Yeasts

Plant Cell, 2004 Nov, 16(11), 2967 - 83 Epub 2004 Oct 19.
Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy; Yoshimoto K et al.; Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components . Thus far, plant autophagy has been studied primarily using morphological analyses . A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants . It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy . To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome . In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation . To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8) . In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions . By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions . In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor . Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins . The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.

Nucleic Acids Res, 2004 Oct 19, 32(18), 5636 - 48 Print 2004.
Characterization of dRFX2, a novel RFX family protein in Drosophila; Otsuki K et al.; A transcriptional regulatory element was identified in the region between URE (upstream regulatory element) and DRE (DNA replication-related element) in the Drosophila PCNA gene promoter . This element plays an important role in promoter activity in living flies . A yeast one-hybrid screening using this element as a bait allowed isolation of a cDNA encoding a protein which binds to the element in vitro . Nucleotide sequence analyses revealed that the cDNA encodes a novel protein containing a characteristic DNA-binding domain conserved among the regulatory factor X (RFX) family proteins . We termed this protein Drosophila RFX2 (dRFX2) and this element dRFX2 site . To investigate the function of dRFX2 in vivo, we took the strategy of analyzing the dominant negative effects against the endogenous dRFX2 . Transgenic flies were established in which expression of HA-dRFX(202-480) carrying the amino acid sequences from 202 to 480 containing the RFX domain (DNA-binding domain) of dRFX2 was targeted to the cells in the eye imaginal discs . In the eye imaginal disc expressing the HA-dRFX(202-480), the G1-S transition and/or the progression of S phase were/was interrupted, and the ectopic apoptosis was induced, though photoreceptor cells differentiated normally . These results indicate that dRFX2 plays a role in G1-S transition and/or in progression of S phase.

J Biol Chem, 2004 Dec 24, 279(52), 54808 - 16 Epub 2004 Oct 19.
The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs; Bilodeau PS et al.; Ubiquitin (Ub) attachment to membrane proteins can serve as a sorting signal for lysosomal delivery . Recognition of Ub as a sorting signal can occur at the trans-Golgi network and is mediated in part by the clathrin-associated Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding proteins (GGA) . GGA proteins bind Ub via a three-helix bundle subdomain in their GAT (GGA and target of Myb1 protein) domain, which is also present in the Ub binding domain of target of Myb1 protein . Ubiquitin binding by yeast Ggas is required to direct sorting of ubiquitinated proteins such as general amino acid permease (Gap1) from the trans-Golgi network to endosomes . Using affinity chromatography and nuclear magnetic resonance spectroscopy, we have found that the human GGA3 GAT domain contains two Ub binding motifs that bind to the same surface of ubiquitin . These motifs are found within different helices within the three-helix GAT subdomain . When functionally analyzed in yeast, each motif was sufficient to mediate trans-Golgi network to endosomal sorting of Gap1, and mutation of both motifs resulted in defective Gap1 sorting without defects in other GGA-dependent processes.

Cell Signal, 2005 Feb, 17(2), 153 - 66
Sumoylation of internally initiated Sp3 isoforms regulates transcriptional repression via a Trichostatin A-insensitive mechanism; Spengler ML et al.; Sp3 is a ubiquitously expressed member of the Sp family of transcription factors that encodes three proteins, Sp3, M1 and M2, with differing capacities to stimulate or repress transcription . As part of ongoing efforts to study the functions of Sp3 isoforms, we employed a yeast "two-hybrid" screen to identify Sp3-binding proteins . This screen resulted in the identification of Ubc9, a SUMO-1 conjugating enzyme, as an M2-binding protein, and consistent with these results sequence analyses identified consensus sumoylation motifs within several Sp family members . Western blots probed with anti-Sp3 detected a high molecular weight Sp3 isoform that is stabilized by a SUMO-1 hydrolase inhibitor, and this protein is also bound by anti-SUMO-1 antiserum . Transient transfection assays with epitope-tagged-SUMO-1 and GFP-SUMO-1 fusion proteins confirmed that Sp3, M1 and M2 proteins are sumoylated in vivo . Substitution of arginine for lysine at one putative site of sumoylation, lysine(551), blocked sumoylation of all Sp3 isoforms in vivo and led to a marginal increase in Sp3-mediated trans-activation in insect and mammalian cells . In contrast, introduction of this amino acid substitution within M1 converted it into a potent transcriptional trans-activator . We conclude that Sp3 isoforms are sumoylated in vivo and this post-translational modification plays an important role in the regulation of Sp3-mediated transcription.

Biochem Soc Trans, 2004 Nov, 32(Pt 5), 878 - 80
Interactions between G-protein-coupled receptors and periplakin: a selective means to regulate G-protein activation; Milligan G et al.; A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens . Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated . In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.

Biochem Soc Trans, 2004 Nov, 32(Pt 5), 868 - 70
Determining calmodulin binding to metabotropic glutamate receptors with distinct protein-interaction methods; Lidwell K et al.; mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that modulate synaptic transmission . The eight mammalian mGluRs form three groups based on sequence and functional similarities: group I (1 and 5), group II (2 and 3) and group III (4, 6-8) mGluRs . In the present study, we used a Y2H (yeast two hybrid) screen to identify proteins that interact with the C-terminal intracellular tail of mGluR3 . Prominent among the candidate receptor interacting proteins was calmodulin, a Ca(2+) sensor known to bind identifiable sequences in group I and III mGluRs . The Y2H method was used to investigate calmodulin binding to mGluRs but failed to confirm the documented interaction with group III mGluRs . Furthermore, subsequent biochemical analysis showed that calmodulin does not interact with group II mGluRs . This illustrates that certain Ca(2+)-dependent interactions are not recapitulated in yeast . Moreover, it highlights the necessity for supporting biochemical data to substantiate interactions identified with Y2H methods.

Biochem Soc Trans, 2004 Nov, 32(Pt 5), 707 - 11
Pleckstrin homology domains: not just for phosphoinositides; Lemmon MA; PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to phosphoinositides . Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains . Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx . 33%) appear, most often, to require both phosphoinositide and non-phosphoinositide determinants for their subcellular localization . Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization . Some others have described a signalling role for PH domains in regulating small GTPases, although phosphoinositides may also play a role . These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.

Biol Chem, 2004 Sep, 385(9), 801 - 8
Non-muscle alpha-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1); Magdolen U et al.; PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling . In addition to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or alpha(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis . To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed . Screening of a human placenta cDNA library identified--in addition to the C-terminal region of cytokeratin 18 (CK18(182-430))--a large C-terminal fragment of alpha-actinin-4 (Act-4) as a binding partner for PAI-1 . Two different cDNA clones encoding Act-4(287-911) and Act-4(330-911) respectively, were isolated . An Act-4(330-911)/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1 . In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4(330-911)/GST . Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity . Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells . However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation . We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis.

J Assoc Res Otolaryngol, 2004 Sep, 5(3), 295 - 304 Epub 2004 Jun 24.
A comparative study of Eya1 and Eya4 protein function and its implication in branchio-oto-renal syndrome and DFNA10; Zhang Y et al.; Allele variants of EYA1 and EYA4, two members of the vertebrate Eya gene family, underlie two types of inherited human deafness, branchio-oto-renal (BOR) syndrome and DFNA10, respectively . To clarify how mutations in these two genes and their encoded proteins impact the normal biology of hearing, we completed a number of functional studies using the yeast-two-hybrid system . We verified that bait constructs of the homologous region ( Eya1HR and Eya4HR) interact with Six1 prey constructs, although no interaction with Dach1 prey was demonstrable . To compare interaction affinities, we evaluated alpha-galactosidase activity after cotransformation of Eya1HR/Six1 and Eya4HR/Six1 and found that the latter interaction was weaker . By immunofluorescence staining, we showed Eya4HR localization to the cytoplasm . After coexpression of Six1, Eya4HR was translocated to the nucleus . Results with Eya1HR were similar . Translation of mutant constructs ( Eya4HR(R564X) and Eya1HR(R539X)) could not be demonstrated . Using dual Eya-containing constructs (with two wild-type alleles or wild-type and mutant alleles), we confirmed no translation of the mutant allele, even if the mutation was nontruncating . These results are consistent with clinical data and implicate haploinsufficiency as the cause of BOR syndrome and DFNA10.

Int J Oncol, 2004 Nov, 25(5), 1249 - 56
The PDZ protein Tip-1 is a gain of function target of the HPV16 E6 oncoprotein; Hampson L et al.; Previous work has indicated that the PDZ domain Tax interacting protein 1 (Tip-1) is a target of the HTLV1 Tax protein and is a potential RhoA effector . We have used the yeast two-hybrid system to show that Tip-1 also interacts with the HPV16 E6 protein . This interaction was confirmed by co-immunoprecipitation from E6 expressing C33A cervical carcinoma cells (C33A-E6) which showed that Tip-1 was not degraded by interaction with the HPV16 E6 oncoprotein . During routine passage we observed that C33A-E6 had a less compact morphology and were less adherent than control vector transfected cells C33A-V cells - a known effect of GTP-RhoA . Comparison of C33A-E6 to C33A-V demonstrated that E6 expressing cells had higher levels of phosphorylated myosin light chains (MLC) and increased cell motility, which was inhibited by antisense silencing of Tip-1 expression and by the RhoA kinase (ROCK) inhibitor Y27632 . Both C33A-E6 and C33A-V cells were shown to express GTP activated RhoA . Since ROCKs can be activated by GTP RhoA these data indicate that E6 may increase cell motility by augmenting GTP RhoA mediated activation of ROCKs and that this is dependent on the expression of the Tip-1 protein.

Int J Oncol, 2004 Nov, 25(5), 1213 - 21
Ubiquilin-1 is a novel HASH-1-complexing protein that regulates levels of neuronal bHLH transcription factors in human neuroblastoma cells; Persson P et al.; The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homologue-1 (MASH-1 in mouse and HASH-1 in humans) is expressed in specific subsets of embryonic neuronal precursors of both the peripheral and central nervous systems . This gene is essential for development of olfactory and most peripheral autonomic neurons . Neuro-blastoma is a pediatric malignancy derived from sympathetic nervous system precursors and HASH-1 is expressed in a majority of neuroblastoma tumors and cell lines, indicating the immature phenotype of these cells . Using a human neuroblastoma cDNA library and the yeast two-hybrid system to identify novel HASH-1-interacting proteins, we isolated ubiquilin-1 (DA41, hPLIC-1), a gene that contains multiple ubiquitin-related domains . Further analyses showed that ubiquilin-1 interacts not only with HASH-1, but also with other tissue-specific bHLH proteins, including HES-1 . Overexpression of ubiquilin-1 led to accumulation of HASH-1 and HES-1 . Moreover, ubiquilin-1 was translocated from the cytoplasm to the nucleus upon co-expression with HASH-1 . These results indicate that ubiquilin-1 plays an active role in the precise regulation of HASH-1 and of other tissue-specific bHLH proteins.

Mech Ageing Dev, 2004 Sep, 125(9), 591 - 4
Role of sirtuin proteins in life extension by caloric restriction; Masoro EJ; The deacetylase activity of sirtuin proteins may play a key role in the life extending action of caloric restriction in organisms ranging from yeast to mammals . Recent research has been focused on the possible afferent pathway by which caloric restriction increases the deacetylase activity and on the efferent pathway by which the increased deacetylase activity extends life . Further research is needed to firmly establish the role of sirtuin proteins in life extension by caloric restriction in mammals.

BMC Bioinformatics . 2004 Oct 18;5(1):154.
Information assessment on predicting protein-protein interactions; Lin N et al.; BACKGROUND: Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell . Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions . In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation . Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information . RESULTS: Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast . In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality . We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions . CONCLUSIONS: In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO) functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al . Random forests based on the MIPS and GO information alone can give highly accurate classifications . In this particular subset of complete information, adding other genomic data does little for improving predictions . We also found that the data discretizations used in the Bayesian methods decreased classification performance.

Br J Dermatol, 2004 Oct, 151(4), 898 - 902
Sodium benzoate-induced repeated episodes of acute urticaria/angio-oedema: randomized controlled trial; Nettis E et al.; BACKGROUND: Sodium benzoate (E 211) is widely used to delay yeast spoilage of acidic foods and beverages . Numerous cases of adverse reactions to benzoate have been recorded, but most of the studies that have been conducted lacked proper placebo controls or blinding . OBJECTIVE: The aim of this study is to determine the incidence of intolerance to sodium benzoate among subjects who experienced repeated episodes of acute urticaria/angio-oedema following the ingestion of a meal or a product containing this substance . METHODS: This was a retrospective study based on the analysis of data from patients reported to have experienced episodes of urticaria, with or without angio-oedema, after ingesting meals or products containing sodium benzoate . At the first visit to the outpatients clinic, a careful history was taken . Patients were then given the following diagnostic tests: tests for IgE for common inhalant allergens and food allergens, and a double-blind, placebo-controlled challenge with sodium benzoate . RESULTS: A total of 47 subjects were enrolled in the study; five (11%) showed at least one relevant positive reaction to an IgE test for food allergy . Only one subject (2%) had a reaction after the ingestion of 75 mg of sodium benzoate without an adverse reaction to placebo . CONCLUSION: This study shows that the percentage of repeated episodes of acute urticaria/angio-oedema reactions induced by sodium benzoate is very low (2%) . In view of our results, we suggest that when faced with patients who have suffered adverse reactions that could be attributed to sodium benzoate, physicians should also carefully evaluate other possible causes.

Genetics . 2004 Oct 16; {Epub ahead of print}
A Torrid Zone on Mouse Chromosome 1 Containing a Cluster of Recombinational Hotspots; Paigen K; Within the 2.38 Mb Ath1 region of mouse Chromosome 1, 42 of 45 genetic crossovers from crosses between C57BL/6J (B6) and either C3H/HeJ (H) or Mus spretus (SPRET) occurred in four zones (A-D); zone A, 100 Kb long, contained a cluster of at least four recombination hot spots . F1 sperm assays indicate that within this "torrid zone" the most active hot spot (A3) can initiate recombination on H and SPRET but not B6 chromosomes . The A3 DNA sequence contains a (G/C)TTT repeat, long stretches of A or T, and a cyclic variation in AT content . Recombination was drastically reduced in a cross between B6 and a B6.SPRET Ath1 congenic strain, but was unaffected in a B6 x B6.H Ath1 congenic cross . Similar non-random clustering of hot spots has been observed in yeast and the major histocompatibility complexes of human and mouse . To the extent that torrid zones are a general feature of mammalian genomes, they have considerable implications for genetic mapping strategies in both human populations and mouse crosses.

J Biol Chem, 2004 Dec 24, 279(52), 54069 - 78 Epub 2004 Oct 15.
Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I; Colley WC et al.; Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT) . Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality . Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates . Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage . We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants . Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality . Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype . The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis . Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp . In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.

Biophys J, 2005 Jan, 88(1), 639 - 46 Epub 2004 Oct 15.
Control of glycolytic oscillations by temperature; Mair T et al.; External control of oscillatory glycolysis in yeast extract has been performed by application of either homogeneous temperature oscillations or stationary, spatial temperature gradients . Entrainment of the glycolytic oscillations by the 1/2- and 1/3-harmonic, as well as the fundamental input frequency, could be observed . From the phase response curve to a single temperature pulse, a distinct sensitivity of NADH-oxidizing processes, compared with NAD-reducing processes, is visible . Determination of glycolytic intermediates shows that the feedback-regulated phosphofructokinase as well as the glyceraldehyde-3-phosphate dehydrogenase are the most temperature-sensitive steps of glycolysis . We also find strong concentration changes in ATP and AMP at varying temperatures and, accordingly, in the energy charge . Construction of a feedback loop for spatial control of temperature by means of a Peltier element allowed us to apply a temperature gradient to the yeast extract . With this setup it is possible to initiate traveling waves and to control the wave velocity.

Genes Dev, 2004 Oct 15, 18(20), 2557 - 70
The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis; Higgins JD et al.; MSH4, a meiosis-specific member of the MutS-homolog family of genes, is required for normal levels of recombination and fertility in budding yeast, mouse, and Caenorhabditis elegans . In this paper, we report the identification and characterization of the Arabidopsis homolog of MSH4 (AtMSH4) . We demonstrate that AtMSH4 expression can only be detected in floral tissues, consistent with a role in reproduction . Immunofluorescence studies indicate that its expression is limited to early meiotic prophase I, preceding the synapsis of homologous chromosomes . A T-DNA insertional mutant (Atmsh4) exhibited normal vegetative growth but a severe reduction in fertility, consistent with a meiotic defect; this was confirmed by cytological analysis of meiosis . RNAi-induced down-regulation of the MSH4 gene resulted in a similar fertility and meiotic phenotype . We demonstrate that prophase I chromosome synapsis is delayed and may be incomplete in Atmsh4, and metaphase I chiasma frequency is greatly reduced to approximately 15% of wild type, leading to univalence and nondisjunction . We show that these residual chiasmata are randomly distributed among cells and chromosomes . These features of chiasma frequency and distribution in Atmsh4 show close parallels to MSH4-independent crossovers in budding yeast that have been proposed to originate by a separate pathway . Furthermore, the characteristics of the MSH4-independent chiasmata in the Atmsh4 mutant closely parallel those of second-pathway crossovers that have been postulated from Arabidopsis crossover analysis and mathematical modeling . Taken together, this evidence strongly indicates that Arabidopsis possesses two crossover pathways.

Mol Hum Reprod, 2004 Dec, 10(12), 917 - 24 Epub 2004 Dec.
Association between MSH4 (MutS homologue 4) and the DNA strand-exchange RAD51 and DMC1 proteins during mammalian meiosis; Neyton S et al.; During meiotic prophase, chromosomes must undergo highly regulated recombination events, some of which lead to reciprocal exchanges . In yeast, MSH4, a meiosis-specific homologue of the bacterial MutS protein, is required for meiotic recombination . In mice, disruption of the Msh4 gene results in male and female infertility due to meiotic failure . To date, the implication of MSH4 mutations has not been established in human sterility . However, it is noteworthy that mutant mice exhibit a defect in the chromosome synapsis, strikingly similar to the clinical observations found in human infertility . As a step towards understanding the molecular mechanisms underlying the role of MSH4 in human gametogenesis, we decided to determine whether this protein interacts with recombination machinery enzymes . Our results provide biochemical evidence indicating that the human MSH4 protein physically interacts with both RAD51 and DMC1, two RecA homologues known to initiate DNA strand-exchange between homologous chromosomes . Immunolocalization analyses show that some MSH4 foci, located on mouse meiotic chromosomes, colocalize with DMC1/RAD51 complexes . Our data support the view that MSH4 is associated with the early meiotic recombination machinery in mammals . We consider the possibility that MSH4 is involved in the regulation of recombination events by exerting a function closely after DNA strand-exchange has been initiated.

FEMS Yeast Res, 2004 Nov, 5(2), 127 - 32
Mitochondria damage checkpoint in apoptosis and genome stability; Singh KK; Mitochondria perform multiple cellular functions including energy production, cell proliferation and apoptosis . Studies described in this paper suggest a role for mitochondria in maintaining genomic stability . Genomic stability appears to be dependent on mitochondrial functions involved in maintenance of proper intracellular redox status, ATP-dependent transcription, DNA replication, DNA repair and DNA recombination . To further elucidate the role of mitochondria in genomic stability, I propose a mitochondria damage checkpoint (mitocheckpoint) that monitors and responds to damaged mitochondria . Mitocheckpoint can coordinate and maintain proper balance between apoptotic and anti-apoptotic signals . When mitochondria are damaged, mitocheckpoint can be activated to help cells repair damaged mitochondria, to restore normal mitochondrial function and avoid production of mitochondria-defective cells . If mitochondria are severely damaged, mitocheckpoint may not be able to repair the damage and protect cells . Such an event triggers apoptosis . If damage to mitochondria is continuous or persistent such as damage to mitochondrial DNA resulting in mutations, mitocheckpoint may fail which can lead to genomic instability and increased cell survival in yeast . In human it can cause cancer . In support of this proposal we provide evidence that mitochondrial genetic defects in both yeast and mammalian systems lead to impaired DNA repair, increased genomic instability and increased cell survival . This study reveals molecular genetic mechanisms underlying a role for mitochondria in carcinogenesis in humans.

Exp Gerontol, 2004 Sep, 39(9), 1369 - 78
Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology; Yoon IK et al.; The aging process is known to be regulated by specific genes in various organisms, including yeast, the nematode C . elegans, fruitflies and mice . To explore the novel genes involved in aging process, we applied cDNA microarray technology to a replicative senescence model of human dermal fibroblasts (HDF) . Eighty-four genes, including inflammatory genes, cell cycle regulatory genes, cytoskeletal genes, and metabolic genes were found to show more than two fold expressional differences in young and old fibroblasts . Furthermore, 31 genes were confirmed to be up- or down-regulated during replicative senescence by semi-quantitative RT-PCR . The overexpressions of several genes including CD36, putative lymphocyte G0/G1 switch gene (G0S2), tumor protein D52-like 1 (TPD52L1), chemokine (C-X-C motif) ligand 6, myxovirus resistant gene 1 (MX1), and the down-regulation of the immunoglobulin superfamily containing leucine-rich repeat (ISLR), neurotrimin, insulin-like growth factor 2 associated protein (IGF2A), and apoptosis-related RNA binding protein (NAPOR3) were newly identified . These results suggest that fibroblasts show the deregulation of various cellular processes, such as inflammatory response, mitosis, cell adhesion, transport, signal transduction, and metabolism during replicative senescence .

Methods Enzymol, 2004, 390, 53 - 64
Analysis of the regulation of microtubule dynamics by interaction of RGSZ1 (RGS20) with the neuronal stathmin, SCG10; Nixon AB et al.; Regulators of G protein signaling (RGS proteins) are a diverse family of proteins that act to negatively regulate signaling by heterotrimeric G proteins; however, recent data have implied additional functions for RGS proteins . Previously, we employed the yeast two-hybrid system and identified the microtubule-destabilizing protein, superior cervical ganglia neural-specific 10 protein (SCG10), as a potential effector protein of RGSZ1 . This article describes the expression and biochemical purification of both RGSZ1 and SCG10 and details the development of various in vitro assays to evaluate microtubule polymerization?depolymerization . Both turbidimetric and microscopy-based assays can be employed to study the impact that RGS proteins have on SCG10 function . The application of these in vitro assays may help identify a novel role for RGS proteins in regulating the cytoskeletal network.

Methods Enzymol, 2004, 390, 31 - 52
Analysis of RGSZ1 protein interaction with Galphai subunits; Wang Y et al.; RGSZ1 has been reported to interact with G-protein subunits of the Galphai family and function as a GTPase-accelerating protein on intrinsic Galphai GTPase activity . This article describes several experimental approaches and assays used to investigate the effect of RGSZ1 on Galphai subunits . The formats described here include physical and functional interaction assays by which the association of RGSZ1 with Galphai is explored both in vitro and in vivo . The methods analyzing physical interaction include pull-down and coimmunoprecipitation assays . We also apply yeast two-hybrid techniques to detect RGSZ1 protein interaction with Galpha subunits . Additionally, we developed several functional assay systems to identify the functional relationship between RGSZ1 and Galphai, such as the single turnover GTPase assay, yeast pheromone response assay, mitogen-activated protein kinase assay, and serum response element reporter assay.

Rev Iberoam Micol, 2001 Sep, 18(3), 133 - 6
Sporotrichosis in patient with AIDS: report of a case and review; Rocha MM et al.; Although sporotrichosis is not an AIDS-defining infection, reports of sporotrichosis in individuals infected with HIV are increasing . We report an unusual case of this co-infection in a man with progressive deep cutaneous ulcerations with numerous pleomorphic yeast cells of Sporothrix schenckii . In addition a review of the literature on this subject was carried out and commented upon.

Rev Iberoam Micol, 2001 Sep, 18(3), 118 - 22
Selection of strains of Lentinula edodes and Lentinula boryana adapted for efficient mycelial growth on wheat straw; Mata G et al.; Mycelial growth rates are presented for 11 strains of Lentinula edodes and six strains of Lentinula boryana cultivated on solid media: derived from malt extract (MEA); malt yeast extract (YMEA); and, YMEA plus soluble lignin derivatives (YMEA+WSLD) . The results were compared with data for mycelial growth rates, of the same strains cultivated on substrates derived from wheat straw treated at different temperatures (50, 65, 75 and autoclaving at 121 degrees C) . In general, the addition of WSLD significantly reduced mycelial growth rates in both species . The greatest mycelial growth rate was obtained on sterilized straw at 121 degrees C for the majority of strains . However, this growth was not significantly different from that obtained at 75 degrees C . L . edodes showed greater growth rates than L . boryana . The feasibility of using estimates of mycelial growth rate on YMEA and YMEA+WSLD are discussed as possible indicators of a strain's potential for mycelial growth on substrates derived from wheat straw.

J Trace Elem Med Biol, 2004, 18(1), 69 - 74
A report of high-dose selenium supplementation: response and toxicities; Reid ME et al.; Concerns about the toxicity of selenium has limited the doses used in chemoprevention . Based on previous studies, intakes of 400 microg/day and plasma selenium of 1000 ng/ml (Dietary Reference Intakes, Academy Press, New York, 2000, p . 384) were established as the no observed adverse effect level (NOAEL) . This investigation summarizes the plasma response and toxicity reports from 24 men with biopsy-proven prostate cancer who were randomized to either 1600 or 3200 microg/day of selenized yeast as part of a controlled clinical trial testing selenium as a chemopreventive agent for prostate cancer progression . Subjects were on these doses for averages of almost 12 months . Plasma selenium levels were monitored throughout the course of follow-up . Symptoms of selenium toxicity were assessed by patient interview with specific questions regarding breath, hair and nail changes . Several liver and kidney function tests and hematology were measured at 6-month intervals . 8 subjects were randomized to the 1600 microg/day and 16 to the 3200 microg/day group . The mean plasma selenium levels achieved with supplementation were 492.2 ng/ml (SD = 188.3) and 639.7 ng/ml (SD = 490.7) for the 1600 and 3200 microg/ day doses, respectively . The 3200 microg/day group reported more selenium-related side effects . Blood chemistry and hematology results were all within normal limits for both treatment groups . More subjects on 3200 microg/day reported symptoms of selenium toxicity; however, these reports did not correspond to peaks in plasma selenium levels . We observed no obvious selenium-related serious toxicities . As selenium is used in more chemoprevention and therapeutic settings, additional information on selenium species, sequestration of selenium in specific organs, excretion, and toxicities is needed.

Mycopathologia, 2004 Jul, 158(1), 131 - 5
Synergistic mixtures of fungitoxic monochloro and dichloro-8-quinolinols against five fungi; Gershon H et al.; Fourteen mono- and dichloro-8-quinolinols were tested against five fungi (Aspergillus niger, A . oryzae, Myrothecium verrucaria, Trichoderma viride, and Mucor circinelloides) and compared with the fungitoxicity of 8-quinolinol in Yeast Nitrogen Base containing 1% D-glucose and 0.088% L-asparagine . All of the compounds were more fungitoxic than 8-quinolinol except for the surprising activity of 8-quinolinol against A . oryzae . Mixtures of the MICs of monochloro- and dichloro-8-quinolinols in which the halogens were in different positions of the quinoline ring showed synergism . Comparable mixtures in which one position of each compound was occupied by the same halogen showed additive activity . In a different study we showed that 3,5,6-, 3,5,7-, 4,5,7-, and 5,6,7-trichloro-8-quinolinols were not toxic to M . circinelloides, whereas the combinations of the correspondingly substituted mono- and dichloro-8-quinolinols as well as 3,6-dichloro- and 5,7-dichloro-8-quinolinols were inhibitory . This indicated that a steric factor can be involved in affecting fungitoxicity.

Circ Res, 2004 Nov 12, 95(10), 971 - 80 Epub 2004 Oct 14.
Silent information regulator 2alpha, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes; Alcendor RR et al.; Yeast silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide-dependent histone deacetylase (HDAC) and founding member of the HDAC class III family, functions in a wide array of cellular processes, including gene silencing, longevity, and DNA damage repair . We examined whether or not the mammalian ortholog Sir2 affects growth and death of cardiac myocytes . Cardiac myocytes express Sir2alpha predominantly in the nucleus . Neonatal rat cardiac myocytes were treated with 20 mmol/L nicotinamide (NAM), a Sir2 inhibitor, or 50 nmol/L Trichostatin A (TSA), a class I and II HDAC inhibitor . NAM induced a significant increase in nuclear fragmentation (2.2-fold) and cleaved caspase-3, as did sirtinol, a specific Sir2 inhibitor, and expression of dominant-negative Sir2alpha . TSA also modestly increased cell death (1.5-fold) but without accompanying caspase-3 activation . Although TSA induced a 1.5-fold increase in cardiac myocyte size and protein content, NAM reduced both . In addition, NAM caused acetylation and increases in the transcriptional activity of p53, whereas TSA did not . NAM-induced cardiac myocyte apoptosis was inhibited in the presence of dominant-negative p53, suggesting that Sir2alpha inhibition causes apoptosis through p53 . Overexpression of Sir2alpha protected cardiac myocytes from apoptosis in response to serum starvation and significantly increased the size of cardiac myocytes . Furthermore, Sir2 expression was increased significantly in hearts from dogs with heart failure induced by rapid pacing superimposed on stable, severe hypertrophy . These results suggest that endogenous Sir2alpha plays an essential role in mediating cell survival, whereas Sir2alpha overexpression protects myocytes from apoptosis and causes modest hypertrophy . In contrast, inhibition of endogenous class I and II HDACs primarily causes cardiac myocyte hypertrophy and also induces modest cell death . An increase in Sir2 expression during heart failure suggests that Sir2 may play a cardioprotective role in pathologic hearts in vivo.

Nucleic Acids Res, 2004 Oct 14, 32(18), 5570 - 81 Print 2004.
DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51; Doe CL et al.; In budding yeast most Rad51-dependent and -independent recombination depends on Rad52 . In contrast, its homologue in fission yeast, Rad22, was assumed to play a less critical role possibly due to functional redundancy with another Rad52-like protein Rti1 . We show here that this is not the case . Rad22 like Rad52 plays a central role in recombination being required for both Rhp51-dependent and -independent events . Having established this we proceed to investigate the involvement of the Mus81-Eme1 endonuclease in these pathways . Mus81 plays a relatively minor role in the Rhp51-dependent repair of DNA damage induced by ultraviolet light . In contrast Mus81 has a key role in the Rad22-dependent (Rhp51-independent) repair of damage induced by camptothecin, hydroxyurea and methyl-methanesulfonate . Furthermore, spontaneous intrachromosomal recombination that gives rise to deletion recombinants is impaired in a mus81 mutant . From these data we propose that a Rad22-Mus81-dependent (Rhp51-independent) pathway is an important mechanism for the repair of DNA damage in fission yeast . Consistent with this we show that in vitro Rad22 can promote strand invasion to form a D-loop that can be cleaved by Mus81.

Plant Cell, 2004 Nov, 16(11), 2954 - 66 Epub 2004 Oct 14.
The plant-specific kinase CDKF;1 is involved in activating phosphorylation of cyclin-dependent kinase-activating kinases in Arabidopsis; Shimotohno A et al.; Cyclin-dependent kinases (CDKs) play essential roles in coordinate control of cell cycle progression . Activation of CDKs requires interaction with specific cyclin partners and phosphorylation of their T-loops by CDK-activating kinases (CAKs) . The Arabidopsis thaliana genome encodes four potential CAKs . CAK2At (CDKD;3) and CAK4At (CDKD;2) are closely related to the vertebrate CAK, CDK7/p40MO15; they interact with cyclin H and phosphorylate CDKs, as well as the C-terminal domain (CTD) of the largest subunit of RNA polymerase II . CAK1At (CDKF;1) shows cyclin H-independent CDK-kinase activity and can activate a heterologous CAK, Mcs6, in fission yeast . In Arabidopsis, CAK1At is a subunit of a protein complex of 130 kD, which phosphorylates the T-loop of CAK2At and CAK4At and activates the CTD-kinase activity of CAK4At in vitro and in root protoplasts . These results suggest that CAK1At is a novel CAK-activating kinase that modulates the activity of CAK2At and CAK4At, thereby controlling CDK activities and basal transcription in Arabidopsis.

Mol Cell Biol, 2004 Nov, 24(21), 9305 - 16
Genetic steps of mammalian homologous repair with distinct mutagenic consequences; Stark JM et al.; Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements . We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR) . For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted . We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased . In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair . Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR . Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining . Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1 . Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA . Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption . We also examined the role of RAD52 in homologous repair . In contrast to yeast, Rad52(-)(/)(-) mouse cells had no detectable HDR defect, although SSA was decreased . These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

J Biol Chem, 2004 Dec 31, 279(53), 55895 - 904 Epub 2004 Oct 12.
Biochemical and cell biological analyses of a mammalian septin complex, Sept7/9b/11; Nagata K et al.; Septins are members of a conserved family of cytoskeletal GTPases present in organisms as diverse as yeast and mammals . Unlike lower eukaryotic cells, the physiological significance of mammalian septin complexes is largely unknown . Using specific antibodies, we found at least five septins, Sept2, Sept7, Sept8, Sept9b, and Sept11, in septin complexes affinity-purified with anti-Sept7 antibody-conjugated column from rat embryonic fibroblast REF52 cells . Immunofluorescence studies revealed co-localization of Sept7, Sept9b, and Sept11 along stress fibers in REF52 cells . Biochemical and immunoprecipitation analyses revealed that the three septins directly bind with each other through their N- or C-terminal divergent regions . These septins per se formed distinct and characteristic filament structures when transiently expressed in COS7 cells . When two of the three septins were co-expressed in COS7 cells, combination-dependent filament elongation, bundling, or disruption was observed . Taken together, our results suggest that septin filament structures may be affected by interactions with other septins included in the complex.

