO-1 attachment . As previously reported, though, HIV-1 bearing the p6-K27R mutation replicated just like the wild type . Overproduction of SUMO-1 in HIV-1 producer cells had no apparent effect on virion release or on virion protein or RNA content . Infectivity of the resulting virions, though, was decreased, with the defect occurring after membrane fusion, at the time of viral cDNA synthesis . HIV-1 bearing the p6-K27R mutation was insensitive to SUMO-1 overexpression, suggesting that covalent attachment of SUMO-1 to p6 is detrimental to HIV-1 replication.

Biofizika, 2004 Nov-Dec, 49(6), 987 - 94
{The interaction of the GroEL chaperone with early kinetic intermediates of renaturing proteins inhibits the formation of their native structure}; Reining in cytokinesis with a septin corral; Biology Department, Rensselaer Polytechnic Institute, 110 8th St . Troy, NY 12180Septins are a family of conserved GTP-binding proteins that function in cytokinesis in fungi and animals . In budding yeast, septins form scaffolds for assembly of the actomyosin contractile ring at the cleavage plane, a role that does not appear to be conserved in other organisms . The septins form an hourglass-shaped collar at the mother-bud neck, which splits into two rings flanking the division plane at cytokinesis . A recent study1 demonstrates that these two septin rings constitute diffusion barriers that create a cytokinetic compartment to retain cortical cytokinetic factors in proximity to the cleavage plane . BioEssays 27:5-8, 2005 . (c) 2004 Wiley Periodicals, Inc.

Cell Cycle . 2005 Jan 10;4(1) {Epub ahead of print}
Cdc14 and the Temporal Coordination Between Mitotic Exit and Chromosome Segregation; Torres-Rosell J et al.; Cell division involves the inheritance of a complete set of the genome in the form of chromosomes . One of the strategies employed by eukaryotic cells is to maintain replicated sister chromatids together until the anaphase onset . A protein complex named cohesin holds sisters together following replication until anaphase when cleavage of cohesin by the protease separase initiates segregation . Recent studies in budding yeast have shown that cohesin cleavage alone is not sufficient for the segregation of the entire genome . Instead, repetitive regions, such as the ribosomal DNA (rDNA) array and telomeres, require additional mechanisms during mitotic disjunction . The segregation of such chromosome regions is delayed and needs specific cell cycle regulators such as the FEAR network and the conserved phosphatase Cdc14, all of which orchestrate the timely completion of chromosome segregation before mitotic exit . Future studies will be targeted towards unravelling the nature of the additional segregation requirements for repetitive regions and the specifics of its cell cycle control.

Cell Cycle . 2005 Jan 10;4(1) {Epub ahead of print}
Nucleolus in the Spotlight; Hernandez-Verdun D; The recent contributions on chromatid segregation at the metaphase/anaphase transition demonstrate two distinct pathways in budding yeast . While segregation of most of the genome is a direct consequence of cohesin cleavage by separase, rDNA segregation requires a novel pathway involving Cdc14 phosphatase activation . This activation induces targeting of condensin to rDNA which in association with Aurora B kinase modulates rDNA compaction during anaphase . The resolution of rDNA sequences occurs after this step.

Cell Cycle . 2005 Jan 29;4(1) {Epub ahead of print}
Cell Cycle Dependent Regulation of the Origin Recognition Complex; Depamphilis ML; The eukaryotic origin recognition complex (ORC) not only selects the sites where prereplication complexes are assembled and DNA replication begins, it is the first in a series of multiple coherent pathways that determines when prereplication complexes are assembled . Data from yeast, frogs, flies and mammals present a compelling case that one or more of the six ORC subunits undergoes cell cycle dependent modifications involving phosphorylation and ubiquitination that repress ORC activity during S, G(2) and M-phases . ORC activity is not restored until mitosis is complete and a nuclear membrane is present . In yeast, frogs and mammals, the same cyclin-dependent protein kinase {Cdk1(Cdc2)} that initiates mitosis also inhibits assembly of functional ORC/chromatin sites . In yeast, ORC remains bound to chromatin throughout cell division, but in the metazoa either ORC or the Orc1 subunit appears to cycle on and off the chromatin . Thus, this "ORC cycle" is the premier step in preventing rereplication of DNA during a single cell division cycle.

J Cell Biol, 2004 Dec 20, 167(6), 1087 - 98
Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles; Fratti RA et al.; Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein-protein interactions (e.g., coated vesicles) . During docking, yeast vacuoles assemble "vertex" ring-shaped microdomains around the periphery of their apposed membranes . Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)-VpsC Rab effector complex, SNAREs, and actin . Membrane fusion initiates at vertex microdomains . We now find that the "regulatory lipids" ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner . Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS . Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment . Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles . Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

Genetics, 2004 Dec, 168(4), 2207 - 15
The effect of sequence divergence on recombination between direct repeats in Arabidopsis; Opperman R et al.; It is well established that sequence divergence has an inhibitory effect on homologous recombination . However, a detailed analysis of this relationship is missing for most higher eukaryotes . We have measured the rate of somatic recombination between direct repeats as a function of the number, type, and position of divergent nucleotides in Arabidopsis . We show that a minor divergence level of 0.16% (one mutation in otherwise identical 618 bp) has a profound effect, decreasing the recombination rate approximately threefold . A further increase in the divergence level affects the recombination rate to a smaller extent until a "divergence saturation" effect is reached at relatively low levels of divergence ( approximately 0.5%) . The type of mismatched nucleotide does not affect recombination rates . The decrease in the rate of recombination caused by a single mismatch was not affected by the position of the mismatch along the repeat . This suggests that most recombination intermediate tracts contain a mismatch and thus are as long as the full length of the 618-bp repeats . Finally, we could deduce an antirecombination efficiency of approximately 66% for the first mismatch in the repeat . Altogether, this work shows some degree of conservation across kingdoms when compared to previous reports in yeast; it also provides new insight into the effect of sequence divergence on homologous recombination.

J Biol Chem . 2004 Dec 16; {Epub ahead of print}
Ubc9 and PIAS1 activate COUP-TFI-mediated human CYP11B2 gene transcription; Kurihara I et al.; Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells . Using electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter . Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity . However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation . To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1 which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively . The co-immunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells . Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells . Co-expression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI . However, SUMOylation defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that SUMOylation activity are separable from coactivator ability . In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9 and PIAS1 were recruited to an endogenous CYP11B2 promoter . Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI . Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.

J Biol Chem . 2004 Dec 17; {Epub ahead of print}
The ammonium transporter RhBG : Requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells; Lopez C et al.; RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters . The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells . We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis tyrosine-based signal and an association with ankyrin-G, respectively . We first show, using a model of polarized epithelial MDCK cells, that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C-terminus of the protein . Secondly, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G . The biological relevance of this interaction is supported by two observations: i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections, ii) the disruption of the FLD binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected MDCK cells . Mutation of both targeting signal and ankyrin-G binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

J Biol Chem . 2004 Dec 20; {Epub ahead of print}
Essential role of synoviolin in embryogenesis; Yagishita N et al.; We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect . For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno-/-) mice by gene-targeted disruption . Strikingly, all fetuses lacking syno died in utero around E13.5, though Hrd1p, a yeast orthologue of Synoviolin, is nonessential for survival . Histologically, hypocellularity and aberrant apoptosis were noted in the syno-/- fetal liver . Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno-/- embryos, causing death in utero . Cultured embryonic fibroblasts derived from syno-/- mice were more susceptible to ER stress-induced apoptosis than those from syno+/+ mice, but the susceptibility was rescued by overexpression of synoviolin . Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

J Biol Chem . 2004 Dec 20; {Epub ahead of print}
Nte1p mediated deacylation of phosphatidylcholine functionally interacts with Sec14p; Murray JP et al.; Deciphering the function of the essential yeast Sec14p protein has revealed a regulatory interface between cargo secretion from Golgi and lipid homeostasis . Abrogation of the CDP-choline (CDP-Cho) pathway for phosphatidylcholine (PC) synthesis allows for life in absence of the otherwise essential Sec14p . Nte1p, the product of ORF YML059c, is an integral membrane phospholipase against CDP-Cho derived PC producing intracellular glycerophosphocholine (GPCho) and free fatty acids . We monitored Nte1p activity in through in vivo PC turnover measurements and observed that intracellular GPCho accumulation is decreased in a sec14ts strain shifted to 37 masculineC in 10 mM choline (Cho)-containing medium, in comparison with a Sec14p proficient strain . Overexpression of two Sec14p homologues Sfh2p and Sfh4p in sec14ts cells restored secretion and growth at the restrictive temperature but did not restore GPCho accumulation . Instead, newly synthesized PC was degraded by phospholipase D (Spo14p) . Similar analysis performed in a sec14D background confirmed these observations . These results imply that the ability of Sfh2p and Sfh4p to restore secretion and growth is not through a shared function with Sec14p in the regulation of PC turnover via Nte1p . Furthermore, our analyses revealed a profound alteration of PC metabolism triggered by the absence of Sec14p: Nte1p unresponsiveness; Spo14p activation, and; deregulation of Pct1p . Sfh2p and Sfh4p overexpressing cells coped with the absence of Sec14p by controlling the rate of phosphocholine formation, limiting the amount of Cho available for this reaction, and actively excreting Cho from the cell . Increased Sfh4p also significantly reduced uptake of exogenous Cho . Beyond the new PC metabolic control features we ascribe to Sfh2p and Sfh4p we also describe a second role for Sec14p in mediating PC homeostasis . Sec14p acts as a positive regulator of Nte1p mediated PC deacylation with the functional consequence of increased Nte1p activity increasing the permissive temperature for growth of sec14ts cells.

J Biol Chem . 2004 Dec 15; {Epub ahead of print}
Overlapping but separable determinants of DNA binding and nuclear localization map to the C-terminal end of the C . elegans DAF-12 DNA binding domain; Shostak Y et al.; Proteins are commonly viewed as modular assemblies of functional domains . We analyzed a loss of function mutation in the Caenorhabditis elegans intracellular receptor (IR) DAF-12, a conservative substitution of an arginine to a lysine at position 197 (R197K) . R197 resides in region similar to a nuclear localization signal (NLS), just downstream of the receptor minimal zinc finger DNA binding domain (DBD) core . We found that the R197K, but not mutations of neighboring arginine or lysine residues, dramatically reduced DAF-12 transcriptional regulatory activity in a yeast reporter assay . This reduction in regulatory activity correlated with greatly decreased DNA binding affinity in vitro, suggesting a role for the DAF-12 DBD C-terminal region (dbdC), and specifically for R197, in DNA binding . Remarkably, three basic residues immediately contiguous with R197 played little role in DNA binding, but rather affected nuclear localization; in contrast, R197 itself was dispensable for nuclear localization . Thus, DAF-12 dbdC harbors overlapping but separable determinants of DNA binding and nuclear localization in a single small region.

Chem Biol, 2004 Dec, 11(12), 1677 - 87
Using a small molecule inhibitor of Peptide: N-glycanase to probe its role in glycoprotein turnover; Misaghi S et al.; Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol . Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo . Through chemical synthesis we determined that the P(1) residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition . We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different . Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.

Mol Biochem Parasitol, 2005 Jan, 139(1), 91 - 7
Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR); Banerjee S et al.; The protozoan parasite Entamoeba histolytica express a cytosine-5 DNA methyltransferase (Ehmeth) that belongs to the DNMT2 protein family . The biological function of members of this DNMT2 family is unknown . In the present study, we have demonstrated that Ehmeth is a nuclear matrix protein . Indeed, we showed by south-western analysis and yeast one-hybrid system that Ehmeth binds to EhMRS2, a DNA element which contains the eukaryotic consensus scaffold/matrix attachment regions (S/MAR) bipartite recognition sequences . S/MARs have been implicated in a variety of important functions, such as genome organization and gene expression . The methylation status of cytosine located within EhMRS2 was analyzed by bisulfite genomic sequencing . We observed the presence of methylated cytosine within the 3'-end of EhMRS2 . These data provide the first evidence that a member of the DNMT2 family interacts with a S/MAR containing DNA element.

Mol Biochem Parasitol, 2005 Jan, 139(1), 25 - 32
Novel alleles of Plasmodium falciparum dhfr that confer resistance to chlorcycloguanil; Hunt SY et al.; In Plasmodium falciparum, resistance to folate inhibitors like pyrimethamine is mediated by point mutations in the target gene dihydrofolate reductase (dhfr) . The resistance to pyrimethamine increases with the accumulation of particular point mutations . These mutations also confer increased resistance to chlorcycloguanil, the active metabolite of chlorproguanil and one component of a newly introduced DHFR inhibitor, LapDap((R)) . One genotype (16V/108T) has been previously identified that confers resistance to cycloguanil but not to pyrimethamine . This study was designed to identify novel alleles that might confer resistance to chlorcycloguanil, but escape the surveillance methods currently in place for common pyrimethamine-resistant alleles . Directed mutagenesis was performed using the wild type and the common pyrimethamine-resistant allele, 51I/59R/108N, to determine the effect of the 16V and 108T mutations on enzyme activity and drug resistance . In addition, we randomly mutagenized the 51I/59R/108N allele and identified nine novel alleles that could confer resistance to chlorcycloguanil . These yeast strains were also resistant to pyrimethamine, but retained sensitivity to the experimental DHFR inhibitor, WR99210 . None of the alleles generated in this study was as resistant to chlorcycloguanil as the common quadruple mutant, 51I/59R/108N/164L . In addition, selection of high levels of chlorcycloguanil resistance in parasites that carry the 51I/59R/108N allele will require two directed steps, a change from 108N to 108T followed by a mutation from A16 to 16V . The resulting allele, 16V/51I/59R/108T is highly resistant to chlorcycloguanil, but 200-fold more sensitive to pyrimethamine than the 51I/59R/108N allele.

Mech Ageing Dev, 2005 Jan, 126(1), 171 - 5
Global analysis of protein function using protein microarrays; Smith MG et al.; Protein microarrays containing thousands of proteins arrayed at high density can be prepared and probed for a wide variety of activities, thereby allowing the large scale analysis of many proteins simultaneously . In addition to identifying the activities of many previously uncharacterized proteins, protein microarrays can reveal new activities of well-characterized proteins, thus providing new insights about the functions of these proteins . Below, we describe the construction and use of protein microarrays and their applications using yeast as a model system.

Mol Cell, 2004 Dec 22, 16(6), 1027 - 34
Proteasome involvement in the repair of DNA double-strand breaks; Krogan NJ et al.; Affinity purification of the yeast 19S proteasome revealed the presence of Sem1 as a subunit . Its human homolog, DSS1, was found likewise to copurify with the human 19S proteasome . DSS1 is known to associate with the tumor suppressor protein BRCA2 involved in repair of DNA double-strand breaks (DSBs) . We demonstrate that Sem1 is required for efficient repair of an HO-generated yeast DSB using both homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways . Deletion of SEM1 or genes encoding other nonessential 19S or 20S proteasome subunits also results in synthetic growth defects and hypersensitivity to genotoxins when combined with mutations in well-established DNA DSB repair genes . Chromatin immunoprecipitation showed that Sem1 is recruited along with the 19S and 20S proteasomes to a DSB in vivo, and this recruitment is dependent on components of both the HR and NHEJ repair pathways, suggesting a direct role of the proteasome in DSB repair.

