Yeasts

Exp Cell Res, 2000 May 25, 257(1), 22 - 32
Cloning and characterization of DIP1, a novel protein that is related to the Id family of proteins; Yao Y et al.; Using human cyclin D1 as the "bait" in a yeast two-hybrid system, together with a HL60 cDNA library, we identified a novel human nuclear protein designated DIP1 . This protein is expressed in a variety of cell types, and in fibroblasts its level remains constant throughout the cell cycle . However, the level of this protein increases severalfold during the differentiation of HL60 cells . The DIP1 protein can be phosphorylated in vitro by a cellular kinase and this activity reaches its maximum in extracts obtained from cells in the G1 phase of the cell cycle . DIP1 contains a helix-loop-helix motif but lacks an adjacent basic DNA-binding domain, thus resembling the Id family of proteins . The dip1 gene is located on human chromosome 16p11.2-12, a locus that is amplified in several types of human cancer . These results suggest that DIP1 may be involved in the control of gene expression and differentiation, but its precise function remains to be determined.

Curr Genet, 2000 May, 37(5), 328 - 32
Mutation of a putative AMPK phosphorylation site abolishes the repressor activity but not the nuclear targeting of the fungal glucose regulator CRE1; Vautard-Mey G et al.; In filamentous ascomycetes, glucose repression is mediated by CRE1, a zinc-finger protein related to Miglp from yeast . Five putative AMPK phosphorylation motifs identified in the glucose repressor from the phytopathogenic fungus Sclerotinia sclerotiorum were mutated in a GFP::CRE1 translational fusion . Complementation experiments in Aspergillus nidulans and fluorescence microscopy analyses showed that mutation of one site (Ser266) abolishes the repressor activity of the fusion protein but not its nuclear targeting, suggesting that an AMPK protein kinase may be involved in the function of the fungal glucose repressor.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7178 - 83
A role of transcriptional activators as antirepressors for the autoinhibitory activity of TATA box binding of transcription factor IID; Kotani T et al.; The TATA box-binding activity of transcription factor IID (TFIID) is autoinhibited by the N-terminal domain of the Drosophila TATA box-binding protein- (TBP) associated factor 230/yeast TBP-associated factor 145 subunit, which binds to the TATA box-binding domain of TBP by mimicking the TATA box structure . Here, we propose a mechanism of transcriptional activation that involves antirepression of this autoinhibitory activity by transcriptional activators . Like the autoinhibitory domain of TFIID, various acidic activators interact with the TATA box-binding domain of TBP . Moreover, the autoinhibitory domain of TFIID, which is known to interact with only the TATA box-binding domain of TBP, acts as an activation domain when fused to the GAL4 DNA-binding domain, indicating that interaction with the TATA-binding domain of TBP is crucial for activation of transcription . In a reciprocal fashion, the acidic activation domains can function as the autoinhibitory domain when the latter is replaced by the former within TFIID . These results indicate that activation domains and the autoinhibitory domain of TFIID are interchangeable, supporting a role for transcriptional activators as antirepressors of the autoinhibitory activity of the TATA box binding of TFIID.

Int J Oncol, 2000 Jul, 17(1), 189 - 95
Binding of the growth suppressor p53 protein to the cell cycle regulator phosphatase cdc25C; Rief N et al.; Human p53 is a growth suppressor which not only functions in mammalian cells but also in fission yeast . It was previously shown that the cell cycle regulating phosphatase cdc25C suppresses the p53 induced growth arrest in fission yeast . In the present study we analysed the mechanism of this suppression . We found that cdc25C directly interacts with p53 . By using different deletion mutants the binding region was narrowed down on the polypeptide chain of p53 to amino acids 287-340 . To test the functional significance we analysed the effect of this interaction on the DNA binding activity of p53 . As shown by band shift experiments binding of cdc25C to p53 does not modify the DNA binding activity of p53 . Our data suggest that the observed suppression of the p53 induced growth arrest by cdc25C might be achieved by direct binding of cdc25C to the C-terminus of p53.

J Biol Chem, 2000 Aug 25, 275(34), 26316 - 21
A novel human rad54 homologue, Rad54B, associates with Rad51; Tanaka K et al.; Members of the SNF2/SWI2 family, characterized with sequence motifs similar to those found in DNA and RNA helicases, play roles in various aspects of cellular fundamental processes such as transcriptional regulation, chromosome stability, nucleotide excision repair, and recombination . We have isolated a novel member of the human SNF2/SWI2 family, RAD54B, which is highly homologous to mammalian RAD54 . The RAD54 gene is a member of the RAD52 epistasis group which is involved in the recombinational repair of DNA damage . Here we demonstrate that human Rad54B (hRad54B), like human Rad54 (hRad54), associates with human Rad51 (hRad51) . Both hRad54B and hRad54 associate with hRad51 through their NH(2)-terminal domains, but there are differences in their ways of association with hRad51 . In contrast to Rad54, whose association with Rad51 is induced by ionizing radiation, Rad54B associates with Rad51 constitutively in immunoprecipitation experiments . Also, the failure to detect the interaction between hRad54B and hRad51 in the yeast two-hybrid assay suggests that their interaction, unlike that between hRad54 and hRad51, may be indirect . Immunofluorescence microscopy revealed that hRad54B formed nuclear foci that colocalized with hRad51, hRad54, and BRCA1 . These findings suggest that Rad54B may be functionally distinct from Rad54, although it may play an active role in recombination processes in concert with other members of the RAD52 epistasis group.

J Biol Chem, 2000 Aug 25, 275(34), 26158 - 63
Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells; Trepakova ES et al.; Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC) . Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC . Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells . In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets . The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes . Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches . These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.

Blood, 2000 Jun 15, 95(12), 3945 - 50
Differential expression of a novel C-terminally truncated splice form of SMAD5 in hematopoietic stem cells and leukemia; Jiang Y et al.; SMADs are evolutionarily conserved transducers of the differentiation and growth arrest signals from the transforming growth factor/BMP (TGF/BMP) family of ligands . Upon receptor activation, the ligand-restricted SMADs(1-35) are phosphorylated in the C-terminal MH2 domain and recruit the common subunit SMAD4/DPC-4 gene to the nucleus to mediate target gene expression . Frequent inactivating mutations of SMAD4, or less common somatic mutations of SMAD2 seen in solid tumors, suggest that these genes have a suppressor function . However, there have been no identified mutations of SMAD5, although the gene localizes to the critical region of loss in chromosome 5q31.1 (chromosome 5, long arm, region 3, band 1, subband 1) in myelodysplasia (MDS) and acute myelogenous leukemia (AML) . A ubiquitously expressed novel isoform, SMAD5beta, encodes a 351 amino acid protein with a truncated MH2 domain and a unique C-terminal tail of 18 amino acids, which may be the functional equivalent of inactivating mutations . The levels of SMAD5beta transcripts are higher in the undifferentiated CD34(+) hematopoietic stem cells than in the terminally differentiated peripheral blood leukocytes, thereby implicating the beta form in stem cell homeostasis . Yeast 2-hybrid interaction assays reveal the lack of physical interactions between SMAD5beta and SMAD5 or SMAD4 . The expression of SMAD5beta may represent a novel mechanism to protect pluripotent stem cells and malignant cells from the growth inhibitory and differentiation signals of BMPs . (Blood . 2000;95:3945-3950)

J Neurosci, 2000 Jun 15, 20(12), 4615 - 26
Lens injury stimulates axon regeneration in the mature rat optic nerve; Leon S et al.; In mature mammals, retinal ganglion cells (RGCs) are unable to regenerate their axons after optic nerve injury, and they soon undergo apoptotic cell death . However, a small puncture wound to the lens enhances RGC survival and enables these cells to regenerate their axons into the normally inhibitory environment of the optic nerve . Even when the optic nerve is intact, lens injury stimulates macrophage infiltration into the eye, Muller cell activation, and increased GAP-43 expression in ganglion cells across the entire retina . In contrast, axotomy, either alone or combined with intraocular injections that do not infringe on the lens, causes only a minimal change in GAP-43 expression in RGCs and a minimal activation of the other cell types . Combining nerve injury with lens puncture leads to an eightfold increase in RGC survival and a 100-fold increase in the number of axons regenerating beyond the crush site . Macrophage activation appears to play a key role, because intraocular injections of Zymosan, a yeast cell wall preparation, stimulated monocytes in the absence of lens injury and induced RGCs to regenerate their axons into the distal optic nerve.

J Neurosci, 2000 Jun 15, 20(12), 4596 - 605
Functional interactions between Drosophila bHLH/PAS, Sox, and POU transcription factors regulate CNS midline expression of the slit gene; Ma Y et al.; During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells . CNS midline development is dependent on Single-minded (Sim), a basic-helix-loop-helix (bHLH)-PAS transcription factor . We show here that Fish-hook (Fish), a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression . single-minded, fish-hook, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays revealed genetic interactions between these genes . The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells . Fish-hook directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast . In addition, Fish can form homodimers and also associates with other bHLH-PAS and POU proteins . These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors . Functional interactions between members of these protein families may be important for numerous developmental and physiological processes.