J Biol Chem, 2004 Dec 17, 279(51), 53717 - 24 Epub 2004 Oct 12.
The phosphoinositol 3,4-bisphosphate-binding protein TAPP1 interacts with syntrophins and regulates actin cytoskeletal organization; Hogan A et al.; Syntrophins are scaffold proteins of the dystrophin glycoprotein complex (DGC), which target ion channels, receptors, and signaling proteins to specialized subcellular domains . A yeast two-hybrid screen of a human brain cDNA library with the PSD-95, Discs-large, ZO-1 (PDZ) domain of gamma1-syntrophin yielded overlapping clones encoding the C terminus of TAPP1, a pleckstrin homology (PH) domain-containing adapter protein that interacts specifically with phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) . In biochemical assays, the C terminus of TAPP1 bound specifically to the PDZ domains of gamma1-, alpha1-, and beta2-syntrophin and was required for syntrophin binding and for the correct subcellular localization of TAPP1 . TAPP1 is recruited to the plasma membrane of cells stimulated with platelet-derived growth factor (PDGF), a motogen that produces PI(3,4)P(2) . Cell migration in response to PDGF stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to plasma membrane specializations including peripheral and dorsal circular ruffles . Both TAPP1 and syntrophins were localized to PDGF-induced circular membrane ruffles in NIH-3T3 cells . Ectopic expression of TAPP1 potently blocked PDGF-induced formation of dorsal circular ruffles, but did not affect peripheral ruffling . Interestingly, coexpression of alpha1- or gamma1-syntrophin with TAPP1 prevented the blockade of circular ruffling . In addition to syntrophins, several other proteins of the DGC were enriched in circular ruffles . Collectively, our results suggest syntrophins regulate the localization of TAPP1, which may be important for remodeling the actin cytoskeleton in response to growth factor stimulation.

J Biol Chem, 2004 Dec 24, 279(52), 54629 - 36 Epub 2004 Oct 14.
ARPC1/Arc40 mediates the interaction of the actin-related protein 2 and 3 complex with Wiskott-Aldrich syndrome protein family activators; Pan F et al.; The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex . Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear . In this study we characterized the function of the 40-kDa subunit, ARPC1/Arc40, of the yeast Arp2/3 complex . We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies . Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K(d) of 0.45 mum, close to that of the full complex with VCA (0.30 microm), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA . Using a newly constructed Delta arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA . The Delta arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.

J Biol Chem, 2004 Dec 24, 279(52), 54599 - 609 Epub 2004 Oct 13.
Heterogeneous nuclear ribonucleoprotein K enhances insulin-induced expression of mitochondrial UCP2 protein; Ostrowski J et al.; The uncoupling protein 2, UCP2, is a member of a family of inner mitochondrial membrane ion carriers involved in a host of metabolic processes . UCP2 protein is encoded by nuclear genome, but the protein is found exclusively in the mitochondria . The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein involved in many processes that compose gene expression, including mRNA processing and translation . The yeast three-hybrid screen revealed K protein bound to ucp2 mRNA through sites located in the 3'-untranslated region of the transcript . ucp2 mRNA-K protein complexes were associated with polysome-coated mitochondria . Expression of exogenous K protein augmented the insulin-induced mitochondrial level of UCP2 protein that was not accompanied by a corresponding increase in ucp2 mRNA . These results suggest the insulin stimulates translation of ucp2 mRNA in a process that involves K protein.

J Biol Chem, 2004 Dec 24, 279(52), 54590 - 8 Epub 2004 Oct 13.
Human Zwint-1 specifies localization of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling; Wang H et al.; Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome . Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10) in HeLa cells . Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain . Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZW10 kinetochore localization . In addition, depletion of Zwint-1 affects no mitotic arrest but causes aberrant premature chromosome segregation . These Zwint-1-suppressed cells display chromosome bridge phenotype with sister chromatids inter-connected . Moreover, Zwint-1 is required for stable association of CENP-F and dynamitin but not BUB1 with the kinetochore . Finally, our studies show that Zwint-1 is a new component of the mitotic check-point, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding interphase cells with multinuclei . As ZW10 and Zwint-1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.

J Biol Chem, 2004 Dec 24, 279(52), 54620 - 8 Epub 2004 Oct 13.
Recruitment of thyroid hormone receptor/retinoblastoma-interacting protein 230 by the aryl hydrocarbon receptor nuclear translocator is required for the transcriptional response to both dioxin and hypoxia; Beischlag TV et al.; The aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor (ARNT/HIF-1 beta) mediates an organism's response to various environmental cues, including those to chemical carcinogens, such as 2,3,7,8-tetrachlorodibenzo-rho-dioxin (TCDD or dioxin), via its formation of a functional transcription factor with the ligand activated aryl hydrocarbon receptor (AHR) . Similarly, tissue responses to hypoxia are largely mediated through the HIF-1 heterodimeric transcription factor, comprising hypoxia-inducible factor-1 alpha (HIF-1 alpha) and ARNT . The latter response is essential for a metabolic switch from oxidative phosphorylation to glycolytic anaerobic metabolism as well as for angiogenesis and has been implicated as necessary for growth in many solid tumors . In this report, we demonstrate that the thyroid hormone receptor/retinoblastoma-interacting protein 230 (TRIP230) interacts directly with ARNT and is essential for both hypoxic and TCDD-mediated transcriptional responses . We initially identified TRIP230 as an ARNT-interacting protein in a yeast two-hybrid assay screen . This interaction was confirmed in mammalian cell systems using co-immunoprecipitation and in mammalian two-hybrid assays . Furthermore, TRIP230 could be recorded at sites of activated transcription of either TCDD- or hypoxia-inducible genes in a stimulus-dependent fashion by chromatin immunoprecipitation analysis . Finally, using single-cell microinjection and RNA interference assays, we demonstrate that TRIP230 is indispensable for TCDD- and hypoxia-dependent gene transcription.

Annu Rev Genomics Hum Genet, 2004, 5, 177 - 87
Molecular networks in model systems; Galitski T; Model organisms, especially the budding yeast, are leading systems in the transformation of biology into an information science . With the availability of genome sequences and genome-scale data generation technologies, the extraction of biological insight from complex integrated molecular networks has become a major area of research . Here I examine key concepts and review research developments . I propose specific areas of research effort to drive network analysis in directions that will promote modeling with increasing predictive power.

J Gen Virol, 2004 Nov, 85(Pt 11), 3291 - 303
Vpx proteins of SIVmac239 and HIV-2ROD interact with the cytoskeletal protein alpha-actinin 1; Mueller SM et al.; vpx genes of human immunodeficiency virus type 2 (HIV-2) and immunodeficiency viruses from macaques (SIVmac), sooty mangabeys (SIVsm) and red-capped mangabeys (SIVrcm) encode a 112 aa protein that is packed into virion particles via interaction with the p6 domain of p55(gag) . Vpx localizes to the nucleus when expressed in the absence of other viral proteins . Moreover, Vpx is necessary for efficient nuclear import of the pre-integration complex (PIC) and critical for virus replication in quiescent cells, such as terminally differentiated macrophages and memory T cells . Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs) . Therefore, it is likely that Vpx-dependent import of the PIC is mediated by interaction of Vpx with cellular proteins that do not belong to the classical import pathways . By using a yeast two-hybrid screen, alpha-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx . Interestingly, deletion of the proline-rich C-terminal domain (aa 101-112) of Vpx, which is important for nuclear localization, resulted in loss of interaction with alpha-actinin 1 . These findings suggest that the interaction with alpha-actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.

Rev Iberoam Micol, 2001 Mar, 18(1), 42 - 4
Specific antibody response in a patient with Candida dubliniensis fungemia; Salesa R et al.; We report a case of fungemia caused by Candida dubliniensis in a non-HIV infected patient . Multiple cultures of blood performed over a period of 13 days were positive for this recently described yeast species . The C . dubliniensis isolates recovered were susceptible to fluconazole in vitro and the patient responded to intravenous therapy with this antifungal agent . It was possible to differentiate the fungemia caused by C . dubliniensis in this patient from that caused by C . albicans in other patients on the basis of the analysis of the antibody response since the C . dubliniensis-infected patient exhibited a characteristic and specific antibody response against a cell wall component of 160-170 kDa.

Am J Chin Med, 2004, 32(4), 531 - 9
Evaluation of analgesic, antipyretic activity and toxicity study of Bryonia laciniosa in mice and rats; Sivakumar T et al.; Analgesic, antipyretic activity and toxicity study of the leaves of Bryonia laciniosa Linn . (Family: Cucurbitaceae) was evaluated in the standard animal models . The methanol extract of Bryonia laciniosa (MEBL) was evaluated by hot plate and acetic acid-induced writhing methods to assess analgesic activity . The antipyretic activity of the extract was also evaluated by normal body temperature and yeast-induced hyperpyrexia . The extract showed significant analgesic and antipyretic activity . The MEBL was further evaluated for toxicity at the doses of 125 and 250 mg/kg administered orally for 14 days in rats . At the end of experiments, the blood, liver function and kidney metabolism were observed . The hematological profile and different biochemical parameters such as SGOT, SGPT and ALP were estimated . The present study revealed that MEBL exhibited significant analgesic and antipyretic activity in the tested experimental animal models . The toxicity study indicates that the extract is not toxic at the tested doses.

Yi Chuan Xue Bao, 2004 Aug, 31(8), 822 - 9
{Identification and analysis of a group of highly conserved trs-like genes in rice}; Hu X et al.; There are at least ten transcriptional trs-like genes in rice that have been confirmed by RT - PCR and sequencing, based on the annotation results of rice genome and homologous search . These ten genes correspond to six of the ten known subunits of TRAPP complex in yeast . Four pairs of them are duplicates while the other two are unique according to the known rice genomic sequences . All of the ten genes are constitutively expressed in rice tissues and share phylogenetic homology to some extent with other eukaryotic trs-like genes in their gene structures and protein sequences.

Arch Dermatol Res . 2004 Oct 5; {Epub ahead of print}
Determination of the antioxidant capacity of an antioxidant combination using the fluoroscan assay in vitro and visualization of its effects using histological methods; El Hindi T et al.; The effects of a well-defined combination of antioxidants on oxidative stress were investigated in vitro using classical techniques together and its protective effects against UV damage were investigated using a newly developed skin model . After determining the cytotoxic potential of the combination, its quenching effect on the oxidative stress induced by hydrogen peroxide was quantified by a nonfluorescent (C-H2DCF-DA/AM)/fluorescent (C-DCF) dye system using the fluoroscan assay . Two different skin models consisting of normal human skin fibroblasts and the keratinocyte cell line HaCaT were developed and subsequently used to visualize the protective effects of the combination against UVA damage . No evidence of any cytotoxic potential of the combination could be seen . Supplementation of human skin fibroblasts demonstrated a clear, dose-dependent enhancement of the antioxidative capacity of these cells . Histological findings confirmed the beneficial effects of the antioxidants present in the combination in the skin models used . Supplementation induced morphological changes leading to a thicker epidermal layer providing evidence of the positive effects of the treatment on the viability of the keratinocytes after UVA irradiation . This in vitro study provided convincing evidence of the combined antioxidative action of alpha-tocopherol, beta-carotene, tomato extract, grape seed extract, ascorbic acid and selenium yeast, and indicated a potential beneficial action of the combination against oxidative stress caused by external oxidative stress factors such as UV irradiation.

Oncogene, 2004 Dec 9, 23(57), 9289 - 94
Isolation and characterization of a novel gene CLUAP1 whose expression is frequently upregulated in colon cancer; Takahashi M et al.; To disclose mechanisms of colorectal carcinogenesis and identify novel diagnostic markers and drug targets for treatment of these tumors, we previously analysed the expression profiles of 11 colorectal cancers using a genome-wide cDNA microarray containing 23,040 genes . Among the genes commonly transactivated in the cancers, we identified a novel human gene, which we termed CLUAP1 (clusterin-associated protein 1) . It encodes a nuclear protein of 413 amino acids containing a coiled-coil domain . To investigate its function, we searched for CLUAP1-interacting proteins using yeast two-hybrid system and identified nuclear Clusterin . Expression of CLUAP1 was gradually increased in the late S to G2/M phases of cell cycle and it returned to the basal level in the G0/G1 phases . Suppression of this gene by short interfering RNAs resulted in growth retardation in the transfected cells . These data provide better understanding of colorectal carcinogenesis, and inactivation of CLUAP1 may conceivably serve in the future as a novel therapeutic intervention for treatment of colon cancer.

Oncogene, 2004 Nov 18, 23(54), 8711 - 9
Rap1 mutants with increased affinity for the guanine-nucleotide exchange factor C3G; Shi S et al.; The mutant of Ras protein with serine to asparagine mutation at residue 17 (Ras-17N) is known to interfere with the signaling function of the wild-type Ras protein by sequestering its guanine-nucleotide exchange factors (GEFs) . The similar mutant of another Ras family protein Rap1 (Rap1-17N) fails to effectively interfere with the interaction between the wild-type Rap1 and one of its GEFs, C3G, in vitro . In the present study, we have attempted to isolate Rap1 mutants with increased affinity for C3G using random mutagenesis and yeast two-hybrid screening . Based on the pattern of mutations found among these mutants, we could design a potent C3G-binder, named Rap1-AGE, harboring mutations in three sites (17A, 29G, and 117E) . The association of Rap1-AGE with C3G in the cells was confirmed by co-immunoprecipitation experiments . The ability of Rap1-AGE to inhibit C3G-mediated Rap1-activation and cell spreading was also demonstrated . On the other hand, Rap1 activation mediated by two other GEFs, Epac and smgGDS, was not inhibited by Rap1-AGE . These results suggest that Rap1-AGE acts as a dominant interfering factor against C3G and serves as a useful tool in analyzing the roles of C3G-Rap1 signaling pathway in various biological processes.

J Virol, 2004 Nov, 78(21), 11890 - 903
PCNA interacts with Indian mung bean yellow mosaic virus rep and downregulates Rep activity; Bagewadi B et al.; Proliferative cell nuclear antigen (PCNA), a conserved plant protein as well as an important replication factor, is induced in response to geminivirus infection in the resting cells of the phloem tissues . The biochemical role of PCNA in rolling circle replication (RCR) of geminivirus DNA has not been explored in detail . The initiation of RCR of the bipartite genome of a geminivirus, Indian mung bean yellow mosaic virus (IMYMV), is mainly controlled by viral protein Rep (or AL1 or AC1) . The role of host PCNA in RCR of IMYMV was revealed by studying the physical and functional interactions between recombinant PCNA and recombinant IMYMV Rep . Pea nuclear PCNA as well as recombinant pea PCNA showed binding to recombinant Rep in experiments involving both affinity chromatography and yeast two-hybrid approaches . The contacting amino acid residues of PCNA seemed to be present throughout a wide region of the trimeric protein, while those of Rep appeared to be localized only in the middle part of the protein . The site-specific nicking-closing activity and the ATPase function of IMYMV Rep were impaired by PCNA . These observations lead to interesting speculations about the control of viral RCR and dynamic profiles of protein-protein interactions at the RCR origin of the geminiviruses.

J Virol, 2004 Nov, 78(21), 11551 - 62
Intracellular localization and protein interactions of the gene 1 protein p28 during mouse hepatitis virus replication; Brockway SM et al.; Coronaviruses encode the largest replicase polyprotein of any known positive-strand RNA virus . Replicase protein precursors and mature products are thought to mediate the formation and function of viral replication complexes on the surfaces of intracellular double-membrane vesicles . However, the functions of only a few of these proteins are known . For the coronavirus mouse hepatitis virus (MHV), the first proteolytic processing event of the replicase polyprotein liberates an amino-terminal 28-kDa product (p28) . While previous biochemical studies have suggested that p28 is associated with viral replication complexes, the intracellular localization and interactions of p28 with other proteins during the course of MHV replication have not been defined . We used immunofluorescence confocal microscopy to show that p28 localizes to viral replication complexes in the cytoplasm during early times postinfection . However, at late times postinfection, p28 localizes to sites of M accumulation distinct from the replication complex . Furthermore, by yeast two-hybrid and coimmunoprecipitation analyses, we demonstrate that p28 specifically binds to p10 and p15, two coronavirus replicase proteins of unknown function . Deletion mutagenesis experiments determined that the carboxy terminus of p28 is not required for its interactions with p10 and p15 . These results suggest that p28 may play a part at the replication complex by interacting with p10 and p15 . Moreover, our findings highlight a potential role for p28 at virion assembly sites.

J Cell Biol, 2004 Oct 11, 167(1), 161 - 70
Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration; Chen C et al.; The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation . alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain . alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent . Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain . Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains . The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration . We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

J Cell Biol, 2004 Oct 11, 167(1), 27 - 33
Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response; Lu PD et al.; Stress-induced eukaryotic translation initiation factor 2 (eIF2) alpha phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program . Previous studies implicated the 5' end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation . Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs . In unstressed cells, low levels of eIF2alpha phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR . In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2 . These features are common to regulated translation of GCN4 in yeast . The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details.

J Cell Biol, 2004 Oct 11, 167(1), 23 - 5
Membrane biogenesis and the unfolded protein response; Ron D et al.; In addition to serving as the entry point for newly translated polypeptides making their way through the secretory pathway, the endoplasmic reticulum (ER) also synthesizes many lipid components of the entire endomembrane system . A report published in this issue implicates a signaling pathway known to respond to ER unfolded protein load in the control of phospholipid biosynthesis by the organelle (Sriburi et al., 2004) . The reasonable notion that demand for ER membrane is integrated with protein processing capacity was initially suggested by genetic analysis of yeast . The new data lend direct support for this idea and imply interesting mechanistic possibilities for how this coupling develops.

BMC Genomics . 2004 Oct 12;5(1):79.
Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote; Ward P et al.; BACKGROUND: Malaria, caused by the parasitic protist Plasmodium falciparum, represents a major public health problem in the developing world . The P . falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets . Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets . We report an exhaustive analysis of the P . falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism . RESULTS: Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups . Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite . A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif . The FIKK family seems restricted to Apicomplexa, with 20 members in P . falciparum and just one member in some other Apicomplexan species . CONCLUSION: The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells.

Biochem J, 2004 Dec 1, 384(Pt 2), 233 - 7
The human SNARE protein Ykt6 mediates its own palmitoylation at C-terminal cysteine residues; Veit M; The yeast SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein Ykt6 was shown to mediate palmitoylation of the fusion factor Vac8 in a reaction essential for the fusion of vacuoles . Here I present evidence that hYkt6 (human Ykt6) has self-palmitoylating activity . Incubation of recombinant hYkt6 with {3H}Pal-CoA ({3H}palmitoyl-CoA) leads to covalent attachment of palmitate to C-terminal cysteine residues . The N-terminal domain of human Ykt6 contains a Pal-CoA binding site and is required for the reaction.

Yi Chuan Xue Bao, 2004 May, 31(5), 485 - 8
{Cloning and primary analysis of spt1 as a ncRNA candidate gene}; Sun Q et al.; In a process of screening mouse embryonic cDNA library with suc2 signal sequence trap, a strongly positive clone named spt1 was obtained repeatedly . Analysis of spt1 shows that the inserted sequence is 697 bp containing 37 start codons (ATG) and 80 stop codons (TGA, TAG, TAA), and there is no potential open reading fame in it . Similarity search by BLAST indicates that the sequence locates in the long arm of 17th chromosome, and no known genes show significant similarity with it . Northern blot and RT-PCR give positive signal specifically in ovary tissue, the full length of spt1 is about 4 . 5 approximately 5.0 kb . Yeast transformation and sequence truncation experiment suggested that spt1 could mediate the secretion of invertase . Hence, spt1 may be part of a novel ncRNA gene which is involved in protein secretion.

Cell Cycle, 2004 Oct, 3(10), 1243 - 5 Epub 2004 Oct 03.
Cyclin proteolysis and CDK inhibitors: two redundant pathways to maintain genome stability in mammalian cells; Chibazakura T; Cyclin-dependent kinases (CDKs) are regulated by cyclin proteolysis and CDK inhibitors (CKIs) during mitotic exit and G(1) phase in yeast and Drosophila, and disruption of both regulatory pathways leads to genomic instability . Our study using mouse cell lines that constitutively express a stabilized mutant of cyclin A revealed that three CKIs, p21, p27, and Rb-related p107, are responsible for cyclin proteolysis-independent inactivation of CDK during mitotic exit and G(1) . Enforced expression of cyclin A in the cells lacking all three CKIs induced rapid tetraploidization . Thus, the redundant pathways consisting of cyclin proteolysis and CKIs control CDK activity during mitotic exit and contribute to maintenance of genome stability in mammalian cells.

Cancer Biol Ther . 2004 Dec 14;3(12) {Epub ahead of print}
Genomic Instability is Associated with Lack of Telomerase Activation in Ovarian Cancer; Landen CN et al.; Introduction: Malignant cells are capable of an unlimited number of cell divisions, either through production of telomerase, or through the alternate lengthening of telomere (ALT) mechanism . Yeast cells with genomic instability have been shown to survive in the absence of telomerase by increased recombination events . We hypothesized that ovarian cancers with high microsatellite instability (MSI-H) are more likely to lack telomerase activation . Methods: We examined 104 invasive ovarian cancers for MSI with six microsatellite markers (BAT25, BAT26, D5S346, D2S123, D17S250 and NME1) . Telomerase activity was determined with ELISA, and its subunits human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR) by RT-PCR . Statistical analysis was performed with Chi-square and p<0.05 was considered significant . Results: Telomerase activity was detected in 79 samples (76%) . The hTERT subunit was detected in 85% of samples, and hTR was found in all ovarian cancers . Presence of hTERT was positively associated with telomerase activity (p = 0.001) . High MSI (MSI-H), defined as two or more positive markers, was detected in 15% of ovarian cancers; low MSI (MSI-L), defined as having only one positive marker, was found in 13%; the remaining 72% were microsatellite stable (MSI-S) . Telomerase activity was detected in 83% of MSI-S and 79% of MSI-L tumors, but only 40% of MSI-H tumors (p = 0.002) . Interestingly, hTERT was similar in all three groups (range 73-84%, p = 0.59), demonstrating that the presence of hTERT transcript was not the only determinant of telomerase activity in MSI-H tumors . Conclusions: Ovarian cancers with high MSI are more likely to propagate without the need to produce telomerase.

FEMS Microbiol Lett, 2004 Oct 15, 239(2), 277 - 83
Heterologous complementation of the exopolysaccharide synthesis and carbon utilization phenotypes of Sinorhizobium meliloti Rm1021 polyhydroxyalkanoate synthesis mutants; Aneja P et al.; A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021 . Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain . This phenotype is attributed to reduction in succinoglycan synthesis . We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene . Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B . japonicum genome . The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.

J Mol Biol, 2004 Oct 29, 343(4), 1147 - 55
Molecular basis of distinct interactions between Dok1 PTB domain and tyrosine-phosphorylated EGF receptor; Zhang Y et al.; Phosphotyrosine binding (PTB) domains of the adaptor proteins Doks (downstream of tyrosine kinases) play an important role in regulating signal transduction of cell-surface receptors in cell growth, proliferation and differentiation; however, ligand specificity of the Dok PTB domains has until now remained elusive . In this study, we have investigated the molecular basis of specific association between the Dok1 PTB domain and the tyrosine-phosphorylated EGFR . Using yeast two-hybrid and biochemical binding assays, we show that only the PTB domain from Dok1 but not Dok4 or Dok5 can selectively bind to two known tyrosine phosphorylation sites at Y1086 and Y1148 in EGFR . Our structure-based mutational analyses define the molecular determinants for the two distinct Dok1 PTB domain/EGFR interactions and provide the structural understanding of the specific interactions between EGFR and PTB domains in the divergent Dok homologues.

J Bone Miner Res, 2004 Nov, 19(11), 1892 - 904 Epub 2004 Jul 07.
Growth hormone attenuates the transcriptional activity of Runx2 by facilitating its physical association with Stat3beta; Ziros PG et al.; We document that GH controls osteoblast function by modulating the biological activity of the osteospecific transcription factor Runx2 . Evidence is provided for a physical interaction between Runx2 and Stat3beta, which is enhanced by GH and downregulates the transcriptional properties of this key osteogenic regulator . INTRODUCTION: Growth hormone (GH) signals to bone either through insulin-like growth factor-1 or directly by influencing the function of osteoblasts, the bone-forming cells . This study aimed at exploring the molecular events that underlie the direct biological action of GH on osteoblastic cells, and specifically, the effects that it might exert on the function of the bone-specific transcriptional regulator Runx2 . MATERIALS AND METHODS: The GH-responsive human osteoblastic cell line Saos-2 was used as our experimental system . Western blot analyses were used to monitor the presence of several parameters known to be affected by GH in these cells (i.e., downregulation of GH receptor, induction of STATs, and extracellular signal-regulated kinase {ERK} mitogen-activated protein kinase {MAPK} pathways) . Electrophoretic mobility shift assays were used to assess Runx2 and Stat3 binding activity on an osteoblast-specific element (OSE2) after GH treatment . A combination of yeast two-hybrid and co-immunoprecipitation assays were performed to test for the existence of a physical Runx2.Stat3beta association . Finally, co-transfection experiments were used to investigate the interplay of the two transcription factors on the activity of a p6OSE2-Luc promoter after GH stimulation . RESULTS: We show that GH signaling through Stat3/ERK MAPK potentiates the DNA binding activity of Runx2 but, at the same time, restrains its transcriptional potential . Moreover, a novel physical interaction of Runx2 with transcription factor Stat3beta, which is enhanced by GH stimulation, was documented both in vitro and in vivo . Importantly, this interaction impairs the transcriptional activity of Runx2 without affecting its DNA binding capacity . CONCLUSION: Our data provide the first evidence that GH modulates the transcriptional function of Runx2 in osteoblastic cells by promoting its inhibitory interaction with Stat3beta . Shedding light on such mechanisms will contribute to a better understanding of GH effects on skeletal homeostasis that may impact on decisions at the clinical level, especially in diseases affecting bone quantity and quality (e.g., osteoporosis).

BMC Bioinformatics . 2004 Oct 11;5(1):148.
Rank Difference Analysis of Microarrays (RDAM), a novel approach to statistical analysis of microarray expression profiling data; Martin DE et al.; BACKGROUND: A key step in the analysis of microarray expression profiling data is the identification of genes that display statistically significant changes in expression signals between two biological conditions . RESULTS: We describe a new method, Rank Difference Analysis of Microarrays (RDAM), which estimates the total number of truly varying genes and assigns a p-value to each signal variation . Information on a group of differentially expressed genes includes the sensitivity and the false discovery rate . We demonstrate the feasibility and efficiency of our approach by applying it to a large synthetic expression data set and to a biological data set obtained by comparing vegetatively-growing wild type and tor2-mutant yeast strains . In both cases we observed a significant improvement of the power of analysis when our method is compared to another popular nonparametric method . CONCLUSIONS: This study provided a valuable new statistical method to analyze microarray data . We conclude that the good quality of the results obtained by RDAM is mainly due to the quasi-perfect equalization of variation distribution, which is related to the standardization procedure used and to the measurement of variation by rank difference.

Nat Genet, 2004 Nov, 36(11), 1174 - 80 Epub 2004 Oct 10.
RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing; Noma K et al.; RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing . In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing . Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci . At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer . Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9 . This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.

Cell Biochem Biophys, 2004, 41(2), 295 - 318
EH Proteins: Multivalent Regulators of Endocytosis (and Other Pathways); Miliaras NB et al.; Endocytosis is a protein and lipid-trafficking pathway that occurs in all eukaryotic cells . It involves the internalization of plasma membrane proteins and lipids into the cell and the subsequent degradation of proteins in the lysosome or the recycling of proteins and lipids back to the plasma membrane . Over the past decade, studies in yeast and mammalian cells have revealed endocytosis to be a very complex molecular process that depends on regulated interactions between a variety of proteins and lipids . The Eps15 homology (EH) domain is a conserved, modular protein-interaction domain found in several endocytosis proteins . EH proteins can function as key regulators of endocytosis through their ability to interact with many of the other proteins involved in this process.

Clin Cancer Res, 2004 Oct 1, 10(19), 6744 - 9
Cisplatin rapidly down-regulates its own influx transporter hCTR1 in cultured human ovarian carcinoma cells; Holzer AK et al.; PURPOSE: Cisplatin (DDP)-resistant cells commonly exhibit reduced drug accumulation . Previous studies have shown that the major copper (Cu) influx transporter CTR1 controls the uptake of DDP in yeast and mammalian cells . The goal of this study was to examine the effect of Cu and DDP on the level and subcellular localization of hCTR1 protein in human ovarian carcinoma cells . EXPERIMENTAL DESIGN: Cultured human ovarian carcinoma A2780 cells were exposed to DDP and Cu, and the effect on hCTR1 was determined using Western blot analysis and confocal digital deconvolution microscopy . RESULTS: Loss of hCTR1 was triggered by DDP exposure in a concentration and time-dependent manner . Exposure to 0.5 micromol/L DDP for 5 minutes reduced hCTR1 levels and exposure to DDP concentrations > or =2 micromol/L caused almost complete disappearance . The loss of hCTR1 was observed within 1 minute of the start of exposure to 2 micromol/L DDP . Treatment of cells with 100 micromol/L Cu for 5 minutes produced a smaller effect . Pretreatment of cells with 2 micromol/L DDP for 5 minutes resulted in a 50% decrease in 64Cu uptake, demonstrating that the DDP-induced loss of hCTR1 detected by Western blot analysis and imaging was functionally significant . CONCLUSIONS: DDP down-regulated the amount of its major influx transporter in cultured human ovarian carcinoma cells in a concentration- and time-dependent manner . The effect was observed at DDP concentrations within the range found in the plasma of patients being treated with DDP, and it occurred very quickly relative to the half-life of the drug.

J Biol Chem, 2004 Dec 24, 279(52), 54770 - 82 Epub 2004 Oct 08.
Human Nischarin/imidazoline receptor antisera-selected protein is targeted to the endosomes by a combined action of a PX domain and a coiled-coil region; Lim KP et al.; Around 50 mammalian and 15 yeast proteins are known to contain the phox (PX) domain, the majority (about 30) of which is classified as sorting nexins (SNXs) . The PX domain, a hallmark of these proteins, is a conserved stretch of about 120 amino acids and is recently shown to mediate phosphoinositide binding . A few PX domain proteins (including some SNXs) have been shown to participate in diverse cellular processes such as protein sorting, signal transduction, and vesicle fusion . In this report, we present our results supporting a role of human IRAS to act as a SNX . The mouse homologue, previously identified as Nischarin, has been shown to interact with the alpha(5) subunit of integrin and inhibit cell migration (Alahari, S . K., Lee J . W., and Juliano R . L . (2000) J . Cell Biol . 51, 1141-1154) . Its human homologue (imidazoline receptor antisera-selected (IRAS)), on the other hand, contains an NH(2)-terminal extension and is a larger protein of 1504 amino acids consisting of an NH(2)-terminal PX domain, 5 putative leucine-rich repeats, a predicted coiled-coil domain, and a long COOH-terminal region . We show that it has the ability to homo-oligomerize via its coiled-coil region . The PX domain of IRAS is essential for association with phosphatidylinositol 3-phosphate-enriched endosomal membranes . However, the PX domain of IRAS alone is insufficient for its localization to endosomes, unless the coiled-coil domain was included or it is artificially dimerized by glutathione S-transferase . Interaction of human IRAS with alpha(5) integrin is not affected by the NH(2)-terminal extension, and overexpression of IRAS could cause a redistribution of surface alpha(5) integrin to intracellular endosomal structures.

J Biol Chem, 2004 Dec 17, 279(51), 53056 - 61 Epub 2004 Oct 07.
The mutation F227I increases the coupling of metal ion transport in DCT1; Nevo Y et al.; Metal ion transport by DCT1, a member of the natural resistance-associated macrophage protein family, is driven by protons . The stoichiometry of the proton to metal ion is variable, and under optimal transport conditions, more than 10 protons are co-transported with a single metal ion . To understand this phenomenon better, we used site-directed mutagenesis of DCT1 and analyzed the mutants by complementation of yeast suppressor of mitochondria import function-null mutants and electrophysiology with Xenopus oocytes . The mutation F227I resulted in an increase of up to 14-fold in the ratio between metal ions to protons transported . This observation suggests that low metal ion to proton transport of DCT1 resulting from a proton slippage is not a necessity of the transport mechanism in which positively charged protons are driving two positive charges of the metal ion in the same direction . It supports the idea that the proton slippage has a physiological advantage, and the proton slip was positively selected during the evolution of DCT1.