Mol Cell, 2004 Dec 22, 16(6), 991 - 1002
DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain; Unal E et al.; The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms . A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals . Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion . Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding . Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion . We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

Mol Cell, 2004 Dec 22, 16(6), 979 - 90
Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites; Downs JA et al.; Yeast histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair . We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs) . Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides . A single NuA4 subunit, Arp4, is responsible for the interaction . The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding . Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs . This association again requires Arp4 but also prior NuA4 recruitment and action . Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers . This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair.

Hum Gene Ther, 2004 Nov, 15(11), 1077 - 90
Inhibition of simian virus 40 large tumor antigen expression in human fetal glial cells by an antisense oligodeoxynucleotide delivered by the JC virus-like particle; Wang M et al.; Human JC virus (JCV) is a neurotropic virus, and the etiological agent of progressive multifocal leukoencephalopathy (PML), a fatal neurological disease . Because of its natural infection tropism, it is possible to use the JCV capsid as a gene-transducing vector for therapeutic purposes in neurological disorders . In the current study, a recombinant JCV virus-like particle (VLP) was generated and purified from yeast . VLP was able to accommodate and protect DNA molecules of up to approximately 2000 bp in length . VLP was able to package and deliver an antisense oligodeoxynucleotide (AS-ODN) against simian virus 40 (SV40) large tumor antigen (LT) into SV40-transformed human fetal glial (SVG) cells in order to inhibit expression of the oncoprotein . Subsequently, apoptosis of VLP-AS-ODN-treated cells was demonstrated after the blocking of LT expression . In addition, JCV VLP was able to deliver ODN into human astrocytoma, neuroblastoma, and glioblastoma cells with high efficiency . In vivo delivery of ODN into a human neuroblastoma tumor nodule by VLP was also demonstrated . These findings suggest that JCV VLP is a gene delivery vector with potential therapeutic use for human neurological disorders.

Nat Struct Mol Biol . 2004 Dec 19; {Epub ahead of print}
Crystal structure of bet3 reveals a novel mechanism for Golgi localization of tethering factor TRAPP; Kim YG et al.; Transport protein particle (TRAPP) is a large multiprotein complex involved in endoplasmic reticulum-to-Golgi and intra-Golgi traffic . TRAPP specifically and persistently resides on Golgi membranes . Neither the mechanism of the subcellular localization nor the function of any of the individual TRAPP components is known . Here, the crystal structure of mouse Bet3p (bet3), a conserved TRAPP component, reveals a dimeric structure with hydrophobic channels . The channel entrances are located on a putative membrane-interacting surface that is distinctively flat, wide and decorated with positively charged residues . Charge-inversion mutations on the flat surface of the highly conserved yeast Bet3p led to conditional lethality, incorrect localization and membrane trafficking defects . A channel-blocking mutation led to similar defects . These data delineate a molecular mechanism of Golgi-specific targeting and anchoring of Bet3p involving the charged surface and insertion of a Golgi-specific hydrophobic moiety into the channels . This essential subunit could then direct other TRAPP components to the Golgi.

EMBO Rep, 2005 Jan, 6(1), 39 - 45
How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures; Court R et al.; Human telomeres consist of tandem arrays of TTAGGG sequence repeats that are specifically bound by two proteins, TRF1 and TRF2 . They bind to DNA as preformed homodimers and have the same architecture in which the DNA-binding domains (Dbds) form independent structural units . Despite these similarities, TRF1 and TRF2 have different functions at telomeres . The X-ray crystal structures of both TRF1- and TRF2-Dbds in complex with telomeric DNA (2.0 and 1.8 A resolution, respectively) show that they recognize the same TAGGGTT binding site by means of homeodomains, as does the yeast telomeric protein Rap1p . Two of the three G-C base pairs that characterize telomeric repeats are recognized specifically and an unusually large number of water molecules mediate protein-DNA interactions . The binding of the TRF2-Dbd to the DNA double helix shows no distortions that would account for the promotion of t-loops in which TRF2 has been implicated.

Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D633 - 6
Arabidopsis nucleolar protein database (AtNoPDB); Brown JW et al.; The Arabidopsis Nucleolar Protein Database provides information on 217 proteins identified in a proteomic analysis of nucleoli isolated from Arabidopsis cell culture . The database is organized on the basis of the Arabidopsis gene identifier number . The information provided includes protein description, protein class, whether or not the plant protein has a homologue in the most recent human nucleolar proteome and the results of reciprocal BLAST analysis of the human proteome . In addition, for one-third of the 217 Arabidopsis nucleolar proteins, localization images are available from analysis of full-length cDNA-green fluorescent protein (GFP) fusions and the strength of signal in different parts of the cell-nucleolus, nucleolus-associated structures, nucleoplasm, nuclear bodies and extra-nuclear-is provided . For each protein, the most likely human and yeast orthologues, where identifiable through BLASTX analysis, are given with links to relevant information sources.

Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D605 - 10
AMPDB: the Arabidopsis Mitochondrial Protein Database; Heazlewood JL et al.; The Arabidopsis Mitochondrial Protein Database is an Internet-accessible relational database containing information on the predicted and experimentally confirmed protein complement of mitochondria from the model plant Arabidopsis thaliana . The database was formed using the total non-redundant nuclear and organelle encoded sets of protein sequences and allows relational searching of published proteomic analyses of Arabidopsis mitochondrial samples, a set of predictions from six independent subcellular-targeting prediction programs, and orthology predictions based on pairwise comparison of the Arabidopsis protein set with known yeast and human mitochondrial proteins and with the proteome of Rickettsia . A variety of precomputed physical-biochemical parameters are also searchable as well as a more detailed breakdown of mass spectral data produced from our proteomic analysis of Arabidopsis mitochondria . It contains hyperlinks to other Arabidopsis genomic resources (MIPS, TIGR and TAIR), which provide rapid access to changing gene models as well as hyperlinks to T-DNA insertion resources, Massively Parallel Signature Sequencing (MPSS) and Genome Tiling Array data and a variety of other Arabidopsis online resources . It also incorporates basic analysis tools built into the query structure such as a BLAST facility and tools for protein sequence alignments for convenient analysis of queried results.

Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D413 - 7
3did: interacting protein domains of known three-dimensional structure; Stein A et al.; The database of 3D Interacting Domains (3did) is a collection of domain-domain interactions in proteins for which high-resolution three-dimensional structures are known . 3did exploits structural information to provide critical molecular details necessary for understanding how interactions occur . It also offers an overview of how similar in structure are interactions between different members of the same protein family . The database also contains Gene Ontology-based functional annotations and interactions between yeast proteins from large-scale interaction discovery studies . A web-based tool to query 3did is available at http://3did.embl.de.

Bioinformatics . 2004 Dec 17; {Epub ahead of print}
Clustering of diverse genomic data using information fusion; Kasturi J et al.; MOTIVATION: Genome sequencing projects and high-throughput technologies like DNA and Protein arrays have resulted in a very large amount of information-rich data . Microarray experimental data are a valuable, but limited source for inferring gene regulation mechanisms on a genomic scale . Additional information such as promoter sequences of genes/DNA binding motifs, gene ontologies, and location data, when combined with gene expression analysis can increase the statistical significance of the finding . This paper introduces a machine learning approach to information fusion for combining heterogeneous genomic data . The algorithm uses an unsupervised joint learning mechanism that identifies clusters of genes using the combined data . RESULTS: The correlation between gene expression time-series patterns obtained from different experimental conditions and the presence of several distinct and repeated motifs in their upstream sequences is examined here using publicly available yeast cell-cycle data . The results show that the combined learning approach taken here identifies correlated genes effectively . The algorithm provides an automated clustering method, but allows the user to specify apriori the influence of each data type on the final clustering using probabilities . AVAILABILITY: Software code is available by request from the first author.

Cell, 2004 Dec 17, 119(6), 789 - 802
Two RNAi Complexes, RITS and RDRC, Physically Interact and Localize to Noncoding Centromeric RNAs; Motamedi MR et al.; RNAi-mediated heterochromatin assembly in fission yeast requires the RNA-induced transcriptional silencing (RITS) complex and a putative RNA-directed RNA polymerase (Rdp1) . Here we show that Rdp1 is associated with two conserved proteins, Hrr1, an RNA helicase, and Cid12, a member of the polyA polymerase family, in a complex that has RNA-directed RNA polymerase activity (RDRC, RNA-directed RNA polymerase complex) . RDRC physically interacts with RITS in a manner that requires the Dicer ribonuclease (Dcr1) and the Clr4 histone methyltransferase . Moreover, both complexes are localized to the nucleus and associate with noncoding centromeric RNAs in a Dcr1-dependent manner . In cells lacking Rdp1, Hrr1, or Cid12, RITS complexes are devoid of siRNAs and fail to localize to centromeric DNA repeats to initiate heterochromatin assembly . These findings reveal a physical and functional link between Rdp1 and RITS and suggest that noncoding RNAs provide a platform for siRNA-dependent localization of RNAi complexes to specific chromosome regions.

Cell, 2004 Dec 17, 119(6), 777 - 88
Recruitment of the INO80 Complex by H2A Phosphorylation Links ATP-Dependent Chromatin Remodeling with DNA Double-Strand Break Repair; van Attikum H et al.; The budding yeast INO80 complex is a conserved ATP-dependent nucleosome remodeler containing actin-related proteins Arp5 and Arp8 . Strains lacking INO80, ARP5, or ARP8 have defects in transcription . Here we show that these mutants are hypersensitive to DNA damaging agents and to double-strand breaks (DSBs) induced by the HO endonuclease . The checkpoint response and most transcriptional modulation associated with induction of DNA damage are unaffected by these mutations . Using chromatin immunoprecipitation we show that Ino80, Arp5, and Arp8 are recruited to an HO-induced DSB, where a phosphorylated form of H2A accumulates . Recruitment of Ino80 is compromised in cells lacking the H2A phosphoacceptor S129 . Finally, we demonstrate that conversion of the DSB into ssDNA is compromised in arp8 and H2A mutants, which are both deficient for INO80 activity at the site of damage . These results implicate INO80-mediated chromatin remodeling directly at DSBs, where it appears to facilitate processing of the lesion.

Cell, 2004 Dec 17, 119(6), 767 - 75
INO80 and gamma-H2AX Interaction Links ATP-Dependent Chromatin Remodeling to DNA Damage Repair; Morrison AJ et al.; While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive . We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast . The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX) . This interaction requires Nhp10, an HMG-like subunit of the INO80 complex . The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB . Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast . Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

FEMS Immunol Med Microbiol, 2005 Jan 1, 43(1), 13 - 20
Candida guilliermondii isolated from HIV-infected human secretes a 50 kDa serine proteinase that cleaves a broad spectrum of proteinaceous substrates; Dos Santos AL et al.; Non-albicans Candida species cause 35-65% of all candidemias in the general population, especially in immunosuppressed individuals . Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism . This clinical yeast isolate was identified as Candida guilliermondii through mycological tests . C . guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 degrees C . After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS-PAGE . A 50 kDa proteolytic enzyme was detected with activity at physiological pH . This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class . E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase . Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C . guilliermondii by approximately 50% . Proteinases are a well-known class of enzymes that participate in a vast context of yeast-host interactions . In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components . We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides . Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non-albicans Candida.

Genomics, 2005 Jan, 85(1), 106 - 16
Identification and characterization of human Golgi nucleotide sugar transporter SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family; Ishida N et al.; We report the molecular cloning of SLC35D2, a novel member of the SLC35 nucleotide sugar transporter family . The gene SLC35D2 maps to chromosome 9q22.33 . SLC35D2 cDNA codes for a hydrophobic protein consisting of 337 amino acid residues with 10 putative transmembrane helices . Northern blot analysis revealed the SLC35D2 mRNA as a single major band corresponding to 2.0 kb in length . SLC35D2 was localized in the Golgi membrane and exhibited around 50% similarity with three nucleotide sugar transporters: human SLC35D1 (UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter), fruitfly fringe connection (frc) transporter, and nematode SQV-7 transporter, the latter two being involved in developmental and organogenetic processes . Heterologous expression of SLC35D2 protein in yeast indicated that UDP-N-acetylglucosamine is a candidate for the substrate(s) of the transporter . The sequence similarity, subcellular localization, and transporting substrate suggest that SLC35D2 is a good candidate for the ortholog of frc transporter, which is involved in the Notch signaling system by providing the fringe N-acetylglucosaminyltransferase with the substrate . We also describe the identification and categorization of the human SLC35 gene family.

Bioresour Technol, 2005 May, 96(7), 831 - 42
Generation of organic acids and monosaccharides by hydrolytic and oxidative transformation of food processing residues; Fischer K et al.; Carbohydrate-rich biomass residues, i.e . sugar beet molasses, whey powder, wine yeast, potato peel sludge, spent hops, malt dust and apple marc, were tested as starting materials for the generation of marketable chemicals, e.g . aliphatic acids, sugar acids and mono-/disaccharides . Residues were oxidized or hydrolyzed under acidic or alkaline conditions applying conventional laboratory digestion methods and microwave assisted techniques . Yields and compositions of the oxidation products differed according to the oxidizing agent used . Main products of oxidation by 30% HNO(3) were acetic, glucaric, oxalic and glycolic acids . Applying H(2)O(2)/CuO in alkaline solution, the organic acid yields were remarkably lower with formic, acetic and threonic acids as main products . Gluconic acid was formed instead of glucaric acid throughout . Reaction of a 10% H(2)O(2) solution with sugar beet molasses generated formic and lactic acids mainly . Na(2)S(2)O(8) solutions were very inefficient at oxidizing the residues . Glucose, arabinose and galactose were formed during acidic hydrolysis of malt dust and apple marc . The glucose content reached 0.35g per gram of residue . Important advantages of the microwave application were lower reaction times and reduced reagent demands.

J Biochem Mol Biol, 2004 Nov 30, 37(6), 741 - 8
Systematic identification of hepatocellular proteins interacting with NS5A of the hepatitis C virus; Ahn J et al.; The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas . Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein . This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV . Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1 approximately 447) and its four different derivatives, denoted as NS5A-A (aa 1 approximately 150), -B (aa 1 approximately 300), -C (aa 300 approximately 447) and D (aa 150 approximately 447) . NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and centaurindelta 2 (CENTdelta2) . However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity . Based on an in vitro binding assay, CRABP-1, PI4K, CENTdelta2 and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein . Furthermore, the interactions of CRABP-1, PI4K and CENTdelta2 were not related to the PXXP motif (class II), as judged by a domain analysis . While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.