J Biol Chem, 2000 Aug 25, 275(34), 26458 - 66
Molecular characterization of human acetyl-CoA synthetase, an enzyme regulated by sterol regulatory element-binding proteins; Luong A et al.; Through suppressive subtractive hybridization, we identified a new gene whose transcription is induced by sterol regulatory element-binding proteins (SREBPs) . The gene encodes acetyl-CoA synthetase (ACS), the cytosolic enzyme that activates acetate so that it can be used for lipid synthesis or for energy generation . ACS genes were isolated previously from yeast, but not from animal cells . Recombinant human ACS was produced by expressing the cloned cDNA transiently in human cells . After purification by nickel chromatography, the 701-amino acid cytosolic enzyme was shown to function as a monomer . The recombinant enzyme produced acetyl-CoA from acetate in a reaction that required ATP . As expected for a gene controlled by SREBPs, ACS mRNA was induced when cultured cells were deprived of sterols and repressed by sterol addition . The pattern of regulation resembled the regulation of enzymes of fatty acid synthesis . ACS mRNA was also elevated in livers of transgenic mice that express dominant-positive versions of all three isoforms of SREBP . We conclude that ACS mRNA, and hence the ability of cells to activate acetate, is regulated by SREBPs in parallel with fatty acid synthesis in animal cells.

Genomics, 2000 May 15, 66(1), 110 - 2
Physical map of the region surrounding the OTOFERLIN locus on chromosome 2p22-p23; Yasunaga S et al.; The autosomal recessive form of nonsyndromic deafness DFNB9 has been mapped to a 2-cM region on chromosome 2p22-p23, and the responsible gene, OTOF, has been recently identified by positional cloning combined with a candidate gene approach . In the course of this gene cloning, we established a contig of yeast artificial chromosomes, bacterial artificial chromosomes, and P1 phage artificial chromosomes delimited by polymorphic markers D2S2170 and D2S170, i.e . , extending over approximately 3500 kb . Sixty expressed sequence tags or genes and 14 sequence-tagged sites, 11 of which are polymorphic, were mapped to this contig and assigned to 21 chromosomal intervals .

Genomics, 2000 May 15, 66(1), 104 - 9
Large-insert clone/STS contigs in Xq11-q12, spanning deletions in patients with androgen insensitivity and mental retardation; Schueler MG et al.; An integrated large-insert clone map of the region Xq11-q12 is presented . A physical map containing markers within a few hundred kilobases of the centromeric locus DXZ1 to DXS1125 spans nearly 5 Mb in two contigs separated by a gap estimated to be approximately 100-250 kb . The contigs combine 75 yeast artificial chromosome clones, 12 bacterial artificial chromosome clones, and 17 P1-derived artificial chromosome clones with 81 STS or EST markers . Overall marker density across this region is approximately 1 STS/60 kb . Mapped within the contigs are 12 ESTs as well as 5 known genes, moesin (MSN), hephaestin (HEPH), androgen receptor (AR), oligophrenin-1 (OPHN1), and Eph ligand-2 (EPLG2) . Orientation of the contigs on the X chromosome, as well as marker order within the contigs, was unambiguously determined by reference to a number of X chromosome breakpoints . In addition, the distal contig spans deletions from chromosomes of three patients exhibiting either complete androgen insensitivity (CAI) or a contiguous gene syndrome that includes CAI, impaired vision, and mental retardation .

Genomics, 2000 May 15, 66(1), 48 - 54
Identification and characterization of YME1L1, a novel paraplegin-related gene; Coppola M et al.; A gene responsible for an autosomal recessive form of hereditary spastic paraplegia (SPG7) was recently identified . This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial AAA proteases Afg3p, Rca1p, and Yme1p, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane . By screening the expressed sequence tag database, we identified and characterized a novel human gene, YME1L1 (YME1L1-like1, HGMW-approved symbol) . This gene encodes a predicted protein of 716 amino acids highly similar to all mitochondrial AAA proteases and in particular to yeast Yme1p . Expression and immunofluorescence studies revealed that YME1L1 and paraplegin share a similar expression pattern and the same subcellular localization in the mitochondrial compartment . YME1L1 may represent a candidate gene for other forms of hereditary spastic paraplegia and possibly for other neurodegenerative disorders .

Genomics, 2000 May 15, 66(1), 15 - 25
Chromosome translocations in breast cancer with breakpoints at 8p12; Courtay-Cahen C et al.; Unbalanced chromosome translocations with breakpoints around 8p12, resulting in loss of distal 8p, are common in carcinomas . We have mapped the 8p12 breakpoints in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1, using YACs and PACs between D8S540 and D8S255 by fluorescence in situ hybridization . All three lines had a breakpoint close to D8S505, proximal to HGL . Each breakpoint was distinct, but all were within 0.5 to 1.5 Mb of each other . The T-47D cell line had a straightforward translocation, but in MDA-MB-361 and ZR-75-1 the translocations were accompanied by local rearrangements of surprising complexity . Small regions of 8p from close to the breakpoint were duplicated or amplified as inserts in the attached chromosome fragment . ZR-75-1 also had retained a separate fragment of about 1 Mb, from the region 1 to 3 Mb telomeric to the common breakpoint, that included HGL . This line also had an interstitial deletion several megabases more centromeric . The data suggest that breakpoints on 8p12 are clustered in a small region and show that translocations breaking there may be accompanied by additional rearrangements .

Oncogene, 2000 Jun 1, 19(24), 2812 - 9
Degradation of human Aurora2 protein kinase by the anaphase-promoting complex-ubiquitin-proteasome pathway; Honda K et al.; Human Aurora2 was originally identified by its close homology to yeast IPL1 and fly aurora, which are key regulators of chromosome segregation and a family of serine/threonine kinases . Here we demonstrate that the Aurora2 protein is degraded rapidly after G2/M phase release in mammalian cells . Aurora2 protein has a rapid turnover rate with a half-life of approximately 2 h . In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of unstable proteins . The treatment of mammalian cells with proteasome inhibitors blocks Aurora2 degradation . Furthermore, Aurora2 is polyubiquitinated in vivo and in vitro using anaphase-promoting complex (APC) . These results demonstrate that Aurora2 protein is turned over through the APC-ubiquitin-proteasome pathway . Oncogene (2000) 19, 2812 - 2819

Cell, 2000 May 26, 101(5), 471 - 83
Identification of human Rap1: implications for telomere evolution; Li B et al.; It has been puzzling that mammalian telomeric proteins, including TRF1, TRF2, tankyrase, and TIN2 have no recognized orthologs in budding yeast . Here, we describe a human protein, hRap1, that is an ortholog of the yeast telomeric protein, scRap1p . hRap1 has three conserved sequence motifs in common with scRap1, is located at telomeres, and affects telomere length . However, while scRap1 binds telomeric DNA directly, hRap1 is recruited to telomeres by TRF2 . Extending the comparison of telomeric proteins to fission yeast, we identify S . pombe Taz1 as a TRF ortholog, indicating that TRFs are conserved at eukaryotic telomeres . The data suggest that ancestral telomeres, like those of vertebrates, contained a TRF-like protein as well as Rap1 . We propose that budding yeast preserved Rap1 at telomeres but lost the TRF component, possibly concomitant with a change in the telomeric repeat sequence.

Cancer Res, 2000 Jun 1, 60(11), 2786 - 9
Translocation t(10;14)(q11.2:q22.1) fusing the kinetin to the RET gene creates a novel rearranged form (PTC8) of the RET proto-oncogene in radiation-induced childhood papillary thyroid carcinoma; Salassidis K et al.; Evaluation of 20 cases of radiation-induced childhood papillary thyroid carcinoma using fluorescence in situ hybridization demonstrated the presence of clonal translocations affecting the RET locus . Semiquantitative reverse transcription-PCR indicated overexpression of the RET tyrosine kinase (TK) domain in four cases . In two cases, the RET rearrangements PTC6 and PTC7 were identified and assigned to balanced translocations t(7;10)(q32;q11.2) and t(1;10)(p13;q11.2), respectively . In one case with a balanced translocation t(10;14)(q11.2;q22.1), 5' rapid amplification of cDNA ends revealed a novel type of RET oncogenic activation (PTC8), arising from a fusion of the 5' part of the kinectin (KTN1) gene to the TK domain of the RET gene . The presence of coiled-coil domains in the resulting ktn1/ret fusion protein suggests ligand-independent dimerization and thus constitutive activation of the ret TK domain.

Cancer Res, 2000 Jun 1, 60(11), 2775 - 9
Deletion of 6q16-q21 in human lymphoid malignancies: a mapping and deletion analysis; Jackson A et al.; Two distinct regions of minimal deletion (RMD) have been identified at 6q25-q27 in non-Hodgkin's lymphoma (RMD-1), and at 6q21-q23 in acute lymphoblastic leukemia (ALL; RMD-2) by loss of heterozygosity and fluorescence in situ hybridization studies . In this study, 30 overlapping yeast artificial chromosomes (YACs), 1 expressed sequence tag, and 11 novel YAC ends were identified using bidirectional YAC walks between markers D6S447 (proximal) and D6S246 (distal) in RMD-2 . The genes AF6q21, human homologue of the Drosophila tailless (HTLX), CD24 antigen, the Kruppel-like zinc finger BLIMP1, and cyclin C (CCNC), previously mapped to 6q21, were accurately positioned in a telomere-to-centromere orientation . Approximately 3.5 Mb were found to separate the BLIMP1 (adjacent to D6S447) and AF6q21 genes (telomeric to D6S246) . Deletions of 6q were investigated in 21 cases of ALL using the newly characterized YAC clones in dual-color fluorescence in situ hybridization studies . A region centromeric to D6S447 (containing marker D6S283) and a region telomeric to marker CHLC.GGAT16CO2 (and containing marker D6S268) were identified as distinct and nonoverlapping regions of deletion in ALL.

