Yeasts

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 peripheral blood leukocytes from a clinically active PAPA patient with the A230T PSTPIP1/CD2BP1 mutation and in cell lines transfected with both PAPA-associated mutants.

J Biol Chem, 2004 Feb 6, 279(6), 4829 - 39 Epub 2003 Nov 01.
Centrosomal anchoring of protein kinase C betaII by pericentrin controls microtubule organization, spindle function, and cytokinesis; Chen D et al.; Location is a critical determinant in dictating the cellular function of protein kinase C (PKC) . Scaffold proteins contribute to localization by poising PKC at specific intracellular sites . Using a yeast two-hybrid screen, we identified the centrosomal protein pericentrin as a scaffold that tethers PKC betaII to centrosomes . Co-immunoprecipitation studies reveal that the native proteins interact in cells . Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494-593 . Immunofluorescence analysis reveals that endogenous PKC betaII colocalizes with pericentrin at centrosomes . Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure . Overexpression of this disrupting fragment has no effect in cells lacking PKC betaII, indicating a specific regulatory role of this isozyme in centrosome function . These results reveal a novel role for PKC betaII in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes.

J Biol Chem, 2004 Jan 23, 279(4), 2394 - 402 Epub 2003 Oct 31.
Interaction of human HSP22 (HSPB8) with other small heat shock proteins; Sun X et al.; Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies . Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1) . Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily . sHSPs interact with each other and form homo- and hetero-oligomeric complexes . The function of these complexes is poorly understood . Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i) . HSP22 forms high molecular mass complexes in the heart, (ii) . HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii) . HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners . HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction . HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner.

Biochem J, 2004 Mar 15, 378(Pt 3), 779 - 84
Ligand control of interaction in vivo between ecdysteroid receptor and ultraspiracle ligand-binding domain; Bergman T et al.; Ecdysteroids (Ecs) enhance the formation of Ec receptor-ultraspiracle protein (EcR-USP) heterodimers which regulate gene transcription . To study EcR-USP heterodimerization, fusion proteins were constructed from the LBDs (ligand-binding domains) of Drosophila EcR or USP and the activation or DNA-binding region of GAL4 respectively . Reporter gene ( lacZ ) activation was fully dependent on the co-expression of the two fusion proteins and thus constitutes a reliable measure for the interaction in vivo between the two LBDs in the yeast cell . To identify structures involved in heterodimerization, a total of 27 point mutations were generated in the EcR and USP LBDs at selected sites . Heterodimerization and its inducibility by ligand were mainly affected by mutations in the dimerization interface and in the ligand-binding pocket of EcR respectively . However, also mutations not located in these structures or even in the LBD of USP influenced ligand-dependent heterodimerization . Together with previously reported ligand-binding studies, the existence of such local, intra- and inter-molecular mutation effects suggest that ligand-induced dimerization results from a synergistic interaction between ligand-binding and heterodimerization functions in EcR LBD, and that it depends on global features of the LBDs of EcR and USP and on their mutual surface compatibility.

Prog Cell Cycle Res, 2003, 5, 327 - 34
Polo-like kinases and the microtubule organization center: targets for cancer therapies; Dai W et al.; Studies from eukaryotic model systems, ranging from yeast to human, indicate that Polo and Polo-like kinases (Plks) are essential for the activity of the microtubule organization center . Polo/Plks localize to centrosomes or spindle pole bodies and undergo dramatic subcellular relocation during the cell cycle . Deregulated activities of Plks often result in abnormalities in centrosome duplication, maturation, and/or microtubule dynamics . Genetic and biochemical approaches have identified several candidate genes that either lie in the same pathway as POLO/PLKs or whose products are direct targets of Polo/Plks during the centrosome cycle . Recent studies have demonstrated that mammalian Plks also regulate the function of the Golgi complex, a cellular organelle closely associated with the centrosome and also having microtubule organization activity . Furthermore, deregulated expression of human PLK1 and PLK3 is strongly correlated with the development of many types of malignancies, and ectopic expression of kinase-active Plk3 or Plk1 dominant negative protein leads to rapid cell death . Given that several effective anti-tumor drugs directly interfere with microtubule dynamics, mammalian Plks are excellent targets for the development of anticancer drugs.

Nat Cell Biol, 2003 Nov, 5(11), 1023 - 5
Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1; Herbert M et al.; Disjunction of pairs of homologous chromosomes during the first meiotic division (MI) requires anaphase-promoting complex (APC)-mediated activation of separase in budding yeast and Caenorhabditis elegans, but not Xenopus laevis . It is not clear which model best fits the mammalian system . Here we show that homologue disjunction in mouse oocytes is dependent on proteolysis of the separase inhibitor securin and the Cdk1 regulatory sub-unit cyclin B1 . Proteolysis of both proteins was entirely dependent on their conserved destruction box (D-box) motifs, through which they are targeted to the APC . These data indicate that the mechanisms regulating homologue disjunction in mammalian oocytes are similar to those of budding yeast and C.elegans.

Science, 2003 Dec 19, 302(5653), 2088 - 94 Epub 2003 Oct 30.
Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse; Serizawa S et al.; In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) gene in a monoallelic and mutually exclusive manner . Such expression forms the genetic basis for OR-instructed axonal projection of OSNs to the olfactory bulb of the brain during development . Here, we identify an upstream cis-acting DNA region that activates the OR gene cluster in mouse and allows the expression of only one OR gene within the cluster . Deletion of the coding region of the expressed OR gene or a naturally occurring frame-shift mutation allows a second OR gene to be expressed . We propose that stochastic activation of only one OR gene within the cluster and negative feedback regulation by that OR gene product are necessary to ensure the one receptor-one neuron rule.

J Biol Chem, 2004 Jan 16, 279(3), 2324 - 31 Epub 2003 Oct 30.
Interactions between p-33 and p-55 domains of the Helicobacter pylori vacuolating cytotoxin (VacA); Torres VJ et al.; The VacA toxin secreted by Helicobacter pylori is considered to be an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer . VacA monomers self-assemble into water-soluble oligomeric structures and can form anion-selective membrane channels . The goal of this study was to characterize VacA-VacA interactions that may mediate assembly of VacA monomers into higher order structures . We investigated potential interactions between two domains of VacA (termed p-33 and p-55) by using a yeast two-hybrid system . p-33/p-55 interactions were detected in this system, whereas p-33/p-33 and p-55/p-55 interactions were not detected . Several p-33 proteins containing internal deletion mutations were unable to interact with wild-type p-55 in the yeast two-hybrid system . Introduction of these same deletion mutations into the H . pylori vacA gene resulted in secretion of mutant VacA proteins that failed to assemble into large oligomeric structures and that lacked vacuolating toxic activity for HeLa cells . Additional mapping studies in the yeast two-hybrid system indicated that only the N-terminal portion of the p-55 domain is required for p-33/p-55 interactions . To characterize further p-33/p-55 interactions, we engineered an H . pylori strain that produced a VacA toxin containing an enterokinase cleavage site located between the p-33 and p-55 domains . Enterokinase treatment resulted in complete proteolysis of VacA into p-33 and p-55 domains, which remained physically associated within oligomeric structures and retained vacuolating cytotoxin activity . These results provide evidence that interactions between p-33 and p-55 domains play an important role in VacA assembly into oligomeric structures.

EMBO J, 2003 Nov 3, 22(21), 5863 - 74
An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control; Staeva-Vieira E et al.; Rad51 is a conserved protein essential for recombinational repair of double-stranded DNA breaks (DSBs) in somatic cells and during meiosis in germ cells . Yeast Rad51 mutants are viable but show meiosis defects . In the mouse, RAD51 deletions cause early embryonic death, suggesting that in higher eukaryotes Rad51 is required for viability . Here we report the identification of SpnA as the Drosophila Rad51 gene, whose sequence among the five known Drosophila Rad51-like genes is most closely related to the Rad51 homologs of human and yeast . DmRad51/spnA null mutants are viable but oogenesis is disrupted by the activation of a meiotic recombination checkpoint . We show that the meiotic phenotypes result from an inability to effectively repair DSBs . Our study further demonstrates that in Drosophila the Rad51-dependent homologous recombination pathway is not essential for DNA repair in the soma, unless exposed to DNA damaging agents . We therefore propose that under normal conditions a second, Rad51-independent, repair pathway prevents the lethal effects of DNA damage.

Arch Biochem Biophys, 2003 Nov 15, 419(2), 198 - 206
Superoxide anion generation by the cytochrome bc1 complex; Sun J et al.; We have measured the rates of superoxide anion generation by cytochrome bc(1) complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc(1) complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins . With all of the bc(1) complexes the rate of superoxide anion production was greatest in the absence of bc(1) inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction . Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc(1) complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate . Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc(1) complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol . Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation . A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity . These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc(1) complexes . The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 506 - 13
Molecular cloning of a novel human gene encoding histone acetyltransferase-like protein involved in transcriptional activation of hTERT; Lv J et al.; To isolate proteins involved in hTERT transcriptional regulation, the HeLa cDNA library was screened using the hTERT promoter-based yeast one-hybrid assay . A positive clone was rescued and proved to contain an open reading frame and the upstream coding sequences were obtained by 5'-RACE . The assembled full cDNA consisted of a 2.5 kb reading frame encoding 834 amino acids, in which a conserved N-acetyltransferase domain (GNAT family) was searched out in bioinformatics, and thus named as hALP (human N-acetyltransferase-like protein, GenBank Accession No . AF 489535) . The expression of native hALP was identified in HeLa cells and proved to distribute in the cellular nucleus . The binding potential of hALP to hTERT promoter was confirmed by EMSA and the interacting sequence involved to -201- to -56-nt upstream region of the promoter . On transfection assay, hALP could obviously transactivate hTERT promoter and stimulate endogenous telomerase activity of cells . The analysis on histone acetyltransferase showed that hALP could specifically acetylate free histones in vitro . The investigation suggested that hALP influences the activity of histone acetylation and could up-regulate telomerase activity through transactivation of hTERT promoter.

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 351 - 60
Pub, a novel PU.1 binding protein, regulates the transcriptional activity of PU.1; Hirose S et al.; PU.1 is a member of the Ets family of transcription factors and plays critical roles in the development of hematopoietic cells such as macrophages and B cells . To elucidate the molecular mechanism(s) underlying the regulation of PU.1 function, we screened for PU.1 interacting proteins using a yeast two-hybrid approach . As a result, a novel PU.1 binding factor, which we termed Pub, was isolated . The Pub protein has one B-box zinc finger domain, followed by a coiled-coil region and a B30.2-like domain, these features being characteristic of the tripartite motif (TRIM) family of protein . The PEST domain of PU.1 was found to interact with the N-terminal portion of Pub, a region that includes the TRIM which is considered to mediate protein-protein interactions . Northern blot and RT-PCR analyses demonstrated that Pub is predominantly expressed in hematopoietic tissues and cells where PU.1 is also expressed . Using a luciferase-based assay, we showed that Pub inhibited the transcriptional activity of PU.1 . Moreover, the B-box zinc finger domain of Pub was critical for this inhibitory activity . These data suggest that Pub may be important in regulating the transcriptional activity of PU.1.

Curr Biol, 2003 Oct 28, 13(21), 1882 - 7
The Sac1 lipid phosphatase regulates cell shape change and the JNK cascade during dorsal closure in Drosophila; Wei HC et al.; The Sac1 lipid phosphatase dephosphorylates several phosphatidylinositol (PtdIns) phosphates and, in yeast, regulates a diverse range of cellular processes including organization of the actin cytoskeleton and secretion . We have identified mutations in the gene encoding Drosophila Sac1 . sac1 mutants die as embryos with defects in dorsal closure (DC) . DC involves the migration of the epidermis to close a hole in the dorsal surface of the embryo occupied by the amnioserosa . It requires cell shape change in both the epidermis and amnioserosa and activation of a Jun N-terminal kinase (JNK) MAPK cascade in the leading edge cells of the epidermis {2} . Loss of Sac1 leads to the improper activation of two key events in DC: cell shape change in the amnioserosa and JNK signaling . sac1 interacts genetically with other participants in these two events, and our data suggest that loss of Sac1 leads to upregulation of one or more signals controlling DC . This study is the first report of a role for Sac1 in the development of a multicellular organism.

J Cell Biochem, 2003 Nov 1, 90(4), 819 - 36
Rho exchange factor ECT2 is induced by growth factors and regulates cytokinesis through the N-terminal cell cycle regulator-related domains; Saito S et al.; The ECT2 protooncogene plays a critical role in cytokinesis, and its C-terminal half encodes a Dbl homology-pleckstrin homology module, which catalyzes guanine nucleotide exchange on the Rho family of small GTPases . The N-terminal half of ECT2 (ECT2-N) contains domains related to the cell cycle regulator/checkpoint control proteins including human XRCC1, budding yeast CLB6, and fission yeast Cut5 . The Cut5-related domain consists of two BRCT repeats, which are widespread to repair/checkpoint control proteins . ECT2 is ubiquitously expressed in various tissues and cell lines, but elevated levels of ECT2 expression were found in various tumor cell lines and rapidly developing tissues in mouse embryos . Consistent with these findings, induction of ECT2 expression was observed upon stimulation by serum or various growth factors . In contrast to other oncogenes whose expression is induced early in G1, ECT2 expression was induced later, coinciding with the initiation of DNA synthesis . To test the role of the cell cycle regulator/checkpoint control protein-related domains of ECT2 in cytokinesis, we expressed various ECT2 derivatives in U2OS cells, and analyzed their DNA content by flow cytometry . Expression of the N-terminal half of ECT2, which lacks the catalytic domain, generated cells with more than 4N DNA content, suggesting that cytokinesis was inhibited in these cells . Interestingly, ECT2-N lacking the nuclear localization signals inhibited cytokinesis more strongly than the derivatives containing these signals . Mutational analyses revealed that the XRCC1, CLB6, and BRCT domains in ECT2-N are all essential for the cytokinesis inhibition by ECT2-N . These results suggest that the XRCC1, CLB6, and BRCT domains of ECT2 play a critical role in regulating cytokinesis . Published 2003 Wiley-Liss, Inc.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2307 - 10
A new type of RNase T2 ribonuclease in two Basidiomycetes fungi, Lentinus edodes and Irpex lacteus; Kobayashi H et al.; Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively . The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast . The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.

Expert Rev Mol Med, 1999 Dec 28, 1999, 1 - 12
Genetic control of lifespan: studies from animal models; Van Zant G et al.; The premise that there are genes that wield a strong influence on longevity has, until recently, not been a popular one and there has been no concerted effort to find such genes . However, the finding that single genes can have large effects on the lifespans of yeast, worms and flies raises the possibility that individual genes in mammals may similarly have relatively large effects on longevity . Recent advances in mammalian genetics, many associated with the large-scale efforts to sequence the human and mouse genomes, have accelerated the search for longevity genes, principally in mice . Here, we review results using animal models that have recently shed light on genes regulating longevity and ageing patterns . A large number of genetically defined strains of mice are available and this, together with their established history of use in genetic research and their relatively short lifespans, has made murine models particularly useful . We also review our own work in which genes regulating mouse lifespan and those regulating cell cycling of haematopoietic progenitor cells have been mapped to the same locations in the genome . These results suggest that some of the same genes affect both traits, and further suggest a cause-and-effect relationship between cumulative cell-cycle activity and longevity of an organism.

J Bone Miner Res, 2003 Oct, 18(10), 1863 - 71
Focal adhesion kinase pp125FAK interacts with the large conductance calcium-activated hSlo potassium channel in human osteoblasts: potential role in mechanotransduction; Rezzonico R et al.; Molecular events of mechanotransduction in osteoblasts are poorly defined . We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock . This could convert mechanical signals in biochemical events, leading to osteoblast activation . INTRODUCTION: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage . The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized . The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock . Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK . METHODS: Interaction of FAK with the C terminus of the hSlo alpha-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts . Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins . RESULTS: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo . We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase . FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo . In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo . CONCLUSIONS: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts.

IUBMB Life, 2003 Jul, 55(7), 359 - 66
The PUF family of RNA-binding proteins: does evolutionarily conserved structure equal conserved function?
Spassov DS, Jurecic R.
Drosophila Pumilio (Pum) protein is a founder member of a novel family of RNA-binding proteins, known as the PUF family . The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants, and are characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats . The conserved biochemical features and genetic function of PUF family members have emerged from studies of model organisms . PUF proteins bind to related sequence motifs in the 3' untranslated region (3'UTR) of specific target mRNAs and repress their translation . Frequently, PUF proteins function asymmetrically to create protein gradients, thus causing asymmetric cell division and regulating cell fate specification . Thus, it was recently proposed that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells . Here we review the evolution, conserved genetic and biochemical properties of PUF family of proteins, and discuss protein interactions, upstream regulators and downstream targets of PUF proteins . We also suggest that a conserved mechanism of PUF function extends to the newly described mammalian members of the PUF family (human PUM1 and PUM2, and mouse Pum1 and Pum2), that show extensive homology to Drosophila Pum, and could have an important role in cell development, fate specification and differentiation.

J Biol Chem, 2004 Jan 16, 279(3), 1607 - 14 Epub 2003 Oct 28.
Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes; Yanagida M et al.; Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies . FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain . Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1) . We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB . Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.

J Biol Chem, 2004 Jan 23, 279(4), 2922 - 6 Epub 2003 Oct 28.
The protein kinase SOS2 activates the Arabidopsis H(+)/Ca(2+) antiporter CAX1 to integrate calcium transport and salt tolerance; Cheng NH et al.; The regulation of ions within cells is an indispensable component of growth and adaptation . The plant SOS2 protein kinase and its associated Ca(2+) sensor, SOS3, have been demonstrated to modulate the plasma membrane H(+)/Na(+) antiporter SOS1; however, how these regulators modulate Ca(2+) levels within cells is poorly understood . Here we demonstrate that SOS2 regulates the vacuolar H(+)/Ca(2+) antiporter CAX1 . Using a yeast growth assay, co-expression of SOS2 specifically activated CAX1, whereas SOS3 did not . CAX1-like chimeric transporters were activated by SOS2 if the chimeric proteins contained the N terminus of CAX1 . Vacuolar membranes from CAX1-expressing cells were made to be H(+)/Ca(2+)-competent by the addition of SOS2 protein in a dose-dependent manner . Using a yeast two-hybrid assay, SOS2 interacted with the N terminus of CAX1 . In each of these yeast assays, the activation of CAX1 by SOS2 was SOS3-independent . In planta, the high level of expression of a deregulated version of CAX1 caused salt sensitivity . These findings suggest multiple functions for SOS2 and provide a mechanistic link between Ca(2+) and Na(+) homeostasis in plants.

Physiol Genomics, 2003 Dec 16, 16(1), 131 - 40
Pancreatic triacylglycerol lipase in a hibernating mammal . II . Cold-adapted function and differential expression; Squire TL et al.; Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) exploit the low-temperature activity of pancreatic triacylglycerol lipase (PTL) during hibernation . Lipolytic activity at body temperatures associated with hibernation was examined using recombinant ground squirrel and human PTLs expressed in yeast . Both the human and ground squirrel enzymes displayed high activity at temperatures as low as 0 degrees C and showed Q10 values of 1.2-1.5 over a range of 37-7 degrees C . These studies indicate that low-temperature lipolysis is a general property of PTL and does not require protein modifications unique to mammalian cells and/or the hibernating state . Western blots show elevated levels of PTL protein during hibernation in both heart and white adipose tissue (WAT) . Significant increases in PTL gene expression are seen in heart, WAT, and testes; but not in pancreas, where PTL mRNA levels are highest . Upregulation of PTL in testes is also accompanied by expression of the PTL-specific cofactor, colipase . The multi-tissue expression of PTL during hibernation supports its role as a key enzyme that shows high activity at low temperatures.

Cancer Res, 2003 Oct 15, 63(20), 6847 - 54
Direct and bystander killing of sarcomas by novel cytosine deaminase fusion gene; Ramnaraine M et al.; Soft tissue and bone sarcomas of the extremities can be difficult to eradicate, and standard treatment may require limb amputation . New therapies to decrease tumor size could improve the effectiveness of treatment and decrease the frequency of limb amputation . Cytosine deaminase (CD)-based gene therapy has been shown to be effective in decreasing growth of solid tumors when animals with CD-expressing tumor cells are treated with 5 fluorocytosine (5FC), an inert prodrug that is converted to 5-fluorouracil (5FU) by CD . In this investigation, we used a novel CD-containing fusion gene to determine whether CD-based gene therapy affected soft tissue or bone sarcomas . The novel fusion gene (NGFR-CD) encodes for a protein with extracellular and transmembrane domains of human nerve growth factor receptor (NGFR) and cytoplasmic CD . Murine 2472 (2) sarcoma cells were transduced with fusion genes containing either the bacterial (NGFR-(b)CD) or yeast (NGFR-(y)CD) CD gene . 5FC treatment killed NGFR-(b)CD- and NGFR-(y)CD-transduced sarcoma cells in vitro through direct and bystander effects (P < 0.01) . In contrast, 5FC treatment of mice with s.c . 2NGFR-(b)CD or 2NGFR-(y)CD tumors affected only 2NGFR-(y)CD tumors . 5FC had no effect on growth of NGFR-(b)CD tumors but caused significant decrease in the size of 2NGFR-(y)CD tumors (51 +/- 60 versus 938 +/- 767 mm(3), treated versus control, P < 0.01) . Evaluation of bystander killing in vivo revealed significant tumor killing, with a 5-fold reduction in s.c . tumor volume evident in saline versus 5FC-treated mice when tumors were comprised of 90% 2472 cells and 10% 2NGFR-(y)CD selected for fluorescence-activated cell sorting (P < 0.01) . Bone sarcomas were eliminated in 9 of 10 5FC-treated mice, compared with 11.8 +/- 6.0 mm(2) in saline-treated mice (P < 0.002) . In addition, 5FC treatment of bone sarcomas caused a significant reduction in cancer-induced bone destruction (P < 0.002) and resulted in a reduction in the number of osteoclasts . Finally, 5FC treatment had no effect on animal weight or survival, whereas doses of 5FU providing equivalent tumor reduction as 5FC resulted in treatment-associated deaths and significant weight loss (P < 0.001).

Cancer Res, 2003 Oct 15, 63(20), 6626 - 34
SKI activates Wnt/beta-catenin signaling in human melanoma; Chen D et al.; Overexpression of the oncoprotein SKI correlates with the progression of human melanoma in vivo . SKI is known to curtail the growth inhibitory activity of tumor growth factor beta through the formation of repressive transcriptional complexes with Smad2 and Smad3 at the p21(Waf-1) promoter . Here, we show that SKI also stimulates growth by activating the Wnt signaling pathway . From a yeast two-hybrid screen and immunoprecipitation studies, we identified the protein FHL2/DRAL as a novel SKI binding partner . FHL2, a LIM-only protein, binds beta-catenin and can function as either a transcriptional repressor or activator of the Wnt signaling pathway . SKI enhanced the activation of FHL2 and/or beta-catenin- regulated gene promoters in melanoma cells . Among the SKI targets were microphthalmia-associated transcription factor and Nr-CAM, two proteins associated with melanoma cell survival, growth, motility, and transformation . Transient overexpression of SKI and FHL2 in ski(-/-) melanocytes synergistically enhanced cell growth, and stable overexpression of SKI in a poorly clonogenic human melanoma cell line was sufficient to stimulate rapid proliferation, decreasing the number of cells in the G(1) phase of the cell cycle, and dramatically increasing clonogenicity, colony size and motility . Taken together, these results suggest that by targeting members of the tumor growth factor beta and beta-catenin pathways, SKI regulates crucial events required for melanoma growth, survival, and invasion.

OMICS, 2003 Fall, 7(3), 285 - 99
Modeling regulatory networks at Virginia Tech; Allen NA et al.; The life of a cell is governed by the physicochemical properties of a complex network of interacting macromolecules (primarily genes and proteins) . Hence, a full scientific understanding of and rational engineering approach to cell physiology require accurate mathematical models of the spatial and temporal dynamics of these macromolecular assemblies, especially the networks involved in integrating signals and regulating cellular responses . The Virginia Tech Consortium is involved in three specific goals of DARPA's computational biology program (Bio-COMP): to create effective software tools for modeling gene-protein-metabolite networks, to employ these tools in creating a new generation of realistic models, and to test and refine these models by well-conceived experimental studies . The special emphasis of this group is to understand the mechanisms of cell cycle control in eukaryotes (yeast cells and frog eggs) . The software tools developed at Virginia Tech are designed to meet general requirements of modeling regulatory networks and are collected in a problem-solving environment called JigCell.

