Trends Cell Biol, 2001 May, 11(5), 220 - 30 The Ste20 group kinases as regulators of MAP kinase cascades; Dan I et al.; Ste20p (sterile 20 protein) is a putative yeast mitogen-activated protein kinase kinase kinase kinase (MAP4K) involved in the mating pathway . Its homologs in mammals, Drosophila, Caenorhabditis elegans and other organisms make up a large emerging group of protein kinases including 28 members in human . The Ste20 group kinases are further divided into the p21-activated kinase (PAK) and germinal center kinase (GCK) families . They are characterized by the presence of a conserved kinase domain and a noncatalytic region of great structural diversity that enables the kinases to interact with various signaling molecules and regulatory proteins of the cytoskeleton . This review describes the phylogenetic relationships of the Ste20 group kinases based on discussions with many researchers in this field . With the newly established phylogenetic relationships, crucial arguments can be advanced regarding the functions of these kinases as upstream activators of the MAPK pathways and possible activity as MAP4Ks . Their involvement in apoptosis, morphogenesis and cytoskeletal rearrangements is also discussed. Oncogene, 2001 Mar 1, 20(9), 1128 - 34 Towards a blueprint of the cell cycle; Alberghina L et al.; The understanding of the organisation of cell cycle events is of utmost importance to devise effective therapeutic strategies for cancer . In this article we gather evidences from the literature in support of a system model of the cell cycle, in which a growth-sensitive threshold controls entry into S phase and the sequential activation of cyclin-dependent kinases . The cycle is terminated by an End function, that comprises events from the onset of mitosis to cell division and that may also be modulated by the increase of cell size . This blueprint allows quantitative predictions by computer simulations of steady and transitory states . In fact, we show that the proposed control system applies to budding yeast populations during nutritional shift-up and following hyperactivation of the cAMP signalling pathway . Besides the growth-sensitive control system it is shown to apply to mammalian cells both in the exit from quiescence and in active proliferation . The putative molecular determinants that set the threshold controlling S phase entry are consistently altered in cancer cells . Finally, we discuss an input/output analysis based on the simulated behaviour derived from the blueprint as a new tool to investigate the road to cancer. Oncogene, 2001 Feb 8, 20(6), 739 - 47 Tumor specific modulation of KU70/80 DNA binding activity in breast and bladder human tumor biopsies; Pucci S et al.; The Ku70/80 heterodimer is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) and its DNA-binding activity mediates DNA double-strand breaks repair . Although Ku80 was recently proposed as a caretaker gene involved in the control of genome integrity, no data are available on Ku70/80 DNA-binding activity in human tumors . Heterodimer DNA-binding activity and protein expression were assayed by electrophoretic-mobility-shift-assay (EMSA) and Western blot analysis, in nuclear and cytoplasmic extracts from eight breast, seven bladder primary tumors and three metastatic nodes from breast cancers . Corresponding normal tissues of the same patients were used as controls . Ten out of 15 tumors showed nuclear Ku-binding activity 3-10 times higher than in the normal tissues, irrespective of bladder or breast origin . Conversely, in 5/15 primary tumors and in all the metastatic nodes analysed, nuclear Ku-activity was 1.5-4.5-fold lower than in the corresponding normal tissues . Cytoplasmic heterodimer activity significantly differed between tumor and normal tissues, displaying a 2-10-fold increase in neoplastic tissues . Three different patterns combining both Ku expression and activity with tumor characteristics were identified . In low aggressive breast tumors p70/p80 proteins were expressed in tumor but not in normal tissues . The heterodimer binding-activity matched the protein levels . In non-invasive bladder carcinomas no significant differences in protein expression between tumor and the corresponding normal tissues were found, however heterodimer binding-activity was increased in tumor samples . In breast and bladder tumors, at the advanced stage and in node metastases, the binding activity was strongly reduced in tumor biopsies, however no differences were demonstrated between normal and tumor protein levels . Our results suggest a different modulation of Ku70/80 DNA-binding activity in human neoplastic tissues, possibly related to tumor progression . Findings provide further data on tissue-specific protein expression and post-translational regulation of heterodimer activity. Oncogene, 2001 Jan 18, 20(3), 404 - 9 The monocytic leukemia zinc finger protein MOZ is a histone acetyltransferase; Champagne N et al.; The monocytic leukemia zinc finger protein (MOZ) gene is rearranged in t(8;16)(p11;p13), t(8;22)(p11;q13) and inv(8)(p11q13) associated with acute myeloid leukemia . The other fusion partners involved are CBP, p300 and TIF2, transcriptional coactivators with known or potential histone acetyltransferase (HAT) activity . MOZ itself is a 2004-residue protein containing a putative acetyl CoA-binding motif, so it was hypothesized that MOZ is a HAT . Here we present direct evidence that MOZ has intrinsic HAT activity . Moreover, MOZ possesses a transcriptional repression domain at its N-terminal part and an activation domain at its C-terminal part . The activation domain does not show sequence similarity to any yeast proteins, but when tethered, it is able to activate transcription in yeast . Therefore, MOZ is a HAT with characteristics of a transcriptional coregulator, supporting the hypothesis that aberrant acetylation by abnormal MOZ proteins leads to leukemogenesis. Oncogene, 2001 Jan 18, 20(3), 295 - 302 Runx2: a novel oncogenic effector revealed by in vivo complementation and retroviral tagging; Blyth K et al.; The Runx2 (Cbfa1, Pebp2alphaA, Aml3) gene was previously identified as a frequent target for transcriptional activation by proviral insertion in T-cell lymphomas of CD2-MYC transgenic mice . We have recently shown that over-expression of the full-length, most highly expressed Runx2 isoform in the thymus perturbs T-cell development, leads to development of spontaneous lymphomas at low frequency and is strongly synergistic with Myc . To gain further insight into the relationship of Runx2 to other lymphomagenic pathways, we tested the effect of combining the CD2-Runx2 transgene either with a Pim1 transgene (E(mu)-Pim1) or with the p53 null genotype, as each of these displays independent synergy with Myc . In both cases we observed synergistic tumour development . However, Runx2 appeared to have a dominant effect on the tumour phenotype in each case, with most tumours conforming to the CD3(+), CD8(+), CD4(+/-) phenotype seen in CD2-Runx2 mice . Neonatal infection of CD2-Runx2 mice with Moloney murine leukaemia virus (Moloney MLV) also led to a dramatic acceleration of tumour onset . Analysis of known Moloney MLV target genes in these lymphomas showed a high frequency of rearrangement at c-Myc or N-Myc (82%), and a significant number at Pim1 or Pim2 (23%), and at Pal1/Gfi1 (18%) . These results indicate that Runx2 makes a distinct contribution to T-cell lymphoma development which does not coincide with any of the oncogene complementation groups previously identified by retroviral tagging. Oncogene, 2001 Jan 11, 20(2), 178 - 87 PRCC, the commonest TFE3 fusion partner in papillary renal carcinoma is associated with pre-mRNA splicing factors; Skalsky YM et al.; In papillary renal cell carcinomas the TFE3 transcription factor becomes fused to the PSF and NonO pre-mRNA splicing factors and most commonly to a protein of unknown function designated PRCC . In this study we have examined the ability of the resulting PRCC-TFE3 and NonO-TFE3 fusions to activate transcription from the plasminogen activator inhibitor-1 (PAI-1) promoter . The results show that only fusion to PRCC enhanced transcriptional activation, indicating that the ability to enhance the level of transcription from endogenous TFE3 promoters is not a consistent feature of TFE3 fusions . In investigations of the normal function of PRCC we observed that PRCC expressed as a green fluorescent fusion protein colocalizes within the nucleus with Sm pre-mRNA splicing factors . It was also found that endogenous PRCC is coimmunoprecipitated by antibodies that recognize a variety of pre-mRNA splicing factors including SC35, PRL1 and CDC5 . Association with the cellular splicing machinery is therefore, a common feature of the proteins that become fused to TFE3 in papillary renal cell carcinomas. Gene Ther, 2001 Jan, 8(2), 166 - 71 Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway; Chan CK et al.; Efficient nuclear delivery of plasmid DNA represents a major barrier in nonviral gene transfer . One approach has been to use DNA-binding proteins such as GAL4 from yeast as DNA carriers with nuclear targeting properties . We recently showed, however, that GAL4 is inefficient in targeting DNA to the nucleus because its DNA-binding and nuclear targeting activities are mutually exclusive, which relates to the fact that GAL4 nuclear import occurs via a novel pathway . Here, we 'switch' this pathway to a more conventional one by adding a modified poly-lysine to which an optimized nuclear targeting signal, based on that of the SV40 large T-antigen, is linked . We also use a chimeric GAL4-alpha-melanocyte stimulating hormone (MSH) fusion protein to enable gene transfer to cells expressing the MSH receptor . Switching the nuclear import pathway of the transfecting complex significantly enhances receptor-mediated gene transfer through enabling interaction with desired components of the cellular nuclear import machinery . The present study represents the first demonstration that nuclear targeting signals can enhance receptor-mediated gene delivery, the approaches having important relevance to research and clinical applications, such as in generating transgenic or knock-out animals, or in gene therapy. Cell Death Differ, 2001 Jan, 8(1), 63 - 9 Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c; Bhuyan AK et al.; Observation of the electrical potential difference across the cell membrane is described as a new method for monitoring apoptosis of a single cell . The resting membrane potential (DeltaPsi) of Xenopus oocytes has been recorded in real time following microinjection of cytochrome c . Soon after microinjection, DeltaPsi becomes less negative and attains a new constant value with a half time, t(m), of about 35 (+ /- 5) min at all cytochrome c concentrations greater than 1 microM . The cytosol extract of cytochrome c-injected oocytes shows DEVD proteolytic activity characteristic of aspartate specific proteases, implicating an apoptotic death pathway . In response to the delivery of cytochrome c into the cytosol, caspases are activated within 7 min while the changes in DeltaPsi begin to occur after about 30 min . The method described here will be potentially useful to assess the effectiveness of cell death regulators and modulators of synthetic and biological origin, and the results presented shed light on the currently debated issue of the importance of the redox state of cytochrome c in the initiation of apoptosis. Science, 2001 May 18, 292(5520), 1376 - 8 Epub 2001 Apr 19. A GDP/GTP exchange factor involved in linking a spatial landmark to cell polarity; Kang PJ et al.; In both animal and yeast cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate polarized organization of the actin cytoskeleton . In the budding yeast Saccharomyces cerevisiae, the Ras-like GTPase Bud1/Rsr1 and its guanosine 5'-diphosphate (GDP)/guanosine 5'-triphosphate (GTP) exchange factor Bud5 are involved in the selection of a specific site for growth, thus determining cell polarity . We found that Bud5 is localized at the cell division site and the presumptive bud site . Its localization is dependent on potential cellular landmarks, such as Bud3 and Axl2/Bud10 in haploid cells and Bud8 and Bud9 in diploid cells . Bud5 also physically interacts with Axl2/Bud10, a transmembrane glycoprotein, suggesting that a receptor-like transmembrane protein recruits a GDP/GTP exchange factor to connect an intrinsic spatial signal to oriented cell growth. Science, 2001 Jun 8, 292(5523), 1876 - 82 Epub 2001 Apr 19. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution; Gnatt AL et al.; The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution . Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site . Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site . The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking . The 5'-most residue of the RNA is close to the point of entry to an exit groove . Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid . Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation. Science, 2001 Jun 8, 292(5523), 1863 - 76 Epub 2001 Apr 19. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution; Cramer P et al.; Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution . Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center . In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription . Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription . A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis . The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain. Mol Cell Biol, 2001 May, 21(10), 3564 - 75 Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway; Wang G et al.; The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase . Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain . A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins . We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated . Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites . The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p . The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization . Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein . Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway. Mol Cell Biol, 2001 May, 21(10), 3558 - 63 Role of DNA polymerase zeta in the bypass of a (6-4) TT photoproduct; Johnson RE et al.; UV light-induced DNA lesions block the normal replication machinery . Eukaryotic cells possess DNA polymerase eta (Poleta), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Poleta result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum . Here, we test Poleta for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer . Opposite the 3' T of a (6-4) TT photoproduct, both yeast and human Poleta preferentially insert a G residue, but they are unable to extend from the inserted nucleotide . DNA Polzeta, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3' T of the (6-4) TT lesion by Poleta, and Polzeta inserts the correct nucleotide A opposite the 5' T of the lesion . Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Poleta and Polzeta, wherein Poleta inserts a nucleotide opposite the 3' T of the lesion and Polzeta extends from it . These biochemical observations are in concert with genetic studies in yeast indicating that mutations occur predominantly at the 3' T of the (6-4) TT photoproduct and that these mutations frequently exhibit a 3' T-->C change that would result from the insertion of a G opposite the 3' T of the (6-4) TT lesion. Mol Cell Biol, 2001 May, 21(10), 3514 - 22 A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex; Sutton A et al.; In the yeast Saccharomyces cerevisiae, a and alpha mating-type information is stored in transcriptionally silenced cassettes called HML and HMR . Silencing of these loci, maintained by the formation of a specialized type of heterochromatin, requires trans-acting proteins and cis-acting elements . Proteins required for silencing include the Sir2 NAD(+)-dependent deacetylase, Sir3, and Sir4 . Factors that bind to the cis elements at HMR and HML and that are important for silencing include the origin recognition complex (ORC) . Mutations of any of these Sir proteins or combinations of cis elements result in loss of silencing . SUM1-1 was previously identified as a dominant mutation that restores silencing to HMR in the absence of either the Sir proteins or some of the cis elements . We have investigated the novel mechanism whereby Sum1-1 causes Sir-independent silencing at HMR and present the following findings: Sum1-1 requires the Sir2 homolog, Hst1, for silencing and most probably requires the NAD(+)-dependent deacetylase activity of this protein . Sum1-1 interacts strongly with ORC, and this strong interaction is dependent on HMR DNA . Furthermore, ORC is required for Sum1-1-mediated silencing at HMR . These observations lead to a model for Sum1-1 silencing of HMR in which Sum1-1 is recruited to HMR by binding to ORC . Sum1-1, in turn, recruits Hst1 . Hst1 then deacetylates histones or other chromatin-associated proteins to cause chromatin condensation and transcriptional silencing. Mol Cell Biol, 2001 May, 21(10), 3425 - 35 Evidence for biased holliday junction cleavage and mismatch repair directed by junction cuts during double-strand-break repair in mammalian cells; Baker MD et al.; In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination . In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution . According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes . The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency . However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule . The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting . The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants. Mol Cell Biol, 2001 May, 21(10), 3405 - 15 Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p; Gadal O et al.; Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified . Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants . One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits . We find that thermosensitive nmd3 mutants are impaired in large subunit export . Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p . Moreover, we show that export of 60S subunits is Xpo1p dependent . We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p. J Biol Chem, 2001 Jun 15, 276(24), 20817 - 20 Epub 2001 Apr 19. A new pathway for heavy metal detoxification in animals . Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans; Vatamaniuk OK et al.; Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms . Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, gamma-Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins . Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH . Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism. FEMS Microbiol Lett, 2001 Apr 13, 197(2), 209 - 14 A novel actin-related protein gene of Colletotrichum gloeosporioides f . sp . malvae shows altered expression corresponding with spore production; Li J et al.; A novel actin-related protein (arp) was found in the plant pathogenic fungus, Colletotrichum gloeosporioides f . sp . malvae (Cgm), which causes anthracnose disease of round-leaved mallow (Malva pusilla) . Sequence comparisons showed that this gene, arpA, belongs to the highly divergent 'other arps' category in the current arp classification system . ArpA is most similar to the arp11 gene of Mus musculus but has a unique structure with deletions at the C-terminus similar to that of the arp10 gene of Saccharomyces cerevisiae . A portion of another putative arp gene, arpB, was found immediately downstream of arpA . Expression of arpA was compared to the constitutively expressed Cgm actin gene, actA . In culture, the relative expression of arpA increased when growth conditions favored sporulation . During infection, arpA expression was greatest at the late necrotrophic phase, when sporulation occurred . Arps have been shown to be important in nuclear migration in fungal hyphae, and the expression pattern of arpA indicates that it may have a particular role during sporulation. J Agric Food Chem, 2001 Mar, 49(3), 1607 - 11 Antifungal mechanism of polygodial; Kubo I et al.; The primary antifungal action of polygodial comes in part from its ability to function as a nonionic surfactant, disrupting the lipid-protein interface of integral proteins and denaturing their conformation . As a result, the antifungal mechanism of this sesquiterpene dialdehyde is associated with the membrane functions or derangement of the membrane . For example, the glucose-induced medium acidification process of Saccharomyces cerevisiae was inhibited by polygodial, presumably caused by inhibition of the plasma membrane H(+)-ATPase . However, the potent antifungal activity of polygodial results from its multiple functions. J Virol, 2001 May, 75(10), 4902 - 6 Identification of critical elements in the tRNA acceptor stem and T(Psi)C loop necessary for human immunodeficiency virus type 1 infectivity; Yu Q et al.; A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe), which relies on exogenous yeast tRNA(Phe) as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and T(Psi)C stem-loop required for the tRNA primer selection and use in HIV-1 replication . tRNA(Phe) mutants with two- or four-nucleotide deletions in the 3' end retained the capacity to complement replication of psHIV-Phe . tRNA(Phe) mutants with an extended 5' end had reduced capacity for complementation, which could be restored by extension of the 3' end of these tRNA(Phe) mutants with sequences complementary to the HIV-1 U5 region . Further analysis of mutations in the acceptor stem of tRNA(Phe) suggested that an intact acceptor stem RNA structure is important for complementation . Analysis of single-nucleotide changes in the T(Psi)C stem-loop of tRNA(Phe) revealed an unexpected, essential role of this region for rescue of psHIV-Phe. J Endocrinol, 2001 May, 169(2), 389 - 96 GH gene expression in the submaxillary gland in normal and Ames dwarf mice; Perez-Romero A et al.; High local GH-releasing hormone (GHRH) levels are capable of inducing transdifferentiation in salivary cells to synthesize GH . However, the factors implicated in this process remain unknown . To study this subject, normal and Ames dwarf mice were implanted in the submaxillary gland with a slow release pellet releasing 21 microgram GHRH (1-29)-NH(2)/day for 2 months . Control animals received placebo pellets at the same site . After 60 days, heart blood was collected and submaxillary glands were removed . Circulating levels of GH and IGF-I were significantly decreased (P<0.05) in dwarf mice in comparison with controls, and GHRH treatment did not modify either of these two parameters . Controls carrying GHRH pellets showed a significantly higher GH content (P<0.05) in the submaxillary gland than the placebo-treated normal mice . There were no differences between the IGF-I concentrations of placebo- and GHRH-treated salivary tissue from normal mice . Analysis of GH mRNA by RT-PCR followed by Southern blot revealed that GH transcripts were present in the salivary gland samples carrying the placebo pellets in both normal and dwarf mice . The expression of GH was significantly (P<0.05) increased by the GHRH pellets in salivary tissue from normal mice, but not in submaxillary glands from dwarf mice . Pit-1 mRNA was not detected in the GHRH-treated glands of normal and dwarf mice by RT-PCR or by Southern blot . Using these highly sensitive methods, we have been able to detect the transcription of both GH and Pit-1 in pituitaries from Pit-1-deficient Ames dwarf mice . The present experiment demonstrates that salivary tissue synthesizes GH when it is exposed to the influence of GHRH . Both basal and GHRH-induced salivary GH expression appear to be independent of Pit-1. Gene, 2001 Apr 4, 267(1), 101 - 10 Interaction of MCC2, a novel homologue of MCC tumor suppressor, with PDZ-domain Protein AIE-75; Ishikawa S et al.; AIE-75 is a protein identified as an autoantigen in patients with autoimmune enteropathy and as a colon cancer-related antigen . It has recently been assigned to be a causative gene for Usher type 1C congenital syndromic hearing loss . The novel protein has three PSD-95/Dlg/ZO-1 (PDZ) protein-protein interaction domains and is therefore implicated to function as a molecular anchor or sorter . We have identified a novel protein that binds to AIE-75 by yeast two-hybrid screening . The protein has a high homology to the tumor suppressor MCC (mutated in colon cancer; or MCC1 hereafter) and was named MCC2 . MCC2 protein binds the first PDZ domain of AIE-75 with its C-terminal amino acids -DTFL . Since the MCC1 does not bind to AIE-75 and the MCC2 displays different expression patterns in various organs compared to MCC1, they appear to play distinct roles in cells . The MCC2 gene is located on chromosome 19p13 in the vicinity of APCL gene, while MCC1 maps near to APC tumor suppressor gene . Because of negative expression of MCC2 in a panel of cancer cell-lines compared to the corresponding normal tissues, we suggest that further study is necessary to investigate a possible role of MCC2 as a tumor suppressor. FEBS Lett, 2001 Apr 13, 494(3), 208 - 12 The transcriptional regulator, Arg82, is a hybrid kinase with both monophosphoinositol and diphosphoinositol polyphosphate synthase activity; Zhang T et al.; The Arg82 gene of Saccharomyces cerevisiae encodes a transcriptional regulator that phosphorylates inositol 1,4,5-trisphosphate {Saiardi et al . (1999) Curr . Biol . 