Gene, 2004 Oct 13, 340(2), 179 - 87
Identification and phylogenetic analyses of the protein arginine methyltransferase gene family in fish and ascidians; Hung CM et al.; Protein arginine methyltransferases (PRMT) involved in the regulations of signal transduction, protein subcellular localization, and transcription have been mostly studied in mammals and yeast . In this study orthologues of eight human PRMT genes (PRMT1-7 and HRMT1L3) were identified in both puffer fish Fugu rubripes and zebrafish Danio rerio . The fish PRMT genes appear to be conserved with their mammalian orthologues at the levels of amino acid sequences as well as genomic structures . All vertebrate PRMT genes contain 10-16 coding exons except PRMT6 that contains only one coding exon . Western blot analyses of zebrafish tissue extracts confirmed the expression of some PRMT proteins in zebra fish . We further identified six PRMT members (PRMT1, 3-7) in an invertebrate chordate Ciona intestinalis . Genomic structures of the PRMT orthologues are no more conserved in the ascidians, as PRMT3 and PRMT5 contain only one coding exon while PRMT6 contains six exons . PRMT2 and HRMT1L3 that are missing in Ciona appear to be vertebrate-specific . HRMT1L3 is a PRMT1 paralogue with highly conserved sequences and exact exon junctions, whereas the PRMT2 orthologues are very diverged . Different PRMT orthologues are likely to evolve at different rates and the PRMT1 orthologues appear to be most conserved through evolution . Furthermore, phylogenetic analyses using the core regions of various PRMT genes show that PRMT5 with the type II PRMT activity is separated in one branch . All other PRMT genes including PRMT1, 2, 3, 4, 6, 7 and HRMT1L3 clustered in the other branch, probably represent the genes for the type I activity.

Exp Cell Res, 2004 Nov 1, 300(2), 440 - 54
Calcium binding sequences in calmyrin regulates interaction with presenilin-2; Zhu J et al.; Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands . Calmyrin interacts with a number of proteins, including presenilin-2 (PS2) . However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood . By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium . Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations . By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences . Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min) . By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively . Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin . Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2 . Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.

Exp Cell Res, 2004 Nov 1, 300(2), 379 - 87
Macrophage migration inhibitory factor directly interacts with hepatopoietin and regulates the proliferation of hepatoma cell; Li Y et al.; Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in growth factor-dependent cell proliferation, cell cycle, angiogenesis, and tumorigenesis . Several studies have documented MIF expression in the sera following hepatic resection or in the course of liver cancer progression, but there is a paucity of information regarding the effect of MIF on hepatoma cells and relating mechanisms . In this paper, by {3H} thymidine incorporation, we found that exogenously added MIF could promote the proliferation of HepG2 in a dose-dependent manner . Hepatopoietin (HPO), as a liver-specific regeneration augmenter, could be induced by the expression of MIF in hepatoma cells . The activity of HPO promoter was increased, and its levels were enhanced after MIF was overexpressed in hepatoma cells . The similarities between HPO and MIF in structure and action led us to investigate their interaction and the inducing biological significance . Using yeast two-hybrid identification, we found that HPO interacted with MIF in yeast cells, and their binding ability was higher than that between HPO and JAB1 (Jun activation domain binding protein) or MIF and JAB1 in yeast cells . Their interaction was further verified by His pull-down assay in vitro and coimmunoprecipitation experiment in vivo . They were colocalized in the cytoplasm . Both HPO and MIF could bind to JAB1 and modulate the AP-1 pathway . When HPO and MIF were cotransfected into HepG2 cells, the binding activity of MIF to JAB1 was reduced, and the activity of AP-1 was improved . In contrast, MIF overexpressed in HepG2 was unable to interfere with the binding activity of HPO to JAB1, but its potentiation on AP-1 activity was reduced significantly . Taken together, these results indicate that MIF plays an important role in the proliferation of hepatoma cells, and the effect of MIF is in concert with HPO.

Biochem Biophys Res Commun, 2004 Nov 12, 324(2), 946 - 52
Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression; Aikawa S et al.; Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence . We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene . Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2 . Co-transfection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractive stimulation of reporter gene expression . Immunohistochemistry of adult porcine pituitary confirmed the colocalization of the Prop-1 and FSHbeta subunit . This study is the first to report that Prop-1 participates in the regulation of FSHbeta gene . The present finding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD), since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.

Biochem Biophys Res Commun, 2004 Nov 12, 324(2), 640 - 7
Recruitment of C-terminal Src kinase by the leukocyte inhibitory receptor CD85j; Sayos J et al.; The CD85j inhibitory receptor (also termed ILT2 or LIR-1) is a type-I transmembrane protein that belongs to the Ig superfamily and is expressed by different leukocyte lineages . The extracellular region of CD85j binds HLA class I molecules and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs (ITIM) . Upon tyrosine phosphorylation CD85j recruits the SHP-1 tyrosine phosphatase, involved in negative signaling . In order to identify other molecules to which CD85j might interact with in a phosphotyrosine-dependent manner, a cDNA B-cell library was screened in a three-hybrid system in yeast using the CD85j cytoplasmic tail as bait in the presence of the Src-kinase c-fyn420, 531Y-F, 176R-Q mutant . In this system, the C-terminal Src kinase (Csk) was shown to interact with CD85j . Phosphorylation-dependent recruitment of Csk to the CD85j cytoplasmic tail was confirmed in CD85j-transfected mammalian cells by immunoprecipitation and Western blot analysis . Mutational analyses and phospho-peptide mapping suggested that the SH2 domain of Csk may preferentially bind to ITIM Y562 of CD85j; yet, mutation to phenylalanine of Y533, Y614, and Y644 also significantly reduced Csk recruitment by CD85j . Even though CD85j was detected in both anti-SHP1 and CSK immunoprecipitates, these two molecules did not co-precipitate together with CD85j . Our data support the possibility that Csk regulates the function of CD85j.

DNA Repair (Amst), 2004 Dec 2, 3(12), 1549 - 59
EXO1-A multi-tasking eukaryotic nuclease; Tran PT et al.; Exo1 was first isolated as a 5' --> 3' exonuclease activity induced during meiosis in fission yeast and since that time has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity . Involvement in multiple pathways affecting genomic stability makes EXO1 a logical target for mutation during oncogenesis . Here, we review studies in several experimental systems that shed light on the role of Exo1 in these DNA transaction pathways, particularly those that may relate to oncogenesis.

Gene, 2004 Oct 27, 341, 41 - 7
Serine-arginine-rich nuclear protein Luc7l regulates myogenesis in mice; Kimura E et al.; Using a gene trap technique, we identified a murine homologue of the yeast LUC7-like gene (Luc7l), which is a serine-arginine-rich protein (SR protein) that localizes in the nucleus through its arginine-serine-rich domain (RS domain) at the C-terminus and shows a speckled distribution pattern . Although its transcripts are widely expressed in embryos and adults, they are rarely detected in adult skeletal muscle, and Luc7l expression was found to be negatively regulated during the course of development of limb skeletal muscle, as well as during in vitro differentiation of the myoblast cell lines Sol8 and C2C12 . We also demonstrated that forced expression of Luc7l protein inhibited myogenesis in vitro . Based on our results, Luc7l is thought to play an important role in the regulation of muscle differentiation.

FEBS Lett, 2004 Oct 8, 576(1-2), 231 - 6
A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel; Li Q et al.; The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells . This purification procedure was also applied to study interactions between soluble proteins in mammalian cells . In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease . We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein . Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system . Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells .

Annu Rev Cell Dev Biol, 2004, 20, 395 - 425
Retrovirus budding; Morita E et al.; Human immunodeficiency virus (HIV) and other retroviruses acquire their envelopes and spread infection by budding through the limiting membranes of producer cells . To facilitate budding, retroviruses usurp a cellular pathway that is normally used to create vesicles that bud into late endosomal compartments called multivesicular bodies (MVB) . Research on yeast and human MVB biogenesis has led to the identification of 25 human proteins that are required for vesicle formation and for HIV-1 budding, and has produced a working model for sequential recruitment of these proteins during MVB vesicle formation . Retroviruses can redirect this machinery to the plasma membrane and leave the cell in a single step or, alternatively, can bud directly into MVB compartments and then exit cells via the exosome pathway . Remarkably, virus release from both the plasma membrane and MVB compartments can occur directionally into specialized sites of cell-to-cell contact called virological synapses . Thus retroviruses have evolved elaborate mechanisms for escaping the cell and maximizing their chances of infecting a new host.

J Biomol Struct Dyn, 2004 Dec, 22(3), 267 - 80
Collective motions of RNA polymerases . Analysis of core enzyme, elongation complex and holoenzyme; Yildirim Y et al.; The anisotropic network model (ANM), a coarse-grained normal mode analysis, is used to study the vibrational dynamics of RNA polymerases (RNAP) around the native states . The theoretical temperature factors obtained from ANM are in conformity with the experimental values for yeast and bacterial RNAP structures in free and complex forms . In the low-frequency collective modes that are related to biological function, both bacterial and yeast RNAPs with a crab claw shape display an opening/closing of the cleft due to the rigid-body motion of the clamp (bottom pincer), which has been also predicted by experiments, together with the motion of the top pincer . Even though slightly lower fluctuations are observed in the elongation complex of yeast RNAP, similar clamp motion still exists in collective modes, which should be concerted with the flexible switches and the bridge helix in driving the transcription process, pointing at the possibility of a ratchet-like mechanism . Two different bacterial holoenzyme (HE) structures are studied, which may have functional significance at different stages of transcription initiation . In a specific closed conformation of the HE, the clamp and top pincer are highly immobilized due to interactions with the sigma subunit . In contrast, the deformation of the top pincer is not inhibited in a relatively open conformation of another HE, which may help load the DNA into the cleft during transcription initiation, even though the clamp motion is still inhibited.

J Proteome Res, 2004 Sep-Oct, 3(5), 1073 - 81
Characterization of low abundant membrane proteins using the protein sequence tag technology; Prinz T et al.; About 25% of open reading frames in fully sequenced genomes are estimated to encode transmembrane proteins that represent valuable targets for drugs . However, the global analysis of membrane proteins has been proven to be problematic, e.g., because of their very amphiphilic nature . In this paper, we show that the recently published Protein Sequence Tag (PST) technology combined with an efficient sample preparation is a powerful method to perform protein analysis of highly enriched membrane fractions . The PST approach is a gel-free proteomics tool for the analysis of proteins, which relies on a "sampling" strategy by isolating N-terminal protein sequence tags from cyanogen bromide cleaved proteins . The identification of these N-terminal PST peptides is based on LC-MS/MS . The effectiveness of the technology is demonstrated for a membrane fraction, which was isolated from crude mitochondria of yeast after alkaline sodium carbonate treatment . The PST approach performed on this fraction analyzed 148 proteins, whereas 84% are identified as membrane proteins . More interestingly, among these membrane proteins 56% are predicted to be of low abundance . These encouraging results are an important step toward the development of a quantitative PST approach (qPST) for the differential display of membrane protein analysis.

Med Mycol, 2004 Aug, 42(4), 311 - 8
Development of a novel, simple and rapid molecular identification system for clinical Candida species; Deak R et al.; Identification of clinical yeast isolates causing candidiasis is routinely performed by commercial yeast identification systems based on biochemical, morphological and physiological tests . These systems require 3-5 days and the proportion of identifications that are incorrect is high . Our novel and rapid molecular identification system for clinical Candida species is based on the analysis of restriction patterns obtained from PCR-generated ribosomal DNA sequences using five restriction enzymes . A software package (CandID) was designed to include a database of restriction fragment length polymorphism (RFLP) patterns for 29 Candida species . For 'in-house' validation, 122 clinical isolates that had previously identified in clinical laboratories were typed by this system . These clinical isolates were also independently re-identified by the API 20C AUX system . The ribosomal DNA RFLP database in the context of supporting analytical software allowed simple and rapid (1 work day) identification.

IEEE Trans Nanobioscience, 2004 Sep, 3(3), 172 - 9
Mathematical modeling of complex regulatory networks; Stelling J et al.; Cellular regulation comprises overwhelmingly complex interactions between genes and proteins that ultimately will only be rendered understandable by employing formal approaches . Developing large-scale mathematical models of such systems in an efficient and reliable way, however, requires careful evaluation of structuring principles for the models, of the description of the system dynamics, and of the experimental data basis for adjusting the models to reality . We discuss these three aspects of model development using the example of cell cycle regulation in yeast and suggest that capturing complex dynamic networks is feasible despite incomplete (quantitative) biological knowledge.

Lab Chip, 2004 Oct, 4(5), 481 - 7 Epub 2004 Sep 14.
Microfluidic biosensing systems . Part I . Development and optimisation of enzymatic chemiluminescent micro-biosensors based on silicon microchips; Davidsson R et al.; Chemiluminescent (CL) enzyme-based flow-through microchip biosensors (micro-biosensors) for detection of glucose and ethanol were developed for the purpose of monitoring real-time production and release of glucose and ethanol from microchip immobilised yeast cells . Part I of this study focuses on the development and optimisation of the micro-biosensors in a microfluidic sequential injection analysis (microSIA) system . Glucose oxidase (GOX) or alcohol oxidase (AOX) was co-immobilised with horseradish peroxidase (HRP) on porous silicon flow through microchips . The hydrogen peroxide produced from oxidation of the corresponding analyte (glucose or ethanol) took part in the chemiluminescent (CL) oxidation of luminol catalysed by HRP enhanced by addition of p-iodophenol (PIP) . All steps in the microSIA system, including control of syringe pump, multiposition valve (MPV) and data readout, were computer controlled . The influence of flow rate and luminol- and PIP concentration were investigated using a 2(3)-factor experiment using the GOX-HRP sensor . It was found that all estimated single factors and the highest order of interaction were significant . The optimum was found at 250 microM luminol and 150 microM PIP at a flow rate of 18 microl min(-1), the latter as a compromise between signal intensity and analysis time . Using the optimised system settings one sample was processed within 5 min . Two different immobilisation chemistries were investigated for both micro-biosensors based on 3-aminopropyltriethoxsilane (APTS)- or polyethylenimine (PEI) functionalisation followed by glutaraldehyde (GA) activation . GOX-HRP micro-biosensors responded linear in a log-log format within the range 10-1000 microM glucose . Both had an operational stability of at least 8 days, but the PEI-GOX-HRP sensor was more sensitive . The AOX-HRP micro-biosensors responded linear (log-log) in the range between 1 and 10 mM ethanol, but the PEI-AOX-HRP sensor was in general more sensitive . Both sensors had an operational stability of at least 8 h, but with a half-life of 2-3 days.

Hum Mol Genet, 2004 Dec 1, 13(23), 3017 - 27 Epub 2004 Oct 07.
Interconnections of CLN3, Hook1 and Rab proteins link Batten disease to defects in the endocytic pathway; Luiro K et al.; The endosomal/lysosomal transmembrane protein CLN3 is mutated in the Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) . However, the molecular mechanism of JNCL pathogenesis and the exact function of the CLN3 protein have remained unclear . Previous studies have shown that deletion of BTN1, the yeast orthologue of CLN3, leads to increased expression of BTN2 . BTN2 encodes Btn2p, a proposed homologue to a novel microtubule-binding protein Hook1, which regulates endocytosis in Drosophila . We analysed here the putative interconnection between CLN3 and Hook1 in the mammalian cells and discovered that overexpression of human CLN3 induces aggregation of Hook1 protein, potentially by mediating its dissociation from the microtubules . Using in vitro binding assay we were able to demonstrate a weak interaction between Hook1 and the cytoplasmic segments of CLN3 . We also found receptor-mediated endocytosis to be defective in CLN3-deficient JNCL fibroblasts, connecting CLN3, Hook1 and endocytosis in the mammalian system . Moreover, co-immunoprecipitation experiments showed that Hook1 physically interacts with endocytic Rab7, Rab9 and Rab11, hence delineating a manifold role for mammalian Hook1 in membrane trafficking events . These novel interactions between the microtubule-binding Hook1 and the large family of Rab GTPases also suggest a link between CLN3 function, microtubule cytoskeleton and endocytic membrane trafficking.

J Biol Chem, 2004 Dec 24, 279(52), 54502 - 9 Epub 2004 Oct 06.
Platinated DNA adducts enhance poisoning of DNA topoisomerase I by camptothecin; van Waardenburg RC et al.; Camptothecins constitute a novel class of chemotherapeutics that selectively target DNA topoisomerase I (Top1) by reversibly stabilizing a covalent enzyme-DNA intermediate . This cytotoxic mechanism contrasts with that of platinum drugs, such as cisplatin, which induce inter- and intrastrand DNA adducts . In vitro combination studies using platinum drugs combined with Top1 poisons, such as topotecan, showed a schedule-dependent synergistic activity, with promising results in the clinic . However, whereas the molecular mechanism of these single agents may be relatively well understood, the mode of action of these chemotherapeutic agents in combination necessitates a more complete understanding . Indeed, we recently reported that a functional homologous recombination pathway is required for cisplatin and topotecan synergy yet represses the synergistic toxicity of 1-beta-D-arabinofuranosyl cytidine in combination with topotecan (van Waardenburg, R . C., de Jong, L . A., van Delft, F., van Eijndhoven, M . A., Bohlander, M., Bjornsti, M . A., Brouwer, J., and Schellens, J . H . (2004) Mol . Cancer Ther . 3, 393-402) . Here we provide direct evidence for Pt-1,3-d(GTG) poisoning of Top1 in vitro and demonstrate that persistent Pt-DNA adducts correlate with increased covalent Top1-DNA complexes in vivo . This contrasts with a lack of persistent lesions induced by the alkylating agent bis{chloroethyl}nitrosourea, which exhibits only additive activity with topotecan in a range of cell lines . In human IGROV-1 ovarian cancer cells, the synergistic activity of cisplatin with topotecan requires processive DNA polymerization, whereas overexpression of Top1 enhances yeast cell sensitivity to cisplatin . These results indicate that the cytotoxic activity of cisplatin is due, in part, to poisoning of Top1, which is exacerbated in the presence of topotecan.

Eukaryot Cell, 2004 Oct, 3(5), 1206 - 16
TbDSS-1, an essential trypanosoma brucei exoribonuclease homolog that has pleiotropic effects on mitochondrial RNA metabolism; Penschow JL et al.; Mitochondrial gene expression in trypanosomes is controlled primarily at the levels of RNA processing and RNA stability . This regulation undoubtedly involves numerous ribonucleases . Here we characterize the Trypanosoma brucei homolog of the yeast DSS-1 mitochondrial exoribonuclease, which we term TbDSS-1 . Biochemical fractionation indicates that TbDSS-1 is mitochondrially localized, as predicted by its N-terminal sequence . In contrast to its yeast homolog, TbDSS-1 does not appear to be associated with mitochondrial ribosomes . Targeted downregulation of TbDSS-1 by RNA interference in procyclic-form T . brucei results in a severe growth defect . In addition, TbDSS-1 depletion leads to a decrease in the levels of never edited cytochrome oxidase subunit I (COI) mRNA and both unedited and edited COIII mRNAs, indicating this enzyme functions in the control of mitochondrial RNA abundance . We also observe a considerable reduction in the level of edited apocytochrome b (CYb) mRNA and a corresponding increase in unedited CYb mRNA, suggesting that TbDSS-1 functions, either directly or indirectly, in the control of RNA editing . The abundance of both gCYb{560} and gA6{149} guide RNAs is reduced upon TbDSS-1 depletion, although the reduction in gCYb{560} is much more dramatic . The significant reduction in gCYb levels could potentially account for the observed decrease in CYb RNA editing . Western blot analyses of mitochondrial RNA editing and stability factors indicate that the perturbations of RNA levels observed in TbDSS-1 knock-downs do not result from secondary effects on other mitochondrial proteins . In all, these data demonstrate that TbDSS-1 is an essential protein that plays a role in mitochondrial RNA stability and RNA editing.

Biol Reprod . 2004 Oct 6; {Epub ahead of print}
SRY Associates with the Heterochromatin Protein 1 Complex by Interacting with a KRAB Domain Protein; Oh HJ et al.; In mammals, the SRY/Sry gene on the Y chromosome is necessary and sufficient for a bipotential gonad to develop into testis regardless of its chromosomal sex . SRY/Sry gene encodes a protein belonging to a high mobility group (HMG) box protein family and has been postulated to modulate the expression of genes necessary for male gonadal differentiation . Using a yeast two-hybrid screen, we identified a novel protein containing only a Kruppel associated box (KRAB) domain, which is hereto named KRAB-O (KRAB Only), as an SRY interacting protein . KRAB-O protein is encoded by an alternatively spliced transcript from the Zfp208 locus that also produces another transcript coding for a KRAB-zinc finger protein, ZFP208 . The interaction of the mouse SRY with KRAB-O was further confirmed by GST pull-down assay and co-immunoprecipitation in transfected COS7 cells . The KRAB-O interaction domain in both the human and mouse SRY was mapped to the bridge region outside of the HMG box . Indirect immunofluorescence and confocal microscopy show that the mouse SRY co-localizes with KRAB-O in nuclear dots in transiently transfected COS7 cells and primary fetal mouse gonadal cells . Using similar approaches, we demonstrate that KRAB-O interacts directly with KAP1 (KRAB associated protein 1), the obligatory co-repressor for KRAB domain proteins . Further, we show that the mouse SRY is associated indirectly with KAP1 and heterochromatin protein 1 (HP1) through its interaction with KRAB-O, suggesting that the mouse SRY could utilize the KRAB-KAP1-HP1 organized transcriptional regulatory complex to regulate its yet-to-be identified downstream target genes.

Mol Biol Cell, 2004 Dec, 15(12), 5712 - 23 Epub 2004 Dec.
NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence; Nagahama M et al.; NVL (nuclear VCP-like protein), a member of the AAA-ATPase family, is known to exist in two forms with N-terminal extensions of different lengths in mammalian cells . Here, we show that they are localized differently in the nucleus; NVL2, the major species, is mainly present in the nucleolus, whereas NVL1 is nucleoplasmic . Mutational analysis demonstrated the presence of two nuclear localization signals in NVL2, one of which is shared with NVL1 . In addition, a nucleolar localization signal was found to exist in the N-terminal extra region of NVL2 . The nucleolar localization signal is critical for interaction with ribosomal protein L5, which was identified as a specific interaction partner of NVL2 on yeast two-hybrid screening . The interaction of NVL2 with L5 is ATP-dependent and likely contributes to the nucleolar translocation of NVL2 . The physiological implication of this interaction was suggested by the finding that a dominant negative NVL2 mutant inhibits ribosome biosynthesis, which is known to take place in the nucleolus.

J Biol Chem, 2004 Dec 24, 279(52), 54387 - 97 Epub 2004 Oct 06.
Identification and functional characterization of a novel human misshapen/Nck interacting kinase-related kinase, hMINK beta; Hu Y et al.; Misshapen/NIKs-related kinase (MINK) is a member of the germinal center family of kinases that are homologous to the yeast sterile 20 (Ste20) kinases and regulate a wide variety of cellular processes, including cell morphology, cytoskeletal rearrangement, and survival . Here, we present the cloning and functional characterization of a novel human Misshapen/NIKs-related kinase beta (hMINK beta) that encodes a polypeptide of 1312 amino acids . hMINK beta is ubiquitously expressed in most tissues with at least five alternatively spliced isoforms . Similar to Nck interacting kinase (NIK) and Traf2 and Nck-interacting kinase (TNIK), hMINK beta moderately activates c-Jun N-terminal kinase (JNK) and associates with Nck via the intermediate domain in the yeast two-hybrid system and in a glutathione S-transferase (GST) pull-down assay . Interestingly, overexpression of the kinase domain deleted and kinase-inactive mutants of hMINK beta in human fibrosarcoma HT1080 cells enhanced cell spreading, actin stress fiber formation, and adhesion to extracellular matrix, as well as decreased cell motility and cell invasion . Furthermore, these mutants also promoted cell-cell adhesion in human breast carcinoma MCF7 cells, evidenced with cell growth in clusters and increased membrane localization of beta-catenin, a multifunctional protein involved in E-cadherin-mediated cell adhesion . Finally, hMINK beta protein was found to colocalize with the Golgi apparatus, implicating that hMINK beta might exert its functions, at least in part, through the modulation of intracellular protein transport . Taken together, these results suggest that hMINK beta plays an important role in cytoskeleton reorganization, cell adhesion, and cell motility.

Mol Cell, 2004 Oct 8, 16(1), 93 - 105
Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin; Vaquero A et al.; We characterized human SirT1, one of the human homologs of the budding yeast Sir2p, an NAD+-dependent histone deacetylase involved in establishing repressive chromatin and increased life span . SirT1 deacetylates histone polypeptides with a preference for histone H4 lysine 16 (H4-K16Ac) and H3 lysine 9 (H3-K9Ac) in vitro . RNAi-mediated decreased expression of SirT1 in human cells causes hyperacetylation of H4-K16 and H3-K9 in vivo . SirT1 interacts with and deacetylates histone H1 at lysine 26 . Using an inducible system directing expression of SirT1 fused to the Gal4-DNA binding domain and a Gal4-reporter integrated in euchromatin, Gal4-SirT1 expression resulted in the deacetylation of H4-K16 and H3-K9, recruitment of H1 within the promoter vicinity, drastically reduced reporter expression, and loss of H3-K79 methylation, a mark restricting silenced chromatin . We propose a model for SirT1-mediated heterochromatin formation that includes deacetylation of histone tails, recruitment and deacetylation of histone H1, and spreading of hypomethylated H3-K79 with resultant silencing.

J Biochem Mol Biol, 2004 Jul 31, 37(4), 402 - 7
Human liver specific transcriptional factor TCP10L binds to MAD4; Jiang DJ et al.; A human gene T-complex protein 10 like (TCP10L) was cloned in our lab . A previous study showed that it expressed specifically in the liver and testis . A transcription experiment revealed that TCP10L was a transcription factor with transcription inhibition activity . In this study, the human MAD4 was identified to interact with TCP10L by a yeast two-hybrid screen . This finding was confirmed by immunoprecipitation and subcellular localization experiments . As MAD4 is a member of the MAD family, which antagonizes the functions of MYC and promotes cell differentiation, the biological function of the interaction between TCP10L and MAD4 may be to maintain the differentiation state in liver cells . Also, we propose that the up-regulation of Myc is caused by the down-regulation of TCP10L in human hepatocarcinomas.

Plant J, 2004 Nov, 40(3), 386 - 98
Molecular dissection of plant cytokinesis and phragmoplast structure: a survey of GFP-tagged proteins; Van Damme D et al.; To identify molecular players implicated in cytokinesis and division plane determination, the Arabidopsis thaliana genome was explored for potential cytokinesis genes . More than 100 open reading frames were selected based on similarity to yeast and animal cytokinesis genes, cytoskeleton and polarity genes, and Nicotiana tabacum genes showing cell cycle-controlled expression . The subcellular localization of these proteins was determined by means of GFP tagging in tobacco Bright Yellow-2 cells and Arabidopsis plants . Detailed confocal microscopy identified 15 proteins targeted to distinct regions of the phragmoplast and the cell plate . EB1- and MAP65-like proteins were associated with the plus-end, the minus-end, or along the entire length of microtubules . The actin-binding protein myosin, the kinase Aurora, and a novel cell cycle protein designated T22, accumulated preferentially at the midline . EB1 and Aurora, in addition to other regulatory proteins (homologs of Mob1, Sid1, and Sid2), were targeted to the nucleus, suggesting that this organelle operates as a coordinating hub for cytokinesis.

Oncogene, 2004 Nov 11, 23(53), 8681 - 7
NHERF (Na+/H+ exchanger regulatory factor) gene mutations in human breast cancer; Dai JL et al.; Yeast two-hybrid screening was used to explore novel proteins that interact with a breast tumor or metastasis suppressor, SYK (spleen tyrosine kinase) . The screening yielded NHERF (Na+/H+ exchanger regulatory factor, also known as NHERF1 or EBP-50) that binds to the interdomain B of SYK . NHERF is an estrogen-responsive gene that encodes an inhibitory factor for epithelial Na+/H+ exchanger isoform 3 (NHE3) . We found intragenic mutation of the NHERF gene accompanied by loss of heterzygosity (LOH) in approximately 3% (3/85) of breast cancer cell lines and primary breast tumors . Mutations occurred at the conserved PDZ domains at NHERF NH2-terminus that bound to SYK, or at its COOH-terminus motif that binds to MERLIN, the product of Neurofibromatosis 2 (NF2) tumor suppressor gene . NHERF tumorigenic mutations decreased or abolished its interaction with SYK or MERLIN, suggesting a pathway link among these three molecules that may play a critical role in mammary neoplastic progression . Primary breast tumors with LOH at the NHERF locus had clinical presentations of higher aggressiveness, indicating that deregulated NHERF signaling may be associated with disease progression . Moreover, the LOH was inversely correlated with SYK promoter methylation, suggesting that NHERF and SYK may transduce a common suppressive signal . Taken together, the results indicated NHERF to be a candidate tumor suppressor gene in human breast carcinoma that may be interconnected to the SYK and MERLIN suppressors.

Lab Invest, 2004 Nov, 84(11), 1484 - 90
SYT, a partner of SYT-SSX oncoprotein in synovial sarcomas, interacts with mSin3A, a component of histone deacetylase complex; Ito T et al.; Synovial sarcomas are soft-tissue tumors predominantly affecting children and young adults . They are molecular-genetically characterized by the SYT-SSX fusion gene generated from chromosomal translocation t(X; 18) (p11.2; q11.2) . When we screened new gene products that interact with SYT or SSX proteins by yeast two-hybrid assay, we found that mSin3A, a component of the histone deacetylase complex, interacts with SYT but not with SSX . These results were confirmed by mammalian two-hybrid and pull-down assays . Analyses with sequential truncated proteins revealed a main mSin3A-interaction region on the SYT amino-terminal 93 amino acids, and another one on the region between 187th amino acid and break point . In luciferase assay, mSin3A repressed the transcriptional activity of reporter promoter mediated by SYT and hBRM/BRG1 . Our results suggest that the histone deacetylase complex containing mSin3A may regulate the transcriptional activation mediated by SYT.

Nat Cell Biol, 2004 Nov, 6(11), 1122 - 8 Epub 2004 Oct 03.
Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive; Jacinto E et al.; The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth . In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2 . A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals . Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists . mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor . Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton . mTORC2 is not upstream of the mTORC1 effector S6K . Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.

Cell Cycle, 2004 Oct, 3(10), 1267 - 70 Epub 2004 Oct 06.
Evolutionary conservation of a novel splice variant of the Cds1/CHK2 checkpoint kinase restricted to its regulatory domain; Lemaire M et al.; The Cds1/CHK2 kinase plays a key role in the activation of the G(2) checkpoint after DNA damage . Here we report the existence in fission yeast of a short variant (Sv) of Cds1 that is produced through an alternative splicing mechanism leading to a frame shift and premature termination . This SvCds1 protein consists solely of the regulatory region and lacks the catalytic domain . Expression of SvCds1 increases sensitivity to ionizing radiation and, to a lesser extent, to hydroxyurea, but not to UV radiation . We also report that in the human orthologue of Cds1, CHK2, differential splicing of a cryptic exon leads to a frame shift and premature termination producing a short variant (SvCHK2) . Thus, we have discovered the existence of an evolutionary conserved mechanism ensuring the production of a catalytically inactive variant Cds1/CHK2 that is restricted to SQTQ and FHA domains and that can act as a dominant negative . The role that this short variant of Cds1/CHK2 might play in the response to DNA damage and the physiopathological consequences are discussed.

Cell Cycle, 2004 Oct, 3(10), 1278 - 84 Epub 2004 Oct 06.
Multi-kinase phosphorylation of the APC/C activator Cdh1 revealed by mass spectrometry; Hall MC et al.; Cdh1 contributes to proper exit from mitosis and maintenance of G(1) phase in eukaryotic cells by activating a large ubiquitin ligase called the anaphase-promoting complex, or cyclosome (APC/C) . At the end of G(1), APC/C(Cdh1) is inhibited by cyclin-dependent kinase (CDK) phosphorylation of Cdh1 . The specific Cdh1 phosphorylation sites used to regulate APC/C(Cdh1) activity have not been directly identified . Here, we used a mass spectrometric approach to identify the in vivo phosphorylation sites on yeast Cdh1 . Surprisingly, in addition to several expected CDK phosphorylation sites, we discovered numerous nonCDK phosphorylation sites . In total, at least 19 serine and threonine residues on Cdh1 are phosphorylated in vivo . Seventeen of these sites are located in the N-terminal half of Cdh1, outside the highly conserved WD40 repeats . The pattern of phosphorylation was the same when Cdh1 was purified from yeast cultures arrested in S, early M and late M . Mutation of CDK consensus sequences eliminated detectable phosphorylation at many of the nonCDK sites . In contrast, mutation of nonCDK sites had no significant effect on CDK phosphorylation . We conclude that phosphorylation of CDK sites promotes the subsequent recognition of Cdh1 by at least one additional kinase . The function of nonCDK phosphorylation may differ from CDK phosphorylation because mutation of nonCDK sites did not result in constitutive activation of APC and consequent cell cycle arrest . These results suggest that phosphoregulation of APC/C(Cdh1) activity is much more complex than previously thought.

Glycoconj J, 2004, 21(1-2), 3 - 7
Structure, function and pathology of O-mannosyl glycans; Endo T; Animal cells contain many glycoproteins, i.e . , proteins with covalently liked sugar chains . The major glycans of glycoproteins can be classified into two groups, N-glycans and O-glycans, according to their glycan-peptide linkage regions . Development of sensitive methods for the analyses of glycan structures have revealed a new type of glycosidic linkage to the peptide portion, the O-mannosyl linkage, in mammals, which used to be considered specific to yeast . O-Mannosylation is present in a limited number of glycoproteins of brain, nerve, and skeletal muscle . Recently O-mannosylation has been shown to be important in muscle and brain development . Glycobiology of O-mannosyl glycans is expected to produce remarkable advances in the understanding and treatment of congenital muscular dystrophies . In this article, I describe the structure, biosynthesis, and pathology of O-mannosyl glycans.