Plant Cell Physiol, 2003 Oct, 44(10), 975 - 81
Arabidopsis CAMTA family proteins enhance V-PPase expression in pollen; Mitsuda N et al.; The pollen-specific cis-acting region of the AVP1 gene is involved in the expression of the Arabidopsis V-PPase gene during pollen development . We isolate AtCAMTA5, which binds to the 38-bp pollen-specific cis-acting region, by one-hybrid screening using the cis-acting region as a probe . The green fluorescent protein-fused AtCAMTA5 is specifically localized to the nucleus in Arabidopsis suspension cultured cells . The promoter-beta-glucuronidase reporter experiment shows the expression not only of AtCAMTA5 but also of AtCAMTA1 in pollen . In particular, AtCAMTA1 is specifically expressed in pollen . Both the one-hybrid analysis in the reporter yeast and in vivo transient effector-reporter analysis in Arabidopsis suspension cultured cells revealed that AtCAMTA1 could regulate gene expression depending on the CGCG-box within the 38-bp pollen-specific cis-acting region . These results indicate that AtCAMTA1 as well as AtCAMTA5 possibly enhance AVP1 expression in pollen.

J Virol, 2003 Nov, 77(22), 12193 - 202
Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane; Miller DJ et al.; Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles . We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase . Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain . We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p . Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER . FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels . Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes . Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A . We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.

J Virol, 2003 Nov, 77(22), 12022 - 32
DC-SIGN and L-SIGN can act as attachment receptors for alphaviruses and distinguish between mosquito cell- and mammalian cell-derived viruses; Klimstra WB et al.; C-type lectins such as DC-SIGN and L-SIGN, which bind mannose-enriched carbohydrate modifications of host and pathogen proteins, have been shown to bind glycoproteins of several viruses and facilitate either cis or trans infection . DC-SIGN and L-SIGN are expressed in several early targets of arbovirus infection, including dendritic cells (DCs) and cells of the reticuloendothelial system . In the present study, we show that DC-SIGN and L-SIGN can function as attachment receptors for Sindbis (SB) virus, an arbovirus of the Alphavirus genus . Human monocytic THP-1 cells stably transfected with DC-SIGN or L-SIGN were permissive for SB virus replication, while untransfected controls were essentially nonpermissive . The majority of control THP-1 cells were permissive when attachment and entry steps were eliminated through electroporation of virus transcripts . Infectivity for the DC-SIGN/L-SIGN-expressing cells was largely blocked by yeast mannan, EDTA, or a DC-SIGN/L-SIGN-specific monoclonal antibody . Infection of primary human DCs by SB virus was also dependent upon SIGN expression by similar criteria . Furthermore, production of virus particles in either C6/36 mosquito cells or CHO mammalian cells under conditions that limited complex carbohydrate content greatly increased SB virus binding to and infection of THP-1 cells expressing these lectins . C6/36-derived virus also was much more infectious for primary human DCs than CHO-derived virus . These results suggest that (i) lectin molecules such as DC-SIGN and L-SIGN may represent common attachment receptor molecules for arthropod-borne viruses, (ii) arbovirus particles produced in and delivered by arthropod vectors may preferentially target vertebrate host cells bearing these or similar lectin molecules, and (iii) a cell line has been identified that can productively replicate alphaviruses but is deficient in attachment receptors.

J Biol Chem, 2004 Jan 9, 279(2), 1562 - 9 Epub 2003 Oct 26.
Breast cancer metastasis suppressor 1 (BRMS1) forms complexes with retinoblastoma-binding protein 1 (RBP1) and the mSin3 histone deacetylase complex and represses transcription; Meehan WJ et al.; Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple human and murine cancer cells without inhibiting tumorigenicity . By yeast two-hybrid and co-immunoprecipitation, BRMS1 interacts with retinoblastoma binding protein 1 and at least seven members of the mSin3 histone deacetylase (HDAC) complex in human breast and melanoma cell lines . BRMS1 co-immunoprecipitates enzymatically active HDAC proteins and represses transcription when recruited to a Gal4 promoter in vivo . BRMS1 exists in large mSin3 complex(es) of approximately 1.4-1.9 MDa, but also forms smaller complexes with HDAC1 . Deletion analyses show that the carboxyl-terminal 42 amino acids of BRMS1 are not critical for interaction with much of the mSin3 complex and that BRMS1 appears to have more than one binding point to the complex . These results further show that BRMS1 may participate in transcriptional regulation via interaction with the mSin3.HDAC complex and suggest a novel mechanism by which BRMS1 might suppress cancer metastasis.

J Cell Biol, 2003 Oct 27, 163(2), 223 - 9
BPAG1n4 is essential for retrograde axonal transport in sensory neurons; Liu JJ et al.; Disruption of the BPAG1 (bullous pemphigoid antigen 1) gene results in progressive deterioration in motor function and devastating sensory neurodegeneration in the null mice . We have previously demonstrated that BPAG1n1 and BPAG1n3 play important roles in organizing cytoskeletal networks in vivo . Here, we characterize functions of a novel BPAG1 neuronal isoform, BPAG1n4 . Results obtained from yeast two-hybrid screening, blot overlay binding assays, and coimmunoprecipitations demonstrate that BPAG1n4 interacts directly with dynactin p150Glued through its unique ezrin/radixin/moesin domain . Studies using double immunofluorescent microscopy and ultrastructural analysis reveal physiological colocalization of BPAG1n4 with dynactin/dynein . Disruption of the interaction between BPAG1n4 and dynactin results in severe defects in retrograde axonal transport . We conclude that BPAG1n4 plays an essential role in retrograde axonal transport in sensory neurons . These findings might advance our understanding of pathogenesis of axonal degeneration and neuronal death.

Cancer Genet Cytogenet, 2003 Nov, 147(1), 84 - 6
Identification of a BAC clone overlapping the t(6p12.3) breakpoint in the cell line ESS-1 derived from an endometrial stromal sarcoma; Gunawan B et al.; A major subgroup of endometrial stromal sarcomas (ESS) is characterized by translocations involving chromosome 6 with consistent breakpoints at 6p11 approximately p21 . As part of an ongoing positional cloning effort to identify the genes affected by these translocations, this article reports on the delineation of the 6p breakpoint in the cell line ESS-1 derived from an ESS . The G- and 4',6-diamidino-2-phenylindole-banded karyotypes showed an unbalanced translocation described originally as der(3)t(3;6) (q29;p21.1) . Fluorescence in situ hybridization using probes derived from contigous yeast artificial chromosome, bacterial artificial chromosome (BAC), and P1-derived artificial chromosome clones specific to 6p12.3 approximately p21.1 located the breakpoint at 6p to the BAC clone RP11-337K13 mapping to 6p12.3 . The DNA sequence of the breakpoint region contained in RP11-337K13 will serve as a candidate locus for further molecular genetic analyses to isolate the gene(s) altered in ESS with 6p rearrangement.

Curr Opin Chem Biol, 2003 Oct, 7(5), 648 - 54
Metabonomics: NMR spectroscopy and pattern recognition analysis of body fluids and tissues for characterisation of xenobiotic toxicity and disease diagnosis; Griffin JL; Global profiling tools are required to fully understand the impact of genetic modifications and toxicological interventions on the network of transcripts, proteins and metabolites found within a cell, tissue or organism . High-resolution 1H NMR spectroscopy in conjunction with statistical pattern recognition is one such technique, referred to as metabonomics or metabolomics, which is increasingly being used to globally profile metabolites . This review examines analytical advances in NMR spectroscopy that have aided this development including high-resolution magic angle spinning NMR spectroscopy, cryogenically cooled probes and high-through put systems . This has allowed the approach to identify genetically modified yeast strains and distinguish both disease presence and severity in coronary heart disease.

Biochemistry, 2003 Nov 4, 42(43), 12662 - 8
A C-terminal PDZ motif in NHE3 binds NHERF-1 and enhances cAMP inhibition of sodium-hydrogen exchange; Weinman EJ et al.; NHERF-1, a protein adapter containing two tandem PDZ domains, was first identified as an essential cofactor required for the phosphorylation and downregulation of NHE3 activity in response to elevated intracellular cAMP . NHERF-1 contains multiple protein interaction domains, but the mechanism by which it binds NHE3 remains unknown . Yeast two-hybrid analyses demonstrated that the C-terminal sequence, STHM, of NHE3 constitutes a PDZ motif critical for its association with NHERF-1 . In this assay, NHE3 bound both PDZ-I and PDZ-II when presented as isolated domains, but mutations of the individual PDZ domains in the full-length NHERF-1 suggested a significant preference of NHE3 for the PDZ-II domain . To investigate NHERF-1/NHE3 association in cells, NHERF-1 complexes were isolated from PS120 cells expressing hexahistidine-tagged NHERF-1 and NHE3 using nickel-NTA-agarose . In these experiments, mutating the C-terminal PDZ motif still allowed NHE3 binding to NHERF-1, suggesting the presence of additional mechanisms or components that stabilized a cellular NHE3/NHERF-1 complex . Transport assays in PS120 cells, however, showed that the C-terminal PDZ motif in NHE3 and a functional PDZ-II domain in NHERF-1 were required for maximal inhibition of sodium-hydrogen exchange in response to forskolin and 8-Br-cAMP . Together, the data suggested that the PDZ interaction between the NHE3 C-terminus and a NHERF-1 PDZ domain enhanced the regulation of sodium-hydrogen exchange by cAMP-elevating hormones.

J Neurol, 2003 Oct, 250 Suppl 3, III25 - 9
Pael receptor, endoplasmic reticulum stress, and Parkinson's disease; Takahashi R et al.; Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations of the parkin gene . Parkin is an E3 ubiquitin ligase that specifically recognizes its substrate protein, promoting its ubiquitination and subsequent degradation . Accordingly, we hypothesized that AR-JP may be caused by accumulation of an unidentified neurotoxic protein, which is a substrate of parkin . Based on this hypothesis, we cloned parkin-binding protein using a yeast two-hybrid system and identified a putative G protein-coupled receptor protein,which we named the Pael receptor (Pael-R) . When overexpressed in cells, this receptor became unfolded, insoluble, and ubiquitinated . Accumulation of the insoluble Pael-R subsequently led to endoplasmic reticulum (ER) stress-induced cell death . Parkin specifically ubiquitinates the unfolded Pael-R and promotes its degradation, resulting in suppression of cell death induced by the accumulation of unfolded Pael-R . Moreover, insoluble Pael-R accumulates in the brains of AR-JP patients . It is highly expressed by the dopaminergic neurons of the substantia nigra, strongly suggesting that accumulation of unfolded Pael-R may lead to selective death of dopaminergic neurons in AR-JP.Recently, we identified Hsp70 and its co-chaperone CHIP as novel parkin-binding partners . We found that CHIP enhanced parkinmediated ubiquitination of Pael-R . In concert with Hsp70, CHIP also enhanced the ability of parkin to inhibit cell death induced by Pael-R, indicating that CHIP and Hsp70 are both co-factors of parkin.

J Biol Chem, 2003 Dec 26, 278(52), 52914 - 8 Epub 2003 Oct 24.
Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains; Rodriguez M et al.; Protein phosphorylation by protein kinases may generate docking sites for other proteins . It thus allows the assembly of signaling complexes in response to kinase activation . Several protein domains that bind phosphoserine or phosphothreonine residues have been identified, including the 14-3-3, PIN1, FHA, KIX, WD-40 domain, and polo box (Yaffe, M . B., and Elia, A . E . (2001) Curr . Opin . Cell Biol . 13, 131-138; Elia, A . E., Cantley, L . C., and Yaffe, M . B . (2003) Science 299, 1228-1231) . The BRCA1 COOH-terminal (BRCT) domains are protein modules found in many proteins that regulate DNA damage responses (Koonin, E . V., Altschul, S . F., and Bork, P . (1996) Nat . Genet . 13, 266-268) . Whether BRCT domains can mediate phosphorylation-dependent interactions has not been systematically investigated . We report here that the BRCT domains also recognize phosphopeptides . Oriented peptide library analysis indicated that the BRCT domains from BRCA1, MDC1, BARD1, and DNA Ligase IV preferred distinct phosphoserine-containing peptides . In addition, the interaction between BRCA1 and the BRCT binding motif of BACH1 was required for BACH1 checkpoint activity . Furthermore, BRCT domains of the yeast DNA repair protein Rad9 could bind phosphopeptides, suggesting that the BRCT domains represent a class of ancient phosphopeptide-binding modules . Potential targets of BRCT domains were identified through data base search . Structural analysis of BRCA1 BRCT repeats also predicted conserved residues that may form the phosphopeptide-binding pocket . Thus, the BRCT repeats are a new family of phosphopeptide-binding domains in DNA damage responses.

Yakugaku Zasshi, 2003 Oct, 123(10), 825 - 35
{Finding of O-mannosyl glycan in mammals and congenital muscular dystrophies due to glycosylation defects}; Endo T; Most proteins within living organisms contain glycans . Glycan structures can modulate the biological properties and function of glycoproteins . Developments in glycobiology have revealed a new type of glycosidic linkage to the peptide portion, the O-mannosyl linkage in mammals, although heretofore it had been thought to be specific to yeast . One of the best known O-mannosyl-modified glycoproteins is dystroglycan, which is a central component of dystrophinglycoprotein complex isolated from skeletal muscle membranes . We identify and characterize a glycosyltransferase, UDP-N-acetylglucosamine: protein O-mannose beta 1,2-N-acetylglucosaminyltransferase (POMGnT1), involved in the biosynthesis of mammalian type O-mannosyl glycans . Finally, we find that the POMGnT1 gene is responsible for muscle-eye-brain disease (MEB) . MEB is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities and brain malformation (type II lissencephaly) . Like MEB, recent data suggest that the aberrant protein glycosylation of a specific glycoprotein, alpha-dystroglycan, is the primary cause of some forms of congenital muscular dystrophy . Here I review the new insight into glycobiology of muscular dystrophy and neuronal migration disorder.

Oncogene, 2003 Oct 23, 22(48), 7593 - 9
Breast cancer-specific gene 1 interacts with the mitotic checkpoint kinase BubR1; Gupta A et al.; The abnormal expression of breast cancer-specific gene 1 (BCSG1) in malignant mammary epithelial cells is highly associated with the development and progression of breast cancer . A series of in vitro and in vivo studies performed in our laboratory and others have demonstrated that BCSG1 expression significantly stimulates proliferation, invasion, and metastasis of breast cancer cells . However, currently little is known about how BCSG1 exerts its oncogenic functions . To elucidate the cellular mechanisms underlying the effects of BCSG1 in breast cancer cells, we used a yeast two-hybrid system to screen for proteins that could associate with BCSG1 . Through this screening, we identified the mitotic checkpoint protein BubR1 as a novel binding partner of BCSG1 . The specific association of BCSG1 with BubR1 in breast cancer cells was demonstrated by immunoprecipitation and GST pull-down assays . Intriguingly, experiments conducted in four different cell lines all showed that exogenous expressions of BCSG1 consistently reduce the cellular levels of the BubR1 protein without affecting BubR1 mRNA expression . The tendency of endogenous BCSG1 expression coinciding with lower BubR1 protein levels was also observed in seven out of eight breast cancer cell lines . We further showed that the reducing effect of BCSG1 on BubR1 protein expression could be prevented by treating BCSG1-transfected cells with MG-132, a selective 26S proteasome inhibitor, implying that the proteasome machinery may be involved in the BCSG1-induced reduction of the BubR1 protein . Accompanied with a reduction of BubR1 protein level, BCSG1 expression resulted in multinucleation of breast cancer cells upon treatment with spindle inhibitor nocodazole, indicating an impaired mitotic checkpoint . Taken together, our novel findings suggest that BCSG1 may accelerate the progression of breast cancer at least in part by compromising the mitotic checkpoint control through inactivation of BubR1.

Cell Death Differ, 2004 Feb, 11(2), 175 - 82
VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation; Bozhkov PV et al.; Plant embryogenesis is intimately associated with programmed cell death . The mechanisms of initiation and control of programmed cell death during plant embryo development are not known . Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts . Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue . In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis . We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis . This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling . The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn(2+) concentration . Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1 . In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death . Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation . Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.

J Cell Sci, 2003 Nov 15, 116(Pt 22), 4675 - 85
Mutation of an unusual mitochondrial targeting sequence of SODB2 produces multiple targeting fates in Toxoplasma gondii; Brydges SD et al.; Proteins destined for the mitochondria travel an intricate pathway through two membranes, each with its own receptors and channels . These proteins interact with receptors via N-terminal presequences that form amphipathic helices . Generally, these helices contain abundant positive charges on one face and hydrophobic residues on the other, but share little primary sequence homology . While extensive research on mitochondrial import has been done in yeast and mammalian cells, little is known about import or contents of the single mitochondrion of Toxoplasma gondii, a parasite in the phylum Apicomplexa . We describe here the characterization of TgSODB2, a novel, mitochondrial superoxide dismutase in T . gondii with an unusual targeting sequence consisting of a hydrophobic segment resembling a signal peptide, followed by a presequence . We show that although the hydrophobic segment is competent to target a reporter protein to the secretory system, it is prevented from directing ER translocation when coupled with the presequence . When we mutated the only charged residue in the hydrophobic sequence, ER translocation is restored and the reporter targeted to the apicoplast, a chloroplast-like organelle found in most apicomplexans . The presequence that follows is predicted to form an amphipathic helix, but targeted the cytoplasm when the hydrophobic peptide is removed . In addition to having an unusual targeting sequence, TgSODB2 is only the second mitochondrially imported, iron-containing SOD to be described.

Mol Endocrinol, 2004 Jan, 18(1), 13 - 25 Epub 2003 Oct 23.
Androgen receptor corepressor-19 kDa (ARR19), a leucine-rich protein that represses the transcriptional activity of androgen receptor through recruitment of histone deacetylase; Jeong BC et al.; Androgen receptor (AR) that mediates androgen action is a crucial factor in male reproductive functions . Here, we report a novel AR corepressor ARR19 (androgen receptor corepressor-19 kDa), which has been isolated as a putative androgen-induced gene from murine testis . ARR19 encoding a leucine-rich protein is expressed only in male reproductive organs such as testis and prostate . ARR19 expression in the testis is developmentally regulated . Functional analysis conducted by the transient transfection of mammalian cells shows that ARR19 represses AR transactivation in a dose-dependent manner . Furthermore, yeast two-hybrid and glutathione S-transferase pull-down analyses reveal that ARR19 directly associates with AR through the N-terminal and leucine zipper-containing regions of ARR19 and the DNA binding-hinge domain of AR . Interestingly, ARR19 localized in the cytoplasmic compartment cotranslocates into the nucleus with AR upon androgen exposure . The ARR19 repression of AR transactivation is through the recruitment of histone deacetylase 4 (HDAC4) by ARR19 . Overexpression of HDAC4 further inhibits the ARR19-repressed AR transactivation . In addition, ARR19 directly interacts with HDAC4 in vitro . Furthermore, DNA-protein complex immunoprecipitation assays reveal that HDAC4 is recruited to an androgen-regulated promoter through ARR19 . Taken together, the results suggest that ARR19 may act as an AR corepressor in vivo and play an important role in male reproductive functions.

Plant Cell, 2003 Nov, 15(11), 2647 - 53 Epub 2003 Oct 23.
Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistance; Zhang Y et al.; Arabidopsis nonexpresser of pathogenesis-related (PR) genes (NPR1) is the sole positive regulator that has been shown to be essential for the induction of systemic acquired resistance . In npr1 mutant plants, salicylic acid (SA)-mediated PR gene expression and pathogen resistance are abolished completely . NPR1 has been shown to interact with three closely related TGA transcription factors-TGA2, TGA5, and TGA6-in yeast two-hybrid assays . To elucidate the biological functions of these three TGA transcription factors, we analyzed single and combined deletion knockout mutants of TGA2, TGA5, and TGA6 for SA-induced PR gene expression and pathogen resistance . Induction of PR gene expression and pathogen resistance by the SA analog 2,6-dichloroisonicotinic acid (INA) was blocked in tga6-1 tga2-1 tga5-1 but not in tga6-1 or tga2-1 tga5-1 plants . Loss of INA-induced resistance to Peronospora parasitica Noco2 cosegregated with the tga6-1 mutation in progeny of multiple lines that were heterozygous for tga6-1 and homozygous for tga2-1 tga5-1 and could be complemented by genomic clones of wild-type TGA2 or TGA5, indicating that TGA2, TGA5, and TGA6 encode redundant and essential functions in the positive regulation of systemic acquired resistance . In addition, tga6-1 tga2-1 tga5-1 plants had reduced tolerance to high levels of SA and accumulated higher basal levels of PR-1 under noninducing conditions, suggesting that these TGA factors also are important for SA tolerance and the negative regulation of the basal expression of PR-1.

Plant Physiol, 2003 Dec, 133(4), 1809 - 19 Epub 2003 Oct 23.
An Arabidopsis pex10 null mutant is embryo lethal, implicating peroxisomes in an essential role during plant embryogenesis; Sparkes IA et al.; Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling . In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates . PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast . To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10) . Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds . The embryos in the abnormal seeds are predominantly homozygous for the disruption allele . They show retarded development and some morphological abnormalities . No viable homozygous mutant plants were obtained . AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.

Antonie Van Leeuwenhoek, 2003, 84(3), 185 - 91
Pityrialactone- a new fluorochrome from the tryptophan metabolism of Malassezia furfur; Mayser P et al.; As the main nitrogen source in Malassezia (M.) furfur, tryptophan induces the formation of fluorochromes and pigments, which makes the yeast less sensitive towards UV light . For the investigation of the fluorochromes, M . furfur (CBS1878) was incubated at 32 degrees C for 14 days on a pigment-inducing medium, and the agar extract was purified by column chromatography, preparative TLC and HPLC . The structures of the pure metabolites were determined by mass spectrometry and NMR spectroscopy . A pale yellow compound eluting from the column with 22% acetonitrile was found to exhibit a strong green-yellow fluorescence . The fluorochrome is a new bisindolyl compound (C(20)H(12)N(2)O(3), MW 328.33) named pityrialactone because of its furan-2,3-dione structure . The UV protective properties (lambda(max) 352, 292, 276, 224 nm) of this metabolite were confirmed in a yeast model . As shown by the fluorescence spectrum, pityrialactone appears to be responsible for the green-yellow fluorescence of pityriasis versicolor lesions under Wood light . Pityrialactone is accompanied by the isomeric bisindolylmaleic anhydride (pityriaanhydride), which has not yet been described as a natural product but is a known intermediate in the total synthesis of bisindolylmaleimides.

Antonie Van Leeuwenhoek, 2003, 84(4), 279 - 87
Genetic determination of ploidy level in Xanthophyllomyces dendrorhous; Hermosilla G et al.; Xanthophyllomyces dendrorhous (formely Phaffia rhodozyma) is a basidiomycetous yeast-like fungus that produces carotenoids useful for the food industry . Recently, its sexual cycle was reported but little is known about its genetic constitution . To inquire into the ploidy state of X . dendrorhous, biased mutant spectrum, genetic complementation and mitotic recombination analysis were used . A wild-type strain was subjected to N-methyl-N'-nitro-N-nitrosoguanidine mutagenic treatment . Auxotrophic and carotene mutants were forced to revert to the wild-type phenotype . Pigment producing and prototroph revertants behaved as diploid except for adenine less mutants . These results are in agreement with the limited spectrum of auxotrophs obtained in this strain for the ADE1 locus . To analyze the genetic characteristic of the adenine genetic marker of X . dendrorhous, protoplast fusion experiments with several adenine less mutants were performed . The experiments presented in this work suggest that the ATCC 2430 (UDC 67-385) strain of X . dendrorhous is diploid and a heterozygous constitution is proposed for the ADE1 locus.

