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 addu