Cytogenet Genome Res, 2004, 107(3-4), 216 - 31
Novel and diverse functions of the DNA mismatch repair family in mammalian meiosis and recombination; Kolas NK et al.; The mismatch repair (MMR) family is a highly conserved group of proteins that function in genome stabilization and mutation avoidance . Their role has been particularly well studied in the context of DNA repair following replication errors, and disruption of these processes results in characteristic microsatellite instability, repair defects and, in mammals, susceptibility to cancer . An additional role in meiotic recombination has been described for several family members, as revealed by extensive studies in yeast . More recently, the role of the mammalian MMR family in meiotic progression has been elucidated by the phenotypic analysis of mice harboring targeted mutations in the genes encoding several MMR family members . This review will discuss the phenotypes of the various mutant mouse lines and, drawing from our knowledge of MMR function in yeast meiosis and in somatic cell repair, will attempt to elucidate the significance of MMR activity in mouse germ cells . These studies highlight the importance of comparative analysis of MMR orthologs across species, and also underscore distinct sexually dimorphic characteristics of mammalian recombination and meiosis .

Biol Pharm Bull, 2004 Oct, 27(10), 1515 - 20
Antipyretic, analgesic and anti-inflammatory activities of ketoprofen beta-cyclodextrin inclusion complexes in animals; Lu WL et al.; Ketoprofen is a nonsteroidal anti-inflammatory drug (NSAID) orally effective in treating fever, pain, and inflammation but gastrointestinal side effects were observed . Preparation of ketoprofen beta-cyclodextrin inclusion complexes was to increase the solubility and reduce the irritation . The complexes were prepared and preliminarily confirmed using X-ray diffraction and dissolution test . Antipyretic, analgesic and anti-inflammatory models were induced by 10% yeast using rabbits, 0.8% acetic acid using mice and 1% carrageenin using rats, respectively . Results showed that the dissolution rate of ketoprofen was significantly improved by complexation . X-Ray diffraction pattern of the complexes exhibited a diffuse pattern that differed from that of physical mixture of ketoprofen and beta-cyclodextrin . Ketoprofen markedly inhibited the fever reactions at a single dose of 2 mg/kg as follows: 64.53% (inhibition rate %) at 1 h for ketoprofen, 73.04% at 1 h for ketoprofen beta-cyclodextrin inclusion complexes, respectively . Alleviating pain reaction rates following a single dose of 8 mg/kg at 20 min were 39.25% for the inclusion complexes and 26.72% for ketoprofen, respectively . Inhibition rates to rat edema following a single dose of 5 mg/kg at 1 h were 39.47% for the inclusion complexes and 23.86% for ketoprofen . Results for antipyretic, analgesic and anti-inflammatory activities showed that the rapid and stronger effects were found in the treatment group of ketoprofen beta-cyclodextrin inclusion complexes in comparison with those of free ketoprofen.

Cancer Epidemiol Biomarkers Prev, 2004 Oct, 13(10), 1574 - 82
A case-control study of risk factors for invasive cervical cancer among U.S . women exposed to oncogenic types of human papillomavirus; Shields TS et al.; Oncogenic human papillomavirus (HPV) infections, the necessary cause of most cervical cancers, are common and usually clear within 1 to 2 years . Identifying cofactors that lead to cancer among HPV-infected women has depended mainly on case-control studies defining HPV by DNA testing . DNA testing assesses only current infection; thus, concerns about residual confounding remain . To assess cofactors, we used seropositivity to five oncogenic HPV types as a marker of past exposure and confined our analysis to seropositive controls compared with cancer cases . Study subjects had participated in a multicenter U.S . case-control study conducted in the early 1980s . The detailed questionnaire and stored sera for 235 cases of squamous carcinoma and 486 controls motivated the reanalysis . We measured antibodies to HPV types 16, 18, 31, 45, and 52 . Independent, significant predictors of seropositivity among controls included numbers of sexual partners, Black race, and oral contraceptive use . Condom use was protective . Among HPV-exposed women, Papanicolaou screening, Black race, and yeast infection were significantly associated with reduced cancer risk . Current smoking was associated with a 2-fold increase in risk; there were independent, significant trends of increased risk with numbers of cigarettes smoked (P for trend = 0.003) and years of smoking (P for trend = 0.01) . Other significant predictors of increased risk included low education and income and history of nonspecific genital infection . Unlike recent HPV DNA-based investigations, based on the use of HPV-seropositive controls in this study, oral contraceptive use was unrelated to the risk of cervical cancer and multiparity was only weakly related to risk . It is particularly worth considering further why studies of different designs are inconsistent regarding the effect of oral contraceptive use.

Blood . 2004 Oct 5; {Epub ahead of print}
Endostatin dramatically inhibits endothelial cell migration, vascular morphogenesis, and perivascular cell recruitment in vivo; Skovseth DK et al.; Endostatin is a proteolytic fragment of collagen XVIII that inhibits endothelial cell migration in vitro and experimental tumor growth in vivo . To determine how endostatin affects the in vivo behavior of endothelial cells, we took advantage of a surrogate model of human angiogenesis, in which human endothelial cells are transferred to immunodeficient mice and develop into complex vessels in the course of 30 days . Systemic delivery of human yeast-derived endostatin (serum levels of 30-35 ng/mL) inhibited the number of human vessels dramatically (95% at day 20), as most endothelial cells remained suspended as single cells . The fraction of cells with a migratory phenotype (F-actin-positive, extending pseudopods) was strongly reduced (from 50% to 13% at day 10), while the number of apoptotic and mitotic cells remained unchanged . Endostatin also hampered the recruitment of alpha-smooth muscle actin-expressing perivascular cells and thus reduced the number of mature vessels (from 64.3% to 28.6% at day 30) . Moreover, transcripts of pericyte-recruiting platelet-derived growth factor-B (PDGFB) were strongly reduced in endothelial cells of endostatin-treated mice . Our results are strong evidence that endostatin inhibits angiogenesis at several levels in vivo, including perivascular cell recruitment.

Genetics . 2004 Sep 30; {Epub ahead of print}
A screen for S . pombe mutants defective in re-replication identifies new alleles of rad4+, cut9+, and psf2+
Gomez EB, Angeles VT, Forsburg SL.
Fission yeast mutants defective in DNA replication have widely varying morphological phenotypes . We designed a screen for temperature sensitive mutants defective in the process of replication regardless of morphology by isolating strains unable to re-replicate their DNA in the absence of cyclin B (Cdc13) . Of the 42 re-replication-defective mutants analyzed, we were able to clone complementing plasmids for 10 . This screen identified new alleles of the APC subunit cut9+ and the initiation/checkpoint factor rad4+/cut5+, and the first mutant allele of psf2+, a subunit of the novel GINS replication complex . Other genes identified are likely to play general roles in gene expression and protein localization.

J Biol Chem, 2004 Dec 10, 279(50), 52087 - 94 Epub 2004 Dec 10.
Cell-specific activation of the atrial natriuretic factor promoter by PITX2 and MEF2A; Toro R et al.; The PITX2 homeodomain protein is mutated in patients with Axenfeld-Rieger syndrome and is involved in the development of multiple organ systems, including the heart . We have examined the interaction of PITX2 isoforms with myocyte-enhancing factor 2A (MEF2A), which is a known regulator of cardiac development . A direct interaction between PITX2a and MEF2A was demonstrated using yeast two-hybrid and GST pull-down assays . To study the functional significance of this interaction, we used the atrial natriuretic factor (ANF) promoter . Coexpression of MEF2A and PITX2a or Pitx2c resulted in a strong synergistic activation of the ANF promoter in LS8 oral epithelial cells but not in other cell lines (NIH/3T3, Chinese hamster ovary, or C2C12) . The synergism was dependent on promoter context, because it required MEF2 binding sites and was not seen with two other PITX2 target promoters . DNA binding by MEF2A was required but not sufficient for synergism . Upstream activators of p38 MAP kinases, MKK3 and MKK6, increased PITX2a and Pitx2c activity to yield up to 90-fold activation of the ANF promoter in LS8 cells . Because Axenfeld-Rieger syndrome is autosomal dominant and affects development of the oral epithelium, we tested one of the known PITX2 mutants . The PITX2a-K88E mutant protein suppressed wild type PITX2a synergism with MEF2A . These results demonstrate a promoter- and cell-specific functional interaction between PITX2 and MEF2A and suggest the possibility of coordinate control by these factors in the oral epithelium.

Genome Res, 2004 Oct, 14(10A), 1948 - 56
High-content screening microscopy identifies novel proteins with a putative role in secretory membrane traffic; Starkuviene V et al.; Here we describe the establishment of microscope-based functional screening assays in intact cells that allow us to systematically identify new proteins involved in secretory membrane traffic, and proteins that can influence the integrity of the Golgi complex . We were able to identify 20 new proteins that affected either secretory transport, Golgi morphology, or both, when overexpressed in cells . Control experiments with human orthologs to yeast proteins with a role in membrane traffic, or already well characterized mammalian regulators of the secretory pathway, confirmed the specificity and significance of our results . Proteins localized to the Golgi complex or endoplasmic reticulum (ER) showed preferential interference in our assays . Bioinformatic analysis of the new proteins interfering with membrane traffic and/or Golgi integrity revealed broad functional variety, but demonstrated a bias towards proteins with predicted coiled-coil domains and repeat structures . Extending our approach to a much larger set of novel proteins in the future will be an important step toward a more comprehensive understanding of the molecular basis of the secretory pathway . It will also serve as an example for similar microscope-based screens addressing different biological questions.

Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14931 - 6 Epub 2004 Oct 01.
Conditional mutagenesis using site-specific recombination in Plasmodium berghei; Carvalho TG et al.; Reverse genetics in Plasmodium, the genus of parasites that cause malaria, still faces major limitations . Only red blood cell stages of this haploid parasite can be transfected . Consequently, the function of many essential genes in these and subsequent stages, including those encoding vaccine candidates, cannot be addressed genetically . Here, we establish conditional mutagenesis in Plasmodium by using site-specific recombination and the Flp/FRT system of yeast . Site-specific recombination is induced after cross-fertilization in the mosquito vector of two clones containing either the target sequence flanked by two FRT sites or the Flp recombinase . Parasites that have undergone recombination are recognized in the cross progeny through the expression of a fluorescence marker . This approach should permit to dissect the function of any essential gene of Plasmodium during the haploid phase of its life, i.e., during infection of salivary glands in the mosquito and infection of both the liver and red blood cells in the mammal.

Biophys J, 2004 Dec, 87(6), 3750 - 63 Epub 2004 Dec.
Metabolic control analysis under uncertainty: framework development and case studies; Wang L et al.; Information about the enzyme kinetics in a metabolic network will enable understanding of the function of the network and quantitative prediction of the network responses to genetic and environmental perturbations . Despite recent advances in experimental techniques, such information is limited and existing experimental data show extensive variation and they are based on in vitro experiments . In this article, we present a computational framework based on the well-established (log)linear formalism of metabolic control analysis . The framework employs a Monte Carlo sampling procedure to simulate the uncertainty in the kinetic data and applies statistical tools for the identification of the rate-limiting steps in metabolic networks . We applied the proposed framework to a branched biosynthetic pathway and the yeast glycolysis pathway . Analysis of the results allowed us to interpret and predict the responses of metabolic networks to genetic and environmental changes, and to gain insights on how uncertainty in the kinetic mechanisms and kinetic parameters propagate into the uncertainty in predicting network responses . Some of the practical applications of the proposed approach include the identification of drug targets for metabolic diseases and the guidance for design strategies in metabolic engineering for the purposeful manipulation of the metabolism of industrial organisms.

J Biol Chem, 2004 Dec 10, 279(50), 51958 - 64 Epub 2004 Dec 10.
Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease; Treiber C et al.; The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide . Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels . A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques . Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol . Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels . This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels . The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels . The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold . These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.

J Biol Chem, 2004 Dec 10, 279(50), 51973 - 80 Epub 2004 Dec 10.
Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54; Raschle M et al.; In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination . Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules . Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51 . Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51 . By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured . Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively . Surprisingly, under less stringent conditions, the Rad54 Delta129 protein can interact with Rad51 in affinity pull-down and functional assays . These results highlight the functional importance of the N-terminal Rad51 interaction domain of Rad54 and reveal that Rad54 contacts Rad51 through separable epitopes.

Biochim Biophys Acta, 2004 Oct 11, 1685(1-3), 48 - 62
Consequences of NPC1 and NPC2 loss of function in mammalian neurons; Walkley SU et al.; Genetic deficiency of NPC1 or NPC2 results in a devastating cholesterol-glycosphingolipidosis of brain and other organs known as Niemann-Pick type C (NPC) disease . While NPC1 is a transmembrane protein believed involved in retroendocytic shuttling of substrate(s) to the Golgi and possibly elsewhere in cells as part of an essential recycling/homeostatic control mechanism, NPC2 is a soluble lysosomal protein known to bind cholesterol . The precise role(s) of NPC1 and NPC2 in endosomal-lysosomal function remain unclear, nor is it known whether the two proteins directly interact as part of this function . The pathologic features of NPC disease, however, are well documented . Brain cells undergo massive intracellular accumulation of glycosphingolipids (lactosylceramide, glucosylceramide, GM2 and GM3 gangliosides) and cholesterol and concomitant distortion of neuron shape (meganeurite formation) . In neurons from humans with NPC disease the metabolic defects and storage often lead to extensive growth of new, ectopic dendrites (possibly linked to ganglioside sequestration) as well as formation of neurofibrillary tangles (NFTs) (possibly linked to dysregulation of cholesterol metabolism) . Other features of cellular pathology in NPC disease include fragmentation of the Golgi apparatus and neuroaxonal dystrophy, though reasons for these changes remain largely unknown . As the disease progresses, neurodegeneration is also apparent for neurons in some brain regions, particularly Purkinje cells of the cerebellum, but the basis of this selective neuronal vulnerability is unknown . The NPC1 protein is evolutionarily conserved with homologues reported in yeast to humans; NPC2 is reported in C . elegans to humans . While neurons in mammalian models of NPC1 and NPC2 diseases exhibit many changes that are remarkably similar to those in humans (e.g., endosomal/lysosomal storage, Golgi fragmentation, neuroaxonal dystrophy, neurodegeneration), a reduced degree of ectopic dendritogenesis and an absence of NFTs in these species suggest important differences in the way lower mammalian neurons respond to NPC1/NPC2 loss of function.

Curr Opin Struct Biol, 2004 Oct, 14(5), 601 - 6
Biosynthesis of human-type N-glycans in heterologous systems; Betenbaugh MJ et al.; Insects, yeasts and plants generate widely different N-glycans, the structures of which differ significantly from those produced by mammals . The processing of the initial Glc2Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 in the endoplasmic reticulum shows significant similarities among these species and with mammals, whereas very different processing events occur in the Golgi compartments . For example, yeasts can add 50 or even more Man residues to Man(8-9)GlcNAc2, whereas insect cells typically remove most or all Man residues to generate paucimannosidic Man(3-1)GlcNAc2N-glycans . Plant cells also remove Man residues to yield Man(4-5)GlcNAc2, with occasional complex GlcNAc or Gal modifications, but often add potentially allergenic beta(1,2)-linked Xyl and, together with insect cells, core alpha(1,3)-linked Fuc residues . However, genomic efforts, such as expression of exogenous glycosyltransferases, have revealed more complex processing capabilities in these hosts that are not usually observed in native cell lines . In addition, metabolic engineering efforts undertaken to modify insect, yeast and plant N-glycan processing pathways have yielded sialylated complex-type N-glycans in insect cells, and galactosylated N-glycans in yeasts and plants, indicating that cell lines can be engineered to produce mammalian-like glycoproteins of potential therapeutic value .

Biochem Biophys Res Commun, 2004 Nov 5, 324(1), 70 - 6
Functional interaction between Smad, CREB binding protein, and p68 RNA helicase; Warner DR et al.; The transforming growth factors beta control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region . TGFbeta superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFbeta-responsive genes . Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN . Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific . In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue . The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays . Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFbeta-stimulated reporter gene transcription . Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression . Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP . This offers a means of enhancing TGFbeta-mediated cellular responses in developing orofacial tissue.

J Biotechnol, 2004 Oct 19, 114(1-2), 153 - 64
Links between morphology and physiology of Ganoderma lucidum in submerged culture for the production of exopolysaccharide; Wagner R et al.; Ganoderma lucidum was grown in submerged culture in shake flasks on a medium containing peptone, yeast extract and glucose . In pre-cultures, inoculated from an agar-grown culture, morphological and metabolic events were linked: the pellets originally produced protuberances when glucose was present in the medium, although glucose was not consumed . The protuberances were then liberated into the medium as second-generation pellets, at which time glucose consumption began and the rate of exopolysaccharide (EPS) production increased . The synchrony between events was repeated in cultures fed with either glucose or peptone and yeast extract . In main cultures, inoculated from a 16-day-old pre-culture, the biomass concentration increased linearly, while glucose consumption and EPS production were initially slow but then accelerated . Protuberances were produced and liberated similarly to the pre-culture, but there was less synchrony amongst the pellets . When glucose was added to such a culture on day 10, an EPS concentration of 5.7 g L(-1) was achieved on day 13, this being the highest reliable EPS concentration yet reported for submerged culture of G . lucidum . We conclude that a greater understanding of the morphological and physiological events during the culture of G . lucidum will allow the proposal of culture strategies to improve EPS production.

Hear Res, 2004 Oct, 196(1-2), 39 - 48
Localization of the mitochondrial uncoupling protein family in the rat inner ear; Kitahara T et al.; Uncoupling proteins (UCPs) are a proton transporter family located in the mitochondrial inner membrane . The molecular expression and activity of UCPs in brown adipose tissue and skeletal muscle are regulated by factors as diverse as chronic overeating and cold exposure, suggesting roles in energy expenditure and heat production . Although UCP2, UCP4 and brain mitochondrial carrier protein-1 (BMCP-1, i.e . UCP5) mRNAs are expressed in the central nervous system, their central function is unknown . This study presents the first evidence on localization and quantitative expression of UCPs in the rat inner ear by real-time PCR and immunohistochemistry . Real-time PCR studies revealed that UCP2 mRNA was expressed in the vestibular and spiral ganglia more abundantly than any other UCP . Neocortex, by contrast, contained UCP2 and UCP4 equally . Notably, UCP3 and UCP4 mRNAs were expressed in inner ear ganglia, but brain UCP3 mRNA expression level was undetectable by simple PCR . Immunohistochemical studies confirmed that both UCP2- and UCP3-like immunoreactivities were detected in vestibular and spiral ganglion cells and co-localized with a mitochondrial marker, MitoFluorGreen . According to previous reports, UCP2 and UCP3 are thermogenic in yeast and brain UCP2 has been suggested to modulate pre- and post-synaptic events by axonal thermogenesis . It has also been reported recently that UCP2 and UCP3 responses to superoxide application may be an antioxidant protective mechanism . Therefore, it is suggested that mitochondrial UCPs (UCP2, UCP3, UCP4) may play both a protective role against oxidative damage and a thermal signaling role in the eighth nerve.

Parasitol Today, 1992 Jul, 8(7), 225 - 9
Towards a high-resolution map of the Plasmodium falciparum genome; Triglia T et al.; Until recently very little was known about the genome of Plasmodium falciparum . The situation has changed considerably with the advent of pulsed field gradient electrophoresis and yeast artificial chromosome technologies . It should now be possible to generate a high-resolution map within a few years . Here, Tony Triglia, Thomas Wellems and David Kemp review current knowledge.

Parasitol Today, 1988 Jan, 4(1), 15 - 7
Blastocystis hominis, a long-misunderstood intestinal parasite; Zierdt CH; For over 50 years, Blastocystis hominis has been held to be a harmless intestinal yeast-probably frequent in stool samples from man and other primates, but usually ignored except as a possible source of confusion with Entamoeba histolytica . More recently, its status as a protozoan parasite has been accepted, and it is now increasingly recognized as an agent of intestinal disease - usually self-limiting but occasionally fatal in monkeys . Here, Charles Zierdt reviews the status o f this intriguing protozoan, drawing attention to its unusual biochemistry.

Genome Biol . 2004;5(10):R78 . Epub 2004 Sep 29.
Genomic neighborhoods for Arabidopsis retrotransposons: a role for targeted integration in the distribution of the Metaviridae; Peterson-Burch BD et al.; BACKGROUND: Retrotransposons are an abundant component of eukaryotic genomes . The high quality of the Arabidopsis thaliana genome sequence makes it possible to comprehensively characterize retroelement populations and explore factors that contribute to their genomic distribution . RESULTS: We identified the full complement of A . thaliana long terminal repeat (LTR) retroelements using RetroMap, a software tool that iteratively searches genome sequences for reverse transcriptases and then defines retroelement insertions . Relative ages of full-length elements were estimated by assessing sequence divergence between LTRs: the Pseudoviridae were significantly younger than the Metaviridae . All retroelement insertions were mapped onto the genome sequence and their distribution was distinctly non-uniform . Although both Pseudoviridae and Metaviridae tend to cluster within pericentromeric heterochromatin, this association is significantly more pronounced for all three Metaviridae sublineages (Metavirus, Tat and Athila) . Among these, Tat and Athila are strictly associated with pericentromeric heterochromatin . CONCLUSIONS: The non-uniform genomic distribution of the Pseudoviridae and the Metaviridae can be explained by a variety of factors including target-site bias, selection against integration into euchromatin and pericentromeric accumulation of elements as a result of suppression of recombination . However, comparisons based on the age of elements and their chromosomal location indicate that integration-site specificity is likely to be the primary factor determining distribution of the Athila and Tat sublineages of the Metaviridae . We predict that, like retroelements in yeast, the Athila and Tat elements target integration to pericentromeric regions by recognizing a specific feature of pericentromeric heterochromatin.

Genes Cells, 2004 Oct, 9(10), 967 - 82
Harp (harmonin-interacting, ankyrin repeat-containing protein), a novel protein that interacts with harmonin in epithelial tissues; Johnston AM et al.; Mutations in the triple PDZ domain-containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C . Independently, we identified harmonin in a screen for genes expressed in pancreatic beta cells . Using a yeast two-hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for harmonin-interacting, ankyrin repeat-containing protein . This interaction was confirmed in an over-expression system and in mammalian cells, and shown to be mediated by the three C-terminal amino acids of harp . Harp is expressed in many of the same epithelia as harmonin and co-localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic beta-cell line . Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif . Human harp maps to chromosome 16, and its mouse homologue to chromosome 7 . Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 (js) mutant mouse and in human Usher syndrome type 1G . The functional domain structures of harp and harmonin, their interaction under native conditions and their co-localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.

Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14901 - 6 Epub 2004 Sep 30.
Mediation of Af4 protein function in the cerebellum by Siah proteins; Oliver PL et al.; We have established that the gene AF4, which had long been recognized as disrupted in childhood leukemia, also plays a role in the CNS . Af4 is mutated in the robotic mouse that is characterized by ataxia and Purkinje cell loss . To determine the molecular basis of this mutation, we carried out a yeast two-hybrid screen and show that Af4 binds the E3 ubiquitin ligases Drosophila seven in absentia (sina) homologues (Siah)-1a and Siah-2 in the brain . Siah-1a and Af4 are expressed in Purkinje cells and colocalize in the nucleus of human embryonic kidney 293T and P19 cells . In vitro binding assays and coimmunoprecipitation reveal a significant reduction in affinity between Siah-1a and robotic mutant Af4 compared with wild-type, which correlates with the almost complete abolition of mutant Af4 degradation by Siah-1a . These data strongly suggest that an accumulation of mutant Af4 occurs in the robotic mouse due to a reduction in its normal turnover by the proteasome . A significant increase in the transcriptional activity of mutant Af4 relative to wild-type was obtained in mammalian cells, suggesting that the activity of Af4 is controlled through Siah-mediated degradation . Another member of the Af4 family, Fmr2, which is involved in mental handicap in humans, binds Siah proteins in a similar manner . These results provide evidence that a common regulatory mechanism exists that controls levels of the Af4/Fmr2 protein family . The robotic mouse thus provides a unique opportunity to understand how these proteins play a role in disorders as diverse as leukemia, mental retardation, and neurodegenerative disease.

J Leukoc Biol, 2005 Jan, 77(1), 80 - 9 Epub 2004 Sep 30.
CD93 interacts with the PDZ domain-containing adaptor protein GIPC: implications in the modulation of phagocytosis; Bohlson SS et al.; CD93 was originally identified as a myeloid cell-surface marker and subsequently associated with an ability to modulate phagocytosis of suboptimally opsonized immunoglobulin G and complement particles in vitro . Recent studies using mice deficient in CD93 have demonstrated that this molecule modulates phagocytosis of apoptotic cells in vivo . To investigate signal transduction mechanisms mediated by CD93, CD93 cytoplasmic tail (CYTO)-binding proteins were identified in a yeast two-hybrid screen . Fifteen of 34 positive clones contained a splice variant or a partial cDNA encoding GIPC, a PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein, shown previously to regulate cytoskeletal dynamics . A single clone of the N-terminal kinase-like protein p105 and an uncharacterized stem cell transcript also showed specificity for binding to the CYTO by yeast two-hybrid . Using the yeast two-hybrid system and an in vitro glutathione S-transferase fusion protein-binding assay, the binding of GIPC to the CYTO was shown to involve a newly identified class I PDZ-binding domain in the CD93 carboxyl terminus . Four positively charged amino acids in the juxtamembrane domain of CD93 were shown to be critical in stabilizing these interactions . Treatment of human monocytes with a cell-permeable peptide encoding the C-terminal 11 amino acids of CD93 resulted in an enhancement of phagocytosis, supporting the hypothesis that this protein-protein interaction domain is involved in the modulation of phagocytosis . These protein interactions may participate as molecular switches in modulating cellular phagocytic activity.

J Inorg Biochem, 2004 Oct, 98(10), 1607 - 13
The role of copper transporters in the development of resistance to Pt drugs; Safaei R et al.; Recent studies in yeast, mouse and human cells suggest that the conserved metal binding transporters of the Cu homeostasis pathway can mediate resistance to Pt drugs in cancer cells . This review summarizes the data available from these studies . The observation that cells selected for resistance to Cu or the Pt drugs display bidirectional cross-resistance, parallel defects in the transport of Cu and the Pt drugs and altered expression of Cu transporters is consistent with the concept that the Cu homeostasis proteins regulate sensitivity to the Pt drugs by influencing their uptake, efflux and intracellular distribution . This model is supported by the finding that when mammalian and yeast cells are genetically engineered to express altered levels of the Cu transporters they exhibit altered sensitivity to Pt drugs and are defective in intracellular Pt accumulation due to altered uptake and/or efflux rates . Negative associations between the expression of ATP7A and ATP7B and the outcome of Pt therapy further support the significance of the Cu homeostasis proteins as both markers of and contributors to Pt resistance.

Structure (Camb), 2004 Oct, 12(10), 1877 - 9
Prediction of multiple tandem OB-fold domains in telomere end-binding proteins Pot1 and Cdc13; Theobald DL et al.; The heterodimeric Oxytricha nova telomere end binding protein, the original telomere end binding protein characterized, contains four OB-fold domains used for recognition of single-stranded telomeric DNA . In contrast, only solitary OB-fold domains have been found in the telomere end binding proteins from yeast and higher eukaryotes . Using a sliding-window algorithm coupled with sequence profile-profile analysis, we provide support for the existence of multiple OB-fold domains in two other telomeric ssDNA binding proteins, vertebrate Pot1 and budding yeast Cdc13 . This common usage of multiple, tandem OB-fold domains in telomeric end binding proteins extends the known evolutionary conservation of eukaryotic end-protection mechanisms .

Biochem J, 2005 Feb 1, 385(Pt 3), 755 - 61
Recognition and processing of a nuclear-encoded polyprotein precursor by mitochondrial processing peptidase; Oshima T et al.; The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14 . After the preSDHB-RPS14 polyprotein is transported into the mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14 . Here we report that the general MPP (mitochondrial processing peptidase) plays an essential role in processing of the polyprotein . Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14 . Moreover, the connector region was processed more rapidly than the presequence . When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP processing motif that has also been shown to be present in the N-terminal presequence . Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence . In addition, MPP preferentially recognized the unfolded structure of preSDHB-RPS14 . In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before processing of the presequence.

J Int Med Res, 2004 Sep-Oct, 32(5), 484 - 7
Rapid identification of the Candida species from direct blood cultures by CHROMagar Candida; Birinci A et al.; We evaluated the ability of CHROMagar Candida to identify Candida species isolated directly from blood cultures . A total of 50 clinical isolates of Candida were incubated at 35 degrees C, and once growth was established, an aliquot of each was plated onto CHROMagar Candida medium . A control specimen was plated directly from Sabouraud's dextrose agar . Following incubation at 30 degrees C, all yeast isolates were identified by colony morphology and colour . We were able to identify all isolates of C . albicans (n = 20), C . tropicalis (n = 14), C . glabrata (n = 6), and C . krusei (n = 5), which were isolated from blood or from control cultures . This study demonstrated that CHROMagar Candida reliably isolated and identified yeast taken directly from blood cultures . We conclude that this rapid and easy method of identifying Candida species will enable clinicians to quickly choose the appropriate antifungal agent . This should decrease patient morbidity and mortality.

Mol Reprod Dev, 2004 Dec, 69(4), 387 - 96
KIF2Abeta: A kinesin family member enriched in mouse male germ cells, interacts with translin associated factor-X (TRAX); Bray JD et al.; Translin associated factor X (TRAX) is a binding partner of TB-RBP/Translin . A cDNA encoding the 260 C-terminal amino acids of KIF2Abeta was isolated from mouse testis cDNAs in a yeast two-hybrid library screen for specific TRAX-interacting proteins . KIF2Abeta was expressed predominantly in the mouse testis and enriched in germ cells . The interaction of full-length KIF2Abeta or its C-terminus with TRAX was verified using in vitro synthesized fusion proteins . Deletion mapping of the TRAX-binding region of KIF2Abeta indicated that amino acids 514-659 were necessary and sufficient for the interaction in vivo . Confocal microscopy studies using GFP-fusion proteins demonstrated that KIF2Abeta colocalizes with TRAX in a perinuclear location . KIF2Abeta does not interact with TB-RBP, suggesting that either TRAX can function as an adaptor molecule for motor proteins and TB-RBP, or that this interaction reveals an undescribed role for TRAX in germ cells . The interaction with KIF2Abeta suggests a role for TRAX in microtubule-based functions during spermatogenesis .

EMBO J, 2004 Oct 27, 23(21), 4243 - 52 Epub 2004 Sep 30.
Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo; Kristjuhan A et al.; The mechanism and kinetics of RNA polymerase II transcription and histone acetylation were studied by chromatin immunoprecipitation in yeast . Our results indicate that a significant fraction of polymerases starting transcription never make it to the end of a long GAL-VPS13 fusion gene . Surprisingly, induction of GAL genes results in substantial loss of histone-DNA contacts not only in the promoter but also in the coding region . The loss of nucleosomes is dependent on active transcript elongation, but apparently occurs independently of histone acetylation . In contrast, histones in genes previously shown to require the histone acetyltransferases GCN5 and ELP3 for normal transcription do not lose DNA contacts, but do become acetylated as a result of transcription . Together, these results suggest the existence of at least two distinct mechanisms to achieve efficient transcript elongation through chromatin: a pathway based on loss of histone-DNA contacts, and a histone acetylation-dependent mechanism correlating with little or no net loss of nucleosomes.

J Mol Neurosci, 2004, 24(2), 269 - 76
The splicing regulatory protein p18SRP is down-regulated in Alzheimer's disease brain; Heese K et al.; Alzheimer's disease (AD) is the most common neurodegenerative disorder of aging, accounting for an estimated two-thirds of all cases of senile dementia . Using bioinformatics, the yeast two-hybrid-system, reverse transcription polymerase chain reaction, and fluorescence microscopy analysis, we demonstrate here that the new putative splicing regulatory protein p18SRP is a lysine-rich zinc finger domain-containing protein that interacts with the serine-arginine (SR)-rich splicing regulatory protein SRrp86 . The additional finding of its down-regulation in the brain of AD subjects points to a possible pivotal role of p18SRP in the control of cellular survival.

Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14707 - 12 Epub 2004 Sep 29.
Creation and discovery of ligand-receptor pairs for transcriptional control with small molecules; Schwimmer LJ et al.; The nuclear receptor retinoid X receptor (RXR) is a ligand-activated transcription factor . To create receptors for a new ligand, a structure-based approach was used to generate a library of approximately 380,000 mutant RXR genes . To discover functional variants within the library, we used chemical complementation, a method of protein engineering that uses the power of genetic selection . Wild-type RXR has an EC50 of 500 nM for 9-cis retinoic acid (9cRA) and an EC50 of >10 microM for the synthetic retinoid-like compound LG335 in yeast . The library produced ligand-receptor pairs with LG335 that have a variety of EC50 values (40 nM to >2 microM) and activation levels (10-80% of wild-type RXR with 9cRA) in yeast . The variant I268V;A272V;I310L;F313M has an EC50 for LG335 of 40 nM and an EC50 for 9cRA of >10 microM in yeast . This variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at a 10-fold-lower ligand concentration in yeast . This EC50 is 25-fold lower than the best receptor we have engineered through site-directed mutagenesis, Q275C;I310M;F313I . Furthermore, the variants' EC50 values and activation levels in yeast and mammalian cells correlate . This protein engineering method should be extendable to produce other functional ligand-receptor pairs, which can be selected and characterized from libraries within weeks . Coupling large library construction with chemical complementation could be used to engineer proteins that bind virtually any small molecule for conditional gene expression, applications in metabolic engineering, and biosensors and to engineer enzymes through genetic selection.