Protein Sci, 2003 Nov, 12(11), 2542 - 8
Free-energy landscape of a chameleon sequence in explicit water and its inherent alpha/beta bifacial property; Ikeda K et al.; A sequence in yeast MATalpha2/MCM1/DNA complex that folds into alpha-helix or beta-hairpin depending on the surroundings has been known as "chameleon" sequence . We obtained the free-energy landscape of this sequence by using a generalized-ensemble method, multicanonical molecular dynamics simulation, to sample the conformational space . The system was expressed with an all-atom model in explicit water, and the initial conformation for the simulation was a random one . The free-energy landscape demonstrated that this sequence inherently has an ability to form either alpha or beta structure: The conformational distribution in the landscape consisted of two alpha-helical clusters with different packing patterns of hydrophobic residues, and four beta-hairpin clusters with different strand-strand interaction patterns . Narrow pathways connecting the clusters were found, and analysis on the pathways showed that a compact structure formed at the N-terminal root of the chameleon sequence controls the cluster-cluster transitions . The free-energy landscape indicates that a small conditional change induces alpha-beta transitions . Additional unfolding simulations done with replacing amino acids showed that the chameleon sequence has an advantage to form an alpha-helix . Current study may be useful to understand the mechanism of diseases resulting from abnormal chain folding, such as amyloid disease.

J Gen Virol, 2003 Nov, 84(Pt 11), 3011 - 9
Baculovirus P35 interacts with a subunit of human RNA polymerase II and can enhance promoter activity in human cells; Takramah D et al.; The early protein P35 from the baculovirus Autographa californica nucleopolyhedrovirus is a direct inhibitor of caspases and can block apoptosis in a wide variety of systems . In addition, it has been linked to the regulation of viral gene expression, shut-down of protein synthesis in infected insect cells and malignant transformation of mouse fibroblasts . By yeast-two-hybrid screening we identified the RPB11a subunit of human RNA polymerase II as an interaction partner of P35 . Specificity of the interaction was confirmed by affinity blotting . By immunocytology, P35 was in part found in the nucleus of transfected cells . Homology searches further revealed that P35 has structural similarity with RPB3, the subunit of RNA polymerase II that has been demonstrated to interact directly with RPB11a . When transfected into human colon carcinoma cells, P35 was able to enhance the activity of E-cadherin and beta-actin promoters by about a factor of two as measured by luciferase reporter assay . P35 and hRPB11a together enhanced the E-cadherin activity about three- to fourfold . These data suggest an additional role for P35 in the regulation of cellular transcription.

Mol Pharmacol, 2003 Nov, 64(5), 1092 - 100
Interaction between the mu opioid receptor and filamin A is involved in receptor regulation and trafficking; Onoprishvili I et al.; The carboxyl tail of the human mu opioid receptor was shown to bind the carboxyl terminal region of human filamin A, a protein known to couple membrane proteins to actin . Results from yeast two-hybrid screening were confirmed by direct protein-protein binding and by coimmunoprecipitation of filamin and mu opioid receptor from cell lysates . To investigate the role of filamin A in opioid receptor function and regulation, we used the melanoma cell line M2, which does not express filamin A, and its subclone A7, transfected with human filamin A cDNA . Both cell lines were stably transfected with cDNA encoding myc-tagged human mu opioid receptor . Fluorescent studies, using confocal microscopy, provided evidence that filamin and mu opioid receptors were extensively colocalized on the membranes of filamin-expressing melanoma cells . The immunostaining of mu opioid receptors indicated that the lack of filamin had no detectable effect on membrane localization of the receptors . Moreover, mu opioid receptors function normally in the absence of filamin A, as evidenced by studies of opioid binding and DAMGO inhibition of forskolin-stimulated adenylyl cyclase . However, agonist-induced receptor down-regulation and functional desensitization were virtually abolished in cells lacking filamin A . The level of internalized mu-opioid receptors, after 30-min exposure to agonist, was greatly reduced, suggesting a role for filamin in mu opioid receptor trafficking . During these studies, we observed that forskolin activation of adenylyl cyclase was greatly reduced in filamin-lacking cells . An even more unexpected finding was the ability of long-term treatment with {d-Ala2,N-Me-Phe4,Gly5-ol}-enkephalin of M2 cells, containing mu opioid receptors, to restore normal forskolin activation . The mechanism of this effect is currently unknown . It is postulated that the observed effects on mu opioid receptor regulation by filamin A and, by implication, of the actin cytoskeleton may be the result of its role in mu opioid receptor trafficking.

Mol Pharmacol, 2003 Nov, 64(5), 1085 - 91
Interaction of G protein beta subunit with inward rectifier K(+) channel Kir3; Zhao Q et al.; G protein betagamma subunits bind and activate G protein-coupled inward rectifier K+ (GIRK) channels . This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons . The crystal structure of Gbeta shows a seven-bladed propeller with four beta strands in each blade . The Gbeta/Galpha interacting surface contains sites for activating GIRK channels . Furthermore, our recent investigation using chimeras between Gbeta1 and yeast beta (STE4) suggested that the outer strands of blades 1 and 2 of Gbeta1 could be an interaction area between Gbeta1 and GIRK . In this study, we made point mutations on suspected residues on these outer strands and investigated their ability to activate GIRK1/GIRK2 channels . Mutations at Thr-86, Thr-87, and Gly-131, all located on the loops between beta-strands, substantially reduced GIRK channel activation, suggesting that these residues are Gbeta/GIRK interaction sites . These mutations did not affect the expression of Gbeta1 or its ability to stimulate PLCbeta2 . These residues are surface-accessible and located outside Gbeta/Galpha interaction sites . These results suggest that the residues on the outer surface of blades 1 and 2 are involved in the interaction of Gbetagamma with GIRK channels . Our study suggests a mechanism by which different effectors use different blades to achieve divergence of signaling . We also observed that substitution of alanine for Trp-332 of Gbeta1 impaired the functional interaction of Gbeta1 with GIRK, in agreement with the data on native neuronal GIRK channels . Trp-332 plays a critical role in the interaction of Gbeta1 with Galpha as well as all effectors so far tested.

J Neurosci, 2003 Oct 22, 23(29), 9547 - 56
Cross-repressive interaction of the Olig2 and Nkx2.2 transcription factors in developing neural tube associated with formation of a specific physical complex; Sun T et al.; In developing neural tube, the basic helix-loop-helix (bHLH) transcription factor Olig2 interacts with the homeodomain transcription factor Nkx2.2 at two distinct stages . During neuronogenesis, a cross-repressive interaction appears to establish the precise boundary between the p3 and pMN domains . At later times, a cooperative interaction is noted because Nkx2.2 promotes maturation of oligodendrocyte progenitor cells specified by expression of Olig2 . We show here that the Olig2 protein can form a physical complex with Nkx2.2 protein in mammalian cells and yeast two-hybrid trap assay . This interaction is specific because Olig2 does not bind to a biologically irrelevant homeodomain transcription factor (Nkx6.1), and Nkx2.2 does not interact with a biologically irrelevant bHLH protein (NeuroD) . Deletion mapping analysis suggests that formation of an Olig2-Nkx2.2 physical complex is insufficient for the induction of oligodendrocyte progenitors in developing spine; however, the protein-protein interaction observed might be important for the cross-repressive interaction between Olig2 and Nkx2.2 that helps to establish the pMN-p3 boundary in the developing spinal cord.

Genetics, 2003 Oct, 165(2), 575 - 88
The evolutionary duplication and probable demise of an endodermal GATA factor in Caenorhabditis elegans; Fukushige T et al.; We describe the elt-4 gene from the nematode Caenorhabditis elegans . elt-4 is predicted to encode a very small (72 residues, 8.1 kD) GATA-type zinc finger transcription factor . The elt-4 gene is located approximately 5 kb upstream of the C . elegans elt-2 gene, which also encodes a GATA-type transcription factor; the zinc finger DNA-binding domains are highly conserved (24/25 residues) between the two proteins . The elt-2 gene is expressed only in the intestine and is essential for normal intestinal development . This article explores whether elt-4 also has a role in intestinal development . Reporter fusions to the elt-4 promoter or reporter insertions into the elt-4 coding regions show that elt-4 is indeed expressed in the intestine, beginning at the 1.5-fold stage of embryogenesis and continuing into adulthood . elt-4 reporter fusions are also expressed in nine cells of the posterior pharynx . Ectopic expression of elt-4 cDNA within the embryo does not cause detectable ectopic expression of biochemical markers of gut differentiation; furthermore, ectopic elt-4 expression neither inhibits nor enhances the ectopic marker expression caused by ectopic elt-2 expression . A deletion allele of elt-4 was isolated but no obvious phenotype could be detected, either in the gut or elsewhere; brood sizes, hatching efficiencies, and growth rates were indistinguishable from wild type . We found no evidence that elt-4 provided backup functions for elt-2 . We used microarray analysis to search for genes that might be differentially expressed between L1 larvae of the elt-4 deletion strain and wild-type worms . Paired hybridizations were repeated seven times, allowing us to conclude, with some confidence, that no candidate target transcript could be identified as significantly up- or downregulated by loss of elt-4 function . In vitro binding experiments could not detect specific binding of ELT-4 protein to candidate binding sites (double-stranded oligonucleotides containing single or multiple WGATAR sequences); ELT-4 protein neither enhanced nor inhibited the strong sequence-specific binding of the ELT-2 protein . Whereas ELT-2 protein is a strong transcriptional activator in yeast, ELT-4 protein has no such activity under similar conditions, nor does it influence the transcriptional activity of coexpressed ELT-2 protein . Although an elt-2 homolog was easily identified in the genomic sequence of the related nematode C . briggsae, no elt-4 homolog could be identified . Analysis of the changes in silent third codon positions within the DNA-binding domains indicates that elt-4 arose as a duplication of elt-2, some 25-55 MYA . Thus, elt-4 has survived far longer than the average duplicated gene in C . elegans, even though no obvious biological function could be detected . elt-4 provides an interesting example of a tandemly duplicated gene that may originally have been the same size as elt-2 but has gradually been whittled down to its present size of little more than a zinc finger . Although elt-4 must confer (or must have conferred) some selective advantage to C . elegans, we suggest that its ultimate evolutionary fate will be disappearance from the C . elegans genome.

Biochim Biophys Acta, 2003 Oct 13, 1623(2-3), 88 - 97
The effect of glycation on the structure, function and biological fate of human serum albumin as revealed by recombinant mutants; Nakajou K et al.; Recombinant wild-type human serum albumin (rHSA), the single-residue mutants K199A, K439A and K525A and the triple-residue mutant K199A/K439A/K525A were produced using a yeast expression system . Portions of the rHSA were glycated to different degrees (2.5-250 mM D-glucose) . As detected by far-UV and near-UV CD, intrinsic tryptophan-fluorescence and probed by 1,1'-bis(4-anilino)naphthalene-5,5-disulfonic acid, the single-residue mutations had no effect on albumin conformation, whereas the triple-residue mutation and glycation caused conformational changes . The triple-residue mutation and glycation had comparable increased effects on high-affinity binding of warfarin (site I), but decreased effects on high-affinity binding of dansylsarcosine (site II) and the esterase-like activity of albumin . The relation between plasma half-lives in rats were found to be glycated rHSA (50 mM glucose)<triple-residue mutated rHSA<rHSA . The opposite trend was found for liver and kidney uptakes in mice . Even though the functional and the in vivo properties of rHSA could be effected differently by the minor conformational changes caused by the triple-residue mutation and glycation, the present findings indicate that the effect of glycation can be partly explained by blockage of the positive charges of lysine at positions 199, 439 and 525.

FEBS Lett, 2003 Oct 23, 553(3), 440 - 4
Identification of a very long chain polyunsaturated fatty acid Delta4-desaturase from the microalga Pavlova lutheri; Tonon T et al.; Pavlova lutheri, a marine microalga, is rich in the very long chain polyunsaturated fatty acids (VLCPUFAs) eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids . Using an expressed sequence tag approach, we isolated a cDNA designated Pldes1, and encoding an amino acid sequence showing high similarity with polyunsaturated fatty acid front-end desaturases . Heterologous expression in yeast demonstrated that PlDES1 desaturated 22:5n-3 and 22:4n-6 into 22:6n-3 and 22:5n-6 respectively, and was equally active on both substrates . Thus, PlDES1 is a novel VLCPUFA Delta4-desaturase . Pldes1 expression is four-fold higher during the mid-exponential phase of growth compared to late exponential and stationary phases.

Int J Parasitol, 2003 Nov, 33(13), 1455 - 61
Expression profiles of peroxiredoxin proteins of the rodent malaria parasite Plasmodium yoelii; Kawazu S et al.; Patterns of expression of the 2-Cys and 1-Cys peroxiredoxin (Prx) proteins of the rodent malaria parasite Plasmodium yoelii during its life cycle were observed by immunofluorescent antibody staining and confocal laser scanning microscopy . 2-Cys Prx was expressed in the parasite cytoplasm throughout the life cycle, and the thioredoxin (Trx)-peroxidase activity of 2-Cys Prx revealed with the recombinant protein suggested that the Prx is constitutively expressed and, thus, likely plays a housekeeping role in the parasite's intracellular redox control . In contrast, 1-Cys Prx showed stage-specific expression in blood-stage parasites . The limited expression of 1-Cys Prx in the trophozoite cytoplasm suggests that 1-Cys Prx may be involved in haemoglobin metabolism by the parasite, which generates a prooxidative haem iron and increases intracellular oxidative stress . The antioxidant activity of 1-Cys Prx was tested for its ability to protect yeast enolase against inactivation of the mixed-function oxidation system . Differential expression of the two Prx proteins during the erythrocytic and insect stages suggests the importance of these proteins in protecting parasites against oxidative stress, which is generated by the parasite's metabolism and also from the environment.

Genome Inform Ser Workshop Genome Inform, 2002, 13, 201 - 13
Automatic ontology construction from the literature; Blaschke C et al.; Detailed classifications, controlled vocabularies and organised terminology are widely used in different areas of science and technology . Their relatively recent introduction in molecular biology has been crucial for progress in the analysis of genonics and massive proteomics experiments . Unfortunately the construction of the ontologies, including terminology, classification and entity relations requires considerable effort, including the analysis of massive amounts of literature . We propose here a method that automatically generates classifications of gene-product functions using bibliographic information . The corresponding classification structures mirror the ones constructed by human experts . The analysis of a large structure built for yeast gene-products, and the detailed inspection of various examples, show encouraging properties . In particular, the comparison with the well accepted GO ontology points to different situations in which the automatically derived classification can be useful for assisting human experts in the annotation of ontologies.

Pesqui Odontol Bras, 2003 Apr-Jun, 17(2), 151 - 5 Epub 2003 Oct 10.
Candida spp . occurrence in oral cavities of breastfeeding infants and in their mothers' mouths and breasts; Zollner MS et al.; This study aimed to determine the occurrence of Candida spp . in the oral cavity of predominantly breastfed infants and in their mothers' mouths and breasts, as well as in the oral cavity of bottlefed infants and in non-lactating women . One hundred and sixty nine women and eighty-five milk-fed infants took part in this study and were divided into four groups: 1) infants predominantly on breastfeeding (n = 55) and their mothers (n = 55); 2) infants on bottlefeeding (n = 30); 3) non-lactating women on whom oral collections were performed (n = 80) and, 4) non-lactating women on whom breast collections were performed (n = 34) . Oral and mammary swabs were cultured on Sabouraud agar dextrose with chloramphenicol . The Candida yeast strains found were isolated and identified through morphological and biochemical tests . Candida species were much less frequent in infants who were predominantly breastfed than in those who were bottlefed . Yeasts were much more frequent on the breasts of lactating women, with statistical difference in relation to the control group.

J Med Genet, 2003 Oct, 40(10), 733 - 40
Disruption of a novel member of a sodium/hydrogen exchanger family and DOCK3 is associated with an attention deficit hyperactivity disorder-like phenotype; de Silva MG et al.; BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is a complex condition with high heritability . However, both biochemical investigations and association and linkage studies have failed to define fully the underlying genetic factors associated with ADHD . We have identified a family co-segregating an early onset behavioural/developmental condition, with features of ADHD and intellectual disability, with a pericentric inversion of chromosome 3, 46N inv(3)(p14:q21) . METHODS: We hypothesised that the inversion breakpoints affect a gene or genes that cause the observed phenotype . Large genomic clones (P1 derived/yeast/bacterial artificial chromosomes) were assembled into contigs across the two inversion breakpoints using molecular and bioinformatic technologies . Restriction fragments crossing the junctions were identified by Southern analysis and these fragments were amplified using inverse PCR . RESULTS: The amplification products were subsequently sequenced to reveal that the breakpoints lay within an intron of the dedicator of cytokinesis 3 (DOCK3) gene at the p arm breakpoint, and an intron of a novel member of the solute carrier family 9 (sodium/hydrogen exchanger) isoform 9 (SLC9A9) at the q arm . Both genes are expressed in the brain, but neither of the genes has previously been implicated in developmental or behavioural disorders . CONCLUSION: These two disrupted genes are candidates for involvement in the pathway leading to the neuropsychological condition in this family.

Nucl Recept . 2003 Sep 10;1(1):7.
A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation; Henry II KW et al.; BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes . This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex . Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription . RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene . NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1 . Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary . A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro . Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner . CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression . As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

Arch Pathol Lab Med, 2003 Nov, 127(11), 1413 - 20
Precision in gynecologic cytologic interpretation: a study from the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology; Renshaw AA et al.; CONTEXT: Numerous studies address the accuracy or positive predictive value of cytologic interpretations for defined histopathologic entities . The reproducibility (precision) of cytologic interpretation is less well defined . OBJECTIVE: To establish and compare the reproducibility (precision) of cytologic interpretation in gynecologic cytopathology, as reflected in the educational program of the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology (PAP) . METHODS: The pathologists' interpretations for both validated (25 745 responses) and educational conventional (14 353 responses) slides in the PAP program for 2001 were analyzed . The frequency of exact matches between the reference and pathologists' interpretation for each of the cytologic interpretative categories was identified, and the cumulative distributions of exact match rates were derived . chi2 Tests by reference interpretations were used for cytodiagnostic categories, least and most reproducible groupings, and high-grade (HSIL) versus low-grade (LSIL) squamous intraepithelial lesions . RESULTS: Pathologists' interpretations of negative, Candida, Trichomonas, herpes, and LSIL were characterized by a high degree of exact matching, while interpretations of repair, HSIL, adenocarcinoma, and squamous cell carcinomas were characterized by a lesser degree of exact matching (reproducibility) . Pathologists' cytologic interpretations of HSIL were significantly less reproducible than those of LSIL . CONCLUSION: The cytologic interpretations of the most significant categories (HSIL, squamous cell carcinoma, and adenocarcinoma) are less precise than those of specific infection (Candida, Trichomonas, and yeast), negative, and LSIL categories . Cytologic interpretations of LSIL are made with greater precision than those of HSIL and may represent a more appropriate endpoint to measure the precision performance of gynecologic cytology laboratories.

J Enzyme Inhib Med Chem, 2003 Aug, 18(4), 357 - 70
Inhibition of alpha, betaI, delta, eta, and zeta protein kinase C isoforms by xanthonolignoids; Saraiva L et al.; The effect of the xanthonolignoids trans-(+/-)-kielcorin C, cis-(+/-)-kielcorin C, trans-(+/-)-kielcorin D, trans-(+/-)-isokielcorin D and trans-(+/-)-kielcorin E on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was studied using the yeast phenotypic assay . All the compounds tested revealed an effect compatible with PKC inhibition, similar to that exhibited by the well established PKC inhibitor chelerythrine, and with differences in their potency towards the distinct isoforms tested, being, in general, potent inhibitors of the atypical PKC isoform (PKC-zeta) . PKC inhibition caused by these kielcorins was confirmed using an in vitro kinase assay . The present study constitutes the first attempt to unravel the molecular mechanism of kielcorins activity, and shows that xanthonolignoids are a promising group of compounds to investigate for isoform selective PKC inhibitors.

Pathol Biol (Paris), 2003 Jul, 51(5), 275 - 8
{Normal and pathologic sebaceous function . Research in a shallow milieu?}; Saint-Leger D; Modern therapeutic approaches allow to control sebaceous secretion but knowledge about the sebaceous gland and its precise function within the pilosebaceous unit is still insufficient . Steroid hormones are the principal albeit not exclusive regulators of the sebaceous glands . Three phases may be distinguished in sebaceous physiology: secretion-production, stocking in the follicular reservoir, and excretion . Human "native" intracellular sebum, before secretion, is composed of squalene, waxes, and triglycerides . Once secreted, the sebum is colonised by various xenobiots whose development is controlled by several defensive humoral mechanisms and by the contact with ambient oxygen . Oxygen and micro-organisms transform "native" sebum, lysis of triglycerides to fatty acids being the most pronounced activity . Certain components of this complex mixture of molecules present in the sebum are clearly cytotoxic or irritant, provoking reactive follicular hyperkeratosis and comedone formation--the first step to acne . Some lipophilic organisms like Malassezia yeast may be highly antigenic and induce chronic inflammatory reactions like in seborrhoeic dermatitis . Demodex is an inrafollicular parasite feeding on sebum that frequently causes blepharitis . Sebum is also a vehicle transporting and transmitting several endogenous and exogenous molecules, including potential regulatory factors of hair follicles . Recent development of in vitro cultures of functional sebocytes should help to better understand several aspects of the sebaceous gland's biology.

J Biol Chem, 2003 Dec 26, 278(52), 52848 - 56 Epub 2003 Oct 16.
Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase; Piechotta K et al.; Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK) . Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1 . Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function . In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein . First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity . Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways . Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner . The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged . These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress.

Gene, 2003 Oct 16, 316, 79 - 89
Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria; Mazet F et al.; The Forkhead or Fox gene family encodes putative transcription factors . There are at least four Fox genes in yeast, 16 in Drosophila melanogaster (Dm) and 42 in humans . Recently, vertebrate Fox genes have been classified into 17 groups named FoxA to FoxQ . Here, we extend this analysis to invertebrates, using available sequences from D . melanogaster, Anopheles gambiae (Ag), Caenorhabditis elegans (Ce), the sea squirt Ciona intestinalis (Ci) and amphioxus Branchiostoma floridae (Bf), from which we also cloned several Fox genes . Phylogenetic analyses lend support to the previous overall subclassification of vertebrate genes, but suggest that four subclasses (FoxJ, L, N and Q) could be further subdivided to reflect their relationships to invertebrate genes . We were unable to identify orthologs of Fox subclasses E, H, I, J, M and Q1 in D . melanogaster, A . gambiae or C . elegans, suggesting either considerable loss in ecdysozoans or the evolution of these subclasses in the deuterostome lineage . Our analyses suggest that the common ancestor of protostomes and deuterostomes had a minimum complement of 14 Fox genes.

Biochem Pharmacol, 2003 Nov 1, 66(9), 1755 - 68
Identification and characterization of mechanistically distinct inducers of gamma-globin transcription; Haley JD et al.; Inhibition of HbS polymerization is a major target for therapeutic approaches in sickle cell anemia . Toward this goal, initial efforts at pharmacological elevation of fetal hemoglobin (HbF) has shown therapeutic efficacy . In order to identify well-tolerated, novel agents that induce HbF in patients, we developed a high-throughput screening approach based on induction of gamma-globin gene expression in erythroid cells . We measured gamma-globin transcription in K562 cells transfected with either gamma promoter elements fused with the locus control region hypersensitivity site 2 and luciferase reporter gene (HS2 gamma) or a beta-yeast artificial chromosome in which the luciferase reporter gene was recombined into the gamma-globin coding sequences (gamma YAC) . Corresponding pharmacological increases in HbF protein were confirmed in both K562 cells and in human primary erythroid progenitor cells . Approximately 186,000 defined chemicals and fungal extracts were evaluated for their ability to increase gamma gene transcription in either HS2 gamma or gamma YAC models . Eleven distinct classes of compounds were identified, the majority of which were active within 24-48 hr . The short chain hydroxamate-containing class generally exhibited delayed maximal activity, which continued to increase transcription up to 120 hr . The cyclic tetrapeptide OSI-2040 and the hydroxamates were shown to have histone deacetylase inhibitory activity . In primary hematopoietic progenitor cell cultures, OSI-2040 increased HbF by 4.5-fold at a concentration of only 40 nM, comparable to the effects of hydroxyurea at 100 microM . This screening methodology successfully identifies active compounds for further mechanistic and preclinical evaluation as potential therapeutic agents for sickle cell anemia.