Ther Drug Monit, 2000 Jun, 22(3), 237 - 44
Catalytic activity of three variants (Ile, Leu, and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis; Ieiri I et al.; This study evaluated the catalytic activity of three variants (Ile, Leu, and Thr) at codon 359 of CYP2C9 enzymes expressed in a yeast cDNA expression system, and then established single-strand conformation polymorphism (PCR-SSCP) analysis for simultaneous detection as a screening method . Diclofenac was used for the in vitro experiment, and its hydroxy metabolite (4'-hydroxydiclofenac) was measured by HPLC . To discuss the in vivo effect of the Thr359 variant on the pharmacokinetics of phenytoin, a case report is presented . The efficiency of the SSCP method was evaluated by analyzing DNA samples from a homozygote for Ile359 and a heterozygote for Leu359 or Thr359 . To evaluate the interaction between the P450 level and reductase activity, two batches of the Thr359 variant with a different P450:reductase activity ratio (1:4.0 and 1:1.4) were used . The in vitro study revealed that recombinant Ile359, Leu359, and Thr359 (2 batches) possessed a mean Km of 2.0, 16.5 and (3.8 and 2.9) micromol and Vmax of 12.4, 17.9 and (4.4 and 5.1) nmol/min/nmol P450, respectively . Although the magnitude of the change in catalytic efficiency for the Thr359 variant was close to that of the Leu359 variant, the effect of the two variants on diclofenac 4'-hydroxylation appears to be different because Leu359 variant was associated with a high Km, and Thr359 with a low Vmax . No significant differences in the kinetic data were observed between the two Thr359 enzymes, suggesting that low reductase activity in the Thr359 enzyme was not a major determinant in the present in vitro experiment . Estimated pharmacokinetic parameters of phenytoin obtained by the Bayesian method in an epileptic patient who was a heterozygote carrier for Thr359 variant were: Km = 6.45 microg/mL, Vmax = 5.77 mg/kg/d, and Vmax/Km = 0.89 L/kg/day . The Vmax/Km value in this patient was similar to the population mean value (0.90 L/kg/day) in Japanese heterozygotes for the Leu359 variant . Results for PCR-SSCP were in complete agreement with those obtained using established methods . Thus, the PCR-SSCP approach is useful for identifying these three variants of the CYP2C9 gene.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 1147 - 61
Brazilian bioethanol program; Zanin GM et al.; Brazil is the largest producer of bioethanol, and sugarcane is the main raw material . Bioethanol is produced by both batch and continuous processes, and in some cases, flocculating yeast is used . This article analyzes the Brazilian Ethanol Program . For the 1996-1997 harvest, Brazil produced 14.16 billion L of ethanol and 13.8 million metric t of sugar, from 286 million metric t of sugarcane . These products were produced by 328 industries in activity, with 101 autonomous ethanol plants producing only ethanol, and 227 sugar mills producing sugar and ethanol . The sugar-ethanol market reaches about 7.5 billion US$/yr, accounting for direct and indirect revenues.

Int J Androl, 2000, 23 Suppl 2, 92 - 4
Temporal control of protein synthesis during spermatogenesis; Braun RE; During oogenesis and spermatogenesis transcription ceases prior to the differentiation of the mature cells . To complete germ cell differentiation and initiate early embryogenesis, proteins are synthesized from pre-existing mRNAs that are stored for several days . It is well established that important regulatory elements functioning in spatial localization, temporal translation or messenger RNA stability are located in the 3' untranslated region (3' UTR) of mRNAs . During mammalian spermatogenesis temporal translational regulation of the protamine 1 (Prm1) mRNA is dependent on a highly conserved sequence located in the distal region of its 3' UTR . The 17-nucleotide translational control element (TCE) mediates translational repression of the Prm1 mRNA . Mutation of the TCE causes premature synthesis of protamine protein and sterility . The Prm1 mRNA is stored as a cytoplasmic ribonucleoprotein (mRNP) particle in spermatids . Contained within the particle are several members of the Y box family of nucleic acid binding proteins . In the yeast three-hybrid system the murine Y box proteins MSY1, MSY2 and MSY4 bind in a sequence-dependent manner to a conserved region in the proximal portion of the Prm1 3' UTR . Sequence-specific binding by MSY4 to the Y box recognition sequence (YRS) is dependent on the highly conserved cold shock domain, possibly through the RNP1 and RNP2 motifs present within it . The Y box proteins may function as translational repressors in vivo . Alternatively, their primary function may be to protect mRNAs from degradation during their extended period of storage . Translational activation of stored mRNAs is essential for the completion of gametogenesis . Proper translational activation of the Prm1 mRNA in elongated spermatids requires the cytoplasmic double-stranded RNA binding protein TARBP2 . Tarbp2 is expressed at low levels in many cells but is expressed at robust levels in late stage meiotic cells and in postmeiotic spermatids . Mice mutant for Tarbp2 are defective in proper translational activation of the Prm1 and Prm2 mRNAs and are sterile . Current studies are designed to determine the mechanism by which proteins bound to the 3' UTR communicate with the 5' end of the message to control translational silencing and activation.

Plant J, 2000 May, 22(4), 355 - 65
Isolation and characterization of HsfA3, a new heat stress transcription factor of Lycopersicon peruvianum; Bharti K et al.; Stress-induced transcription of heat shock proteins (Hsps) in eukaryotes is mediated by a conserved class of transcription factors called heat stress transcription factors (Hsfs) . Here we report the isolation and functional characterization of HsfA3, a new member of the Hsf family . HsfA3 was cloned from a tomato heat stress cDNA library by yeast two-hybrid screening, using HsfA1 as a bait . HsfA3 is a single-copy gene with all the conserved sequence elements characteristic of a heat stress transcription factor . The constitutively expressed HsfA3 is mainly found in the cytoplasm under control conditions and in the nucleus under heat stress conditions . Functionally, HsfA3 behaves similarly to the already known members of tomato Hsf family . It is able to substitute yeast Hsf for viability functions and is a strong activator of Hsf-dependent reporter constructs both in tobacco protoplasts and yeast . Finally, similar to the AHA motifs in HsfA1 and HsfA2, the activator function depends on four short peptide motifs with a central tryptophan residue found in the C-terminal domain of HsfA3.

Eur J Biochem, 2000 Jun, 267(12), 3891 - 901
The PKC targeting protein RACK1 interacts with the Epstein-Barr virus activator protein BZLF1; Baumann M et al.; Phorbol esters reactivate Epstein-Barr virus (EBV) from latently infected cells via transcriptional activation of the viral immediate-early gene BZLF1 . BZLF1 is a member of the extended AP-1 family of transcription factors that binds to specific BZLF1-binding motifs within early EBV promoters and to consensus AP-1 sites . Regulation of BZLF1's activity is achieved at the transcriptional level as well as through post-translational modifications . Recently, we reported that the transcriptional activity of BZLF1 is augmented by TPA {Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H . J . & Hammerschmidt, W., (1998) J . Virol . 72, 8105-8114} . The increase of BZLF1's activity depends on a single serine residue (S186) that is phosphorylated by protein kinase C (PKC) in vitro and in vivo after stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) . Here, we identified RACK1 as a binding partner of BZLF1 in a yeast interaction trap assay . RACK stands for receptor of activated C-kinase and is involved in targeting activated PKCs and other signaling proteins . In vivo, RACK1 binds directly to the transactivation domain of BZLF1 . Although a functional relationship between BZLF1 and PKC could be mediated by RACKs, RACK1 did not have a detectable effect on the phosphorylation status of BZLF1 in in vitro or in vivo phosphorylation assays . We suggest that RACK1 may act as a scaffolding protein on BZLF1 independently of activated PKCs.

Eur J Biochem, 2000 Jun, 267(12), 3453 - 60
Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum) new alpha-amyrin-producing enzyme is a multifunctional triterpene synthase; Morita M et al.; Ursane type triterpene is one of the most widespread triterpene aglycones found in plants, together with oleanane type, and these two types often occur together in the same plant . Pisum sativum is known to produce both types of triterpenes . Homology based PCRs with degenerate primers designed from the conserved sequences found in the known beta-amyrin synthases have resulted in cloning of two triterpene synthase cDNAs from immature seeds of P . sativum . They show high sequence identities to each other (78%) and also to the known beta-amyrin synthases (70-90%) . ORFs of the full-length clones named as PSY (2277 bp, codes for 759 amino acids) and PSM (2295 bp, codes for 765 amino acids) were ligated into the yeast expression vector pYES2 under the control of GAL1 promoter . Heterologous expression in yeast revealed PSY to be a P . sativum beta-amyrin synthase . Surprisingly, however, PSM turned out to be a novel mixed amyrin synthase producing both alpha- and beta-amyrin . Several minor triterpenes were also identified as the PSM byproducts . The presence of such multifunctional triterpene synthase would account for the co-occurence of ursane and oleanane type triterpenes in plants.