Sci Aging Knowledge Environ . 2004 Sep 29;2004(39):pe36.
Ras: the other pro-aging pathway; Longo VD; Studies in worms, flies, and mice point to the insulin/insulin-like growth factor-1 (IGF-1)/phosphatidylinositol 3-kinase/Akt-like pathway as a central regulator of longevity . A similar pathway, which includes Sch9, a functional mammalian Akt/protein kinase B homolog, regulates longevity in yeast . Chronological aging in yeast is also regulated by a second pathway that includes Ras, adenylate cyclase, protein kinase A, the transcription factors Msn2 and Msn4, and Sod2 . Although Ras proteins have not been implicated in longevity regulation in worms or flies, the major role of Ras in mammalian IGF-1 signaling raises the possibility that homologs of yeast Ras2 might accelerate aging in mammals . Here I review the data from experiments at both the organismal and cellular levels that support a role for Ras in the regulation of stress resistance and life span in eukaryotes.

Mol Cell Biol, 2004 Oct, 24(20), 8951 - 62
Notch-induced E2A degradation requires CHIP and Hsc70 as novel facilitators of ubiquitination; Huang Z et al.; E2A transcription factors, E12 and E47, are important regulators of lymphocyte development . Notch signaling pathways have been shown to regulate E2A function by accelerating the degradation of E2A proteins through a mitogen-activated protein kinase-dependent and ubiquitin-mediated pathway . To further understand the mechanism underlying E2A ubiquitination and degradation, we conducted a yeast two-hybrid screen and identified the carboxyl terminus of Hsc70-interacting protein (CHIP) as an E47 binding protein . Here, we show that CHIP associates with E2A proteins in vivo and that overexpression of CHIP induces E47 degradation in a phosphorylation-dependent manner . Conversely, knocking down CHIP with small interfering RNA alleviates Notch-induced E47 degradation . CHIP binds E47 through the E protein homology domains 2 and 3 (EHD2 and EHD3) . This interaction between CHIP and E47 is independent of the U-box domain with E3 ubiquitin ligase activity but requires the chaperone binding tetratricopeptide repeats domain . The ability of CHIP to induce E47 ubiquitination and degradation correlates with its ability to bind E47 . We propose that CHIP, together with its partner Hsc70, forms a preubiquitination complex (PUC) with E47 and Skp2, thus facilitating the interaction between E47 and Skp2 . CHIP also associates with Cul1, which introduces PUC to the SCF E3 ligase complex, responsible for E47 ubiquitination . Therefore, CHIP plays a crucial role in the ubiquitination and degradation of E2A proteins.

Mol Cell Biol, 2004 Oct, 24(20), 8917 - 28
The cyclin A1-CDK2 complex regulates DNA double-strand break repair; Muller-Tidow C et al.; Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase . Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood . We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53 . cyclin A1-/- cells showed increased radiosensitivity . To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex . DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells . Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins . Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining . DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective . These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.

J Cell Sci, 2004 Oct 1, 117(Pt 21), 4881 - 8
RNA-directed DNA methylation; Mathieu O et al.; Double-stranded RNAs (dsRNAs) and their 'diced' small RNA products can guide key developmental and defense mechanisms in eukaryotes . Some RNA-directed mechanisms act at a post-transcriptional level to degrade target messenger RNAs . However, dsRNA-derived species can also direct changes in the chromatin structure of DNA regions with which they share sequence identity . For example, plants use such RNA species to lay down cytosine methylation imprints on identical DNA sequences, providing a fundamental mark for the formation of transcriptionally silent heterochromatin . Thus, RNA can feed backwards to modulate the accessibility of information stored in the DNA of cognate genes . RNA triggers for DNA methylation can come from different sources, including invasive viral, transgene or transposon sequences, and in some cases are derived from single-stranded RNA precursors by RNA-dependent RNA polymerases . The mechanism by which RNA signals are translated into DNA methylation imprints is currently unknown, but two plant-specific types of cytosine methyltransferase have been implicated in this process . RNA can also direct heterochromatin formation in fission yeast and Drosophila, but in these organisms the process occurs in the absence of DNA methylation.

J Biol Chem, 2004 Dec 10, 279(50), 52703 - 13 Epub 2004 Dec 10.
Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family; Torban E et al.; Mammalian Vangl1 and Vangl2 are highly conserved membrane proteins that have evolved from a single ancestral protein Strabismus/Van Gogh found in Drosophila . Mutations in the Vangl2 gene cause a neural tube defect (craniorachischisis) characteristic of the looptail (Lp) mouse . Studies in model organisms indicate that Vangl proteins play a key developmental role in establishing planar cell polarity (PCP) and in regulating convergent extension (CE) movements during embryogenesis . The role of Vangl1 in these processes is virtually unknown, and the molecular function of Vangl1 and Vangl2 in PCP and CE is poorly understood . Using a yeast two-hybrid system, glutathione S-transferase pull-down and co-immunoprecipitation assays, we show that both mouse Vangl1 and Vangl2 physically interact with the three members of the cytoplasmic Dishevelled (Dvl) protein family . This interaction is shown to require both the predicted cytoplasmic C-terminal half of Vangl1/2 and a portion of the Dvl protein containing PDZ and DIX domains . In addition, we show that the two known Vangl2 loss-of-function mutations identified in two independent Lp alleles associated with neural tube defects impair binding to Dvl1, Dvl2, and Dvl3 . These findings suggest a molecular mechanism for the neural tube defect seen in Lp mice . Our observations indicate that Vangl1 biochemical properties parallel those of Vangl2 and that Vangl1 might, therefore, participate in PCP and CE either in concert with Vangl2 or independently of Vangl2 in discrete cell types.

J Biol Chem, 2004 Dec 10, 279(50), 52456 - 64 Epub 2004 Dec 10.
MBD3L1 is a transcriptional repressor that interacts with methyl-CpG-binding protein 2 (MBD2) and components of the NuRD complex; Jiang CL et al.; Methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) are transcriptional repressors that contain methyl-CpG binding domains and are components of a CpG-methylated DNA binding complex named MeCP1 . Methyl-CpG-binding protein 3-like 1 (MBD3L1) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG binding domain . MBD3L1 interacts with MBD2 and MBD3 in vitro and in yeast two-hybrid assays . Gel shift experiments with a CpG-methylated DNA probe indicate that recombinant MBD3L1 can supershift an MBD2-methylated DNA complex . In vivo, MBD3L1 associates with and colocalizes with MBD2 but not with MBD3 and is recruited to 5-methylcytosine-rich pericentromeric heterochromatin in mouse cells . In glutathione S-transferase pull-down assays MBD3L1 is found associated with several known components of the MeCP1.NuRD complex, including HDAC1, HDAC2, MTA2, MBD2, RbAp46, and RbAp48, but MBD3 is not found in the MBD3L1-bound fraction . MBD3L1 enhances transcriptional repression of methylated DNA by MBD2 . The data are consistent with a role of MBD3L1 as a methylation-dependent transcriptional repressor that may interchange with MBD3 as an MBD2-interacting component of the NuRD complex . MBD3L1 knockout mice were created and were found to be viable and fertile, indicating that MBD3L1 may not be essential or there is functional redundancy (through MBD3) in this pathway . Overall, this study reveals additional complexities in the mechanisms of transcriptional repression by the MBD family proteins.

Glycobiology, 2005 Feb, 15(2), 193 - 201 Epub 2004 Sep 29.
Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line; Bakker H et al.; Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen . In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis . The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized . In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport . The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs . Despite this low overall identity, the two proteins clearly belong to the same gene family . Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay . The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate . Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family.

Development, 2004 Nov, 131(21), 5263 - 76 Epub 2004 Sep 29.
Interaction of Polycomb-group proteins controlling flowering in Arabidopsis; Chanvivattana Y et al.; In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G) proteins . Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase . Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others . We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway . Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain . We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER (SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants . Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2 . The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal . We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.

Vestn Ross Akad Med Nauk, 2004, (8), 27 - 31
{Effects of the dsRNA interferon inducer on the interaction between macrophages and Mycobacterium tuberculosis in vitro}; Masycheva VI et al.; Effects of the IFN inducer, yeast dsRNA, produced on the interaction between mouse macrophages and phagocytized mycobacteria were experimentally studied in vitro . Mycobacteria were shown to reproduce in macrophages in their initial infection at a ratio of 1:1.25, 1:2.5, 1:5 and 1:10, which was confirmed by an increased insertion of 5.6-{3H}-uracil in M . tuberculosis H37Rv; they also had a destructive impact on macrophages as verified by a higher release of lactic dehydrogenase (LDG) from macrophages . The dsRNA preparation, 40.0, 80.0 and 120.0 microg/ml, was demonstrated to decrease the insertion amount of labeled uracil in phagocytized mycobacteria at a macrophage:mycobacteria ratio of 1:10 and 1:100 . The effect depended on a preparation dose and infection degree of macrophages . A decreased release of specific LDG from infected macrophages was shown under the same conditions . The dsRNA affects the interplay of macrophages and phagocytized mycobacteria through inhibiting the vitality of intracellular mycobacteria and through enhancing the stability of macrophages . Special studies denoted that dsRNA, 40.0 microg/ml and 120 microg/ml, activated the production of peroxidation compounds by neutrophils, which phagocytized the sheep erythrocytes . Finally, a possible mechanism of dsRNA impact on the interaction between macrophages and mycobacteria phagocytized by them is under discussion.

Int J Cancer, 2005 Jan 20, 113(3), 393 - 6
Maternal human polyomavirus infection and risk of neuroblastoma in the child; Stolt A et al.; To investigate if polyomavirus infection during pregnancy is linked to development of neuroblastoma in the child, serum samples of 115 index mothers from the pregnancy where the child eventually developed neuroblastoma were identified and matched with serum samples from 8 control mothers per index mother . The samples were tested for specific IgG and IgM antibodies to BK and JC virus using enzyme immunoassays based on purified yeast-expressed virus-like particles (VLPs) . The serum samples as well as 10 neuroblastoma cell lines were also analyzed using Real Time (TaqMan) PCR for detection and quantification of BK virus DNA . The BK virus IgG seroprevalence was similar among index mothers (80%) and control mothers (83%) {OR 0.8; 95% confidence interval (95% CI): 0.5-1.3} . BK virus IgM was also not associated with neuroblastoma risk (OR was OR = 0.6; 95% with CI, 0.2-1.9) . Also JC virus had no association, neither for IgG (OR = 0.9; 95% CI, 0.6-1.4) nor for IgM (OR = 0.9; 95% CI, 0.4-1.9) . All serum samples and all neuroblastoma cell lines were negative for BKV DNA . In summary, a comprehensive cohort using both serology and polyomavirus DNA detection found no evidence for association between BKV or JCV polyomaviruses and neuroblastoma.

J Insect Sci . 2002;2:26 . Epub 2002 Dec 17.
Characterization of Z/E11- and Z9-desaturases from the obliquebanded leafroller moth, Choristoneura rosaceana; Hao G et al.; A (triangle up) 11-desaturase gene was cloned from the sex pheromone gland of the obliquebanded leafroller moth, Choristoneura rosaceana . The desaturase cDNA sequence spans 1300 nucleotides with an open reading frame encoding a 335 amino-acid protein, which has 81% identity to a Z/E11-desaturase of the redbanded leafroller moth, Argyrotaenia velutinana . A functional assay with a pYES2 yeast expression system demonstrated that the (triangle up) 11-desaturase exhibits unusual substrate and stereospecificities in producing a Z/E11 mixture (7:1) of only C14 acids . A metabolic Z9-desaturase also was cloned from fat body of this species, and proved to be in the class that produces more Z9-16:Acid than Z9-18:Acid.

J Cell Sci, 2004 Oct 15, 117(Pt 22), 5313 - 21 Epub 2004 Sep 28.
Maternal UNC-45 is involved in cytokinesis and colocalizes with non-muscle myosin in the early Caenorhabditis elegans embryo; Kachur T et al.; The Caenorhabditis elegans UNC-45 protein contains tetratricopeptide repeats and a domain with similarity to fungal proteins, and it differentially colocalizes with myosin heavy chain B in the body wall muscles of adult worms . Although it is essential for normal myosin filament assembly in body wall muscle development, strong mutants show a previously unexplained maternal effect . We show here that the UNC-45 protein is maternally contributed and is present in all cells of the early embryo whereas zygotic UNC-45 expression is only detected in the developing muscle cells . Embryos produced from adults with reduced germline expression of UNC-45 exhibit cytokinesis defects suggesting that UNC-45 has a novel role in the early embryo in addition to muscle development . Yeast two-hybrid screens show that UNC-45 can directly interact with NMY-2, a non-muscle type II myosin, and UNC-45 and NMY-2 colocalize at cell boundaries in early embryos . Localization of UNC-45 at these boundaries is dependent upon the presence of NMY-2 . Our results suggest that UNC-45 interacts with more than one type of myosin and functions in the embryo to regulate cytoplasmic myosin assembly and/or stability during cytokinesis.

Genetics, 2004 Sep, 168(1), 489 - 502
Estimating the degree of saturation in mutant screens; Pollock DD et al.; Large-scale screens for loss-of-function mutants have played a significant role in recent advances in developmental biology and other fields . In such mutant screens, it is desirable to estimate the degree of "saturation" of the screen (i.e., what fraction of the possible target genes has been identified) . We applied Bayesian and maximum-likelihood methods for estimating the number of loci remaining undetected in large-scale screens and produced credibility intervals to assess the uncertainty of these estimates . Since different loci may mutate to alleles with detectable phenotypes at different rates, we also incorporated variation in the degree of mutability among genes, using either gamma-distributed mutation rates or multiple discrete mutation rate classes . We examined eight published data sets from large-scale mutant screens and found that credibility intervals are much broader than implied by previous assumptions about the degree of saturation of screens . The likelihood methods presented here are a significantly better fit to data from published experiments than estimates based on the Poisson distribution, which implicitly assumes a single mutation rate for all loci . The results are reasonably robust to different models of variation in the mutability of genes . We tested our methods against mutant allele data from a region of the Drosophila melanogaster genome for which there is an independent genomics-based estimate of the number of undetected loci and found that the number of such loci falls within the predicted credibility interval for our models . The methods we have developed may also be useful for estimating the degree of saturation in other types of genetic screens in addition to classical screens for simple loss-of-function mutants, including genetic modifier screens and screens for protein-protein interactions using the yeast two-hybrid method.

Genetics, 2004 Sep, 168(1), 199 - 214
SNR1 (INI1/SNF5) mediates important cell growth functions of the Drosophila Brahma (SWI/SNF) chromatin remodeling complex; Zraly CB et al.; SNR1 is an essential subunit of the Drosophila Brahma (Brm) ATP-dependent chromatin remodeling complex, with counterparts in yeast (SNF5) and mammals (INI1) . Increased cell growth and wing patterning defects are associated with a conditional snr1 mutant, while loss of INI1 function is directly linked with aggressive cancers, suggesting important roles in development and growth control . The Brm complex is known to function during G1 phase, where it appears to assist in restricting entry into S phase . In Drosophila, the activity of DmcycE/CDK2 is rate limiting for entry into S phase and we previously found that the Brm complex can suppress a reduced growth phenotype associated with a hypomorphic DmcycE mutant . Our results reveal that SNR1 helps mediate associations between the Brm complex and DmcycE/CDK2 both in vitro and in vivo . Further, disrupting snr1 function suppressed DmcycEJP phenotypes, and increased cell growth defects associated with the conditional snr1E1 mutant were suppressed by reducing DmcycE levels . While the snr1E1-dependent increased cell growth did not appear to be directly associated with altered expression of G1 or G2 cyclins, transcription of the G2-M regulator string/cdc25 was reduced . Thus, in addition to important functions of the Brm complex in G1-S control, the complex also appears to be important for transcription of genes required for cell cycle progression.

Genetics, 2004 Sep, 168(1), 141 - 6
Shared forces of sex chromosome evolution in haploid-mating and diploid-mating organisms: Microbotryum violaceum and other model organisms; Hood ME et al.; It is usually posited that the most important factors contributing to sex chromosome evolution in diploids are the suppression of meiotic recombination and the asymmetry that results from one chromosome (the Y) being permanently heterozygous and the other (the X) being homozygous in half of the individuals involved in mating . To distinguish between the roles of these two factors, it would be valuable to compare sex chromosomes in diploid-mating organisms and organisms where mating compatibility is determined in the haploid stage . In this latter group, no such asymmetry occurs because the sex chromosomes are equally heterozygous . Here we show in the fungus Microbotryum violaceum that the chromosomes carrying the mating-type locus, and thus determining haploid-mating compatibility, are rich in transposable elements, dimorphic in size, and carry unequal densities of functional genes . Through analysis of available complete genomes, we also show that M . violaceum is, remarkably, more similar to humans and mice than to yeast, nematodes, or fruit flies with regard to the differential accumulation of transposable elements in the chromosomes determining mating compatibility vs . the autosomes . We conclude that restricted recombination, rather than asymmetrical sheltering, hemizygosity, or dosage compensation, is sufficient to account for the common sex chromosome characteristics.

Eur J Cancer, 2004 Oct, 40(15), 2287 - 92
Methotrexate inhibits the glyoxalase system in vivo in children with acute lymphoid leukaemia; Bartyik K et al.; The inhibition of glyoxalase I leads to antitumour activity through the accumulation of methylglyoxal . Our earlier observations suggested that methotrexate (MTX) may affect the glyoxalase system . This prompted a serial study of the drug on this metabolic pathway . Ten children with acute lymphoid leukaemia (ALL), admitted to our department between January 2002 and July 2003, were enrolled . Plasma D-lactate was assayed before, 24 and 72 h after the start of four consecutive MTX infusions (5 g/m(2)/24 h) in each patient . Inhibition of glyoxalase I was tested in vitro, using human erythrocyte lysates and yeast enzyme . The elevated initial plasma D-lactate levels (P<0.02) fell significantly (P<0.001) in response to 24 h MTX infusions . In vitro, MTX, folic and folinic acids inhibited the activity of glyoxalase I . Thus, MTX seems to affect the alpha-oxoaldehyde metabolism in vivo, as a likely consequence of glyoxalase I inhibition . This action probably contributes to the anticancer activity and toxicity of the drug.

Biochem J . 2004 Sep 29; Pt {Epub ahead of print}
FHL3 negatively regulates the human high affinity IgE receptor beta-chain gene expression by acting as a transcriptional co-repressor of MZF-1; Takahashi K et al.; The high affinity IgE receptor, FcepsilonRI, plays a key role in triggering allergic reaction . We recently reported that human FcepsilonRI beta chain gene expression was downregulated by a transcription factor MZF-1 through an element in the fourth intron . Here we revealed that this transcriptional repression by MZF-1 required FHL3 as a cofactor . Yeast two hybrid and immunoprecipitation assays demonstrated that FHL3 bound MZF-1 in vitro and in vivo . Overexpression of FHL3 in KU812 cells suppressed the beta chain promoter activity via the element in the fourth intron in an MZF-1 dependent manner . Furthermore, results from pull down assays and gel filtration chromatography employing the nuclear extracts indicated that MZF-1 and FHL3 formed a complex of high molecular mass with some additional proteins in the nucleus . Granulocyte-macrophage colony-stimulating factor (GM-CSF), which was reported to decrease FcepsilonRI expression, induced accumulation of FHL3 in the nucleus, in accordance with the repressive role of FHL3 in the beta chain gene expression.

Annu Rev Pharmacol Toxicol . 2004 Sep 28; {Epub ahead of print}
Clinical Development of Histone Deacetylase Inhibitors As Anticancer Agents; Drummond DC et al.; Acetylation is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways . Although histones are the most thoroughly studied of acetylated protein substrates, histone acetyltransferases (HATs) and deacetylases (HDACs) are also responsible for modifying the activity of diverse types of nonhistone proteins, including transcription factors and signal transduction mediators . HDACs have emerged as uncredentialed molecular targets for the development of enzymatic inhibitors to treat human cancer, and six structurally distinct drug classes have been identified with in vivo bioavailability and intracellular capability to inhibit many of the known mammalian members representing the two general types of NAD+-independent yeast HDACs, Rpd3 (HDACs 1, 2, 3, 8) and Hda1 (HDACs 4, 5, 6, 7, 9a, 9b, 10) . Initial clinical trials indicate that HDAC inhibitors from several different structural classes are very well tolerated and exhibit clinical activity against a variety of human malignancies; however, the molecular basis for their anticancer selectivity remains largely unknown . HDAC inhibitors have also shown preclinical promise when combined with other therapeutic agents, and innovative drug delivery strategies, including liposome encapsulation, may further enhance their clinical development and anticancer potential . An improved understanding of the mechanistic role of specific HDACs in human tumorigenesis, as well as the identification of more specific HDAC inhibitors, will likely accelerate the clinical development and broaden the future scope and utility of HDAC inhibitors for cancer treatment . Expected online publication date for the Annual Review of Pharmacology and Toxicology Volume 45 is January 6, 2005 . Please see for revised estimates.

J Virol, 2004 Oct, 78(20), 11303 - 12
Human immunodeficiency virus type 2 Gag interacts specifically with PRP4, a serine-threonine kinase, and inhibits phosphorylation of splicing factor SF2; Bennett EM et al.; Using a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that bind to the human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified PRP4, a serine-threonine protein kinase . Specific interaction of PRP4 and HIV-2 Gag was confirmed in in vitro and in vivo assays . The interacting region of HIV-2 Gag is located in the conserved matrix and capsid domains, while both the RS (arginine-serine-rich) domain and the KS (kinase) domain of PRP4 are able to bind to HIV-2 Gag . PRP4 is not incorporated into virus particles . HIV-2 Gag is able to inhibit PRP4-mediated phosphorylation of the splicing factor SF2 . This is also observed with Gag from simian immunodeficiency virus, a closely related virus, but not with Gag from human T-cell lymphotropic virus type 1 . Our results provide evidence for a novel interaction between Gag and a cellular protein kinase involved in the control of constitutive splicing in two closely related retroviruses . We hypothesize that as Gag accumulates in the cell, down regulation of splicing occurs through reduced phosphorylation of SF2 . At late stages of infection, this interaction may replace the function of the early viral regulatory protein Rev.

J Virol, 2004 Oct, 78(20), 11161 - 71
Interaction of the movement protein NSP and the Arabidopsis acetyltransferase AtNSI is necessary for Cabbage leaf curl geminivirus infection and pathogenicity; Carvalho MF et al.; DNA viruses can modulate the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in order to support virus replication . A role for protein acetylation in regulating the nuclear export of the bipartite geminivirus DNA genome was recently suggested by the findings that the viral movement protein NSP, which shuttles the viral genome between the nucleus and the cytoplasm, interacts with a novel Arabidopsis acetyltransferase, AtNSI, and the increased expression of AtNSI enhances susceptibility to Cabbage leaf curl virus infection . To further investigate the interaction of NSP and AtNSI and to establish the importance of this interaction in virus infections, we used a reverse yeast two-hybrid selection and deletion analysis to identify NSP mutants that were impaired in their ability to bind AtNSI . These mutants identified a 38-amino-acid region of NSP, to which no function had so far been assigned, as being necessary for NSP-AtNSI interaction . Three NSP missense mutants were analyzed in detail and were found to be comparable to wild-type NSP in their levels of accumulation, nucleocytoplasmic shuttling, DNA binding, and cooperative interaction with the viral cell-to-cell movement protein MP . Despite this, Cabbage leaf curl virus that expressed each mutated NSP was defective in its ability to infect Arabidopsis, exhibiting lower levels of infectivity than the wild-type virus, and delayed systemic spread of the virus and attenuated disease symptoms . Our data demonstrate the importance of the interaction of NSP with AtNSI for virus infection and pathogenicity.

J Cell Biol, 2004 Sep 27, 166(7), 1081 - 91
CD44 modulates Smad1 activation in the BMP-7 signaling pathway; Peterson RS et al.; Bone morphogenetic protein 7 (BMP-7) regulates cellular metabolism in embryonic and adult tissues . Signal transduction occurs through the activation of intracellular Smad proteins . In this paper, using a yeast two-hybrid screen, Smad1 was found to interact with the cytoplasmic domain of CD44, a receptor for the extracellular matrix macromolecule hyaluronan . Coimmunoprecipitation experiments confirmed the interaction of Smad1 with full-length CD44-interactions that did not occur when CD44 receptors truncated within the cytoplasmic domain were tested . Chondrocytes overexpressing a truncated CD44 on a background of endogenous full-length CD44 no longer exhibited Smad1 nuclear translocation upon BMP-7 stimulation . Further, pretreatment of chondrocytes with Streptomyces hyaluronidase to disrupt extracellular hyaluronan-cell interactions inhibited BMP-7-mediated Smad1 phosphorylation, nuclear translocation of Smad1 or Smad4, and SBE4-luciferase reporter activation . These results support a functional link between the BMP signaling cascade and CD44 . Thus, changes in hyaluronan-cell interactions may serve as a means to modulate cellular responsiveness to BMP.

Invest Ophthalmol Vis Sci, 2004 Oct, 45(10), 3704 - 12
Conditional knockdown of tubedown-1 in endothelial cells leads to neovascular retinopathy; Wall DS et al.; PURPOSE: Identification of novel proteins involved in retinal neovascularization may facilitate new and more effective molecular-based treatments for proliferative retinopathy . Tubedown-1 (Tbdn-1) is a novel protein that shows homology to the yeast acetyltransferase subunit NAT1 and copurifies with an acetyltransferase activity . Tbdn-1 is expressed in normal retinal endothelium but is specifically suppressed in retinal endothelial cells from patients with proliferative diabetic retinopathy . The purpose of this study was to investigate the importance of Tbdn-1 expression in retinal blood vessels in vivo . METHODS: A bitransgenic mouse model that enables conditional knockdown of Tbdn-1 specifically in endothelial cells was produced and studied using molecular, histologic, and immunohistochemical techniques and morphometric analysis . RESULTS: Tbdn-1-suppressed mice exhibited retinal and choroidal neovascularization with intra- and preretinal fibrovascular lesions similar to human proliferative retinopathies . Retinal lesions observed in Tbdn-1-suppressed mice increased in severity with prolonged suppression of Tbdn-1 . In comparison to normal retina, the retinal lesions displayed alterations in the basement membrane of blood vessels and in the distribution of glial and myofibroblastic cells . Moreover, the pathologic consequences of Tbdn-1 knockdown in endothelium were restricted to the retina and the choroid . CONCLUSIONS: These results indicate that the maintenance of Tbdn-1 expression is important for retinal blood vessel homeostasis and for controlling retinal neovascularization in adults . Restoration of Tbdn-1 protein expression and/or activity may provide a novel approach for treating proliferative retinopathies.

Biochem Biophys Res Commun, 2004 Oct 29, 323(4), 1293 - 8
Immediate early gene X-1 interacts with proteins that modulate apoptosis; Kumar R et al.; Immediate early gene X-1 (IEX-1) modulates apoptosis, cellular growth, mechanical strain-induced cardiac hypertrophy, and vascular intimal hyperplasia . To determine how IEX-1 alters apoptosis, we performed yeast two-hybrid studies using IEX-1 as the "bait" protein, and examined interactions between IEX-1 and proteins expressed by a human kidney cDNA expression library . We found that IEX-1 interacts with several proteins of which at least four are known to play a role in the regulation of apoptosis: (1) calcium-modulating cyclophilin ligand; (2) tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor superfamily, member 10); (3) ML-1 myeloid cell leukemia gene encoded protein; and (4) BAT3, a gene present in the major histo-compatibility complex . Our data suggest that IEX-1 may regulate apoptosis by directly interacting with various proteins involved in the control of apoptotic pathways.

Biochem Biophys Res Commun, 2004 Oct 29, 323(4), 1216 - 22
Identification of a novel BRMS1-homologue protein p40 as a component of the mSin3A/p33(ING1b)/HDAC1 deacetylase complex; Nikolaev AY et al.; Repression of gene transcription is mediated by histone deacetylases containing repressor-co-repressor complexes, which are recruited to promoters of target genes via interactions with sequence-specific transcription factors . The mammalian Sin3A co-repressor complex contains a core of at least seven proteins including the pRb-interacting protein RBP1 and a putative tumor suppressor p33(ING1b) . By biochemical purification and mass spectrometry, we have identified a novel component p40 from this complex . p40 bears homology to both yeast Sds3, a component of yeast histone deacetylase complexes, and its mammalian homologue mSds3 . The p40-associated complex purified from human cells shows a strong histone deacetylase activity . When tethered to a Gal-DNA binding domain, the Gal-p40 is able to significantly repress transcription of a Gal-luciferase promoter . Interestingly, database analysis reveals that p40 is also highly homologous to BRMS1, a breast carcinoma metastasis suppressor, and overexpression of p40 in human cells can significantly inhibit cell growth . Thus, our data indicate that p40 may be critically involved in transcription repression of cell growth-associated gene expression by recruiting the HDAC1 deacetylase complex.

Lancet, 2004 Sep 25, 364(9440), 1173 - 82
Superficial fungal infections; Schwartz RA; Superficial fungal infections arise from a pathogen that is restricted to the stratum corneum, with little or no tissue reaction . In this Seminar, three types of infection will be covered: tinea versicolor, piedra, and tinea nigra . Tinea versicolor is common worldwide and is caused by Malassezia spp, which are human saprophytes that sometimes switch from yeast to pathogenic mycelial form . Malassezia furfur, Malassezia globosa, and Malassezia sympodialis are most closely linked to tinea versicolor . White and black piedra are both common in tropical regions of the world; white piedra is also endemic in temperate climates . Black piedra is caused by Piedraia hortae; white piedra is due to pathogenic species of the Trichosporon genus . Tinea nigra is also common in tropical areas and has been confused with melanoma.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 187 - 93
Blastomyces dermatitidis produces melanin in vitro and during infection; Nosanchuk JD et al.; Melanin is made by several important pathogenic fungi and is implicated in the pathogenesis of a number of mycoses . This study investigates whether the thermally dimorphic fungal pathogen Blastomyces dermatitidis produces melanin . Using techniques developed to study melanization in other fungi, we demonstrate that B . dermatitidis conidia and yeast produce melanin in vitro and that yeast cells synthesize melanin or melanin-like pigment in vivo . Melanization reduced susceptibility to amphotericin B, but not to itraconazole or voriconazole . Since melanin is an important virulence factor in other pathogenic fungi, this pigment may affect the pathogenesis of blastomycosis.

Biochem Pharmacol, 2004 Nov 1, 68(9), 1879 - 88
Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and its 5'-phosphates by mammalian phosphotransferases; Feng JY et al.; Amdoxovir {(-)-beta-D-2,6-diaminopurine dioxolane, DAPD}, the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection . In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5'-nucleotidase (5'-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase . In addition, the metabolism of 14C-labeled DXG was studied in CEM cells . DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7 mM) . Human 5'-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine) . DXG-MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP . DXG-DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase . The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG-DP . In CEM cells incubated with 5 microM DXG for 24 h, 0.015 pmole/10(6) cells (approximately 7.5 nM) of DXG-TP was detected as the primary metabolite . Our study demonstrated that 5'-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.

FEMS Yeast Res, 2004 Sep, 4(8), 789 - 94
Hansenula polymorpha Tup1p is important for peroxisome degradation; Leao-Helder AN et al.; In the yeast Hansenula polymorpha peroxisomes are selectively degraded upon a shift of cells from methanol to glucose-containing media . We identified the H . polymorpha TUP1 gene by functional complementation of the peroxisome degradation deficient mutant pdd2-4 . Tup1 proteins function in transcriptional repression of specific sets of genes involved in various cellular processes . Our combined data indicate that H . polymorpha TUP1 is involved in regulation of the switch between peroxisome biogenesis and selective degradation . The initial DNA fragment that complemented H . polymorpha pdd2-4 contained a second gene, encoding H . polymorpha Vps4p . Deletion of the VPS4 gene did not affect selective peroxisome degradation.

Eur J Neurosci, 2004 Oct, 20(8), 1984 - 94
The contribution of activated phagocytes and myelin degeneration to axonal retraction/dieback following spinal cord injury; McPhail LT et al.; Myelin-derived molecules inhibit axonal regeneration in the CNS . The Long-Evans Shaker rat is a naturally occurring dysmyelinated mutant, which although able to express the components of myelin lacks functional myelin in adulthood . Given that myelin breakdown exposes axons to molecules that are inhibitory to regeneration, we sought to determine whether injured dorsal column axons in a Shaker rat would exhibit a regenerative response absent in normally myelinated Long-Evans (control) rats . Although Shaker rat axons did not regenerate beyond the lesion, they remained at the caudal end of the crush site . Control rat axons, in contrast, retracted and died back from the edge of the crush . The absence of retraction/dieback in Shaker rats was associated with a reduced phagocytic reaction to dorsal column crush around the caudal edge of the lesion . Systemic injection of minocycline, a tetracycline derivative, in control rats reduced both the macrophage response and axonal retraction/dieback following dorsal column injury . In contrast, increasing macrophage activation by spinal injection of the yeast particulate zymosan had no effect on axonal retraction/dieback in Shaker rats . Schwann cell invasion was reduced in minocycline-treated control rats compared with untreated control rats, and was almost undetectable in Shaker rats, suggesting that like axonal retraction/dieback, spinal Schwann cell infiltration is dependent upon macrophage-mediated myelin degeneration . These results indicate that following spinal cord injury the phagocyte-mediated degeneration of myelin and subsequent exposure of inhibitory molecules to the injured axons contributes to their retraction/dieback.