BMC Bioinformatics . 2004 Dec 17;5(1):204.
An SVD-based comparison of nine whole eukaryotic genomes supports a coelomate rather than ecdysozoan lineage; Stuart GW et al.; BACKGROUND: Eukaryotic whole genome sequences are accumulating at an impressive rate . Effective methods for comparing multiple whole eukaryotic genomes on a large scale are needed . Most attempted solutions involve the production of large scale alignments, and many of these require a high stringency pre-screen for putative orthologs in order to reduce the effective size of the dataset and provide a reasonably high but unknown fraction of correctly aligned homologous sites for comparison . As an alternative, highly efficient methods that do not require the pre-alignment of operationally defined orthologs are also being explored . RESULTS: A non-alignment method based on the Singular Value Decomposition (SVD) was used to compare the predicted protein complement of nine whole eukaryotic genomes ranging from yeast to man . This analysis resulted in the simultaneous identification and definition of a large number of well conserved motifs and gene families, and produced a species tree supporting one of two conflicting hypotheses of metazoan relationships . CONCLUSIONS: Our SVD-based analysis of the entire protein complement of nine whole eukaryotic genomes suggests that highly conserved motifs and gene families can be identified and effectively compared in a single coherent definition space for the easy extraction of gene and species trees . While this occurs without the explicit definition of orthologs or homologous sites, the analysis can provide a basis for these definitions.

J Neurochem, 2005 Jan, 92(1), 93 - 102
beta-amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI: binding partners processed by furin; Wickham L et al.; Using a yeast two-hybrid system, we screened a human brain cDNA library for possible interacting proteins with the C-terminal cytosolic tail of the beta-secretase beta-amyloid protein converting enzyme (BACE)1 . This identified seven potential candidates, including the brain-specific type II membrane protein BRI3 . Co-localization and co-immunoprecipitation experiments confirmed that BACE1 and BRI3 co-localize and interact with each other via the cytosolic tail of BACE1 . Furthermore, pulse and pulse-chase analyses revealed that the pro-protein convertases furin, and to a lesser extent PC7 and PC5A, process BRI3 into a C-terminal secreted approximately 4-kDa product . Thus, furin efficiently processes both pro-BACE1 and its novel interacting protein pro-BRI3.

Plant Mol Biol, 2004 Sep, 56(2), 271 - 83
Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from Arabidopsis; Chen K et al.; In Arabidopsis, there is a family of receptor-like protein kinases (RLKs) containing novel cysteine-rich repeats in their extracellular domains . Genes encoding many of these cysteine-rich RLKs (CRKs) are induced by pathogen infection, suggesting a possible role in plant defense responses . We have previously generated Arabidopsis plants expressing four pathogen-regulated CRK genes (CRK5, 6, 10 and 11) under control of a steroid-inducible promoter and found that induced expression of CRK5, but not the other three CRK genes, triggered hypersensitive response-like cell death in transgenic plants . In the present study, we have analyzed the structural relationship of the CRK family and identified three CRKs (CRK4, 19 and 20) that are structurally closely related to CRK5 . Genes encoding these three CRKs are all induced by salicylic acid and pathogen infection . Furthermore, induced expression of CRK4, 19 and 20 all activates rapid cell death in transgenic plants . Thus, the activity of inducing rapid cell death is shared by these structurally closely related CRKs . We have also performed yeast two-hybrid screens and identified proteins that interact with the kinase domains of CRKs . One of the identified CRK-interacting proteins is the kinase-associated type 2C protein phospohatase known to interact with a number of other RLKs through its kinase-interacting FHA domain . Other CRK-interacting proteins include a second protein with a FHA domain and another type 2C protein phosphatase . Interactions of CRKs with these three proteins in vivo were demonstrated through co-immunoprecipitation . These CRK-interacting proteins may play roles in the regulation and signaling of CRKs.

Plant Mol Biol, 2004 Jul, 55(4), 567 - 77
The AtPPT1 gene encoding 4-hydroxybenzoate polyprenyl diphosphate transferase in ubiquinone biosynthesis is required for embryo development in Arabidopsis thaliana; Okada K et al.; 4-Hydroxybenzoate polyprenyl diphosphate transferase (4HPT) is the key enzyme that transfers the prenyl side chain to the benzoquione frame in ubiquinone (UQ) biosynthesis . The Arabidopsis AtPPT1 cDNA encoding 4HPT was cloned by reverse transcription-polymerase chain reaction (RT-PCR) based on the information of the Arabidopsis genomic sequence, and the function of the gene was determined . Heterologous expression of the AtPPT1 gene enabled restoration of the respiratory ability and UQ synthesis in a yeast mutant that was defective in 4HPT activity . The mitochondrial fraction that was prepared from the yeast mutant, which expressed the AtPPT1 gene, exhibited 4HPT enzymatic activity with geranyl diphosphate (GPP) as the prenyl substrate . This indicated that the AtPPT1 gene encodes active 4HPT with a broad substrate specificity in terms of the prenyl donor . The AtPPT1 mRNA was predominantly expressed in the flower cluster, and the green fluorescent protein (GFP) fused with the signal peptide of AtPPT1 was translocated into the mitochondria . T-DNA insertion mutation that disrupts the AtPPT1 gene in Arabidopsis resulted in the arrest of embryo development at an early stage of zygotic embryogenesis . These results demonstrate that the AtPPT1 gene involved in the biosynthesis of mitochondrial UQ plays an essential role in embryo development in Arabidopsis .

Plant Mol Biol, 2004 May, 55(3), 417 - 31
An auxin-responsive SCARECROW-like transcriptional activator interacts with histone deacetylase; Gao MJ et al.; Members of the plant-specific GRAS family of putative transcription factors are involved in various aspects of plant development . SCARECROW (SCR) is a member of this protein family and plays a significant role in the radial patterning of both roots and shoots . However, little is known about the regulation of SCR expression and its mode of action in plants . Here, we report on the isolation and characterization of a Brassica napus SCARECROW-like protein, BnSCL1, isolated by selecting for proteins that interact with the Arabidopsis histone deacetylase AtHDA19 in a yeast two-hybrid screen . BnSCL1 contains domains conserved in the GRAS family of proteins, interacts with AtHDA19 through a VHIID domain, and exerts transcription activation of reporter genes . BnSCL1 is expressed predominantly in the roots, where its expression is regulated by auxin, as it also is in shoots and mature leaves . These results indicate that BnSCL1 is a member of the GRAS family, and suggest that its mode of action in plant auxin response may involve interaction with HDA19.

Plant Mol Biol, 2004 May, 55(2), 239 - 52
Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity; Bhat RA et al.; Maize Opaque-2 (ZmO2), a bZip class transcription factor has been shown to activate the transcription of a series of genes expressed in the maturation phase of endosperm development . Activation requires the presence of one or more enhancer binding sites, which confer the propensity for activation by ZmO2 on heterologous promoters and in heterologous plant cell types, such as tobacco mesophyll protoplasts . The region of ZmO2 required for conferring transcriptional activation has been localised to a stretch of acidic residues in the N-terminal portion of the ZmO2 sequence, which is conserved between O2-related bZip factor sequences . Previously we identified the maize homologues of yeast transcriptional co-activators GCN5 and ADA2 that are implicated in nucleosome modification and transcription . In the present study we have shown that transcriptional modulation by ZmO2 involves the intranuclear interaction of ZmO2 with ZmADA2 and ZmGCN5 . Forster resonance energy transfer (FRET) based techniques have enabled us to estimate the intracellular site of these intermolecular interactions . As a functional readout of these intranuclear interactions, we used the ZmO2 responsive maize b-32 promoter to drive the beta-glucuronidase (GUS) in the presence and absence of ZmGCN5 and ZmADA2 . Our results suggest that the likely recruitment of ZmADA2 and ZmGCN5 modulates the transactivation of b-32 promoter by ZmO2 and that there may be a competition between ZmGCN5 and ZmO2 for binding to the amino-terminal of ZmADA2 . The results may be taken as a paradigm for other processes of transcriptional modulation in planta involving acidic activation domains.

Plant Mol Biol, 2004 May, 55(2), 183 - 92
Ectopic overexpression of tomato JERF3 in tobacco activates downstream gene expression and enhances salt tolerance; Wang H et al.; The ethylene, jasmonic acid and osmotic signaling pathways respond to environmental stimuli and in order to understand how plants adapt to biotic and abiotic stresses it is important to understand how these pathways interact each other . In this paper, we report a novel ERF protein--jasmonate and ethylene-responsive factor 3 (JERF3)--that unites these pathways . JERF3, which functions as an in vivo transcription activator in yeast, binds to the GCC box, an element responsive to ethylene/JA signaling, as well as to DRE, a dehydration-responsive element that responds to dehydration, high salt and low-temperature . Expression of JERF3 in tomato is mainly induced by ethylene, JA, cold, salt or ABA . Constitutive expression of JERF3 in transgenic tobacco significantly activated expression of pathogenesis-related genes that contained the GCC box, resulting in enhanced tolerance to salt . These results indicate that JERF3 functions as a linker in ethylene- and osmotic stress-signaling pathways.

Plant Mol Biol, 2004 May, 55(2), 153 - 64
Functional characterization of tobacco transcription factor TGA2.1; Kegler C et al.; Activation sequence-1 (as-1)-like regulatory cis elements mediate transcriptional activation in response to increased levels of plant signalling molecules auxin and salicylic acid (SA) . Our earlier work has shown that tobacco cellular as-1-binding complex SARP (salicylic acid responsive protein) is primarily comprised of bZIP protein TGA2.2 and of minor amounts of a protein that cross-reacts with an antibody directed against related bZIP factor TGA2.1 . As this protein was significantly smaller than recombinant TGA2.1, the origin of this protein had remained unresolved . Here we demonstrate that it corresponds to a distinct cleavage product of TGA2.1 generated during extract preparation . Overexpression of TGA2.1 led to increased levels of the TGA2.1/TGA2.2 heterodimer which was as effective with regard to enhancing the SA-inducibility of as-1 containing target gene Nt103 as corresponding amounts of the TGA2.2 homodimer . Thus, the TGA2.1 specific N-terminal domain, which had revealed transcriptional activation potential in yeast, did not show enhanced transcriptional activation in planta . TGA2.1 even had a negative effect on the SA-induced expression of the truncated CaMV 35S (-90) promoter that contains an isolated as-1-element upstream of the TATA-box . Plants expressing a TGA mutant deficient in DNA binding (TGA2.1trd) showed reduced levels of SA-inducible Nt103 expression, thus resembling plants expressing the analogous TGA2.2 derivative TGA2.2trd . In contrast to TGA2.2trd, TGA2.1trd did not reduce auxin-induced expression of Nt103 and SA-induced expression of pathogenesis related protein PR-1a, indicating that TGA2.1trd and TGA2.2trd differ in their capacity to outcompete regulatory factors involved in these regulatory pathways.

Cancer Res, 2004 Dec 15, 64(24), 9080 - 5
Adenoviral E1A targets Mdm4 to stabilize tumor suppressor p53; Li Z et al.; The adenoviral protein E1A associates with multiple anticancer activities, including stabilization of p53 tumor suppressor, and has been tested through gene therapy approaches in clinical trials . To identify potential E1A-binding proteins involved in E1A's anticancer activities, we screened a yeast two-hybrid library and identified Mdm4, an Mdm2-related p53-binding protein, as a novel E1A-binding protein . The NH(2)-terminal region of Mdm4 and the CR1 domain of E1A were required for the interaction between E1A and Mdm4 . E1A preferentially bound to Mdm4 rather than Mdm2 and formed a complex with p53 in the presence of Mdm4, resulting in the stabilization of p53 in a p14(ARF)-independent manner . E1A failed to stabilize p53 in the absence of Mdm4, showing that Mdm4 was required for p53 stabilization by E1A . Moreover, E1A-mediated stabilization of p53 occurred in nucleus . Although it had no effect on the p53-Mdm2 interaction, E1A facilitated Mdm4 binding to p53 and inhibited Mdm2 binding to Mdm4, resulting in decreased nuclear exportation of p53 . Thus, our findings highlighted a novel mechanism, whereby E1A stabilized the p53 tumor suppressor through Mdm4.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Dec, 24(12), 1373 - 7
{Purification and separation of mannan-binding lectin (MBL) and MBL-associated serine proteases complex from human plasma.}; Chen Y et al.; OBJECTIVE: To separate mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) from human plasma . METHODS: A two-step affinity chromatography on underivatized sepharose 4B was employed for purification of MBL-MASP complex, followed by gel filtration on a Sephacryl S-300 column for separation of MBL and MASPs from the complex . The purification procedures were performed at 4 degrees Celsius with the addition of two proteolytic inhibitors, phenyl methylsulfonyl fluoride and 1,10-phenanthroline during affinity chromatography but not in the gel filtration buffer . RESULTS: Preparations of highly purified MBL and proenzyme MASPs were obtained . The purified MBL was shown by SDS-PAGE and Western blotting to be a functional multimer composed of 28,000 and 32,000 peptide chains, with high bioactivity as demonstrated by ligand-binding assay and yeast agglutination experiment . CONCLUSION: A simple and convenient procedure is established successfully for the purification of MBL and the proenzyme MASPs.

Neuron, 2004 Dec 16, 44(6), 987 - 96
Identification of PSD-95 palmitoylating enzymes; Fukata M et al.; Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system . Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity . The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast . Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo . These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells . Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission . This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.

Proteomics . 2004 Dec 15; {Epub ahead of print}
A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells; Drakas R et al.; The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast . The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used . We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures . The recovery allows protein identification by mass spectrometry.

Proc Natl Acad Sci U S A, 2004 Dec 28, 101(52), 18135 - 40 Epub 2004 Dec 15.
Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I; Kedar V et al.; Muscle-specific RING finger protein 1 (MuRF1) is a sarcomere-associated protein that is restricted to cardiac and skeletal muscle . In skeletal muscle, MuRF1 is up-regulated by conditions that provoke atrophy, but its function in the heart is not known . The presence of a RING finger in MuRF1 raises the possibility that it is a component of the ubiquitin-proteasome system of protein degradation . We performed a yeast two-hybrid screen to search for interaction partners of MuRF1 in the heart that might be targets of its putative ubiquitin ligase activity . This screen identified troponin I as a MuRF1 partner protein . MuRF1 and troponin I were found to associate both in vitro and in vivo in cultured cardiomyocytes . MuRF1 reduced steady-state troponin I levels when coexpressed in COS-7 cells and increased degradation of endogenous troponin I protein in cardiomyocytes . The degradation of troponin I in cardiomyocytes was associated with the accumulation of ubiquitylated intermediates of troponin I and was proteasome-dependent . In vitro, MuRF1 functioned as a ubiquitin ligase to catalyze ubiquitylation of troponin I through a RING finger-dependent mechanism . In isolated cardiomyocytes, MuRF1 reduced indices of contractility . In cardiomyocytes, these processes may determine the balance between hypertrophic and antihypertrophic signals and the regulation of myocyte contractile responses in the setting of heart failure.