Mol Biol Cell, 2000 Jun, 11(6), 2069 - 83
Histone deacetylase inhibitors trigger a G2 checkpoint in normal cells that is defective in tumor cells; Qiu L et al.; Important aspects of cell cycle regulation are the checkpoints, which respond to a variety of cellular stresses to inhibit cell cycle progression and act as protective mechanisms to ensure genomic integrity . An increasing number of tumor suppressors are being demonstrated to have roles in checkpoint mechanisms, implying that checkpoint dysfunction is likely to be a common feature of cancers . Here we report that histone deacetylase inhibitors, in particular azelaic bishydroxamic acid, triggers a G2 phase cell cycle checkpoint response in normal human cells, and this checkpoint is defective in a range of tumor cell lines . Loss of this G2 checkpoint results in the tumor cells undergoing an aberrant mitosis resulting in fractured multinuclei and micronuclei and eventually cell death . This histone deacetylase inhibitor-sensitive checkpoint appears to be distinct from G2/M checkpoints activated by genotoxins and microtubule poisons and may be the human homologue of a yeast G2 checkpoint, which responds to aberrant histone acetylation states . Azelaic bishydroxamic acid may represent a new class of anticancer drugs with selective toxicity based on its ability to target a dysfunctional checkpoint mechanism in tumor cells.

Mol Biol Cell, 2000 Jun, 11(6), 2007 - 18
Interaction of the tau2 transcriptional activation domain of glucocorticoid receptor with a novel steroid receptor coactivator, Hic-5, which localizes to both focal adhesions and the nuclear matrix; Yang L et al.; Hic-5 (hydrogen peroxide-inducible clone-5) is a focal adhesion protein that is involved in cellular senescence . In the present study, a yeast two-hybrid screen identified Hic-5 as a protein that interacts with a region of the glucocorticoid receptor that includes a nuclear matrix-targeting signal and the tau2 transcriptional activation domain . In transiently transfected mammalian cells, overexpression of Hic-5 potentiated the activation of reporter genes by all steroid receptors, excluding the estrogen receptor . The activity of the estrogen receptor and the thyroid hormone receptor was stimulated by Hic-5 in the presence but not in the absence of coexpressed coactivator GRIP1 . In biochemical fractionations and indirect immunofluorescence assays, a fraction of endogenous Hic-5 in REF-52 cells and transiently expressed Hic-5 in Cos-1 cells was associated with the nuclear matrix . The C-terminal region of Hic-5, which contains seven zinc fingers arranged in four LIM domains, was required for interaction with focal adhesions, the nuclear matrix, steroid receptors, and the tau2 domain of glucocorticoid receptor . The N-terminal region of Hic-5 possesses a transcriptional activation domain and was essential for the coactivator activity of Hic-5 . Given the coexisting cytoplasmic and nuclear distributions of Hic-5 and its role in steroid receptor-mediated transcriptional activation, it is proposed that Hic-5 might transmit signals that emanate at cell attachment sites and regulate transcription factors, such as steroid receptors.

Cell, 2000 Apr 28, 101(3), 271 - 81
act up controls actin polymerization to alter cell shape and restrict Hedgehog signaling in the Drosophila eye disc; Benlali A et al.; Cells in the morphogenetic furrow of the Drosophila eye disc undergo a striking shape change immediately prior to their neuronal differentiation . We have isolated mutations in a novel gene, act up (acu), that is required for this shape change . acu encodes a homolog of yeast cyclase-associated protein, which sequesters monomeric actin; we show that acu is required to prevent actin filament polymerization in the eye disc . In contrast, profilin promotes actin filament polymerization, acting epistatically to acu . However, both acu and profilin are required to prevent premature Hedgehog-induced photoreceptor differentiation ahead of the morphogenetic furrow . These findings suggest that dynamic changes in actin filaments alter cell shape to control the movement of signals that coordinate a wave of differentiation.

Mol Endocrinol, 2000 Jun, 14(6), 915 - 25
Activating protein-1, nuclear factor-kappaB, and serum response factor as novel target molecules of the cancer-amplified transcription coactivator ASC-2; Lee SK et al.; ASC-2 was recently discovered as a cancer-amplified transcription coactivator molecule of nuclear receptors, which interacts with multifunctional transcription integrators steroid receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP)/p300 . Herein, we report the identification of three mitogenic transcription factors as novel target molecules of ASC-2 . First, the C-terminal transactivation domain of serum response factor (SRF) was identified among a series of ASC-2-interacting proteins from the yeast two-hybrid screening . Second, ASC-2 specifically interacted with the activating protein-1 (AP-1) components c-Jun and c-Fos as well as the nuclear factor-kappaB (NFkappaB) components p50 and p65, as demonstrated by the glutathione S-transferase pull-down assays as well as the yeast two-hybrid tests . In cotransfection of mammalian cells, ASC-2 potentiated transactivations by SRF, AP-1, and NFkappaB in a dose-dependent manner, either alone or in conjunction with SRC-1 and p300 . In addition, ASC-2 efficiently relieved the previously described transrepression between nuclear receptors and either AP-1 or NFkappaB . Overall, these results suggest that the nuclear receptor coactivator ASC-2 also mediates transactivations by SRF, AP-1, and NFkappaB, which may contribute to the putative, ASC-2-mediated tumorigenesis.

Mol Microbiol, 2000 Jun, 36(5), 1148 - 55
Interacting interfaces of the P4 antirepressor E and the P2 immunity repressor C; Eriksson SK et al.; Antirepressors have been identified as proteins interacting with transcriptional repressors leading to expression of the repressed genes . The defective satellite phage/plasmid P4 has the capacity to derepress the unrelated prophage P2 after infection, thereby getting access to the late functions of the helper that are required for P4 lytic growth . The derepression of prophage P2 is mediated by the P4 E protein that function as an antirepressor by binding to the P2 immunity repressor C . A P2 mutant, sos, has been isolated that is insensitive to the action of the P4 E protein . In the present study, we show that sos is a point mutation in the P2 immunity repressor gene C and that it makes P4 E unable to turn the transcriptional switch of P2 from the lysogenic state to the lytic mode in a two plasmid reporter system . Furthermore, the interaction between C and E, when analysed in the yeast two-hybrid system, is blocked by the sos mutation . An analysis of C mutants indicates that the dimerization function of C is located in the C-terminal part of the protein and the dimerization defective mutants are unable to bind to their operator DNA . The sos mutation does not affect the capacity of the protein to dimerize . Using the yeast two-hybrid system, compensatory E mutants have been isolated that can interact with Sos, but they are unable to turn the transcriptional switch controlled by the Sos repressor . However, one point mutation in the E protein is shown to be unable to turn the transcriptional switch controlled by the wild-type C repressor.

Kidney Int, 2000 Jun, 57(6), 2221 - 8
Identification of novel genes expressed during metanephric induction through single-cell library screening; Abidari JM et al.; BACKGROUND: Development of the mature kidney is dependent on a series of inductive events between a portion of the epithelial bud at the distal end of the nephric duct and a neighboring domain of committed metanephric mesenchyme . Several genes have been identified to date that are critical in the inductive process . For example, the deletion of Bmp7 from the mouse genome results in dysgenesis or agenesis of the kidney . These findings suggest that Bmp7 controls the expression of genes important for nephrogenesis, but the identity of these genes has remained largely undetermined . METHODS: Single cells were isolated from mouse metanephric mesenchyme during the time of induction (between E11.0 and E11.5) and cDNA libraries constructed from induced and uninduced tissue . Subtractive hybridization was performed to isolate genes that were expressed during E11.5 but not E11.0 . RESULTS: Using this approach, we identified eight previously known genes, three of which were known to be involved in metanephric induction, thus validating our approach, and nine novel genes . Eight of these genes were completely novel, whereas one was similar to a member of the yeast Anaphase Promoting Complex . CONCLUSIONS: Through subtractive hybridization of mouse E11.0 and E11.5 metanephric mesenchyme single-cell cDNA libraries, we have identified novel genes that are candidates for involvement in nephrogenesis through their up-regulation during the inductive process.

J Immunol, 2000 Jun 15, 164(12), 6287 - 95
Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function; Junn E et al.; As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen . Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding . mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm . Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX . When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced . TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1 . Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress . To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells . When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells . In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated . Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.

J Immunol, 2000 Jun 15, 164(12), 6188 - 92
Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation; Ling X et al.; Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents . A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein . A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry . Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo . Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro . These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.

Dev Dyn, 2000 Jun, 218(2), 300 - 15
Tubedown-1, a novel acetyltransferase associated with blood vessel development; Gendron RL et al.; We have used an embryonic endothelial cell line (IEM cells) as an experimental system for identifying and characterizing new molecules which are regulated during blood vessel development . A novel gene isolated from IEM cells, tubedown-1 (tbdn-1), is expressed at high levels in unstimulated IEM cells and is downregulated during formation of capillary tube structures by the IEM cells induced by basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) in vitro . Tbdn-1 is also downregulated in M1 myeloid leukemia cells after differentiation in response to LIF in vitro . Tbdn-1 is homologous to the yeast NAT-1 N-terminal acetyltransferases and encodes a novel protein of approximately 69 kDa associated with an acetyltransferase activity . Levels and distribution of tbdn-1 expression are regulated in both endothelial and hematopoietic cells during development in tissues such as the yolk sac blood islands, heart, and liver blood vessels . In the adult, tbdn-1 expression is low or undetected in most organs examined with the exception of the atrial endocardium, the endothelial and myeloid compartments of bone marrow, and the remodeling vascular bed of atretic ovarian follicles . The distribution and regulation of expression of tbdn-1 suggest that this novel acetyltransferase may be involved in regulating vascular and hematopoietic development and physiologic angiogenesis .