Adolesc Med Clin, 2004 Jun, 15(2), 235 - 51
Vaginitis in adolescents; Syed TS et al.; Vaginitis is a common complaint of adolescent females . It can cause extreme distress for some patients, especially those with recurrent symptoms . Thus, it is important to take care when evaluating these patients and to acknowledge their frustration when appropriate . A thoughtful and thorough history will determine most causes, with the most common being yeast, trichomoniasis, and BV.

Appl Opt, 2004 Sep 1, 43(25), 4831 - 7
Optical manipulation in combination with multiphoton microscopy for single-cell studies; Goksor M et al.; We demonstrate how optical tweezers can be incorporated into a multiphoton microscope to achieve three-dimensional imaging of trapped cells . The optical tweezers, formed by a cw 1064 nm Nd:YVO4 laser, were used to trap live yeast cells in suspension while the 4',6-diamidino-2-phenylindole-stained nucleus was imaged in three dimensions by use of a pulsed femtosecond laser . The trapped cell was moved in the axial direction by changing the position of an external lens, which was used to control the divergence of the trapping laser beam . This gives us a simple method to use optical tweezers in the laser scanning of confocal and multiphoton microscopes . It is further shown that the same femtosecond laser as used for the multiphoton imaging could also be used as laser scissors, allowing us to drill holes in the membrane of trapped spermatozoa.

J Gen Virol, 2004 Oct, 85(Pt 10), 3135 - 47
Functional analysis of the Cucumber mosaic virus 2b protein: pathogenicity and nuclear localization; Wang Y et al.; The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor and pathogenicity determinant in solanaceous hosts, but a movement determinant in cucumber . In addition, synergistic interactions between CMV and Zucchini yellow mosaic virus (ZYMV) have been described in several cucurbit species . Here, it was shown that deletion of the 2b gene from CMV prevented extensive systemic movement of the virus in zucchini squash, which could not be complemented by co-infection with ZYMV . Thus, ZYMV expressing a silencing suppressor with a different target could not complement the CMV 2b-specific movement function . Expression of the 2b protein from an attenuated ZYMV vector resulted in a synergistic response, largely restoring infection symptoms of wild-type ZYMV in several cucurbit species . Deletion or alteration of either of two nuclear localization signals (NLSs) did not affect nuclear localization in two assays, but did affect pathogenicity in several cucurbit species, whilst deletion of both NLSs led to loss of nuclear localization . The 2b protein interacted with an Arabidopsis thaliana karyopherin alpha protein (AtKAPalpha) in the yeast two-hybrid system, as did each of the two single NLS-deletion mutants . However, 2b protein containing a deletion of both NLSs was unable to interact with AtKAPalpha . These data suggest that the 2b protein localizes to the nucleus by using the karyopherin alpha-mediated system, but demonstrate that nuclear localization was insufficient for enhancement of the 2b-mediated pathogenic response in cucurbit hosts . Thus, the sequences corresponding to the two NLSs must have another role leading to pathogenicity enhancement.

J Gen Virol, 2004 Oct, 85(Pt 10), 3115 - 22
Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA; Rubino L et al.; Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV . DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA . Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells . However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells . These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.

J Biol Chem, 2004 Dec 3, 279(49), 50857 - 63 Epub 2004 Dec 3.
Atf1-Pcr1-M26 complex links stress-activated MAPK and cAMP-dependent protein kinase pathways via chromatin remodeling of cgs2+; Davidson MK et al.; Although co-ordinate interaction between different signal transduction pathways is essential for developmental decisions, interpathway connections are often obscured and difficult to identify due to cross-talk . Here signals from the fission yeast stress-activated MAPK Spc1 are shown to regulate Cgs2, a negative regulator of the cAMP-dependent protein kinase (protein kinase A) pathway . Pathway integration is achieved via Spc1-dependent binding of Atf1-Pcr1 heterodimer to an M26 DNA site in the cgs2+ promoter, which remodels chromatin to regulate expression of cgs2+ and targets downstream of protein kinase A . This direct interpathway connection co-ordinates signals of nitrogen and carbon source depletion to affect a G0 cell-cycle checkpoint and sexual differentiation . The Atf1-Pcr1-M26 complex-dependent chromatin remodeling provides a molecular mechanism whereby Atf1-Pcr1 heterodimer can function differentially as either a transcriptional activator, or as a transcriptional repressor, or as an inducer of meiotic recombination . We also show that the Atf1-Pcr1-M26 complex functions as both an inducer and repressor of chromatin remodeling, which provides a way for various chromatin remodeling-dependent effector functions to be regulated.

Am J Clin Nutr, 2004 Oct, 80(4), 911 - 8
Determining bioavailability of food folates in a controlled intervention study; Hannon-Fletcher MP et al.; BACKGROUND: The concept of dietary folate equivalents (DFEs) in the United States recognizes the differences in bioavailability between natural food folates and the synthetic vitamin, folic acid . However, many published reports on folate bioavailability are problematic because of several confounding factors . OBJECTIVE: We compared the bioavailability of food folates with that of folic acid under controlled conditions . To broadly represent the extent to which natural folates are conjugated in foods, we used 2 natural sources of folate, spinach (50% polyglutamyl folate) and yeast (100% polyglutamyl folate) . DESIGN: Ninety-six men were randomly assigned according to their screening plasma homocysteine (tHcy) concentration to 1 of 4 treatment groups for an intervention period of 30 d . Each subject received (daily under supervision) either a folate-depleted "carrier" meal or a drink plus 1) placebo tablet, 2) 200 microg folic acid in a tablet, 3) 200 microg natural folate provided as spinach, or 4) 200 microg natural folate provided as yeast . RESULTS: Among the subjects who completed the intervention, responses (increase in serum folate, lowering of tHcy) relative to those in the placebo group (n = 18) were significant in the folic acid group (n = 18) but not in the yeast folate (n = 19) or the spinach folate (n = 18) groups . Both natural sources of folate were significantly less bioavailable than was folic acid . Overall estimations of folate bioavailability relative to that of folic acid were found to be between 30% (spinach) and 59% (yeast) . CONCLUSION: Relative bioavailability estimates were consistent with the estimates from the metabolic study that were used as a basis to derive the US DFE value.

Plant J, 2004 Oct, 40(2), 291 - 301
Formation of an SCF complex is required for proper regulation of circadian timing; Han L et al.; The circadian timing system involves an autoregulatory transcription/translation feedback loop that incorporates a diverse array of factors to maintain a 24-h periodicity . In Arabidopsis a novel F-box protein, ZEITLUPE (ZTL), plays an important role in the control of the free-running period of the circadian clock . As a class, F-box proteins are well-established components of the Skp/Cullin/F-box (SCF) class of E3 ubiquitin ligases that link the target substrates to the core ubiquitinating activity of the ligase complex via direct association with the Skp protein . Here we identify and characterize the SCFZTL complex in detail . Yeast two-hybrid tests demonstrate the sufficiency and necessity of the F-box domain for Arabidopsis Skp-like protein (ASK) interactions and the dispensability of the unique N-terminal LOV domain in this association . Co-immunoprecipitation of full-length (FL) ZTL with the three known core components of SCF complexes (ASK1, AtCUL1 and AtRBX1) demonstrates that ZTL can assemble into an SCF complex in vivo . F-box-containing truncated versions of ZTL (LOV-F and F-kelch) can complex with SCF components in vivo, whereas stably expressed LOV or kelch domains alone cannot . Stable expression of F-box-mutated FL ZTL eliminates the shortened period caused by mild ZTL overexpression and also abolishes ASK1 interaction in vivo . Reduced levels of the core SCF component AtRBX1 phenocopy the long period phenotype of ztl loss-of-function mutations, demonstrating the functional significance of the SCFZTL complex . Taken together, our data establish SCFZTL as an essential SCF class E3 ligase controlling circadian period in plants.

Chemphyschem, 2004 Aug 20, 5(8), 1159 - 70
Identification of biotic and abiotic particles by using a combination of optical tweezers and in situ Raman spectroscopy; Gessner R et al.; A highly versatile setup, which introduces an optical gradient trap into a Raman spectrometer, is presented . The particular configuration, which consists of two lasers, makes trapping independent from the Raman excitation laser and allows a separate adjustment of the trapping and excitation wavelengths . Thus, the excitation wavelength can be chosen according to the needs of the application . We describe the successful application of an optical gradient trap on transparent as well as on reflective, metal-coated microparticles . Raman spectra were recorded from optically trapped polystyrene beads and from single biological cells (e.g., erythrocytes, yeast cells) . Also, metal-coated microparticles were trapped and used as surface enhanced Raman spectroscopy (SERS) substrates for tests on yeast cells . Furthermore, the optical gradient trap was combined with a SERS fiber probe . Raman spectra were recorded from trapped red blood cells using the SERS fiber probe for excitation.

J Cell Biochem, 2004 Nov 15, 93(5), 1033 - 47
Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor; Rath U et al.; We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor . Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle . During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor . However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure . Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization . Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein . Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects . These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis . (c) 2004 Wiley-Liss, Inc.

Protein Sci, 2004 Oct, 13(10), 2829 - 31
Arm-domain interactions can provide high binding cooperativity; Schleif R et al.; Peptidyl arms extending from one protein domain to another protein domain mediate many important interactions in biology . A well-studied example of this type of protein-protein interaction occurs between the yeast homeodomain proteins, MAT alpha2 and MAT a1, which form a high-affinity heterodimer on DNA . The carboxyl-terminal arm extending from MAT alpha2 to MAT a1 has been proposed to produce an allosteric conformational change in the a1 protein that generates a very large increase in the DNA binding affinity of a1 . Although early studies lent some support to this model, a more recent crystal structure determination of the free a1 protein argues against any allosteric change . This note presents a thermodynamic argument that accounts for the proteins' binding behavior, so that allosteric conformational changes are not required to explain the large affinity increase . The analysis presented here should be useful in analyzing binding behavior in other systems involving arm interactions.

FEBS Lett, 2004 Sep 24, 575(1-3), 35 - 40
Association of the Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) with the p85 subunit of phosphoinositide 3-kinase; Shim EK et al.; To investigate additional functions of the T cell adaptor, Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD (SLP-76), we performed a yeast two-hybrid assay using the N-terminal region of SLP-76 fused with the kinase domain of Syk . By screening a human leukemia cDNA library, we identified the p85 subunit of phosphoinositide 3-kinase (PI3K) as one of the interacting molecules . Unlike the SH2 domain of Vav or Nck, tyrosine phosphorylation of SLP-76 at position 113 or 128 was sufficient for it to associate with the N-terminal SH2 of p85 . Collectively, these data suggest that SLP-76 may play a role in PI3K signaling pathways.

J Anim Physiol Anim Nutr (Berl), 2004 Oct, 88(9-10), 340 - 7
The selenium requirement of the puppy; Wedekind KJ et al.; Current selenium (Se) recommendations for the puppy are based on extrapolation from other species (0.11 mg Se/kg diet) . The purpose of this study was to experimentally determine the Se requirement in puppies . Thirty beagle puppies (average = 8.8 weeks old) were utilized in a randomized complete block design with age, litter and gender used as blocking criteria . Puppies were fed a low Se (0.04 mg Se/kg diet) torula yeast-based diet for 14 days (pre-test period) after which this same diet was supplemented with five levels of Na2SeO3 for 21 days (experimental period) to construct a response curve (0, 0.13, 0.26, 0.39 or 0.52 mg Se/kg diet) . Response variables included Se concentrations and Se-dependent glutathione peroxidase activities (GSHpx) in serum as well as serum total triiodothyronine (TT3), serum total thyroxine (TT4) and serum free T4 (FT4) . No significant changes in food intake and body weight gain occurred, and no clinical signs of Se deficiency were observed . A breakpoint for serum GSHpx could not be determined in our study due to analytical difficulties . A broken-line, two-slope response in serum Se occurred with a breakpoint at 0.17 mg Se/kg diet . When Se from the basal diet was added to this estimate, the breakpoint for serum Se equated to 0.21 mg Se/kg diet . TT3 increased linearly with increasing Se intake, whereas TT4 was unchanged . However, the ratio of TT4 : TT3 decreased linearly in response to supplemental Se . In summary, although we estimated the selenium requirement for the puppy based on serum Se, our 0.21 mg Se/kg diet estimate is higher than that seen for adult dogs, kittens, rats or poultry (0.13, 0.15, 0.15 and 0.15 mg Se/kg diet respectively) . This difference may be due to the fact that GSHpx was used as the biomarker of Se status.

EMBO J, 2004 Sep 29, 23(19), 3758 - 68 Epub 2004 Sep 23.
The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9; Monferran S et al.; The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair . Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood . From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components . Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies . In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides . Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.

J Biol Chem, 2004 Dec 17, 279(51), 53435 - 41 Epub 2004 Sep 22.
Eukaryotic CTR copper uptake transporters require two faces of the third transmembrane domain for helix packing, oligomerization, and function; Aller SG et al.; Members of the copper uptake transporter (CTR) family from yeast, plants, and mammals including human are required for cellular uptake of the essential metal copper . Based on biochemical data, CTRs have three transmembrane domains and have been shown to oligomerize in the membrane . Among individual members of the family, there is little amino acid sequence identity, raising questions as to how these proteins adopt a common fold, oligomerize, and participate in copper transport . Using site-directed mutagenesis, tryptophan scanning, genetic complementation, subcellular localization, chemical cross-linking, and the yeast unfolded protein response, we demonstrated that at least half of the third transmembrane domain (TM3) plays a vital role in CTR structure and function . The results of our analysis showed that TM3 contains two functionally distinct faces . One face bears a highly conserved Gly-X-X-X-Gly (GG4) motif, which we showed to be essential for CTR oligomerization . Moreover, we showed that steric constraints reach past the GG4-motif itself including amino acid residues that are not conserved throughout the CTR family . A second face of TM3 contains three amino acid positions that, when mutated to tryptophan, cause predominantly abnormal localization but are still partially functional in growth complementation experiments . These mutations cluster on the face opposite to the GG4-bearing face of TM3 where they may mediate interactions with the remaining two transmembrane domains . Taken together, our data support TM3 as being buried within trimeric CTR where it plays an essential role in CTR assembly.

Hum Mol Genet, 2004 Nov 15, 13(22), 2823 - 8 Epub 2004 Sep 22.
Evidence and characteristics of putative human alpha recombination hotspots; Zhang J et al.; Understanding recombination rate variation is very important for studying genome diversity and evolution, and for investigation of phenotypic association and genetic diseases . Recombination hotspots have been observed in many species and are well studied in yeast . Recent study demonstrated that recombination hotspots are also a ubiquitous feature of the human genome . But the nature of human hotspots remains largely unknown . We have developed and validated a novel computational method for testing the existence of hotspots as well as for localizing them with either unphased or phased genotyping data . To study the characteristics of hotspots within or close to genes, we scanned for unusually high levels of recombination using the European population samples in the SeattleSNPs database, and found evidence for the existence of human alpha hotspots similar to those of yeast . This type of hotspots, found at promoter regions, accounts for about half of the total detected and appears to depend on some specific transcription factor binding sites (such as CGCCCCCGC) . These characteristics can explain the observed weak correlation between hotspots and GC-content, and their variation may contribute to the diversity of hotspot distribution among different individuals and species . These long-sought putative human alpha recombination hotspots should deserve further experimental investigations.

Biol Reprod, 2005 Jan, 72(1), 188 - 94 Epub 2004 Sep 22.
CDC6 Requirement for Spindle Formation During Maturation of Mouse Oocytes; Anger M et al.; A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication . Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals . Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G(2)/M-phase transition . We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G(2)/M-phase border . Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis . The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.

Dev Biol, 2004 Oct 15, 274(2), 426 - 35
Enkurin is a novel calmodulin and TRPC channel binding protein in sperm; Sutton KA et al.; The TRPC cation channel family has been implicated in receptor- or phospholipase C (PLC)-mediated Ca2+ entry into animal cells . These channels are present in mammalian sperm and are assigned a role in ZP3-evoked Ca2+ influx that drives acrosome reactions . However, the mechanisms controlling channel activity and coupling Ca2+ entry through these channels to cellular responses are not well understood . A yeast two-hybrid screen was carried out to identify TRPC-interacting proteins that would be candidate regulators or effectors . We identified a novel protein, enkurin, that is expressed at high levels in the testis and vomeronasal organ and at lower levels in selected other tissues . Enkurin interacts with several TRPC proteins (TRPC1, TRPC2, TRPC5, but not TRPC3) and colocalizes with these channels in sperm . Three protein-protein interaction domains were identified in enkurin: a C-terminal region is essential for channel interaction; an IQ motif binds the Ca2+ sensor, calmodulin, in a Ca2+-dependent manner; and a proline-rich N-terminal region contains predicted ligand sequences for SH3 domain proteins, including the SH3 domain of the p85 regulatory subunit of 1-phosphatidylinositol-3-kinase . We suggest that enkurin is an adaptor that functions to localize a Ca2+ sensitive signal transduction machinery in sperm to a Ca2+-permeable ion channel.

J Econ Entomol, 2004 Aug, 97(4), 1269 - 77
Feeding activity and attraction of blueberry maggot (Diptera: Tephritidae) to protein baits, ammonium acetate, and sucrose; Barry JD et al.; Attraction and feeding assays were conducted on blueberry maggot, Rhagoletis mendax Curran, to three protein baits, ammonium acetate, and sucrose . Flies fed significantly longer on concentrations of 25 and 50% SolBait than they did on any of the concentrations tested for Nu-Lure, AY50% (Mauri Yeast Australia), or a water control . The number of flies arriving at SolBait in an attraction assay was significantly higher than for Nu-Lure and a water control but was not different from AY50% . Flies fed less on aqueous solutions of 1 and 4% ammonium acetate, a known fruit fly attractant, than they did on either 0.25% ammonium acetate or water . Aqueous concentrations of 8, 16, and 32% sucrose elicited greater feeding responses from flies than either 4% sucrose or water . These findings suggest that SolBait is a superior protein bait based on attraction and feeding assays . Development of alternative baits should contain at least 8% sucrose, as a significant feeding stimulant, and some amount of ammonium acetate as an attractant . Future work should determine whether the feeding deterrence of ammonium acetate could be reduced or even eliminated in the presence of sucrose.

PLoS Biol . 2004 Oct;2(10):e321 . Epub 2004 Sep 21.
Mechanism of prion propagation: amyloid growth occurs by monomer addition; Collins SR et al.; Abundant nonfibrillar oligomeric intermediates are a common feature of amyloid formation, and these oligomers, rather than the final fibers, have been suggested to be the toxic species in some amyloid diseases . Whether such oligomers are critical intermediates for fiber assembly or form in an alternate, potentially separable pathway, however, remains unclear . Here we study the polymerization of the amyloidogenic yeast prion protein Sup35 . Rapid polymerization occurs in the absence of observable intermediates, and both targeted kinetic and direct single-molecule fluorescence measurements indicate that fibers grow by monomer addition . A three-step model (nucleation, monomer addition, and fiber fragmentation) accurately accounts for the distinctive kinetic features of amyloid formation, including weak concentration dependence, acceleration by agitation, and sigmoidal shape of the polymerization time course . Thus, amyloid growth can occur by monomer addition in a reaction distinct from and competitive with formation of potentially toxic oligomeric intermediates.

J Cell Sci, 2004 Oct 1, 117(Pt 21), 5043 - 57 Epub 2004 Sep 21.
Smooth muscle archvillin: a novel regulator of signaling and contractility in vascular smooth muscle; Gangopadhyay SS et al.; The mechanisms by which protein kinase C (PKC) and extracellular-signal-regulated kinases (ERK1/2) govern smooth-muscle contractility remain unclear . Calponin (CaP), an actin-binding protein and PKC substrate, mediates signaling through ERK1/2 . We report here that CaP sequences containing the CaP homology (CH) domain bind to the C-terminal 251 amino acids of smooth-muscle archvillin (SmAV), a new splice variant of supervillin, which is a known actin- and myosin-II-binding protein . The CaP-SmAV interaction is demonstrated by reciprocal yeast two-hybrid and blot-overlay assays and by colocalization in COS-7 cells . In differentiated smooth muscle, endogenous SmAV and CaP co-fractionate and co-translocate to the cell cortex after stimulation by agonist . Antisense knockdown of SmAV in tissue inhibits both the activation of ERK1/2 and contractions stimulated by either agonist or PKC activation . This ERK1/2 signaling and contractile defect is similar to that observed in CaP knockdown experiments . In A7r5 smooth-muscle cells, PKC activation by phorbol esters induces the reorganization of endogenous, membrane-localized SmAV and microfilament-associated CaP into podosome-like structures that also contain F-actin, nonmuscle myosin IIB and ERK1/2 . These results indicate that SmAV contributes to the regulation of contractility through a CaP-mediated signaling pathway, involving PKC activation and phosphorylation of ERK1/2.

J Cell Sci, 2004 Oct 1, 117(Pt 21), 5023 - 33 Epub 2004 Sep 21.
Ubiquitin ligase Rad18Sc localizes to the XY body and to other chromosomal regions that are unpaired and transcriptionally silenced during male meiotic prophase; van der Laan R et al.; In replicative damage bypass (RDB) in yeast, the ubiquitin-conjugating enzyme RAD6 interacts with the ubiquitin ligase RAD18 . In the mouse, these enzymes are represented by two homologs of RAD6, HR6a and HR6b, and one homolog of RAD18, Rad18Sc . Expression of these genes and the encoded proteins is ubiquitous, but there is relatively high expression in the testis . We have studied the subcellular localization by immunostaining Rad18Sc and other RDB proteins in mouse primary spermatocytes passing through meiotic prophase in spermatogenesis . The highest Rad18Sc protein level is found at pachytene and diplotene, and the protein localizes mainly to the XY body, a subnuclear region that contains the transcriptionally inactivated X and Y chromosomes . In spermatocytes that carry translocations for chromosomes 1 and 13, Rad18Sc protein concentrates on translocation bivalents that are not fully synapsed . The partly synapsed bivalents are often localized in the vicinity of the XY body, and show a very low level of RNA polymerase II, indicating that the chromatin is in a silent configuration similar to transcriptional silencing of the XY body . Thus, Rad18Sc localizes to unsynapsed and silenced chromosome segments during the male meiotic prophase . All known functions of RAD18 in yeast are related to RDB . However, in contrast to Rad18Sc, expression of UBC13 and poleta, known to be involved in subsequent steps of RDB, appears to be diminished in the XY body and regions containing the unpaired translocation bivalents . Taken together, these observations suggest that the observed subnuclear localization of Rad18Sc may involve a function outside the context of RDB . This function is probably related to a mechanism that signals the presence of unsynapsed chromosomal regions and subsequently leads to transcriptional silencing of these regions during male meiotic prophase.

J Cell Sci, 2004 Oct 1, 117(Pt 21), 5013 - 22 Epub 2004 Sep 21.
A novel partner for Dictyostelium filamin is an alpha-helical developmentally regulated protein; Knuth M et al.; The filamins are a family of highly homologous actin-crosslinking proteins that stabilize three-dimensional actin networks, link them to membrane proteins and direct intracellular signaling reactions to the actin scaffold through interaction with various binding partners . Here, we describe the first Dictyostelium filamin-interacting protein to be isolated--FIP, a 229.8 kDa protein with two alpha-helical coiled coil domains . FIP was identified in a yeast two-hybrid screen using the rod domain of filamin as bait . FIP can also be coimmunoprecipitated with filamin from cellular extracts . Deletion analysis located the interaction domain of FIP to a C-terminal region; by contrast, in filamin rods, repeats 2-4 interacted with the recombinant FIP protein . The 7 kb transcript of FIP is upregulated during early development . Monoclonal antibodies raised against a bacterially expressed FIP polypeptide recognize a 230 kDa developmentally regulated protein in western blots . Immunofluorescence analysis shows a punctate staining pattern in the cytosol and, in cell fractionation experiments, FIP is mainly found in the cytosolic fraction . A fusion protein composed of GFP and the C-terminal part localizes to the plasma membrane and is associated with the cytoskeleton . Expression of the fusion protein affects development and influences the size of the multicellular aggregates and the phototactic behavior of slugs . Thus, FIP might provide a candidate link between the dynamic actin cytoskeleton and signal transduction events during the multicellular stages of Dictyostelium amoebae.

Exp Cell Res, 2004 Oct 15, 300(1), 121 - 33
Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts; Wegmann F et al.; Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ) . Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM . MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells . MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction . The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments . The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated . We found MAGI-1 to be associated with cell-cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM . In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM . Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.

Mol Cell, 2004 Sep 24, 15(6), 853 - 65
A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington's disease; Goehler H et al.; Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function . Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD) . The network contains 186 PPIs among 35 bait and 51 prey proteins . It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments . The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles . Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions . Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis .

J Virol Methods, 2004 Nov, 121(2), 247 - 57
A novel strategy for creating recombinant infectious RNA virus genomes; Fernandez-Delmond I et al.; Reverse transcriptases with RNase H activity are particularly apt to switch templates and generate recombinant molecules in vitro . This property has been exploited for the first time to create a library of recombinant RNAs 3 between two strains of Cucumber mosaic virus (CMV) or between CMV and Tomato aspermy virus (TAV), which share 75 and 63% sequence identity, respectively . The recombination events were almost entirely of the precise homologous type, and occurred at the same sites as those previously identified in co-infected plants, making it possible to use this strategy to create numerous cDNA fragments with crossovers similar to those occurring in vivo . Sub-cloning of recombinant fragments into an infectious full-length clone was accomplished by homologous recombination in yeast, alleviating the need for in vitro ligation at common restriction sites . Most of the recombinant genomes were infectious . Association of these two methods constitutes an efficient and practical means for generating numerous infectious viral genomes equivalent to ones that might arise by precise homologous recombination between two parental viral genomes in nature.

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 1084 - 90
Human kallikrein 13 involvement in extracellular matrix degradation; Kapadia C et al.; The human kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4 and shares a high degree of homology . These proteolytic enzymes have diverse physiological functions in many different tissues . Growing evidence suggests that many kallikreins are differentially expressed in cancer and may play a role in metastasis . Human kallikrein gene 13 (KLK13) is a member of this family and codes for a trypsin-like, secreted serine protease (hK13) that is overexpressed in ovarian cancer patients . The aim of this study was to determine if hK13 can degrade extracellular matrix components . Recombinant hK13 was produced in yeast and purified using cation exchange and reverse-phase chromatography . The protein was used as an immunogen to generate mouse monoclonal antibodies . Enzymatic activity of hK13 was verified by using synthetic tri-peptide fluorogenic substrates and gelatin zymography . Active hK13 was incubated with biotinylated extracellular matrix (ECM) proteins and degradation was evaluated by Western blot analysis . hK13-secreting cancer cell lines were treated in a chemotaxis invasion chamber that was coated with various ECM proteins, to determine if hK13 plays a role in tumor cell migration and invasion . Assay with the synthetic substrates and zymography have shown that recombinant hK13 was enzymatically active . The Western blot results showed that hK13 was able to cleave the major components of the extracellular matrix . In the chemotaxis invasion chamber experiment, it was found that ovarian cancer cell lines that secreted hK13 and were treated with an hK13 neutralizing antibody migrated less than untreated cells . Human kallikrein13 may play a role in tissue remodeling and/or tumor invasion and metastasis . Targeting hK13 activity with neutralizing antibodies may have therapeutic applications.

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 796 - 801
Characterisation of the NUCKS gene on human chromosome 1q32.1 and the presence of a homologous gene in different species; Grundt K et al.; The NUCKS gene is located on human chromosome 1q32.1 and consists of seven exons and six introns . The gene lacks a TATA box but contains two Inr elements, two GC boxes, and one consensus-binding site for E2F-1 . NUCKS is expressed in all human adult and foetal tissues investigated, and has all the features of being a housekeeping gene . Both data searches and Western immunoblotting experiments show that a homologous protein is present in fish, amphibians, and birds but not in insects and yeast, suggesting that NUCKS is a vertebrate specific gene . In all the species investigated, the protein contains several consensus phosphorylation sites for cyclin-dependent kinases and CK-2, and we have shown that the fish protein (like mammalian NUCKS) indeed is a substrate for CDK1 and CK-2 in vitro . The NUCKS protein is also conserved with respect to a DNA-binding domain previously characterised in mammals, and two putative bipartite nuclear localisation signals.

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 731 - 8
Characterization of the P5 subfamily of P-type transport ATPases in mice; Schultheis PJ et al.; In mammals, the most poorly understood P-type ATPases are those of the P(5) subfamily . To begin characterization of the mammalian P(5)-ATPases, BLAST searches of DNA sequence databases were performed . Five genes were identified in the mouse, human, dog, and rat genomes, and the coding sequences of the mouse genes, termed Atp13a1-Atp13a5, were determined . The intron/exon organization of Atp13a1 differs entirely from those of Atp13a2-5, which are closely related . Amino acid sequence comparisons between the five mouse and two yeast P(5)-ATPases suggest that Atp13a1 is orthologous to the yeast Cod1 gene and that Atp13a2-5 are orthologous to yeast Yor291w . Northern blot analysis showed that Atp13a1, Atp13a2, and Atp13a3 mRNAs were expressed in all mouse tissues, whereas Atp13a4 and Atp13a5 mRNAs were restricted to brain and stomach . While the substrate specificity of these transporters is unknown, their importance is underscored by the presence of homologs in fish, insects, worms, and other eukaryotes.

Biol Cell, 2004 Sep, 96(7), 509 - 17
Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B' regulatory subunit; Dozier C et al.; Chk2 is a key player of the DNA damage signalling pathway . To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B' regulatory subunit of protein phosphatase PP2A . In vitro GST-Chk2 pulldowns demonstrated that B'gamma isoforms bound to Chk2 with the strongest apparent affinity . This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells . The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A . In vitro kinase assays showed that B'gamma3 was a potent Chk2 substrate . This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2 . Finally, we demonstrated that overexpressing B'gamma3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells .

Chem Biol, 2004 Sep, 11(9), 1269 - 77
Amphidinolide h, a potent cytotoxic macrolide, covalently binds on actin subdomain 4 and stabilizes actin filament; Usui T et al.; The actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs . Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton . AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro . AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins . Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers . These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin.

DNA Repair (Amst), 2004 Nov 2, 3(11), 1503 - 14
Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein; Tissier A et al.; The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome . One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site . This process entails DNA polymerase switch between replicative and specialized DNA polymerases . Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1 . To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen . In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota . Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures . This hREV1 nuclear localization occurs independently of the presence of hpol eta . Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.

Curr Biol, 2004 Sep 21, 14(18), R754 - 6
Protein degradation: recognition of ubiquitinylated substrates; Hartmann-Petersen R et al.; A cell-free system has been developed in budding yeast that provides direct evidence that the Dsk2/Dph1, Rad23/Rhp23 and Rpn10/Pus1 multi-ubiquitin-binding proteins, long implicated in substrate recognition and presentation to the 26S proteasome, actually fulfil such a role.

Biochemistry, 2004 Sep 28, 43(38), 12113 - 22
Fetal Alz-50 clone 1 interacts with the human orthologue of the Kelch-like Ech-associated protein; Strachan GD et al.; The fetal Alz-50 reactive clone 1 (FAC1) protein exhibits altered expression and subcellular localization during neuronal development and neurodegenerative diseases such as Alzheimer's disease . Using the yeast two-hybrid screen, the human orthologue of Keap1 (hKeap1) was identified as a FAC1 interacting protein . Keap1 is an important regulator of the oxidative stress response pathway through its interaction with the Nrf family of transcription factors . An interaction between full-length FAC1 and hKeap1 proteins has been demonstrated, and the FAC1 binding domain of hKeap1 has been identified as the Kelch repeats . In addition, FAC1 colocalizes with endogenous Keap1 within the cytoplasm of PT67 cells . Exogenously introduced eGFP:hKeap1 fusion protein redistributed FAC1 to colocalize with eGFP:hKeap1 in perinuclear, spherical structures . The interaction between FAC1 and hKeap1 is reduced by competition with the Nrf2 protein . However, competition by Nrf2 for hKeap1 is reduced by diethylmaleate (DEM), a known disrupter of the Nrf2:Keap1 interaction . DEM does not affect the ability of FAC1 to bind hKeap1 in our assay . These results suggest that hKeap1 regulates FAC1 in addition to its known role in control of Nrf2 . Furthermore, the observed competition between FAC1 and Nrf2 for binding hKeap1 indicates that the interplay between these three proteins has important implications for neuronal response to oxidative stress.

Proteomics, 2004 Dec, 4(12), 3765 - 75
A mass spectrometric "Western blot" to evaluate the correlations between histone methylation and histone acetylation; Zhang K et al.; Histone acetylation, methylation, and phosphorylation occur predominantly in the unstructured N-terminal domains or histone "tails" . These modifications and others comprise a "histone code" that directly facilitates or antagonizes association of regulatory proteins with nucleosomes to mediate changes in chromatin structure and activity . Methylation of histone H3 outside of the tail region at lysine 79 has been reported for a variety of species ranging from yeast to humans and in some gene-specific cases appears to be associated with active chromatin and transcription . Whether methylation of lysine 79 is associated with other post-translational modifications of the H3 tail is unknown . Using mass spectrometric relative quantitation, a mass spectrometric "Western blot", we compare methylation at lysines 4, 9, and 79 with acetylation of human histone H3 . We find that the total levels of lysine 4 and 79 methylation (combined mono-, di-, and trimethylation) in the H3 population increase with the degree of H3 tail acetylation . The total amount of lysine 4 methylation increases progressively from less than 10% in the nonacetylated H3 to greater than 90% in the penta-acetylated H3 . In addition, significant levels of lysine 4 trimethylation also occur in combination with the penta-acetylated H3 species . In contrast, the level of H3 lysine 9 trimethylation is greatest for the monoacetylated species while H3 lysine 9 acetylation occurs predominantly in hyperacetylated (tetra- and penta-acetylated) H3 isoforms . Together, these results indicate that methylation of lysine 4 and 79 as well as the switch from lysine 9 methylation to acetylation are coordinated synchronously with H3 hyperacetylation as marks of active chromatin.