9, 1323-1326} . However, some controversy has surrounded the nature of the reaction products . We now show that Arg82 phosphorylates inositol 1,3,4,5-tetrakisphosphate to inositol pentakisphosphate, which is itself converted to two isomers of diphosphoinositol tetrakisphosphate, one of which has never previously been identified . One of the diphosphoinositol phosphates was further phosphorylated by a yeast cell lysate . We propose that Arg82 is an ancestral precursor of two distinct and specific enzyme families: inositol 1,4,5-trisphosphate kinases and diphosphoinositol polyphosphate synthases. Nature, 2001 Apr 19, 410(6831), 955 - 9 Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability; Rao H et al.; Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin . At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K) . The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule . Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway . Overexpression of a long-lived derivative of the SCC1 fragment is lethal . In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss . The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment . This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue . A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway. Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 5122 - 7 Epub 2001 Apr 17. The 3'-->5' exonuclease of DNA polymerase delta can substitute for the 5' flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability; Jin YH et al.; Many DNA polymerases (Pol) have an intrinsic 3'-->5' exonuclease (Exo) activity which corrects polymerase errors and prevents mutations . We describe a role of the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5' flap endonuclease . A yeast rad27 null allele was lethal in combination with Pol delta mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol delta . The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol delta Exo I and Exo II motifs . However, rad27-p Pol delta Exo III double mutants were viable . They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability . Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5' flaps in the lagging strand . These results suggest that the 3'-->5' Exo activity of Pol delta is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand. J Cell Biol, 2001 Apr 16, 153(2), 251 - 62 Nuclear import of histone H2A and H2B is mediated by a network of karyopherins; Mosammaparast N et al.; The first step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones . The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers . We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family . An abundant complex of H2A, H2B, and Kap114p was detected in cytosol . In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B . Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B . We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail . Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains . In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo . Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association . These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role. J Biol Chem, 2001 Jun 29, 276(26), 24203 - 11 Epub 2001 Apr 17. A novel snare N-terminal domain revealed by the crystal structure of Sec22b; Gonzalez LC Jr et al.; Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable alpha-helical bundles . Many SNAREs, in addition to their alpha-helical regions, contain N-terminal domains that likely have essential regulatory functions . To better understand this regulation, we have determined the 2.4-A crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking . The domain consists of a mixed alpha-helical/beta-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules . The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro . An analysis of surface conserved residues reveals a potential protein interaction site . Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains . Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7. J Biol Chem, 2001 Jun 29, 276(26), 24323 - 30 Epub 2001 Apr 17. Accurate in vitro end joining of a DNA double strand break with partially cohesive 3'-overhangs and 3'-phosphoglycolate termini: effect of Ku on repair fidelity; Chen S et al.; To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3'-overhangs and a one-base gap in each strand, was constructed . In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence . In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3' --> 5' resection before ligation, was formed . Similar results were seen for a substrate with 5'-overhangs and recessed 3'-phosphoglycolate ends . Supplementation of the xrs6 extracts with purified Ku restored accurate end joining . In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity . The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation. Mol Microbiol, 2001 Apr, 40(2), 361 - 75 The Aspergillus nidulans GATA transcription factor gene areB encodes at least three proteins and features three classes of mutation; Conlon H et al.; In Aspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA-binding proteins . In seeking additional GATA factors, we have cloned areB, which was originally identified via a genetic screen for suppressors of areA loss-of-function mutations . Based on our analysis, areB is predicted to encode at least three distinct protein products . These arise from the use of two promoters, differential splicing and translation initiating at AUG and non-AUG start codons . All the putative products include a GATA domain and a putative Leu zipper . These regions show strong sequence similarity to regulatory proteins from Saccharomyces cerevisiae (Dal80p and Gzf3p), Penicillium chrysogenum (NREB) and Neurospora crassa (ASD4) . We have characterized three classes of mutation in areB; the first are loss-of-function mutations that terminate the polypeptides within or before the GATA domain . The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain . The third class fuses novel gene sequences to areB with the potential to produce putative chimeric polypeptides . These novel gene fusions transform the putative negative-acting transcription factor into an activator that can partially replace areA. J Agric Food Chem, 2001 Apr, 49(4), 2030 - 6 Effect of chemical and genetic attachment of polysaccharides to proteins on the production of IgG and IgE; Arita K et al.; To investigate the effect of polysaccharide attachment to proteins on the production of IgG and IgE, the genetic attachment of polysaccharide to lysozymes (G49N and R21T) using the yeast expression system (Saccharomyces cerevisiae AH 22) and the Maillard-type polysaccharide attachment to native lysozyme and soybean P34 protein were attempted . The production of IgG and IgE was investigated by using mice immunized with the protein-polysaccharide conjugates or native proteins . The attachment of polysaccharide to lysozyme using the yeast expression system greatly suppressed the production level of IgG and IgE . The attachment of polysaccharide to native lysozyme and soybean P34 protein using the Maillard-type reaction was also found to be effective in reducing the production level of IgE compared to IgG. Biol Chem, 2001 Feb, 382(2), 161 - 77 Congenital disorders of glycosylation: glycosylation defects in man and biological models for their study; Marquardt T et al.; Several inherited disorders affecting the biosynthetic pathways of N-glycans have been discovered during the past years . This review summarizes the current knowledge in this rapidly expanding field and covers the molecular bases of these disorders as well as their phenotypical consequences. J Protein Chem, 2000 Nov, 19(8), 649 - 62 Expression and properties of recombinant HbA2 (alpha2delta2) and hybrids containing delta-beta sequences; Inagaki K et al.; Hemoglobin A2 (alpha2delta2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic . However, reports on its functional properties regarding O2 binding are conflicting . We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties . The construct expressing HbA2 contains only alpha and delta genes so that the extensive purification required to isolate natural HbA2 is circumvented . Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant alpha2delta2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the alpha + beta or the alpha + gamma genes, respectively, are present on the construct . Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA . However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin . Molecular modeling studies which attempt to understand these properties of HbA2 are described. J Biol Chem, 2001 Jul 6, 276(27), 24654 - 60 Epub 2001 Apr 16. Functional analysis of the protein-interacting domains of chloroplast SRP43; Jonas-Straube E et al.; The chloroplast signal recognition particle (cpSRP) consists of an evolutionarily conserved 54-kDa subunit (cpSRP54) and a dimer of a unique 43-kDa subunit (cpSRP43) . cpSRP binds light-harvesting chlorophyll proteins (LHCPs) to form a cpSRP/LHCP transit complex, which targets LHCP to the thylakoid membrane . Previous studies showed that transit complex formation is mediated through the binding of the L18 domain of LHCP to cpSRP43 . cpSRP43 is characterized by a four-ankyrin repeat domain at the N terminus and two chromodomains at the C terminus . In the present study we used the yeast two-hybrid system and in vitro binding assays to analyze the function of different domains of cpSRP43 in protein complex formation . We report here that the first ankyrin repeat binds to the 18-amino acid domain on LHCP that binds to cpSRP43, whereas the third and fourth ankyrin repeats are involved in the dimerization of cpSRP43 . We show further that the interaction of cpSRP43 with cpSRP54 is mediated via binding of the methionine-rich domain of cpSRP54 to the C-terminally located chromodomains of cpSRP43 . Both chromodomains contain essential elements for binding cpSRP54, indicating that the closely spaced chromodomains together create a single binding site for cpSRP54 . In addition, our data demonstrate that the interaction of cpSRP54 with the chromodomains of cpSRP43 is enhanced indirectly by the dimerization motif of cpSRP43. J Biol Chem, 2001 Jun 22, 276(25), 22595 - 603 Epub 2001 Apr 16. An essential role for Mad homology domain 1 in the association of Smad3 with histone deacetylase activity*; Liberati NT et al.; The Smads are a family of sequence-specific DNA-binding proteins that modulate transcription in response to transforming growth factor beta (TGFbeta) by recruiting transcriptional activators like the histone acetyltransferase, p300/CBP, or repressors like the histone deacetylase, HDAC1, to TGFbeta target genes . The association of Smads and HDAC1 is mediated in part by direct binding of Smads to the HDAC1-associated proteins, TG-interacting factor, c-ski, and SnoN . Although ectopic expression of these proteins inhibits Smad-activated transcription, the contribution of histone deacetylase enzymatic activity to transcriptional repression by TGFbeta is unknown . Here, the biological requirements for the interaction between Smads and endogenous histone deacetylase activity are investigated . We identify residues in Mad homology domain 1 of Smad3 that are required for association with histone deacetylase activity . An amino acid change at one of these critical residues does not disrupt the association of Smad3 with c-ski, SnoN, and transforming growth-interacting factor but does abrogate the ability of Smad3 to repress transcription . These findings indicate that the association of Smad3 and histone deacetylase activity relies on additional protein mediators that make contact with Smad3 at its amino terminus . Moreover, these data suggest that the suppressive effect of Smad3 on transcription is dependent upon its association with histone deacetylase enzymatic activity. J Biol Chem, 2001 Jun 22, 276(25), 22439 - 45 Epub 2001 Apr 16. Cloning and characterization of MST4, a novel Ste20-like kinase; Qian Z et al.; MST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized . Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3 . Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase . Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situ hybridization . Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase . An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues . MST4a lacks an exon encoding kinase subdomains IX-XI that stabilizes substrate binding . The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase. EMBO Rep, 2001 Apr, 2(4), 287 - 91 Homologous recombination in planta is stimulated in the absence of Rad50; Gherbi H et al.; Chromosomal double-strand DNA breaks must be repaired; in the absence of repair the resulting acentromeric (and telomereless) fragments may be lost and/or the broken DNA ends may recombine causing general chromosomal instability . The Rad50/Mre11/Xrs2 protein complex acts at DNA ends and is implicated in both homologous and non-homologous recombination . We have isolated a rad50 mutant of the plant Arabidopsis thaliana and show here that it has a somatic hyper-recombination phenotype in planta . This finding supports the hypothesis of a competition between homologous and illegitimate recombination in higher eukaryotes . To our knowledge, this is the first direct in vivo support for the role of this complex in chromosomal recombination in a multicellular organism and the first description of a mutation of a known gene leading to hyper-recombination in plants. Cell Growth Differ, 2001 Mar, 12(3), 157 - 67 Signaling mediated by the closely related mammalian Rho family GTPases TC10 and Cdc42 suggests distinct functional pathways; Murphy GA et al.; The mammalian Rho family GTPases TC10 and Cdc42 share many properties . Activated forms of both proteins stimulate transcription mediated by nuclear factor kappaB, serum response factor, and the cyclin D1 promoter; activate c-Jun NH2-terminal kinase; cooperate with activated Raf to transform NIH-3T3 cells; and, by a mechanism independent of all of these effects, induce filopodia formation . In contrast, previously reported differences between TC10 and Cdc42 are not striking . We now present studies of TC10 and Cdc42 in cell culture that reveal clear functional differences: (a) wild-type TC10 localizes predominantly to the plasma membrane and less extensively to a perinuclear membranous compartment, whereas wild-type Cdc42 localizes predominantly to this compartment and less extensively to the plasma membrane; (b) expression of Rho guanine nucleotide dissociation inhibitor alpha results in a redistribution of wild-type Cdc42 to the cytosol but has no effect on the plasma membrane localization of wild-type TC10; (c) TC10 fails to rescue a Saccharomyces cerevisiae cdc42 mutation, unlike mammalian Cdc42; (d) dominant negative Cdc42, but not dominant negative TC10, inhibits neurite outgrowth in PC12 cells stimulated by nerve growth factor; and (e) activation of nuclear factor kappaB-dependent transcription by Cdc42, but not by TC10, is inhibited by sodium salicylate . These findings point to distinct pathways in which TC10 and Cdc42 may act and distinct modes of regulation of these proteins. Chem Biol, 2001 Mar, 8(3), 231 - 41 Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities; Kasanov J et al.; Introduction: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins . Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains . Class I WW domains bind Pro-Pro-Xxx-Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity.Results: Fourteen WW domains selected Leu/Pro-Pro-Xxx-Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains . These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5 . Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains . Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not . Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands.Conclusions: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands . The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands. Trends Endocrinol Metab, 2001 Apr, 12(3), 127 - 34 The TRAP/SMCC/Mediator complex and thyroid hormone receptor function; Ito M et al.; The TRAP/SMCC/Mediator complex is a mammalian transcriptional regulatory complex that contains over 25 polypeptides and is, in part, phylogenetically conserved . It was originally isolated as a thyroid hormone receptor (TR)-associated protein (TRAP) complex that mediates TR-activated transcription from DNA templates in conjunction with the general transcription machinery, and probably acts in vivo after the action of other receptor-interacting coactivators involved in chromatin remodeling . Subsequently, the TRAP complex was identified as a more broadly used coactivator complex for a wide variety of activators . The TRAP220 subunit mediates ligand-dependent interactions of the complex with TR and other nuclear receptors; and genetic ablation of murine TRAP220 has revealed that it is essential both for optimal TR function and for a variety of early developmental and adult homeostasis events in mice, but not for cell viability per se. Trends Cell Biol, 2001 Mar, 11(3), 136 - 41 Congenital disorders of glycosylation: genetic model systems lead the way; Aebi M et al.; N-linked glycosylation is the most frequent modification of secretory proteins in eukaryotic cells . The highly conserved glycosylation process is initiated in the endoplasmic reticulum (ER), where the Glc(3)Man(9)GlcNAc(2) oligosaccharide is assembled on the lipid carrier dolichylpyrophosphate and then transferred to selected asparagine residues of polypeptide chains . In recent years, several inherited human diseases, congenital disorders of glycosylation (CDG), have been associated with deficiencies in this pathway . The ER-associated glycosylation pathway has been studied in the budding yeast Saccharomyces cerevisiae, and this model system has been invaluable in elucidating the molecular basis of novel types of CDG. Trends Cell Biol, 2001 Mar, 11(3), 102 - 6 Towards an understanding of complex protein networks; Tucker CL et al.; Large-scale two-hybrid screens have generated a wealth of information describing potential protein--protein interactions . When compiled with data from systematic localizations of proteins, mutant screens and other functional tests, a network of interactions among proteins and between proteins and other components of eukaryotic cells can be deduced . These networks can be viewed as maps of the cell, depicting potential signaling pathways and interactive complexes . Most importantly, they provide potential clues to the function of previously uncharacterized proteins . Focusing on recent experiments, we explore these protein-interaction studies and the maps derived from such efforts. Genome Biol . 2001;2(4):REVIEWS1013 . Epub 2001 Apr 04. Genome-wide analysis of protein-DNA interactions in living cells; Pugh BF et al.; Understanding the regulation of gene expression requires an analysis of gene-specific transcription factors . This review highlights recent work that uses protein-DNA crosslinking, immunoprecipitation and DNA microarrays to determine the binding sites for specific transcription factors throughout the yeast genome. Genome Biol . 2001;2(4):REVIEWS0003 . Epub 2001 Apr 05. A tale of histone modifications; Grant PA; The modification of chromatin structure is important for a number of nuclear functions, exemplified by the regulation of transcription . This review discusses recent studies of covalent histone modifications and the enzymatic machines that generate them. Biochemistry, 2001 Apr 24, 40(16), 4893 - 903 Enzymes of sphingolipid metabolism: from modular to integrative signaling; Hannun YA et al.; Many enzymes of sphingolipid metabolism are regulated in response to extra- and intracellular stimuli and in turn serve as regulators of levels of bioactive lipids (such as sphingosine, ceramide, sphingosine 1-phosphate, and diacylglycerol), and as such, they serve a prototypical modular function in cell regulation . However, lipid metabolism is also closely interconnected in that a product of one enzyme serves as a substrate for another . Moreover, many cell stimuli regulate more than one of these enzymes, thus adding to the complexity of regulation of lipid metabolism . In this paper, we review the status of enzymes of sphingolipid metabolism in cell regulation and propose a role for these enzymes in integration of cell responses, a role that builds on the modular organization while also taking advantage of the complexity and interconnectedness of lipid metabolism, thus providing for a combinatorial mechanism of generating diversity in cell responses . This may be a general prototype for the involvement of metabolic pathways in cell regulation. Anal Chem, 2001 Mar 15, 73(6), 1307 - 15 Nanoflow gradient generator coupled with mu-LC-ESI-MS/MS for protein identification; Le Bihan T et al.; The large-scale identification of proteins from proteomes of complex organisms, and the availability of various types of protein and DNA databases, increasingly require the additional information provided by tandem mass spectrometry . HPLC and microLC coupled to ESI-MS/MS presently dominate the field of protein identification by tandem mass spectrometry and database searching . The analysis of protein digests is typically performed using HPLC or LC columns with 50-100-microm diameters, requiring the delivery of solvent gradients at low to mid nanoliter per minute flow rates . This has been typically achieved using expensive generic HPLC pumping systems for the delivery of microliter per minute gradients that were either flow-split or sampled . Here we present an alternative system for the delivery of nanoliter per minute gradients . The inexpensive nanoflow gradient generator (etagrad) described here can be modulated to reproducibly deliver selected gradients . The performance of the etagrad on-line with a microLC-ESI-MS/MS system has been demonstrated for the identification of standard protein digests . Moreover, the performance of the etagrad-microLC-ESI-MS/MS system, with protein prefractionation by IPG isoelectric focusing, was also evaluated for rapid study of yeast and human proteomes. J Biol Chem, 2001 Jun 15, 276(24), 20973 - 80 Epub 2001 Apr 13. Coactivator p300 acetylates the interferon regulatory factor-2 in U937 cells following phorbol ester treatment; Masumi A et al.; Interferon regulatory factor-2 (IRF-2) is a transcription factor of the IRF family that represses interferon-mediated gene expression . In the present study, we show that human monocytic U937 cells express truncated forms of IRF-2 containing the DNA binding domain but lacking much of the C-terminal regulatory domain . U937 cells are shown to respond to phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce expression of histone acetylases p300 and p300/CBP-associated factor (PCAF) . In addition, TPA treatment led to the appearance of full-length IRF-2, along with a reduction of the truncated protein . Interestingly, full-length IRF-2 in TPA-treated U937 cells occurred as a complex with p300 as well as PCAF and was itself acetylated . Consistent with these results, recombinant IRF-2 was acetylated by p300 and to a lesser degree by PCAF in vitro . Another IRF member, IRF-1, an activator of interferon-mediated transcription, was also acetylated in vitro by these acetylases . Finally, we demonstrate that the addition of IRF-2 but not IRF-1 inhibits core histone acetylation by p300 in vitro . The addition of IRF-2 also inhibited acetylation of nucleosomal histones in TPA-treated U937 cells . Acetylated IRF-2 may affect local chromatin structure in vivo by inhibiting core histone acetylation and may serve as a mechanism by which IRF-2 negatively regulates interferon-inducible transcription. Biochem Biophys Res Commun, 2001 Apr 20, 282(5), 1244 - 50 Rpg1p/Tif32p, a subunit of translation initiation factor 3, interacts with actin-associated protein Sla2p; Palecek J et al.; The yeast two-hybrid system was used to screen for proteins that interact in vivo with Saccharomyces cerevisiae Rpg1p/Tif32p, the large subunit of the translation initiation factor 3 core complex (eIF3) . Eight positive clones encoding portions of the SLA2/END4/MOP2 gene were isolated . They overlapped in the region of amino acids 318-550 . Subsequent deletion analysis of Sla2p showed that amino acids 318-373 were essential for the two-hybrid protein-protein interaction . The N-terminal part of Rpg1p (aa 1-615) was essential and sufficient for the Rpg1p-Sla2p interaction . A coimmunoprecipitation assay provided additional evidence for the physical interaction of Rpg1p/Tif32p with Sla2p in vivo . Using immunofluorescence microscopy, Rpg1p and Sla2p proteins were colocalized at the patch associated with the tip of emerging bud . Considering the essential role of Rpg1p as the large subunit of the eIF3 core complex and the association of Sla2p with the actin cytoskeleton, a putative role of the Rpg1p-Sla2p interaction in localized translation is discussed . Biochem Biophys Res Commun, 2001 Apr 20, 282(5), 1211 - 9 The role of heat shock protein 70 in vitamin D receptor function; Lutz W et al.; We previously demonstrated that the 1alpha,25-dihydroxyvitamin D(3) receptor (VDR) interacts with the constitutive heat shock protein, hsc70 in vitro, and with DnaK (Biochem . Biophys . Res . Commun . 