Toxicol Sci . 2004 Dec 15; {Epub ahead of print}
Structural Requirements for the Interaction of 91 Hydroxylated Polychlorinated Biphenyls with Estrogen and Thyroid Hormone Receptors; Arulmozhiraja S et al.; Estrogenic and thyroid activities of 91 monohydroxylated PCBs were measured with two-hybrid assays using yeast cells containing the human estrogen receptor ERalpha or human thyroid receptor TRalpha . Estrogenic activity of 30 of the 91 compounds, including all compounds active in the yeast two-hybrid assay, were also measured by a reporter gene assay employing Chinese hamster ovary cells . The mammalian cell assay was more sensitive than the yeast assay but the rank order of estrogenicities of the compounds were in broad agreement for the two assays . Results for estrogenicity and thyroid activity were analyzed by inspection and those for estrogenicity by a theoretical treatment . Inspection indicated para- hydroxyl was more likely to be estrogenically active than meta-, which was more likely to be active than ortho-; one ortho- chlorine was important for activity but additional ortho- chlorines did not increase activity; and 2 lateral chlorines or 2,4,6-chloro- substitution of the non-phenol ring were favorable . In contrast, thyroid activity appeared not to depend strongly on the position of the hydroxyl group although ortho- hydroxyls occurred in the most active compounds . Activity was usually associated with at least one ortho- chlorine, with 2 chlorines in the phenolic ring and, importantly, two chlorines in the non-phenolic ring, and with 1 or 2 chlorines ortho to the hydroxyl group . Examination of the torsion angle between the rings, in the theoretical examination of estrogenicity, suggested that perpendicular orientation (i.e., rigidity) was not essential for activity . Intramolecular hydrogen bonding of the phenolic groups to adjacent chlorines or to the pi-electron cloud of the non-phenol ring possibly decreased activity - the hydroxyl should be free of intramolecular interactions for maximum activity . It was difficult to predict the estrogenic activity of a congener from its obtained potential energy curve (PEC) . In general, estrogenically active congeners had large values for the sum of the atomic charges on the carbon atoms of the hydroxylated ring, and on the oxygen atom . Hydroxyl substitution at the para- position allowed the compounds to become more polarizable in the x-axis (molecular axis), whereas OH substitution at the ortho-position made the congeners less polarizable in the same direction . However, no general statement about polarizability and estrogenic activity was possible.

Hum Mol Genet, 2005 Feb 1, 14(3), 437 - 45 Epub 2004 Dec 15.
MAN1, an integral protein of the inner nuclear membrane, binds Smad2 and Smad3 and antagonizes transforming growth factor-{beta} signaling; Lin F et al.; MAN1 (also known as LEMD3) is an integral protein of the inner nuclear membrane . Recently, mutations in MAN1 have been shown to result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis . We show that the nucleoplasmic, C-terminal domain of human MAN1 binds to Smad2 and Smad3 and antagonizes signaling by transforming growth factor-beta (TGF-beta) . In a yeast two-hybrid screen using the C-terminal domain of MAN1 as bait, eight positive clones were obtained that encoded Smad3 . In direct two-hybrid assays, this portion of MAN1 bound to Smad2 and Smad3 . In glutathione-S-transferase precipitation assays, the C-terminal domain of MAN1 bound to Smad2 and Smad3 under stringent conditions . Antibodies against MAN1 were able to co-immunoprecipiate Smad2 from cells, demonstrating that they reside in the same complex in vivo . TGF-beta treatment stimulated transcription from a reporter gene in control cells, but reporter gene stimulation was significantly inhibited in cells overexpressing MAN1 or its C-terminal domain but not its N-terminal domain . TGF-beta-induced cell proliferation arrest was also inhibited in stable cell lines overexpressing MAN1 . These results show that the nuclear envelope regulates a signal transduction pathway and have implications for how mutations in nuclear envelope proteins cause different human diseases.

Funct Integr Genomics, 2005 Jan, 5(1), 28 - 31 Epub 2004 Sep 09.
Nonparametric methods for analyzing replication origins in genomewide data; Ghosh D; Due to the advent of high-throughput genomic technology, it has become possible to monitor cellular activities on a genomewide basis . With these new methods, scientists can begin to address important biological questions . One such question involves the identification of replication origins, which are regions in the chromosomes where DNA replication is initiated . One hypothesis is that their locations are nonrandom throughout the genome . In this article, we analyze data from a recent yeast study in which candidate replication origins were profiled using cDNA microarrays to test this hypothesis . We find no evidence for such clustering.

Genes Genet Syst, 2004 Oct, 79(5), 301 - 5
Isolation of mutants with aberrant mitochondrial morphology from Arabidopsis thaliana; Feng X et al.; To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana . Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence . From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations . All mitochondria in these mutants were longer and/or larger than wild-type mitochondria . The approximate chromosomal loci of the mutations of seven mutants that grew well were determined . The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant . Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci . At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology.

Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Apr, 30(2), 229 - 33
{Expression of some genes in Tamarix androssowii plant under NaHCO3 stress}; Yang CP et al.; Differential display technique was used to study the gene expression of Tamarix androssowii treated with NaHCO(3) solution . Seventeen differentially expressed genes were obtained through Northern detection . The result of BLASTX analysis shows that two of these genes (GenBank accession number CD028574 and CD028579) are homologous to the F-box protein family member, which can regulate diverse cellular processes, including cell cycle transition, transcription regulation and signal transduction, by playing roles in Skp1p-cullin-F-box protein (SCF) complexes or non-SCF complexes . F-box protein plays a very important role in plant defence system . One gene (GenBank accession number CD028589) is highly homologous to the translation initiation factor eIF, which regulates the proteins translation and is an important determinant of sodium tolerance in yeast and plants . Additionally, two genes (GenBank accession numbers CD028584 and CD028586) that are homologous to secretory peroxidase and peroxidases were identified, peroxidases are familiar proteins expressed in plant under stress . Five new genes closely related to salt and alkali resistance were obtained, which were expressed quite differentially when treated by NaHCO(3) solution . Seven differentially expressed genes were obtained which have highly or some homologous to the known genes, their function in stress need to be further studied . These seventeen genes were accepted as novel cDNA sequences by GenBank and were given them new accession numbers, their accession numbers were CD028574- CD028581 and CD028583-CD028591.

Plant Cell, 2005 Jan, 17(1), 204 - 18 Epub 2004 Dec 14.
Arabidopsis POLYOL TRANSPORTER5, a New Member of the Monosaccharide Transporter-Like Superfamily, Mediates H+-Symport of Numerous Substrates, Including myo-Inositol, Glycerol, and Ribose; Klepek YS et al.; Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery {Apium graveolens}, common plantain {Plantago major}, or sour cherry {Prunus cerasus}) . The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols . Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters . Transient expression of an AtPLT5-green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H(+)-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol . Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters . RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs . The potential physiological role of AtPLT5 is discussed.

Bioorg Med Chem, 2005 Jan 17, 13(2), 455 - 62
Structure-activity relationship study on 13-deoxytedanolide, a highly antitumor macrolide from the marine sponge Mycale adhaerens; Nishimura S et al.; To obtain information of structure-activity relationships (SARs) of 13-deoxytedanolide, its chemical transformation has been carried out, targeting on such functional groups as an epoxide, hydroxyls, ketones, and olefins . A total of 10 derivatives have been prepared and their cytotoxicity against P388 murine leukemia cells and inhibitory activity of polypeptide elongation in yeast cell lysate provided some important SARs; the southern hemisphere comprises the pharmacophore, while the epoxide-bearing side chain is essential for the activity.

Bioorg Med Chem, 2005 Jan 17, 13(2), 449 - 54
13-Deoxytedanolide, a marine sponge-derived antitumor macrolide, binds to the 60S large ribosomal subunit; Nishimura S et al.; 13-Deoxytedanolide is a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens . In spite of its remarkable activity, the mode of action of 13-deoxytedanolide has not been elucidated . {11-(3)H}-(11S)-13-Deoxydihydrotedanolide derived from the macrolide was used for identifying the target molecule from the yeast cell lysate . Fractionation of the binding protein revealed that the labeled 13-deoxytedanolide derivative strongly bound to the 80S ribosome as well as to the 60S large subunit, but not to the 40S small subunit . In agreement with this observation, 13-deoxytedanolide efficiently inhibited the polypeptide elongation . Interestingly, competition studies demonstrated that 13-deoxytedanolide shared the binding site on the 60S large subunit with pederin and its marine-derived analogues . These results indicate that 13-deoxytedanolide is a potent protein synthesis inhibitor and is the first macrolide to inhibit the eukaryotic ribosome.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Jun, 22(3), 160 - 3
{Screening of cDNA clone for putative RNA polymerase subunit of Cysticercus cellulosae}; Luo XN et al.; OBJECTIVE: To obtain related genes of Cysticercus cellulosae from spliced leader (SL) cDNA library . METHODS: Spliced leader library of Cysticercus cellulosae was constructed using SL specific primer and oligo (dT) 15 with M13M4 primer, and positive clones were then screened randomly, identified with enzyme restriction, followed by sequencing and homologous analysis . RESULTS: The amino acid sequence, encoded by the positive clone with a poly (A)22 tail and a complete open reading frame (ORF), was with homology of RNA polymerase subunit genes of human, B . napus, fission yeast, A . thaliana, C . elegans and fruit fly up to 71.6% . CONCLUSION: The protein, RNA polymerase subunit encoded putatively by the clone, is high conservative in different species.

J Mol Recognit . 2004 Dec 14; {Epub ahead of print}
The structural basis of recognition and removal of cellular mRNA 7-methyl G 'caps' by a viral capsid protein: a unique viral response to host defense; Tang J et al.; The single segment, double-stranded RNA genome of the L-A virus (L-A) of yeast encodes two proteins: the major coat protein Gag (76 kDa) and the Gag-Pol fusion protein (180 kDa) . The icosahedral L-A capsid is formed by 120 copies of Gag and has architecture similar to that seen in the reovirus, blue tongue virus and rice dwarf virus inner protein shells . Gag chemically removes the m(7)GMP caps from host cellular mRNAs . Previously we identified a trench on the outer surface of Gag that included His154, to which caps are covalently attached . Here we report the refined L-A coordinates at 3.4 A resolution with additional structural features and the structure of L-A with bound m(7)GDP at 6.5 A resolution, which shows the conformational change of the virus upon ligand binding . Based on site-directed mutations, residues in or adjacent to the trench that are essential (or dispensable) for the decapping reaction are described here . Along with His154, the reaction requires a cluster of positive charge adjoining the trench and residues Tyr 452, Tyr150 and either Tyr or Phe at position 538 . A tentative mechanism for decapping is proposed . Copyright (c) 2004 John Wiley & Sons, Ltd.

J Virol, 2005 Jan, 79(1), 245 - 56
The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor; Liao G et al.; Herpesviruses encode a set of core proteins essential for lytic replication of their genomes . Three of these proteins form a tripartite helix-primase complex that, in the case of Epstein-Barr virus (EBV), consists of the helicase BBLF4, the primase BSLF1, and the linker protein BBLF2/3 . BBLF2/3 and its homologs in the other herpesviruses remain relatively poorly characterized . To better understand the contribution to replication made by BBLF2/3, a yeast two-hybrid screen was performed with BBLF2/3 as the bait protein . This screen identified as interactors a number of cell replication-related proteins such as DNA polymerase beta and subunits of DNA polymerase delta along with the EBV-encoded DNase BGLF5 . The screen also identified the DNA binding zinc finger protein ZBRK1 and the ZBRK1 corepressor KAP-1 as BBLF2/3 interactors . Interaction between BBLF2/3 and ZBRK1 and KAP-1 was confirmed in coimmunoprecipitation assays . A binding site for ZBRK1 in the EBV oriLyt enhancer was identified by electrophoretic mobility shift assay . ZBRK1, KAP-1, and the ZBRK1 binding protein BRCA1 were shown by indirect immunofluorescence to be present in replication compartments in lytically induced D98-HR1 cells, and additionally, chromatin immunoprecipitation assays determined that these proteins associated with oriLyt DNA . Replication of an oriLyt plasmid and a variant oriLyt (DeltaZBRK1) plasmid was examined in lytically induced D98-HR1 cells . Exogenous ZBRK1, KAP-1, or BRCA1 increased the efficiency of oriLyt replication, while deletion of the ZBRK1 binding site impaired replication . These experiments identify ZBRK1 as another cell protein that, through BBLF2/3, provides a tethering point on oriLyt for the EBV replication complex . The data also suggest that BBLF2/3 may serve as a contact interface for cell proteins involved in replication of EBV oriLyt.

Crit Rev Biochem Mol Biol, 2004 Jul-Aug, 39(4), 217 - 59
Structure-function relationships in unusual nonvertebrate globins; Shikama K et al.; Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study . For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail . These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure . These proteins are not grouped by any common features other than the fact they have globin domains and heme groups . As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear . In this review, special emphasis is placed on the stability properties of the heme-bound O2 . Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo . However, the reversible and stable binding of O2 to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion . The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive . In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures . Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II) . Such comparative studies of the stability of MbO2 or HbO2 are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.

J Biol Chem . 2004 Dec 13; {Epub ahead of print}
Protein NPM3 interacts with the multifunctional nucleolar protein B23/NPM and inhibits ribosome biogenesis; Huang N et al.; Protein B23/nucleophosmin is a multifunctional protein that plays roles in ribosome biogenesis, control of centrosome duplication and regulation of p53 expression . A yeast two hybrid screen was performed in a search for interaction partners of B23 . The cDNA for a highly acidic protein, nucleoplasmin 3 (NPM3), was found in multiple positive clones . Protein NPM3 and its interaction with B23 were further characterized . Endogenous B23 was able to be co-immunoprecipitated with NPM3 and this complex was resistant to ribonuclease treatment and high concentrations of salt . The N-terminal 35 - 90 amino acids of B23 were found to be required for their interaction . Separate co-immunoprecipitation studies of B23 and NPM3 suggested the existence of two different complexes: one containing B23 and 28S rRNA, and another composed of B23, NPM3 and other proteins, but no RNA . NPM3 was localized in the nucleolus and its nucleolar localization depended on active rRNA transcription . In the cells overexpressing NPM3, there were decreased rates of pre-rRNA synthesis and processing . Overexpression of a mutant of NPM3 that did not interact with B23 did not alter pre-rRNA synthesis and processing, suggesting that the interaction of NPM3 with B23 plays a role in the ribosome biogenesis.

J Biol Chem . 2004 Dec 13; {Epub ahead of print}
Autoinhibition of p50 Rho GTPase-activating protein (GAP) is released by prenylated small GTPases; Moskwa P et al.; Interaction of p50 Rho GTPase activating protein (p50RhoGAP) with Rho family small GTPases was investigated in a yeast two-hybrid system, by radioactive GAP assay and in a Rac-regulated enzymatic reaction, superoxide production by the phagocytic NADPH oxidase . The yeast two-hybrid system revealed an interaction between the C-terminal GAP domain and the N-terminal part of p50RhoGAP . The first 48 amino acids play a special role both in the stabilization of the intramolecular interaction and in recognition of the prenyl tail of small GTPases . The GAP assay and the NADPH oxidase activity indicate that the GTPase activating effect of full-length p50RhoGAP is lower on non-prenylated than on prenylated small GTPase . Removal of amino acids 1-48 and 169-197 of p50RhoGAP increases the GAP effect on non-prenylated Rac, whereas prenylated Rac reacts equally well with the full-length and the truncated proteins . We suggest that p50RhoGAP is in an autoinhibited conformation stabilized by the stretches 1-48 and 169-197 and the prenyl group of the small GTPase plays a role in releasing this intramolecular restraint.