J Cell Biochem, 2004 Oct 15, 93(3), 579 - 87
WDR26: a novel Gbeta-like protein, suppresses MAPK signaling pathway; Zhu Y et al.; WD40 repeat proteins play important roles in a variety of cellular functions, including cell growth, proliferation, apoptosis, and intracellular signal transduction . Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation . Previous studies revealed that G-protein coupled receptors (GPCRs) play important roles in the signal transduction from extracellular stimuli to MAPKs and the WD40-containing Gbeta proteins as well as Gbeta-like proteins are involved in the stimulation and regulation of the MAPK signaling pathways . Here we report the identification and characterization of a novel human WD40 repeat protein, WD40 repeat protein 26 (WDR26) . The cDNA of WDR26 is 3,729 bp, encoding a Gbeta-like protein of 514 amino acids in the cytoplasm . The protein is highly conserved in evolution across different species from yeast, Drosophila, mouse, to human . Northern blot analysis indicates that WDR26 is expressed in most of the examined human tissues, especially at a high level in skeletal muscle . Overexpression of WDR26 in the cell inhibits the transcriptional activities of ETS proteins, ELK-1 and c-fos serum response element (SRE), mediated by MEKK1 . These results suggest that WDR26 may act as a negative regulator in MAPK signaling pathway and play an important role in cell signal transduction .

Am J Med Genet, 2004 Oct 15, 130A(3), 288 - 94
Supernumerary ring chromosome 8: clinical and molecular cytogenetic characterization in a case report; Demori E et al.; We report on a 3-year-old male with developmental delay, autistic behavior, and minor abnormalities consistent with trisomy 8 syndrome whose cytogenetic analysis revealed mosaicism for a supernumerary ring chromosome (SRC) . Fluorescence in situ hybridization (FISH) studies, using centromeric and yeast artificial chromosome (YAC) probes, were performed to characterize further the supernumerary chromosome . The ring origin has been detected from the short arm of chromosome 8, resulting in r(8)(p10p23.1) . Moreover, uniparental disomy (UPD) using microsatellite analysis was excluded . To our knowledge a total of 25 cases, confirmed by FISH, have been reported with either supernumerary marker or ring chromosome 8 . We present a detailed clinical and molecular cytogenetic characterization of this additional case in order to better define the genotype-phenotype correlation.

J Basic Microbiol, 2004, 44(5), 331 - 8
Decolourisation of diverse industrial dyes by some Phlebia spp . and their comparison with Phanerochaete chrysosporium; Arora DS et al.; Three species of Phlebia, viz . P . brevispora, P . fascicularia and P . floridensis have been evaluated for their potential to decolourise eight industrial dyes including; reactive yellow, reactive orange, reactive red, rathidol scarlet, coracryl black, coracryl pink, coracryl violet and coracryl red . The cultures used for the present study were pre adapted by growing these on yeast glucose agar medium supplemented with Poly-R 478, a reference dye . The fungal cultures were grown in mineral salts broth and harvested after different incubation periods to obtain their cell free enzyme extracts which were then used to assess their ability to decolourise the above mentioned dyes . The extracts obtained from the cultures grown for six days significantly decolourised the tested dyes . The study revealed Phlebia spp . to be better dye decolourisers than Phanerochaete chrysosporium .

Nat Struct Mol Biol, 2004 Oct, 11(10), 968 - 74 Epub 2004 Sep 19.
A phenylalanine zipper mediates APS dimerization; Dhe-Paganon S et al.; The APS, SH2-B and LNK proteins are adapters that activate and modulate receptor tyrosine kinase and JAK/STAT signaling . We now show that a conserved N-terminal domain mediates APS homodimerization . We determined the crystal structure of the dimerization domain at a resolution of 1.7 A using bromide ion MAD phasing . Each molecule contributes two helices to a compact four-helix bundle having a bisecting-U topology . Its most conspicuous feature is a stack of interdigitated phenylalanine side chains at the domain core . These residues create a new motif we refer to as a 'phenylalanine zipper,' which is critical to dimerization . A newly developed bridging yeast tri-hybrid assay showed that APS dimerizes JAK2, insulin receptor and IGF1 receptor kinases using its SH2 and dimerization domains . Dimerization via the phenylalanine zipper domain provides a mechanism for activating and modulating tyrosine kinase activity even in the absence of extracellular ligands.

Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14725 - 30 Epub 2004 Sep 17.
Insertional assembly of actin filament barbed ends in association with formins produces piconewton forces; Kovar DR et al.; Formins are large multidomain proteins required for assembly of actin cables that contribute to the polarity and division of animal and fungal cells . Formin homology-1 (FH1) domains bind profilin, and highly conserved FH2 domains nucleate actin filaments . We characterized the effects of two formins, budding yeast Bni1p and fission yeast Cdc12p, on actin assembly . We used evanescent wave fluorescence microscopy to observe assembly of actin filaments (i) nucleated by soluble formin FH1FH2 domains and (ii) associated with formin FH1FH2 domains immobilized on microscope slides . Bni1p(FH1FH2)p and Cdc12p(FH1FH2)p nucleated new actin filaments or captured the barbed ends of preformed actin filaments that grew by insertion of subunits between the immobilized formin and the barbed end of the filament . Both formins remained bound to growing actin filament barbed ends for >1,000 sec . Elongation of a filament between an immobilized formin and a second anchor point buckled filament segments as short as 0.7 microm, demonstrating that polymerization of single actin filaments produces forces of >1 piconewton, close to the theoretical maximum . After buckling, further growth produced long loops that did not supercoil, suggesting that formins do not stair step along the two subunits exposed on the growing barbed end . In agreement, Arp2/3 complex branched filaments did not rotate as they grew from formins attached to the slide surface . Formins are not mechanistically identical because barbed end elongation from Cdc12(FH1FH2)p, but not Bni1(FH1FH2)p, requires profilin . However, profilin increased the rate of Bni1(FH1FH2)p-mediated barbed end elongation from 75% to 100% of full-speed.

Protein Eng Des Sel, 2004 Aug, 17(8), 635 - 46 Epub 2004 Aug.
Characterization of the UDP-N-acetylgalactosamine binding domain of bovine polypeptide alphaN-acetylgalactosaminyltransferase T1; Duclos S et al.; UDP-GalNAc:polypeptide alphaN-acetylgalactosaminyltransferases (ppGaNTases) transfer GalNAc from UDP-GalNAc to Ser or Thr . Structural features underlying their enzymatic activity and their specificity are still unidentified . In order to get some insight into the donor substrate recognition, we used a molecular modelling approach on a portion of the catalytic site of the bovine ppGaNTase-T1 . Fold recognition methods identified as appropriate templates the bovine alpha1,3galactosyltransferase and the human alpha1,3N-acetylgalactosaminyltransferase . A model of the ppGaNTase-T1 nucleotide-sugar binding site was built into which the UDP-GalNAc and the Mn2+ cation were docked . UDP-GalNAc fits best in a conformation where the GalNAc is folded back under the phosphates and is maintained in that special conformation through hydrogen bonds with R193 . The ribose is found in van der Waals contacts with F124 and L189 . The uracil is involved in a stacking interaction with W129 and forms a hydrogen bond with N126 . The Mn2+ is found in coordination both with the phosphates of UDP and the DXH motif of the enzyme . Amino acids in contact with UDP-GalNAc in the model have been mutated and the corresponding soluble forms of the enzyme expressed in yeast . Their kinetic constants confirm the importance of these amino acids in donor substrate interactions.

Plant Cell, 2004 Oct, 16(10), 2795 - 808 Epub 2004 Sep 17.
Spotted leaf11, a negative regulator of plant cell death and defense, encodes a U-box/armadillo repeat protein endowed with E3 ubiquitin ligase activity; Zeng LR et al.; The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens . Here, we have isolated Spl11 via a map-based cloning strategy . The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection . The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively . Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions . A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein . Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice-blast interactions . In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.

Bioinformatics, 2005 Jan 15, 21(2), 227 - 38 Epub 2004 Sep 17.
Superiority of network motifs over optimal networks and an application to the revelation of gene network evolution; Ott S et al.; MOTIVATION: Estimating the network of regulative interactions between genes from gene expression measurements is a major challenge . Recently, we have shown that for gene networks of up to around 35 genes, optimal network models can be computed . However, even optimal gene network models will in general contain false edges, since the expression data will not unambiguously point to a single network . RESULTS: In order to overcome this problem, we present a computational method to enumerate the most likely m networks and to extract a widely common subgraph (denoted as gene network motif) from these . We apply the method to bacterial gene expression data and extensively compare estimation results to knowledge . Our results reveal that gene network motifs are in significantly better agreement to biological knowledge than optimal network models . We also confirm this observation in a series of estimations using synthetic microarray data and compare estimations by our method with previous estimations for yeast . Furthermore, we use our method to estimate similarities and differences of the gene networks that regulate tryptophan metabolism in two related species and thereby demonstrate the analysis of gene network evolution . AVAILABILITY: Commercial license negotiable with Gene Networks Inc . (cherkis@gene-networks.com) CONTACT: sascha-ott@gmx.net.

Annu Rev Plant Biol, 2004 Jun 2, 55, 373 - 399
REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction; Apel K et al.; Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism . Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms . Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior . This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions . New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants . Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

Cell Biochem Funct . 2004 Sep 17; {Epub ahead of print}
EP24.15 interacts with the angiotensin II type I receptor and bradykinin B(2) receptor; Shivakumar BR et al.; The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 receptor (AT(1)) is known to interact with several classes of intracellular proteins that may modulate receptor function . Employing yeast two-hybrid screening of a human embryonic kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the AT(1) receptor as a bait, we have isolated EP24.15 (EC 3.4.24.15, thimet oligopeptidase) as a potentially interacting protein . EP24.15 is widely distributed and is known to degrade bioactive peptides such as angiotensin I and II and bradykinin . In addition, EP24.15 was previously identified as a putative soluble angiotensin II binding protein . Two-hybrid screening also determined that EP24.15 can interact with the B(2) bradykinin receptor . Transient expression of EP24.15 in a porcine kidney epithelial cell line stably expressing full length AT(1) and full length B(2) followed by affinity chromatography and co-immunoprecipitation confirmed EP24.15 association with both AT(1) and B2 receptors . EP24.15 was also co-immunoprecipitated with AT(1) and B(2) in rat kidney brush border membranes (BBM) and basolateral membranes (BLM) . Both AT(1) and B(2) undergo ligand-induced endocytosis . Analysis of endosomal fractions following immunoprecipitation with AT(1) or B(2) antibodies detected strong association of EP24.15 with the receptors in both light and heavy endosomal populations . Therefore, the present study indicates that EP24.15 associates with AT(1) and B(2) receptors both at the plasma membrane and after receptor internalization and suggests a possible mechanism for endosomal disposition of ligand that may facilitate receptor recycling .

Chromosoma, 2004 Oct, 113(4), 177 - 87 Epub 2004 Jul 30.
The Drosophila meiotic kleisin C(2)M functions before the meiotic divisions; Heidmann D et al.; Stepwise and regionally controlled resolution of sister chromatid cohesion is thought to be crucial for faithful chromosome segregation during meiotic divisions . In yeast, the meiosis-specific alpha-kleisin subunit of the cohesin complex, Rec8, is protected from cleavage by separase but only during meiosis I and specifically within the pericentromeric region . While the Drosophila genome does not contain an obvious Rec8 orthologue, as other animal and plant genomes, it includes c(2)M, which encodes a distant alpha-kleisin family member involved in female meiosis . C(2)M associates in vivo with the Smc3 cohesin subunit, as previously shown for yeast Rec8 . In contrast to Rec8, however, C(2)M accumulates predominantly after the pre-meiotic S-phase . Moreover, after association with the synaptonemal complex, it disappears again and cannot be detected on meiotic chromosomes by metaphase I . C(2)M cleavage fragments are not observed during completion of the meiotic divisions, and mutations within putative separase cleavage sites do not interfere with meiotic chromosome segregation . Therefore, C(2)M appears to function within the synaptonemal complex during prophase I but possibly not thereafter . This suggests that C(2)M may not confer sister chromatid cohesion needed for meiosis I and II chromosome segregation.

J Biol Chem, 2004 Oct 29, 279(44), 45308 - 11 Epub 2004 Sep 16.
p150(Glued), Dynein, and microtubules are specifically required for activation of MKK3/6 and p38 MAPKs; Cheung PY et al.; To look for regulators of the mitogen-activated protein kinase (MAPK) kinase 6 (MKK6), a yeast two-hybrid screen was initiated using MKK6 as bait . p150(Glued) dynactin, a key component of the cytoplasmic dynein-dynactin motor complex, was found to specifically interact with MKK6 and its close homologue MKK3 . Silencing of p150(Glued) expression by small interference RNA reduced the stimulus-induced phosphorylation of MKK3/6 and p38 MAPKs . The similar adverse effect was also seen when the cytoplasmic dynein motor was disrupted by other means . Like p150(Glued), MKK3/6 directly associate with microtubules . Disruption of microtubules prior to cell stimulation specifically inhibits the stimulus-induced phosphorylation of both MKK3/6 and p38 MAPKs . Our unexpected findings reveal a specific requirement for p150(Glued)/dynein/functional microtubules in activation of MKK3/6 and p38 MAPKs in vivo.

Bioinformatics . 2004 Sep 16; {Epub ahead of print}
Identifying time-lagged gene clusters on gene expression data; Ji L et al.; MOTIVATION: Analysis of gene expression data can provide insights into the time-lagged co-regulations of genes/gene clusters . However, existing methods such as Event Method and Edge Detection Method are inefficient as they only compare two genes each time . More importantly, they lose some important information due to their scoring criterion . In this paper, we propose an efficient algorithm to identify time-lagged co-regulated gene clusters . The algorithm facilitates localized comparison and processes several genes simultaneously to generate detailed and complete time-lagged information between genes/gene clusters . RESULTS: We experimented with the time series Yeast gene dataset and compared our scheme with the Event Method . Our results show that our scheme is not only efficient, but delivers more reliable and detailed information of time-lagged co-regulation between genes/gene clusters . AVAILABILITY: The software is available upon request . SUPPLEMENTARY INFORMATION: Supplementary tables and figures for this paper can be found at http://www.comp.nus.edu.sg/~jiliping/p2.htm.

Peptides, 2004 Sep, 25(9), 1477 - 90
Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies; Chapman T et al.; The study of insect seminal fluid proteins provides a unique window upon adaptive evolution in action . The seminal fluid of Drosophila melanogaster contains over 80 proteins and peptides, which are transferred together with sperm by mating males . The functions of many of these substances are not yet known . However, those that have been characterized have marked effects on the reproductive success of males and females . For example, seminal fluid proteins and peptides can decrease female receptivity, can increase egg production and can increase sperm storage, and are necessary for sperm transfer and success in sperm competition . In this review we focus on the currently known functions of seminal fluid molecules and on new technologies and approaches that are enabling novel questions about their form and function to be addressed . We discuss how techniques for disrupting the production of seminal fluid proteins, such as homologous recombination and RNA interference, along with the use of microarrays and yeast two hybrid systems, should allow us to address ever more sophisticated questions about seminal fluid protein function . These and similar techniques promise to reveal the function of naturally-occurring variants of these proteins and hence the evolutionary significance of genetic variation for them.

J Ethnopharmacol, 2004 Nov, 95(1), 83 - 5
Evaluation of anti-pyretic and analgesic activity of Emblica officinalis Gaertn; Perianayagam JB et al.; The present study was designed to investigate the anti-pyretic and analgesic activity of ethanol (EEO) and aqueous (AEO) extracts of Emblica officinalis fruits in several experimental models . A single oral dose of EEO and AEO (500 mg/kg, i.p.) showed significant reduction in brewer's yeast induced hyperthermia in rats . EEO and AEO also elicited pronounced inhibitory effect on acetic acid-induced writhing response in mice in the analgesic test . Both, EEO and AEO did not show any significant analgesic activity in the tail-immersion test . These findings suggest that extracts of Emblica officinalis fruits possessed potent anti-pyretic and analgesic activity . Preliminary phytochemical screening of the extracts showed the presence of alkaloids, tannins, phenolic compounds, carbohydrates and amino acids, which may be responsible for anti-pyretic and analgesic activities.

EMBO J, 2004 Sep 29, 23(19), 3747 - 57 Epub 2004 Sep 16.
Genome-wide lethality screen identifies new PI4,5P(2) effectors that regulate the actin cytoskeleton; Audhya A et al.; To further understand the roles played by the essential phosphoinositide PI4,5P(2), we have used a synthetic lethal analysis, which systematically combined the mss4(ts) mutation, partially defective in PI4P 5-kinase activity, with each of approximately 4700 deletion mutations . This genomic screening technique uncovered numerous new candidate effectors and regulators of PI4,5P(2) in yeast . In particular, we identified Slm1 (Yil105c), a previously uncharacterized PI4,5P(2) binding protein . Like Mss4, Slm1 and its homolog Slm2 (Ynl047c) were required for actin cytoskeleton polarization and viability . Co-immunoprecipitation experiments revealed that Slm1 interacts with a component of TORC2, a Tor2 kinase-containing complex, which also regulates the actin cytoskeleton . Consistent with these findings, phosphorylation of Slm1 and Slm2 was dependent on TORC2 protein kinase activity, both in vivo and in vitro, and Slm1 localization required both PI4,5P(2) and functional TORC2 . Together, these data suggest that Slm1 and Slm2 function downstream of PI4,5P(2) and the TORC2 kinase pathway to control actin cytoskeleton organization.

Mol Biol Cell, 2004 Dec, 15(12), 5383 - 98 Epub 2004 Dec.
Stress granule assembly is mediated by prion-like aggregation of TIA-1; Gilks N et al.; TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress . The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD) . Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells . The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation . Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs . Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite . Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.

J Biol Chem, 2004 Nov 19, 279(47), 49251 - 8 Epub 2004 Sep 14.
A novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis; Li AL et al.; Although eukaryotic translation initiation factor 5A (eIF5A) was originally designated as an "initiation factor," recent data have shown it to be also involved in apoptosis . However, the actual function of eIF5A in apoptosis is still unknown . In this study, we performed yeast two-hybrid screens to identify eIF5A-interacting proteins to help us understand the mechanisms of eIF5A . Our results demonstrated that eIF5A and syntenin could engage in a specific interaction both in vitro and in vivo and functioned collaboratively to regulate p53 activity . Our findings, for the first time, revealed a new biological activity for eIF5A as the regulator of p53 . Overexpression of eIF5A or its EFP domain resulted in up-regulation of p53, and silencing eIF5A by small interfering RNA reduced the p53 protein level . Further analysis by reverse transcription PCR showed eIF5A-activated p53 transcription . The effect of eIF5A on p53 transcriptional activity was further demonstrated by the increasing expressions of p21 and Bax, well known target genes of p53 . In contrast, a point mutant of eIF5A, hypusination being abolished, was revealed to be functionally defective in p53 up-regulation . Overexpression of eIF5A led to a p53-dependent apoptosis or sensitized cells to induction of apoptosis by chemotherapeutic agents . However, when eIF5A interacted with its novel partner, syntenin, the eIF5A-induced increase in p53 protein level was significantly inhibited . Therefore, eIF5A seems to be a previously unrecognized regulator of p53 that may define a new pathway for p53-dependent apoptosis, and syntenin might regulate p53 by balancing the regulation of eIF5A signaling to p53 for apoptosis.

Genes Dev, 2004 Sep 15, 18(18), 2269 - 82
Identification of a novel telomerase repressor that interacts with the human papillomavirus type-16 E6/E6-AP complex; Gewin L et al.; The critical immortalizing activity of the human papillomavirus (HPV) type-16 E6 oncoprotein is to induce expression of hTERT, the catalytic and rate-limiting subunit of telomerase . Additionally, E6 binds to a cellular protein called E6-associated protein (E6-AP) to form an E3 ubiquitin ligase that targets p53 for proteasome-dependent degradation . Although telomerase induction and p53 degradation are separable and distinct functions of E6, binding of E6 to E6-AP strongly correlated with the induction of hTERT . Here, we demonstrate using shRNAs to reduce E6-AP expression that E6-AP is required for E6-mediated telomerase induction . A yeast two-hybrid screen to find new targets of the E6/E6-AP E3 ubiquitin ligase complex identified NFX1 . Two isoforms of NFX1 were found: NFX1-123, which coactivated with c-Myc at the hTERT promoter, and NFX1-91, which repressed the hTERT promoter . NFX1-91 was highly ubiquitinated and destabilized in epithelial cells expressing E6 . Furthermore, knockdown of NFX1-91 by shRNA resulted in derepression of the endogenous hTERT promoter and elevated levels of telomerase activity . We propose that the induction of telomerase by the HPV-16 E6/E6-AP complex involves targeting of NFX1-91, a newly identified repressor of telomerase, for ubiquitination and degradation.

Development, 2004 Oct, 131(20), 4965 - 75 Epub 2004 Sep 15.
The Arabidopsis thaliana SNF2 homolog AtBRM controls shoot development and flowering; Farrona S et al.; Chromatin remodeling is essential for the reprogramming of transcription associated with development and cell differentiation . The SWI/SNF complex was the first chromatin remodeling complex characterized in yeast and Drosophila . In this work we have characterized an Arabidopsis thaliana homolog of Brahma, the ATPase of the Drosophila SWI/SNF complex . As its Drosophila counterpart, Arabidopsis thaliana BRAHMA (AtBRM) is a nuclear protein present in a high molecular mass complex . Furthermore, the N terminus of AtBRM interacts, in the two-hybrid system, with CHB4 (AtSWI3C), an Arabidopsis homolog of the yeast SWI/SNF complex subunit SWI3 . The AtBRM gene is primarily expressed in meristems, organ primordia and tissues with active cell division . Silencing of the expression of the AtBRM gene by RNA interference demonstrated that AtBRM is required for vegetative and reproductive development . The AtBRM silenced plants exhibited a reduction in overall plant size with small and curled leafs, as well as a reduction in the size of the inflorescence meristem . In the absence of AtBRM, Arabidopsis flowers have small petals and stamens, immature anthers, homeotic transformations and reduced fertility . The AtBRM silenced plants flower earlier than wild-type plants both under inductive and non-inductive photoperiods . Furthermore, levels of CO, FT and SOC1 transcripts were up-regulated under non-inductive conditions suggesting that AtBRM is a repressor of the photoperiod-dependent flowering pathway.

Acta Derm Venereol, 2004, 84(5), 339 - 45
Malassezia sympodialis stimulation differently affects gene expression in dendritic cells from atopic dermatitis patients and healthy individuals; Gabrielsson S et al.; It is known that 28-84% of patients with atopic dermatitis exhibit IgE and/or T-cell reactivity to the opportunistic yeast Malassezia sympodialis, which can be taken up by immature monocyte-derived dendritic cells (MDDCs), resulting in MDDC maturation . The aim of this study was to investigate whether MDDCs from patients with atopic dermatitis respond differently to M . sympodialis compared to MDDCs from healthy individuals . Immature MDDCs were stimulated with M . sympodialis and the gene expression profiles were analysed with cDNA arrays containing 406 genes . Our results show that M . sympodialis differently affected MDDCs from patients with atopic dermatitis, and more so in severely ill patients, compared with healthy individuals . Six genes were more than fivefold up-regulated in MDDCs from more than one patient with atopic dermatitis, coding for CD54, CD83, IL-8, monocyte-derived chemokine (MDC), BTG1 and IL-1R antagonist . In healthy individuals this was true only for BTG1 . Up-regulations of IL-8 and MDC were confirmed at the protein level . Our findings might reflect an increased trafficking and stimulatory capacity in MDDCs from the patients, which is likely to result in a stronger inflammatory response to M . sympodialis.

Arch Biochem Biophys, 2004 Oct 15, 430(2), 198 - 209
Membrane lipid biosynthesis in Chlamydomonas reinhardtii: ethanolaminephosphotransferase is capable of synthesizing both phosphatidylcholine and phosphatidylethanolamine; Yang W et al.; Phosphatidylethanolamine, but not phosphatidylcholine, is found in Chlamydomonas reinhardtii . A cDNA coding for diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase (EPT) was cloned from C . reinhardtii . The C . reinhardtii EPT appears phylogenetically more similar to mammalian aminoalcoholphosphotransferases than to those of yeast and the least close to those of plants . Similar membrane topography was found between the C . reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast, and plants . A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C . reinhardtii EPT gene . Enzymatic assays of C . reinhardtii EPT from the complemented yeast microsomes demonstrated that the C . reinhardtii EPT synthesized both PC and PE in the transformed yeast . The addition of either unlabeled CDP-ethanolamine or CDP-choline to reactions reduced incorporation of radiolabeled CDP-choline and radiolabeled CDP-ethanolamine into phosphatidylcholine and phosphatidylethanolamine . EPT activity from the transformed yeast or C . reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine, and CMP when {14C}CDP-choline was used as the primary substrate, but differentially by unlabeled CDP-choline and CDP-ethanolamine when {14C}CDP-ethanolamine was the primary substrate . The Km value of the enzyme for CDP-choline was smaller than that for CDP-ethanolamine . This provides evidence that C . reinhardtii EPT, similar to plant aminoalcoholphosphotransferase, is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine in the Kennedy pathway.

Biochem Biophys Res Commun, 2004 Oct 15, 323(2), 499 - 504
Regulation of Dyrk1A kinase activity by 14-3-3; Kim D et al.; Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome . In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity . We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p . We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo . The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status . Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro . In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7 . In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3 .

Cell, 2004 Sep 17, 118(6), 715 - 29
Mis16 and Mis18 are required for CENP-A loading and histone deacetylation at centromeres; Hayashi T et al.; Centromeres contain specialized chromatin that includes the centromere-specific histone H3 variant, spCENP-A/Cnp1 . Here we report identification of five fission yeast centromere proteins, Mis14-18 . Mis14 is recruited to kinetochores independently of CENP-A, and, conversely, CENP-A does not require Mis14 to associate with centromeres . In contrast, Mis15, Mis16 (strong similarity with human RbAp48 and RbAp46), Mis17, and Mis18 are all part of the CENP-A recruitment pathway . Mis15 and Mis17 form an evolutionarily conserved complex that also includes Mis6 . Mis16 and Mis18 form a complex and maintain the deacetylated state of histones specifically in the central core of centromeres . Mis16 and Mis18 are the most upstream factors in kinetochore assembly as they can associate with kinetochores in all kinetochore mutants except for mis18 and mis16, respectively . RNAi knockdown in human cells shows that Mis16 function is conserved as RbAp48 and RbAp46 are both required for localization of human CENP-A.

Chembiochem, 2004 Sep 6, 5(9), 1256 - 66
Binding mode of TMC-95A analogues to eukaryotic 20S proteasome; Kaiser M et al.; The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy . The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities . The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities . X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome . The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically . However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites . This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.

Mol Cell Biol, 2004 Oct, 24(19), 8556 - 66
Subcellular localization of RPB5-mediating protein and its putative functional partner; Delgermaa L et al.; We previously identified a novel cellular protein, RPB5-mediating protein (RMP), that retains corepressor activity and functionally antagonizes transcriptional modulation via hepatitis B virus X protein . The subcellular localization of RMP was examined using green fluorescent protein-fused protein forms . We found that a nuclear localization signal (NLS) and a coiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS) are important for the subcellular localization of RMP . The CLS apparently acts dominantly, since RMP was mostly localized in the cytoplasm with weak and diffuse signals in the nucleus, and the NLS was indispensable for the nuclear localization of RMP only in the absence of the CLS . Using a yeast two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1-associating protein (DMAP1), which was found to bind to the CC domain of RMP . DMAP1 facilitated the nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent manner by interacting with the CC domain of RMP . These results are discussed in light of a recent paper showing a novel evolutionarily conserved role of URI in the TOR signaling pathway.

J Virol, 2004 Oct, 78(19), 10574 - 81
Ring finger protein ZIN interacts with human immunodeficiency virus type 1 Vif; Feng F et al.; Virion infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary human CD4 T lymphocytes and macrophages . Vif overcomes the HIV-inhibitory effects of cellular factor APOBEC3G, which has cytidine deaminase activity . We previously reported the isolation of a Vif-interacting ring finger protein, Triad 3, from a human leukocyte cDNA library, using the yeast two-hybrid system . The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NF-kappaB (ZIN) . Sequence analysis indicates that Triad 3 protein contains all four major ring-like motifs of ZIN . We report here that ZIN binds to purified Vif in vitro and that Triad 3/ZIN interacts with HIV-1 Vif in transfected human 293T cells, as demonstrated by coimmunoprecipitation . To test the biological relevance of this interaction, we produced infectious HIV-1 NL4.3 in the presence or absence of cotransfected ZIN . HIV-1 NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were twofold less infectious in a single-cycle infectivity assay than virus produced in the absence of exogenous ZIN . It was further shown that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were impaired in viral DNA synthesis by twofold . The impairment in viral reverse transcription and the reduction in single-cycle viral infectivity were both shown to be dependent on the presence of Vif in the virus producer cells . The possible mechanisms by which ZIN interferes with the early events of HIV-1 replication are discussed.

Funct Integr Genomics . 2004 Sep 9; {Epub ahead of print}
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.

J Clin Microbiol, 2004 Sep, 42(9), 4408 - 9
Cost-effective method for identification of dimorphic fungi; Hughes AD et al.; Traditional methods to identify dimorphic fungi dictate that the mold be converted to the yeast phase at 35 to 37 degrees C . We present a time- and cost-saving method of confirming the identification of a dimorph by using special stains to demonstrate the yeast phase directly growing in the original clinical specimens.

J Clin Microbiol, 2004 Sep, 42(9), 4319 - 20
In vitro susceptibilities of isolates of Sporothrix schenckii to itraconazole and terbinafine; Kohler LM et al.; Thirty isolates of the yeast form of Sporothrix schenckii were evaluated for in vitro susceptibility to itraconazole and terbinafine by the recommended NCCLS modified technique (M27-A2) . The MICs of itraconazole obtained oscillated between 0.062 and 4.0 microg/ml, and those of terbinafine oscillated between 0.007 and 0.50 microg/ml; therefore, terbinafine showed greater in vitro activity.

J Biol Chem, 2004 Nov 12, 279(46), 47815 - 21 Epub 2004 Sep 13.
Identification of Tim40 that mediates protein sorting to the mitochondrial intermembrane space; Naoe M et al.; Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and sorted to one of the four mitochondrial subcompartments . Here we identified a new inner membrane protein, Tim40, that mediates sorting of small Tim proteins to the intermembrane space . Tim40 is essential for yeast cell growth, and its function in vivo requires six conserved Cys residues but not anchoring of the protein to the inner membrane by its N-terminal hydrophobic segment . Depletion of Tim40 impairs the import of small Tim proteins into mitochondria both in vivo and in vitro . In wild-type mitochondria, Tim40 forms a translocation intermediate with small Tim proteins prior to their assembly in the intermembrane space in vitro . These results suggest the essential role of Tim40 in sorting/assembly of small Tim proteins.

J Biol Chem, 2004 Nov 12, 279(46), 47783 - 91 Epub 2004 Sep 10.
CD22 is a functional ligand for SH2 domain-containing protein-tyrosine phosphatase-1 in primary T cells; Sathish JG et al.; The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes . The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved . The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation . Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains . Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1 . Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation . Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells.

J Biol Chem, 2004 Nov 12, 279(46), 48079 - 84 Epub 2004 Sep 09.
The coxsackievirus and adenovirus receptor interacts with the multi-PDZ domain protein-1 (MUPP-1) within the tight junction; Coyne CB et al.; The coxsackievirus and adenovirus receptor (CAR) is a component of the epithelial cell tight junction . In a yeast two-hybrid screen we identified the multi-PDZ domain protein MUPP1 as an interaction partner for the CAR cytoplasmic domain . CAR and MUPP1 were found to colocalize at the tight junction, to coprecipitate from epithelial cells, and to interact in vitro . The interaction was found to specifically involve the PDZ-binding motif within the CAR C terminus and MUPP1 PDZ domain 13 . In transfected cells, CAR recruited MUPP1 to cell-cell contacts . The inhibition of CAR expression with small interfering RNA inhibited MUPP1 localization to the tight junction . The results indicated that CAR interacts with MUPP1 and is involved in MUPP1 recruitment to the tight junction.

J Mol Biol, 2004 Oct 1, 342(5), 1559 - 67
Cofilin induced conformational changes in F-actin expose subdomain 2 to proteolysis; Muhlrad A et al.; Cofilin/ADF affects strongly the structure of actin filaments and especially the intermolecular contacts of the DNase I binding loop (D-loop) in subdomain 2 . In G-actin, the D-loop is cleaved by subtilisin between Met47 and Gly48, while in F-actin this cleavage is inhibited . Here, we report that yeast cofilin, which is resistant to both subtilisin and trypsin, accelerates greatly the rate of subtilisin cleavage of this loop in F-actin at pH 6.8 and at pH 8.0 . Similarly, cofilin accelerates strongly the tryptic cleavage in F-actin of loop 60-69 in subdomain 2, at Arg62 and Lys68 . The acceleration of the loops' proteolysis cannot be attributed to an increased treadmilling of F-actin for the following reasons: (i) the rate of subtilisin cleavage is independent of pH between pH 6.8 and 8.0, unlike F-actin depolymerization, which is pH-dependent; (ii) at high concentrations of protease the cleavage rate of F-actin in the presence of cofilin is faster than the rate of monomer dissociation from the pointed end of TRC-labeled F-actin, which limits the rate of treadmilling; and (iii) cofilin also accelerates the rate of subtilisin cleavage of F-actin in which the treadmilling is blocked by interprotomer cross-linking of the D-loop to the C terminus on an adjacent protomer . This suggests a substantial flexibility of the D-loop in the cross-linked F-actin . The increased cleavage rates of the D-loop and loop 60-69 reveal extensive exposure of subdomain 2 in F-actin to proteolytic enzymes by cofilin.

