J Nutr Biochem, 2003 Nov, 14(11), 637 - 47 The chemical form of selenium affects insulinomimetic properties of the trace element: investigations in type II diabetic dbdb mice; Mueller AS et al.; The objective of the present study was to investigate the effects of oral selenate application in comparison to selenium deficiency and selenite treatment on the development of the diabetic status (glucose tolerance, insulin resistance and activities of glycolytic and gluconeogenic marker enzymes) in dbdb mice, representing a type II diabetic animal model . Therefore 21 adult male dbdb mice were assigned to 3 experimental groups of 7 animals each and put on a selenium deficient diet (< 0.03 mg/kg diet) based on torula yeast . Group 0Se was kept on selenium deficiency for 10 weeks while the mice of the groups SeIV and SeVI were supplemented daily with 15% of their individual LD(50) of sodium selenite or sodium selenate in addition to the diet . After 10 weeks a distinct melioration of the diabetic status indicated by a corrected glucose tolerance and a lowered insulin resistance was measured in selenate treated mice (group SeVI) in comparison to their selenium deficient and selenite treated companions and to their initial status . Activities of the glycolytic marker enzymes hexokinase, phosphofructokinase and pyruvate kinase were increased 1.7 to 3-fold in liver and/or adipose tissue by selenate treatment as compared to mice on selenium deficiency and mice with selenite administration . In contrast selenate treatment (SeVI) repressed the activity of liver pyruvate carboxylase the first enzyme in gluconeogenesis by about 33% in comparison to the selenium deficient (0Se) and selenite treated mice (SeIV) . However the current study revealed an insulinomimetic role for selenate (selenium VI) also in type II diabetic animals due to a melioration of insulin resistance . In contrast selenium deficiency and especially selenite (selenium IV) impaired the diabetic status of dbdb mice, demonstrating the need for investigations on the insulinomimetic action of selenium due to the metabolism of different selenium compounds. Oncogene, 2003 Nov 20, 22(52), 8422 - 31 Carom: a novel membrane-associated guanylate kinase-interacting protein with two SH3 domains; Ohno H et al.; MAGI-1 and CASK are membrane-associated guanylate kinases of epithelial junctions . MAGI-1 is localized at tight junctions in polarized epithelial cells, whereas CASK is localized along the lateral membranes . We obtained the KIAA0769 gene product through the yeast two-hybrid screening using MAGI-1 as a bait and named it Carom . Carom has a coiled-coil domain in the middle region, and two src homology 3 domains and a PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif in the C-terminal region . Carom binds to the fifth PDZ domain of MAGI-1 and the calmodulin kinase domain of CASK in vitro . MAGI-1 and CASK bind to the distinct sequences in the C-terminal region of Carom, but still compete with each other for Carom binding . The study using a stable transformant of Madine Darby canine kidney (MDCK) cells expressing GFP-Carom revealed that Carom was partially overlapped by MAGI-1 in MDCK cells, which have not yet established mature cell junctions, but became separated from MAGI-1 and colocalized with CASK in polarized cells . Carom was highly resistant to Triton X-100 extractions and recruited CASK to the Triton X-100-insoluble structures . Carom is a binding partner of CASK, which interacts with CASK in polarized epithelial cells and may link it to the cytoskeleton. Development, 2003 Dec, 130(26), 6431 - 9 Epub 2003 Nov 19. The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root; Bernhardt C et al.; The position-dependent specification of the hair and non-hair cell types in the Arabidopsis root epidermis provides a simple model for the study of cell fate determination in plants . Several putative transcriptional regulators are known to influence this cell fate decision . Indirect evidence from studies with the maize R gene has been used to suggest that a bHLH transcription factor also participates in this process . We show that two Arabidopsis genes encoding bHLH proteins, GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), act in a partially redundant manner to specify root epidermal cell fates . Plants homozygous for mutations in both genes fail to specify the non-hair cell type, whereas plants overexpressing either gene produce ectopic non-hair cells . We also find that these genes are required for appropriate transcription of the non-hair specification gene GL2 and the hair cell specification gene CPC, showing that GL3 and EGL3 influence both epidermal cell fates . Furthermore, we show that these bHLH proteins require a functional WER MYB protein for their action, and they physically interact with WER and CPC in the yeast two-hybrid assay . These results suggest a model in which GL3 and EGL3 act together with WER in the N cell position to promote the non-hair cell fate, whereas they interact with the incomplete MYB protein CPC in the H position, which blocks the non-hair pathway and leads to the hair cell fate. Biol Reprod, 2004 Mar, 70(3), 775 - 84 Epub 2003 Nov 19. Identification and characterization of human VCY2-interacting protein: VCY2IP-1, a microtubule-associated protein-like protein; Wong EY et al.; VCY2 is a testis-specific protein that locates in a frequently deleted azoospermia factor c region on chromosome Yq . Although its genomic structure has been characterized, the function of VCY2 is still unknown . To gain insight regarding the likely function of VCY2, we investigated the proteins that interact with VCY2 using the yeast two-hybrid system . We identified a novel VCY2 interaction partner, named VCY2IP-1, that encodes an open reading frame of 1059 amino acids . The amino acid sequence of VCY2IP-1 shows 59.3% and 41.9% homology to two human microtubule-associated proteins (MAPs), MAP1B and MAP1A, respectively . VCY2IP-1 has an extensive homology to the N-terminus and C-terminus regions of MAP1B and MAP1A, placing it within a large family of MAPs . We mapped VCY2IP-1 to chromosome 19p13.11 . The VCY2IP-1 gene spans 15 kilobases (kb) and consists of seven exons . Northern blot analysis identified a single, intense band of approximately 3.2-kb VCY2IP-1 transcript, predominantly expressed in human testis . In situ hybridization of human testicular sections showed the localization of VCY2IP-1 transcripts in germ cells, and reverse transcription-polymerase chain reaction analysis demonstrated the presence of VCY2 and VCY2IP-1 transcripts in human ejaculated spermatozoa . Our expression data support the involvement of VCY2 and VCY2IP-1 in spermatogenesis . Based on the high homology of VCY2IP-1 with MAPs, we propose the involvement of VCY2 in the cytoskeletal network via interaction with VCY2IP-1. Cell Stress Chaperones, 2003 Summer, 8(2), 114 - 9 Cdc37 goes beyond Hsp90 and kinases; MacLean M et al.; Cdc37 is a relatively poorly conserved and yet essential molecular chaperone . It has long been thought to function primarily as an accessory factor for Hsp90, notably directing Hsp90 to kinases as substrates . More recent discoveries challenge this simplistic view . Cdc37 client proteins other than kinases have now been found, and Cdc37 displays a variety of Hsp90-independent activities both in vitro and in vivo . It can function as a molecular chaperone by itself, interact with other Hsp90 cochaperones in the absence of Hsp90, and even support yeast growth and protein folding without its Hsp90-binding domain . Thus, for many substrates, there may be many alternative chaperone pathways involving Cdc37, Hsp90, or both. Lakartidningen, 2003 Oct 23, 100(43), 3408 - 12 {Preventive vaccines against papillomavirus and cervix cancer will soon enter clinical practice}; Lehtinen M et al.; A vaccine may protect against cancers caused by high-risk human papillomaviruses (HPVs) . Sexually transmitted high-risk HPV types are almost always found in cervical cancer . Incidents of HPV type 16 and cervical cancer has more than doubled after the 1980's in Finland . HPV L1 capsid protein can be produced in the yeast, after which it assembles into virus-like-particles (VLP) and can be readily used for vaccine production . HPV VLP vaccine is well tolerated and induces ten-fold higher serum antibody levels as compared to natural infection . HPV16 VLP vaccine has shown to be 91% protective effect against HPV16 infections in the first phase III study . Neutralizing HPV antibodies, induced by HPV VLP vaccination, effectively reduce the viral load even though total elimination of the virus may not be needed . It is not known for how long the vaccine induced protection will last . Recruitment of adolescents into population-based phase III vaccination studies should be large to allow reliable cancer registry based evaluation of protective effect against grave dysplasia and cervical cancer. Cell Mol Life Sci, 2003 Nov, 60(11), 2325 - 33 Telomerase-independent mechanisms of telomere elongation; Biessmann H et al.; The ends of linear chromosomes must be elongated in a DNA-replication-independent fashion . For chromosome end elongation the majority of eukaryotes use a specialized reverse transcriptase, telomerase, which adds a short, tandemly repeated DNA sequence motif to chromosome ends . Chromosome elongation can also be achieved, however, by mechanisms other than telomerase . Such elongation events have been detected under conditions where telomerase has been inactivated experimentally and in the few organisms that naturally lack telomerase . We will summarize current knowledge on these telomerase-independent elongation mechanisms in yeast and mammalian cells and will discuss in more detail the telomere elongation mechanism by retrotransposons in Drosophila melanogaster. J Biol Chem, 2004 Feb 20, 279(8), 7275 - 86 Epub 2003 Nov 18. Novel nuclear shuttle proteins, HDBP1 and HDBP2, bind to neuronal cell-specific cis-regulatory element in the promoter for the human Huntington's disease gene; Tanaka K et al.; Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the HD gene, and the expression level of either normal or mutant huntingtin is implicated in the pathogenesis of HD . However, a molecular base of the HD gene transcription has not been elucidated as yet . In this study, we identified two proteins, HDBP1 and HDBP2, which bind to the promoter region for the HD gene using a yeast one-hybrid system . Amino acid sequence analysis of the proteins deduced the presence of nuclear localization signal, nuclear export signal, zinc finger, serine/proline-rich region, and highly conserved C-terminal region . In vitro DNA binding assay indicated that the C-terminal conserved regions of the proteins were responsible for binding to the unique promoter DNA sequences of the HD gene . The DNA sequence protected from DNase I digestion was a 7-bp consensus sequence (GCCGGCG), which resides in triplicate at intervals of 13 bp within and proximal to the 20-bp direct repeat sequences of the HD promoter region . The mutation of 7-bp consensus sequence abolishes the HD promoter function in a neuronal cell line (IMR32) . In human cultured cells, ectopically expressed green fluorescent protein-fused HDBP1 and HDBP2 localized in the cytoplasm, but both proteins totally shift from cytoplasm to nucleus by the treatment with an inhibitor of the nuclear export, leptomycin B, and mutagenesis of the putative nuclear export signals . Taken together, HDBP1 and HDBP2 are novel transcription factors shuttling between nucleus and cytoplasm and bind to the specific GCCGGCG, which is an essential cis-element for HD gene expression in neuronal cells. Arch Microbiol, 2004 Jan, 181(1), 35 - 44 Epub 2003 Nov 18. The alternative D-galactose degrading pathway of Aspergillus nidulans proceeds via L-sorbose; Fekete E et al.; The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway . In contrast, Aspergillus nidulans mutants in galactokinase ( galE) can still grow on d-galactose in the presence of ammonium-but not nitrate-ions as nitrogen source . A . nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate . The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A . nidulans loss-of-function mutant in this enzyme ( araA1) did not show NAD(+)-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol . The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A . nidulans hexokinase ( frA1) mutant . l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A . nidulans was unable to grow on d-galactose . The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A . nidulans that involves reduction of the d-galactose to galactitol and NAD(+)-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose. Curr Opin Infect Dis, 2003 Dec, 16(6), 553 - 8 Molecular markers for drug resistance in malaria: use in treatment, diagnosis and epidemiology; Wernsdorfer WH et al.; PURPOSE OF REVIEW: Malaria and the increasing role of drug resistance as an obstacle to its control are global problems . The identification and implications of molecular markers for antimalarial drug resistance - the subject of this review - are key issues in elucidating and eventually controlling resistance . RECENT FINDINGS: Recent achievements include the successful expression of the Plasmodium falciparum chloroquine resistance transporter gene, pfcrt, in yeast, the identification of polymorphisms on the gamma-glutamylcysteine synthetase gene, ggcs, as potential determinants of chloroquine and mefloquine resistance, and the usefulness of a combined Plasmodium falciparum dihydrofolate reductase gene, pfdhfr, 59ARG and Plasmodium falciparum dihydropteroate synthase gene, pfdhps, 540GLU marker in reliably representing resistance to antifolates . Moreover, treatment with sulfadoxine-pyrimethamine in the presence of pfdhfr 108ASP alone delayed parasite clearance and increased gametocytogony without an overt loss of the overall therapeutic efficacy of the drug . SUMMARY: The use of pfdhfr and pfdhps markers in determining antifolate resistance of Plasmodium falciparum has been consolidated . Similar progress has been made with pfcrt markers for chloroquine resistance, auguring well the operational deployment of molecular techniques . Regarding the molecular basis of resistance to arylaminoalcohols, related drugs, and artemisinin and its derivatives, answers remain elusive, but there are promising new leads. Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13892 - 7 Epub 2003 Nov 17. Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A; Cans C et al.; Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1 . To better characterize the function of TCTP, a yeast two-hybrid hunt was performed . Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bbeta, as TCTP-interacting partners . In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bbeta and eEF1A . Additionally, MS analysis also identified eEF1A as a TCTP interactor . Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A . Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bbeta . These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis. J Biol Chem, 2004 Feb 20, 279(8), 7014 - 23 Epub 2003 Nov 17. DAMAGE, a novel alpha-dystrobrevin-associated MAGE protein in dystrophin complexes; Albrecht DE et al.; Mice rendered null for alpha-dystrobrevin, a component of the dystrophin complex, have muscular dystrophy, despite the fact that the sarcolemma remains relatively intact (Grady, R . M., Grange, R . W., Lau, K . S., Maimone, M . M., Nichol, M . C., Stull, J . T., and Sanes, J . R . (1999) Nat . Cell Biol . 1, 215-220) Thus, alpha-dystrobrevin may serve a signaling function that is important for the maintenance of muscle integrity . We have identified a new dystrobrevin-associated protein, DAMAGE, that may play a signaling role in brain, muscle, and peripheral nerve . In humans, DAMAGE is encoded by an intronless gene located at chromosome Xq13.1, a locus that contains genes involved in mental retardation . DAMAGE associates directly with alpha-dystrobrevin, as shown by yeast two-hybrid, and co-immunoprecipitates with the dystrobrevin-syntrophin complex from brain . This co-immunoprecipitation is dependent on the presence of alpha-dystrobrevin but not beta-dystrobrevin . The DAMAGE protein contains a potential nuclear localization signal, 30 12-amino acid repeats, and two MAGE homology domains . The domain structure of DAMAGE is similar to that of NRAGE, a MAGE protein that mediates p75 neurotrophin receptor signaling and neuronal apoptosis (Salehi, A . H., Roux, P . P., Kubu, C . J., Zeindler, C., Bhakar, A., Tannis, L . L., Verdi, J . M., and Barker, P . A . (2000) Neuron 27, 279-288) . DAMAGE is highly expressed in brain and is present in the cell bodies and dendrites of hippocampal and Purkinje neurons . In skeletal muscle, DAMAGE is at the postsynaptic membrane and is associated with a subset of myonuclei . DAMAGE is also expressed in peripheral nerve, where it localizes along with other members of the dystrophin complex to the perineurium and myelin . These results expand the role of dystrobrevin and the dystrophin complex in membrane signaling and disease. J Biol Chem, 2004 Feb 20, 279(8), 6401 - 13 Epub 2003 Nov 17. Purification of the Arabidopsis 26 S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms; Yang P et al.; The 26 S proteasome is a multisubunit protease complex responsible for degrading a wide range of intracellular proteins in eukaryotes, especially those modified with polyubiquitin chains . It is composed of a self-compartmentalized core protease (CP) that houses the peptidase active sites appended on either or both ends by a regulatory particle (RP) that identifies appropriate substrates and translocates them into the lumen of the CP for breakdown . Here, we describe the molecular and biochemical properties of the 26 S proteasome from the plant Arabidopsis thaliana . Like the CP and the ATPase ring of the RP, the RP non-ATPase subunits are often encoded by two transcriptionally active genes with some pairs displaying sufficient sequence divergence to suggest functional differences . Most RPN subunits could functionally replace their yeast counterparts, implying that they have retained their positions and activities within the complex . A method was developed to purify the 26 S proteasome intact from whole Arabidopsis seedlings . These preparations are biochemically indistinguishable from those from yeast and mammals, including the need for ATP to maintain integrity and a strong sensitivity to the inhibitors MG115, MG132, lactacystin, and epoxomicin . Mass spectrometric analysis of the complex detected the presence of almost all CP and RP subunits . In many cases, both products of paralogous genes were detected, demonstrating that each isoform assembles into the mature particle . As with the yeast and animal 26 S proteasomes, attenuation of individual RP genes induces a coordinated up-regulation of many of the other 26 S proteasome genes, suggesting that plants contain a negative feedback mechanism to regulate the 26 S proteasome levels . The incorporation of paralogous subunits into the Arabidopsis holoprotease raises the intriguing possibility that plants synthesize multiple 26 S proteasome types with unique properties and/or target specificities. Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 877 - 83 hSGT interacts with the N-terminal region of myostatin; Wang H et al.; Myostatin is a new member of the transforming growth factor-beta (TGFbeta) superfamily and functions as a negative regulator of skeletal muscle growth . Herein, we report the identification of a myostatin-associated protein hSGT (human small glutamine-rich tetratricopeptide repeat-containing protein) by using a yeast two-hybrid system . The physical interaction between hSGT and myostatin was further confirmed by pull-down and co-immunoprecipitation experiments . To identify regions involved in the interaction between hSGT and myostatin, we constructed various deletion mutants of hSGT and myostatin, respectively, and examined their interactions in yeast cells . Our results showed that the N-terminal signal peptide of myostatin is essential for its association with hSGT and the C-terminal region of hSGT containing the third TPR motif was indispensable for its interaction with myostatin . Recent studies indicate that hSGT probably functions as a molecular chaperone involved in protein folding and processing . These findings suggest that hSGT may play a role in the regulation of myostatin secretion and activation. J Mol Biol, 2003 Nov 28, 334(3), 445 - 58 The solution structure of human mitochondria fission protein Fis1 reveals a novel TPR-like helix bundle; Suzuki M et al.; Fis1 in yeast localizes to the outer mitochondrial membrane and facilitates mitochondrial fission by forming protein complexes with Dnm1 and Mdv1 . Fis1 orthologs exist in higher eukaryotes, suggesting that they are functionally conserved . In the present study, we cloned the human Fis1 ortholog that was predicted in a database, and determined the protein structure using NMR spectroscopy . Following a flexible N-terminal tail, six alpha-helices connected with short loops construct a single core domain . The C-terminal tail containing a transmembrane segment appears to be disordered . In the core domain, each of two sequentially adjacent helices forms a hairpin-like conformation, resulting in a six helix assembly forming a slightly twisted slab similar to that of a tandem array of tetratrico-peptide repeat (TPR) motif folds . Within this TPR-like core domain, no significant sequence similarity to the typical TPR motif is found . The structural analogy to the TPR-containing proteins suggests that Fis1 binds to other proteins at its concave hydrophobic surface . A simple composition of Fis1 comprised of a binding domain and a transmembrane segment indicates that the protein may function as a molecular adaptor on the mitochondrial outer membrane . In HeLa cells, however, increased levels in mitochondria-associated Fis1 did not result in mitochondrial translocation of Drp1, a potential binding partner of Fis1 implicated in the regulation of mitochondrial fission, suggesting that the interaction between Drp1 and Fis1 is regulated. FEBS Lett, 2003 Nov 20, 554(3), 455 - 61 Identification of HMG-5 as a double-stranded telomeric DNA-binding protein in the nematode Caenorhabditis elegans; Im SH et al.; Many protein components of telomeres, the multifunctional DNA-protein complexes at the ends of eukaryotic chromosomes, have been identified in diverse species ranging from yeast to humans . In Caenorhabditis elegans, CEH-37 has been identified by a yeast one hybrid screen to be a double-stranded telomere-binding protein . However, the role of CEH-37 in telomere function is unclear because a deletion mutation in this gene does not cause severe telomere defects . This observation raises the possibility of the presence of genetic redundancy . To identify additional double-stranded telomere-binding proteins in C . elegans, we used a different approach, namely, a proteomic approach . Affinity chromatography followed by Finnigan LCQ ion trap mass spectrometer analysis allowed us to identify several candidate proteins . We further characterized one of these, HMG-5, which is encoded by F45E4.9 . HMG-5 bound to double-stranded telomere in vitro as shown by competition assays . At least two telomeric DNA repeats were needed for this binding . HMG-5 was expressed in the nuclei of the oocytes and all embryonic cells, but not in the hatched larvae or adults . HMG-5 mainly localized to the chromosomal ends, indicating that HMG-5 also binds to telomeres in vivo . These observations suggest that HMG-5 may participate, together with CEH-37, in early embryogenesis by acting at the telomeres. FEBS Lett, 2003 Nov 20, 554(3), 289 - 94 Direct interaction between alpha-actinin and hepatitis C virus NS5B; Lan S et al.; It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication . By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library . For one of these, alpha-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis . Experiments with deletion mutants showed that domains NS5B(84-95), NS5B(466-478), and alpha-actinin(621-733) are responsible for the interaction . Studies of the HCV subgenomic replicon system with small interference RNA indicate that alpha-actinin is essential for HCV RNA replication . Our results suggest alpha-actinin may be a component of the HCV replication complex. Eur J Neurosci, 2003 Nov, 18(9), 2425 - 32 Developmental elimination of ectopic projection sites for the transgenic OR gene that has lost zone specificity in the olfactory epithelium; Nakatani H et al.; In rodents, olfactory receptor (OR) genes are expressed in one of four zones in the olfactory epithelium (OE), and olfactory sensory neurons (OSNs) expressing the same OR project their axons to a specific set of glomeruli on the olfactory bulb (OB) . Using the yeast artificial chromosome (YAC) transgenic system, we have analysed the expression of the murine OR gene MOR29A of the MOR28 cluster located on chromosome 14 . Although expression of the endogenous MOR29A was restricted to the most dorsomedial zone, the transgenic MOR29A (Tg MOR29A) was expressed in all four zones of the OE . When the OB of the transgenic mouse was analysed, the axons of the OSNs expressing Tg MOR29A were found to project not only to the dorsal side but also to the ventral side of the OB as well . The ectopic projection sites on the ventral side gradually disappear during postnatal development . Naris occlusion prevents this elimination, suggesting that odorant stimulation is involved in eliminating the ectopic projection sites. Biochemistry, 2003 Nov 25, 42(46), 13762 - 71 Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state; Platt GW et al.; A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability . Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment . However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn . The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations . Surprising, alcoholic cosolvents {5-10% (v/v) TFE} are shown to accelerate the folding rate of the NPDG mutant . We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process . It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics . However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity. Biochemistry, 2003 Nov 25, 42(46), 13476 - 83 An NF-kappaB-specific inhibitor, IkappaBalpha, binds to and inhibits cyclin-dependent kinase 4; Li J et al.; IkappaBalpha, a protein composed of six ankyrin repeats, is a specific inhibitor of nuclear factor kappaB (NF-kappaB) and functions in signal transductions in many different cell types . Using both in vivo yeast two-hybrid assays and in vitro activity and binding assays, we showed that IkappaBalpha binds to cyclin-dependent kinase 4 (CDK4) specifically and inhibits its kinase activity . The potencies of binding and inhibition of IkappaBalpha are comparable to those of INK4 proteins, the specific CDK4 inhibitors that also contain ankyrin repeats . Furthermore, we showed that INK4 proteins and IkappaBalpha compete with each other for binding to CDK4 . These results led us to propose a hypothesis that there is cross talk between the NF-kappaB/IkappaBalpha pathway and the p16/CDK4/Rb pathway in cells, and that IkappaBalpha could substitute for the CDK4-inhibiting function of p16, a tumor suppressor frequently inactivated in human tumors . To further understand the structural basis of IkappaBalpha-CDK binding, we used different mutants of CDK4 to show that there are notable differences between IkappaBalpha and INK4 proteins in CDK4 binding since the binding is affected differently by different CDK4 mutations . We also demonstrated that the interaction of IkappaBalpha with CDK4 is different from that with its NF-kappaB . While most of the contacts contributing to NF-kappaB binding are located within the last two C-terminal ankyrin repeats and the loop region bridging them, the first four ankyrin repeats at the N-terminus are responsible for CDK4 binding and inhibition. Biochemistry, 2003 Nov 25, 42(46), 13363 - 70 Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin-interacting protein 1: new blades for twisted filaments; Ono S; Actin depolymerizing factor (ADF)/cofilin enhances turnover of actin filaments by severing and depolymerizing filaments . A number of proteins functionally interact with ADF/cofilin to modulate the dynamics of actin filaments . Actin-interacting protein 1 (AIP1) has emerged as a conserved WD-repeat protein that specifically enhances ADF/cofilin-induced actin dynamics . Interaction of AIP1 with actin was originally characterized by a yeast two-hybrid system . However, biochemical studies revealed its unique activity on ADF/cofilin-bound actin filaments . AIP1 alone has negligible effects on actin filament dynamics, whereas in the presence of ADF/cofilin, AIP1 enhances filament fragmentation by capping ends of severed filaments . Studies in model organisms demonstrated that AIP1 genetically interacts with ADF/cofilin and participates in several actin-dependent cellular events . The crystal structure of AIP1 revealed its unique structure with two seven-bladed beta-propeller domains . Thus, AIP1 is a new class of actin regulatory proteins that selectively enhances ADF/cofilin-dependent actin filament dynamics. Int J Cancer, 2004 Jan 1, 108(1), 152 - 7 Constitutive activation of the androgen receptor by a point mutation in the hinge region: a new mechanism for androgen-independent growth in prostate cancer; Ceraline J et al.; Androgen receptor (AR) mutations that modify both the ligand binding and the transactivation capacities of the AR represent one of the mechanisms involved in the transition of prostate cancer (PCa) from androgen-dependent to androgen-independent growth . We use a yeast-based functional assay to detect and analyze mutant ARs in PCa . We report the detection of 2 different mutant ARs within the same metastatic tumour sample harvested in a patient with advanced PCa who had escaped androgen deprivation . Concomitantly to the widely described T877A mutant AR, we identified an additional double mutant AR harboring the nonsense mutation Q640Stop just downstream the DNA binding domain together with the T877A point mutation . This type of mutation, which leads to a c-terminal truncated AR, has not been described yet in PCa . Using luciferase reporter assays we demonstrated that this truncated AR exhibited constitutive transactivation properties . In conclusion, our data suggest that mutation-induced constitutive activation of the AR could be a mechanism used by PCa cells to escape androgen deprivation . Virus Genes, 2003 Dec, 27(3), 237 - 47 Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts with human myeloid cell nuclear differentiation antigen induced by interferon alpha; Fukushi M et al.; Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpes virus type 8 (HHV-8) is tightly linked to the development of Kaposi's sarcoma, primary effusion lymphoma (PEL) and some cases of multicentric Castleman's disease . Latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in these diseases as well as in KSHV-infected cell lines derived from PEL, and has been shown to play crucial role in persistence of KSHV genomes in the infected cells . In this study, we explored the cellular factors that interact with LANA using yeast two-hybrid screening, and isolated a part of gene encoding human myeloid cell nuclear differentiation antigen (MNDA) . MNDA is a hematopoietic interferon-inducible nuclear proteins with a HIN-200 family member with conserved 200-amino acid repeats . Immunoprecipitation assay revealed that LANA interacted with MNDA in a mammalian embryonic kidney cell line . MNDA transcript was undetectable in three PEL cell lines by reverse-transcription polymerase chain reaction, but it was induced by interferon alpha (IFNalpha) . Moreover, LANA and MNDA were co-localized in the nuclei of MNDA-expressing PEL cells . Our results suggest that LANA interacts with MNDA in KSHV-infected cells exposed to IFNalpha . Such interaction may modulate IFN-mediated host defense activities. Biogerontology, 2003, 4(5), 275 - 87 RecQ helicases and topoisomerase III in cancer and aging; Laursen LV et al.; RecQ helicases have in recent years attracted increasing attention due to the important roles they play in maintaining genomic integrity, which is essential for the life of a cell and the survival of a species . Humans with mutations in RecQ homologues are cancer prone and suffer from premature aging . A great effort has therefore been made to understand the molecular mechanisms and the biological pathways, in which RecQ helicases are involved . It has become clear that these enzymes work in close concert with DNA topoisomerase III, and studies in both yeast and mammalian systems point to a role of the proteins in processes involving homologous recombination . In this review we discuss the genetic and biochemical evidence for possible functions of RecQ helicases and DNA topoisomerase III in multiple cellular processes such as DNA recombination, DNA replication, and cell cycle checkpoint control. Mol Biol Cell, 2004 Feb, 15(2), 696 - 705 Epub 2003 Nov 14. Interactions of GIPC with dopamine D2, D3 but not D4 receptors define a novel mode of regulation of G protein-coupled receptors; Jeanneteau F et al.; The C-terminus domain of G protein-coupled receptors confers a functional cytoplasmic interface involved in protein association . By screening a rat brain cDNA library using the yeast two-hybrid system with the C-terminus domain of the dopamine D(3) receptor (D(3)R) as bait, we characterized a new interaction with the PDZ domain-containing protein, GIPC (GAIP interacting protein, C terminus) . This interaction was specific for the dopamine D(2) receptor (D(2)R) and D(3)R, but not for the dopamine D(4) receptor (D(4)R) subtype . Pull-down and affinity chromatography assays confirmed this interaction with recombinant and endogenous proteins . Both GIPC mRNA and protein are widely expressed in rat brain and together with the D(3)R in neurons of the islands of Calleja at plasma membranes and in vesicles . GIPC reduced D(3)R signaling, cointernalized with D(2)R and D(3)R, and sequestered receptors in sorting vesicles to prevent their lysosomal degradation . Through its dimerization, GIPC acts as a selective scaffold protein to assist receptor functions . Our results suggest a novel function for GIPC in the maintenance, trafficking, and signaling of GPCRs. Mol Biol Cell, 2004 Feb, 15(2), 552 - 62 Epub 2003 Nov 14. RNA interference inhibition of Mus81 reduces mitotic recombination in human cells; Blais V et al.; Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex . In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function . Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo . We show that recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro . By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1 . Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo . Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells . The recombination defect is rescued by expression of a bacterial Holliday junction resolvase . These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo. Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13970 - 5 Epub 2003 Nov 14. IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion; Song SW et al.; Our previous studies have shown that insulin-like growth factor binding protein 2 (IGFBP-2) is frequently overexpressed in the highly invasive glioblastoma multiforme (GBM) . By using a yeast two-hybrid system, we identified a gene, invasion inhibitory protein 45 (IIp45), whose protein product bound to IGFBP-2 through the thyroglobulin-RGD region of the C terminus of IGFBP-2 . The IIp45 gene is located on chromosome 1p36 and has nine exons . The IIp45 protein has three SEG (segment of low compositional complexity) domains and an integrin-binding RGD motif . The IIp45 protein was not expressed in some GBMs . Functional studies showed that IIp45 inhibited GBM cell invasion both in vitro and in xenograft model . Gene expression profiling studies showed that IIp45 consistently inhibited the expression of cell invasion-associated genes, such as the transcriptional NFkappaB, and its downstream target gene, intercellular adhesion molecule 1 . Thus, we report here the isolation and characterization of a gene, IIp45, whose protein product binds to IGFBP-2 and inhibits glioma cell invasion. Traffic, 2003 Dec, 4(12), 891 - 901 Compromise of clathrin function and membrane association by clathrin light chain deletion; Wang J et al.; While clathrin heavy chains from different species are highly conserved in amino acid sequence, clathrin light chains are much more divergent . Thus clathrin light chain may have different functions in different organisms . To investigate clathrin light chain function, we cloned the clathrin light chain, clcA, from Dictyostelium and examined clathrin function in clcA-mutants . Phenotypic deficiencies in development, cytokinesis, and osmoregulation showed that light chain was critical for clathrin function in Dictyostelium . In contrast with budding yeast, we found the light chain did not influence steady-state levels of clathrin, triskelion formation, or contribute to clathrin over-assembly on intracellular membranes . Imaging GFP-CHC in clcA- mutants showed that the heavy chain formed dynamic punctate structures that were remarkably similar to those found in wild-type cells . However, clathrin light chain knockouts showed a decreased association of clathrin with intracellular membranes . Unlike wild-type cells, half of the clathrin in clcA- mutants was cytosolic, suggesting that the absence of light chain compromised the assembly of triskelions onto intracellular membranes . Taken together, these results suggest a role for the Dictyostelium clathrin light chain in regulating the self-assembly of triskelions onto intracellular