Biochem Biophys Res Commun, 2005 Jan 21, 326(3), 594 - 9
Cloning and characterization of mouse disabled 2 interacting protein 2, a mouse orthologue of human NOSTRIN; Choi YJ et al.; The mouse disabled 2 interacting protein 2 (mDaIP2) had been obtained through yeast two hybrid system . It consists of 506 amino acids and its calculated molecular weight is 57.7 kDa . The protein contains N-terminal FCH domain and C-terminal SH3 domain . The SH3 domain interacts with the proline rich domain of mDab2 which had been identified to possess a transcriptional activation function . In RA-treated F9 teratocarcinoma cell, the mDaIP2 and mDab2 genes were differentially expressed in a RA-responsive manner and both were detected to localize in cytoplasm and nucleus . Homology search of all NCBI sequences indicated that the amino acid sequence of mDaIP2 shares 82% identity with human NOSTRIN which controls activity, trafficking, and targeting of nitric oxide synthase (eNos) . The eNos was not detected in RA-treated F9 cell . These results suggest that mDaIP2 somehow functions in a different fashion from NOSTRIN in F9 cell differentiation and that its function may be concerted with that of mDab2.

J Exp Clin Cancer Res, 2004 Sep, 23(3), 507 - 12
Effects of expression of hRFI on adenoma formation and tumor progression in colorectal adenoma-carcinoma sequence; Sasaki S et al.; For colorectal carcinomas as well as colonic polyps we investigated the expression of a newly discovered gene, hRFI, which is isolated by the yeast two-hybrid screening using hTid as a bait and expressed highly in esophageal carcinomas . Immunohistochemical staining was performed on 48 colorectal carcinomas and 77 colorectal polyps consisting of 70 adenomas and 7 hyperplastic polyps using the antibody of hRFI . We analyzed the expression of hRFI and the correlation between the percentage of staining of each and their clinico-pathological characteristics . Protein coding by hRFI was specifically and diffusely expressed in most of the cancerous regions of the colorectum . Also, in the early stage of colorectal adenomas, staining of hRFI was focal, and the percentage area of diffuse staining increased as the degree of dysplasia progressed . Although all normal colorectal glands and most hyperplastic polyps (71.4%) showed no staining of hRFI, most colorectal adenomas and carcinomas (93.2%) showed a focal or diffuse staining (P<0.001) . Furthermore, the percentage of diffuse staining in carcinomas (81.3%) was significantly higher than in adenomas (5.7%) (P<0.001) . hRFI is highly expressed in colorectal carcinomas . In the adenoma-carcinoma sequence, hRFI is involved at the initial tumor formation and its diffuse expression is associated with colorectal carcinogenesis . This evidence suggests that hRFI may act as an oncogenic molecule affecting the apoptotic pathway.

Arch Virol . 2004 Dec 10; {Epub ahead of print}
Identification of phosphoprotein:phosphoprotein and phosphoprotein:nucleocapsid protein interaction domains of the Newcastle disease virus; Jahanshiri F et al.; The yeast two-hybrid system has been used to identify domains of the Newcastle disease virus (NDV) phosphoprotein (P) involved in self-association and interaction with the nucleocapsid protein (NP) . Deletion analysis was used to map the domain(s) of the P protein involved in P:P and P:NP interactions . The C-terminal 45 amino acids (residues 247-291) were shown to play a major role in both of the interactions . Comparison of these findings with other reports suggests that paramyxoviruses are different with respect to interaction domain(s) between these two essential viral proteins involved in genome replication.

Planta . 2004 Dec 9; {Epub ahead of print}
The Arabidopsis SERK1 protein interacts with the AAA-ATPase AtCDC48, the 14-3-3 protein GF14lambda and the PP2C phosphatase KAPP; Rienties IM et al.; Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction . The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development . By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14lambda, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors . In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14lambda . In yeast, AtCDC48 interacts with GF14lambda and with the PP2C phosphatase KAPP . In plant protoplasts AtSERK1 interacts with GF14lambda.

Chromosoma . 2004 Dec 9; {Epub ahead of print}
The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID; Jager H et al.; The centromere-specific histone H3 variant CENP-A plays a crucial role in kinetochore specification and assembly . We chose a genetic approach to identify interactors of the Drosophila CENP-A homolog CID . Overexpression of cid in the proliferating eye imaginal disk results in a rough eye phenotype, which is dependent on the ability of the overexpressed protein to localize to the kinetochore . A screen for modifiers of the rough eye phenotype identified mutations in the Drosophila condensin subunit gene Cap-G as interactors . Yeast two-hybrid experiments also reveal an interaction between CID and Cap-G . While chromosome condensation in Cap-G mutant embryos appears largely unaffected, massive defects in sister chromatid segregation occur during mitosis . Taken together, our results suggest a link between the chromatin condensation machinery and kinetochore structure.

Oncogene . 2004 Dec 13; {Epub ahead of print}
HTLV-I Tax induces and associates with Crk-associated substrate lymphocyte type (Cas-L); Iwata S et al.; Crk-associated substrate lymphocyte type (Cas-L) is a docking protein that is heavily tyrosine phosphorylated by the engagement of beta1 integrins in T cells . In the present study, we attempted to evaluate the role of Cas-L in the pathophysiology of adult T-cell leukemia (ATL) . Examination of peripheral blood mononuclear cells from ATL patients as well as ATL-derived T cell lines showed an elevation of Cas-L in these cells . We showed that tyrosine phosphorylation as well as expression of Cas-L was markedly elevated through the induction of human T-lymphotropic virus type I (HTLV-I) Tax in JPX-9 cells, with these cells showing marked motile behavior on the ligands for integrins . We next performed yeast two-hybrid screening of cDNA library from an HTLV-I-transformed T cell line, which resulted in the identification of Tax as a putative binding partner for Cas-L . Co-precipitation experiments revealed that the serine-rich region of Cas-L might serve as the binding site with the highest affinity for Tax . Co-localization study showed that Tax and Cas-L partly merged in the cytoplasm . Finally, we showed that exogenous Cas-L inhibited Tax-mediated transactivation of nuclear factor kappaB (NF-kappaB), while Tax-independent activation of NF-kappaB remained intact, hence indicating that Cas-L might specifically regulate Tax-NF-kappaB pathway.Oncogene advance online publication, 13 December 2004; doi:10.1038/sj.onc.1208261.

Nat Struct Mol Biol . 2004 Dec 12; {Epub ahead of print}
Conformational changes in the Arp2/3 complex leading to actin nucleation; Rodal AA et al.; The two actin-related subunits of the Arp2/3 complex, Arp2 and Arp3, are proposed to form a pseudo actin dimer that nucleates actin polymerization . However, in the crystal structure of the inactive complex, they are too far apart to form such a nucleus . Here, we show using EM that yeast and bovine Arp2/3 complexes exist in a distribution among open, intermediate and closed conformations . The crystal structure docks well into the open conformation . The activator WASp binds at the cleft between Arp2 and Arp3, and all WASp-bound complexes are closed . The inhibitor coronin binds near the p35 subunit, and all coronin-bound complexes are open . Activating and loss-of-function mutations in the p35 subunit skew conformational distribution in opposite directions, closed and open, respectively . We conclude that WASp stabilizes p35-dependent closure of the complex, holding Arp2 and Arp3 closer together to nucleate an actin filament.

EMBO Rep, 2005 Jan, 6(1), 83 - 9
Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks; Zheng L et al.; Restarting stalled replication forks partly depends on the break-induced recombination pathway, in which a DNA double-stranded break (DSB) is created on the stalled replication fork to initiate the downstream recombination cascades . Single-stranded DNA gaps accumulating on stalled replication forks are potential targets for endonucleases to generate DSBs . However, it is unclear how this process is executed and which nucleases are involved in eukaryotic cells . Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork . In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage . Replication protein A facilitates FEN-1 interaction with DNA bubble structures . Human FEN-1, but not the GEN-deficient mutant, E178A, was shown to rescue the defect in resistance to UV and camptothecin in a yeast FEN-1 null mutant.

J Biomed Sci, 2004 Nov-Dec, 11(6), 902 - 10
Abi enhances Abl-mediated CDC2 phosphorylation and inactivation; Lin TY et al.; Abelson tyrosine kinase (Abl) is a non-receptor tyrosine kinase which is frequently coupled with adaptor proteins to interact with its substrates for the regulation of cytoskeleton rearrangement, cell growth and apoptosis in response to a variety of biological stimuli . The Abl interactor (Abi) family members were first identified as adaptor proteins of Abl for regulating Abl transforming and kinase activity . In the present study, we used a yeast two-hybrid screen to identify Cdc2 as a novel Abi-binding protein . This finding led us to investigate the role of Abi in linking Abl and Cdc2 . These three proteins formed a trimeric complex in Drosophila and mammalian cells . The expression of Abi in cells greatly enhanced the formation of the Abl-Cdc2 complex, suggesting that Abi functions as an adaptor protein facilitating the binding between Abl and Cdc2 . We show that Abi promotes Abl-mediated phosphorylation of Cdc2 at tyrosine 15 and inactivation of Cdc2 kinase activity . Furthermore, coexpression of Abl and Abi in Drosophila S2 cells led to suppression of cell growth . These data suggest that Abl signaling may be involved in the downregulation of Cdc2 kinase in cell cycle control . 2004 National Science Council, ROC and S . Karger AG, Basel

J Biomed Sci, 2004 Nov-Dec, 11(6), 874 - 85
A novel lectin (Morniga M) from mulberry (Morus nigra) bark recognizes oligomannosyl residues in N-glycans; Wu AM et al.; Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra) . In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection . From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine alpha1-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast . The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer {Man alpha1-->6(Man alpha1-->3) Man}, Penta-Man oligomer {Man alpha1-->6(Man alpha1-->3)Man alpha1-->6(Man alpha1-->3) Man} > or = Man alpha1-->2, 3 or 6 Man > Man > GlcNAc, Glc >> L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans . 2004 National Science Council, ROC and S . Karger AG, Basel

Mol Biol Cell . 2004 Dec 9; {Epub ahead of print}
The Tricornered Ser/Thr Protein Kinase Is Regulated by Phosphorylation and Interacts with Furry during Drosophila Wing Hair Development; He Y et al.; Monitoring Editor: Keith Mostov The Trc/Ndr/Sax1/Cbk1 family of ser/thr kinases plays a key role in the morphogenesis of polarized cell structures in flies, worms and yeast . Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase, is required for the normal morphogenesis of epidermal hairs, bristles, laterals and dendrites . We obtained in vivo evidence that Trc function was regulated by phosphorylation and that mutations in key regulatory sites resulted in dominant negative alleles . We found that wild-type, but not mutant Trc, is found in growing hairs and we failed to detect Trc in pupal wing nuclei implying that in this developmental context Trc functions in the cytoplasm . The furry gene and its homologues in yeast and C . elegans have previously been implicated as being essential for the function of the Ndr kinase family . We found that Fry is also found in growing hairs, that it's subcellular localization is dependent on Trc function and that it can be coimmunoprecipitated with Trc . Our data suggests a feedback mechanism involving Trc activity regulates the accumulation of Fry in developing hairs.

J Biol Chem . 2004 Dec 9; {Epub ahead of print}
FKBP51 and FKBP52 differentially regulate dynein interaction and nuclear translocation of the glucocorticoid receptor in mammalian cells; Wochnik GM et al.; We used a cellular system to elucidate the molecular determinants of the large immunophilins FKBP51 and FKBP52 for their action on the glucocorticoid receptor in mammalian cells . Increasing the levels of FKBP51 reduced transcriptional activity of the receptor, as reported . Elevated levels of FKBP52 per se showed no effect, but mitigated the inhibition of the receptor induced by FKBP51 . We discovered that nuclear translocation of the glucocorticoid receptor was delayed by FKBP51 . This correlates with the reduced interaction of FKBP51 with the motor protein dynein, compared to FKBP52 . From mutational analyses we conclude that three features of the immunophilins are required for efficient receptor signaling in mammalian cells: hsp90 interaction, dynein association, and PPIase enzyme activity . The relevance of dynein for receptor function was substantiated by several experiments: 1 . Coexpression of dynamitin, which disrupts the transport complex, reduced receptor activity . 2 . Coexpression of the PPIase domain fragment of FKBP52, which is known to disrupt interaction of the receptor to dynein, reduced GR function, in contrast to the corresponding fragment of FKBP51 . 3 . Swapping the PPIase domains of FKBP51 and FKBP52 reversed the respective activity . We conclude from our results that the mechanisms of the regulatory system FKBP51/FKBP52 discovered in yeast also operate in mammals to modulate hormone binding of the receptor . In addition, differential regulation of dynein association and nuclear translocation contributes to the effects of the two immunophilins on the glucocorticoid receptor in mammals.

Eukaryot Cell, 2004 Dec, 3(6), 1433 - 44
Potential link between the NIMA mitotic kinase and nuclear membrane fission during mitotic exit in Aspergillus nidulans; Davies JR et al.; We have isolated TINC as a NIMA-interacting protein by using the yeast two-hybrid system and have confirmed that TINC interacts with NIMA in Aspergillus nidulans . The TINC-NIMA interaction is stabilized in the absence of phosphatase inhibitors and in the presence of kinase-inactive NIMA, suggesting that the interaction is enhanced when NIMA is not fully activated . TINC is a cytoplasmic protein . TINC homologues and a TINC-like protein (A . nidulans HETC) are conserved in other filamentous fungi . Neither deletion of tinC nor deletion of both tinC and A . nidulans hetC is lethal, but deletion of tinC does produce cold sensitivity as well as osmotic sensitivity . Expression of an amino-terminal-truncated form of TINC (DeltaN-TINC) inhibits colony growth in Aspergillus and localizes to membrane-like structures within the cell . Examination of cell cycle progression in these cells reveals that they progress through multiple defective mitoses . Many cells contain large polyploid single nuclei, while some appear to have separated masses of DNA . Examination of the nuclear envelopes of cells containing more than one DNA mass reveals that both DNA masses are contained within a single nuclear envelope, indicating that nuclear membrane fission is defective . The ability of these cells to separate DNA segregation from nuclear membrane fission suggests that this coordination is normally a regulated process in A . nidulans . Additional experiments demonstrate that expression of DeltaN-TINC results in premature NIMA disappearance in mitotic samples . We propose that TINC's interaction with NIMA and the cell cycle defects produced by DeltaN-TINC expression suggest possible roles for TINC and NIMA during nuclear membrane fission.