260, 446-452, 1999) . The biological significance of VDR-heat shock protein interactions, however, is unknown . To examine the role of such interactions in eukaryotic cells, we heterologously expressed VDR and RXRalpha together with a vitamin D-responsive reporter system in Saccharomyces cerevisiae and examined the consequences of heat shock protein 70 gene (SSA) deletion in these cells . We show that heterologously expressed VDR associates with the yeast cytosolic hsp70 protein, Ssa1p . Deletion of the SSA2, SSA3, and SSA4 genes and reduction of Ssa1p activity, reduces the intracellular concentrations of the VDR and its heterodimeric partner, RXRalpha and reduces the activity of a vitamin D-dependent gene . Hsp70-like chaperone proteins play a role in controlling concentrations of the VDR within the cell . Exp Cell Res, 2001 May 1, 265(2), 195 - 202 Histone acetylation and chromatin remodeling; Gregory PD et al.; Chromatin represents a repressive barrier to the process of transcription . This molecular obstacle is a highly dynamic structure, able to compact the DNA of the entire genome into the confines of a nucleus, and yet it allows access to the genetic information held within . The acetylation of histones has emerged as a regulatory mechanism capable of modulating the properties of chromatin and thus the competence of the DNA template for transcriptional activation . The role of acetylation in chromatin remodeling is therefore of paramount importance to our understanding of gene regulation in vivo . Eur J Cell Biol, 2001 Feb, 80(2), 126 - 38 Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p; Yang X et al.; Saccharomyces cerevisiae delta3,delta2-enoyl-CoA isomerase (Eci1p), encoded by ECI1, is an essential enzyme for the betaoxidation of unsaturated fatty acids . It has been reported, as well as confirmed in this study, to be a peroxisomal protein . Unlike many other peroxisomal proteins, Ecilp possesses both a peroxisome targeting signal type 1 (PTS1)-like signal at its carboxy-terminus (-HRL) and a PTS2-like signal at its amino-terminus (RIEGPFFIIHL) . We have found that peroxisomal targeting of a fusion protein consisting of Eci1p in front of green fluorescent protein (GFP) is not dependent on Pex7p (the PTS2 receptor), ruling out a PTS2 mechanism, but is dependent on Pex5p (the PTS1 receptor) . This Pex5p-dependence was unexpected, since the putative PTS1 of Ecilp is not at the C-terminus of the fusion protein; indeed, deletion of this signal (-HRL-) from the fusion did not affect the Pex5p-dependent targeting . Consistent with this, Pex5p interacted in two-hybrid assays with both Eci1p and Eci1PdeltaHRL . Ecilp-GFP targeting and Eci1pdeltaHRL interaction were abolished by replacement of Pex5p with Pex5p(N495K), a point-mutated Pex5p that specifically abolishes the PTS1 protein import pathway . Thus, Eci1p peroxisomal targeting does require the Pex5p-dependent PTS1 pathway, but does not require a PTS1 of its own . By disruption of ECI1 and DCI1, we found that Dci1p, a peroxisomal PTS1 protein that shares 50% identity with Eci1p, is necessary for Eci1p-GFP targeting . This suggests that the Pex5p-dependent import of Eci1p-GFP is due to interaction and co-import with Dci1p . Despite the dispensability of the C-terminal HRL for import in wild-type cells, we have also shown that this tripeptide can function as a PTS1, albeit rather weakly, and is essential for targeting in the absence of Dci1p . Thus, Eci1p can be targeted to peroxisomes by its own PTS1 or as a hetero-oligomer with Dcilp . These data demonstrate a novel, redundant targeting pathway for Eci1p. J Biol Chem, 2001 Jun 29, 276(26), 24261 - 7 Epub 2001 Apr 11. Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals; Benaroudj N et al.; The disaccharide trehalose, which accumulates dramatically during heat shock and stationary phase in many organisms, enhances thermotolerance and reduces aggregation of denatured proteins . Here we report a new role for trehalose in protecting cells against oxygen radicals . Exposure of Saccharomyces cerevisiae to a mild heat shock (38 degrees C) or to a proteasome inhibitor (MG132) induced trehalose accumulation and markedly increased the viability of the cells upon exposure to a free radical-generating system (H(2)O(2)/iron) . When cells were returned to normal growth temperature (28 degrees C) or MG132 was removed from the medium, the trehalose content and resistance to oxygen radicals decreased rapidly . Furthermore, a mutant unable to synthesize trehalose was much more sensitive to killing by oxygen radicals than wild-type cells . Providing trehalose exogenously enhanced the resistance of mutant cells to H(2)O(2) . Exposure of cells to H(2)O(2) caused oxidative damage to amino acids in cellular proteins, and trehalose accumulation was found to reduce such damage . After even brief exposure to H(2)O(2), the trehalose-deficient mutant exhibited a much higher content of oxidatively damaged proteins than wild-type cells . Trehalose accumulation decreased the initial appearance of damaged proteins, presumably by acting as a free radical scavenger . Therefore, trehalose accumulation in stressed cells plays a major role in protecting cellular constituents from oxidative damage. Bioinformatics, 2001 Apr, 17(4), 309 - 18 Validating clustering for gene expression data; Yeung KY et al.; MOTIVATION: Many clustering algorithms have been proposed for the analysis of gene expression data, but little guidance is available to help choose among them . We provide a systematic framework for assessing the results of clustering algorithms . Clustering algorithms attempt to partition the genes into groups exhibiting similar patterns of variation in expression level . Our methodology is to apply a clustering algorithm to the data from all but one experimental condition . The remaining condition is used to assess the predictive power of the resulting clusters-meaningful clusters should exhibit less variation in the remaining condition than clusters formed by chance . RESULTS: We successfully applied our methodology to compare six clustering algorithms on four gene expression data sets . We found our quantitative measures of cluster quality to be positively correlated with external standards of cluster quality. Exp Hematol, 2001 Apr, 29(4), 490 - 8 Novel expression of cyclin-dependent kinase inhibitors in human B-cell precursors; Fink JR et al.; Eukaryotic cell division is regulated by cyclins, cyclin-dependent kinases (CDK), and cyclin-dependent kinase inhibitors (CKI) . Genes encoding these proteins are mutated or deleted in many types of cancer . For example, 20%-30% of B-lineage acute lymphoblastic leukemias (ALL) have deletions in the CKI known as INK4a . The contribution of INK4a deletions to the progression of B-lineage ALL is uncertain, partially due to a paucity of data on expression in normal B-cell precursors . We therefore conducted a comparative analysis of normal and leukemic human B-cell development for the expression of cyclins, CDK, and CKI.Specific stages of human B-cell development from normal bone marrow were purified by fluorescence-activated cell sorting . The sorted populations and B-lineage ALL cell lines (BLIN-1, 2, 3, 4) were examined for expression of cyclins, CDK, and CKI by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.RT-PCR analysis showed that cyclin D2, cyclin D3, CDK4, and CDK6 were ubiquitously expressed in normal B-cell development and in the BLIN ALL cell lines . The p19(INK4d) CKI was the most commonly expressed member of the INK4 family, whereas p16(INK4a) was more weakly and variably expressed . Expression of the p57(KIP2) CKI varied as a function of the stage of B-cell development . Analysis of normal B-cell precursors by Western blotting indicated that CDK4, CDK6, p19(INK4d), and p57(KIP2) were expressed, whereas p16(INK4a) was not detected.Cyclin D/CDK expression in normal and leukemic human B-cell precursors is similar to expression of these proteins in human and murine mature B cells . In contrast, the ubiquitous expression of p19(INK4d) has not been previously described in human or murine B-lineage cells . Our results suggest that loss of INK4a may only minimally contribute to tumor cell progression in B-lineage ALL, since expression of INK4d could provide a compensatory function as a cyclin-dependent kinase inhibitor. Plant Physiol, 2001 Apr, 125(4), 1813 - 20 ANT1, an aromatic and neutral amino acid transporter in Arabidopsis; Chen L et al.; A new amino acid transporter was identified from the Arabidopsis expressed sequence tag cDNAs by expressing the cDNA in a yeast amino acid transport mutant . Transport analysis of the expressed protein in yeast showed that it transports aromatic and neutral amino acids, as well as arginine . This transporter (ANT1, aromatic and neutral transporter) also transports indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid . The cDNA is 1.6 kb in length with an open reading frame that codes for a protein with 432 amino acids and a calculated molecular mass of 50 kD . Hydropathy analysis showed ANT1 is an integral membrane protein with 11 putative membrane-spanning domains . Southern analysis and a BLAST search of the Arabidopsis genome database suggests that ANT1 is part of a small gene family containing at least five members . Phylogenetic comparisons with other known amino acid transporters in plants suggests that ANT1 represents a new class of amino acid transporter . RNA gel-blot analysis showed that this transporter is expressed in all organs with highest abundance in flowers and cauline leaves. Eur J Neurosci, 2001 Apr, 13(7), 1339 - 48 Corticotropin-releasing factor triggers neurite outgrowth of a catecholaminergic immortalized neuron via cAMP and MAP kinase signalling pathways; Cibelli G et al.; Corticotropin-releasing factor (CRF), a neuropeptide of 41 amino acids, acts as the major physiological regulator of the basal and stress-induced release of corticotropin (ACTH), beta-endorphin and other proopiomelanocortin-derived peptides from the anterior pituitary gland . In addition to its endocrine activity, CRF displays extrahypophysiotropic effects, mainly as a regulator of stress responses . We show here that CRF may additionally function as a differentiating factor in immortalized noradrenergic neuronal CATH.a cells that express CRF receptor type I and resemble locus coeruleus-derived neurons . CRF triggers morphological changes in CATH.a cells including the appearance of extended long, slender neurites with prominent growth cones . CRF-treated CATH.a cells exhibit a morphology similar to locus coeruleus neurons in primary culture . CRF-induced neurite outgrowth of CATH.a cells was blocked by addition of inhibitors for cAMP-dependent protein kinase or extracellular signal-regulated protein kinase (ERK), a subtype of the mitogen-activated protein kinases . The participation of ERK within the CRF signalling cascade was further confirmed by Western blot experiments, with antibodies directed against the phosphorylated form of ERK, and also with transcription-based assays . We conclude that CRF functions as a differentiating factor of CATH.a cells via the cAMP and the MAP kinase signalling pathways. Eur J Biochem, 2001 Apr, 268(8), 2512 - 9 Multisite control of the Crabtree effect in ascites hepatoma cells; Rodriguez-Enriquez S et al.; AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively . To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined . The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8) . Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively) . The cytosolic concentrations of Ca2+ and Mg2+ were not modified . The addition of galactose or glycerol did not modify the concentrations of the metabolites . Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation . The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose. Eur J Immunol, 2001 Apr, 31(4), 1239 - 46 Tumor-associated antigens identified by mRNA expression profiling induce protective anti-tumor immunity; Mathiassen S et al.; Defined tumor-associated antigens (TAA) are attractive targets for anti-tumor immunotherapy . Here, we describe a novel genome-wide approach to identify multiple TAA from any given tumor . A panel of transplantable thymomas was established from an inbred p53-/- mouse strain . The resulting tumors were examined for gene expression by mRNA microarray scanning . This analysis revealed heterogeneity of the tumors in agreement with the assumption that they represent different tumorigenic events . Several genes were overexpressed in one or more of the tumors . To examine whether overexpressed genes might be used to identify TAA, mice were immunized with mixtures of peptides representing putative cytotoxic T cell epitopes derived from one of the gene products . Indeed, such immunized mice were partially protected against subsequent tumor challenge . Despite being immunized with bona fide self antigens, no clinical signs of autoimmune reactions were observed . Thus, it appears possible to evaluate the entire metabolism of any given tumor and use this information rationally to identify multiple epitopes of value in the generation of tumor-specific immunotherapy . We expect that human tumors express similar tumor-specific metabolic imprints, which may be used to identify patient-specific arrays of TAA . This may enable a multi-epitope based immunotherapy with improved prospects of clinical tumor rejection. Curr Opin Struct Biol, 2001 Apr, 11(2), 163 - 73 Structural insights into the molecular mechanism of calcium-dependent vesicle-membrane fusion; Brunger AT; The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions . Recently determined structures of the SNARE complex, synaptotagmin III, nSec1, domains of the NSF chaperone and its adaptor (SNAP), and Rab3 and some of its effectors provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models . Ultimately, knowledge of the structures of higher-order complexes and their dynamic behavior will be required to obtain a full understanding of the vesicle fusion protein machinery. FEBS Lett, 2001 Apr 6, 494(1-2), 90 - 4 Functional characterization of sphingolipid C4-hydroxylase genes from Arabidopsis thaliana; Sperling P et al.; In the genome of Arabidopsis thaliana, two genes were identified encoding isoenzymes for C4-hydroxylation of long chain bases (LCB) in plant sphingolipids . Both predicted proteins consist of 258 amino acid residues (77% identity) which show sequence similarity to di-iron-binding enzymes, such as Sur2p and Erg3p from yeast, involved in oxygen-dependent lipid modifications . Heterologous expression of these genes in a yeast sur2Delta-null mutant lacking C4-LCB hydroxylation resulted in the formation of D-ribo-C(18)- and -C(20)-phytosphinganine . The identity and stereochemical configuration of the isolated trihydroxybases was confirmed by electrospray ionization-mass spectroscopy, gas-liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance spectroscopy . These results represent the first functional identification of SUR2 genes from plants as well as from any organism other than yeast. FEBS Lett, 2001 Apr 6, 494(1-2), 64 - 8 Why Ppr1p is a weak activator of transcription; Patzold AJ et al.; Upon uracil depletion, the transcriptional activator Ppr1p stimulates expression of the Saccharomyces cerevisiae URA3 gene only four-fold . We performed a split-ubiquitin screen with Tup1p as bait, and we found that the global repressor Tup1p interacts with the transcriptional activator Ppr1p both in vivo and in vitro . The interaction is biologically significant, since the deletion of the TUP1 gene as well as the removal of the Tup1p-binding domain from Ppr1p results in an increased expression of the URA3 gene . Our results suggest that Tup1p blocks Ppr1p directly, and that Ppr1p is a weak activator of transcription because of its interaction with Tup1p . Thus we were able to demonstrate that the global repressor Tup1p can modulate transcription by interacting with an activator. FEBS Lett, 2001 Apr 6, 494(1-2), 38 - 43 The second cysteine-rich domain of Mac1p is a potent transactivator that modulates DNA binding efficiency and functionality of the protein; Voutsina A et al.; Mac1p is a Saccharomyces cerevisiae DNA binding transcription factor that activates genes involved in copper uptake . A copper-induced N-C-terminal intramolecular interaction and copper-independent homodimerization affect its function . Here, we present a functional analysis of Mac1p deletion derivatives that attributes new roles to the second cysteine-rich (REPII) domain of the protein . This domain exhibits the copper-responsive potent transactivation function when assayed independently and, in the context of the entire protein, modulates the efficiency of Mac1p binding to DNA . The efficiency of binding to both copper-response promoter elements can determine the in vivo functionality of Mac1p independent of homodimerization. FEBS Lett, 2001 Apr 6, 494(1-2), 6 - 10 A putative proton binding site of plasma membrane H(+)-ATPase identified through homology modelling; Bukrinsky JT et al.; We have used the 2.6 A structure of the rabbit sarcoplasmic reticulum Ca(2+)-ATPase isoform 1a, SERCA1a {Toyoshima, C., Nakasako, M., Nomura, H . and Ogawa, H . (2000) Nature 405, 647-655}, to build models by homology modelling of two plasma membrane (PM) H(+)-ATPases, Arabidopsis thaliana AHA2 and Saccharomyces cerevisiae PMA1 . We propose that in both yeast and plant PM H(+)-ATPases a strictly conserved aspartate in transmembrane segment (M)6 (D684(AHA2)/D730(PMA1)), and three backbone carbonyls in M4 (I282(AHA2)/I331(PMA1), G283(AHA2)/I332(PMA1) and I285(AHA2)/V334(PMA1)) comprise a binding site for H3O(+), suggesting a previously unknown mechanism for transport of protons . Comparison with the structure of the SERCA1a made it feasible to suggest a possible receptor region for the C-terminal auto-inhibitory domain extending from the phosphorylation and anchor domains into the transmembrane region. FEBS Lett, 2001 Apr 6, 494(1-2), 1 - 5 The rotor in the membrane of the ATP synthase and relatives; Arechaga I et al.; In recent years, structural information on the F(1) sector of the ATP synthase has provided an insight into the molecular mechanism of ATP catalysis . The structure strongly supports the proposal that the ATP synthase works as a rotary molecular motor . Insights into the membrane domain have just started to emerge but more detailed structural information is needed if the molecular mechanism of proton translocation coupled to ATP synthesis is to be understood . This review will focus mainly on the ion translocating rotor in the membrane domain of the F-type ATPase, and the related vacuolar and archaeal relatives. J Clin Endocrinol Metab, 2001 Apr, 86(4), 1539 - 44 Novel assay for determination of androgen bioactivity in human serum; Raivio T et al.; We have developed a mammalian cell (COS-1) bioassay, which can measure androgen bioactivity directly from a small amount (10 microL) of human serum . The recombinant assay is based on androgen-dependent interaction between the ligand-binding domain and the N-terminal region of the androgen receptor (AR), which were fused to Gal4 DNA-binding domain of Saccharomyces cerevisiae and transcriptional activation domain of herpes simplex VP16 protein, respectively . The interaction is amplified by coexpression of AR-interacting protein 3 in the cells . The reporter plasmid contains 5 Gal4-binding sites upstream of the luciferase gene; luciferase activity in cell lysates is derived from androgen bioactivity in human serum . Saturating concentration of testosterone in FCS induced more than 700-fold induction in relative luciferase activity . The sensitivity was less than 1.0 nmol/L testosterone in FCS . The intra- and interassay coefficients of variation were 8.3% and 21%, respectively . Interaction between the AR termini was blocked by nonsteroidal antiandrogens, and the assay exhibited minimal cross-reactivity with 17 beta-estradiol . Serum androgen bioactivity was studied in 23 boys (13.9--16.8 yr old) with constitutional delay of puberty and in 9 prepubertal boys with cryptorchidism (1.0--6.4 yr old) . Androgen bioactivity was detectable in 15 boys with constitutional delay of puberty and in all boys with cryptorchidism during treatment with human CG (range, 1.0-14.5 nmol/L testosterone equivalents) . Serum androgen bioactivity measured by the bioassay correlated strongly with serum testosterone concentration (r = 0.93, P < 0.0001, n = 22) but not to 5 alpha-dihydrotestosterone, dehydroepiandrosterone, or androstenedione levels . We conclude that our novel bioassay enables quantitation of mammalian cell response to bioactive androgens in human serum, even in pediatric patients with relatively low androgen levels. J Biol Chem, 2001 Apr 27, 276(17), 14257 - 63 Epub 2001 Jan 31. N-methyl-D-aspartate receptors regulate a group of transiently expressed genes in the developing brain; Sugiura N et al.; Mammalian brain development requires the transmission of electrical signals between neurons via the N-methyl-d-aspartate (NMDA) class of glutamate receptors . However, little is known about how NMDA receptors carry out this role . Here we report the first genes shown to be regulated by physiological levels of NMDA receptor function in developing neurons in vivo: NMDA receptor-regulated gene 1 (NARG1), NARG2, and NARG3 . These genes share several striking regulatory features . All three are expressed at high levels in the neonatal brain in regions of neuronal proliferation and migration, are dramatically down-regulated during early postnatal development, and are down-regulated by NMDA receptor function . NARG2 and NARG3 appear to be novel, while NARG1 is the mammalian homologue of a yeast N-terminal acetyltransferase that regulates entry into the G(o) phase of the cell cycle . The results suggest that highly specific NMDA receptor-dependent regulation of gene expression plays an important role in the transition from proliferation of neuronal precursors to differentiation of neurons. J Biol Chem, 2001 May 11, 276(19), 16520 - 7 Epub 2001 Jan 31. 5-Lipoxygenase interacts with coactosin-like protein; Provost P et al.; We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait . In this report, we demonstrate a direct interaction between 5LO and CLP . 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner . Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates . In reciprocal experiments, 5LO was detected in CLP immunoprecipitates . Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca(2+)-independent manner . Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding . In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments . However, no F-actin-CLP.5LO ternary complex was observed . In contrast, 5LO appeared to compete with F-actin for the binding of CLP . Moreover, 5LO was found to interfere with actin polymerization . Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics. Biochemistry, 2001 Mar 27, 40(12), 3657 - 65 Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2; Rockwell NC et al.; Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2 . These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity . Pre-steady-state studies have shown that both Kex2 and furin exhibit an initial burst of 7-amino-4-methylcoumarin release in cleavage of peptidyl methylcoumarinamide substrates that are based on physiological cleavage sites . Thus, in cleavage of such substrates, formation of the acylenzyme intermediate is fast relative to some later step (deacylation or N-terminal product release) . This behavior is significant, because Kex2 also exhibits burst kinetics in cleavage of peptide bonds . k(cat) for cleavage of a tetrapeptidyl methylcoumarinamide substrate based on the physiological yeast substrate pro-alpha-factor exhibits a weak solvent isotope effect, but neither this isotope effect nor temperature dependence studies with this substrate conclusively identify the rate-limiting step for Kex2 cleavage of this substrate . We therefore developed an assay to measure deacylation directly by pulse-chase incorporation of H(2)(18)O in a rapid-quenched-flow mixer followed by mass spectrometric quantitation . The results given by this assay rule out rate-limiting product release for cleavage of this substrate by Kex2 . These experiments demonstrate that cleavage of the acylenzyme ester bond, as opposed to either the initial attack on the amide bond or product release, is rate-limiting for the action of Kex2 at physiological sequences . This work demonstrates a fundamental difference in the catalytic strategy of proprotein processing enzymes and degradative subtilisins. Biochemistry, 2001 Mar 27, 40(12), 3544 - 52 Energetics of coiled coil folding: the nature of the transition states; Bosshard HR et al.; Coiled coils are simple models for studying the association of two polypeptide chains to form a folded protein . Previous work has shown that the folding of a coiled coil can be described by a two-state transition between two unfolded monomeric peptide chains and a folded coiled coil dimer . Here we report the thermodynamic activation parameters for the folding and unfolding of two unrelated coiled coils: C62GCN4 and A(2) . C62GCN4 corresponds to the 62 C-terminal residues of yeast transcription factor GCN4 . The peptide forms a dimeric coiled coil through its 33 C-terminal residues . A(2) is a designed 30-residue dimeric coiled coil whose folding is induced by low pH {Durr, E., Jelesarov, I., and Bosshard, H . R . (1999) Biochemistry 38, 870-880} . Folding and unfolding were assessed under identical native buffer conditions so that the microscopic reversibility applied and the transition state was the same for folding and unfolding . The time course of folding was followed from the self-quenching of a C-terminal fluorescent label (Texas Red) . The overall folding of both peptides is enthalpy-driven and opposed by a loss of entropy . The main energetic changes occur after the system has passed the transition state . In the folding of C62GCN4, only 10-20% of the heat capacity change is attained between the monomeric state and the dimeric transition state . For coiled coil A(2), the fractional heat capacity change preceding the transition state is 30-40% . The results indicate that the activated states of folding of coiled coils are not well structured and differ considerably from the folded coiled coil conformation . These findings are in agreement with a rate-limiting transition state in which the coiled coil helices and the hydrophobic coiled coil interface are poorly developed. EMBO J, 2001 Apr 17, 20(8), 2028 - 40 MOT1-catalyzed TBP-DNA disruption: uncoupling DNA conformational change and role of upstream DNA; Darst RP et al.; SNF2/SWI2-related ATPases employ ATP hydrolysis to disrupt protein-DNA interactions, but how ATP hydrolysis is coupled to disruption is not understood . Here we examine the mechanism of action of MOT1, a yeast SNF2/SWI2-related ATPase that uses ATP hydrolysis to remove TATA binding protein (TBP) from DNA . MOT1 function requires a 17 bp DNA 'handle' upstream of the TATA box, which must be double stranded . Remarkably, MOT1-catalyzed disruption of TBP-DNA does not appear to require DNA strand separation, DNA bending or twisting of the DNA helix . Thus, TBP-DNA disruption is accomplished in a reaction apparently not driven by a change in DNA structure . MOT1 action is supported by DNA templates in which the handle is connected to the TATA box via single-stranded DNA, indicating that the upstream duplex DNA can be conformationally uncoupled from the TATA box . Combining these results with proposed similarities between SNF2/SWI2 ATPases and helicases, we suggest that MOT1 uses ATP hydrolysis to translocate along the handle and thereby disrupt interactions between TBP and DNA. EMBO J, 2001 Apr 17, 20(8), 1984 - 92 Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity; Soutoglou E et al.; CREB-binding protein (CBP) and CBP-associated factor (P/CAF) are coactivators possessing an intrinsic histone acetyltransferase (HAT) activity . They are positioned at promoter regions via association with sequence-specific DNA-binding factors and stimulate transcription in a gene-specific manner . The current view suggests that coactivator function depends mainly on the strength and specificity of transcription factor-coactivator interactions . Here we show that two dominant-negative mutants of hepatocyte nuclear factor-1alpha (HNF-1alpha), P447L and P519L, occurring in maturity onset diabetes of the young (MODY3) patients, exhibit paradoxically stronger interactions than the wild-type protein with either CBP or P/CAF . However, CBP and P/CAF recruited by these mutants lack HAT activity . In contrast, wild-type HNF-1alpha and other transcription factors, such as Sp1 or HNF-4, stimulated the HAT activity of CBP . The results suggest a more dynamic role for DNA-binding proteins in the transcription process than was considered previously . They are not only required for the recruitment of coactivators to the promoter but they may also modulate their enzymatic activity. Cancer Lett, 2001 May 10, 166(1), 55 - 64 Paradoxical effects of trichostatin A: inhibition of NF-Y-associated histone acetyltransferase activity, phosphorylation of hGCN5 and downregulation of cyclin A and B1 mRNA; Nair AR et al.; Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), is widely used to study the role of histone acetylation in gene expression, since genes that use histone acetylation as a means of regulating expression may be up regulated when TSA is added . In this study, however, we show that TSA has an unexpected paradoxical effect leading to inhibition of NF-Y-associated histone acetyl transferase (HAT) activity and phosphorylation of the HAT, hGCN5 . TSA treatment of cells resulted in diminished levels of NF-Y-associated HAT activity without changes in NF-Y(A) amount . hGCN5 is one of the HATs known to associate with NF-Y . The association of hGCN5 with NF-Y was not altered by TSA treatment . The enzymatic activity of hGCN5 is known to be inhibited by phosphorylation . TSA treatment of Hela cells resulted in phosphorylation of hGCN5 . Exposure of the NF-Y immunoprecipitates from TSA-treated cells to a phosphatase resulted in enhanced HAT activity . We have also shown that the mRNA levels of several genes, cyclin B1 and cyclin A, are downregulated by TSA; these effects do not require protein synthesis and the downregulation of cyclin B1 by TSA occurs through transcription . These results suggest that TSA can have contradictory effects, on one hand stimulating HAT activity in general by inhibition of HDACs, but also resulting in inhibition of NF-Y-associated HAT activity and phosphorylation of hGCN5. Brain Res Mol Brain Res, 2001 Mar 31, 88(1-2), 124 - 34 Localization of mRNAs for six ARFs (ADP-ribosylation factors) in the brain of developing and adult rats and changes in the expression in the hypoglossal nucleus after its axotomy; Suzuki I et al.; ADP-ribosylation factors (ARFs) play crucial roles in the intracellular vesicular transport and in regulation of phospholipid-modifying enzyme activities and cytoskeletons . Using in situ hybridization histochemistry, the gene expression for six isoforms of ARF was examined during normal development of rats and in the hypoglossal nucleus following its axotomy . In the embryonic brain, the expression for ARF-1, -4, -5, -6 mRNAs was distinct in the ventricular germinal zone while that for ARF-3, -4, -5 in the mantle zone . In early postnatal brain, the expression for six ARFs was seen widely in various loci of the gray matter with different intensity, and the expression of ARF-4, -5, -6 mRNAs was evident in the cerebellar external granule cell layer . In the adult brain, the gene expression for all ARF isoforms decreased more or less in most gray matters and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex . The expression levels of ARF-2 and -4 mRNAs in affected hypoglossal nucleus increased after 24 h up to 7 days following axotomy of the hypoglossal nerve, while no such changes were seen in the expression levels for the other ARFs . The present findings suggest that ARFs are differentially involved in some processes essential to nerve regeneration as well as neuronal differentiation and maturation. Mol Biol Cell, 2001 Apr, 12(4), 1093 - 101 O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation; Harty C et al.; Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes . It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins . We asked whether misfolded secretory proteins are covalently modified in the ER before export . We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pmt2p) . O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane . Deletion of PMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells . In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen . Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage . We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel. Mol Biol Cell, 2001 Apr, 12(4), 1035 - 45 The ADP ribosylation factor-nucleotide exchange factors Gea1p and Gea2p have overlapping, but not redundant functions in retrograde transport from the Golgi to the endoplasmic reticulum; Spang A et al.; The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors . Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p . We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant . SEC21 encodes the gamma-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat . GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable . Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions . The two genes do not serve completely overlapping functions because a Deltagea1 Deltaarf1 mutant is not more sickly than a Deltaarf1 strain, whereas Deltagea2 Deltaarf1 is inviable . Biochemical experiments revealed similar distributions and activities for the two proteins . Gea1p and Gea2p exist both in membrane-bound and in soluble forms . The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum . In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking. J Biol Chem, 2001 Jun 22, 276(25), 22788 - 96 Epub 2001 Apr 09. Activation and functional characterization of the mosaic receptor SorLA/LR11; Jacobsen L et al.; We previously isolated and sequenced the approximately 250-kDa type 1 receptor sorLA/LR11, a mosaic protein with elements characterizing the Vps10p domain receptor family as well as the low density lipoprotein receptor family . The N terminus of the Vps10p domain comprises a consensus sequence for cleavage by furin ((50)RRKR(53)) that precedes a truncation found in sorLA isolated from human brain . Here we show that sorLA, like sortilin-1/neurotensin receptor-3, whose lumenal domain consists of a Vps10p domain only, is synthesized as a proreceptor that is cleaved by furin in late Golgi compartments . We show that the truncation conditions the Vps10p domain for propeptide inhibitable binding of neuropeptides and the receptor-associated protein . We further demonstrate that avid binding of the receptor-associated protein, apolipoprotein E, and lipoprotein lipase not inhibited by propeptide occurs to sites located in other lumenal domains . In transfected cells, about 10% of full-length sorLA were expressed on the cell surface capable of mediating endocytosis . However, the major pool of receptors was found in late Golgi compartments, suggesting possible interaction with newly synthesized ligands . The results show that sorLA, following activation by truncation, binds multiple ligands and may mediate both endocytosis and sorting. J Biol Chem, 2001 Jun 22, 276(25), 22810 - 8 Epub 2001 Apr 05. Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery; Suvorova ES et al.; The roles of the components of the Sec34p protein complex in intracellular membrane trafficking, first identified in the yeast Saccharomyces cerevisiae, have yet to be characterized in higher eukaryotes . We cloned a human cDNA whose predicted amino acid sequence showed 41% similarity to yeast Sec34p with homology throughout the entire coding region . Affinity-purified antibodies raised against the human SEC34 protein (hSec34p) recognized a cellular protein of 94 kDa in both soluble and membrane fractions . Like yeast Sec34p, cytosolic hSec34p migrated with an apparent molecular mass of 300 kDa on a glycerol velocity gradient, suggesting that it is part of a protein complex . Immunofluorescence microscopy localized hSec34p to the Golgi compartment in cells of all species examined, where it co-localized well with the cis/medial Golgi marker membrin and partially co-localized with cis-Golgi network marker p115 and trans-Golgi marker TGN38 . The co-localization with membrin was maintained at 15 degrees C and after microtubule depolymerization with nocodazole . During transport of the tsO45 vesicular stomatitis virus G protein through the Golgi, there was significant overlap with the hSec34p compartment . Green fluorescent protein-hSec34 expressed in HeLa cells was restricted to Golgi cisternae, and its membrane association was sensitive to brefeldin A treatment . Taken together, our findings indicate that hSec34p is part of a peripheral membrane protein complex localized on cis/medial Golgi cisternae where it may participate in tethering intra-Golgi transport vesicles. J Immunol Methods, 2001 May 1, 251(1-2), 53 - 61 A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera; Jafri F et al.; Sera from patients with systemic lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease are frequently characterized by the presence of high levels of autoantibodies directed against linked sets of nuclear proteins . One of these autoantigen systems is made up of Ku and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), proteins that are essential for double-strand DNA break repair and for the related process of V(D)J recombination . Ku and DNA-PKcs bind avidly to DNA ends in vivo and in vitro and form an active protein kinase complex . One hypothesis is that this assembled nucleoprotein particle, rather than its component proteins, is a primary trigger for the autoimmune response and thus a major target for the resulting autoantibodies . To screen for particle-specific antibodies, we developed an assay in which the fully native nucleoprotein particle is reconstituted in vitro and is tethered to the surface of an ELISA plate via a streptavidin-biotin linkage . These particles are recognized efficiently by monoclonal antibodies and by autoantibodies present in patient sera . The assay may detect a broader spectrum of epitopes than a conventional ELISA in which Ku and DNA-PKcs are adsorbed directly to a plastic surface . The method will be advantageous for high-throughput screening for antibodies and other ligands that bind the assembled DNA-dependent protein kinase complex. Semin Cell Dev Biol, 2001 Apr, 12(2), 183 - 91 Phosphatidylinositol transfer proteins couple lipid transport to phosphoinositide synthesis; Cockcroft S; Phosphatidylinositol transfer proteins (PITPs) are lipid binding proteins that can catalyse the transfer of phosphatidylinositol (PI) from membranes enriched in PI to PI-deficient membranes . Three soluble forms of PITP of 35--38 kDa (PITP alpha, PITP beta and rdgB beta) and two larger integral proteins of 160 kDa (rdgB alpha I and II), which contain a PITP domain, are found in mammalian cells . PITPs are intimately associated with the compartmentalised synthesis of different phosphorylated inositol lipids . PI is the primary inositol lipid that is synthesised at the endoplasmic reticulum and is further phosphorylated in distinct membrane compartments by many specific lipid kinases to generate seven phosphorylated inositol lipids which are required for both signalling and for membrane traffic . PITPs play essential roles in both signalling via phospholipase C and phosphoinositide 3-kinases and in multiple aspects of membrane traffic including regulated exocytosis and vesicle biogenesis . J Mol Biol, 2001 Apr 13, 307(5), 1427 - 50 Socket: a program for identifying and analysing coiled-coil motifs within protein structures; Walshaw J et al.; The coiled coil is arguably the simplest protein-structure motif and probably the most ubiquitous facilitator of protein-protein interactions . Coiled coils comprise two or more alpha-helices that wind around each other to form "supercoils" . The hallmark of most coiled coils is a regular sequence pattern known as the heptad repeat . Despite this apparent simplicity and relatedness at the sequence level, coiled coils display a considerable degree of structural diversity: the helices may be arranged parallel or anti-parallel and may form a variety of oligomer states . To aid studies of coiled coils, we developed SOCKET, a computer program to identify these motifs automatically in protein structures . We used SOCKET to gather a set of unambiguous coiled-coil structures from the RCSB Protein Data Bank . Rather than searching for sequence features, the algorithm recognises the characteristic knobs-into-holes side-chain packing of coiled coils; this proved to be straightforward to implement and was able to distinguish coiled coils from the great majority of helix-helix packing arrangements observed in globular domains . SOCKET unambiguously defines coiled-coil helix boundaries, oligomerisation states and helix orientations, and also assigns heptad registers . Structures retrieved from the Protein Data Bank included parallel and anti-parallel variants of two, three and four-stranded coiled coils, one example of a parallel pentamer and a small number of structures that extend the classical description of a coiled coil . We anticipate that our structural database and the associated sequence data that we have gathered will be of use in identifying principles for coiled-coil assembly, prediction and design . To illustrate this we give examples of sequence and structural analyses of the structures that are possible using the new data bases, and we present amino acid profiles for the heptad repeats of different motifs . J Mol Biol, 2001 Apr 13, 307(5), 1247 - 60 Supporting the structural basis of prion strains: induction and identification of {PSI} variants; King CY; The {PSI} genetic element, which enhances the nonsense suppression efficiency in the yeast Saccharomyces cerevisiae, is thought to be amyloid-like aggregates of the Sup35 protein, and to self-propagate by a prion-like mechanism . Analogous to strains of the mammalian prion, variants of {PSI}, with different nonsense suppression efficiencies and mitotic stabilities, can be isolated from the same yeast genetic background . In the framework of the "protein-only" hypothesis, variants of prion are assumed to be distinct conformers of the same prion polypeptide . This study aims to provide further support for the structural basis of {PSI} variation . Three variants of {PSI} were induced and distinguished by a panel of 11 single point mutations of the Sup35 protein . The variant phenotypes are intrinsically associated with {PSI} elements, presumably structurally different amyloids, rather than produced from variations in the genetic background . Differential incorporation to {PSI} variants of a Sup35 point mutation as well as N and C-terminally truncated Sup35 fragments is further demonstrated in vivo, suggesting that distinct patches of amino acid residues are involved in the assembly of {PSI} variants . These results establish a method for {PSI} variant-typing and indicate that heritable variations of amyloid structures can be derived from the same polypeptide . Immunol Rev, 2001 Feb, 179, 156 - 62 Regulation of eosinophil and neutrophil apoptosis--similarities and differences; Simon HU; Apoptosis is the most common form of physiologic cell death and a necessary process to maintain cell numbers in multicellular organisms . In many chronic inflammatory diseases, reduced cell death of different types of granulocytes is one important mechanism for cell accumulation . Granulocytes are constantly produced in large amounts in the bone marrow and the same numbers die, under normal circumstances, within a defined time period . Changing the rate of apoptosis rapidly changes cell numbers in such systems . Overexpression of IL-5 appears to be crucial for delaying eosinophil apoptosis in many allergic disorders, whereas overexpression of GM-CSF and G-CSF is associated with suppression of neutrophil apoptosis in bacterial and non-bacterial inflammations . Cytokine withdrawal leads to the induction of apoptosis both in vitro and in vivo . In contrast to the role of survival cytokines, little is known about the role of death factors and their receptors in the regulation of granulocyte apoptosis . Recent observations suggest a role for mitochondria in both eosinophil and neutrophil apoptosis, although the mechanisms that trigger mitochondria to release pro-apoptotic factors remain to be determined . Besides similarities, there are differences in the regulation of apoptosis between these granulocyte subtypes that include both expression and function of Bcl-2 and caspase family members . The identification of differences in the apoptosis regulation may help to define new molecular targets that allow specific induction of either eosinophil or neutrophil apoptosis by pharmacological means. J Neurobiol, 2001 May, 47(2), 81 - 92 Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons; Kitamoto T; Behavior is a manifestation of temporally and spatially defined neuronal activities . To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit . I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila . In this method, a temperature-sensitive allele of shibire (shi(ts1)) is overexpressed in neuronal subsets using the GAL4/UAS system . Because the shi gene product is essential for synaptic vesicle recycling, and shi(ts1) is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi(ts1) expressing neurons . When shi(ts1) expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30 degrees C . This temperature-induced paralysis was reversible . After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min . When shi(ts1) was expressed in photoreceptor cells, adults and larvae exhibited temperature-dependent blindness . These observations show that the GAL4/UAS system can be used to express shi(ts1) in a specific subset of neurons to cause temperature-dependent changes in behavior . Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals . Biotechnol Bioeng, 2000-2001, 71(3), 217 - 22 Detection of toxic chemicals with high sensitivity by measuring the quantity of induced P450 mRNAs based on surface plasmon resonance; Oyama M et al.; In this study we describe a novel sensor system to detect toxic chemicals based on measurement of the quantity of Saccharomyces cerevisiae P450 mRNAs induced by them . Detection was conducted using a flow-injection-type sensor system based on surface plasmon resonance (SPR) . The DNA and peptide nucleic acid (PNA) probes containing a complementary sequence to a part of P450 mRNA were immobilized on the sensor chip and the P450 mRNAs hybridized to the probes were quantified . We succeeded in detecting 10 ng/L (10 ppt) of atrazine using both DNA and PNA probes . Using this sensor system, we were able to detect bisphenol A in addition to atrazine . Furthermore, we achieved higher sensitivity by amplifying the target P450 mRNA based on nucleic acid sequence-based amplification (NASBA) . This method allows for sensitive, rapid, and easy detection of some toxic chemicals . Genetics, 2001 Apr, 157(4), 1639 - 48 Transgenic analysis of the Smad family of TGF-beta signal transducers in Drosophila melanogaster suggests new roles and new interactions between family members; Marquez RM et al.; Smad signal transducers are required for transforming growth factor-beta-mediated developmental events in many organisms including humans . However, the roles of individual human Smad genes (hSmads) in development are largely unknown . Our hypothesis is that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad) . We expressed six hSmad and four dSmad transgenes in Drosophila melanogaster using the Gal4/UAS system and compared their phenotypes . Phylogenetically related human and Drosophila Smads induced similar phenotypes supporting the hypothesis . In contrast, two nearly identical hSmads generated distinct phenotypes . When expressed in wing imaginal disks, hSmad2 induced oversize wings while hSmad3 induced cell death . This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles . Our observations also suggest new roles for the dSmads, Med and Dad, in dActivin signaling and potential interactions be