membranes, and demonstrate a crucial contribution of the light chain to clathrin function in vivo. Plant J, 2003 Nov, 36(3), 382 - 9 Molecular cloning and characterization of a sodium-pump ATPase of the moss Physcomitrella patens; Benito B et al.; Physcomitrella patens grew slowly at 600 mm Na+, pH 6.0, affected by the low water potential but without signs of suffering Na+ toxicity . At pH 8.0, tolerance seemed to be lower but it grew at 200 mm Na+, again without signs of Na+ toxicity . The resistance of Physcomitrella cells to the toxic effects of Na+ can be accounted for by their capacity to keep high K+:Na+ ratios and to extrude Na+ by a system that is not dependent on DeltapH . Physcomitrella expresses two P-type ATPases similar in sequence to fungal ENA-type Na+-ATPases . A functional study in yeast demonstrated that one of these ATPases, PpENA1, is an Na+-pump . We also found that P . patens has a plant-type SOS1 Na+/H+ antiporter . We discuss that Na+-ATPases existed in early land plants but that they were lost during the evolution of bryophytes to flowering plants. Annu Rev Genet, 2003, 37, 329 - 48 Genetics of aging in the fruit fly, Drosophila melanogaster; Helfand SL et al.; Research into the mechanisms underlying the process of aging is emerging as an exciting area of biomedical research . Observations challenging the fundamental assumptions of aging have begun to rejuvenate the field, opening up aging research to fresh ideas and approaches . Genetic approaches, which have been successfully used to understand other complex biological phenomena, are beginning to reveal important patterns and conservations between the processes of aging in a variety of species including yeast, nematodes, flies, and mice . A combination of candidate and random gene alteration approaches, particularly in the fruitfly model system, Drosophila melanogaster, should prove to be especially valuable for elucidating the primary physiological systems involved in aging and life span determination. Nippon Ishinkin Gakkai Zasshi, 2003, 44(4), 299 - 306 Histoplasma capsulatum variety duboisii isolated in Japan from an HIV-infected Ugandan patient; Sharmin S et al.; A strain of Histoplasma capsulatum var . duboisii (deposited as IFM 50954 in Chiba University) was isolated from the cerebrospinal fluid of a female Ugandan patient infected with HIV . The isolate had in vitro urease activity on Christensen's urea agar slants, although the common belief is that H . capsulatum var . duboisii is urease negative, and is, considered one of the characteristic markers that distinguishes the three varieties of H . capsulatum . Forty H . capsulatum var . capsulatum, five H . capsulatum var . duboisii, and five H . capsulatum var . farciminosum isolates were evaluated for urease activity on Christensen's urea agar slants and for other qualitative and quantitative urease assays of activity . All 50 isolates of H . capsulatum used in this study were positive for urease activity, suggesting that urease activity may be universal characteristic of H . capsulatum . We also compared the urease activity and pathogenicity of seven H . capsulatum isolates that convert into yeast-form cells . Although isolate IFM 50954 showed moderate virulence in mice and moderate urease activity among tested H . capsulatum isolates, there was no correlation between level of urease activity and pathogenicity . In addition, scanning electron microscopy revealed that some microconidia of isolate IFM 50954 formed "double-cell" configurations that were attached to each other by narrow bases. J Biomed Biotechnol, 2003, 2003(4), 249 - 255 Selective Enrichment of Membrane Proteins by Partition Phase Separation for Proteomic Studies; Qoronfleh MW et al.; The human proteome project will demand faster, easier, and more reliable methods to isolate and purify protein targets . Membrane proteins are the most valuable group of proteins since they are the target for 70-80% of all drugs . Perbio Science has developed a protocol for the quick, easy, and reproducible isolation of integral membrane proteins from eukaryotic cells . This procedure utilizes a proprietary formulation to facilitate cell membrane disruption in a mild, nondenaturing environment and efficiently solubilizes membrane proteins . The technique utilizes a two-phase partitioning system that enables the class separation of hydrophobic and hydrophilic proteins . A variety of protein markers were used to investigate the partitioning efficiency of the membrane protein extraction reagents (Mem-PER) (Mem-PER is a registered trademark of Pierce Biotechnology, Inc) system . These included membrane proteins with one or more transmembrane spanning domains as well as peripheral and cytosolic proteins . Based on densitometry analyses of our Western blots, we obtained excellent solubilization of membrane proteins with less than 10% contamination of the hydrophobic fraction with hydrophilic proteins . Compared to other methodologies for membrane protein solubilization that use time-consuming protocols or expensive and cumbersome instrumentation, the Mem-PER reagents system for eukaryotic membrane protein extraction offers an easy, efficient, and reproducible method to isolate membrane proteins from mammalian and yeast cells. Plant Cell, 2003 Dec, 15(12), 2826 - 42 Epub 2003 Nov 13. A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis; Weber I et al.; In the early stages of plant infection, yeast-like haploid sporidia of Ustilago maydis respond to pheromone secreted by compatible partners by forming conjugation tubes . These then fuse to generate a dikaryotic hypha that forms appressoria to penetrate the host plant . As a first step toward understanding the structural requirements for these transitions, we have identified myo5, which encodes a class-V myosin . Analysis of conditional and null mutants revealed that Myo5 plays nonessential roles in cytokinesis and morphogenesis in sporidia and is required for hyphal morphology . Consistent with a role in morphogenesis, a functional green fluorescent protein-Myo5 fusion protein localized to the bud tip and the hyphal apex as well as to the septa and the spore wall during later stages of infection . However, the loss of Myo5 did not affect the tip growth of hyphae and sporidia . By contrast, Myo5 was indispensable for conjugation tube formation . Furthermore, myo5 mutants were impaired in the perception of pheromones, which indicates a particular importance of Myo5 in the mating process . Consequently, few mutant hyphae were formed that penetrated the plant epidermis but did not continue invasive growth . These results indicate a crucial role of Myo5 in the morphogenesis, dimorphic switch, and pathogenicity of U . maydis. Lab Invest, 2003 Nov, 83(11), 1555 - 67 Potent phagocytic activity discriminates metastatic and primary human malignant melanomas: a key role of ezrin; Lugini L et al.; Features of phagocytosis have been observed in human tumors, but the phagocytic apparatus of tumor cells and the mechanism(s) underlying this phenomenon have yet to be defined . To address the phenomenon of phagocytosis, its underlying mechanism(s), and its possible role in tumor biology, we used human melanoma cells as a prototypic model . Our results showed that a process of phagocytosis of apoptotic cells occurs in vivo in human melanoma . This finding was consistent with evidence that human melanoma cells in vitro express all of the known lysosomal and phagocytic markers on their cytoplasmic vesicles and that a process of phagocytosis occurs in these vesicles . However, exclusively human melanoma cells deriving from metastatic lesions possess an efficient phagocytic machinery responsible for a macrophage-like activity against latex beads, yeast, and apoptotic cells of different origins, which was comparable to that of human primary macrophages . Moreover, the actin-binding protein ezrin was expressed on phagocytic vacuoles of melanoma cells and of cells deriving from a human adenocarcinoma; both treatment with cytochalasin B and specific inhibition of ezrin synthesis strongly affected the phagocytic activity of melanoma cells . This suggests that the association with the actin cytoskeleton is a crucial requirement for the development of this phenomenon . Hence our data provide evidence for a potent phagocytic activity exerted by metastatic melanoma cells possibly involved in determining the level of aggressiveness of human melanoma . This suggests that the assessment of phagocytic activity may be exploited as a new tool to evaluate the malignancy of human melanoma . Moreover, our data suggest that gene therapy or drug treatments aimed at inhibiting actin assembly to the phagosomal membranes may be proposed as a new strategy for the control of tumor aggressiveness. Alcohol, 2003 Aug-Oct, 31(1-2), 19 - 24 Use of an "acetaldehyde clamp" in the determination of low-KM aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells; Moncada C et al.; The high-affinity (K(M)<1 microM) mitochondrial class 2 aldehyde dehydrogenase (ALDH2) metabolizes most of the acetaldehyde generated in the hepatic oxidation of ethanol . H4-II-E-C3 rat hepatoma cells have been found to express ALDH2 . We report a method to assess ALDH2 activity in intact hepatoma cells that does not require mitochondrial isolation . To determine only the high-affinity ALDH2 activity it is necessary to keep constant low concentrations of acetaldehyde in the cells to minimize its metabolism by high-K(M) aldehyde dehydrogenases . To maintain both low and constant concentrations of acetaldehyde we used an "acetaldehyde clamp," which keeps acetaldehyde at a concentration of 4.2+/-0.4 microM . The clamp is attained by addition of excess yeast alcohol dehydrogenase, 14C-ethanol, and oxidized form of nicotinamide adenine dinucleotide (NAD(+)) to the hepatoma cell culture medium . The concentration of 14C-acetaldehyde attained follows the equilibrium constant of the alcohol dehydrogenase reaction . Thus, 14C-acetate is generated virtually by the low-K(M) aldehyde dehydrogenase activity . 14C-acetate is separated from the culture medium by an anionic resin and its radioactivity is determined . We showed that (1) acetate production is linear for 120 min, (2) addition of 160 microM cyanamide to the culture medium leads to a 75%-80% reduction of acetate generated, and (3) ALDH2 activity is dependent on cell-to-cell contact and increases after cells reach confluence . The clamp system allows the determination of ALDH2 activity in less than one million H4-II-E-C3 rat hepatoma cells . The specificity and sensitivity of the "acetaldehyde clamp" assay should be of value in evaluation of the effects of new agents that modify Aldh2 gene expression, as well as in the study of ALDH2 regulation in intact cells. Curr Biol, 2003 Nov 11, 13(22), 2004 - 8 LKB1 is the upstream kinase in the AMP-activated protein kinase cascade; Woods A et al.; Inactivating mutations in the protein kinase LKB1 lead to a dominantly inherited cancer in humans termed Peutz-Jeghers syndrome . The role of LKB1 is unclear, and only one target for LKB1 has been identified in vivo {3} . AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a pivotal role in energy homeostasis . AMPK may have a role in protecting the body from metabolic diseases including type 2 diabetes, obesity, and cardiac hypertrophy . We previously reported the identification of three protein kinases (Elm1, Pak1, and Tos3 {9}) that lie upstream of Snf1, the yeast homologue of AMPK . LKB1 shares sequence similarity with Elm1, Pak1, and Tos3, and we demonstrated that LKB1 phosphorylates AMPK on the activation loop threonine (Thr172) within the catalytic subunit and activates AMPK in vitro {9} . Here, we have investigated whether LKB1 corresponds to the major AMPKK activity present in cell extracts . AMPKK purified from rat liver corresponds to LKB1, and blocking LKB1 activity in cells abolishes AMPK activation in response to different stimuli . These results identify a link between two protein kinases, previously thought to lie in unrelated, distinct pathways, that are associated with human diseases. Curr Biol, 2003 Nov 11, 13(22), 1921 - 9 The C . elegans Tousled-like kinase (TLK-1) has an essential role in transcription; Han Z et al.; BACKGROUND: The Tousled kinases comprise an evolutionarily conserved family of proteins that have been previously implicated in chromatin remodeling, DNA replication, and DNA repair . Here, we used RNA mediated interference (RNAi) to determine the function of the C . elegans Tousled kinase (TLK-1) during embryonic development . RESULTS: TLK-1-deficient embryos arrested with a phenotype reminiscent of embryos that are broadly