J Biol Chem . 2004 Dec 6; {Epub ahead of print}
Isolation and characterization of a humoral factor that stimulates transcription of the acyl-CoA binding protein in the pheromone gland of the silkmoth, bombyx mori; Ohnishi A et al.; Acyl-CoA binding protein (ACBP) is a highly conserved 10-kDa intracellular lipid-binding protein that binds straight-chain (C14-C22) acyl-CoA esters with high affinity and is expressed in a wide variety of species ranging from yeast to mammals . Functionally, ACBP can act as an acyl-CoA carrier or as an acyl-CoA pool maker within the cell . While numerous work on the biochemical properties regarding the ACBP has been performed using various vertebrate and plant tissues, as well as different types of cells in culture, the regulatory mechanisms underlying ACBP gene expression have remained poorly understood . By exploiting the unique sex pheromone production system in the moth pheromone gland (PG), we report that transcription of a specific ACBP termed pheromone gland ACBP (pgACBP) is triggered by a hemolymph based humoral factor . Following purification and structure elucidation by means of high-resolution electrospray-ionization mass spectrometry (HR-ESIMS) and NMR analyses, in conjunction with stereochemical analyses using acid hydrolysates, the humoral factor was identified to be -D-glucosyl-O-L-tyrosine . Examination of the hemolymph titers during development revealed that the amount of -D-glucosyl-O-L-tyrosine dramatically rose prior to eclosion and reached a maximum of 5 mg/mL (about 1 mg/pupa) on the day preceding eclosion, which was consistent with the effective dose of -D-glucosyl-O-L-tyrosine in stimulating pgACBP transcription in vivo . Furthermore, in vitro assays using trimmed PG indicated that -D-glucosyl-O-L-tyrosine acts directly on the PG . These results provide the first evidence that transcription of some ACBPs can be triggered by specific humoral factors.

J Biol Chem . 2004 Dec 7; {Epub ahead of print}
Specificity and mechanism of RNA cap guanine-N2 methyltransferase (Tgs1); Hausmann S et al.; The 2,2,7-trimethylguanosine (TMG) cap structure is characteristic of certain eukaryotic small nuclear and small nucleolar RNAs . Prior studies have suggested that cap trimethylation might be contingent on cis-acting elements in the RNA substrate, protein components of a ribonucleoprotein complex, or intracellular localization of the RNA substrate . However, the enzymatic requirements for TMG cap formation remain obscure because TMG synthesis has not been reconstituted in vitro from defined components . Tgs1 is a conserved eukaryal protein that was initially identified as being required for RNA cap trimethylation in vivo in budding yeast . Here we show that purified recombinant fission yeast Tgs1 catalyzes methyl transfer from AdoMet to m7GTP and m7GDP . Tgs1 also methylates the cap analog m7GpppA, but is unreactive with GTP, GDP, GpppA, m2,2,7GTP, m2,2,7GDP, ATP, CTP, UTP, and ITP . The products of methyl transfer to m7GTP and m7GDP formed under conditions of excess methyl acceptor are 2,7-dimethyl GTP and 2,7-dimethyl GDP, respectively . Under conditions of limiting methyl acceptor, the initial m2,7GDP product is converted to m2,2,7GDP in the presence of excess AdoMet . We conclude that Tgs1 is guanine-specific, that N7 methylation must precede N2 methylation, that Tgs1 acts via a distributive mechanism, and that the chemical steps of TMG synthesis do not require input from RNA or protein cofactors.

J Biol Chem . 2004 Dec 6; {Epub ahead of print}
Periplakin interferes with G protein activation by the melanin-concentrating hormone receptor-1 by binding to the proximal segment of the receptor C-terminal tail; Murdoch H et al.; In mice genetic ablation of expression of either melanin concentrating hormone or the melanin concentrating hormone-1 receptor results in alterations in energy metabolism and a lean phenotype . There is thus great interest in the function and regulation of this receptor . Using the yeast two-hybrid system we identified an interaction of the actin- and intermediate filament-binding protein periplakin with the intracellular C-terminal tail of the melanin concentrating hormone-1 receptor . Direct association of these proteins was verified in pull-down and co-immunoprecipitation experiments . Truncations and internal deletions delineated the site of interaction to a group of eleven amino acids proximal to transmembrane helix VII that was distinct from the binding site for the melanin concentrating hormone-1 receptor interacting zinc-finger protein . Immunohistochemistry demonstrated co-expression of periplakin with melanin concentrating hormone-1 receptor in specific cells of the piriform cortex, amygdala and other structures of the adult mouse brain . Co-expression of the melanin concentrating hormone-1 receptor with periplakin in HEK293 cells did not prevent agonist-mediated internalization of the receptor but did interfere with activation of the G protein Gao1 and the elevation of {Ca2+}i . Co-expression of the receptor with the interacting zinc-finger protein did not modulate receptor internalization or G protein activation . The interaction of periplakin with receptors was selective . Co-expression of periplakin with the IP prostanoid receptor did not result in co-immunoprecipitation nor interfere with agonist-mediated G protein activation . Periplakin is the first protein described to modify the capacity of the melanin concentrating hormone-1 receptor to initiate signal transduction.

Biochim Biophys Acta, 2004 Dec 6, 1742(1-3), 133 - 40
Evolutionary perspective on annexin calcium-binding domains; Morgan RO et al.; Molecular systematic analysis of the annexin gene superfamily characterized the evolutionary origin, frequency and range of structural variation in calcium interaction domains that are considered intrinsic for membrane targeting and ion channel function . Approximately 36% of annexin repeat domains in an estimated 100 distinct subfamilies contained amino acid changes consistent with the functional loss of type two calcium-binding sites . At least 11% of annexin domains contained a novel K/H/RGD motif conserved in particular subfamilies and manifest in all phyla, apparently via convergent evolution . The first yeast annexin from Yarrowia lipolytica was classified in the ANXC1 subfamily with fungal and mycetozoan representatives . This clade had intact calcium-binding sites but disruption of the normally well-conserved, mid-repeat 4 region implicated in calcium channel regulation . Conversely, a tandem pair of novel annexins from the amphioxus Branchiostoma floridae resembled annexin A13 in gene structure and conserved the charged amino acids associated with the internal hydrophilic pore, but were devoid of external type 2 calcium-binding sites and incorporated K/RGD motifs instead, like annexin A9 . The selective erosion of calcium-binding sites in annexin domains and the occurrence of alternate ligands in the same exposed, interhelical loops are pervasive features of the superfamily . This suggests greater complexity than previously appreciated in the mechanisms controlling annexin membrane interaction and calcium channel operation.

FEBS Lett, 2004 Dec 17, 578(3), 311 - 5
Interactions of putative telomere-binding proteins in Arabidopsis thaliana: identification of functional TRF2 homolog in plants; Kuchar M et al.; Telomere-binding proteins are required for forming the functional structure of chromosome ends and regulating telomerase action . Although a number of candidate proteins have been identified by homology searches to plant genome databases and tested for their affinity to telomeric DNA sequences in vitro, there are minimal data relevant to their telomeric function . To address this problem, we made a collection of cDNAs of putative telomere-binding proteins of Arabidopsis thaliana to analyse their protein-protein interactions with the yeast two-hybrid system . Our results show that one myb-like protein, AtTRP1, interacts specifically with AtKu70, the latter protein having a previously described role in plant telomere metabolism . In analogy to the interaction between human Ku70 and TRF2 proteins, our results suggest that AtTRP1 is a likely homolog of TRF2 . The AtTRP1 domain responsible for AtKu70 interaction occurs between amino acid sequence positions 80 and 269 . The protein AtTRB1, a member of the single myb histone (Smh) family, shows self-interaction and interactions to the Smh family proteins AtTRB2 and AtTRB3 . Protein AtTRB1 also interacts with AtPot1, the Arabidopsis homolog of oligonucleotide-binding-fold-containing proteins which bind G-rich telomeric DNA . In humans, the TRF1-complex recruits hPot1 to telomeres by protein-protein interactions where it is involved in telomere length regulation . Possibly, AtTRB1 has a similar role in recruiting AtPot1.

Free Radic Biol Med, 2005 Jan 1, 38(1), 146 - 7
Cross-compartment protection by SOD1; Liochev SI et al.; The absence of SOD1 in yeast has been found to result in inactivation of Lys4p . This {4Fe-4S}-containing dehydratase is in the pathway of biosynthesis of lysine, hence the oxygen-dependent lysine auxotrophy seen in this case . O(2)(-) is known to oxidize and thus destabilize the {Fe-4S} clusters of dehydratases; hence, this would make perfect sense were it not for the fact that SOD1 localizes to the cytosol and the intermembrane space of mitochondria, whereas Lys4p localizes to the mitochondrial matrix . How could SOD1 in one compartment protect against O(2)(-) attack in a different compartment? We suggest that the relatively high levels of O(2)(-) in the cytosol and intermembrane space of the SOD1 mutant may react with endogenous NO, forming HOONO that can diffuse into the mitochondrial matrix and there inactivate Lys4p and other {4Fe-4S}-containing dehydratases.

Curr Biol, 2004 Dec 14, 14(23), 2143 - 8
Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis; Stumpff J et al.; Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle . Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs . Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1 . Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis . The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases . Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality . These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.

Curr Biol, 2004 Dec 14, 14(23), 2096 - 106
DNA breaks promote genomic instability by impeding proper chromosome segregation; Kaye JA et al.; BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) can result in the whole or partial loss of chromosomes . Previously, we showed that the ends of broken chromosomes remain associated . Here, we have examined the machinery that holds broken chromosome ends together, and we have explored the behavior of broken chromosomes as they pass through mitosis . RESULTS: Using GFP-localized arrays flanking an HO endonuclease site, we examined the association of broken chromosome ends in yeast cells that are checkpoint-arrested in metaphase . This association is partially dependent upon Rad50 and Rad52 . After 6-8 hr, cells adapted to the checkpoint and resumed mitosis, segregating the broken chromosome . When this occurred, we found that the acentric fragments cosegregated into either the mother or daughter cell 95% of the time . Similarly, pedigree analysis showed that postmitotic repair of a broken chromosome (rejoining the centric and acentric fragments) occurred in either the mother or daughter cell, but rarely both, consistent with a model in which both acentric sister chromatid fragments are passaged into the same nucleus . CONCLUSIONS: These data suggest two related phenomena: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister chromatids to each other and promotes their missegregation . Strikingly, the interchromosomal association of DNA breaks also promotes the missegregation of centromeric chromosomal fragments, albeit to a lesser extent than acentric fragments . The DNA break-induced missegregation of acentric and centric chromosome fragments provides a novel mechanism for the loss of heterozygosity that precedes tumorigenesis in mammalian cells.

Fungal Genet Biol, 2005 Jan, 42(1), 61 - 72
Fusarium oxysporum G-protein beta subunit Fgb1 regulates hyphal growth, development, and virulence through multiple signalling pathways; Delgado-Jarana J et al.; The vascular wilt fungus Fusarium oxysporum causes disease in a wide variety of crops . A signalling cascade controlled by the extracellular-regulated mitogen-activated protein kinase (MAPK) Fmk1 was previously found to be required for plant infection . To investigate the role of the heterotrimeric G-protein beta subunit Fgb1 as a putative upstream component of the Fmk1 signalling cascade, we generated F . oxysporum strains carrying either a Deltafgb1 loss-of-function allele or an fgb1(W115G) allele that mimicks the yeast STE4(W136G) mutation resulting in insensitivity to the cognate G-protein alpha subunit . Both types of mutants showed reduced virulence on tomato plants, similar to Deltafmk1 strains . However, in contrast to the latter, Deltafgb1 mutants displayed an abnormal hyphal growth phenotype with highly elongated cells, increased tip growth, a completely straight hyphal growth axis, and reduced subapical branching . Exogenous cAMP reversed part but not all of the Deltafgb1 growth phenotypes . Likewise, expression of the fgb1(W115G) allele only partly reversed growth phenotypes and failed to restore virulence on plants, whereas reintroduction of a functional fgb1 allele fully restored the wild type phenotype . Immunoblot analysis showed that levels of Fmk1 phosphorylation in fgb1 mutants were comparable to those in the wild type strain . Our results support a model in which Fgb1 controls hyphal growth, development and virulence in F . oxysporum both through cAMP-dependent and -independent pathways.

Exp Hematol, 2004 Dec, 32(12), 1173 - 81
Protein partners of C/EBPepsilon; Chih DY et al.; CCAAT-enhancer binding protein-epsilon (C/EBPepsilon) is a nuclear transcription factor implicated in the regulation of terminal myeloid differentiation . Using a yeast two-hybrid screen, potential interaction partners of C/EBPepsilon involved in myeloid development were identified . C/EBPepsilon was found to associate with other C/EBP family members, including C/EBPepsilon and CHOP as well as other proteins that are known to contain a leucine-zipper protein interaction motif including CREB2, LDOC1, E6TP1, and AF-17 . In addition, C/EBPepsilon demonstrated the potential for interaction with proteins that do not possess a leucine-zipper motif, including proteins that may be involved in sumoylation (protein inhibitor of activated STAT1 {PIAS1} and ubiquitin-conjugating enzyme E2I) . As expected, the association of C/EBPepsilon with other C/EBP family members depends on the presence of a functional leucine-zipper motif . Mapping studies of C/EBPepsilon with PIAS1 (as an example of a nonleucine-zipper-containing protein) showed that C/EBPepsilon interacts with the amino-terminal domain of PIAS1 . The function of C/EBPepsilon interacting proteins was further investigated . Co-expression of C/EBPepsilon with C/EBPdelta resulted in potent transactivation in a lactoferrin reporter system . A gel mobility shift assay suggests that C/EBPepsilon, C/EBPalpha, and C/EBPdelta proteins can bind as heterodimers to a C/EBP consensus DNA-binding site . As CHOP is known to represent a transcriptional repressor, the functional interaction between C/EBPepsilon and CHOP was investigated . Co-expression of C/EBPepsilon and c-Myb with CHOP caused marked transcriptional repression of target reporter genes . Our results suggest heterodimeric partners of C/EBPepsilon modulate the function of C/EBPepsilon in mediating gene transcription during myelopoiesis.

Biochim Biophys Acta, 2004 Dec 1, 1703(1), 63 - 7
Solvent isotope effects on alpha-glucosidase; O'donnell AH et al.; The solvent kinetic isotope effects (SKIE) on the yeast alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 degrees C . With p-nitrophenyl-D-glucopyranoside (pNPG), the dependence of k(cat)/K(m) on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, (DOD)(k(cat)/K(m)), of 1.9 (+/-0.3) . The two pK(a)s characterizing the pH profile were increased in D(2)O . The shift in pK(a2) of 0.6 units is typical of acids of comparable acidity (pK(a)=6.5), but the increase in pK(a1) (=5.7) of 0.1 unit in going from H(2)O to D(2)O is unusually small . The initial velocities show substrate inhibition (K(is)/K(m) approximately 200) with a small solvent isotope effect on the inhibition constant {(DOD)K(is)=1.1 (+/-0.2)} . The solvent equilibrium isotope effects on the K(is) for the competitive inhibitors D-glucose and alpha-methyl D-glucoside are somewhat higher {(DOD)K(i)=1.5 (+/-0.1)} . Methyl glucoside is much less reactive than pNPG, with k(cat) 230 times lower and k(cat)/K(m) 5 x 10(4) times lower . The solvent isotope effect on k(cat) for this substrate {=1.11 (+/-0 . 02)} is lower than that for pNPG {=1.67 (+/-0.07)}, consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.