Adv Exp Med Biol, 2003, 530, 75 - 84
The influence of a clear layer on near infrared spectrophotometry: comparison of measurements in a liquid neonatal head phantom to infants in vivo; Wolf M et al.; Near infrared spectrophotometric (NIRS) algorithms to determine the tissue oxygen saturation (TOI) assume a semi-infinite, homogenous tissue geometry . At the head, the clear cerebrospinal fluid (CSF) layer may violate this assumption . The aim was to estimate the error in the TOI values caused by the CSF layer in vitro and to confirm the results in vivo . The liquid phantom mimicking the neonatal head, consisted of a spherical shell of silicone filled with a liquid solution (1% Intralipid, 60 mumol/l haemoglobin, yeast) and a clear layer imitating CSF . The solution was oxygenated and deoxygenated, while measuring its TOI and pO2 . Without clear layer the mean TOI was 90.9 +/- 0.5% at pO2 > 18 kPa and decreased to 26.0 +/- 1.3% at pO2 = 0 kPa . With a clear layer the TOI increased from 27.8 +/- 0.8% at pO2 > 18 kPa to 68.0 +/- 0.8% at pO2 = 0 kPa . The clear layer caused a large error in the TOI . In ten mechanically ventilated infants (postnatal age 0.03 to 8 months) the TOI (at the head) and arterial oxygen saturation (SaO2) were measured while the inspired oxygen fraction was altered . The TOI was always positively correlated with the SaO2 (mean slope linear regression = 0.89, r2 = 0.62) . Thus an adverse effect of the CSF layer on TOI measurements can be excluded for infants . The CSF layer is not modelled correctly in the phantom.

J Biol Chem, 2004 Jan 2, 279(1), 407 - 13 Epub 2003 Oct 15.
Photoreceptor cGMP phosphodiesterase delta subunit (PDEdelta) functions as a prenyl-binding protein; Zhang H et al.; Bovine PDEdelta was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues . Other studies showed that PDEdelta can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the N-terminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated . We show by immunocytochemistry with a PDEdelta-specific antibody that PDEdelta is present in rods and cones . We find by yeast two-hybrid screening with a PDEdelta bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction . In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDEdelta fusion protein . Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDEdelta and dansylated prenyl cysteines as fluorescent ligands, we show that PDEdelta specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 and 0.70 microm, respectively . Our experiments establish that PDEdelta functions as a prenyl-binding protein interacting with multiple prenylated proteins.

J Clin Invest, 2003 Oct, 112(8), 1164 - 75
Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum; Nosanchuk JD et al.; A protective role for antibodies has not previously been described for host defense against the pathogenic fungus Histoplasma capsulatum (Hc) . Mouse mAb's were generated from mice immunized with Hc yeast that binds the cell surface of Hc . Administration of mAb's before Hc infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model . Protection mediated by mAb's was associated with enhanced levels of IL-4, IL-6, and IFN-gamma in the lungs of infected mice . The mAb's increased phagocytosis of yeast by J774.16 cells through a CR3-dependent process . Ingestion of mAb-opsonized Hc by J774.16 macrophage-like cells was associated with yeast cell growth inhibition and killing . The mAb's bound to a 17-kDa antigen expressed on the surface of Hc . The antigen was identified as a histone H2B-like protein . This study establishes that mAb's to a cell surface protein of Hc alter the intracellular fate of the fungus and mediate protection in a murine model of lethal histoplasmosis, and it suggests a new candidate antigen for vaccine development.

Development, 2003 Dec, 130(24), 5885 - 94 Epub 2003 Oct 15.
A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis; Esch JJ et al.; Previously characterized Arabidopsis gl3 mutants have trichomes that are smaller, less branched and undergo fewer rounds of endoreplication than wild-type trichomes . A new gl3 mutant, called gl3-sst, has oddly shaped trichomes that over expand during early development, undergo more endoreduplication and that have a striking nuclear morphology . The mutant nuclei consist of many interconnected lobes; however, only a single set of polytene-like chromosomes reside in the mutant nuclei . The predicted gl3-sst polypeptide has a Leu to Phe substitution (codon 78) within a region responsible for protein-protein interaction . Yeast interaction assays comparing GL3 with gl3-sst proteins show that the mutant protein interaction with GL1 and TTG1 is decreased by 75% and 50%, respectively, but there is no difference in its interaction with TRY . Furthermore, TRY has the ability to prevent the GL1 GL3 interaction and the GL1 gl3-sst interaction is even more sensitive to TRY . Analysis of plants expressing functional GFP-tagged versions of GL1, GL3 and TRY show that the proteins are localized in trichome nuclei . These results have been used to model trichome initiation in terms of protein interactions and threshold levels of activator complex.

Curr Biol, 2003 Oct 14, 13(20), 1814 - 9
A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo; Drengk A et al.; The endocytic pathway depends on the actin cytoskeleton . Actin contributes to internalization at the plasma membrane and to subsequent trafficking steps like propulsion through the cytoplasm, fusion of phagosomes with early endosomes, and transport from early to late endosomes . In vitro studies with mammalian endosomes and yeast vacuoles implicate actin in membrane fusion . Here, we investigate the function of the actin coat that surrounds late endosomes in Dictyostelium . Latrunculin treatment leads to aggregation of these endosomes into grape-like clusters and completely blocks progression of endocytic marker . In addition, the cells round up and stop moving . Because this drug treatment perturbs all actin assemblies in the cell simultaneously, we used a novel targeting approach to specifically study the function of the cytoskeleton in one subcellular location . To this end, we constructed a hybrid protein targeting cofilin, an actin depolymerizing protein, to late endosomes . As a consequence, the endosomal compartments lost their actin coats and aggregated, but these cells remained morphologically normal, and the kinetics of endocytic marker trafficking were unaltered . Therefore, the actin coat prevents the clustering of endosomes, which could be one safeguard mechanism precluding their docking and fusion.

Curr Biol, 2003 Oct 14, 13(20), 1797 - 802
The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity; Terret ME et al.; Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development . The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division . In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase . Their disjunction is mediated by Separase, which cleaves the cohesin . The activation of Separase requires prior degradation of its associated inhibitor, called securin . Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome . After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase . In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity . Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes . We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.

Philos Trans R Soc Lond B Biol Sci, 2003 Oct 29, 358(1438), 1755 - 71
Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis; Tunnacliffe A et al.; In 1702, Van Leeuwenhoek was the first to describe the phenomenon of anhydrobiosis in a species of bdelloid rotifer, Philodina roseola . It is the purpose of this review to examine what has been learned since then about the extreme desiccation tolerance in rotifers and how this compares with our understanding of anhydrobiosis in other organisms . Remarkably, much of what is known today about the requirements for successful anhydrobiosis, and the degree of biostability conferred by the dry state, was already determined in principle by the time of Spallanzani in the late 18th century . Most modern research on anhydrobiosis has emphasized the importance of the non-reducing disaccharides trehalose and sucrose, one or other sugar being present at high concentrations during desiccation of anhydrobiotic nematodes, brine shrimp cysts, bakers' yeast, resurrection plants and plant seeds . These sugars are proposed to act as water replacement molecules, and as thermodynamic and kinetic stabilizers of biomolecules and membranes . In apparent contradiction of the prevailing models, recent experiments from our laboratory show that bdelloid rotifers undergo anhydrobiosis without producing trehalose or any analogous molecule . This has prompted us to critically re-examine the association of disaccharides with anhydrobiosis in the literature . Surprisingly, current hypotheses are based almost entirely on in vitro data: there is very limited information which is more than simply correlative in the literature on living systems . In many species, disaccharide accumulation occurs at approximately the same time as desiccation tolerance is acquired . However, several studies indicate that these sugars are not sufficient for anhydrobiosis; furthermore, there is no conclusive evidence, through mutagenesis or functional knockout experiments, for example, that sugars are necessary for anhydrobiosis . Indeed, some plant seeds and micro-organisms, like the rotifer, exhibit excellent desiccation tolerance in the absence of high intracellular sugar concentrations . Accordingly, it seems appropriate to call for a re-evaluation of our understanding of anhydrobiosis and to embark on new experimental programmes to determine the key molecular mechanisms involved.

Biochem J, 2004 Jan 15, 377(Pt 2), 347 - 55
In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death; Azoulay-Zohar H et al.; In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating . The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood . Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis . In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time . Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c . The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways . Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I . The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I . Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death . These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

J Med Chem, 2003 Oct 23, 46(22), 4687 - 95
Molecular determinants of steroid inhibition for the mouse constitutive androstane receptor; Jyrkkarinne J et al.; The constitutive androstane receptor (CAR) regulates drug and steroid metabolism through binding to cytochrome P450 2B, 2C, and 3A gene enhancers . Uniquely among nuclear receptors, mouse CAR (mCAR) can be suppressed by androstenol and activated by structurally diverse drugs, pesticides, and environmental pollutants . To gain insight into presently ill-defined structural requirements of mCAR ligands, we employed a mCAR inhibition assay in mammalian HEK293 cells to create a QSAR model that could well predict the inhibition by three unknown steroids . Two novel mCAR inhibitors were thus identified . Yeast two-hybrid assays indicated that steroids inhibit mCAR primarily by promoting association of mCAR with the corepressor NCoR, with only minor contribution from other mechanisms . Analysis of chimeric and mutant mCAR constructs suggested that androstenol sensitivity is controlled by residues between amino acids 201-263 (helices 5-7) and it does not depend on the residue 350 within helix 12, as previously suggested.

Curr Top Microbiol Immunol, 2004, 279, 199 - 213
Modulation of the protein kinase activity of mTOR; Lawrence JC et al.; mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase . mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I . The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo . Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast . A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity . In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others . Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin . Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K . The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope.

Curr Top Microbiol Immunol, 2004, 279, 169 - 97
mTOR signaling to translation; Gingras AC et al.; Over the past few years, the target of rapamycin (TOR) pathway has been implicated in the control of translation, both in yeast and in higher eukaryotes . In this review, we provide an overview of translation in eukaryotes, and discuss the mechanisms and advantages of the regulation of translation . We then describe how the TOR pathway can modulate translation in yeast and in mammals, through the modulation of the phosphorylation of key translation components, and the regulation of the abundance of ribosomes and translation factors.

Curr Top Microbiol Immunol, 2004, 279, 115 - 38
TOR action in mammalian cells and in Caenorhabditis elegans; Long X et al.; The p70 S6 kinase (p70 S6K) was the first signaling element in mammalian cells shown to be inhibited by rapamycin . The activity of the p70 S6K in mammalian cell is upregulated by extracellular amino acids (especially leucine) and by signals from receptor tyrosine kinases (RTKs), primarily through activation of the type 1A PI-3 kinase . The amino acid-/rapamycin-sensitive input and the PI-3 kinase input are co-dominant but largely independent, in that deletion of the amino-terminal and carboxy-terminal noncatalytic sequences flanking the p70 S6K catalytic domain renders the kinase insensitive to inhibition by both rapamycin and by withdrawal of amino acids, whereas this p70 S6K mutant remains responsive to activation by RTKs and to inhibition by wortmannin . At a molecular level, this dual control of p70 S6K activity is attributable to phosphorylation of the two p70 S6K sites: The Ptd Ins 3,4,5P3-dependent kinasel (PDK1) phosphorylates p70 S6K at a Thr on the activation loop, whereas mTOR phosphorylates a Thr located in a hydrophobic motif carboxyterminal to the catalytic domain . Together these two phosphorylations engender a strong, positively cooperative activation of p70 S6K, so that each is indispensable for physiologic regulation . Like RTKs, the p70 S6K appears early in metazoan evolution and comes to represent an important site at which the more ancient, nutrient-responsive TOR pathway converges with the RTK/PI-3 kinase pathway in the control of cell growth . Dual regulation of p70 S6K is seen in Drosophila; however, this convergence is not yet evident in Caenorhabditis elegans, wherein nutrient activation of the insulin receptor (InsR) pathway negatively regulates dauer development and longevity, whereas the TOR pathway regulates overall mRNA translation through effectors distinct from p70 S6K, as in yeast . The C . elegans TOR and InsR pathways show none of the cross- or convergent regulation seen in mammalian cells . The nature of the elements that couple nutrient sufficiency to TOR activity remain to be discovered, and the mechanisms by which RTKs influence TOR activity in mammalian cells require further study . One pathway for RTK control involves the tuberous sclerosis complex, which is absent in C . elegans, but of major importance in Drosophila and higher metazoans.

Curr Top Microbiol Immunol, 2004, 279, 97 - 113
Plant growth and the TOR pathway; Menand B et al.; In mammalian, insect, and yeast cells, TOR proteins are essential regulators of cell growth in response to environmental signals including nutrients, mitogens, and stresses . Although many aspects of the TOR-dependent signalling pathway are conserved between animals and fungi, important differences have also been found and are likely to be related to the ecophysiological adaptations of these organisms . The TOR protein also exists in plants . This review will first discuss specific aspects of plants concerning the contribution of cell growth to overall growth, as well as their responses to nutrient starvation, with emphasis on recent results obtained through genetic analysis in the model plant Arabidopsis thaliana . This is followed by the current status of the genetic analysis of the TOR gene in this plant and the search for potential members of a TOR pathway in the Arabidopsis genome.

Mol Cell Biol, 2003 Nov, 23(21), 7861 - 74
Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1; Noguchi E et al.; The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks . It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival . In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53 . Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching . Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1 . Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site . Swi1 also has Cds1-independent functions . Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse . Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants . Swi1 is recruited to chromatin during S phase . We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.

Mol Cell Biol, 2003 Nov, 23(21), 7698 - 707
Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 are essential for sister chromatid cohesion in meiosis and mitosis; Wang F et al.; Sister chromatid cohesion is fundamental for the faithful transmission of chromosomes during both meiosis and mitosis . Proteins involved in this process are highly conserved from yeasts to humans . In screenings for sterile animals with abnormal vulval morphology, mutations in the Caenorhabditis elegans evl-14 and scc-3 genes were isolated . Defects in cell divisions were observed in germ line as well as in vulval and somatic gonad lineages . Through positional cloning of these genes, we have shown that EVL-14 and SCC-3 are likely the only C . elegans homologs of the yeast sister chromatid cohesion proteins Pds5 and Scc3, respectively . Both evl-14 and scc-3 mutants displayed defects in the meiotic germ line . In evl-14 mutants, synaptonemal complexes (SCs) were detectable but more than the usual six DAPI (4',6'-diamidino-2-phenylindole)-positive structures were seen at diakinesis, suggesting that EVL-14/PDS-5 is important for the maintenance of sister chromatid cohesion in late prophase . In scc-3 mutant animals, normal SCs were not visible and approximately 24 DAPI-positive structures were seen at diakinesis, indicating that SCC-3 is necessary for sister chromatid cohesion . Immunostaining revealed that localization of REC-8, a homolog of the yeast meiotic cohesin subunit Rec8, to the chromosomes depends on the presence of SCC-3 but not that of EVL-14/PDS-5 . scc-3 RNA interference (RNAi)-treated embryos were 100% lethal and displayed defects in cell divisions . evl-14 RNAi caused a range of phenotypes . These results indicate that EVL-14/PDS-5 and SCC-3 have functions in both mitosis and meiosis.

Mol Cell Biol, 2003 Nov, 23(21), 7498 - 509
Modulation of retinoid signaling by a cytoplasmic viral protein via sequestration of Sp110b, a potent transcriptional corepressor of retinoic acid receptor, from the nucleus; Watashi K et al.; Hepatitis C virus (HCV) core protein (core) plays a significant role in the development of chronic liver diseases caused by HCV infection . We have discovered that the core sensitized all-trans-retinoic acid (ATRA)-induced cell death in MCF-7 cells . Activation of retinoic acid receptor alpha (RARalpha)-mediated transcription by the core was also seen in all the cell lines tested . By use of a yeast two-hybrid system, we identified Sp110b as a candidate for a core-interacting cellular factor . Although the function of Sp110b has remained unknown, we observed that Sp110b interacts with RARalpha and suppresses RARalpha-mediated transcription . These data suggest that Sp110b is a transcriptional cofactor negatively regulating RARalpha-mediated transcription . RNA interference-mediated reduction of endogenous Sp110b levels depressed the ability of the core to activate RARalpha-mediated transcription, suggesting an essential role for Sp110b in this pathway . The normal nuclear subcellular localization of Sp110b was altered by molecular interaction with the core to the cytoplasmic surface of the endoplasmic reticulum . This evidence suggests a model in which the core sequesters Sp110b from the nucleus and inactivates its corepressor function to activate RARalpha-mediated transcription . These findings likely describe a novel system in which a cytoplasmic viral protein regulates host cell transcription.

Mol Cell Biol, 2003 Nov, 23(21), 7437 - 47
Regulation of alternative splicing by SRrp86 and its interacting proteins; Li J et al.; SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region . Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing . These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins . To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry . We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72 . In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86 . Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

J Biol Chem, 2003 Dec 26, 278(52), 52116 - 23 Epub 2003 Oct 14.
Regulation of SPIN90 phosphorylation and interaction with Nck by ERK and cell adhesion; Lim CS et al.; SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation . We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, betaPIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins . It appears that betaPIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix . The betaPIX.WASP.SPIN90.Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes . The SPIN90.betaPIX.WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions . In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90.Nck and SPIN90.betaPIX interactions, resulting in detachment of cells from extracellular matrix . SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor . Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90.betaPIX.WASP complex and Nck . It thus appears that the interaction of the betaPIX.WASP.SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation.

J Biol Chem, 2003 Dec 26, 278(52), 52307 - 14 Epub 2003 Oct 13.
Silencing of RNA helicase II/Gualpha inhibits mammalian ribosomal RNA production; Henning D et al.; The intricate production of ribosomal RNA is well defined in yeast, but its complexity in higher organisms is barely understood . We recently showed that down-regulation of nucleolar protein RNA helicase II/Gualpha (RH-II/Gualpha or DDX21) in Xenopus oocytes inhibited processing of 20 S rRNA to 18 S and contributed to degradation of 28 S rRNA (Yang, H., Zhou, J., Ochs, R . L., Henning, D., Jin, R., and Valdez, B . C . (2003) J . Biol . Chem . 278, 38847-38859) . Since no nucleolar RNA helicase has been functionally characterized in mammalian cells, we used short interfering RNA to search for functions for RH-II/Gualpha and its paralogue RH-II/Gubeta in rRNA production . Silencing of RH-II/Gualpha by more than 80% in HeLa cells resulted in an almost 80% inhibition of 18 and 28 S rRNA production . This inhibition could be reversed by exogenous expression of wild type RH-II/Gualpha . A helicase-deficient mutant form having ATPase activity was able to rescue the production of 28 S but not 18 S rRNA . A phenotype exhibiting inhibition of 18 S and 28 S rRNA production was also observed when the paralogue RH-II/Gubeta was overexpressed . Both down-regulation of RH-II/Gualpha and overexpression of RH-II/Gubeta slowed cell proliferation . The opposite effects of the two paralogues suggest antagonistic functions.

J Biol Chem, 2003 Dec 26, 278(52), 52988 - 96 Epub 2003 Oct 13.
Involvement of a chaperone regulator, Bcl2-associated athanogene-4, in apolipoprotein B mRNA editing; Lau PP et al.; Apobec-1 is the catalytic subunit of a multicomponent editosome complex that mediates apolipoprotein B (apoB) mRNA editing . We isolated a novel apobec-1-interacting protein by yeast two-hybrid cloning and identified the protein as BAG-4 . BAG-4, a chaperone-regulating protein, also known as SODD (silencer of death domains), is a member of the BAG family of proteins . In this report, we found that apobec-1 is localized in the perinucleolar compartment in HepG2 cells and rat liver MCR-RH7777 cells . BAG-4 binds to apobec-1 via its N-terminal region independent of the BAG domain . It is ubiquitously expressed with predominant occurrence in human pancreas, heart, brain, and placenta . Immunoprecipitation experiments confirmed that BAG-4 interacts with Hsc70/Hsp90 in HepG2 cells . BAG-4 tagged with green fluorescent protein (GFP) or FLAG was localized both in cytoplasm of mouse BNLCL.2 liver cells and human liver hepatoma HepG2 cells . After heat shock, GFP-BAG-4 co-localizes with Hsc70 in the nucleus in HepG2 cells, whereas GFP-BAG-4 mutants lacking the BAG domain remain perinuclear . BAG-4 has no effects on apoB mRNA editing in vitro . However, unlike other apobec-1 complementation factors studied to date, antisense knockdown of BAG-4 in BNLCL.2 cells and in MCR-RH7777 cells increases rather than decreases endogenous apoB mRNA editing . Overexpression of BAG-4 in MCR-RH7777 cells also suppresses apoB mRNA editing . ApoB-48 production also increases with antisense BAG-4 expression in MCR-RH7777 cells . We previously showed that apoB mRNA editing is an intranuclear event (Lau, P . P., Xiong, W . J., Zhu, H . J., Chen, S . H., and Chan, L . (1991) J . Biol . Chem . 266, 20550-20554) . Thus, BAG-4 overexpression down-regulates apoB mRNA editing by shuttling apobec-1 from the intranuclear perinucleolar compartment to the cytoplasm . We propose that BAG-4 functions as a negative regulator for apobec-1-mediated apoB mRNA editing through its ability to suppress the Hsp/Hsc70 chaperone activity and thereby editosome formation and, as a consequence, prevents nuclear localization of the apobec-1 editosome.

Biochem Biophys Res Commun, 2003 Oct 31, 310(4), 1254 - 65
Type 1 angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membrane; Guo DF et al.; The carboxyl terminus of the type 1 angiotensin II receptor (AT(1)) plays an important role in receptor phosphorylation, desensitization, and internalization . The yeast two-hybrid system was employed to isolate proteins associated with the carboxyl terminal region of the AT(1A) receptor . In the present study, we report the isolation of a novel protein, ARAP1, which promotes recycling of AT(1A) to the plasma membrane in HEK-293 cells . ARAP1 cDNA encodes a 493-amino-acid protein and its mRNA is ubiquitously expressed in rat tissues . A complex of ARAP1 and AT(1A) was observed by immunoprecipitation and Western blotting in HEK-293 cells . In the presence of ARAP1, recycled AT(1A) showed a significant Ca(2+) release response to a second stimulation by Ang II 30 min after the first treatment . Immunocytochemical analysis revealed co-localization of recycled AT(1A) and ARAP1 in the plasma membrane 45 min after the initial exposure to Ang II . Taken together, these results indicate a role for ARAP1 in the recycling of the AT(1) receptor to the plasma membrane with presumable concomitant recovery of receptor signal functions.

Biochem Biophys Res Commun, 2003 Oct 31, 310(4), 1117 - 23
Interaction of Smads with collagen types I, III, and V; Ellis LR et al.; Ontogenesis of the mammalian orofacial region is controlled by numerous developmental signals, including those initiated by the transforming growth factors beta (TGFbetas) . Targeted deletion of the genes encoding several of the TGFbetas in mice has been shown to result in clefts of the secondary palate . Members of the TGFbeta family of growth factors utilize intracellular Smads as signal transducers . Smads 2 and 3 are transcriptional regulators that bind DNA through their conserved MH1 domains and activate/inhibit transcription of TGFbeta-responsive genes through their MH2 domains . Using a yeast two-hybrid screen of a cDNA expression library constructed from fetal murine orofacial tissue, we have identified three types of collagens (types I, III, and V) that are capable of binding to the MH2 domain of Smad 3 . These interactions were confirmed by glutathione S-transferase (GST) pull-down assays in which the MH2 domain of Smad 3 fused to GST interacted strongly with in vitro translated, 35S-labeled collagen types I, III, and V . Each collagen also bound to the MH2 domains of Smads 4 and 7 and, to a lesser extent, full-length Smads 1, 2, 3, and 4 . Binding of Smads to collagen is a novel observation . Moreover, TGFbeta is a potent regulator of collagen synthesis and turnover during mammalian orofacial development . These data thus suggest an important means of feedback regulation of the TGFbeta signaling cascade.

Trends Biochem Sci, 2003 Oct, 28(10), 548 - 57
Holliday junctions in the eukaryotic nucleus: resolution in sight?
Heyer WD, Ehmsen KT, Solinger JA.
The Holliday junction is a key recombination intermediate whose resolution generates crossovers . Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism . Holliday junction resolvases in the eukaryotic nucleus have long eluded identification . The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively . Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers . Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.