defective in transcription, and the expression of several reporter genes was dramatically reduced in tlk-1(RNAi) embryos . Furthermore, posttranslational modifications of RNA polymerase II (RNAPII) and histone H3 that have been correlated with transcription elongation, phosphorylation of the RNAPII CTD at Serine 2, and methylation of histone H3 at Lysine 36 were found at significantly reduced levels in tlk-1(RNAi) embryos as compared to wild-type . CONCLUSIONS: These results reveal a surprising requirement for a Tousled-like kinase in transcriptional regulation during development, likely during the elongation phase . In addition, our results confirm that the link between RNAPII phosphorylation and histone H3 methylation previously observed in budding yeast is functionally conserved in metazoans. Oncogene, 2003 Nov 13, 22(51), 8283 - 92 ATM/ATR-independent inhibition of cyclin B accumulation in response to hydroxyurea in nontransformed cell lines is altered in tumour cell lines; Florensa R et al.; The DNA replication checkpoint is an inhibitory pathway ensuring that mitosis occurs only after completion of DNA synthesis . Its function may be relevant to the stability of the genome . The essential elements of this checkpoint are ATM/ATR kinases that indirectly lead to the phosphorylation and inhibition of the mitosis-promoting factor (Cdc2/cyclin B1) . The function of this checkpoint was analysed in diverse nontransformed and tumour-derived cell lines . All cell lines tested arrested mitosis entry when DNA synthesis was inhibited by hydroxyurea (HU) treatment . But, unlike what has been described in yeast and Xenopus, in normal rat kidney (NRK) cells and NIH 3T3 fibroblasts, the arrest induced by HU treatment was not abrogated by caffeine, an ATM and ATR inhibitor . This indicated the presence of an ATM/ATR-independent response to DNA synthesis inhibition in these nontransformed mammalian cell lines . Interestingly, the behaviour of different tumour cell lines after caffeine treatment varied . While SW480, NP29, NP18 and HeLa cells did not enter mitosis in the presence of caffeine after HU treatment, in CaCo2, DLD1, HCT116 and HT29 caffeine abrogated the checkpoint response . In nontransformed cell lines, lack of cyclin B1 accumulation was observed when DNA synthesis was inhibited . This response was not abrogated by caffeine . In the tumour cell lines, a good correlation between the ability to arrest cell cycle when DNA synthesis was inhibited in the presence of caffeine and the lack of cyclin B1 accumulation was observed . Thus, there is an ATM/ATR-independent checkpoint response that leads to a decrease in cyclin B1 accumulation . However, this response is not functional in some tumour cell lines . Using inhibitors of p38alpha and beta, Mek1, 2 and p53-/- knocked-out fibroblasts, we showed that these proteins were also not involved in this particular checkpoint response . Lack of cyclin B1 accumulation after DNA synthesis inhibition in NRK cells was not due to increased degradation of the protein, but correlated with a decrease in mRNA accumulation. Oncogene, 2003 Nov 13, 22(51), 8255 - 62 Protein kinase CKIIalpha interacts with the Bcr moiety of Bcr/Abl and mediates proliferation of Bcr/Abl-expressing cells; Mishra S et al.; The Bcr protein was originally identified because of its fusion to Abl as a consequence of the Philadelphia chromosome translocation found in chronic myelogenous and acute lymphoblastic leukemias . The Bcr moiety is essential for the transforming activity of the Bcr/Abl oncogene . In search of physiologically relevant Bcr and Bcr/Abl-interacting proteins, we performed an interaction screen in yeast using the entire Bcr protein as bait . We here report that the alpha catalytic subunit of protein kinase CKII strongly and specifically forms a complex with Bcr in yeast in mouse lysates . The region in Bcr responsible for CKIIalpha binding was localized to residues 242-413 . CKIIalpha was previously shown to be involved in leukemogenesis and tumorigenesis using different experimental approaches including mouse models . Inhibition of Bcr/Abl P190 in lymphoma cells from Bcr/Abl transgenic mice using imatinib reduced CKIIalpha activity . A highly selective inhibitor of CKIIalpha, 4,5,6,7-tetrabromo-2-benzotriazole, inhibited the growth of murine lymphoid cells with induced P210 Bcr/Abl expression and of P190 lymphoma cells . Our results demonstrate that CKIIalpha plays an important role in the proliferation of Bcr/Abl expressing cells, and suggests that inhibitors of CKIIalpha may have therapeutic potential in the treatment of Bcr/Abl-positive leukemia patients. J Biol Chem, 2004 Feb 6, 279(6), 4670 - 9 Epub 2003 Nov 12. Endofin recruits TOM1 to endosomes; Seet LF et al.; Endofin is an endosomal protein implicated in regulating membrane trafficking . It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule . To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system . Three clones that encode TOM1 were recovered . TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family . Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown . Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1 . Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1 . Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2) . The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody . This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin . It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses . In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1 . A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxyl-terminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking. FEMS Yeast Res, 2003 Nov, 4(2), 131 - 9 Peroxisome homeostasis in Hansenula polymorpha; Leao AN et al.; Peroxisomes are essential organelles in many eukaryotes . Until recently, the main focus of the investigations concerning these important organelles was to understand the biogenesis of the peroxisome (induction, proliferation and matrix protein import) . However, when peroxisomes become redundant they are quickly degraded by highly selective processes known as pexophagy . The first molecular studies on pexophagy have indicated that this process shares many features with certain transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole targeting and endocytosis) . Nevertheless, recent data demonstrate that in addition to common genes also unique genes are required for these transport processes . The main focus for the future should therefore be on identifying the unique determinants of pexophagy . Earlier results suggest that in the methylotrophic yeast Hansenula polymorpha proteins located on the peroxisome itself are required for pexophagy . Thus, it has become essential to study in detail the role of peroxisomal membrane proteins in the degradation process . This review highlights the main achievements of the last few years, with emphasis on H . polymorpha. J Biol Chem, 2004 Jan 30, 279(5), 3370 - 4 Epub 2003 Nov 11. FAD transport and FAD-dependent protein thiol oxidation in rat liver microsomes; Varsanyi M et al.; The transport of FAD and its effect on disulfide bond formation was investigated in rat liver microsomal vesicles . By measuring the intravesicular FAD-accessible space, we observed that FAD permeates across the microsomal membrane and accumulates in the lumen . Rapid filtration experiments also demonstrated the uptake and efflux of the compound, which could be inhibited by atractyloside and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid . FAD entering the lumen promoted the oxidation of protein thiols and increased the intraluminal oxidation of glucose-6-phosphate . These findings support the notion that, similar to yeast, free FAD may have a decisive role in the mechanism of oxidative protein folding in the endoplasmic reticulum lumen of mammalian cells. Curr Opin Plant Biol, 2003 Dec, 6(6), 611 - 6 Feedback from the wall; Pilling E et al.; The ability of cells to perceive changes in the composition and mechanical properties of their cell wall is crucial for plants to achieve coordinated growth and development . Evidence is accumulating to show that the plant cell wall, like its yeast counterpart, is capable of triggering multiple signalling pathways . The components of the cell wall that are responsible for initiating these signal responses remain unknown; however, recent technological advances in cell wall analysis may now facilitate the identification of these components and accelerate the characterisation of changes that occur in cell wall mutants. Biochem J, 2004 Mar 1, 378(Pt 2), 353 - 62 The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins; Paces-Fessy M et al.; Sufu (Suppressor of fused) is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors . However, its function remains poorly understood . In the present study, we determined the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein . We identified several mSufu variants of which some were phosphorylated . A yeast two-hybrid screen with mSufu as bait allowed us to identify several nuclear proteins as potential partners of mSufu . Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing . We confirmed the mSufu-SAP18 and mSufu-Galectin3 interactions by independent biochemical assays . Using a cell transfection assay, we also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B . Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment . In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu . Our findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus. Planta, 2004 Jan, 218(3), 327 - 36 Epub 2003 Nov 11. The plant nuclear envelope; Rose A et al.; This review summarizes our present knowledge about the composition and function of the plant nuclear envelope . Compared with animals or yeast, our molecular understanding of the nuclear envelope in higher plants is in its infancy . However, fundamental differences in the structure and function of the plant and animal nuclear envelope have already been found . Here, we compare and contrast these differences with respect to nuclear pore complexes, targeting of Ran signaling to the nuclear envelope, inner nuclear envelope proteins, and the role and fate of the nuclear envelope during mitosis . Further investigation of the emerging fundamental differences as well as the similarities between kingdoms might illuminate why there appears to be more than one blueprint for building a nucleus. J Virol, 2003 Dec, 77(23), 12819 - 28 Membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand RNA virus RNA replication protein; Lee WM et al.; Multifunctional RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus, localizes to the cytoplasmic face of endoplasmic reticulum (ER) membranes and induces ER lumenal spherules in which viral RNA synthesis occurs . We previously showed that BMV RNA replication in yeast is severely inhibited prior to negative-strand RNA synthesis by a single-amino-acid substitution in the ole1w allele of yeast Delta9 fatty acid (FA) desaturase, which converts saturated FAs (SFAs) to unsaturated FAs (UFAs) . Here we further define the relationships between 1a, membrane lipid composition, and RNA synthesis . We show that 1a expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous 1a-induced spherules are enveloped by invaginations of the outer ER membrane . 1a did not alter total membrane lipid composition in wt or ole1w yeast, but the ole1w mutation selectively depleted 18-carbon, monounsaturated (18:1) FA chains and increased 16:0 SFA chains, reducing the UFA-to-SFA ratio from approximately 2.5 to approximately 1.5 . Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity . The ole1w mutation did not alter 1a-induced membrane synthesis, 1a localization to the perinuclear ER, or colocalization of BMV 2a polymerase, nor did it block spherule formation . Moreover, BMV RNA replication templates were still recovered from cell lysates in a 1a-induced, 1a- and membrane-associated, and nuclease-resistant but detergent-susceptible state consistent with spherules . However, unlike nearby ER membranes, the membranes surrounding spherules in ole1w cells were not distinctively stained with osmium tetroxide, which interacts specifically with UFA double bonds . Thus, in ole1w cells, spherule-associated membranes were locally depleted in UFAs . This localized UFA depletion helps to explain why BMV RNA replication is more sensitive than cell growth to reduced UFA levels . The results imply that 1a preferentially interacts with one or more types of membrane lipids. J Virol, 2003 Dec, 77(23), 12507 - 22 The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1; Violot S et al.; Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R . Peytavi et al., J . Biol . Chem . 