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6334 - 9
Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase; Tachedjian G et al.; HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA . The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity . To analyze this process at the genetic level, we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system . Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction . However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66 . A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system . Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization . In vitro binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits . The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process.

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6316 - 21
Zinc-bundle structure of the essential RNA polymerase subunit RPB10 from Methanobacterium thermoautotrophicum; Mackereth CD et al.; The RNA polymerase subunit RPB10 displays a high level of conservation across archaea and eukarya and is required for cell viability in yeast . Structure determination of this RNA polymerase subunit from Methanobacterium thermoautotrophicum reveals a topology, which we term a zinc-bundle, consisting of three alpha-helices stabilized by a zinc ion . The metal ion is bound within an atypical CX(2)CX(n)CC sequence motif and serves to bridge an N-terminal loop with helix 3 . This represents an example of two adjacent zinc-binding Cys residues within an alpha-helix conformation . Conserved surface features of RPB10 include discrete regions of neutral, acidic, and basic residues, the latter being located around the zinc-binding site . One or more of these regions may contribute to the role of this subunit as a scaffold protein within the polymerase holoenzyme.

Bioseparation, 2000, 9(1), 37 - 41
Contamination of an anion-exchange membrane by glutathione; Gotoh T et al.; Electrodialysis, which can separate electrolytes under mild conditions by using ion-exchange membranes, is a strong candidate for separation of GSH from yeast extracts, because GSH is unstable and easily oxidized forming a disulfide bond especially under alkali conditions . In this paper, sorption behavior of GSH on an anion-exchange membrane, in the pH 3-6 region that is expected to be the most preferable for its electro-dialytic separation, was examined . Sorption of GSH on a Selemion-AMV anion-exchange membrane was accelerated as the pH of the membrane-contact solution increased, and there was a good correlation between the sorbed amounts and the molar fraction of monovalent anionic species of GSH . However, the amounts of GSH desorbed from the membrane by a NaCl desorbing solution were much lower than the initial sorbed amounts, and the difference between them was enlarged with increasing pH . The GSH which was lost could be recovered by the addition of DTT in the membrane-contact and desorbing solutions . Similar results were also obtained with Cys . We thus concluded that an anion-exchange membrane would be contaminated by thiol compounds, such as GSH and Cys, through oxidative binding of the thiol group with the membrane, the local OH- concentration in which was enhanced due to attraction by the positively charged anion-exchange membrane.

Bioseparation, 2000, 9(1), 29 - 36
Expanded bed chromatography of proteins in small-diameter columns . II . Methods development and scale up; Ghose S et al.; The scaled down system developed in Part I of this series was further validated by using a 1-cm diameter column for method development studies for the separation of two model proteins, alcohol dehydrogenase and alpha-glucosidase, from unclarified yeast homogenate by hydrophobic interaction expanded bed chromatography based on the STREAMLINE matrix . The efficacy of solids removal and establishment of optimal binding and separation condition by stepwise elution were investigated . Equilibration of the EBA column and loading at high salt strengths affected the subsequent recovery of the two target proteins . Although good resolution between the target proteins could be achieved, peak tailing was found to be a consistent problem . The optimised separation protocol was scaled up 25-fold to a column diameter of 5.0 cm . The results were in good agreement with the run conducted in the 1-cm column, indicating the potential of using the small columns as an viable approach for method scouting and development studies.

Biochem J, 2000 Jun 15, 348 Pt 3, 585 - 90
A new method for the selection of protein interactions in mammalian cells; Rojo-Niersbach E et al.; In the present study we present a new method that allows for the selection of protein interactions in mammalian cells . We have used this system to verify two interactions previously characterized in vitro . (1) The interaction between human TATA-binding protein 1 and nuclear factor kappaB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha, a protein implicated in chromatin remodelling . We observe for the first time that these interactions also occur in vivo . One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue . Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes . The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway . In the human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium . This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.

Rev Bras Biol, 2000 Feb, 60(1), 45 - 52
Comparison of artificial diets for rearing Corcyra cephalonica (Stainton, 1865) (Lep., Pyralidae) for Trichogramma mass production; Bernardi EB et al.; The objective of this research was, based on biological studies, to determine and adequate diet for rearing Corcyra cephalonica (Stainton, 1865) in the laboratory so as to permit the rearing of this factitious host for Trichogramma mass production . The research was conducted at 25 +/- 1 degrees C, RH 60 +/- 10% and photophase of 14 hours . Six artificial diets were compared: a) whole wheat flour (48.5%), ground rice (48.5%) and sugar (3%); b) ground rice (97%) and sugar (3%); c) whole wheat flour (48.5%), rice flour (48.5%) an sugar (3%); d) whole wheat flour (97%) and yeast (3%); e) wheat germ (97%) and yeast (3%); f) rice bran (94%), sugar (3%) and yeast (3%); f) rice bran (94%), sugar (3%) and yeast (3%) . All of the diets studied permitted the development of C . cephalonica although the diets with wheat germ and yeast and that consisting of rice bran, sugar and yeast proved to be the most adequate for rearing the moth . These diets reduced the total (egg-adult) cycle, shortened the egg laying period, and produced heavier adults . Studies on the fertility life tables showed that higher net reproduction rates (Ro) and finite ratio of increase (lambda) were obtained from adults reared on these diets.

J Biol Chem, 2000 Aug 18, 275(33), 25255 - 61
MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain; Bae J et al.; MCL-1 (myeloid cell leukemia-1) is an antiapoptotic BCL-2 family protein discovered as an early induction gene during myeloblastic leukemia cell differentiation . This survival protein has the BCL-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region . We identified a short splicing variant of the MCL-1 mRNA in the human placenta encoding a protein, termed MCL-1 short (MCL-1S), with an altered C terminus as compared with the full-length MCL-1 long (MCL-1L), leading to the loss of BH1, BH2, and the transmembrane domains . Analysis of the human MCL-1 gene indicated that MCL-1S results from the splicing out of exon 2 during mRNA processing . MCL-1S, unlike MCL-1L, does not interact with diverse proapoptotic BCL-2-related proteins in the yeast two-hybrid system . In contrast, MCL-1S dimerizes with MCL-1L in the yeast assay and coprecipitates with MCL-1L in transfected mammalian cells . Overexpression of MCL-1S induces apoptosis in transfected Chinese hamster ovary cells, and the MCL-1S action was antagonized by the antiapoptotic MCL-1L . Thus, the naturally occurring MCL-1S variant represents a new proapoptotic BH3 domain-only protein capable of dimerizing with the antiapoptotic MCL-1L . The fate of MCL-1-expressing cells could be regulated through alternative splicing mechanisms and interactions of the resulting anti- and proapoptotic gene products.

J Biol Chem, 2000 Aug 18, 275(33), 25700 - 10
Molecular anatomy of the peroxin Pex12p: ring finger domain is essential for Pex12p function and interacts with the peroxisome-targeting signal type 1-receptor Pex5p and a ring peroxin, Pex10p; Okumoto K et al.; The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders . Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109 . This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes . The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting . Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays . The RING finger of Pex12p bound to Pex10p and the PTS1-receptor Pex5p . Pex10p also interacted with Pex2p and Pex5p in vitro . Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where Pex5p was not associated with the Pex12p-Pex10p complex . This observation suggested that Pex5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes . Hence, the RING finger peroxins are most likely to be involved in Pex5p-mediated matrix protein import into peroxisomes.

Curr Opin Plant Biol, 2000 Jun, 3(3), 205 - 10
Delivering copper within plant cells; Himelblau E et al.; Two genes recently identified in Arabidopsis thaliana may be involved in sequestering free copper ions in the cytoplasm and delivering copper to post-Golgi vesicles . The genes COPPER CHAPERONE and RESPONSIVE TO ANTAGONIST1 are homologous to copper-trafficking genes from yeast and humans . This plant copper-delivery pathway is required to create functional ethylene receptors . The pathway may also facilitate the transport of copper from senescing leaf tissue . In addition, several other genes have been identified recently that may have a role in copper salvage during senescence.

Curr Opin Plant Biol, 2000 Jun, 3(3), 211 - 6
Phytochelatin biosynthesis and function in heavy-metal detoxification; Cobbett CS; Plants respond to heavy-metal toxicity via a number of mechanisms . One such mechanism involves the chelation of heavy metals by a family of peptide ligands, the phytochelatins . Molecular genetic approaches have resulted in important advances in our understanding of phytochelatin biosynthesis . In particular, genes encoding the enzyme phytochelatin synthase have been isolated from plant and yeast species . Unexpectedly, genes with similar sequences to those encoding phytochelatin synthase have been identified in some animal species.