Trends Biochem Sci, 2003 Oct, 28(10), 541 - 7
More than folding: localized functions of cytosolic chaperones; Young JC et al.; Compared with other chaperone systems, heat shock proteins Hsp70 and Hsp90 interact with a larger variety of co-chaperone proteins that regulate their activity or aid in the folding of specific substrate proteins . Although many co-chaperones are soluble cytosolic proteins, co-chaperone domains are also found in modular adaptor proteins, which are often localized to intracellular membranes or elements of the cytoskeleton . These specialized co-chaperones include auxilin, cysteine string protein, Tom70, UNC-45 and homologs of Bag-1 . The localized co-chaperones can harness the ATP-dependent mechanisms of Hsp70 and Hsp90 to do conformational work in diverse functional contexts, including vesicle secretion and recycling, protein transport and the regulated assembly and/or disassembly of protein complexes . Such flexibility is unique to the cytosolic Hsp70 and Hsp90 chaperone system.

Brain Res Mol Brain Res, 2003 Oct 7, 117(2), 179 - 89
SEPT5_v2 is a parkin-binding protein; Choi P et al.; Mutations in parkin are associated with various inherited forms of Parkinson's disease (PD) . Parkin is a ubiquitin ligase enzyme that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins destined for proteasomal degradation . The substrates of parkin-mediated ubiquitination have yet to be completely identified . Using a yeast two-hybrid screen, we isolated the septin, human SEPT5_v2 (also known as cell division control-related protein 2), as a putative parkin-binding protein . SEPT5_v2 is highly homologous to another septin, SEPT5, which was recently identified as a target for parkin-mediated ubiquitination . SEPT5_v2 binds to parkin at the amino terminus and in the ring finger domains . Several lines of evidence have validated the putative link between parkin and SEPT5_v2 . Parkin co-precipitates with SEPT5_v2 from human substantia nigra lysates . Parkin ubiquitinates SEPT5_v2 in vitro, and both SEPT5_v1 and SEPT5_v2 accumulate in brains of patients with ARJP, suggesting that parkin is essential for the normal metabolism of these proteins . These findings suggest that an important relationship exists between parkin and septins.

Biochem J, 2004 Feb 1, 377(Pt 3), 607 - 16
Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate; Ito M et al.; Treatment with PTH (parathyroid hormone) or a high-P(i) diet causes internalization of the type IIa sodium-dependent phosphate (Na/P(i) IIa) co-transporter from the apical membrane and its degradation in the lysosome . A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval . To elucidate the mechanism of internalization of Na/P(i) IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening . We found a strong interaction of the Na/P(i) IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p) . PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues . PEX19 is highly expressed in mouse and rat kidney . Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component) . Overexpression of PEX19 stimulated the endocytosis of the Na/P(i) IIa co-transporter in opossum kidney cells in the absence of PTH . In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/P(i) IIa co-transporter.

J Virol, 2003 Nov, 77(21), 11809 - 21
Adenovirus E1B 55-kilodalton oncoprotein binds to Daxx and eliminates enhancement of p53-dependent transcription by Daxx; Zhao LY et al.; The adenovirus E1B 55-kDa protein impairs the p53 pathway and enhances transformation, although the underlying mechanisms remain to be defined . We found that Daxx binds to the E1B 55-kDa protein in a yeast two-hybrid screen . The two proteins interact through their C termini . Mutation of three potential phosphorylation sites (S489/490 and T494 to alanine) within the E1B 55-kDa protein did not affect its interaction with Daxx, although such mutations were previously shown to inhibit E1B's ability to repress p53-dependent transcription and to enhance transformation . In addition to their coimmunoprecipitation in 293 extracts, purified Daxx interacted with the E1B 55-kDa protein in vitro, indicating their direct interaction . In 293 cells, Daxx colocalized with the E1B 55-kDa protein within discrete nuclear dots, where p53 was also found . Such structures were distinct from PML (promyelocytic leukemia protein) bodies, and it appeared that Daxx was displaced from PML bodies . Thus, the Daxx concentration was diminished in dots with a prominent presence of PML and vice versa . Indeed, PML overexpression led to dramatic redistribution of Daxx from p53-E1B 55-kDa protein complexes to PML bodies . Additionally, expression of the E1B 55-kDa protein in Saos2 osteosarcoma cells reduced the number of PML bodies . Our data suggest that E1B and PML compete for available Daxx in the cell . Surprisingly, Daxx significantly augmented p53-mediated transcription and the E1B 55-kDa protein eliminated this effect . Thus, it is likely that the E1B 55-kDa protein sequesters Daxx and p53 in specific nuclear locations, where p53 cannot activate transcription . One consequence of the Daxx-E1B interaction might be an alteration of normal interactions of Daxx, PML, and p53, which may contribute to cell transformation.

Circulation, 2003 Nov 4, 108(18), 2264 - 9 Epub 2003 Oct 13.
Ras induces vascular smooth muscle cell senescence and inflammation in human atherosclerosis; Minamino T et al.; BACKGROUND: Vascular cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence . The functional changes associated with cellular senescence are thought to contribute to human aging and age-related vascular disorders . Ras, an important signaling molecule involved in atherogenic stimuli, is known to promote aging in yeast and cellular senescence in primary human fibroblasts . The aim of this study was to investigate the role of Ras-induced vascular smooth muscle cell (VSMC) senescence in atherogenesis . METHODS AND RESULTS: We introduced an activated ras allele (H-rasV12) into human VSMCs using retroviral infection . Introduction of H-rasV12 induced a growth arrest with phenotypic characteristics of cellular senescence, such as enlarged cell shapes and increases in expression of cyclin-dependent kinase inhibitors and senescence-associated beta-galactosidase (SA-beta-gal) activity . Activation of Ras drastically increased expression of proinflammatory cytokines, in part through extracellular signal-regulated kinase activation . To determine whether Ras activation induces cellular senescence in vivo, we transduced the adenoviral vector encoding H-rasV12 into rat carotid arteries injured by a balloon catheter . Introduction of Ras into the arteries enhanced vascular inflammation and senescence compared with mock-infected injured arteries . Moreover, SA-beta-gal-positive VSMCs were detected in the intima of advanced human atherosclerotic lesions and exhibited increased levels of extracellular signal-regulated kinase activity and proinflammatory cytokine expression . CONCLUSIONS: Our results suggest that atherogenic stimuli mediated by Ras induce VSMC senescence and vascular inflammation, thereby contributing to atherogenesis . This novel mechanism of atherogenesis may provide insights into a new antisenescence treatment for atherosclerosis.

Clin Microbiol Rev, 2003 Oct, 16(4), 730 - 97
Current perspectives on ophthalmic mycoses; Thomas PA; Fungi may infect the cornea, orbit and other ocular structures . Species of Fusarium, Aspergillus, Candida, dematiaceous fungi, and Scedosporium predominate . Diagnosis is aided by recognition of typical clinical features and by direct microscopic detection of fungi in scrapes, biopsy specimens, and other samples . Culture confirms the diagnosis . Histopathological, immunohistochemical, or DNA-based tests may also be needed . Pathogenesis involves agent (invasiveness, toxigenicity) and host factors . Specific antifungal therapy is instituted as soon as the diagnosis is made . Amphotericin B by various routes is the mainstay of treatment for life-threatening and severe ophthalmic mycoses . Topical natamycin is usually the first choice for filamentous fungal keratitis, and topical amphotericin B is the first choice for yeast keratitis . Increasingly, the triazoles itraconazole and fluconazole are being evaluated as therapeutic options in ophthalmic mycoses . Medical therapy alone does not usually suffice for invasive fungal orbital infections, scleritis, and keratitis due to Fusarium spp., Lasiodiplodia theobromae, and Pythium insidiosum . Surgical debridement is essential in orbital infections, while various surgical procedures may be required for other infections not responding to medical therapy . Corticosteroids are contraindicated in most ophthalmic mycoses; therefore, other methods are being sought to control inflammatory tissue damage . Fungal infections following ophthalmic surgical procedures, in patients with AIDS, and due to use of various ocular biomaterials are unique subsets of ophthalmic mycoses . Future research needs to focus on the development of rapid, species-specific diagnostic aids, broad-spectrum fungicidal compounds that are active by various routes, and therapeutic modalities which curtail the harmful effects of fungus- and host tissue-derived factors.

Biochem J, 2004 Feb 15, 378(Pt 1), 63 - 72
Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi; D'Angelo MA et al.; Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals . In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi . TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes . Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein . This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors . TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast . Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme . Confocal laser scanning of T . cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite . The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments . The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

Bioinformatics, 2003 Oct 12, 19(15), 1909 - 16
Topology of gene expression networks as revealed by data mining and modeling; Lukashin AV et al.; MOTIVATION: Interpretation of high-throughput gene expression profiling requires a knowledge of the design principles underlying the networks that sustain cellular machinery . Recently a novel approach based on the study of network topologies has been proposed . This methodology has proven to be useful for the analysis of a variety of biological systems, including metabolic networks, networks of protein-protein interactions, and gene networks that can be derived from gene expression data . In the present paper, we focus on several important issues related to the topology of gene expression networks that have not yet been fully studied . RESULTS: The networks derived from gene expression profiles for both time series experiments in yeast and perturbation experiments in cell lines are studied . We demonstrate that independent from the experimental organism (yeast versus cell lines) and the type of experiment (time courses versus perturbations) the extracted networks have similar topological characteristics suggesting together with the results of other common principles of the structural organization of biological networks . A novel computational model of network growth that reproduces the basic design principles of the observed networks is presented . Advantage of the model is that it provides a general mechanism to generate networks with different types of topology by a variation of a few parameters . We investigate the robustness of the network structure to random damages and to deliberate removal of the most important parts of the system and show a surprising tolerance of gene expression networks to both kinds of disturbance.

Bioinformatics, 2003 Oct 12, 19(15), 1875 - 81
Learning to predict protein-protein interactions from protein sequences; Gomez SM et al.; In order to understand the molecular machinery of the cell, we need to know about the multitude of protein-protein interactions that allow the cell to function . High-throughput technologies provide some data about these interactions, but so far that data is fairly noisy . Therefore, computational techniques for predicting protein-protein interactions could be of significant value . One approach to predicting interactions in silico is to produce from first principles a detailed model of a candidate interaction . We take an alternative approach, employing a relatively simple model that learns dynamically from a large collection of data . In this work, we describe an attraction-repulsion model, in which the interaction between a pair of proteins is represented as the sum of attractive and repulsive forces associated with small, domain- or motif-sized features along the length of each protein . The model is discriminative, learning simultaneously from known interactions and from pairs of proteins that are known (or suspected) not to interact . The model is efficient to compute and scales well to very large collections of data . In a cross-validated comparison using known yeast interactions, the attraction-repulsion method performs better than several competing techniques.

Bioinformatics, 2003 Oct 12, 19(15), 1869 - 74
Greedily building protein networks with confidence; Bader JS; MOTIVATION: With genome sequences complete for human and model organisms, it is essential to understand how individual genes and proteins are organized into biological networks . Much of the organization is revealed by proteomics experiments that now generate torrents of data . Extracting relevant complexes and pathways from high-throughput proteomics data sets has posed a challenge, however, and new methods to identify and extract networks are essential . We focus on the problem of building pathways starting from known proteins of interest . RESULTS: We have developed an efficient, greedy algorithm, SEEDY, that extracts biologically relevant biological networks from protein-protein interaction data, building out from selected seed proteins . The algorithm relies on our previous study establishing statistical confidence levels for interactions generated by two-hybrid screens and inferred from mass spectrometric identification of protein complexes . We demonstrate the ability to extract known yeast complexes from high-throughput protein interaction data with a tunable parameter that governs the trade-off between sensitivity and selectivity . DNA damage repair pathways are presented as a detailed example . We highlight the ability to join heterogeneous data sets, in this case protein-protein interactions and genetic interactions, and the appearance of cross-talk between pathways caused by re-use of shared components . SIGNIFICANCE AND COMPARISON: The significance of the SEEDY algorithm is that it is fast, running time O{(E + V) log V} for V proteins and E interactions, a single adjustable parameter controls the size of the pathways that are generated, and an associated P-value indicates the statistical confidence that the pathways are enriched for proteins with a coherent function . Previous approaches have focused on extracting sub-networks by identifying motifs enriched in known biological networks . SEEDY provides the complementary ability to perform a directed search based on proteins of interest . AVAILABILITY: SEEDY software (Perl source), data tables and confidence score models (R source) are freely available from the author.

Eukaryot Cell, 2003 Oct, 2(5), 1003 - 8
Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei; Matovu E et al.; Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa . Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness . In T . brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents . The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast . Here we investigate the role of TbAT1 in drug uptake and drug resistance in T . brucei by genetic knockout of TbAT1 . Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals . However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced . Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol . Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals . High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

Eukaryot Cell, 2003 Oct, 2(5), 867 - 75
D-xylose metabolism in Hypocrea jecorina: loss of the xylitol dehydrogenase step can be partially compensated for by lad1-encoded L-arabinitol-4-dehydrogenase; Seiboth B et al.; With the goal of the genetic characterization of the D-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal D-xylose catabolism . This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+ . The enzyme catalyzed the NAD-dependent oxidation of xylitol and D-sorbitol and the NADH-dependent reduction of D-xylulose and D-fructose . No activity was observed with NADP, L-arabinose, or L-arabinitol . A single 1.4-kb transcript was formed during growth on xylan, D-xylose, L-arabinose, L-arabinitol and, at a lower abundance, xylitol, D-galactose, galactitol, and lactose but not on D-glucose and glycerol . xdh1 deletion mutants exhibited 50% reduced growth rates on D-xylose, whereas growth rates on xylitol remained unaltered . These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase . This activity was shown to be due to lad1-encoded L-arabinitol-4-dehydrogenase, because H . jecorina xdh1 lad1 double-deletion strains failed to grow on D-xylose or xylitol . In contrast, lad1 deletion strains of H . jecorina grew normally on these carbon sources . These results show that H . jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of D-xylose and that lad1-encoded L-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function.

Int J Radiat Biol, 2003 Aug, 79(8), 663 - 9
Nucleotide excision repair proteins and their importance for radiation-enhanced transfection; Nimura Y et al.; PURPOSE: Irradiated cells transfect more efficiently than unirradiated cells because of a radiation-induced increase in plasmid integration . However, the molecular mechanism is unclear . Because of recent observations that nucleotide excision repair (NER) proteins can be involved in certain types of recombination in yeast, it was hypothesized that NER proteins might play a role in this radiation-enhanced integration . MATERIALS AND METHODS: Hamster and human cells with inactivating mutations in NER genes were irradiated at doses from 0 to 6 Gy and then immediately transfected with a linearized selectable marker plasmid . Transfection-enhancement ratios (TERs) were calculated as the ratio of the number of drug-resistant colonies in unirradiated cells to the number of transfectants in irradiated cells, corrected for cytotoxicity from radiation . RESULTS: Transfection into unirradiated rodent cells was unaffected by NER mutation status . Transfection into unirradiated human cells, however, was increased by NER mutation . The TERs were 5 and 100 for CHO and primary human fibroblasts, respectively, after exposure of the cells to 6 Gy . Mutations in ERCC1, XPA, XPB, XPC, XPF, XPG and CSB dramatically reduced TER . Mutations in ERCC1, XPC, XPF, XPG and CSB suppressed transfection so that the TER was significantly below 1 . CONCLUSIONS: The mechanism of radiation-enhanced plasmid integration was distinct from that of plasmid integration in unirradiated cells, and NER gene products were critical for enhanced integration to occur.

Virology, 2003 Sep 30, 314(2), 591 - 600
Functional analysis of the interaction of the human immunodeficiency virus type 1 Rev nuclear export signal with its cofactors; Kiss A et al.; Human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export of viral RNAs involves the interaction of its leucine-rich nuclear export sequence (NES) with nuclear cofactors . In yeast two-hybrid screens of a human lymph node derived cDNA expression library, we identified the human nucleoporin Nup98 as a highly specific and potent interactor of the Rev NES . Using an extensive panel of nuclear export positive and negative mutants of the functionally homologous NESs of the HIV-1 Rev, human T cell leukemia virus type 1 (HTLV-1) Rex, and equine infectious anemia virus (EIAV) Rev proteins, physiologically significant interaction of hNup98 with the various NESs was demonstrated . Missense mutations in the yeast nuclear export factor Crm1p that abrogated Rev NES interaction with the XXFG repeat-containing nucleoporin, Rab/hRIP, had minimal effects on the interaction of GLFG repeat-containing hNup98 . Functional analysis of Nup98 domains required for nuclear localization demonstrated that the entire ORF was required for efficient incorporation into the nuclear envelope . A putative nuclear localization signal was identified downstream of the GLFG repeat region . Whereas overexpression of both full-length Nup98 and the amino-terminal GLFG repeat region, but not the unique carboxy-terminal region, induced significant suppression of HIV unspliced RNA export, lower levels of exogenous Nup98 expression resulted in a relatively modest increase in unspliced RNA export . These results suggest a physiological role for hNup98 in modulating Rev-dependent RNA export during HIV infection.

Environ Toxicol Chem, 2003 Oct, 22(10), 2275 - 9
Synthesis and estrogenic activity of bisphenol a mono- and di-beta-D-glucopyranosides, plant metabolites of bisphenol A; Morohoshi K et al.; The syntheses and characterization of bisphenol A mono- and di-beta-D-glucopyranosides were undertaken to confirm that these compounds are major plant metabolities of bisphenol A (BPA) and to allow an assessment of their estrogenicity . Synthesis involved the glucosidation of unprotected BPA with glucose penta-acetate with phosphorus oxychloride as catalyst . The estrogenic activity of BPA and its mono- and di-beta-D-glucopyranosides were measured with an enzyme-linked immunosorbent assay (ELISA)-based estrogen receptor competitive binding assay and with a yeast two-hybrid assay adapted to a chemiluminescent reporter gene (for beta-galactosidase) . Both methods showed that the estrogenicity of BPA was eliminated by formation of the di-glucoside, but whereas the ELISA-based method indicated that reduced activity remained in the monoglucoside, the yeast two-hybrid method showed the monoglucoside to be inactive . Presumably these results reflect the more complex interactions of test compound and cellular components required to demonstrate estrogenicity in the yeast two-hybrid assay . As these processes parallel those in mammalian cells, the yeast two-hybrid method is likely to be the more realistic assay . The uptake and metabolism of BPA by plants offers the possibility of phytoremediation of contaminated water, but also provides an additional route for the compound to enter the human food chain.

Environ Toxicol Chem, 2003 Oct, 22(10), 2243 - 50
A toxicity identification evaluation approach to studying estrogenic substances in hog manure and agricultural runoff; Burnison BK et al.; Spreading liquid manure on agricultural fields is a routine way of disposing of animal manure and optimizing the use of nutrients for crops . Limited studies suggest that these wastes may contain a variety of endocrine-disrupting compounds (EDCs) that may be released into aquatic environments through runoff . The purpose of this study was to apply a toxicity identification and evaluation approach to isolate and identify estrogenic compounds in hog manure . A recombinant yeast estrogen screen bioassay was used to detect estrogenicity of high-performance liquid chromatography--separated hog manure fractions . Further analytical analyses of the fractions and comparison to authentic standards resulted in the identification of the endogenous estrogens 17 beta-estradiol (E2) and estrone, and the phytoestrogen metabolite, equol . High levels of equol (6.9-16.6 ppm) were found to be present in manure that was stored for several months . The endocrine-disrupting potential of equol was characterized further by using fish hormone estrogen receptor (ER), sex hormone binding protein (SSBP), and goldfish androgen receptor (AR) radioligand binding assays . Equol was found to be approximately 1,000- and 200-fold less potent that E2 in competing for binding sites of the SSBP and ER, respectively . Equol's potency was 2,200-fold less than testosterone for the AR . Additional studies confirmed the presence of compounds with estrogenic activity in tile drain water after application of hog manure to an agriculture field . In this case, the contribution of equol to the total estrogenicity of the tile drain water was minimal relative to that of natural estrogens . Overall, this study indicates that further work is warranted to assess the impact that EDCs that originate from agricultural runoff may have on the ecology or physiology of exposed biota.

FEBS Lett, 2003 Oct 9, 553(1-2), 183 - 9
BRCA1 interacts with FHL2 and enhances FHL2 transactivation function; Yan J et al.; Germ-line mutations in BRCA1 are associated with an increased lifetime risk of developing breast and/or ovarian tumors . The BRCA1 gene product is a 220-kDa protein that contains a tandem of two BRCA1 C-terminal (BRCT) domains required for transcription . In an attempt to understand how BRCA1 exerts its function through BRCT domains, we search for partners of the BRCT domains of BRCA1 . Using the yeast two-hybrid system, we identified the four and a half LIM-only protein 2 (FHL2) as a novel BRCA1 interacting protein . We demonstrate that BRCA1 and FHL2 can physically associate in vitro, in yeast, and in human cells . BRCA1 interacted with FHL2 through its second BRCT domain and the interaction of FHL2 with BRCA1 requires the last three LIM domains of FHL2 . BRCA1 enhanced FHL2-mediated transcriptional activity in transient transfections . Tumor-derived transactivation-deficient BRCA1 mutants showed a reduced ability to enhance transactivation by FHL2 . Lack of BRCA1 binding sites in the FHL2 completely abolished the FHL2 transactivation function . Reverse transcription polymerase chain reaction analysis showed that FHL2 mRNA levels may be downregulated in many breast cancer cell lines . These results suggest that the BRCA1-FHL2 interaction may be involved in transcriptional regulation and play a significant role in cancer cell growth.

Mech Dev, 2003 Sep, 120(9), 1033 - 43
Identification of neurabin II as a novel doublecortin interacting protein; Tsukada M et al.; The neuronal migration protein doublecortin (DCX) that associates with microtubules through a tandem DCX repeat, is required for the development of the complex architecture of the human cerebral cortex . Using a yeast two-hybrid screen with Dcx as bait, we have isolated neurabin II/spinophilin, an F-actin binding protein known to play a role in dendritic spine formation . The coiled-coil domain of neurabin II binds to a DCX region encompassing the C-terminal portion of the second DCX repeat and the N-terminal portion of the Ser/Pro-rich domain . Immunoprecipitation experiments with brain extracts show that neurabin II and Dcx interact in vivo . Several Dcx constructs that mimic human DCX mutant alleles failed to interact with neurabin II . Since Dcx and neurabin II colocalized in the developing and adult brain, a neurabin II-DCX heterodimer may be involved in neuronal migration and dendritic spine formation.

Biochem Biophys Res Commun, 2003 Oct 24, 310(3), 1032 - 8
Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer; Rao PS et al.; Prostate cancer (PC) patients die from progression to androgen independence (AI) and chemoresistance (CR) . Protein kinase Cmu (PKCmu) a novel member of the PKC family of signal transduction proteins is downregulated in AI PC . Studying PKCmu interactors in the yeast two-hybrid system identified metallothionein 2A (MT 2A) as an interactor of PKCmu kinase domain (KD) in PC, which was quantified by beta-gal assay, confirmed in PC cells by immunoprecipitation, and PKCmu-MT 2A co-localization in vivo by immunofluorescence studies . PKCmu domain interaction studies revealed that MT 2A interacted strongly with KD, relatively weakly with C1, and failed to interact with C2, PH or kinase mutant domains . Peptide library and in silico analysis strongly suggest that MT 2A is a novel substrate of PKCmu and our data indicate that the PKCmu-MT 2A interaction depends on PKCmu kinase activity . Because metallothioneins are associated with cell proliferation and CR, the PKCmu-MT 2A interaction may contribute to CR and/or AI in PC.

Biochem Biophys Res Commun, 2003 Oct 24, 310(3), 720 - 4
Identification of a nuclear protein that promotes NF-kappaB activation; Chen D et al.; Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappaB activation . In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified a novel protein designated as NKAP . Although NKAP interacts with RIP in yeast, NKAP does not interact with RIP in mammalian cells in co-immunoprecipitation experiments . When overexpressed in 293 cells, NKAP activated NF-kappaB in a dose-dependent manner . Moreover, down-regulation of NKAP by antisense RNA significantly inhibited TNF- and IL-1-induced NF-kappaB activation . Immunofluorescent staining indicated that NKAP was localized in the nucleus . Our findings suggest that NKAP is a novel nuclear regulator of TNF- and IL-1-induced NF-kappaB activation.