274:1635-1645, 1999) . In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast . In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264 . In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats . EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner . In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.) . Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle . Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1. EMBO J, 2003 Nov 17, 22(22), 6137 - 47 Eme1 is involved in DNA damage processing and maintenance of genomic stability in mammalian cells; Abraham J et al.; Yeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication . We identified mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3'-flap structures and replication forks rather than Holliday junctions in vitro . We demonstrate that Eme1-/- embryonic stem (ES) cells are hypersensitive to the DNA cross-linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment . Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE) . Unlike Blm-deficient ES cells, increased SCE was seen only following induced DNA damage in Eme1-deficient cells . Most importantly, Eme1 deficiency led to spontaneous genomic instability . These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity. EMBO J, 2003 Nov 17, 22(22), 6115 - 26 Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila; Araki M et al.; The initiation of DNA synthesis is thought to occur at sites bound by a heteromeric origin recognition complex (ORC) . Previously, we have shown that in Drosophila, the level of the large subunit, ORC1, is modulated during cell cycle progression and that changes in ORC1 concentration alter origin utilization during development . Here, we investigate the mechanisms underlying cell cycle-dependent degradation of ORC1 . We show that signals in the non-conserved N-terminal domain of ORC1 mediate its degradation upon exit from mitosis and in G1 phase by the anaphase-promoting complex (APC) in vivo . Degradation appears to be the result of direct action of the APC, as the N-terminal domain is ubiquitylated by purified APC in vitro . This regulated proteolysis is potent, sufficient to generate a normal temporal distribution of protein even when transcription of ORC1 is driven by strong constitutive promoters . These observations suggest that in Drosophila, ORC1 regulates origin utilization much as does Cdc6 in budding yeast. Virus Res, 2003 Dec, 98(1), 83 - 91 Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules; Lee BH et al.; We performed yeast two-hybrid screening of a human kidney cell cDNA library to study the biological role of the hantavirus nucleocapsid protein (NP) . We found that Seoul virus (SEOV) and Hantaan virus (HTNV) NPs were associated with small ubiquitin-like modifier (SUMO)-1-interacting proteins PIAS1, PIASxbeta, HIPK2, CHD3, and TTRAP, which interacted with the SUMO-1 conjugating enzyme (Ubc-9) and SUMO-1 in the yeast two-hybrid assay . Interactions between the HIPK2, CHD3, and TTRAP proteins and SEOV NP were also shown in a mammalian two-hybrid assay . However, there was no interaction between PIAS proteins and NP, which was probably due to the inhibitory effect of PIAS on transcription in the mammalian two-hybrid assay . Nevertheless, a co-expression experiment suggested the existence of a PIAS-NP interaction in the cytoplasm . The region spanning amino acids 100-125 of SEOV NP, which represents a critical region for NP-NP polymerization, was found to be responsible for the interaction with SUMO-1-related molecules in both the yeast and mammalian two-hybrid assays . These results add to the information on interactions of hantavirus NP and host cellular proteins. Virus Res, 2003 Dec, 98(1), 57 - 61 The Tat protein of the human immunodeficiency virus type 1 (HIV-1) interacts with the EGF-like repeats of the Notch proteins and the EGF precursor; Shoham N et al.; Employing the yeast two-hybrid system, the Tat protein of the human immunodeficiency virus (HIV) was shown to interact with a region spanning the EGF-like repeats 1-6 of the mouse Notch1, the human Notch2 and the Drosophila Notch . This observation was confirmed in mammalian cells by demonstrating an interaction between the HIV Tat and the EGF-like repeats 1-6 of the various Notch proteins . The HIV Tat protein interacted also with the full-length mouse Notch1 receptor when co-expressed in mammalian cells . Moreover, the HIV Tat protein interacted also with the EGF-like repeats 1-4-spanning domain of the human EGF precursor . The ability of the HIV Tat protein to interact with the Notch proteins and possibly with other EGF-like repeats-bearing proteins, suggests that such interactions might modulate their physiological functions, thus affecting various AIDS-associated pathologies. Arch Pharm Res, 2003 Oct, 26(10), 846 - 54 Homo- or hetero-dimerization of muscarinic receptor subtypes is not mediated by direct protein-protein interaction through intracellular and extracellular regions; Kang YK et al.; The oligomerization of G-proteincoupled receptors (GPCRs) has been shown to occur by various mechanisms, such as via disulfide covalent linkages, noncovalent (ionic, hydrophobic) interactions of the N-terminal, and/or transmembrane and/or intracellular domains . Interactions between GPCRs could involve an association between identical proteins (homomers) or non-identical proteins (heteromers), or between two monomers (to form dimers) or multiple monomers (to form oligomers) . It is believed that muscarinic receptors may also be arranged into dimeric or oigomeric complexes, but no systematic experimental evidence exists concerning the direct physical interaction between receptor proteins as its mechanism . We undertook this study to determine whether muscarinic receptors form homomers or a heteromers by direct protein-protein interaction within the same or within different subtypes using a yeast two-hybrid system . Intracellular loops (i1, i2 and i3) and the C-terminal cytoplasmic tails (C) of human muscarinic (Hm) receptor subtypes, Hm1, Hm2 and Hm3, were cloned into the vectors (pB42AD and pLexA) of a two-hybrid system and examined for heteromeric or homodimeric interactions between the cytoplasmic domains . No physical interaction was observed between the intracellular domains of any of the Hm/Hm receptor sets tested . The results of our study suggest that the Hm1, Hm2 and Hm3 receptors do not form dimers or oligomers by interacting directly through either the hydrophilic intracellular domains or the C-terminal tail domains . To further investigate extracellular domain interactions, the N-terminus (N) and extracellular loops (o1 and o2) were also cloned into the two-hybrid vectors . Interactions of Hm2N with Hm2N, Hm2o1, Hm2o2, Hm3N, Hm3o1 or Hm3o2 were examined . The N-terminal domain of Hm2 was found to have no direct interaction with any extracellular domain . From our results, we excluded the possibility of a direct interaction between the muscarinic receptor subtypes (Hm1, Hm2 and Hm3) as a mechanism for homo- or hetero-meric dimerization/oligomerization . On the other hand, it remains a possibility that interaction may occur indirectly or require proper conformation or subunit formation or hydrophobic region involvement. Genome, 2003 Oct, 46(5), 745 - 52 Structure and evolution of the Cinful retrotransposon family of maize; Sanz-Alferez S et al.; A maize cDNA clone was isolated by virtue of its intense hybridization to total maize genomic DNA, indicating homology to highly repetitive sequences . Genomic homologues were identified and subcloned from an adh1-bearing maize yeast artificial chromosome (YAC) . Sequencing revealed that the expressed sequence was part of a Ty3-gypsy-type retrotransposon . We discovered and sequenced two complete retrotransposons of this family, and named them Cinful elements because they are members of a family of maize retrotransposons including Zeon-1 and the first plant transposable element sequenced, the solo long terminal repeat (LTR) called Cin1 . All are defective, as Cinful-1 and Cinful-2 elements lack gag and Zeon-1 lacks pol homology . Despite the apparent lack of an intact "autonomous" element, the Cinful family has expanded to a copy number of about 18 000, representing just under 9% of the maize genome . Both point mutations and major rearrangements, including possible gene acquisition, differentiate members of the Cinful family . Cinful family members were found to have an unusual feature that we also observed in two other Ty3-class retrotransposons of teosinte and tobacco: related tandem repeats that separate their internal domains with a gag- or pol-containing homology from a 3' segment of unknown function . The conserved and variable features identified provide insights into the origin, mutational history, and functional components of this major constituent of the maize genome. Nat Biotechnol, 2003 Dec, 21(12), 1509 - 12 Epub 2003 Nov 09. Analyzing antibody specificity with whole proteome microarrays; Michaud GA et al.; Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins . Furthermore, new applications such as antibody arrays and monoclonal antibody therapeutics have increased the demand for more specific antibodies to reduce cross-reactivity and side effects . An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity, because it allows the simultaneous screening of thousands of proteins for possible cross-reactivity . As an initial test of this approach, we screened 11 polyclonal and monoclonal antibodies to approximately 5,000 different yeast proteins deposited on a glass slide and found that, in addition to recognizing their cognate proteins, the antibodies cross-reacted with other yeast proteins to varying degrees . Some of the interactions of the antibodies with noncognate proteins could be deduced by alignment of the primary amino acid sequences of the antigens and cross-reactive proteins; however, these interactions could not be predicted a priori . Our findings show that proteome array technology has potential to improve antibody design and selection for applications in both medicine and research. J Immunol, 2003 Nov 15, 171(10), 5320 - 7 Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta regulate IgE receptor-triggered exocytosis in cultured mast cells; Kapp-Barnea Y et al.; We examined the possible occurrence and function of neuronal Ca(2+) sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3 . This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells . We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein . Both proteins associate in vivo with phosphatidylinositol 4-kinase beta (PI4Kbeta) and colocalize with the enzyme in the Golgi region . We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kbeta, increases IgE receptor (FcepsilonRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), and stimulates FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis . Conversely, expression of a kinase-dead mutant of PI4Kbeta reduces PI4Kbeta activity, decreases FcepsilonRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis . Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kbeta, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P(2) that serves as substrate for the generation of lipid-derived second messengers in FcepsilonRI-triggered cells . We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kbeta and positive regulation of its activity. J Immunol, 2003 Nov 15, 171(10), 5064 - 70 DQ 65-79, a peptide derived from HLA class II, mimics p21 to block T cell proliferation; Dong C et al.; DQ 65-79, a peptide derived from residues 65-79 of the alpha-chain HLA class II molecule DQA03011, blocks T cell proliferation and induces T cell apoptosis . Using a yeast two-hybrid assay, we previously identified proliferating cell nuclear Ag (PCNA) as an intracellular ligand for DQ 65-79 . In this study, we show that three regions of PCNA, residues 81-100, 121-140, and 241-261, interact with DQ 65-79 . Residues 241-261 of PCNA also interact with the C terminus (residues 139-160) of the cell cycle regulator, p21, suggesting that DQ 65-79 and p21 might function similarly . We show here that DQ 65-79 competitively inhibits binding of p21 to PCNA and that both DQ 65-79 and p21 139-160 induce T cell apoptosis, suggesting that DQ 65-79 and p21 act similarly to inhibit cell growth. Trends Biochem Sci, 2003 Nov, 28(11), 612 - 8 SUMO: ligases, isopeptidases and nuclear pores; Melchior F et al.; Small ubiquitin-related modifier (SUMO) proteins are reversibly coupled to numerous intracellular targets and modulate their interactions, localization, activity or stability . Recent advances in the SUMO field have uncovered the first SUMO E3 ligases and point to a complex family of isopeptidases . SUMO has been linked to many different pathways, including nucleocytoplasmic transport . Modifying enzymes and an isopeptidase have been detected at nuclear pore complexes . In addition, studies in yeast suggest a requirement of SUMO conjugation for nuclear protein import, and specific SUMO targets depend on modification for nuclear import or export. Plant Physiol, 2003 Dec, 133(4), 1643 - 53 Epub 2003 Nov 06. CYP72B1 inactivates brassinosteroid hormones: an intersection between photomorphogenesis and plant steroid signal transduction; Turk EM et al.; Active brassinosteroids, such as brassinolide (BL) and castasterone, are growth promoting plant hormones . An Arabidopsis cytochrome p450 monooxygenase encoded by CYP72B1 has been implicated in brassinosteroid catabolism as well as photomorphogenesis . We expressed CYP72B1 in yeast, coupled with brassinosteroid feeding, and established the biochemical function to be the hydroxylation of BL and castasterone, to give 26-hydroxybrassinolide and 26-hydroxycastasterone, respectively . Brassinosteroid feeding experiments with wild-type Arabidopsis, a CYP72B1 null mutant, and a CYP72B1 overexpression line demonstrated that carbon 26 hydroxylation of active brassinosteroids is an endogenous function of CYP72B1 . Seedling growth assays demonstrated that 26-hydroxybrassinolide is an inactive brassinosteroid . Genetic and physiological analysis of the hypocotyl response to exogenous BL and varying intensities of white and monochromatic light suggested that CYP72B1 modulates photomorphogenesis primarily through far-red light and to a lesser extent through blue- and red-light pathways . CYP72B1 transcript accumulation in dark-grown seedlings was organ specific and down-regulated after 1 h of illumination in dim white, red, and blue light, but not far-red light . CYP72B1 translational fusions with the beta-glucuronidase reporter gene demonstrated that protein levels increased in the hypocotyl elongation zone when shifted from the dark to far-red light, but not blue or red light . We propose a model in which Arabidopsis seedling development switches from dark-grown development (skotomorphogenesis) to light-grown development (photomorphogenesis) in part by rapid modulation of brassinosteroid sensitivity and levels . CYP72B1 provides an intersection between the light and brassinosteroid pathways mainly by far-red-light-dependent modulation of brassinosteroid levels. J Clin Microbiol, 2003 Nov, 41(11), 5333 - 6 Allergic fungal sinusitis associated with Trichoderma longibrachiatum; Tang P et al.; We describe allergic fungal sinusitis caused by Trichoderma longibrachiatum in a patient with a history of atopy and asthma . A Gram stain of a sinus biopsy specimen was initially thought to contain yeast cells, but when Trichoderma was recovered in culture, these cells were subsequently recognized as chlamydospores . The patient was successfully managed with a combination of sinus lavage, oral corticosteroids, itraconazole, and allergen immunotherapy . This case also points out that careful scrutiny of direct smears is required to ensure that fungal structures are not misinterpreted. J Clin Microbiol, 2003 Nov, 41(11), 5250 - 3 Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures; Majoros L et al.; We identified 29 yeast isolates from 22 patients using the API ID32C panel . Twenty-eight of these isolates were Candida norvegensis and one was C . inconspicua . Although C . norvegensis is considered a pseudohypha-producing species, only one isolate produced pseudohyphae . Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C . inconspicua. J Biol Chem, 2004 Feb 20, 279(8), 6863 - 73 Epub 2003 Nov 05. Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions; Yoo D et al.; The ROMK subtypes of inward rectifier K+ channels (Kir 1.1, KCNJ1) mediate potassium secretion and regulate NaCl reabsorption in the kidney . In the present study, the role of the PDZ binding motif in ROMK function is explored . Here we identify the Na/H exchange regulatory factors, NHERF-1 and NHERF-2, as PDZ domain interaction partners of the ROMK channel . Characterization of the basis and consequences of NHERF association with ROMK reveals a PDZ interaction-dependent trafficking process and a coupling mechanism for linking ROMK to a channel modifier protein, the cystic fibrosis transmembrane regulator (CFTR) . As measured by antibody binding of external epitope-tagged forms of Kir 1.1 in intact cells, NHERF-1 or NHERF-2 coexpression increased cell surface expression of ROMK . Channel interaction with NHERF proteins and effects of NHERF on ROMK localization were dependent on the presence of the PDZ domain binding motif in ROMK . Both NHERF proteins contain two PDZ domains; recombinant protein-protein binding assays and yeast-two-hybrid studies revealed that ROMK preferentially associates with the second PDZ domain of NHERF-1 and with the first PDZ domain of NHERF-2, precisely opposite of what has been reported for CFTR . Consistent with the scaffolding capacity of the NHERF proteins, coexpression of NHERF-2 with ROMK and CFTR dramatically increases the amount of ROMK protein that coimmunopurifies and functionally interacts with CFTR . Thus NHERF facilitates assembly of a ternary complex containing ROMK and CFTR . These observations raise the possibility that PDZ-based interactions may underscore physiological regulation and membrane targeting of ROMK in the kidney. Gene, 2003 Oct 23, 317(1-2), 111 - 5 A comparative study of the porin genes encoding VDAC, a voltage-dependent anion channel protein, in Anopheles gambiae and Drosophila melanogaster; Sardiello M et al.; The protein called voltage-dependent anion-selective channel (VDAC), or mitochondrial porin, forms channels that provide the major pathway for small metabolites across the mitochondrial outer membrane . We have identified and sequenced agporin, a gene of the malaria vector mosquito Anopheles gambiae that conceptually encodes a protein with 73% identity to the VDAC protein encoded by the porin gene in Drosophila melanogaster . By in situ hybridization, we have localized agporin at region 35D on the right arm of A . gambiae chromosome 3, which is homologous to the 2L chromosomal arm of D . melanogaster where the porin gene resides . The comparison of agporin with its putative Drosophila counterpart revealed that both the nucleotide sequence and the structural organization of the two genes are strikingly conserved even though the ancestral lines of A . gambiae and D . melanogaster are thought to have diverged about 250 million years ago . Our results suggest that, while in yeast, plants, and mammals, VDAC isoforms are encoded by small multigene families and are able to compensate for each other at least partially, in A . gambiae a single gene encodes the VDAC protein. Sci Aging Knowledge Environ . 2002 Jun 19;2002(24):pe10. Oxygen? No thanks, I'm on a diet; Longo VD; In yeast and worms, mutations that extend longevity appear to simulate starvation conditions . The daf-2 pathway in worms plays a major role in life-span extension and in entry into the starvation-resistant and low-metabolism dauer phase . In a recent study published in Science Express on 13 June 2002, researchers screened for Caenorhabditis elegans mutants that survive in a low-oxygen environment and identified a number of daf-2 mutants that are resistant to hypoxia . The implications of these results are discussed in this Perspective. J Cell Sci, 2003 Dec 1, 116(Pt 23), 4847 - 56 Beta-dystrobrevin interacts directly with kinesin heavy chain in brain; Macioce P et al.; Beta-dystrobrevin, a member of the dystrobrevin protein family, is a dystrophin-related and -associated protein restricted to non-muscle tissues and is highly expressed in kidney, liver and brain . Dystrobrevins are now thought to play an important role in intracellular signal transduction, in addition to providing a membrane scaffold in muscle, but the precise role of beta-dystrobrevin has not yet been determined . To study beta-dystrobrevin's function in brain, we used the yeast two-hybrid approach to look for interacting proteins . Four overlapping clones were identified that encoded Kif5A, a neuronal member of the Kif5 family of proteins that consists of the heavy chains of conventional kinesin . A direct interaction of beta-dystrobrevin with Kif5A was confirmed by in vitro and in vivo association assays . Co-immunoprecipitation with a monoclonal kinesin heavy chain antibody precipitated both alpha- and beta-dystrobrevin, indicating that this interaction is not restricted to the beta-dystrobrevin isoform . The site for Kif5A binding to beta-dystrobrevin was localized in a carboxyl-terminal region that seems to be important in heavy chain-mediated kinesin interactions and is highly homologous in all three Kif5 isoforms, Kif5A, Kif5B and Kif5C . Pull-down and immunofluorescence experiments also showed a direct interaction between beta-dystrobrevin and Kif5B . Our findings suggest a novel function for dystrobrevin as a motor protein receptor that might play a major role in the transport of components of the dystrophin-associated protein complex to specific sites in the cell. Malar J . 2003 Sep 19;2(1):31. Polymorphism in the Plasmodium falciparum chloroquine-resistance transporter protein links verapamil enhancement of chloroquine sensitivity with the clinical efficacy of amodiaquine; Warhurst DC; BACKGROUND: Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion . Changes in protein PfCRT in the digestive vacuole membrane of growing intra-erythrocytic stages of Plasmodium falciparum are crucial for resistance . Expressed in yeast, PfCRT resembles an anion channel . Depressed anion channel function could increase intralysosomal pH to reduce entry of basic drug, or enhanced function could reduce drug interaction with target haematin . The most important resistance-associated change is from positively-charged lysine-76 to neutral threonine which could facilitate drug efflux through a putative channel . It has been proposed that the resistance-reversing effect of verapamil is due to hydrophobic binding to the mutated PfCRT protein, and replacement of the lost positive charge, which repels the access of 4-aminoquinoline cations, thus partially restoring sensitivity . Desethylamodiaquine, the active metabolite of amodiaquine, which has significant activity in chloroquine-resistance, may also act similarly on its own . METHODS: Changes in physicochemical parameters in different CQ-resistant PfCRT sequences are analysed, and correlations with drug activity on lines transfected with different alleles of the pfcrt gene are examined . RESULTS AND CONCLUSIONS: The results support the idea that PfCRT is a channel which, in resistant parasites, can allow efflux of chloroquine from the digestive vacuole . Activity of the chloroquine/verapamil combination and of desethylamodiaquine both correlate with the mean hydrophobicity of PfCRT residues 72-76 . This may partly explain clinical-resistance to amodiaquine found in the first chloroquine-resistant malaria cases from South America and enables tentative prediction of amodiaquine's clinical activity against novel haplotypes of PfCRT. Biochem J, 2004 Feb 15, 378(Pt 1), 161 - 8 AlphaII-spectrin is an in vitro target for caspase-2, and its cleavage is regulated by calmodulin binding; Rotter B et al.; The spectrin-actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains . These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape . Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold . It is also one of the major processes involved in membrane remodelling . Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and micro-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis) . In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases . Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to alphaII-spectrin, while the short isoform does not . Like caspase-3, caspase-2 cleaves alphaII-spectrin in vitro at residue Asp-1185 . This study emphasizes a role of executioner caspase for caspase-2 . We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro . This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin . In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176 . These results indicate that alphaII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton. Vopr Virusol, 2003 Sep-Oct, 48(5), 26 - 9 {The impact of compound of double-stranded RNA and hyaluronic acid on the parameters of non-specific resistance in mice}; Masycheva VI et al.; The effect of yeast double-stranded RNA (dsRNA) and of hyaluronic acid (HA) compositions produced on the interferon synthesis, peritoneal phagocytic activity of macrophages and on the hematological parameters were studied in an experiment with white noninbred mice . HA was shown to enhance the dsRNA-induced interferon synthesis, to inhibit the leukopenic reaction and to produce no effect on the phagocytosis-stimulating activity . The data obtained are indicative of that HA is a promising preparation regarding its use within the interferon-inducing compositions based on dsRNA. Am J Med Genet A, 2003 Dec 1, 123(2), 153 - 63 Deletion of the distal long arm of chromosome 10; is there a characteristic phenotype? A report of 15 de novo and familial cases; Irving M et al.; It has been suggested previously that patients with terminal deletions of chromosome 10q have a recognizable phenotype including a characteristic facial appearance combined with other abnormalities including mental retardation, cardiac and anogenital anomalies . We report the largest published series of new cases of terminal 10q deletion, including eight familial and four de novo cases and three cases with interstitial deletions involving chromosome bands 10q25.2-26.3 . The deleted regions were defined by FISH using YAC probes, as well as standard karyotyping . The most consistent clinical features in our cases are cranial anomalies including facial asymmetry, prominent nose and nasal bridge, prominent ears, thin upper lip, along with growth retardation, developmental delay, and digital abnormalities . Visceral abnormalities were only identified in a small number of the patients, with renal involvement in three cases and structural cardiac malformations in two others . Learning difficulties of varying severity were found in 11 cases and behavioral problems described in four . Candidate genes for behavioral and learning difficulties within the deleted region include Calcyon . Other genes in the region that might have a role in causing the phenotype include the genes coding for fibroblast growth factor receptor type 2 (FGFR2) and C-terminal binding protein 2 (CTBP2) . Planta Med, 2003 Sep, 69(9), 856 - 8 Antiandrogenic activity of the phytoestrogens naringenin, 6-(1,1-dimethylallyl)naringenin and 8-prenylnaringenin; Zierau O et al.; Naturally occurring naringenin derivatives, known for their estrogenic activity, were tested in two independent (anti-)androgen screening assays . Using a yeast-based androgen receptor assay relatively strong antiandrogen activities were demonstrated for 6-(1,1-dimethylallyl)naringenin and 8-prenylnaringenin, while the parent compound naringenin did not show recognizable antiandrogen activity . In an androgen receptor activity assay based on the analysis of prostate specific antigen (PSA) concentrations in the supernatants of treated PC3(AR)2 cells the antiandrogenic activity of 6-(1,1-dimethylallyl)naringenin was detected at concentrations of 10 (-5) M . 8-Prenylnaringenin or naringenin have no detectable antiandrogenic effect . In summary, for the first time we provide evidence of the antiandrogenic activity of 6-DMA-N in two independent model systems . In conclusion, we demonstrated the ability of prenylated naringenins not only to act via the estrogen receptor but also through the androgen receptor. Pflugers Arch, 2004 Feb, 447(5), 689 - 709 Epub 2003 Nov 04. The mitochondrial transporter family (SLC25): physiological and pathological implications; Palmieri F; The mitochondrial carriers (MCs) shuttle a variety of metabolites across the inner mitochondrial membrane (i.m.m.) . In man they are encoded by the SLC25 genes . Some MCs have isoforms encoded by different SLC25 genes, whereas the phosphate carrier has two variants arising from an alternative splicing of SLC25A3 . Six MCs have been sequenced after purification, and many more have been identified from their transport and kinetic properties following heterologous over-expression and reconstitution into liposomes . All MCs of known function belong to the same protein family, since their polypeptide chains consist of three tandemly related sequences of about 100 amino acids, and the repeats of the different carriers are homologous . They probably function as homodimers, each monomer being folded in the membrane into six transmembrane segments . The functional information obtained in studies with mitochondria and/or the reconstituted system has helped to gain an insight into the physiological role of the MCs in cell metabolism, as have tissue distribution, the use of knock-out mice (and/or yeast) and over-expression in human cell lines (or yeast) of individual carriers and isoforms . At the same time, the cloning and functional identification of many SLC25 genes has made it possible (i) to identify the genes (and their defects) responsible for some diseases, e.g . Stanley syndrome and Amish microcephaly, and (ii) where the genes were already known, to characterize the function of the gene products and hence understand the molecular basis and the symptoms of the diseases, e.g . hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) syndrome and type II citrullinemia . It is likely that further extension and functional characterization of the SLC25 gene family will elucidate other diseases caused by MC deficiency. Neurol Sci, 2003 Oct, 24(3), 159 - 60 DJ-1( PARK7), a novel gene for autosomal recessive, early onset parkinsonism; Bonifati V et al.; Four chromosomal loci ( PARK2, PARK6, PARK7, and PARK9) associated with autosomal recessive, early onset parkinsonism are known . We mapped the PARK7 locus to chromosome 1p36 in a large family from a genetically isolated population in the Netherlands, and confirmed this linkage in an Italian family . By positional cloning within the refined PARK7 critical region we recently identified mutations in the DJ-1 gene in the two PARK7-linked families . The function of DJ-1 remains largely unknown, but evidence from genetic studies on the yeast DJ-1 homologue, and biochemical studies in murine and human cell lines, suggests a role for DJ-1 as an antioxidant and/or a molecular chaperone . Elucidating the role of DJ-1 will lead to a better understanding of the pathogenesis of DJ-1-related and common forms of Parkinson's disease. Gene, 2003 Nov 27, 320, 41 - 8 Silencing the Drosophila ribosomal protein L14 gene using targeted RNA interference causes distinct somatic anomalies; Enerly E et al.; The Drosophila Minutes are haploinsufficient mutations that are defective in ribosomal protein (rp) production, resulting in short, thin bristles, delayed development and recessive lethality . In a Minute fly, the amount of rp gene messenger RNA (mRNA) is reduced to >or=50% of the normal amount of gene product, and becomes rate limiting for ribosome biogenesis, cell proliferation and growth . Haploinsufficiency increases the vulnerability to complete loss of gene function (homozygous null state) if hit by a second mutation . Because of the homozygous lethality, it has only been possible to study the effects of Minute mutations in heterozygous animals . To be able to study the consequences of a loss-of-function of an rp gene (0%>mRNA<50%) in developing and differentiated cells we used heritable RNA interference (RNAi) in combination with the yeast GAL4/UAS binary system to spatiotemporally knock down the ribosomal protein L14 (RpL14) gene . We show, at the RNA and phenotypic levels, that RNAi efficiently reduces RpL14 gene expression throughout development, causing lethality and distinct and dramatic somatic anomalies in both developing and differentiated cells. Gene, 2003 Nov 13, 319, 33 - 41 SNCF, a SoxNeuro interacting protein, defines a novel protein family in Drosophila melanogaster; Bonneaud N et al.; The involvement of the Sox family of transcription factors in the development of the central nervous system (CNS) appears to be conserved in invertebrates and vertebrates . In Drosophila, SoxNeuro (SoxN) was recently shown to be involved in the formation of neuroblasts {Development 129 (2002) 4193; Development 129 (2002) 4219} . Through a yeast two-hybrid assay searching for proteins interacting with SoxN, we have isolated a novel protein in Drosophila, SoxNeuro Co-Factor (SNCF) . The expression of the SNCF gene was detected during early embryogenesis at the blastoderm stages, and stopped just at the beginning of gastrulation . In transfected cells, the protein localised to nuclei, and strongly accumulated in nucleoli . SNCF was able to enhance SoxN mediated transcriptional activity in transfected cells, suggesting that SNCF might act as a SoxN co-activator . Finally, data are presented showing the existence in Drosophila of several proteins with a domain of homology to SNCF, which are all expressed early in embryogenesis at the blastoderm stage. FEBS Lett, 2003 Nov 6, 554(1-2), 30 - 4 MALS is a binding partner of IRSp53 at cell-cell contacts; Hori K et al.; Insulin receptor substrate p53 (IRSp53) is a key player in cytoskeletal dynamics, interacting with the actin modulators WAVE2 and Mena . Here, we identified a PDZ protein, MALS, as an IRSp53-interacting protein using a yeast two-hybrid screen . A pull-down assay showed that IRSp53 and MALS interact through the PDZ domain of MALS and the C-terminal PDZ-binding sequence of IRSp53 . Their interaction in MDCK cells was also demonstrated by co-immunoprecipitation . Immunocytochemistry showed the colocalization of IRSp53 and MALS at cell-cell contacts . Cytochalasin D induced the redistribution of both proteins to the cytosol . Thus, MALS is a partner of IRSp53 anchoring the actin-based membrane cytoskeleton at cell-cell contacts. Electrophoresis, 2003 Oct, 24(19-20), 3305 - 13 Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium; Chery CC et al.; The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied . Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb) . Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations . This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE) . The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se . Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)) . In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas) . Carbon monoxide was found to offer the best performance . The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation. Mol Biol Cell, 2004 Feb, 15(2), 934 - 45 Epub 2003 Oct 31. The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium; Noegel AA et al.; Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity . We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant . Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton . We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact . However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced . The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton. Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13501 - 6 Epub 2003 Oct 31. Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway; Shoham NG et al.; Pyrin, the familial Mediterranean fever protein, is found in association with the cytoskeleton in myeloid/monocytic cells and modulates IL-1beta processing, NF-kappaB activation, and apoptosis . These effects are mediated in part through cognate interactions with the adaptor protein ASC, which shares an N-terminal motif with pyrin . We sought additional upstream regulators of inflammation by using pyrin as the bait in yeast two-hybrid assays . We now show that proline serine threonine phosphatase-interacting protein {PSTPIP1, or CD2-binding protein 1 (CD2BP1)}, a tyrosine-phosphorylated protein involved in cytoskeletal organization, also interacts with pyrin . Recently, PSTPIP1/CD2BP1 mutations were shown to cause the syndrome of pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA), a dominantly inherited autoinflammatory disorder mediated predominantly by granulocytes . Endogenous PSTPIP1/CD2BP1 and pyrin are coexpressed in monocytes and granulocytes and can be coimmunoprecipitated from THP-1 cells . The B box segment of pyrin was necessary and the B box/coiled-coil segment sufficient for this interaction, whereas the SH3 and coiled-coil domains of PSTPIP1/CD2BP1 were both necessary, but neither was sufficient, for pyrin binding . The Y344F PSTPIP1/CD2BP1 mutation, which blocks tyrosine phosphorylation, was associated with a marked reduction in pyrin binding in pervanadate-treated cells . PAPA-associated A230T and E250Q PSTPIP1/CD2BP1 mutations markedly increased pyrin binding as assayed by immunoprecipitation and, relative to WT, these mutants were hyperphosphorylated when coexpressed with c-Abl kinase . Consistent with the hypothesis that these mutations exert a dominant-negative effect on the previously reported activity of pyrin, we found increased IL-1beta production by peripher