Biotechnol Adv, 2004 Nov, 22(8), 621 - 31
ORC-associated replication factors as biomarkers for cancer; Semple JW et al.; Early detection and treatment of cancer are of central importance to improving patient prognoses . Traditional biomarkers of cell proliferation, such as Ki-67 and PCNA, have had a mixed clinical track record, proving to be good indicators of certain types of cancers but of limited use for many others . Recently, human counterparts of replication factors originally identified in budding yeast have shown great promise as new cancer biomarkers . Each of these factors has been shown to interact with the origin recognition complex (ORC) in yeast, and each has an essential role in the initiation of DNA replication . Studies with minichromosome maintenance (MCM) family proteins show that their levels are upregulated in tumor cells and are much better indicators of a wide variety of cancers than traditional biomarkers . Similarly encouraging results have been obtained in preliminary studies examining Cdc6 protein and Cdc7 kinase transcript levels in normal and cancerous cells.

Int Rev Cytol, 2004, 238, 227 - 74
Electrophysiological approaches to the study of protein translocation in mitochondria; Grigoriev SM et al.; Electrophysiological techniques have been integral to our understanding of protein translocation across various membranes, and, in particular, the mitochondrial inner and outer membranes . Descriptions of various methodologies (for example, patch clamp, planar bilayers, and tip dip, and their past and potential contributions) are detailed within . The activity of protein import channels of native mitochondrial inner and outer membranes can be studied by directly patch clamping mitochondria and mitoplasts (mitochondria stripped of their outer membrane by French pressing) from various genetically manipulated strains of yeast and mammalian tissue cultured cells . The channel activities of TOM, TIM23, and TIM22 complexes are compared with those reconstituted in proteoliposomes and with those of the recombinant proteins Tom40p, Tim23p, and Tim22p, which play major roles in protein translocation . Studies of the mechanism(s) and the role of channels in protein translocation in mitochondria are prototypes, as the same principles are likely followed in all biological membranes including the endoplasmic reticulum and chloroplasts . The ability to apply electrophysiological techniques to these channels is now allowing investigations into the role of mitochondria in diverse fields such as neurotransmitter release, long-term potentiation, and apoptosis.

Pathophysiology, 2004 Oct, 11(2), 113 - 120
Atherogenesis and vascular calcification in mice expressing the human LPA gene; Teivainen PA et al.; Background: Lp(a) lipoprotein (Lp(a)) contains polymorphic glycoprotein, apolipoprotein(a) (apo(a)) and low density lipoprotein (LDL) . The extensive homology between apo(a) and plasminogen is believed to contribute to the pathogenicity of apo(a), but the precise mechanisms by which Lp(a) participates in atherogenesis is still unknown . We used LPA-yeast artificial chromosome (LPA-YAC) transgenic mice with or without the human APOB (hAPOB) gene to study pathogenicity of apo(a)/Lp(a) and illucidate its role in regulation of serum lipid levels . Methods: Middle-aged (1-year-old) mice were fed a control (AIN-76), a high-cholesterol (HC) or a high-cholesterol/high-fat (HCHF) diet for 7 weeks . For the study of serum total apo(a) and lipid levels, mice were sampled prior to the experiment, at 2 weeks and at 7 weeks when the animals were sacrificed . Hearts with ascending aorta were fixed in formalin, embedded in gelatine and prepared for sections on a cryostat . Livers were washed in ice cold saline and submerged in RNAlater trade mark buffer and stored at -70 degrees C until mRNA analysis . Results: Wild type mice fed the control diet did not develop aortic lesions . Presence of the LPA gene was sufficient to induce development of aortic lesions, but neither coexpression of the hAPOB gene nor feeding the HC diet or the HCHF diet augmented the development of aortic lesions in LPA-YAC transgenic mice . On the control diet transgenic females had larger aortic lesion size than transgenic males . Furthermore, aortic lesions in transgenic females were associated with calcification more often than in transgenic males . Serum total cholesterol levels were higher both in wild type and LPA-YAC transgenic males than in females mainly because of higher serum high-density lipoprotein cholesterol levels . HC and HCHF feeding had more pronounced effect on total cholesterol levels in LPA-YAC/hAPOB transgenic mice than in either wild type or LPA-YAC transgenic mice, due to increased low density lipoprotein cholesterol levels . Furthermore, these diets reduced serum total apo(a) levels in both transgenic mouse lines . Conclusion: Expression of the human LPA gene in mice is sufficient to trigger development of aortic lesions . Similar frequency of calcified lesions in LPA-YAC transgenic mice with or without hAPOB gene may suggest that apo(a) is the part of the Lp(a) molecule that causes aortic calcification . The basis for reduced serum total apo(a) level in response to cholesterol feeding is not clear, but interplay between LPA and factors involved in cholesterol or bile acid homeostasis is worth of future studies.

J Theor Biol, 2004 Oct 21, 230(4), 563 - 79
A model for restriction point control of the mammalian cell cycle; Novak B et al.; Inhibition of protein synthesis by cycloheximide blocks subsequent division of a mammalian cell, but only if the cell is exposed to the drug before the "restriction point" (i.e . within the first several hours after birth) . If exposed to cycloheximide after the restriction point, a cell proceeds with DNA synthesis, mitosis and cell division and halts in the next cell cycle . If cycloheximide is later removed from the culture medium, treated cells will return to the division cycle, showing a complex pattern of division times post-treatment, as first measured by Zetterberg and colleagues . We simulate these physiological responses of mammalian cells to transient inhibition of growth, using a set of nonlinear differential equations based on a realistic model of the molecular events underlying progression through the cell cycle . The model relies on our earlier work on the regulation of cyclin-dependent protein kinases during the cell division cycle of yeast . The yeast model is supplemented with equations describing the effects of retinoblastoma protein on cell growth and the synthesis of cyclins A and E, and with a primitive representation of the signaling pathway that controls synthesis of cyclin D.

Anal Chem, 2004 Sep 15, 76(18), 5431 - 5
Quantitative on-line monitoring of cellular glucose and lactate metabolism in vitro with slow perfusion; Leegsma-Vogt G et al.; An on-line in vitro perfusion technique is described that allows the continuous quantification of cellular glucose metabolism in vitro . Using biosensor technology, we measure glucose and lactate metabolism at a minute-to-minute time resolution for periods up to several days . The application of our perfusion-detection technique for in vitro monitoring is demonstrated in a wide variety of cells, including primary neuronal and astroglia cultures, yeast cells, and human lymphocytes . The method shows that variations in oxygen delivery or exposure to a noncompetitive pseudosubstrate (here 2-deoxyglucose) affects normal glucose metabolism . The innovative advantage of the present system is that, in contrast to other devices including a recently described system, metabolism per cell can be quantified . The potential of in vitro on-line monitoring is discussed for application in studying normal and abnormal metabolism, toxic and nontoxic drug effects, and human tissue biopsies.

Biochemistry, 2004 Sep 21, 43(37), 11818 - 27
Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii; Tang XF et al.; Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea . Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays . A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200) . Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay . The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit . A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200) . Since the peptide could increase the 3'-5' exonuclease activity of DP1 {Shen et al . (2004) Nucleic Acids Res . 32, 158}, the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200) . Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200) . DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.

PLoS Biol . 2004 Sep;2(9):E273 . Epub 2004 Sep 07.
Continued colonization of the human genome by mitochondrial DNA; Ricchetti M et al.; Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs) is an ongoing process that shapes nuclear genomes . In yeast this process depends on double-strand-break repair . Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus . From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4-6 million years . Thus, we measured the fixation rate of NUMTs in the human genome . Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics . We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs . Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns . Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor . Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences . This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults . We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.

Oncogene, 2004 Nov 4, 23(52), 8411 - 8
The regulation of CHK2 in human cancer; Craig AL et al.; Exceptional progress has been made in the past two decades in mapping oncogenes and tumour suppressors, defining a function for these master switches, and identifying novel anti-cancer drug targets . The p53 tumour suppressor is a central component of a DNA-damage-inducible pathway controlled by the ataxia telangiectasia mutated (ATM) and CHK2 protein kinases that have a central role in cancer suppression . One limitation of current human cancer research is the difficulty in developing genetic models that reveal the post-translational regulation of a growth suppressor like CHK2 within the microenvironment of a human tumour . Gaining such insights is important since yeast models and human tissue culture cell lines do not necessarily predict how enzymes like CHK2 are regulated in vivo, and therefore what factors can affect CHK2 tumour suppressor function . Translational cancer research aims to link basic research methodologies and clinical biology by uncovering cancer-specific pathways not revealed by other approaches . This approach is exemplified by two studies in this edition of Oncogene: both use a set of well-characterized human cancers with the objective of identifying novel post-translational control of the tumour suppressor CHK2 . The authors have revealed two unexpected epigenetic modifications of the CHK2 pathway in vivo: (1) constitutive phosphorylation of CHK2 at its ATM-activated site in the absence of exogenous DNA damage; and (2) the production of hyper-spliced and inactive isoforms of CHK2 . These studies highlight the need to develop model systems to understand why CHK2-activating pathways are being triggered or suppressed in different human cancers and whether the splicing machinery can be manipulated to control the activity of CHK2 for therapeutic benefit.

J Exp Bot, 2004 Oct, 55(406), 2155 - 68 Epub 2004 Sep 10.
Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3; Okumoto S et al.; Amino acids are regarded as the nitrogen 'currency' of plants . Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem . AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids . To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis . Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem . Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves . GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3 . When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane . Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study . In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips . The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1 . Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation . Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.

J Exp Bot, 2004 Oct, 55(406), 2291 - 303 Epub 2004 Sep 10.
Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds: analysis of tobacco seed development and effects of overexpressing apoplastic invertase; Tomlinson KL et al.; Wild-type tobacco (Nicotiana tabacum L.) seed development was characterized with respect to architecture and carbohydrate metabolism . Tobacco seeds accumulate oil and protein in the embryo, cellular endosperm and inner layer of the seed coat . They have high cell wall invertase (INV) and hexoses in early development which is typical of seeds . INV and the ratio of hexose to sucrose decline during development, switching from high hex to high suc, but not until most oil and all protein accumulation has occurred . The oil synthesis which coincides with the switch is mostly within the embryo . INV activity is greater than sucrose synthase activity throughout development, and both activities exceed the demand for carbohydrate for dry matter accumulation . To investigate the role of INV-mediated suc metabolism in oilseeds, genes for yeast INV and/or hexokinase (HK) were expressed under a seed-specific napin promoter, targeting activity to the apoplast and cytosol, respectively . Manipulating the INV pathway in an oilseed could either increase oil accumulation and sink strength, or disrupt carbohydrate metabolism, possibly through sugar-sensing, and decrease the storage function . Neither effect was found: transgenics with INV and/or HK increased 30-fold and 10-fold above wild-type levels had normal seed size and composition . This contrasted with dramatic effects on sugar contents in the INV lines.

Am J Respir Cell Mol Biol, 2005 Jan, 32(1), 35 - 43 Epub 2004 Sep 10.
Pleiomorphic Adenoma Gene-Like-2, a Zinc Finger Protein, Transactivates the Surfactant Protein-C Promoter; Yang MC et al.; Expression of surfactant protein (SP)-C occurs principally in type II pneumocytes located in the distal lung alveolae . SP-C expression is thought to be primarily regulated by thyroid transcription factor (TTF)-1 and its associated proteins interacting with a previously defined promoter region between -197 and -158 in mice . We screened a human lung cDNA library using a modified yeast one-hybrid system and identified pleiomorphic adenoma gene-like (PLAGL)-2, a ubiquitously expressed zinc finger protein, as a transfactor of the SP-C promoter . The PLAGL2 DNA-binding site was located in the SP-C promoter proximal region close to the TTF-1 sites . This site was demonstrated to be functional by use of electrophoresis mobility shift assay, mutagenesis analysis, and transfection studies . PLAGL2 bound to DNA via its N-terminus zinc fingers and activated the SP-C promoter in a TTF-1-independent manner . Both human and mouse SP-C promoters, but not the SP-B promoter, could be activated by PLAGL2 in transfected human embryonic kidney-293 (HEK293) cells as well as in murine type II (MLE12) cells . The expression of PLAGL2 in isolated human embryonic lung type II cells and its transactivation activity on the SP-C promoter suggest that PLAGL2 may modulate SP-C expression during lung development.

Plant J, 2004 Oct, 40(1), 60 - 74
Selective expression of a novel high-affinity transport system for acidic and neutral amino acids in the tapetum cells of Arabidopsis flowers; Lee YH et al.; Within the flower, microsporogenesis represents a major sink for nitrogen, but knowledge on how the imported nitrogen is transferred from the anther cell layers to developing pollen is lacking . Here, we provide information on characterization of a transporter (AtLHT2) that might play an important role in partitioning of amino acids for microspore development . Biochemical analysis in yeast showed that AtLHT2 transports proline and aspartate with high affinity . However, other neutral and acidic amino acids act as strong competitors for proline and aspartate uptake indicating that AtLHT2 generally transports uncharged and negatively charged amino acids . Comparison of the apparent K(m) values of AtLHT2 with previously characterized amino acid transporters clearly demonstrated that AtLHT2 represents a novel high-affinity system for neutral and acidic amino acids . Northern blot analysis showed strong expression of the amino acid transporter in flower buds . Cellular expression could be resolved by using RNA in situ hybridization and in situ RT-PCR methods, which localized AtLHT2 specifically to the tapetum tissue of the anthers . Developing pollen grains are symplasmically isolated from the sporophytic tissue and rely on the nutrients and other compounds secreted from the tapetum cells . Thus, the functional characterization of AtLHT2, together with our expression and localization studies, strongly suggest that in Arabidopsis flowers, AtLHT2 has a critical function in import of neutral and acidic amino acids into the tapetum cells for synthesis of compounds important for microspore structure and in transfer of organic nitrogen to the locule for pollen development.

Medinfo, 2004, 2004, 798 - 802
Clustering gene expression data with temporal abstractions; Sacchi L et al.; This paper describes a new technique for clustering short time series coming from gene expression data . The technique is based on the labelling of the time series through temporal trend abstractions and a consequent clustering of the series on the basis of their labels . Clustering is performed at three dif-ferent levels of aggregation of the original time series, so that the results are organized and visualized as a three-levels hier-archical tree . Results on simulated and on yeast data are shown . The technique appears robust and efficient and the re-sults obtained are easy to be interpreted.

J Proteome Res, 2004 Jul-Aug, 3(4), 801 - 6
Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS; Du Y et al.; Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS) . Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension . Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold . The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications . Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.

J Bioinform Comput Biol, 2004 Sep, 2(3), 441 - 58
Reconstructing genetic networks from time ordered gene expression data using Bayesian method with global search algorithm; Wang SC; Different genes of an organism are expressed to different levels at different times during the life cycle and in response to various environmental stresses . Elucidating the network of gene-gene interactions responsible for the expression helps understand living processes . Microarray technology allows concurrent genomic scale measurement of an organism's mRNA levels . We describe a power-law formalism to model the combinatorial effect of regulators on gene transcription . The dynamic model allows delayed transcription . We employ a principled network reconstruction approach that accounts for the high noise and low replicate characteristics of present day microarray data . An important feature of our approach is that the detail of the reconstructed network is limited to the noise level of the data . We apply the methodology to a microarray dataset of yeast cells grown in glucose and experiencing a diauxic transition upon glucose depletion . The reconstructed transcriptional regulations of yeast glycolytic genes are consistent with published findings.

Mol Cells, 2004 Aug 31, 18(1), 10 - 6
Regulation of calcineurin, a calcium/calmodulin-dependent protein phosphatase, in C . elegans; Bandyopadhyay J et al.; Calcineurin is a calcium/calmodulin-dependent serine/ threonine protein phosphatase . It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, and a regulatory calcium-binding subunit, calcineurin B . The primary sequence of both subunits and heterodimeric structure is highly conserved from yeast to mammals . Calcineurin has long been implicated in various signaling pathways . Calcineurin genes (cna-1/tax-6 and cnb-1) have been identified in the nematode Caenorhabditis elegans, which share high homology with their Drosophila and mammalian counterparts . C . elegans calcineurin binds calcium and functions as a heterodimeric protein phosphatase establishing its biochemical conservation . Calcineurin expresses in diverse tissues implicating its important role in various physiological processes . This review will focus in brief on the expression pattern and regulation of calcineurin including its effect on growth and development, locomotion, egg-laying, and sensory responses.

Proc Natl Acad Sci U S A, 2004 Sep 21, 101(38), 13774 - 9 Epub 2004 Sep 09.
A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins; Katiyar S et al.; Peptide:N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway . The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis . In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) homologs . Immunofluorescence studies revealed that hPNGase, hHR23B, and hS4 are present in close proximity to the endoplasmic reticulum (ER) when calnexin was used as an ER marker in HeLa cells . Subcellular fractionation suggests not only cytoplasmic but also ER association of hPNGase in HeLa cells . Immunoprecipitation analysis revealed the interaction of h/mPNGase with the 19S proteasome subunit, hS4, through hHR23B . Using an in vitro GST pull-down assay, we also have shown that recombinant mPNGase requires its N terminus and middle domain for interaction with mHR23B . Finally, using misfolded yeast carboxypeptidase Y and chicken ovalbumin as glycoprotein substrates, we have established that mHR23B acts as a receptor for deglycosylated proteins . Based on this finding, we propose that after deglycosylation of misfolded glycoproteins by PNGase, the aglyco forms of these proteins are recognized by HR23B and targeted for degradation.

Hum Mol Genet, 2004 Oct 1, 13 Spec No 2, R217 - 24
Environmental genomics: a key to understanding biology, pathophysiology and disease; Schwartz DA et al.; Recent advances in human and molecular genetics provide an unparalleled opportunity to understand how genes and genetic changes interact with environmental stimuli to either preserve health or cause disease . The fields of environmental genetics and environmental genomics has enormous potential to affect our ability to accurately assess the risk of developing disease, identify and understand basic pathogenic mechanisms that are critical to disease progression, and to more precisely phenotype disease subtypes . However, the application of genetics and genomics to problems in environmental health is only the beginning yet, by itself, represents a potentially effective strategy to substantially impact morbidity and mortality . Collaborative approaches that team together environmental scientists with molecular biologists, geneticists, physiologists and physician scientists are critical to the investigation of environmental aspects of human health . Moreover, exploiting eukaryotic model systems (yeast, Caenorhabditis elegans, zebrafish, Drosophila and rodents) will accelerate our understanding of environmental exposures on human health.

FEBS Lett, 2004 Sep 10, 574(1-3), 31 - 6
Aquaporin homologues in plants and mammals transport ammonia; Jahn TP et al.; Using functional complementation and a yeast mutant deficient in ammonium (NH4+) transport (Deltamep1-3), three wheat (Triticum aestivum) TIP2 aquaporin homologues were isolated that restored the ability of the mutant to grow when 2 mM NH4+ was supplied as the sole nitrogen source . When expressed in Xenopus oocytes, TaTIP2;1 increased the uptake of NH4+ analogues methylammonium and formamide . Furthermore, expression of TaTIP2;1 increased acidification of the oocyte-bathing medium containing NH4+ in accordance with NH3 diffusion through the aquaporin . Homology modeling of TaTIP2;1 in combination with site directed mutagenesis suggested a new subgroup of NH3-transporting aquaporins here called aquaammoniaporins . Mammalian AQP8 sharing the aquaammoniaporin signature also complemented NH4+ transport deficiency in yeast.

FEBS Lett, 2004 Sep 10, 574(1-3), 25 - 30
Oligomerization activity of a double-stranded RNA-binding domain; Hitti EG et al.; Xenopus laevis RNA-binding protein A (Xlrbpa) is a highly conserved, ubiquitously expressed hnRNP- and ribosome-associated RNA-binding protein that contains three double stranded RNA-binding domains (dsRBDs) in tandem arrangement . A two-hybrid screen with Xlrbpa as a bait recovered Xlrbpa itself as the strongest interaction partner, indicating multimerization of this protein . To search for regions responsible for the observed interaction, we conducted two-hybrid assays with Xlrbpa deletion constructs and identified the third dsRBD of Xlrbpa as the exclusive interacting domain . Additionally, these results were confirmed by coimmunoprecipitation experiments with truncated proteins expressed both in yeast and Xenopus oocytes . In PACT, the human homologue of Xlrbpa, we could demonstrate that the third dsRBD displays the same multimerization activity . Interestingly, this domain is essential for the activation of the dsRNA-activated protein kinase PKR . Addition of RNAses to coimmunoprecipitation experiments did not affect the dimerization, suggesting that the interaction is independent of RNA-binding . We report here a homomultimerization activity of a type B dsRBD and suggest possible implications that include a model for PKR activation by PACT.

Trends Plant Sci, 2004 Aug, 9(8), 378 - 84
Learning the lipid language of plant signalling; van Leeuwen W et al.; Plant cells respond to different biotic and abiotic stresses by producing various uncommon phospholipids that are believed to play key roles in cell signalling . We can predict how they work because animal and yeast proteins have been shown to have specific lipid-binding domains, which act as docking sites . When such proteins are recruited to the membrane locations where these phospholipids are synthesized, the phospholipids activate them directly, by inducing a conformational change, or indirectly, by juxtaposing them with an activator protein . The same lipid-binding domains are present in Arabidopsis proteins . We believe that they represent an untapped well of information about plant lipid signalling.

Curr Opin Microbiol, 2004 Aug, 7(4), 400 - 6
Foamy viruses--a world apart; Delelis O et al.; Foamy viruses (FVs) or spumaviruses were described for the first time in the early 1950s in cell cultures derived from monkey kidneys . Later, FVs were isolated in several mammal species such as cats, cattle and horses . Highly prevalent in non-human primates they are not naturally present in humans, although several cases of simian-to-human transmissions have been described . Interestingly, the replication strategy of FVs differs in many aspects from that of other retroviruses, presenting features that are closely related to pararetroviruses, exemplified by the hepatitis B virus (HBV), but also characteristics that are closely related to yeast retrotransposons . These characteristics led to the creation of a distinct viral subfamily by the International Committee on Virus Taxonomy in 2002; the Spumaretrovirinae.

Pigment Cell Res, 2004 Oct, 17(5), 498 - 505
Rab27b is up-regulated in human Griscelli syndrome type II melanocytes and linked to the actin cytoskeleton via exon F-Myosin Va transcripts; Westbroek W et al.; Patients with the autosomal recessive Griscelli-Prunieras syndrome type II are immunologically impaired and have an unusual silvery-grey hypopigmented colour of scalp hair, eyelashes and eyebrows but no noteworthy pigmentary abnormalities of the skin . In most Griscelli patients, the RAB27A gene, which encodes a small GTPase that is associated with the melanosome membrane in melanocytes, is mutated . Here we discuss a genomic RAB27A deletion found in a 21-month-old Moroccan Griscelli patient . Additionally, we provide evidence that the loss of functional Rab27a in melanocytes of this Griscelli patient is partially compensated by the up-regulation of Rab27b, a homologue of Rab27a . By real-time quantitative PCR and western blot analysis, we found that Rab27b mRNA and protein, expressed at low levels in normal human melanocytes, is significantly up-regulated in melanocytes derived from this patient . Our immunofluorescence and yeast two-hybrid screening studies reveal that Rab27b can form a tripartite complex on the melanosome membrane with Melanophilin, a Rab27a effector, and protein products of Myosin Va transcripts that contain exon F . Our data suggest that up-regulated Rab27b in melanocytes of the Griscelli patient can partially take over the function of Rab27a, which could explain the fact that this patient had an evenly pigmented skin and was able to tan.

J Appl Microbiol, 2004, 97(4), 783 - 91
Optimization of extracellular endoxylanase, endoglucanase and peroxidase production by Streptomyces sp . F2621 isolated in Turkey; Tuncer M et al.; AIMS: To determine the effect of environmental conditions on the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp . F2621 and to assess the potential use of these enzymes in the hydrolysis of lignocellulose material . METHODS AND RESULTS: The production of extracellular lignocellulose-degrading enzymes, endoxylanase, endoglucanase and peroxidase during the growth of Streptomyces sp . F2621 in basal salts-yeast extract medium containing different carbon sources and the effect of a number of environmental parameters (e.g . carbon sources and concentrations, pH and temperature) were investigated . The highest endoxylanase (22.41 U ml(-1)) and peroxidase (0.58 U ml(-1)) activities were obtained after 2-4 days of incubation at 30 degrees C in a basal salts medium containing 0.4% (w/v) oat spelt xylan and 0.6% (w/v) yeast extract, corresponding to C : N ratio of 6 : 1 . Cell-free extracellular enzyme preparations from the strain were capable of releasing both sugar and aromatic compounds during incubation with eucalyptus paper pulp, straw and xylan . Overall, 9.3% hydrolysis of xylan occurred after 24-h incubation . However the rates of hydrolysis of paper pulp and straw were approximately twofold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate (24.5% and 16.3%, respectively) was greater than xylan hydrolysis . CONCLUSIONS: The high levels of enzyme production achieved under batch cultivation conditions, coupled with no significant production of endoglucanase during the growth phase of organism and the release of both sugar and aromatic compounds from paper pulp and straw signify the suitability for these enzymes for industrial applications such as pulp and paper production . SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight the environmental conditions for the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp . F2621 and suggest the use of streptomycetes and/or their enzymes in industrial processes.

Rev Neurosci, 2004, 15(3), 185 - 98
Expression and function of brain specific small RNAs; Rogelj B et al.; Small non-messenger RNAs (snmRNAs) are a heterogeneous group of non-coding RNAs with a variety of regulatory functions including regulation of protein expression and guidance in RNA modifications . They are actively being investigated in Archaebacteria, yeast, invertebrates and mammals . Brain-specific snmRNAs have been identified in mammals and they seem to contribute to neuronal differentiation during development and to brain functions subserving learning and memory . Here we review the current knowledge of the properties, expression and functions of three groups of brain-specific snmRNAs: small nucleolar RNAs, BC1/BC200 RNAs and microRNAs.

J Parasitol, 2004 Aug, 90(4), 908 - 13
Molecular characterization of the Leishmania braziliensis L6 ribosomal protein; Thomas MC et al.; By screening a Leishmania braziliensis complementary DNA library with a pool of sera from leishmaniasis patients, the gene coding for L6 ribosomal protein was isolated . The sequence, genomic organization, and transcription of this gene are described in this article . The sequence analysis of the L . braziliensis L6 gene shows a single open reading frame, which codes for a protein of 192 amino acids (aa) with a hypothetical molecular mass of 20.9 kDa . The protein exhibits significant sequence similarity to L6 ribosomal proteins from higher eukaryotes and yeast . Thus, the L . braziliensis L6 protein contains 4 functional motifs, which are located at equivalent positions in other L6 ribosomal proteins described previously . Interestingly, the L6 ribosomal protein from L . braziliensis contains a specific region of 14 aa and a tyrosine kinase motif, which is absent in human and C . elegans L6 protein . The locus coding the L . braziliensis L6 ribosomal protein is formed by 2 gene copies arranged in tandem and located in a chromosome of approximately 0.9 . Mb . The genes are actively transcribed as 2 polyadenylated transcripts of approximately 1.15 and 0.85 kb, which differ in their steady-state level and stability.

Plant Cell Physiol, 2004 Aug, 45(8), 1042 - 52
Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L; Qin F et al.; The transcription factors DREB1s/CBFs specifically interact with the DRE/CRT cis-acting element (core motif: G/ACCGAC) and control the expression of many stress-inducible genes in Arabidopsis . We isolated a cDNA for a DREB1/CBF homolog, ZmDREB1A in maize using a yeast one-hybrid system . The ZmDREB1A proteins specifically bound to DRE and the highly conserved valine at the 14th residue in the ERF/AP2 DNA binding domain was a key to determining the specific interaction between this protein and the DRE sequence . Expression of ZmDREB1A was induced by cold stress and slightly increased by high-salinity stress . This gene was also transiently expressed by mechanical attack . ZmDREB1A activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts . Overexpression of ZmDREB1A in transgenic Arabidopsis induced overexpression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought and freezing stresses . This indicated that ZmDREB1A has functional similarity to DREB1s/CBFs in Arabidopsis . The structure of the ERF/AP2 domain of ZmDREB1A in maize is closely related to DREB1-type ERF/AP2 domains in the monocots as compared with that in the dicots . ZmDREB1A is suggested to be potentially useful for producing transgenic plants that is tolerant to drought, high-salinity and/or cold stresses.

Nucleic Acids Res, 2004 Sep 08, 32(16), 4786 - 803 Print 2004.
Portrait of transcriptional responses to ultraviolet and ionizing radiation in human cells; Rieger KE et al.; To understand the human response to DNA damage, we used microarrays to measure transcriptional responses of 10 000 genes to ionizing radiation (IR) and ultraviolet radiation (UV) . To identify bona fide responses, we used cell lines from 15 individuals and a rigorous statistical method, Significance Analysis of Microarrays (SAM) . By exploring how sample number affects SAM, we rendered a portrait of the human damage response with a degree of accuracy unmatched by previous studies . By showing how SAM can be used to estimate the total number of responsive genes, we discovered that 24% of all genes respond to IR and 32% respond to UV, although most responses were less than 2-fold . Many genes were involved in known damage-response pathways for cell cycling and proliferation, apoptosis, DNA repair or the stress response . However, the majority of genes were involved in unexpected pathways, with functions in signal transduction, RNA binding and editing, protein synthesis and degradation, energy metabolism, metabolism of macromolecular precursors, cell structure and adhesion, vesicle transport, or lysosomal metabolism . Although these functions were not previously associated with the damage response in mammals, many were conserved in yeast . These insights reveal new directions for studying the human response to DNA damage.

J Biol Chem, 2004 Nov 12, 279(46), 47704 - 10 Epub 2004 Sep 07.
Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes; Tanida I et al.; Human light chain 3/MAP1LC3B, an autophagosomal ortholog of yeast Atg8, is conjugated to phospholipid (PL) via ubiquitylation-like reactions mediated by human Atg7 and Atg3 . Since human Atg4B was found to cleave the carboxyl terminus of MAP1LC3B in vitro, we hypothesized that this exposes its carboxyl-terminal Gly(120) . It was recently reported, however, that when Myc-MAP1LC3B-His is expressed in HEK293 cells, its carboxyl terminus is not cleaved . (Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., and Kominami, E . (2004) J . Biol . Chem . 279, 36268-36276) . To clarify this contradiction, we sought to determine whether the carboxyl terminus of MAP1LC3B is cleaved to expose Gly(120) for further ubiquitylation-like reactions . When MAP1LC3B-3xFLAG and Myc-MAP1LC3B-His were expressed in HEK293 cells, their carboxyl termini were cleaved, whereas there was little cleavage of mutant proteins MAP1LC3B(G120A)-3xFLAG and Myc-MAP1LC3B(G120A)-His, containing Ala in place of Gly(120) . An in vitro assay showed that Gly(120) is essential for carboxyl-terminal cleavage by human Atg4B as well as for formation of the intermediates Atg7-MAP1LC3B (ubiquitin-activating enzyme-substrate) and Atg3-MAP1LC3B (ubiquitin carrier protein-substrate) . Recombinant MAP1LC3B-PL was fractionated into the 100,000 x g pellet in a manner similar to that shown for endogenous MAP1LC3B-PL . RNA interference of MAP1LC3B mRNA resulted in a decrease in both endogenous MAP1LC3B-PL and MAP1LC3B . These results indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly(120) for further ubiquitylation-like reactions.

Biochem J, 2005 Jan 1, 385(Pt 1), 21 - 8
Regulation of all members of the antizyme family by antizyme inhibitor; Mangold U et al.; ODC (ornithine decarboxylase) is the rate-limiting enzyme in polyamine biosynthesis . Polyamines are essential for cellular growth and differentiation but enhanced ODC activity is associated with cell transformation . Post-translationally, ODC is negatively regulated through members of the antizyme family . Antizymes inhibit ODC activity, promote ODC degradation through the 26 S proteasome and regulate polyamine transport . Besides the ubiquitously expressed antizymes 1 and 2, there is the tissue-specific antizyme 3 and an yet uncharacterized antizyme 4 . Antizyme 1 has been shown to be negatively regulated through the AZI (antizyme inhibitor) that binds antizyme 1 with higher affinity compared with ODC . In the present study, we show by yeast two- and three-hybrid protein-protein interaction studies that AZI interacts with all members of the antizyme family and is capable of disrupting the interaction between each antizyme and ODC . In a yeast-based ODC complementation assay, we show that human ODC is able to complement fully the function of the yeast homologue of ODC . Co-expression of antizymes resulted in ODC inhibition and cessation of yeast growth . The antizyme-induced growth inhibition could be reversed by addition of putrescine or by the co-expression of AZI . The protein interactions could be confirmed by immunoprecipitation of the human ODC-antizyme 2-AZI complexes . In summary, we conclude that human AZI is capable of acting as a general inhibitor for all members of the antizyme family and that the previously not yet characterized antizyme 4 is capable of binding ODC and inhibiting its enzymic activity similar to the other members of the antizyme family.

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