Mol Imaging Biol, 2002 May, 4(3), 193 - 200
Characterization of uptake of 2-deoxy-2-{18F}fluoro-D-glucose by fungal-associated inflammation: the differential uptake ratio for blastomyces-associated lesions is as high as for lymphoma and higher than for turpentine abscesses in experimentally induced lesions in rats; Bassett CL et al.; PURPOSE: The objective of this investigation was to determine the biologic basis and the significance of uptake of 2-deoxy-2-{18F}fluoro-D-glucose (FDG) using experimentally created fungal lesions in rats . PROCEDURES: Uptake of FDG by experimentally induced Blastomyces granulomas was compared with uptake by turpentine abscesses (Group 1) and by lymphomas (Group 2) using the differential uptake ratio (DUR) measured one hour after administration of 2 mCi FDG intravenously . Frozen tissue sections of Blastomyces lesions and turpentine abscesses were placed in contact with radiographic film for macroautoradiography . RESULTS: In rats in Group 1, the median (range) DUR for the Blastomyces granulomas was 1.9 (1.1-2.6) and was significantly higher than the DUR for turpentine abscesses 0.9 (0.6-1.4) and muscle 0.2 (0.1-0.5; P < 0.001) . In Group 2, the median (range) DUR for the Blastomyces granulomas, lymphomas, and muscle from the rats in Group 2 were 1.8 (1.2-3.4), 1.9 (1.0-4.0), and 0.2 (0.1-0.3), respectively . There was no significant difference between the DUR of Blastomyces granulomas and lymphomas . Macroautoradiographs of the Blastomyces granulomas revealed intense uptake of FDG in the region occupied by the yeast organisms and the granulomatous inflammation . CONCLUSIONS: Blastomyces granulomas typically have high uptake of FDG associated with the region composed of the granulomatous inflammatory reaction and Blastomyces yeast organisms.

Mol Cell, 2003 Aug, 12(2), 509 - 16
Transcription within a functional human centromere; Saffery R et al.; Recent data in yeast and Drosophila suggest a domain-like centromere structure with a modified chromatin core and flanking regions of heterochromatin . We have analyzed a functional human centromere and defined a region of increased chromosome scaffold/matrix attachment that overlaps three other distinct and nonoverlapping domains for constitutive centromere proteins CENP-A and CENP-H, and heterochromatin protein HP1 . Transcriptional competency is intact throughout the S/MAR-enriched region and within the CENP-A- and CENP-H-associated chromatin . These results provide insights into the relationship between centromeric chromatin and transcriptional competency in vivo, highlighting the permissibility of transcription within the constitutively modified, nonheterochromatic chromatin of a functional eukaryotic centromere.

Mol Cell, 2003 Aug, 12(2), 307 - 19
Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex; Schnaufer A et al.; The Trypanosoma brucei editosome catalyzes the maturation of mitochondrial mRNAs through the insertion and deletion of uridylates and contains at least 16 stably associated proteins . We examined physical and functional associations among these proteins using three different approaches: purification of complexes via tagged editing ligases TbREL1 and TbREL2, comprehensive yeast two-hybrid analysis, and coimmunoprecipitation of recombinant proteins . A purified TbREL1 subcomplex catalyzed precleaved deletion editing in vitro, while a purified TbREL2 subcomplex catalyzed precleaved insertion editing in vitro . The TbREL1 subcomplex contained three to four proteins, including a putative exonuclease, and appeared to be coordinated by the zinc finger protein TbMP63 . The TbREL2 subcomplex had a different composition, contained the TbMP57 terminal uridylyl transferase, and appeared to be coordinated by the TbMP81 zinc finger protein . This study provides insight into the molecular architecture of the editosome and supports the existence of separate subcomplexes for deletion and insertion editing.

Biochem J, 2004 Jan 15, 377(Pt 2), 449 - 57
A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin; Alahari SK et al.; In a previous study {Alahari, Lee and Juliano (2000) J . Cell Biol . 151, 1141-1154}, we have identified a novel protein, nischarin, that specifically interacts with the cytoplasmic tail of the alpha5 integrin subunit . Overexpression of this protein profoundly affects cell migration . To examine the nischarin-alpha5 interaction in detail, and to find the minimal region required for the interaction, several mutants of nischarin and alpha5 were created . The results obtained for the yeast two-hybrid system indicate that a 99-aminoacid region of nischarin (from residues 464 to 562) is indispensable for the interaction . Also, we demonstrate that the membrane proximal region (from residues 1017 to 1030) of the alpha5 cytoplasmic tail is essential for the interaction . To characterize more directly the properties of the interaction between nischarin and alpha5, we performed surface-plasmon resonance studies in which peptides were immobilized on the surface of a sensor chip, and the recombinant nischarin protein fragments were injected . Consistent with the two-hybrid results, recombinant nischarin binds well to immobilized alpha5 peptides . In addition, mutational analysis revealed that residues Tyr(1018) and Lys(1022) are crucial for alpha5-nischarin interactions . These results provide evidence that nischarin is capable of directly and selectively binding to a portion of the alpha5 cytoplasmic domain . Further studies demonstrated that the minimal alpha5 binding region of nischarin does not affect cell migration.

Bioinformatics, 2003, 19 Suppl 2, II206 - II214
Finding optimal degenerate patterns in DNA sequences; Shinozaki D et al.; Motivation: The problem of finding transcription factor binding sites in the upstream regions of given genes is algorithmically an interesting and challenging problem in computational biology . A degenerate pattern over a finite alphabet Sigma is a sequence of subsets of Sigma . A string over IUPAC nucleic acid codes is also a degenerate pattern over Sigma = {A, C, G, T}, and is used as one of the major patterns modeling transcription factor binding sites in the upstream regions of genes . However, it is known that the problem of finding a degenerate pattern consistent with both positive and negative string sets is in general NP-complete . Our aim is to devise a heuristic algorithm to find a degenerate pattern which is optimal for positive and negative string sets w.r.t . a given score function . Results: We have proposed an enumerative algorithm called SUPERPOSITION for finding optimal degenerate patterns with a pruning technique, which works with most all reasonable score functions . The performance score of the algorithm has been compared with those of other popular motif-finding algorithms YMF, MEME and AlignACE on various sets of co-regulated genes of yeast . In the computational experiment, SUPERPOSITION has outperformed the others on several gene sets . Availability: The python script SUPERPOSITION is available at Contact: om@math.kyushu-u.ac.jp

Bioinformatics . 2003 Oct;19 Suppl 2:II57.
Probabilistic models for identifying regulation networks; Friedman N; Microarray-based hybridization methods techniques allow to simultaneously measure the expression level for thousands of genes . Such measurements contain information about many different aspects of gene regulation and function, and indeed this type of experiments has become a central tool in biological research . A major computational challenge is finding ways to extract new biological understanding from this wealth of data . Our goal is to uncover the causal structure of the interactions between genes, with the aim of understanding the regulatory processes that bring about the observed expression patterns . I will argue that one way of addressing this question is a Bayesian framework, where we treat the measured expression level of each gene as a random variable and each regulatory interaction as a probabilistic dependency between such variables . In my talk, I will describe an ongoing project to use Bayesian networks and extensions of them to model such dependencies . In the talk I will explain the basic foundations of the approach, the possible choices in defining the modeling language, the methods we use to learn models from data, and finally how we interpret the learned models . This latter stage includes validation against known biology, and constructing new hypotheses about the role of unknown genes . I will present a progression of models that capture different aspect of gene regulation, and an assessment of their performance on several large scale yeast gene expression experiments . This is joint work with Dana Pe'er, Iftach Nachman, Aviv Regev, Gal Elidan, Eran Segel, Micha Shapira, David Botstein, and Daphne Koller . Contact: nir@cs.huji.ac.il . http://www.cs.huji.ac.il/~nir

Adv Exp Med Biol, 2001, 491, 207 - 14
Glycosyl phosphatidylinositol-linked glycoconjugates: structure, biosynthesis and function; Hwa KY; The purpose of this review is to summarize the most recent advances on GPI research . Structural studies on GPI-linked glycoconjugates indicate that there are significant variations in different organisms, although there is a conserved core structure . Furthermore, structural studies suggest that in different cell types, there is an army of glycosyltransferases dedicated to the synthesis of GPI-linked glycoconjugates . Biochemical studies on the synthesis of these GPI-linked glycoconjugates suggest that not only many different enzymes are involved but also that enzymes from different cell types, involving in the conserved core structure can have different substrate specificity . Genetic cloning of the yeast genes involved in synthesizing the core structure suggests that many of these enzymes also have human homologues . However, paroxysmal nocturnal hemogobinuria (PNH) is the only known human disease associated with the synthesis of GPI-linked glycoconjugates . Functional studies suggest that GPI-anchor can act as a signal for protein sorting and localization . Furthermore, GPI-linked receptors play an important role in T-cell activation.

Folia Microbiol (Praha), 2003, 48(4), 555 - 8
The detection of anti-Candida antibodies on experimental animal models; Pilipcinec E et al.; The levels of anti-Candida antibodies were determined in experimental animals immunized with 4 different yeast doses (0.3, 0.6, 1.2, 2.4 x 10(8) CFU) at weekly intervals . After immunization (sampling intervals 1, 2, and 3 months), the intravenous blood was examined for the presence of serum anti-Candida antibodies . After one month, the titers of anti-Candida antibodies reached 1:40-1: 1280 and remained at the same level after two months in the majority of animals; only in a few of them the titers increased or were detected de novo . After three months, when the animals were no longer immunized, a decreasing trend in antibody titers was detected in the majority of animals.

Cell, 2003 Oct 3, 115(1), 109 - 21
Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells; Ono T et al.; The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans . We report here the identification of a second condensin complex (condensin II) from vertebrate cells . Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits . siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology . Simultaneous depletion of both complexes causes the severest defect . In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes . Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro . We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.

Cell, 2003 Oct 3, 115(1), 83 - 95
The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain; Elia AE et al.; Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis . Plks are characterized by a C-terminal noncatalytic region containing two tandem Polo boxes, termed the Polo-box domain (PBD), which has recently been implicated in phosphodependent substrate targeting . We show that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs . The 1.9 A X-ray structure of a human Plk1 PBD-phosphopeptide complex shows that the Polo boxes each comprise beta6alpha structures that associate to form a 12-stranded beta sandwich domain . The phosphopeptide binds along a conserved, positively charged cleft located at the edge of the Polo-box interface . Mutations that specifically disrupt phosphodependent interactions abolish cell-cycle-dependent localization and provide compelling phenotypic evidence that PBD-phospholigand binding is necessary for proper mitotic progression . In addition, phosphopeptide binding to the PBD stimulates kinase activity in full-length Plk1, suggesting a conformational switching mechanism for Plk regulation and a dual functionality for the PBD.

Kidney Int, 2003 Nov, 64(5), 1746 - 54
PDZK1: II . an anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells; Gisler SM et al.; BACKGROUND: PDZK1, a multiple PDZ protein, was recently found to interact with the type IIa Na/Pi cotransporter (NaPi-IIa) in renal proximal tubular cells . In a preceding study, yeast two-hybrid screens using single PDZ domains of PDZK1 as baits were performed . Among the identified proteins, a C-terminal fragment of the dual-specific A-kinase anchoring protein 2 (D-AKAP2) was obtained by screening PDZ domain 4 . METHODS: After its molecular cloning by means of RACE, the renal expression of D-AKAP2 was analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry . Protein interactions were characterized by overlays, pull-downs, and immunoprecipitations from transfected opossum kidney (OK) cells . RESULTS: Based on 5'-RACE and PDZK1 overlays of mouse kidney cortex separated by two-dimensional electrophoresis, it was suggested that the renal isoform of D-AKAP2 in mouse comprises 372 amino acids and exists as a protein of >40 kD . Immunohistochemistry and real-time PCR localized D-AKAP2 only to the subapical pole of proximal tubular cells in mouse kidney . In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity . Similarly, His-tagged D-AKAP2 specifically retained PDZK1 from mouse kidney cortex homogenate . In addition, myc-tagged D-AKAP2 and HA-tagged PDZK1 co-immunoprecipitated from transfected OK cells . CONCLUSION: We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1 . Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA.

Kidney Int, 2003 Nov, 64(5), 1733 - 45
PDZK1: I . a major scaffolder in brush borders of proximal tubular cells; Gisler SM et al.; BACKGROUND: In proximal tubular cells, PDZK1 (NaPi-Cap1) has been implicated in apical expression of the Na+-dependent phosphate cotransporter (NaPi-IIa) via interaction with its C-terminus . PDZK1 represents a multidomain protein consisting of four PDZ domains and thus is believed to have a broader specificity besides NaPi-IIa . METHODS: We subjected single PDZ domains derived from PDZK1 either to yeast two-hybrid screens or yeast trap assays . Different pull-down assays and blot overlays were applied to corroborate the PDZK1-mediated interactions in vitro . Co-localization of interacting proteins with PDZK1 in proximal tubular cells was assessed by immunohistochemistry . RESULTS: In the yeast screens, the most abundant candidate protein to interact with PDZK1 was the membrane-associated protein of 17 kD (MAP17) . Besides MAP17, C-terminal parts of following transporters were also identified: NaPi-IIa, solute carrier SLC17A1 (NaPi-I), Na+/H+ exchanger (NHE-3), organic cation transporter (OCTN1), chloride-formate exchanger (CFEX), and urate-anion exchanger (URAT1) . In addition, other regulatory factors were found among the clones, such as a protein kinase A (PKA)-anchoring protein (D-AKAP2) and N+/H+ exchanger regulator factor (NHERF-1) . All interactions of itemized proteins with PDZK1 were affirmed by in vitro techniques . Apart from PDZK1, strong in vitro interactions of NHERF-1 were also observed with the solute transporters (excluding MAP17) and D-AKAP2 . All identified proteins were immunolocalized in proximal tubular cells, wherein all membrane proteins co-localized with PDZK1 in brush borders . CONCLUSION: We hypothesize that PDZK1 and NHERF-1 establish an extended network beneath the apical membrane to which membrane proteins and regulatory components are anchored.

Biochem J, 2003 Dec 15, 376(Pt 3), 595 - 605
Characterization of a novel binding partner of the melanocortin-4 receptor: attractin-like protein; Haqq AM et al.; The gene dosage effect of the MC4-R (melanocortin 4 receptor) on obesity suggests that regulation of MC4-R expression and function is critically important to the central control of energy homoeostasis . In order to identify putative MC4-R regulatory proteins, we performed a yeast two-hybrid screen of a mouse brain cDNA library using the mouse MC4-R intracellular tail (residues 303-332) as bait . We report here on one positive clone that shares 63% amino acid identity with the C-terminal part of the mouse attractin gene product, a single-transmembrane-domain protein characterized as being required for agouti signalling through the melanocortin 1 receptor . We confirmed a direct interaction between this ALP (attractin-like protein) and the C-terminus of the mouse MC4-R by glutathione S-transferase pulldown experiments, and mapped the regions involved in this interaction using N- and C-terminal truncation constructs; residues 303-313 in MC4-R and residues 1280-1317 in ALP are required for binding . ALP is highly expressed in brain, but also in heart, lung, kidney and liver . Furthermore, co-localization analyses in mice showed co-expression of ALP in cells expressing MC4-R in a number of regions known to be important in the regulation of energy homoeostasis by melanocortins, such as the paraventricular nucleus of hypothalamus and the dorsal motor nucleus of the vagus.

Bioprocess Biosyst Eng, 2003 Nov, 26(1), 63 - 7 Epub 2003 Oct 03.
Batch and continuous cultures of Mannheimia succiniciproducens MBEL55E for the production of succinic acid from whey and corn steep liquor; Lee PC et al.; Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce a large amount of succinic acid in a medium containing glucose, peptone, and yeast extract . In order to reduce the cost of the medium, whey and corn steep liquor (CSL) were used as substrates for the production of succinic acid by M . succiniciproducens MBEL55E . Anaerobic batch cultures of M . succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in the production of succinic acid with a yield of 71% and productivity of 1.18 g/l/h, which are similar to those obtained in a whey-based medium containing yeast extract (72% and 1.21 g/l/h) . Anaerobic continuous culture of M . succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in a succinic acid yield of 69% and a succinic acid productivity as high as 3.90 g/l/h . These results show that succinic acid can be produced efficiently and economically by M . succiniciproducens MBEL55E from whey and CSL.

Nucleic Acids Res, 2003 Oct 15, 31(20), 5941 - 8
Sox6 regulation of cardiac myocyte development; Cohen-Barak O et al.; A mouse mutation (p100H/p100H) has been identified that is associated with cardioskeletal myopathy, heart block, delayed growth and early postnatal death . The gene that is disrupted in this mutation encodes the transcription factor Sox6 . P19CL6 cells were used as an in vitro cardiomyocyte differentiation system and revealed that Sox6 is expressed exclusively when the cells are committed to differentiate to beating cardiac myocytes . We used the yeast two-hybrid system to identify the Prtb (Proline-rich transcript of the brain) protein as a Sox6 interactor, and subsequently confirmed the interaction by co-immunoprecipitation . Prtb expression in P19CL6 cells increased with differentiation to beating cardiomyocytes . Using the P19CL6 cells stably transfected with noggin, an antagonist of BMP (Bone Morphogenic Protein), we found that BMP expression is required for Sox6 expression in cardiomyocyte differentiation . Surprisingly, the expression of the alpha1c-subunit gene of the L-type Ca2+ channel decreased in P19CL6 cells as they differentiated to beating cardiac cells . Ectopic expression of Sox6 or Prtb alone in P19CL6 cells caused down-regulation of L-type Ca2+ alpha1c expression, but when Sox6 and Prtb were co-transfected to the cells, L-type Ca2+ alpha1c remained at basal levels . A similar relationship of Sox6 and L-type Ca2+ alpha1c expression was seen in vivo (comparing wild-type and p(100H)/p(100H) mutant mice) . Thus, Sox6 is within the BMP pathway in cardiac differentiation, interacts with Prtb and may play a critical role in the regulation of a cardiac L-type Ca2+ channel.

J Biol Chem, 2003 Dec 19, 278(51), 50863 - 71 Epub 2003 Oct 06.
Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport factor Rab9 effector p40; Ikonomov OC et al.; PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function . Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval . Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells . The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40 . Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction . Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction . GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity . A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells . Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport.

Neuropharmacology, 2003 Nov, 45(6), 849 - 56
Interaction of transmembrane AMPA receptor regulatory proteins with multiple membrane associated guanylate kinases; Dakoji S et al.; Surface expression of AMPA type glutamate receptors requires stargazin or a related transmembrane AMPA receptor regulatory protein (TARP) . Furthermore, interaction of the cytosolic tail of TARPs with PDZ domains of PSD-95 targets AMPA receptors to postsynaptic densities . Here, we screened for additional proteins that might interact with the cytosolic domain of TARPs . Screening a rat brain cDNA library with the yeast two-hybrid system yielded six PDZ proteins that can bind tail of TARPs . These PDZ proteins include the four neuronal membrane associated guanylate kinases, PSD-95/SAP-90, PSD-93/Chapsyn-110, SAP-97/hDLG and SAP-102; the multi-PDZ protein, MUPP1; and the mitochondrial PDZ protein, OMP-25 . Although all of these proteins can bind to TARPs in vitro, only two of these, PSD-95 and PSD-93 associate with TARPs in brain . We also found that all three PDZ domains from PSD-95 associate with the TARP C-termini with similar affinities . This work identifies biochemical promiscuity for interaction of the TARP C-termini with PDZ domains in vitro, but also shows that only specific synaptic PDZ proteins associate with TARPs in brain.

Nat Immunol, 2003 Nov, 4(11), 1074 - 82 Epub 2003 Oct 05.
Enhancement of CIITA transcriptional function by ubiquitin; Greer SF et al.; Although increasing evidence indicates that there is a direct link between ubiquitination and mono-ubiquitination and transcription in yeast, this link has not been demonstrated in higher eukaryotes . Here we show that the major histocompatibility complex (MHC) class II transactivator (CIITA), which is required for expression of genes encoding MHC class II molecules, is ubiquitinated . This ubiquitination enhanced the association of CIITA with both MHC class II transcription factors and the MHC class II promoter, resulting in an increase in transactivation function and in the expression of MHC class II mRNA . The degree of CIITA ubiquitination was controlled by histone acetylases (HATs) and deacetylases (HDACs), indicating that the crucial cellular processes mediated by these enzymes are linked to regulate transcription . Thus, ubiquitin positively regulates a mammalian coactivator by enhancing its assembly at the promoter.

Mol Cell, 2003 Sep, 12(3), 761 - 74
Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81-Eme1 in meiosis; Osman F et al.; The double Holliday junction (dHJ) is generally regarded to be a key intermediate of meiotic recombination, whose resolution is critical for the formation of crossover recombinants . In fission yeast, the Mus81-Eme1 endonuclease has been implicated in resolving dHJs . Consistent with this role, we show that Mus81-Eme1 is required for generating meiotic crossovers . However, purified Mus81-Eme1 prefers to cleave junctions that mimic those formed during the transition from double-strand break to dHJ . Crucially, these junctions are cleaved by Mus81-Eme1 in precisely the right orientation to guarantee the formation of a crossover every time . These data demonstrate how crossovers could arise without forming or resolving dHJs using an enzyme that is widely conserved amongst eukaryotes.

Mol Cell, 2003 Sep, 12(3), 747 - 59
The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism; Gaillard PH et al.; Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps . Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1 . Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products . Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate . Recombinant Mus81-Eme1 is ineffective at making the first cut . We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.

Annu Rev Biochem, 2003, 72, 783 - 812
Proteomics; Zhu H et al.; Fueled by ever-growing DNA sequence information, proteomics-the large scale analysis of proteins-has become one of the most important disciplines for characterizing gene function, for building functional linkages between protein molecules, and for providing insight into the mechanisms of biological processes in a high-throughput mode . It is now possible to examine the expression of more than 1000 proteins using mass spectrometry technology coupled with various separation methods . High-throughput yeast two-hybrid approaches and analysis of protein complexes using affinity tag purification have yielded valuable protein-protein interaction maps . Large-scale protein tagging and subcellular localization projects have provided considerable information about protein function . Finally, recent developments in protein microarray technology provide a versatile tool to study protein-protein, protein-nucleic acid, protein-lipid, enzyme-substrate, and protein-drug interactions . Other types of microarrays, though not fully developed, also show great potential in diagnostics, protein profiling, and drug identification and validation . This review discusses high-throughput technologies for proteome analysis and their applications . Also discussed are the approaches used for the integrated analysis of the voluminous sets of data generated by proteome analysis conducted on a global scale.

Microbiol Immunol, 2003, 47(8), 601 - 11
Hepatitis C virus nonstructural protein NS3 binds to Sm-D1, a small nuclear ribonucleoprotein associated with autoimmune disease; Iwai A et al.; Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and leads to hepatocellular carcinoma . Nonstructural protein 3 (NS3) of HCV possesses protease, nucleoside triphosphatase, and helicase activities . Using the yeast two hybrid assay, we identified Sm-D1, a host protein that binds to NS3 . Sm-D1 is a component of small nuclear ribonucleoprotein (snRNP) complexes which are associated with autoimmune disease . Sm-D1 has Gly-Arg (GR) repeats at the C terminus, which contains dimethylarginine modified by post-translational modification and may constitute an immunoreactive determinant . Deletion mutants revealed that the C-terminal region of Sm-D1 containing the GR repeats was the binding region for NS3, and the expression feature of Sm-D1 was affected by co-expression of NS3 . Immunostaining assay demonstrated that NS3 was also present in the nucleus of cells overexpressing Sm-D1, although it was usually found in cytoplasm . The localization of NS3 could change following interaction with Sm-D1 and affect the function of Sm-D1.

J Cell Biochem, 2003 Oct 15, 90(3), 573 - 85
Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation; Chan KK et al.; SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY . Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans . A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities . Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems . Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified . We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays . To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter . We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines . The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients . Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter . Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter . While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder .

J Cell Biochem, 2003 Oct 15, 90(3), 534 - 47
Hpr6.6 protein mediates cell death from oxidative damage in MCF-7 human breast cancer cells; Hand RA et al.; Reactive oxygen species (ROS) cause cell death and are associated with a variety of maladies, from trauma and infection to organ degeneration and cancer . Cells mount a complex response to oxidative damage that includes signaling from transmembrane receptors and intracellular kinases . We have analyzed the response to oxidative damage in human breast cancer cells expressing the Hpr6.6 (human membrane progesterone receptor) protein . Although Hpr6.6 is related to a putative progesterone-binding protein, Hpr6.6 is widely expressed in epithelial tissues and shares close homology with a budding yeast damage response protein called Dap1p (damage response protein related to membrane progesterone receptor) . We report here that the Hpr6.6 protein regulates the response to oxidative damage in breast cancer cells . Expression of Hpr6.6 in MCF-7 cells sensitized the cells to death following long-term/low dose or short-term/high dose treatment with hydrogen peroxide . Cell death did not occur through a typical apoptotic mechanism and corresponded with hyperphosphorylation of the Akt and IkappaB proteins . However, inhibition of Akt activation and IkappaB degradation had no effect on Hpr6.6-mediated cell death, suggesting that Hpr6.6 regulates cell death through a novel oxidative damage response pathway . Our work indicates a key regulatory function for Hpr6.6 in epithelial tissues exposed to oxidative damage .

Curr Genet, 2003 Dec, 44(5), 231 - 60 Epub 2003 Oct 02.
Nucleo-cytoplasmic partitioning of proteins in plants: implications for the regulation of environmental and developmental signalling; Merkle T; Considerable progress has been made in the past few years in characterising Arabidopsis nuclear transport receptors and in elucidating plant signal transduction pathways that employ nucleo-cytoplasmic partitioning of a member of the signal transduction chain . This review briefly introduces the major principles of nuclear transport of macromolecules across the nuclear envelope and the proteins involved, as they have been described in vertebrates and yeast . Proteins of the plant nuclear transport machinery that have been identified to date are discussed, the focus being on Importin beta-like nuclear transport receptors . Finally, the importance of nucleo-cytoplasmic partitioning as a regulatory tool for signalling is highlighted, and different plant signal transduction pathways that make use of this regulatory potential are presented.

Cell Mol Life Sci, 2003 Sep, 60(9), 1990 - 7
Differential regulation of the Sir2 histone deacetylase gene family by inhibitors of class I and II histone deacetylases; Kyrylenko S et al.; The Sir2 histone deacetylase gene family consists of seven mammalian sirtuins (SIRTs) which are NAD-dependent histone/protein deacetylases . Sir2 proteins regulate, for instance, genome stability by chromatin silencing in yeast . In mammals, their function is still largely unknown . Due to the NAD+ dependency, Sir2 might be the link between metabolic activity and histone/protein acetylation . Regulation of gene expression also seems to play an important role in Sir2 functions, since increasing the dosage of Sir2 genes increases genome stability in yeast and Caenorhabditis elegans . We observed that the modification of histone/protein acetylation status by several class I and II histone deacetylase (HDAC) inhibitors induces differential changes in gene expression profiles of seven SIRT mRNAs in cultured neuronal cells . SIRT2, SIRT4 and SIRT7 were upregulated, whereas SIRT1, SIRT5 and SIRT6 were downregulated by trichostatin A (TSA) and n-butyrate . The upregulation of SIRT mRNAs was inhibited by actinomycin D . Interestingly, the regulation of SIRT mRNAs was highly similar both in mouse Neuro-2a neuroblastoma cells and post-mitotic rat primary hippocampal and cerebellar granule neurons . Using a chromatin immunoprecipitation technique, we showed that the upregulation of SIRT2 expression with TSA is related to the hyperacetylation of DNA-bound histone H4 within the first 500 bp upstream of the transcription start site of the SIRT2 gene . Chemically different types of HDAC inhibitors, such as TSA, apicidin, SAHA, M344 and n-butyrate induced remarkably similar responses in SIRT1-7 mRNA expression patterns . Differential responses in SIRT mRNA expression profiles indicate that the expression of the Sir2 family of genes is selectively regulated and dependent on histone/protein acetylation status.

J Biol Chem, 2003 Dec 19, 278(51), 51957 - 67 Epub 2003 Oct 01.
Identification and characterization of GCP16, a novel acylated Golgi protein that interacts with GCP170; Ohta E et al.; GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH2-terminal region (positions 137-237) . Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170 . The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed . Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region . Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein . Labeling experiments with {3H}palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys69 and Cys72, accounting for its tight association with the membrane . A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16 . Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface . Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface.

Biochim Biophys Acta, 2003 Oct 1, 1629(1-3), 109 - 13
Molecular characterization of a ribosome-associated Hsp70-homologous gene from Rhizopus nigricans; Cernila B et al.; A ribosome-associated Hsp70-homologous gene (Rnssb-1) was isolated from the genomic library of the filamentous zygomycete fungus Rhizopus nigricans . The nucleotide sequence of a genomic clone encoded the N-terminal part of a protein with high similarity to the yeast SSB ribosome-associated chaperones . The missing 3' end of the gene was obtained by 3' RACE . The Northern blot analysis showed that the Rnssb-1 gene is constitutively expressed and is not induced upon heat shock at 37 degrees C . The primary structure analyses revealed that the coding region of the Rnssb-1 gene is interrupted by at least four introns . Their splicing was not inhibited by exposure of the organism to heat shock as proven by RT-PCR . A Southern blot analysis of R . nigricans genomic DNA confirmed the presence of two additional gene copies of ribosome-associated Hsp70 genes in the fungal genome.

Biochem Biophys Res Commun, 2003 Oct 17, 310(2), 398 - 404
Apoptosis-associated tyrosine kinase is a Cdk5 activator p35 binding protein; Honma N et al.; A 3(')-terminal fragment of a splice variant of KIAA0641, a human homologue of apoptosis-associated tyrosine kinase (AATYK), was screened from human brain cDNA libraries by a yeast two-hybrid system using a Cdk5 activator p35 as a bait . The cloned cDNA encoded 477 amino acids, composed of internal 458 amino acids of KIAA0641 and 19 amino acids unique to this variant after splicing, then referred to this clone as hAATYKs-p35BP (human AATYK short isoform-p35 binding polypeptide) . Using GST-fusion protein, hAATYKs-p35BP was shown to bind to Cdk5/p35 in a rat brain extract . hAATYKs made by fusing the kinase domain of KIAA0641 to the N-terminus of hAATYKs-p35BP was used for binding to Cdk5/p35 in HEK293 cells . Both hAATYKs and KIAA0641 bound to and were phosphorylated by Cdk5/p35 . These results suggest that both isoforms of hAATYK are novel Cdk5/p35-binding and substrate proteins.

Nucl Recept . 2003 Sep 6;1(1):6.
PLZF is a negative regulator of retinoic acid receptor transcriptional activity; Martin PJ et al.; BACKGROUND: Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation . Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment . Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner . RESULTS: We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay . We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR . The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment . The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF . A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor . We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells . CONCLUSION: Thus RAR and PLZF interact physically and functionally . Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins . Our observations therefore define a novel mechanism by which RARs activity may be controlled.

Planta, 2003 Jul, 217(3), 507 - 16 Epub 2003 Mar 18.
Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana; Smith MA et al.; Expression of a cDNA encoding the castor bean ( Ricinus communis L.) oleate Delta12-hydroxylase in the developing seeds of Arabidopsis thaliana (L.) Heynh . results in the synthesis of four novel hydroxy fatty acids . These have been previously identified as ricinoleic acid (12-hydroxy-octadec- cis-9-enoic acid: 18:1-OH), densipolic acid (12-hydroxy-octadec- cis-9,15-enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos- cis-11-enoic acid: 20:1-OH) and auricolic acid (14-hydroxy-eicos- cis-11,17-enoic acid: 20:2-OH) . Using mutant lines of Arabidopsis that lack the activity of the FAE1 condensing enzyme or FAD3 ER Delta-15-desaturase, we have shown that these enzymes are required for the synthesis of C20 hydroxy fatty acids and polyunsaturated hydroxy fatty acids, respectively . Analysis of the seed fatty acid composition of transformed plants demonstrated a dramatic increase in oleic acid (18:1) levels and a decrease in linoleic acid (18:2) content correlating to the levels of hydroxy fatty acid present in the seed . Plants in which FAD2 (ER Delta12-desaturase) activity was absent showed a decrease in 18:1 content and a slight increase in 18:2 levels corresponding to hydroxy fatty acid content . Expression of the castor hydroxylase protein in yeast indicates that this enzyme has a low level of fatty acid Delta12-desaturase activity . Lipase catalysed 1,3-specific lipolysis of triacylglycerol from transformed plants demonstrated that ricinoleic acid is not excluded from the sn-2 position of triacylglycerol, but is the only hydroxy fatty acid present at this position.

Planta, 2003 Jul, 217(3), 457 - 65 Epub 2003 Mar 14.
Plant-specific regulation of replication protein A2 (OsRPA2) from rice during the cell cycle and in response to ultraviolet light exposure; Marwedel T et al.; DNA replication is a process that is highly conserved among eukaryotes . Nonetheless, little is known about the proteins involved in it in plants . Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein with several functions in DNA metabolism in humans and yeast and supposedly also in plants . Here we report on the regulation of OsRPA2, the 32-kDa subunit of RPA from rice ( Oryza sativa L.) . We found conserved regulation mechanisms at the level of gene expression between animal and plant RPA2 genes and distinct features of OsRPA2 regulation at the level of protein expression . Unlike in animals or in yeast, protein abundance in rice was regulated in a cell cycle phase-specific manner and was altered after UV-C light exposure . On the other hand, posttranslational modification through phosphorylation did not appear to play a pivotal role in rice as it does in animal cells . Our results indicate that plant-specific mechanisms of regulation have evolved for RPA2 within the generally well-conserved process of DNA replication, suggesting specific requirements for regulation of DNA metabolism in plants as compared to other eukaryotes.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12183 - 8 Epub 2003 Sep 30.
Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo; Hoek M et al.; De novo chromatin assembly maintains histone density on the daughter strands in the wake of the replication fork . The heterotrimer chromatin assembly factor 1 (CAF-1) couples DNA replication to histone deposition in vitro, but is not essential for yeast cell proliferation . Depletion of CAF-1 in human cell lines demonstrated that CAF-1 was required for efficient progression through S-phase . Cells lacking CAF-1 accumulated in early and mid S-phase and replicated DNA slowly . The checkpoint kinase Chk1, but not Chk2, was phosphorylated in response to CAF-1 depletion, consistent with a DNA replication defect . CAF-1-depleted cell extracts completely lacked DNA replication-coupled chromatin assembly activity, suggesting that CAF-1 is required for efficient S-phase progression in human cells . These results indicate that, in contrast to yeast, human CAF-1 is necessary for coupling chromatin assembly with DNA replication.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12414 - 9 Epub 2003 Sep 30.
Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins; Martin-Serrano J et al.; The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs . PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I) . However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I . Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein-protein interactions . In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1 . Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner . Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function . Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.

J Biol Chem, 2003 Dec 12, 278(50), 50682 - 90 Epub 2003 Sep 30.
Group I metabotropic glutamate receptors bind to protein phosphatase 1C . Mapping and modeling of interacting sequences; Croci C et al.; The modulation of neurotransmitter receptors by kinases and phosphatases represents a key mechanism in controlling synaptic signal transduction . However, molecular determinants involved in the specific targeting and interactions of these enzymes are largely unknown . Here, we identified both catalytic gamma-isoforms of protein phosphatase 1C (PP1gamma1 and PP1gamma2) as binding partners of the group I metabotropic glutamate receptors type 1a, 5a, and 5b in yeast cells and pull-down assays, using recombinant and native protein preparations . The tissue distribution of interacting proteins was compared, and protein phosphatase 1C was detected in dendrites of retinal bipolar cells expressing the respective interacting glutamate receptors . We mapped interacting domains within binding partners and identified five amino acids in the intracellular C termini of the metabotropic glutamate receptors type 1a, 5a, 5b, and 7b being both necessary and sufficient to bind protein phosphatase 1C . Furthermore, we show a dose-dependent competition of these C termini in binding the enzyme . Based on our data, we investigated the structure of the identified amino acids bound to protein phosphatase 1C by homology-based molecular modeling . In summary, these results provide a molecular description of the interaction between protein phosphatase 1C and metabotropic glutamate receptors and thereby increase our understanding of glutamatergic signal transduction.

J Natl Cancer Inst, 2003 Oct 1, 95(19), 1477 - 81
Selenium supplementation and secondary prevention of nonmelanoma skin cancer in a randomized trial; Duffield-Lillico AJ et al.; The Nutritional Prevention of Cancer Trial was a double-blind, randomized, placebo-controlled clinical trial designed to test whether selenium as selenized yeast (200 microg daily) could prevent nonmelanoma skin cancer among 1312 patients from the Eastern United States who had previously had this disease . Results from September 15, 1983, through December 31, 1993, showed no association between treatment and the incidence of basal and squamous cell carcinomas of the skin . This report summarizes the entire blinded treatment period, which ended on January 31, 1996 . The association between treatment and time to first nonmelanoma skin cancer diagnosis and between treatment and time to multiple skin tumors overall and within subgroups, defined by baseline characteristics, was evaluated . Although results through the entire blinded period continued to show that selenium supplementation was not statistically significantly associated with the risk of basal cell carcinoma (hazard ratio {HR} = 1.09, 95% confidence interval {CI} = 0.94 to 1.26), selenium supplementation was associated with statistically significantly elevated risk of squamous cell carcinoma (HR = 1.25, 95% CI = 1.03 to 1.51) and of total nonmelanoma skin cancer (HR = 1.17, 95% CI = 1.02 to 1.34) . Results from the Nutritional Prevention of Cancer Trial conducted among individuals at high risk of nonmelanoma skin cancer continue to demonstrate that selenium supplementation is ineffective at preventing basal cell carcinoma and that it increases the risk of squamous cell carcinoma and total nonmelanoma skin cancer.

Prostaglandins Other Lipid Mediat, 2003 Jul, 71(3-4), 85 - 96
The distribution of 3-hydroxy oxylipins in fungi; Kock JL et al.; One of the best-kept secrets by fungi especially yeast is the function of the different shapes and surface structures of their vegetative and sexual cells . They definitely do not produce these shapes (e.g . round, elongated, kidney, needle, hat, saturnoid, etc.) and surfaces (e.g . smooth, rough, hairy, warty, etc.) for our curiosity or to be classified, but surely produce these for their own benefit . This mini-review will show that a large variety of 3-hydroxy oxylipins are widely distributed in the fungal domain and closely associated with these surface ornamentations . In concert with nano-scale surface structures, they probably play a role in cell aggregation as well as spore release from sexual structures such as asci.

Electrophoresis, 2003 Sep, 24(18), 3289 - 95
Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection; Mao Y et al.; A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping . cRPLC, the first dimension, provided high-resolution separations for salt-free proteins . CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment . Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension . Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest . A peak capacity of more than 10 000 had been achieved . A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest . FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.

Hum Mutat, 2003 Nov, 22(5), 420 - 1
Identification of a frequent variant in ALG6, the cause of Congenital Disorder of Glycosylation-Ic; Westphal V et al.; Congenital Disorder of Glycosylation (CDG) type Ic is caused by mutations in ALG6 . This gene encodes an alpha1,3 glucosyltransferase used for synthesis of the lipid linked oligosaccharide (LLO) precursor of the protein N-glycosylation pathway . CDG-Ic patients have moderate to severe psychomotor retardation, seizures, hypotonia, strabismus, and feeding difficulties . We previously identified a typical patient with a heterozygous point mutation, c.391T>C (p.Tyr131His) in ALG6 . Using complementation analysis of ALG6-deficient yeast, we show that this alteration is as severe as the most common disease-causing mutation, c998C>T (p . Ala333Val), which occurs in over half of all known CDG-Ic patients . The frequency of c.391T>C (p.Tyr131His) in the US population, is 0.0214, suggesting that homozygotes would occur at a rate of& tilde;1:2,200 . We identified one patient with typical CDG-Ic symptoms and a homozygous p.Tyr131His alteration in ALG6 . However, in contrast to most CDG patients, her LLO and plasma transferrin glycosylation appeared normal . Thus, it is unclear whether c.391T>C causes CDG-Ic or contributes to the symptoms . Genotyping additional patients with CDG-like symptoms will be required to resolve this issue .

Mol Cell Biol, 2003 Oct, 23(20), 7271 - 84
Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex; Enninga J et al.; Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC) . At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown . We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13 . By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites . In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus . Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs . Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec13, demonstrating the interaction of Sec13 and Nup96 in vivo . Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal . These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.

EMBO J, 2003 Oct 1, 22(19), 5208 - 19
Regulation of poly(A) polymerase by 14-3-3epsilon; Kim H et al.; Poly(A) polymerase (PAP) is a key enzyme responsible for the addition of the poly(A) at the 3' end of pre-mRNA . The C-terminal region of mammalian PAP carries target sites for protein-protein interaction with the 25 kDa subunit of cleavage factor I and with splicing factors U1A and U2AF65 . We used a yeast two-hybrid screen to identify 14-3-3epsilon as an additional protein binding to the C-terminal region of PAP . Interaction between PAP and 14-3-3epsilon was confirmed by both in vitro and in vivo binding assays . This interaction is dependent on PAP phosphorylation . Deletion analysis of PAP suggests that PAP contains multiple binding sites for 14-3-3epsilon . The binding of 14-3-3epsilon to PAP inhibits the polyadenylation activity of PAP in vitro, and overexpression of 14-3-3epsilon leads to a shorter poly(A) mRNA tail in vivo . In addition, the interaction between PAP and 14-3-3epsilon redistributes PAP within the cell by increasing its cytoplasmic localization . These data suggest that 14-3-3epsilon is involved in regulating both the activity and the nuclear/ cytoplasmic partitioning of PAP through the phosphorylation-dependent interaction.

Virology, 2003 Sep 15, 314(1), 423 - 31
The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity; Prasad CK et al.; Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection . Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP . c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development . Skin is the natural host tissue for both HPV and AAV . In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift) . Addition of anti-Rep78 antibodies inhibited the EMSA supershift . Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays . Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts . Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78 . The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process.

Mech Dev, 2002 Dec, 119 Suppl 1, S253 - 60
Identification and expression of Ima, a novel Ral-interacting Drosophila protein; Beller M et al.; We report the identification of Ima, a novel Drosophila MAGUK-like protein, which contains two WW and four PDZ protein interaction domains and interacts with the small GTPase dRal in the yeast two-hybrid system and pull-down assays . The gene is expressed in distinct spatiotemporal patterns throughout embryonic development . Overexpression of Ima interferes with normal Drosophila development, indicating that the gene functions in a tissue specific manner.

Biol Chem, 2003 Sep, 384(9), 1293 - 8
Phosphatidylinositol-3,5-Bisphosphate is a potent and selective inhibitor of acid sphingomyelinase; Kolzer M et al.; Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide . Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin . Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation . Here, we present a potent and selective novel inhibitor of A-SMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2), a naturally occurring substance detected in mammalian, plant and yeast cells . The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P2 is 0.53 microM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells . Even at concentrations of up to 50 microM, PtdIns3,5P2 neither decreased plasma membrane-associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase . Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase . Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition . Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.

Rapid Commun Mass Spectrom, 2003, 17(19), 2177 - 87
Closely spaced external standard: a universal method of achieving 5 ppm mass accuracy over the entire MALDI plate in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Moskovets E et al.; Close deposition of the sample and external standard was used in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to achieve mass accuracy equivalent to that obtained with an internal standard across the entire MALDI plate . In this work, the sample and external standard were deposited by continuous deposition in separate traces, each approximately 200 micro m wide . The dependence of the mass accuracy on the distance between the sample and standard traces was determined across a MALDI target plate with dimensions of 57.5 mm x 57.0 mm by varying the gap between the traces from 100 micro m to 4 mm . During acquisition, two adjacent traces were alternately irradiated with a 200-Hz laser, such that the peaks in the resulting mass spectra combined the sample and external standard . Ion suppression was not observed even when the peptide concentrations in the two traces differed by more than two orders of magnitude . The five peaks from the external standard trace were used in a four-term mass calibration of the masses of the sample trace . The average accuracy across the whole plate with this method was 5 ppm when peaks of the sample trace had signal-to-noise ratios of at least 30 and the gap between the traces was approximately 100 micro m . This approach was applied to determining peptide masses of a reversed-phase liquid chromatographic (LC) separation of a tryptic digest of beta-galactosidase deposited as a long serpentine trace across the MALDI plate, with accuracy comparable to that obtainable using internal calibration . In addition, the eluent from reversed-phase LC separation of a strong cation-exchange fraction containing tryptic peptides from a yeast lysate along with the closely placed external standard was deposited on the MALDI plate . The data obtained in the MS and MS/MS modes on a MALDI-TOF/TOF mass spectrometer were combined and used in database searching with MASCOT . Since the significant score is a function of mass accuracy in the MS mode, database searching with high mass accuracy reduced the number of false positives and also added peptides which otherwise would have been eliminated at lower mass accuracy (false negatives) .

Rapid Commun Mass Spectrom, 2003, 17(19), 2163 - 7
Probing cell chemistry with time-of-flight secondary ion mass spectrometry: development and exploitation of instrumentation for studies of frozen-hydrated biological material; Cliff B et al.; Imaging static secondary ion mass spectrometry (SIMS) offers a powerful method of obtaining molecular information from biological systems with good spatial resolution . However, the technique needs further development to make it suitable for routine analysis of cells . We report here the development of a new freeze-facture device to facilitate the manipulation and analysis of biological cell material, with the cell chemistry preserved intact by rapid freezing . We illustrate performance characteristics with high-contrast images of freeze-fractured, frozen-hydrated liposomes with the drug clofazamine constrained within the lipid bilayer providing a marker to determine the fracture plane across the liposome structure . By monitoring and imaging clofazamine on the surface of yeast cells in the frozen-hydrated state, and demonstrating its absence within molecular information from a cell fractured to reveal the cell ultrastructure, we demonstrate that the molecule does not penetrate the cell wall .

Acta Biochim Pol, 2003, 50(3), 857 - 64
Using capillary electrophoresis to study methylation effect on RNA-peptide interaction; Mucha P et al.; Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation . Its functional signification is only beginning to be understood . A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented . Two RNA-peptide systems were chosen for the study . The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA(Phe) anticodon stem and loop domain (ASL(Phe)) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL) . A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA . In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL(Phe) shifted from 18.16 to 20.90 min . Formation of the complex was not observed when an unmethylated version of ASL(Phe) was used . The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)) . In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed . Methylation of a residue Arg(52)-->Arg(Me)(2), crucial for TAR binding, strongly disrupted formation of the complex . Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed . CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Cell Biochem Biophys, 2003, 39(2), 119 - 32
Distinct domains of human CDC5 direct its nuclear import and association with the spliceosome; Liu L et al.; Genetic studies have shown that CDC5 proteins are essential for G2 progression and mitotic entry . CDC5 homologs in yeast and mammals are essential for pre-messenger ribonucleic acid (mRNA) processing . Other gene products also have been shown to play roles in both pre-mRNA splicing and cell cycle regulation, prompting the description of several models to explain the mechanism(s) linking these two processes . In this study, we demonstrate that the amino-terminus of human CDC5 directs nuclear import independent of consensus nuclear localization signals or phosphorylation, and that the carboxyl-terminus of human CDC5 preferentially associates with spliceosomal complexes in proximity of RNA transcription during interphase . hCDC5 colocalizes with Sm proteins in a cell cycle- and domain-dependent manner, and several proteins in the human CDC5-associated complex are identified that suggest potential roles for the complex in coupling pre-mRNA splicing to transcriptional activation and protein translation . These results indicate that human CDC5 may function in pre-mRNA processing and cell cycle progression through more than one mechanism.