Exp Cell Res, 2000 Jun 15, 257(2), 272 - 80
The nuclear DEAD box RNA helicase p68 interacts with the nucleolar protein fibrillarin and colocalizes specifically in nascent nucleoli during telophase; Nicol SM et al.; The DEAD box protein, p68, is an established RNA-dependent ATPase and RNA helicase in vitro, but neither the physiological function of this protein nor the macromolecules with which it interacts are known . Using a yeast two-hybrid screen, we identified the nucleolar protein, fibrillarin, as a protein that interacts with p68 . Coimmunoprecipitation experiments confirmed that p68 and fibrillarin can form complexes in cellular extracts, and deletion analysis identified regions in each protein responsible for mediating the interaction . Immunofluorescence studies using confocal microscopy revealed that, in interphase cells, while fibrillarin is predominantly nucleolar, p68 shows a diffuse granular nuclear staining but is largely excluded from the nucleoli . Strikingly, both proteins colocalize in nascent nucleoli during late telophase . These data are consistent with a role for p68 either in postmitotic nucleolar reassembly or in the activation of ribosomal DNA transcription/preribosomal RNA processing during telophase and suggest that differential subnuclear compartmentalization may be a mechanism by which interaction of p68 with fibrillarin is regulated in the cell .

J Mol Evol, 2000 Jun, 50(6), 497 - 509
Anopheles stephensi Dox-A2 shares common ancestry with genes from distant groups of eukaryotes encoding a 26S proteasome subunit and is in a conserved gene cluster; Garvey CF et al.; The sequence of a cloned Anopheles stephensi gene showed 72% inferred amino acid identity with Drosophila melanogaster Dox-A2 and 93% with its putative ortholog in Anopheles gambiae . Dox-A2 is the reported but herein disputed structural locus for diphenol oxidase A2 . Database searches identified Dox-A2 related gene sequences from 15 non-insect species from diverse groups . Phylogenetic trees based on alignments of inferred protein sequences, DNA, and protein motif searches and protein secondary structure predictions produced results consistent with expectations for genes that are orthologous . The only inconsistency was that the C-terminus appears to be more primitive in the yeasts than in plants . In mammals, plants, and yeast these genes have been shown to code for a non-ATPase subunit of the PA700 (19S) regulatory complex of 26S proteasome . The analyses indicated that the insect genes contain no divergent structural features, which taken within an appraisal of all available data, makes the reported alternative function highly improbable . A plausible additional role, in which the 26S proteasome is implicated in regulation of phenol oxidase, would also apply to at least the mammalian genes . No function has yet been reported for the other included sequences . These were from genome projects and included Caenorhabiditus elegans, Arabidopsis thaliana, Fugu rubripes, and Toxoplasma gondii . A consensus of the results predicts a protein containing exceptionally long stretches of helix with a hydrophilic C-terminus . Phosphorylation site motifs were identified at two conserved positions . Possible SRY and GATA-1 binding motifs were found at conserved positions upstream of the mosquito genes . The location of A . stephensi Dox-A2 was determined by in situ hybridization at 34D on chromosome arm 3R . It is in a conserved gene cluster with respect to the other insects . However, the A . stephensi cluster contains a gene showing significant sequence identity to human and pigeon carnitine acetyltransferase genes, therefore showing divergence with the distal end of the D . melanogaster cluster.

Genetics, 2000 Jun, 155(2), 803 - 12
Physical mapping of male fertility and meiotic drive quantitative trait loci in the mouse t complex using chromosome deficiencies; Planchart A et al.; The t complex spans 20 cM of the proximal region of mouse chromosome 17 . A variant form, the t haplotype (t), exists at significant frequencies in wild mouse populations and is characterized by the presence of inversions that suppress recombination with wild-type (+) chromosomes . Transmission ratio distortion and sterility are associated with t and affect males only . It is hypothesized that these phenomena are caused by trans-acting distorter/sterility factors that interact with a responder locus (Tcr(t)) and that the distorter and sterility factors are the same because homozygosity of the distorters causes male sterility . One factor, Tcd1, was previously shown to be amorphic using a chromosome deletion . To overcome limitations imposed by recombination suppression, we used a series of deletions within the t complex in trans to t chromosomes to characterize the Tcd1 region . We find that the distorter activity of Tcd1 is distinct from a linked sterility factor, originally called tcs1 . YACs mapped with respect to deletion breakpoints localize tcs1 to a 1.1-Mb interval flanked by D17Aus9 and Tctex1 . We present evidence for the existence of multiple proximal t complex regions that exhibit distorter activity . These studies demonstrate the utility of chromosome deletions for complex trait analysis.

J Mol Biol, 2000 Jun 9, 299(3), 813 - 25
Mg(2+) binding to tRNA revisited: the nonlinear Poisson-Boltzmann model; Misra VK et al.; Our current understanding of Mg(2+) binding to RNA, in both thermodynamic and structural terms, is largely based on classical studies of transfer RNAs . Based on these studies, it is clear that magnesium ions are crucial for stabilizing the folded structure of tRNA . We present here a rigorous theoretical model based on the nonlinear Poisson-Boltzmann (NLPB) equation for understanding Mg(2+) binding to yeast tRNA(Phe) . We use this model to interpret a variety of experimental Mg(2+) binding data . In particular, we find that the NLPB equation provides a remarkably accurate description of both the overall stoichiometry and the free energy of Mg(2+) binding to yeast tRNA(Phe) without any fitted parameters . In addition, the model accurately describes the interaction of Mg(2+) with localized regions of the RNA as determined by the pK(a) shift of differently bound fluorophores . In each case, we find that the model also reproduces the univalent salt-dependence and the anticooperativity of Mg(2+) binding . Our results lead us to a thermodynamic description of Mg(2+) binding to yeast tRNA(Phe) based on the NLPB equation . In this model, Mg(2+) binding is simply explained by an ensemble of ions distributed according to a Boltzmann weighted average of the mean electrostatic potential around the RNA . It appears that the entire ensemble of electrostatically bound ions superficially mimics a few strongly coordinated ions . In this regard, we find that Mg(2+) stabilizes the tertiary structure of yeast tRNA(Phe) in part by accumulating in regions of high negative electrostatic potential . These regions of Mg(2+) localization correspond to bound ions that are observed in the X-ray crystallographic structures of yeast tRNA(Phe) . Based on our results and the available thermodynamic data, there is no evidence that specifically coordinated Mg ions have a significant role in stabilizing the native tertiary structure of yeast tRNA(Phe) in solution .

J Mol Biol, 2000 Jun 9, 299(3), 667 - 80
Selection of homeotic proteins for binding to a human DNA replication origin; de Stanchina E et al.; We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene . Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence . All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators . We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene . The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays . In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells . We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation .

Biotechnol Prog, 2000 May-Jun, 16(3), 435 - 41
Process options in hepatitis B surface antigen extraction from transgenic potato; Dogan B et al.; The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined . The effects of temperature, the reducing agent beta-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined . Sedimentation profiles were performed to characterize HBsAg assembly into virus-like particles . Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity . The optimum temperature was around 50 degrees C . A 3-fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg . When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained . Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions . BME increased the antigenic activity of HBsAg up to 4-fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies . This observation suggests that BME affects the structure or presentation of the a epitope . In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained . Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases . Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity . Different proteinase inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME . The sedimentation profile of potato-produced HBsAg was determined in 5-30% sucrose gradients . Yeast-derived recombinant HBsAg was used as a positive control . The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles . BME treatment did not change the sedimentation profile.

Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 466 - 71
Interaction of Vesl-1L/Homer 1c with syntaxin 13; Minakami R et al.; Vesl-1S/Homer 1a, reported originally as Vesl/Homer, was isolated as a synaptic plasticity-regulated gene . The expression of Vesl-1S/Homer 1a is regulated during long-term potentiation in the hippocampus . Vesl-1L/Homer 1c, which appears to be formed by a splicing event, shares the N-terminus with Vesl-1S/Homer 1a and also contains additional amino acids at the C-terminus . The short form and the long form of the family members both interact with group 1 metabotropic glutamate receptors (mGluRs) . We herein report the identification of syntaxin 13 as a molecule that interacts with Vesl-1L using yeast two-hybrid screening . Syntaxin 13 is a member of the syntaxin family and is regarded as soluble N-ethylmaleimide-sensitive attachment proteins receptors (SNAREs) in the endosomal membranes . The interaction of Vesl-1L and syntaxin 13 was biochemically confirmed by in vitro binding assays . The coexpression of the two proteins in the transfected cells resulted in a colocalization in the intracellular vesicle-like structures . We thus propose that the association of Vesl-1L with syntaxin 13 plays a role in the translocation of Vesl-1L to the intracellular organelles .

Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 400 - 2
Identification of six CAG repeat domains into the human chromosomic region 12q24.1; Aguiar J; Six different domains of CAG repeats from a human chromosome 12-specific cosmid library were identified, cloned, and sequenced . These CAG repeat domains were localized into the human chromosomic region 12q24.1 . Five of them constitute repeat candidates for expansions in autosomal dominant neurological disorders with genetic anticipation, and they can also contribute to the chromosome walking in the human genome project .