Phytochemistry, 2004 Aug, 65(15), 2189 - 96
Expression of a Stokesia laevis epoxygenase gene; Hatanaka T et al.; Epoxy fatty acids have a number of important uses and there is interest in enzymes catalyzing their synthesis from renewable sources . Both cytochrome P450 monooxygenases and divergent forms of di-iron desaturases are known to produce epoxy fatty acids in plants . Degenerate primers based on conserved sequences of delta12 desaturase-like genes led to the isolation of an epoxygenase gene from Stokesia laevis . The cDNA is 1.4 kb and it encodes 378 amino acids . The similarities of this gene at the amino acid sequence level with epoxygenases of Vernonia and Crepis, and the delta12 desaturases of soybean, FAD2-1 and FAD2-2, are 84%, 69%, 49%, and 55%, respectively . When the vector, pYES2, was used to transform yeast, epoxy fatty acid formation was observed in the cells . The effects of electron donors in the yeast expression system were tested but cytochrome b5 and cytochrome b5 reductase genes from Arabidopsis thaliana co-expressed with the epoxygenase had little effect on vernolic acid accumulation in the yeast . Finally, this gene, driven by a seed-specific phaseolin promoter, was cloned into a TDNA-vector and transferred into Arabidopsis plants . The results showed that T2 seeds of transgenic Arabidopsis expressing the Stokesia gene accumulated vernolic acid but no vernolic acid was detected in control plants . Northern blot analysis indicates this S . laevis epoxygenase gene is expressed mainly in developing seeds and no transcript was detected in leaves or roots.

Nucleic Acids Res, 2004, 32(21), 6425 - 36 Print 2004.
Basic helix-loop-helix transcription factor Tcfl5 interacts with the Calmegin gene promoter in mouse spermatogenesis; Siep M et al.; In mouse spermatogenesis, differentiating germ line cells initiate expression of specific genes at subsequent developmental steps . The Calmegin (Clgn) gene is first expressed in meiotic prophase, in primary spermatocytes, and encodes a protein that acts as a chaperone . To identify testis-specific transcription factors that control expression of the Clgn gene in spermatogenesis, we performed a yeast one-hybrid screening with a Clgn promoter sequence as bait DNA . This screening resulted in the identification of mouse Tcfl5 as a candidate Clgn promoter-binding protein . Tcfl5 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and mouse Tcfl5 shows 83% amino acid sequence identity with human TCFL5 . Gel-shift and yeast one-hybrid experiments showed that Tcfl5 interacts with a non-canonical CACGCG site that is present in the Clgn promoter . By using northern blot, RT-PCR and in situ hybridization, mouse Tcfl5 mRNA was detected only in testis, with the highest expression level in primary spermatocytes and round spermatids . The highest level of Tcfl5 protein was found in primary spermatocytes at the diplotene stage of meiotic prophase, where the protein colocalizes with transcriptionally active chromatin.

J Med Microbiol, 2004 Dec, 53(Pt 12), 1207 - 14
Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR; Gutzmer R et al.; The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs . LightCycler PCR assays were established for seven primer sets specific for fungal DNA . For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA . A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points . Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts . Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture . Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.

J Anim Physiol Anim Nutr (Berl), 2004 Dec, 88(11-12), 393 - 400
Changes in selected serum parameters of broiler chicken fed supplemental chromium; Kroliczewska B et al.; Summary The present study was conducted to evaluate the effect of chromium (Cr) from Cr yeast on the growth performance and total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides, glucose, total protein and Cr concentration in the serum of broiler chicken . The birds were fed a control diet or a control diet supplemented with Cr at a level of 300, 500 mug/kg Cr . The supplementation of 500 mug/kg Cr increased body weight, weight gain and feed efficiency (p < 0.05) . In addition, supplementation with Cr decreased the serum total cholesterol, LDL cholesterol (p < 0.05), triglycerides (p < 0.05) and glucose (p < 0.05) concentrations whereas serum HDL cholesterol increased . Serum total protein and serum Cr concentration slightly but not significantly increased in both Cr groups . The study suggest that Cr supplementation particularly at 500 mug/kg Cr from Cr yeast can influence on carbohydrate and lipid metabolism of broiler chicken and can be used as additives in animal diet but it still needs more investigations.

Biochem J . 2004 Dec 7; {Epub ahead of print}
Aha1 competes with Hop, p50 and p23 for binding to the molecular chaperone Hsp90 and contributes to kinase and hormone receptor activation; Harst A et al.; The ATP dependent molecular chaperone Hsp90 is essential for the maturation of hormone receptors and protein kinases . During the process of client protein activation, Hsp90 cooperates with cofactors/cochaperones of unique sequence like Aha1 (activator of Hsp90 ATPase), p23 or p50 and with cofactors containing tetratricopeptide repeat (TPR) domains like Hop, immunophilins or cyclophilins . Although the binding sites for these different types of cofactors are distributed along the three domains of Hsp90, sterical overlap and competition for binding sites restricts the combinations of cofactors that can bind to Hsp90 at the same time . The recently discovered cofactor Aha1 associates with the middle domain of Hsp90 but its relationship with other cofactors of the molecular chaperone is poorly understood . Therefore, we analyzed whether complexes of Aha1, p23, p50, Hop and a cyclophilin with Hsp90 are disrupted by the respective other four cofactors by gel permeation chromatography using purified proteins . It turned out, that Aha1 competes with the early cofactors Hop and p50 but can bind to Hsp90 in the presence of cyclophilins, suggesting that Aha1 acts as a late cofactor of Hsp90 . In contrast to p50, which can bind to Hop, Aha1 does not interact directly with any of the other four cofactors . In vivo studies in yeast and in mammalian cells revealed that Aha1 is not specific for kinase activation but also contributes to maturation of hormone receptors, proposing a general role for this cofactor in the activation of Hsp90 dependent client proteins.

J Am Chem Soc, 2004 Dec 15, 126(49), 16077 - 86
A library of spirooxindoles based on a stereoselective three-component coupling reaction; Lo MM et al.; A collection of structurally complex and chemically diverse small molecules is a useful tool to explore cell circuitry . In this article, we report the split-pool synthesis of more than 3000 spirooxindoles on high capacity macrobeads . The key reaction to assemble the spirooxindole core stereoselectively is a Lewis acid variant of the Williams' three-component coupling . After formation, the skeleton was elaborated using Sonogashira couplings, amide forming reactions, and N-acylations of gamma-lactams . The final library was analyzed by sampling individual macrobeads and by using binomial confidence limits . It was determined that at least 82% of the library compounds should have better than 80% purity . To demonstrate the utility of our discovery process, a high-throughput chemical genetic modifier screen was performed using stock solutions of the resultant products . A number of positives were identified as enhancers of the cellular actions of latrunculin B, an actin polymerization inhibitor . Through resynthesis, we confirmed one of the positives and demonstrated that, in yeast cells, it has an EC50 in the sub-micromolar range.

J Chromatogr A, 2004 Nov 19, 1057(1-2), 107 - 13
Fully automated online multi-dimensional protein profiling system for complex mixtures; Fujii K et al.; For high throughput proteome analysis of highly complex protein mixtures, we have constructed a fully automated online system for multi-dimensional protein profiling, which utilizes a combination of two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC-MS-MS), based on our well-established offline system described previously {K . Fujii, T . Nakano, T . Kawamura, F . Usui, Y . Bando, R . Wang, T . Nishimura, J . Proteome Res . 3 (2004) 712} . A two-valve switching system on a programmable auto sample injector is utilized for online two-dimensional chromatography with strong cation-exchange (SCX) and reversed-phase (RP) separations . The SCX separation is carried out during the equilibration of RP chromatography and the entire sequence of analysis was performed under fully automated conditions within 4 h, based on six SCX fractionations, and 40 min running time for the two-dimensional RP chromatography . In order to evaluate its performance in the detection and identification of proteins, digests of six standard proteins and yeast 20S proteasome have been analyzed and their results were compared to those obtained by the one-dimensional reversed-phase chromatography system (ID-LC-MS-MS) . The 2D-LC-MS-MS system demonstrated that both the number of peptide fragments detected and the protein coverage had more than doubled . Furthermore, this multi-dimensional protein profiling system was also applied to the human 26S proteasome, which is one of the highly complex protein mixtures . Consequently, 723 peptide fragments were identified as 31 proteasome components, together with other coexisting proteins in the sample . The identification could be comprehensively performed with a 63% sequence coverage on an average, and additionally, with modifications at the N-terminus . These results indicated that the online 2D-LC-MS-MS system being described here is capable of analyzing highly complex protein mixtures in a high throughput manner, and that it would be applicable to dynamic proteomics.

J Chromatogr A, 2004 Nov 19, 1057(1-2), 101 - 6
Protein separation with surfactant-coated octadecylsilyl silica involving Cibacron blue 3GA-conjugated nonionic surfactant; Saitoh T et al.; A novel medium for protein separation, namely affinity admicelle, was prepared by mixing of octadecylsilyl (ODS) silica gels, a polyoxyethylene-type nonionic surfactant (Triton X-100), and a surfactant-conjugated substrate (affinity ligand) in an aqueous solution . The ligand was synthesized by mixing a triazine dye (Cibacron Blue 3GA, CB) and a polyethylene glycol monooleyl ether (C18EO7, C18EO10, or C18EO20) having different length of polyoxyethylene moiety in weakly alkaline solutions . The amount of Triton X-100 sorbed on 1 g of ODS silica was 0.2 mmol . Affinity ligands having highly hydrophobic oleyl group were predominantly sorbed on ODS silica . The losses of Triton X-100 and affinity ligand were within 0.3% and negligible by washing the admicelles were with a 25-fold volume of 1 mM Tris-HCl solution (pH 7.4) . The coating ODS silica with Triton X-100 was effective to prevent the irreversible sorption of albumin (bovine, serum) . An NADH-dependent enzyme, alcohol dehydrogenase (ADH, yeast), was successfully collected on the admicelles involving CB-conjugated ligands (CB-C18EO20) . The maximum collection of ADH to 90 mg/ml of affinity admicelles was 68+/-4% . However, CB-C18EO7 and CB-C18EO10 having shorter polyoxyethylene unit were not available, suggesting the requirement of the spacer moiety in the affinity ligand . The recovery and purification factor based on the ratio of activity (unit)/protein (mg) from Whatman DE52-treated yeast extract was 27% and 12, respectively.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2364 - 7 Epub 2004 Dec.
Expression, purification and preliminary crystallographic characterization of a novel segment from the neurofibromatosis type 1 protein; Bonneau F et al.; Neurofibromin (MW 320 kDa) is the protein responsible for the pathogenesis of neurofibromatosis type 1 (NF1), one of the most common genetic diseases worldwide . The neurofibromin GAP-related domain (GRD, MW 38 kDa) possess a Ras-specific GTPase-activating protein property, which is at present its only clear biochemical function . This article describes the study of the bacterial production and preliminary X-ray crystallographic analysis of a neurofibromin fragment located at the C-terminal end of the GRD, which contains a region reported to be homologous to the yeast Sec14p lipid exchange protein . Of the three crystal variants obtained, a tetragonal form diffracted to a resolution of at least 2.3 A.

J Cell Biol, 2004 Dec 6, 167(5), 841 - 9
DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7; Yamada A et al.; G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1 . Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated . In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited . We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint . Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction . Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase . These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

J Biol Chem . 2004 Dec 6; {Epub ahead of print}
Alteration of lithium pharmacology through manipulation of phosphoadenosine phosphate metabolism; Spiegelberg BD et al.; Bisphosphate 3'-nucleotidase (BPNT1 in mammals and Met22/Hal2 in yeast) is one of five members of a family of signaling phosphatases united through a common tertiary structure and inhibition by subtherapeutic doses of the antibipolar drug lithium . Here we report a role for 3'-nucleotidase and its substrate, 3'-phosphoadenosine 5'-phosphate (PAP), in mediating the cellular effects of lithium . Lithium-induced inhibition of growth in yeast cells may be overcome by dose-dependent heterologous expression of human BPNT1 . Disruption of the yeast 3'-nucleotidase gene or treatment of cells with lithium results in greater than 80-fold accumulation of PAP and leads to potent growth inhibition . These data indicate that accumulation of a 3'-nucleotidase substrate, such as PAP, mediates the toxicity of lithium . To further probe this model we examined the growth inhibitory effects of lithium under conditions in which PAP biosynthetic machinery was concomitantly downregulated . Disruption of met3 or met14 genes (ATP sulfurylase or phosphosulfate kinase), transcriptional downregulation of MET3 through methionine addition, or administration of chlorate, a widely used cell-permeable sulfurylase inhibitor, function to reduce lithium-induced intracellular PAP accumulation and lithium toxicity; all of which were reversed by heterologous expression of human sulfurylase and kinase . Collectively our data support a role for 3'-nucleotidase activity and PAP metabolism in aspects of lithium's mechanism of action and provide a platform for development of novel pharmacological modulators aimed at improving therapies for treatment of bipolar disorder.

Spectrochim Acta A Mol Biomol Spectrosc, 2005 Jan 14, 61(3), 361 - 6
Determination of nucleic acids at nanogram level using resonance light scattering technique with Congo Red; Wu X et al.; Based on the enhancement of the resonance light scattering (RLS) of Congo Red (CR) by nucleic acid, a new quantitative method for nucleic acid is developed . In the Tris-HCl buffer (pH 10.5), the weak light scattering of CR is greatly enhanced by addition of nucleic acid and CTMAB, the maximum peak is at 560 nm and the enhanced intensity of RLS is in proportion to the concentration of nucleic acid . The linear range is 1.0 x 10(-9) to 1.0 x 10(-6) g ml(-1), 7.5 x 10(-8) to 1.0 x 10(-6) g ml(-1) and 7.5 x 10(-8) to 2.5 x 10(-6) g ml(-1) for herring sperm DNA, calf thymus DNA and yeast RNA, and the detection limits are 0.019, 0.89 and 1.2 ng ml(-1) (S/N = 3), respectively . Actual biological samples were satisfactorily determined.

Anal Biochem, 2005 Jan 1, 336(1), 46 - 50
Denaturing RNA electrophoresis in TAE agarose gels; Masek T et al.; Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals . We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species . We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels . In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules . We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis . Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.