Carcinogenesis, 2004 Jan, 25(1), 61 - 7 Epub 2003 Sep 26.
Induction of cytochrome P450, generation of oxidative stress and in vitro cell-transforming and DNA-damaging activities by glucoraphanin, the bioprecursor of the chemopreventive agent sulforaphane found in broccoli; Paolini M et al.; The reduced cancer risk that appears to be linked to a diet rich in fruits and vegetables has fueled the belief that regular intake of isolated phytochemicals could potentially prevent cancer . In recent years, the glucosinolate metabolites derived from cruciferous vegetables, such as the isothiocyanate sulforaphane in broccoli, have gained much attention as potential cancer chemopreventive agents . The protective effect of sulforaphane, which is liberated from its glucosinolate precursor glucoraphanin (GRP) by myrosinase hydrolysis, is conventionally thought to involve the induction of Phase-II metabolizing enzymes . These Phase-II enzymes are implicated in the detoxication of many carcinogens and reactive oxygen species (ROS), thereby protecting cells against DNA damage and subsequent malignant transformation . While the induction of Phase-II enzymes is usually considered beneficial, in some cases these enzymes also bioactivate several hazardous chemicals . Furthermore, despite its projected benefits, the unknown effect of sulforaphane on Phase-I enzyme systems, which are involved in the bioactivation of a variety of carcinogens, should not be overlooked . Here we show that, in rat lungs, while GRP, the bioprecursor of the chemopreventive agent sulforaphane, slightly induced Phase-II detoxifying enzymes, it powerfully induced Phase-I carcinogen-activating enzymes, including activators of carcinogenic polycyclic aromatic hydrocarbons (PAHs) . Concomitant with this Phase-I induction, GRP also over-generated ROS . Additionally, in a cell-transforming assay, GRP facilitated the metabolic activation of the PAH benzo{a}pyrene to reactive carcinogenic forms and in a yeast genotoxicity test it damaged DNA . This suggests that regular administration of GRP could actually increase rather than decrease cancer risk, especially in individuals exposed to environmental mutagens and carcinogens such as those found in tobacco smoke and in certain industrial settings.

Mol Genet Genomics, 2003 Sep, 269(6), 789 - 96 Epub 2003 Jul 24.
Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O . novo-ulmi; Tadesse Y et al.; Cerato-ulmin is a surface protein that belongs to the class of fungal proteins known as hydrophobins . This class II hydrophobin is produced throughout the life cycle and in all developmental stages of Ophiostoma novo-ulmi and O . ulmi; the aggressive and non-aggressive pathogens responsible for Dutch elm disease . Since yeast/mycelial transitions are often important to pathogenesis in dimorphic fungi such as Ophiostoma, we have examined the levels and abundance of cu mRNA in the yeast and mycelial stages of this fungus . The fungus contains one copy of the cu gene per haploid genome, located on chromosome IV . Our studies have been done using phosphoimager-based Northern analysis and real-time quantitative RT-PCR (qRT-PCR) to measure levels of cu mRNA . These measurements were made in both yeast-like and mycelial stages of the pathogen . Two wild-type, aggressive, strains of O . novo-ulmi (VA30 and H327) and one wild type non-aggressive strain of O . ulmi (H5) were analysed . As controls, we have utilized two types of mutants that we had previously generated, the null cu mutants THEK5-8 and THEK5-8-1, and a cu over-expression mutant, H5-tf16 . Data generated by both Northern hybridization and real-time qRT-PCR analyses demonstrate that there is no cu mRNA transcription in the null mutants . The Northern analysis clearly showed that the over-expressing mutant H5-tf16 produces much more cu mRNA than the non-aggressive or aggressive strains . The quantitative data generated using qRT-PCR demonstrated that mycelium generally had 20-60% more cu mRNA than the yeast form . The non-aggressive strain of O . ulmi (H5) produces one-tenth as much cu mRNA as the aggressive strains (VA30 and H327) . When transformed with additional copies of the cu gene, this same non-aggressive strain (H5-tf16) expressed about 20 times more cu mRNA than the wild type H5 strain . These data were consistently generated in multiple real-time qRT-PCR experiments with different RNA preparations, clearly demonstrating that the quantitative abundance values obtained were reproducible . Our study represents the first report on the use of real-time qRT-PCR to compare and quantify gene transcription in different growth phases of a fungal pathogen.

Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 14 - 8
The cyclin-dependent kinase 11(p46) isoform interacts with RanBPM; Mikolajczyk M et al.; We identified Ran-binding protein (RanBPM) as an interacting partner of the caspase-processed C-terminal domain of cyclin-dependent kinase 11 (CDK11(p46)) by using the yeast two-hybrid system . CDK11(p110) protein kinases are members of the cyclin-dependent kinase superfamily . During staurosporine-, Fas-, and tumor necrosis factor alpha-induced apoptosis caspase-processed activated CDK11(p46) is generated from larger CDK11(p110) isoforms . CDK11(p46) promotes apoptosis when it is ectopically expressed in human cells . However, the mechanism of signal transduction through CDK11(p46) is still unclear . In this study, we demonstrate that CDK11(p46) directly interacts with RanBPM in vitro and in human cells . RanBPM contains a conserved SPRY (repeats in splA and Ryr) domain and is localized both in the nucleus and cytoplasm . The SPRY domain of RanBPM is responsible for the association between CDK11(p46) and RanBPM . Furthermore, we show that CDK11(46) phosphorylates RanBPM.

J Biol . 2003;2(4):28 . Epub 2003 Sep 24.
Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade; Hawley SA et al.; BACKGROUND: The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation . Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified . Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK . Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome . We recently showed that LKB1 exists as a complex with two accessory subunits, STRAD alpha/beta and MO25 alpha/beta . RESULTS: We report the following observations . First, two AMPKK activities purified from rat liver contain LKB1, STRAD alpha and MO25 alpha, and can be immunoprecipitated using anti-LKB1 antibodies . Second, both endogenous and recombinant complexes of LKB1, STRAD alpha/beta and MO25 alpha/beta activate AMPK via phosphorylation of Thr172 . Third, catalytically active LKB1, STRAD alpha or STRAD beta and MO25 alpha or MO25 beta are required for full activity . Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1 . Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos . CONCLUSIONS: These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK . Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

Nucleic Acids Res Suppl, 2003, (3), 299 - 300
Utilization efficiency of the oxidized purine nucleotide analogs by DNA polymerase eta; Nishimoto N et al.; To elucidate the behavior of DNA polymerase eta against the oxidized purine nucleotides, we determined the utilization efficiency of 2-hydroxy-dATP and 8-hydroxy-dGTP by the recombinant yeast DNA polymerase eta using the primer extension assay with the synthetic oligonucleotide template-primers, and compared those by DNA polymerase alpha . Results indicate that DNA polymerase eta incorporates 2-hydroxy-dATP opposite template G in addition to template T and 8-hydroxy-dGTP opposite A in addition to C, respectively . Kinetic analysis revealed that the rate of mutation caused by 2-OH-dATP and 8-OH-dGTP with DNA polymerase eta should be much higher than those with DNA polymerase alpha.

J Prosthodont, 2003 Sep, 12(3), 162 - 7
Clinical evaluation of resilient denture liners . Part 2: Candida count and speciation; Brosky ME et al.; PURPOSE: The purpose of this study was to count and to speciate Candida isolated from 2 resilient denture liners, Molloplast-B and MPDS-SL . MATERIALS AND METHODS: A group of 20 patients each had 1 maxillary denture and 2 mandibular dentures fabricated . One mandibular denture was lined with Molloplast-B, and 1 was lined with MPDS-SL . Each denture was used for 3 months . At the end of the 3-month period, the mandibular denture was surrendered, and a 5 x 5-mm circular resilient liner sample was obtained from the tissue surface of the lingual flange . Samples were processed, and Candida was isolated and counted . Speciation of Candida was performed using CHROMagar Candida and API 20C AUX strips . RESULTS: Molloplast-B had, on average, 5 times as many CFU/sample as MPDSL-SL, but this difference was not significant (p = 0.26) . A sign test gave a similar nonsignificant trend (p = 0.057) . CHROMagar identified several Candida species, and confirmation was made using API 20C AUX strips . One patient was lost to follow-up . Of 19 Molloplast-B samples, 7 had no growth, 4 grew C . albicans, 3 grew C . parapsilosis, 2 grew C . glabrata, 1 grew C . tropicalis, 2 grew a Trichosporon spp., and 2 grew a nonidentifiable colony . The analogous counts for 19 MPDS-SL samples were 10, 4, 1, 3, 0, 1, and 1 (p = 0.45 for culture positively, exact McNemar test) . CONCLUSIONS: Candida growth on Molloplast-B was not significantly different from growth on MPDS-SL . Several yeast species were cultured from each material . The rates of culture-positive testing did not differ between the 2 resilient denture liners.

Plant Cell, 2003 Oct, 15(10), 2370 - 82 Epub 2003 Sep 24.
The Arabidopsis anaphase-promoting complex or cyclosome: molecular and genetic characterization of the APC2 subunit; Capron A et al.; In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins . In plants, the function, regulation, and substrates of the APC/C are poorly understood . To gain more insight into the roles of the plant APC/C, we characterized at the molecular level one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis . We show that the Arabidopsis gene is able to partially complement a budding yeast apc2 ts mutant . By yeast two-hybrid assays, we demonstrate an interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23 . A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene . apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin-beta-glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems . The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated . Finally, we report intriguing differences in APC2 protein subcellular localization compared with that in other systems . Our observations support a conserved function of the APC/C in plants but a different mode of regulation.

Plant Cell, 2003 Oct, 15(10), 2308 - 19 Epub 2003 Sep 24.
A nuclear protease required for flowering-time regulation in Arabidopsis reduces the abundance of SMALL UBIQUITIN-RELATED MODIFIER conjugates; Murtas G et al.; The Arabidopsis mutant early in short days4 (esd4) shows extreme early flowering and alterations in shoot development . We have identified ESD4 and demonstrate that it encodes a nuclear protein located predominantly at the periphery of the nucleus . ESD4 contains a segment of >200 amino acids with strong similarity to yeast and animal proteases that are specific for the protein modifier SMALL UBIQUITIN-RELATED MODIFIER (SUMO) . ESD4 shows a similar function to these proteases in vitro and processes the precursor of Arabidopsis SUMO (AtSUMO) to generate the mature form . This activity of ESD4 is prevented by mutations that affect the predicted active site of the protease or the cleavage site of the AtSUMO precursor . In yeast, these proteases also recycle SUMO from conjugates, and this appears to be the major role of ESD4 in vivo . This is suggested because esd4 mutants contain less free AtSUMO and more SUMO conjugates than wild-type plants, and a transgene expressing mature SUMO at high levels enhanced aspects of the esd4 phenotype . ESD4 defines an important role for protein modification by AtSUMO in the regulation of flowering.

J Biol Chem, 2003 Dec 5, 278(49), 49348 - 57 Epub 2003 Sep 24.
GIPC binds to the human lutropin receptor (hLHR) through an unusual PDZ domain binding motif, and it regulates the sorting of the internalized human choriogonadotropin and the density of cell surface hLHR; Hirakawa T et al.; By using a yeast two-hybrid screen we identified GIPC (GAIP-interacting protein C terminus), a protein with a type I PDZ domain as a novel human lutropin receptor (hLHR) binding partner . Pull-down and immunoprecipitation assays confirmed this interaction and showed that it is dependent on the PDZ domain of GIPC and the C-terminal tetrapeptide of the hLHR . To characterize the functional consequences of the GIPC-hLHR interaction, we used a small interfering RNA against GIPC to generate a clonal cell line that is deficient in GIPC . Studies with this cell line reveal that GIPC is partially responsible for the recycling of the hormone that is internalized by the hLHR and also for maintaining a relatively constant level of hLHR at the cell surface during hormone internalization.

Am J Pathol, 2003 Oct, 163(4), 1633 - 44
A human-mouse chimera of the alpha3alpha4alpha5(IV) collagen protomer rescues the renal phenotype in Col4a3-/- Alport mice; Heidet L et al.; Collagen IV is a major structural component of basement membranes . In the glomerular basement membrane (GBM) of the kidney, the alpha3, alpha4, and alpha5(IV) collagen chains form a distinct network that is essential for the long-term stability of the glomerular filtration barrier, and is absent in most patients affected with Alport syndrome, a progressive inherited nephropathy associated with mutation in COL4A3, COL4A4, or COL4A5 genes . To investigate, in vivo, the regulation of the expression, assembly, and function of the alpha3alpha4alpha5(IV) protomer, we have generated a yeast artificial chromosome transgenic line of mice carrying the human COL4A3-COL4A4 locus . Transgenic mice expressed the human alpha3 and alpha4(IV) chains in a tissue-specific manner . In the kidney, when expressed onto a Col4a3(-/-) background, the human alpha3(IV) chain restored the expression of and co-assembled with the mouse alpha4 and alpha5(IV) chains specifically at sites where the human alpha3(IV) was expressed, demonstrating that the expression of all three chains is required for network assembly . The co-assembly of the human and mouse chains into a hybrid network in the GBM restores a functional GBM and rescues the Alport phenotype, providing further evidence that defective assembly of the alpha3-alpha4-alpha5(IV) protomer, caused by mutations in any of the three chains, is the pathogenic mechanism responsible for the disease . This line of mice, humanized for the alpha3(IV) collagen chain, will also provide a valuable model for studying the pathogenesis of Goodpasture syndrome, an autoimmune disease caused by antibodies against this chain.

Am J Pathol, 2003 Oct, 163(4), 1395 - 404
Down-regulation of a novel actin-binding molecule, skeletrophin, in malignant melanoma; Takeuchi T et al.; In the present study, we cloned and characterized a novel actin-binding molecule, designated skeletrophin, from aggregated neuroblastoma cells . The putative amino acid sequence of human skeletrophin cDNA contained a cysteine-rich zinc-finger motif which was also found in dystrophin and five ankyrin repeats . Northern blot analysis revealed that the 3.2-kb skeletrophin mRNA was expressed in normal skeletal muscle, and to a lesser extent in heart, brain, and kidney . Specific antibody was prepared to human skeletrophin peptide, and a single protein band with an approximate molecular weight of 70 kd was detected in tissue extracts by immunoblotting using the antibody . To better understand the biological properties of skeletrophin, we used a yeast two-hybrid system to screen for molecules interacting with skeletrophin and found that skeletrophin bound to actin monomer . Co-immunoprecipitation experiments also demonstrated that skeletrophin was able to bind to actin monomer . Fluorescence in situ hybridization mapped the skeletrophin gene on human chromosome 1p36.2-36.3, in which putative tumor suppressor genes for malignant melanoma have been postulated to exist . We therefore immunohistochemically stained benign nevi and malignant melanoma tissues . Notably, 23 of 25 benign nevi expressed skeletrophin in cytoplasm, but 18 of 38 cases of primary skin melanoma appeared to lack skeletrophin expression . Treatment with a demethylating agent, 5'-aza-2-deoxycytidine, restored skeletrophin expression in cultured Mewo melanoma cells . The present findings suggest that skeletrophin may be a novel actin-binding cytoskeleton-related molecule, expression of which is silenced in a considerable number of melanoma specimens.

J Anim Physiol Anim Nutr (Berl), 2003 Oct, 87(9-10), 315 - 23
Determination of the selenium requirement in kittens; Wedekind KJ et al.; The purpose of this study was to determine the selenium (Se) requirement in kittens . Thirty-six specific-pathogen-free kittens (9.8 weeks old) were utilized in a randomized complete block design to determine the Se requirement in cats with gender and weight used as blocking criteria . Kittens were fed a low Se (0.02 mg/kg Se) torula yeast-based diet for 5 weeks (pre-test) after which an amino acid-based diet (0.027 mg Se/kg diet) was fed for 8 weeks (experimental period) . Six levels of Se (0, 0.05, 0.075, 0.10, 0.20 and 0.30 mg Se/kg diet) as Na2SeO3 were added to the diet and were used to construct a response curve . Response variables included Se concentrations and Se-dependent glutathione peroxidase activities (GSHpx) in plasma and red blood cells (RBC) as well as plasma total T3 (TT3) and total T4 (TT4) . No significant changes in food intake, weight gain or clinical signs of Se deficiency were noted . Estimates of the kitten's Se requirement (i.e . breakpoints) were determined for RBC and plasma GSHpx (0.12 and 0.15 mg Se/kg diet, respectively), but no definitive breakpoint was determined for plasma Se . Plasma TT3 increased linearly, whereas plasma TT4 and the ratio of TT4 : TT3 decreased in a quadratic fashion to dietary Se concentration . The requirement estimate determined in this study (0.15 mg Se/kg) for kittens is in close agreement with other species . As pet foods for cats contain a high proportion of animal protein with a Se bioavailability of 30%, it is recommended that commercial diets for cats contain 0.5 mg Se/kg DM.

Eur J Clin Nutr, 2003 Oct, 57(10), 1254 - 61
Lower glucose-dependent insulinotropic polypeptide (GIP) response but similar glucagon-like peptide 1 (GLP-1), glycaemic, and insulinaemic response to ancient wheat compared to modern wheat depends on processing; Bakhoj S et al.; OBJECTIVE: To test the hypothesis that bread made from the ancient wheat Einkorn (Triticum monococcum) reduces the insulin and glucose responses through modulation of the gastrointestinal responses of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) compared to the responses to bread of modern wheat (Triticum aestivum) . DESIGN: The 3-h postprandial insulinaemic, glycaemic, GIP, and GLP-1 responses to bread made from Einkorn were compared to responses to a traditional Danish wheat loaf . The bread from Einkorn was prepared by 3 different processing methods: leavening with honey-salt added, leavening crushed whole grain, and conventional leavening with yeast added . Bread made from modern wheat was prepared by conventional leavening with yeast added . SUBJECTS: A total of 11 healthy young men . RESULTS: The postprandial GIP response was significantly (P<0.001) reduced by the Einkorn breads processed with honey-salt leavening and by using crushed whole grain bread compared to the yeast leavened bread made from modern wheat or from Einkorn . No significant differences were found in the responses of GLP-1, insulin or glucose . CONCLUSION: Einkorn honey-salt leavened and Einkorn whole grain bread elicit a reduced gastrointestinal response of GIP compared to conventional yeast bread . No differences were found in the glycaemic, insulinaemic and GLP-1 responses . Processing of starchy foods such as wheat may be a powerful tool to modify the postprandial GIP response.

J Biol Chem, 2003 Dec 19, 278(51), 51484 - 93 Epub 2003 Sep 23.
Myosin phosphatase-Rho interacting protein . A new member of the myosin phosphatase complex that directly binds RhoA; Surks HK et al.; Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone . Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis . Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction . Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively . G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase . We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA . This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments . M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins . An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner . M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells . M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.

J Biol Chem, 2003 Dec 19, 278(51), 51504 - 14 Epub 2003 Sep 23.
Siah-1 facilitates ubiquitination and degradation of synphilin-1; Nagano Y et al.; Parkinson's disease is a common neurodegenerative disorder characterized by loss of dopaminergic neurons and appearance of Lewy bodies, cytoplasmic inclusions that are highly enriched with ubiquitin . Synphilin-1, alpha-synuclein, and Parkin represent the major components of Lewy bodies and are involved in the pathogenesis of Parkinson's disease . Synphilin-1 is an alpha-synuclein-binding protein that is ubiquitinated by Parkin . Recently, a mutation in the synphilin-1 gene has been reported in patients with sporadic Parkinson's disease . Although synphilin-1 localizes close to synaptic vesicles, its function remains unknown . To investigate the proteins that interact with synphilin-1, the present study performed a yeast two-hybrid screening and identified a novel interacting protein, Siah-1 ubiquitin ligase . Synphilin-1 and Siah-1 proteins were endogenously expressed in the central nervous system and were found to coimmunoprecipitate each other in rat brain homogenate . Confocal microscopic analysis revealed colocalization of both proteins in cells . Siah-1 was found to interact with the N terminus of synphilin-1 through its substrate-binding domain and to specifically ubiquitinate synphilin-1 via its RING finger domain . Siah-1 facilitated synphilin-1 degradation via the ubiquitin-proteasome pathway more efficiently than Parkin . Siah-1 was found to not facilitate ubiquitination and degradation of wild type or mutant alpha-synuclein . Synphilin-1 inhibited high K+-induced dopamine release from PC12 cells . Siah-1 was found to abrogate the inhibitory effects of synphilin-1 on dopamine release . Such findings suggest that Siah-1 might play a role in regulation of synphilin-1 function.

J Biol Chem, 2003 Dec 5, 278(49), 49063 - 71 Epub 2003 Sep 23.
Cellular localization, oligomerization, and membrane association of the hereditary spastic paraplegia 3A (SPG3A) protein atlastin; Zhu PP et al.; Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord . The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases . The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus . In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi . Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer . Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm . Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.

Hum Mol Genet, 2003 Nov 15, 12(22), 2949 - 56 Epub 2003 Sep 23.
Genetic background regulates beta-amyloid precursor protein processing and beta-amyloid deposition in the mouse; Lehman EJ et al.; Alzheimer's disease (AD) is a multigenic neurodegenerative disorder characterized by distinct neuropathological hallmarks including deposits of the beta-amyloid (A beta) peptide . A beta is a 39- to 43-amino acid peptide derived from the proteolytic processing of the amyloid precursor protein (APP) . While increasing evidence suggests that altered APP processing and A beta metabolism is a common feature of AD, the relationship between the levels of A beta and various APP products and the onset of AD remains unclear . We have undertaken a screen to characterize genetic factors that modify APP processing, A beta metabolism and A beta deposition in a genomic-based yeast artificial chromosome (YAC) transgenic mouse model of AD . A mutant human APP YAC transgene was transferred to three inbred mouse strains . Despite similar levels of holo-APP expression in the congenic strains, the levels of APP C-terminal fragments as well as brain and plasma A beta in young animals varied by genetic background . Furthermore, we demonstrate that age-dependent A beta deposition in the APP YAC transgenic model is dramatically altered depending on the congenic strain examined . These studies demonstrate that APP processing, A beta metabolism and A beta deposition are regulated by genetic background and that analysis of these phenotypes in mice should provide new insights into the factors that regulate AD pathogenesis.

Hum Mol Genet, 2003 Nov 15, 12(22), 2941 - 8 Epub 2003 Sep 23.
Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes; Navas PA et al.; High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5 . Of these, HS3 contains seven GT motifs that are essential for its activity . One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells . We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice . Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated . The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis . During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls . We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver . Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3252 - 9
Pentamidine is active in vitro against Fusarium species; Lionakis MS et al.; Fusariosis is an emerging opportunistic mycosis against which currently used antifungals have limited activity . Here, we investigated the in vitro activities of pentamidine (PNT) against 10 clinical isolates of Fusarium species (five Fusarium solani isolates and five non-F . solani isolates) by using the National Committee for Clinical Laboratory Standards microdilution method in three different media (RPMI, RPMI-2, and a yeast nitrogen base medium), disk diffusion testing, and viability dye staining . PNT had significant activities against all 10 Fusarium isolates . Non-F . solani isolates were more susceptible than F . solani isolates (P < 0.05) . Additionally, PNT was fungicidal against all non-F . solani isolates, whereas it had fungistatic effects against four of the five F . solani isolates . PNT also exhibited greater activity against conidial than against hyphal development of the fungus . This fungicidal activity against non-F . solani Fusarium isolates was confirmed microscopically after staining of PNT-treated Fusarium oxysporum hyphae with the fluorescent viability dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC) . The MICs at which 50% of the isolates were inhibited (2 micro g/ml for non-F . solani isolates and 4 micro g/ml for F . solani isolates) and the minimum fungicidal concentration at which 50% of the isolates were killed (8 micro g/ml for non-F . solani isolates) were much lower than the PNT tissue concentrations previously reported in humans using conventional daily intravenous PNT dosing . Finally, PNT was more active against Fusarium isolates in a hypoxic environment of in vitro growth (P < 0.05) . This finding may be clinically significant, because Fusarium, an angiotropic mold, causes tissue infarcts with resultant low tissue perfusion . Our findings suggest that PNT may have a role in the management of Fusarium infections . Future in vivo studies are needed to verify these in vitro findings.

J Biol Inorg Chem, 2003 Sep, 8(7), 699 - 706 Epub 2003 Jul 09.
Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase; Feng M et al.; Cytochrome c peroxidase (C cP) variants with an engineered Mn(II) binding site, including MnC cP {C cP(MI, G41E, V45E, H181D)}, MnC cP(W191F), and MnC cP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy . Analysis of the Raman bands in the 200-700 cm(-1) and 1300-1650 cm(-1) regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of C cP(MI), the recombinant yeast C cP containing additional Met-Ile residues at the N-terminus . At neutral pH the frequencies of the nu(3) mode indicate that a pure five-coordinate heme iron exists in C cP(MI) whereas a six-coordinate low-spin iron is the dominant species in the C cP variants with the engineered Mn(II) binding site . The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes . Raman spectra of the variants C cP(MI, W191F) and C cP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites . The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP . Such structural features, with a loosened distal water, may facilitate the binding of H(2)O(2) and increase the rate constant for compound I formation . This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnC cP(W191F, W51F).

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