Mol Genet Metab, 2000 May, 70(1), 69 - 74
The tissue-specific, alternatively spliced single ATG exon of the type 3 voltage-dependent anion channel gene does not create a truncated protein isoform in vivo; Decker WK et al.; Voltage-dependent anion channels (VDACs) are small, integral membrane proteins that traverse the outer mitochondrial membrane and conduct ATP and other small metabolites . They are known to bind several kinases of intermediary metabolism in a tissue-specific fashion, have been found in close association with the adenine nucleotide translocator of the inner mitochondrial membrane, and are hypothesized to form part of the mitochondrial permeability transition pore, which results in the release of cytochrome c at the onset of apoptotic cell death . VDACs are found throughout all strata of eukaryotic evolution and exhibit biophysical characteristics that are well conserved from yeast to mammals . The mammalian VDAC gene family consists of three isoforms, each of which shares approximately 70% sequence identity with the other two family members . Recently, we reported that a single codon (ATG) exon is alternatively spliced into the transcript of the type 3 voltage-dependent anion channel (VDAC3) in a tissue-specific fashion . This unusual splicing event was shown to be conserved between mouse and human, and we theorized that the spliced exon could lead to the creation of an alternative translational initiation site . Here we report that a highly specific polyclonal VDAC3 antibody was unable to detect the truncated protein isoform indicative of this putative downstream start site . From these in vivo studies, we conclude that the alternatively spliced exon results in the insertion of a single methionine residue at amino acid position 39 of the mature VDAC3 protein . Additionally, we have used a cross-species genomic sequence comparison to identify conserved regions that may be involved in the regulation of small exon splicing .

J Biochem (Tokyo), 2000 Jun, 127(6), 941 - 3
New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver; Tomisugi Y et al.; 20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast . To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source . Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them . The crystals belong to the same space group but have different cell dimensions . One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution . The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316 . 0 A, and diffracts to 4.0 A resolution . The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.

Microbiology, 2000 May, 146 ( Pt 5), 1045 - 51
Two distinct 18S rRNA secondary structures in Dipodascus (Hemiascomycetes); Ueda-Nishimura K et al.; The nucleotide sequences of the 18S rRNA gene from ascomycetous yeast-like fungi in the genera Dipodascus, Galactomyces and Geotrichum were determined and the tested strains were separated into two groups by sequence length . In group 1, the length and secondary structure of 18S rRNA corresponded to those of typical eukaryotes . In group 2, the 18S rRNA gene sequences were about 150 nt shorter than those of most other eukaryotes and the predicted secondary structure lacked helices 10 and E21-5 . Many substitutions and some deletions in group 2 18S rRNA gene were not only found in variable regions, but also in regions that are highly conserved among ascomycetes . Despite the considerable differences in 18S rRNA gene sequence and secondary structure between group 2 and other fungi, including group 1, phylogenetic analysis revealed that groups 1 and 2 are closely related . These findings suggest that a number of deletions occurred in the 18S rRNA of the common ancestor of group 2 strains.

Gene, 2000 May 16, 249(1-2), 135 - 44
Site-specific integration of targeted DNA into animal cell genomes; Koch KS et al.; Novel genetically engineered retroviral vectors and targeting plasmids are described that enable the site-specific targeting of exogenous DNA into the genomes of cultured animal cells . The protocol involves the transduction of competent cells by a chimeric retroviral vector containing a transcription unit composed of two linked cassettes: an upstream marker gene under the control of the viral 5' LTR; and a downstream reporter trap containing a strong promoter 5' to a 48bp yeast FRT element . When cells containing such integrated units are co-transfected with a plasmid encoding yeast FLP recombinase and a promoterless targeting plasmid containing a reporter cDNA tract 3' to an homologous FRT element, the targeting plasmid recombines at the chromosomally preconfigured FRT site, and a new hemizygous function is introduced into the downstream cassette . These reagents provide a new portable system for site-specific targeting of chemically modified genes into uniform and unique sites in genomically integrated transcription units.

J Inorg Biochem, 2000 Apr, 79(1-4), 11 - 9
Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein; Stillman MJ et al.; The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I) . Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein . CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures . Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data . Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I) . The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells . We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I) . Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM . Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.

Endocrinology, 2000 Jun, 141(6), 1977 - 88
Stat 5B, activated by insulin in a Jak-independent fashion, plays a role in glucokinase gene transcription; Sawka-Verhelle D et al.; Stat proteins are SH2 domain-containing transcription factors that are activated by various cytokines and growth factors . In a previous work, we have identified Stat 5B as a substrate of the insulin receptor based on yeast two-hybrid and mammalian cell transfection studies . In the present study, we have approached the biological relevance of the interaction between the insulin receptor and the transcription factor Stat 5B . Firstly, we show that both insulin and insulin-like growth factor I lead to tyrosine phosphorylation of Stat 5B, and this promotes binding of the transcription factor to the beta-casein promoter containing a Stat 5 binding site . Further, we demonstrate that insulin stimulates the transcriptional activity of Stat 5B . Activation of Stat 5B by insulin appears to be Jak2-independent, whereas Jak2 is required for GH-induced Stat 5B activation . Hence the pathway by which Stat 5B is activated by insulin is different from that used by GH . In addition, by using Jak1- and Tyk2-deficient cells we exclude the involvement of both Jak1 and Tyk2 in Stat 5B activation by insulin . Taken together, our results strengthen the notion that insulin receptor can directly activate Stat 5B . More importantly, we have identified a Stat 5 binding site in the human hepatic glucokinase promoter, and we show that insulin leads to a Stat 5B-dependent increase in transcription of a reporter gene carrying this promoter . These observations favor the idea that Stat 5B plays a role in mediating the expression of the glucokinase gene induced by insulin . As a whole, our results provide evidence for the occurrence of a newly identified circuit in insulin signaling in which the cell surface receptor is directly linked to nuclear events through a transcription factor . Further, we have revealed an insulin target gene whose expression is, at least in part, dependent on Stat 5B activation and/or binding.

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6350 - 5
A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing; Polydorides AD et al.; The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing . We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB) . The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons . brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei . We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor alpha2 (GlyRalpha2) pre-mRNA, and this binding is implicated in Nova-dependent regulation of splicing . Cotransfection assays with a GlyRalpha2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyRalpha2 E3A . brPTB binds to a 90-nt GlyRalpha2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova . When a putative binding site for brPTB on the GlyRalpha2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears . These results suggest that brPTB is a tissue-restricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.

J Biol Chem, 2000 Sep 1, 275(35), 27212 - 20
Fibrillarin genes encode both a conserved nucleolar protein and a novel small nucleolar RNA involved in ribosomal RNA methylation in Arabidopsis thaliana; Barneche F et al.; Fibrillarin is a key nucleolar protein in eukaryotes which associates with box C/D small nucleolar RNAs (snoRNAs) directing 2'-O-ribose methylation of the rRNA . In this study we describe two genes in Arabidopsis thaliana, AtFib1 and AtFib2, encoding nearly identical proteins conserved with eukaryotic fibrillarins . We demonstrate that AtFib1 and AtFib2 proteins are functional homologs of the yeast Nop1p (fibrillarin) and can rescue a yeast NOP1-null mutant strain . Surprisingly, for the first time in plants, we identified two isoforms of a novel box C/D snoRNA, U60.1f and U60.2f, nested in the fifth intron of AtFib1 and AtFib2 . Interestingly after gene duplication the host intronic sequences completely diverged, but the snoRNA was conserved, even in other crucifer fibrillarin genes . We show that the U60f snoRNAs accumulate in seedlings and that their targeted residue on the 25 S rRNA is methylated . Our data reveal that the three modes of expression of snoRNAs, single, polycistronic, and intronic, exist in plants and suggest that the mechanisms directing rRNA methylation, dependent on fibrillarin and box C/D snoRNAs, are evolutionarily conserved in plants.

Cytogenet Cell Genet, 2000, 88(3-4), 289 - 95
Delineation and physical separation of novel translocation breakpoints on chromosome 1p in two genetically closely associated childhood tumors; Steenman MJ et al.; Sporadic childhood tumors associated with Beckwith-Wiedemann syndrome (BWS) all show abnormalities of the same region on chromosome 11 . In addition to chromosome 11, other chromosome regions are affected in some of these tumor types . In this study we analyzed the region on chromosome 1p involved in the etiology of BWS-associated tumors, Wilms tumor, rhabdomyosarcoma, and hepatoblastoma . For this purpose we determined the location of two novel translocation breakpoints in this chromosome region in cells from a Wilms tumor and cells from a rhabdomyosarcoma . We constructed a map of the region and found that both breakpoints are separated by at least 875 kb . We identified a PAC clone which crosses the rhabdomyosarcoma breakpoint and found several exons within this clone . We established that this breakpoint is located proximal to the PAX7 gene and, therefore, identified a new region involved in the etiology of rhabdomyosarcomas .

Cytogenet Cell Genet, 2000, 88(3-4), 249 - 52
Chromosome mapping of the human genes encoding the MAP kinase kinase MEK1 (MAP2K1) to 15q21 and MEK2 (MAP2K2) to 7q32; Meloche S et al.; Activation of the ERK mitogen-activated protein (MAP) kinase pathway has been implicated in the regulation of cell growth, differentiation and senescence . In this pathway, the MAP kinases ERK1/ERK2 are phosphorylated and activated by the dual-specificity kinases MEK1 and MEK2, which in turn are activated by serine phosphorylation by a number of MAP kinase kinase kinases . We report here the chromosomal localization of the human genes encoding the MAP kinase kinase isoforms MEK1 and MEK2 . Using a combination of fluorescence in situ hybridization, somatic cell hybrid analysis, DNA sequencing and yeast artificial chromosome (YAC) clone analysis, we have mapped the MEK1 gene (MAP2K1) to chromosome 15q21 . We also present evidence for the presence of a MEK1 pseudogene on chromosome 8p21 . The MEK2 gene (MAP2K2) was mapped to chromosome 7q32 by fluorescence in situ hybridization and YAC clone analysis .