J Am Coll Cardiol, 2004 Dec 7, 44(11), 2192 - 201
Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy; Hayashi T et al.; OBJECTIVES: We sought to explore the relationship between a Tcap gene (TCAP) abnormality and cardiomyopathy . BACKGROUND: Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) cause severe heart failure and sudden death . Recent genetic investigations have revealed that mutations of genes encoding Z-disc components, including titin and muscle LIM protein (MLP), are the primary cause of both HCM and DCM . The Z-disc plays a role in establishing the mechanical coupling of sarcomeric contraction and stretching, with the titin/Tcap/MLP complex serving as a mechanical stretch sensor . Tcap interacts with the calsarcin, which tethers the calcineurin to the Z-disc . METHODS: The TCAP was analyzed in 346 patients with HCM (236 familial and 110 sporadic cases) and 136 patients with DCM (34 familial and 102 sporadic cases) . Two different in vitro qualitative assays-yeast two-hybrid and glutathion S-transferase pull-down competition-were performed in order to investigate functional changes in Tcap's interaction with MLP, titin, and calsarcin-1 caused by the identified mutations and a reported DCM-associated mutation, R87Q . RESULTS: Two TCAP mutations, T137I and R153H, were found in patients with HCM, and another TCAP mutation, E132Q, was identified in a patient with DCM . It was demonstrated by the qualitative assays that the HCM-associated mutations augment the ability of Tcap to interact with titin and calsarcin-1, whereas the DCM-associated mutations impair the interaction of Tcap with MLP, titin, and calsarcin-1 . CONCLUSIONS: These observations suggest that the difference in clinical phenotype (HCM or DCM) may be correlated with the property of altered binding among the Z-disc components.

Comp Biochem Physiol B Biochem Mol Biol, 2004 Dec, 139(4), 543 - 59
Nutrient sensing and metabolic decisions; Lindsley JE et al.; Cells have several sensory systems that detect energy and metabolic status and adjust flux through metabolic pathways accordingly . Many of these sensors and signaling pathways are conserved from yeast to mammals . In this review, we bring together information about five different nutrient-sensing pathways (AMP kinase, mTOR, PAS kinase, hexosamine biosynthesis and Sir2), highlighting their similarities, differences and roles in disease.

Biochemistry, 2004 Dec 14, 43(49), 15379 - 92
Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants; Melet A et al.; The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model . Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues . Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition . Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes {paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac} . Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin . Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation . Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates . For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel . The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8 . Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently {Schoch, G . A., et al . (2004) J . Biol . Chem . 279, 9497}, were consistent with key roles for S100, S114, and F205 residues in substrate binding . The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'.

Dev Genes Evol . 2004 Dec 3; {Epub ahead of print}
Drosophila Bys is nuclear and shows dynamic tissue-specific expression during development; Stewart MJ et al.; Although the bys-like family of genes has been conserved from yeast to humans, it is not apparent to what extent the function of Bys-like proteins has been conserved across phylogenetic groups . Human Bystin is thought to function in a novel cell adhesion complex involved in embryo implantation . The product of the yeast bys-like gene, Enp1, is nuclear and has a role in pre-ribosomal RNA (pre-rRNA) splicing and ribosome biogenesis . To gain insight into the function of the Drosophila melanogaster bys-like family member, termed bys, we examined bys mRNA expression and the localization of Bys protein . In embryos, bys mRNA is expressed in a tissue-specific pattern during gastrulation . In the larval wing imaginal disc, bys mRNA is expressed in the ventral and dorsal regions of the wing pouch, regions that give rise to epithelia that adhere to one another after the wing disc everts . The bys mRNA expression patterns could be interpreted as being consistent with a role for Bys in events requiring cell-cell interactions . However, embryonic bys mRNA expression patterns mirror those of genes that are potential targets of the growth regulator Myc and encode nucleolar proteins implicated in cell growth . Additionally, in Schneider line 2 (S2) cells, an epitope-tagged Bys protein is localized to the nucleus, suggesting that Drosophila Bys function may be conserved with that of yeast Enp1.

Planta . 2004 Oct 2; {Epub ahead of print}
A stress-inducible plasma membrane protein 3 (AcPMP3) in a monocotyledonous halophyte, Aneurolepidium chinense, regulates cellular Na(+) and K(+) accumulation under salt stress; Inada M et al.; Regulation of ion homeostasis is fundamental to physiological activities in plants . Here, we report on the functional characterization of AcPMP3 { A neurolepidium c hinense (a monocotyledonous halophyte) plasma membrane protein 3} under salt stress . Expression of AcPMP3-1 and AcPMP3-2 genes was highly induced by various abiotic stresses, such as salt, cold and drought . Furthermore, abscisic acid, H(2)O(2) and salicylic acid also triggered expression of AcPMP3 genes . In the Deltanha1 Deltapmr2 Delta pmp3 yeast mutant, which lacks the major Na(+) efflux systems (Na(+)/H(+) antiporter and Na(+)-ATPase), its salt-sensitive phenotype was restored by expressing the AcPMP3-1 gene, and the transformants accumulated lesser amounts of Na(+) and K(+) than mutant cells under 50 mM NaCl and 500 mM KCl conditions, respectively . These results suggested that AcPMP3-1 plays a role as a regulator of both Na(+) and K(+) accumulation in the cells . In situ hybridization showed that the AcPMP3-1 transcript was localized in cells of the root cap and root epidermis, which strongly suggested that AcPMP3-1 is essential for regulating Na(+)/K(+) transportation between plant roots and the outer environment under salt stress.

Oncogene . 2004 Dec 06; {Epub ahead of print}
Parafibromin, product of the hyperparathyroidism-jaw tumor syndrome gene HRPT2, regulates cyclin D1/PRAD1 expression; Woodard GE et al.; Parafibromin is the 531-amino-acid protein product encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal dominant hyperparathyroidism-jaw tumor familial cancer syndrome, sporadic parathyroid cancer, and a minority of families with isolated hyperparathyroidism . Parafibromin contains no identified functional domains but bears sequence homology to Cdc73p, a budding yeast protein component of the RNA polymerase II-associated Paf1 complex . This study addressed the expression and functional properties of human parafibromin . A survey of human and mouse tissues analysed with polyclonal antibodies to parafibromin showed specific immunoreactivity in adrenal and parathyroid glands, kidney, heart, and skeletal muscle . Subcellular fractionation and laser confocal microscopy of normal human parathyroid gland demonstrated expression of parafibromin in both the cytoplasmic and nuclear compartments . Parafibromin was expressed in four parathyroid adenomas but was absent from two parathyroid carcinomas . Transient overexpression of wild-type parafibromin, but not its Leu64Pro missense mutant implicated in parathyroid cancer and familial isolated hyperparathyroidism, inhibited cell proliferation, and blocked expression of cyclin D1, a key cell cycle regulator previously implicated in parathyroid neoplasia . These results demonstrate that human parafibromin is a nucleocytoplasmic protein with functions consistent with its postulated role as a tumor suppressor protein.Oncogene advance online publication, 6 December 2004; doi:10.1038/sj.onc.1208274.

J Biol Chem . 2004 Dec 4; {Epub ahead of print}
Human bloom protein stimulates flap endonuclease 1 activity by resolving DNA secondary structure; Wang W et al.; Flap endonuclease (FEN1) participates in removal of RNA primers of Okazaki fragments, several DNA repair pathways, and genome stability maintenance . Defects in yeast FEN1 produce chromosomal instability, hyper-recombination and sequence duplication . These occur because flaps produced during replication are not promptly removed . Long-lived flaps sustain breaks and form mis-aligned bubble structures that produce duplications . Flaps that can form secondary structure inhibit even wild-type FEN1 and are more likely to form bubbles . Although PCNA stimulates FEN1, it cannot resolve secondary structures . Bloom protein (BLM) is a 3'-5' helicase, mutated in Bloom syndrome . BLM has been reported to interact with and stimulate FEN1 independent of helicase function . In fact, activation of the helicase by ATP did not alter BLM stimulation of cleavage of unstructured flaps . However, BLM stimulation of FEN1 cleavage of foldback flaps, bubbles or triplet repeats was increased by an additional increment when ATP was added . Helicase-dependent stimulation of FEN1 cleavage was robust over a range of sizes of the single-stranded part of bubbles . However, increasing the length of the 5' annealed region of the bubble, ultimately counteracted the stimulatory capacity of the BLM helicase . Moderate helicase dependent stimulation was observed with both fixed and equilibrating CTG flaps . Our results suggest that BLM suppresses genome instability by aiding FEN1 cleavage of structure-containing flaps.

J Biol Chem . 2004 Dec 3; {Epub ahead of print}
Isolation of a gene encoding a 1,2-diacyl-sn-glycerol: Acetyl-CoA acetyltransferase from developing seeds of Euonymus alata; Milcamps A et al.; 1,2-Diacyl-3-acetyl-sn-glycerols (ac-TAG) are unusual triacylglycerols that constitute the major storage lipid in the seeds of Euonymus alata (Burning Bush) . These ac-TAGs have long chain acyl groups esterified at both sn-1 and sn-2 positions of glycerol . Cell free extracts of developing seeds of Euonymus alata contain both long-chain acyl-CoA and acetyl-CoA sn-1,2-diacylglycerol acyltransferase (DGAT) activity . We have isolated a gene from developing seeds of Euonymus alata which shows a very high sequence similarity to the members of the DGAT1 gene family (i.e . related to acyl-CoA:cholesterol acyltransferases) . This Euonymus DGAT1 gene, when expressed in wild type yeast, results in a 5-fold enhancement of long-chain triacylglycerol accumulation (lc-TAG), as well as the appearance of low levels of ac-TAG . Hydrogenated ac-TAG molecular species were identified by GC-MS . Microsomes isolated from this transformed yeast show diacylglycerol:acetyl-CoA acetyltransferase activity which is about 40-fold higher than that measured in microsomes prepared from yeast transformed with the empty vector or with the Arabidopsis thaliana DAGT1 gene . The specific activity of this microsomal acetyltransferase activity is of the same order of magnitude as the microsomal long-chain DGAT activities measured for yeast lines transformed with the empty vector, or either the Arabidopsis or Euonymus DGAT1 genes . Despite this, ac-TAG accumulation in yeast transformed with the Euonymus DGAT1 gene was very low (0.26% of lc-TAG), whereas lc-TAG accumulation was enhanced . Possible reasons for this anomaly are discussed . Expression of the Euonymus DGAT1-like gene in yeast lines where endogenous TAG-synthesis has been deleted confirmed that the gene product has both long-chain acyl- and acetyl-transferase activity.

Genetics, 2004 Nov, 168(3), 1539 - 55
Testing predictions of the double-strand break repair model relating to crossing over in Mammalian cells; Birmingham EC et al.; In yeast, four-stranded, biparental "joint molecules" containing a pair of Holliday junctions are demonstrated intermediates in the repair of meiotic double-strand breaks (DSBs) . Genetic and physical evidence suggests that when joint molecules are resolved by the cutting of each of the two Holliday junctions, crossover products result at least most of the time . The double-strand break repair (DSBR) model is currently accepted as a paradigm for acts of DSB repair that lead to crossing over . In this study, a well-defined mammalian gene-targeting assay was used to test predictions that the DSBR model makes about the frequency and position of hDNA in recombinants generated by crossing over . The DSBR model predicts that hDNA will frequently form on opposite sides of the DSB in the two homologous sequences undergoing recombination {half conversion (HC); 5:3, 5:3 segregation} . By examining the segregation patterns of poorly repairable small palindrome genetic markers, we show that this configuration of hDNA is rare . Instead, in a large number of recombinants, full conversion (FC) events in the direction of the unbroken chromosomal sequence (6:2 segregation) were observed on one side of the DSB . A conspicuous fraction of the unidirectional FC events was associated with normal 4:4 marker segregation on the other side of the DSB . In addition, a large number of recombinants displayed evidence of hDNA formation . In several, hDNA was symmetrical on one side of the DSB, suggesting that the two homologous regions undergoing recombination swapped single strands of the same polarity . These data are considered within the context of modified versions of the DSBR model.

Genetics, 2004 Nov, 168(3), 1231 - 48
Mutation and evolution of microsatellite Loci in neurospora; Dettman JR et al.; The patterns of mutation and evolution at 13 microsatellite loci were studied in the filamentous fungal genus Neurospora . First, a detailed investigation was performed on five microsatellite loci by sequencing each microsatellite, together with its nonrepetitive flanking regions, from a set of 147 individuals from eight species of Neurospora . To elucidate the genealogical relationships among microsatellite alleles, repeat number was mapped onto trees constructed from flanking-sequence data . This approach allowed the potentially convergent microsatellite mutations to be placed in the evolutionary context of the less rapidly evolving flanking regions, revealing the complexities of the mutational processes that have generated the allelic diversity conventionally assessed in population genetic studies . In addition to changes in repeat number, frequent substitution mutations within the microsatellites were detected, as were substitutions and insertion/deletions within the flanking regions . By comparing microsatellite and flanking-sequence divergence, clear evidence of interspecific allele length homoplasy and microsatellite mutational saturation was observed, suggesting that these loci are not appropriate for inferring phylogenetic relationships among species . In contrast, little evidence of intraspecific mutational saturation was observed, confirming the utility of these loci for population-level analyses . Frequency distributions of alleles within species were generally consistent with the stepwise mutational model . By comparing variation within species at the microsatellites and the flanking-sequence, estimated microsatellite mutation rates were approximately 2500 times greater than mutation rates of flanking DNA and were consistent with estimates from yeast and fruit flies . A positive relationship between repeat number and variance in repeat number was significant across three genealogical depths, suggesting that longer microsatellite alleles are more mutable than shorter alleles . To test if the observed patterns of microsatellite variation and mutation could be generalized, an additional eight microsatellite loci were characterized and sequenced from a subset of the same Neurospora individuals.

J Invertebr Pathol, 2004 Oct-Nov, 87(2-3), 77 - 83
Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates; Rangel DE et al.; Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent . Studies were conducted to determine if growth conditions, viz . artificial (agar media or rice grain) or natural (infected insects) substrates for conidial production affect two traits that directly influence performance of conidia after field application: tolerance to UV-B radiation and conidial germination speed . Conidia of two isolates (ARSEF 23 and ARSEF 2575) of M . anisopliae var . anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation . Conidia of both isolates when produced on insect cadavers were significantly more sensitive to UV-B radiation than conidia produced on PDAY . Also, conidia from insect cadavers germinated slower than those from PDAY cultures . A comparison of conidia from artificial substrates showed that conidia produced on Czapek's and Emerson's YpSs agar media or rice grains had higher tolerance to UV-B radiation and germinated faster than conidia raised on PDA and PDAY . Accordingly, the growth substrate and nutritional environment in which conidia are produced influences M . anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.

Protein Pept Lett, 2004 Dec, 11(6), 543 - 6
Pressure stability of proteins at their isoelectric points; Kidman G et al.; Yeast alcohol dehydrogenase is slowly denatured at moderate hydrostatic pressure (t(1/2) approximately equals 30 min at 2 kbar and pH 7) . The extent of denaturation is pH dependent with maximal stability near the isoelectric point of the protein, pH = 5.4 . While not a surprising finding, it appears that this phenomenon has not been documented before (or at least not identified) despite many investigations into the pressure stability of proteins . Consideration of changes in the net charge of proteins far from their isoelectric points may explain other pressure effects as well.

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