Cytogenet Cell Genet, 2000, 88(3-4), 225 - 9
Expression map of human chromosome region 17p13.3, spanning the RP13 dominant retinitis pigmentosa locus, the Miller-Dieker lissencephaly syndrome (MDLS) region, and a putative tumour suppressor locus; McHale JC et al.; Chromosome region 17p13.3 is rich in genes, with 223 expressed sequence tags (ESTs) within the last 15 cM (7 Mb) of chromosome 17p in the GeneMap database . Loci for dominant retinitis pigmentosa (RP13), central areolar choroidal dystrophy (CACD), anterior polar cataract (CTAA2), Miller-Dieker lissencephaly syndrome (MDLS), and a region of tumour loss of heterozygosity (LOH) distinct from TP53 all map into the region adjacent to the 17p telomere . To date, however, there is no physical map of the region, which has resisted the efforts of the CEPH and Whitehead physical mapping programmes to generate contiguous clones across it . We have created a physical map covering approximately 3.5 Mb (6 cM)in this region, spanning the RP13 interval and extending distally to the gene MDCR (formerly, LIS1), which, when deleted, leads to the MDLS phenotype . The region covered is also the point of maximum LOH in lung cancer and has been implicated in the pathogenesis of many other human cancers . The map orders 47 sequence tagged sites, including 32 genes or ESTs, nine genetic markers, four anonymous sequences, and two YAC end clones, and highlights new candidate ESTs for involvement in RP13, MDLS, CTAA2, and a tumour-susceptibility gene .

Hum Reprod, 2000 Jun, 15(6), 1289 - 94
Increased frequency of mutations in DNA from infertile men with meiotic arrest; Nudell D et al.; In diverse organisms from yeast to mice, mutations in numerous genes required for DNA repair may lead to defects in meiosis . Although it is likely that meiosis is conserved throughout evolution, little is known about the genetics of meiosis in humans even though meiotic arrest associated with azoospermia is common . In this work, we compared the sequence fidelity of a polymorphic marker amplified from DNA of two groups of patients: those with testis biopsy suggesting meiotic arrest and those with normal spermatogenesis who were obstructed . We demonstrated that mutations are more common in DNA from testicular tissue derived from men with meiotic arrest than in DNA from testicular tissue derived from men with normal spermatogenesis and physical obstruction (P < 0.05) . No mutations were observed in blood tissue from either group of men . This suggests the possibility that defects in genes required in DNA repair could contribute to meiotic arrest in men just as has been observed in other organisms.

J Biol Chem, 2000 Jun 2, 275(22), 16827 - 36
Interaction of GRASP, a protein encoded by a novel retinoic acid-induced gene, with members of the cytohesin family of guanine nucleotide exchange factors; Nevrivy DJ et al.; A novel, retinoic acid-induced gene, GRP1-associated scaffold protein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy . GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary . The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell . Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells . Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1 (GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins . GRASP . GRP1 interactions were also demonstrated in vitro and in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane . Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases.

J Biol Chem, 2000 Aug 18, 275(33), 25820 - 30
Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin; Ireton GC et al.; Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine . Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains . Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer . The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits . The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive . However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme . The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker . Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.

Am J Hum Genet, 2000 Jul, 67(1), 14 - 22 Epub 2000 May 25.
Additional copies of the proteolipid protein gene causing Pelizaeus-Merzbacher disease arise by separate integration into the X chromosome; Hodes ME et al.; The proteolipid protein gene (PLP) is normally present at chromosome Xq22 . Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD) . Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22 . In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene . The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis . Another three affected males from the family had similar findings . In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome . In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22 . A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern . The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders.

J Biol Chem, 2000 Aug 18, 275(33), 25411 - 7
NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry; Boudrez A et al.; NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles . We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module . A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing . CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells . Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments . The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g . by cyclin E-Cdk2 . When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition . However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts . A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects . We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 337 - 46
Modestobacter multiseptatus gen . nov., sp . nov., a budding actinomycete from soils of the Asgard Range (Transantarctic Mountains); Mevs U et al.; Oligotrophic PYGV medium, inoculated with soils from Linnaeus Terrace (1600 m, Antarctica), yielded four aerobic actinomycetes with short rods, multiple and irregular septa and often motile buds . Cells were 1.0-2.8 x 1.0-3.0 microm and colonies were beige to pink . The isolates were nearly identical in physiological and biochemical tests . Three strains grew from 0 degrees C to 25-28 degrees C, but one was psychrophilic with a maximum growth temperature of 20 degrees C . Carbon sources utilized were D-glucose, D-galactose, lactose, sucrose or mannitol; malate, succinate, fumarate, pyruvate or glutarate were decarboxylated aerobically . Peptone and yeast extract were the preferred nitrogen sources . Nitrate was reduced aerobically or anaerobically . Cell walls contained meso-diaminopimelic acid, glutamate, alanine, glycine, galactose, glucose and ribose . Major fatty acids of strains AA-802, -824, -825 and -826T were n18:1, i16:0 and ai17:0 . Major respiratory quinones were MK-9(H4) and MK-8(H4) . Polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol . Phosphatidylglycerol was found in most strains . The DNA G+C contents were 68-70 mol% . In 16S rDNA analyses, similarity values obtained for 500 nucleotides from the 5' terminus were > 99.5% . Almost complete sequences from AA-826T and -825 were 99.9% similar . Strain AA-826T belonged to a novel cluster of desert soil and rock isolates within the Geodermatophilaceae and was equidistantly related to members of Geodermatophilus and to a Blastococcus lineage . The four isolates represent a new genus, Modestobacter gen . nov., with Modestobacter multiseptatus sp . nov . as the type species . The type strain, Modestobacter multiseptatus AA-826T, was deposited in the DSMZ as DSM 44406T.

Eur J Cell Biol, 2000 Apr, 79(4), 240 - 51
Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus; Behrend L et al.; Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis . We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules . Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation . In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage . The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.

J Cell Sci, 2000 Jun, 113 ( Pt 12), 2273 - 84
Hrs interacts with SNAP-25 and regulates Ca(2+)-dependent exocytosis; Kwong J et al.; Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis . To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen . Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) . Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet . The association of Hrs and SNAP-25 is mediated via coiled-coil interactions . Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions . Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools . Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells . Moreover, overexpression of Hrs in PC12 cells inhibits Ca(2+)-dependent exocytosis . These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.

J Cell Sci, 2000 Jun, 113 ( Pt 12), 2221 - 31
Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription; Javed A et al.; The Runt related transcription factors RUNX (AML/CBF(alpha)/PEBP2(alpha)) are key regulators of hematopoiesis and osteogenesis . Using co-transfection experiments with four natural promoters, including those of the osteocalcin (OC), multi drug resistance (MDR), Rous Sarcoma Virus long terminal repeat (LTR), and bone sialoprotein (BSP) genes, we show that each of these promoters responds differently to the forced expression of RUNX proteins . However, the three RUNX subtypes (i.e . AML1, AML2, and AML3) regulate each promoter in a similar manner . Although the OC promoter is activated in a C terminus dependent manner, the MDR, LTR and BSP promoters are repressed by three distinct mechanisms, either independent of or involving the AML C terminus, or requiring only the conserved C-terminal pentapeptide VWRPY . Using yeast two hybrid assays we find that the C terminus of AML1 interacts with a Groucho/TLE/R-esp repressor protein . Co-expression assays reveal that TLE proteins repress AML dependent activation of OC gene transcription . Western and northern blot analyses suggest that TLE expression is regulated reciprocally with the levels of OC gene expression during osteoblast differentiation . Digital immunofluorescence microscopy results show that TLE1 and TLE2 are both associated with the nuclear matrix, and that a significant subset of each colocalizes with AML transcription factors . This co-localization of TLE and AML proteins is lost upon removing the C terminus of AML family members . Our findings indicate that suppression of AML-dependent gene activation by TLE proteins involves functional interactions with the C terminus of AML at the nuclear matrix in situ . Our data are consistent with the concept that the C termini of AML proteins support activation or repression of cell-type specific genes depending on the regulatory organization of the target promoter and subnuclear localization.

Mol Cell Biol, 2000 Jun, 20(12), 4462 - 73
The oncoprotein kinase chaperone CDC37 functions as an oncogene in mice and collaborates with both c-myc and cyclin D1 in transformation of multiple tissues; Stepanova L et al.; CDC37 encodes a 50-kDa protein that targets intrinsically unstable oncoprotein kinases including Cdk4, Raf-1, and v-src to the molecular chaperone Hsp90, an interaction that is thought to be important for the establishment of signaling pathways . CDC37 is required for proliferation in budding yeast and is coexpressed with cyclin D1 in proliferative zones during mouse development, a finding consistent with a positive role in cell proliferation . CDC37 expression may not only be required to support proliferation in cells that are developmentally programmed to proliferate but may also be required in cells that are inappropriately induced to initiate proliferation by oncogenes . Here we report that mouse mammary tumor virus (MMTV)-CDC37 transgenic mice develop mammary gland tumors at a rate comparable to that observed previously in MMTV-cyclin D1 mice . Moreover, CDC37 was found to collaborate with MMTV-c-myc in the transformation of multiple tissues, including mammary and salivary glands in females and testis in males, and also collaborates with cyclin D1 to transform the female mammary gland . These data indicate that CDC37 can function as an oncogene in mice and suggests that the establishment of protein kinase pathways mediated by Cdc37-Hsp90 can be a rate-limiting event in epithelial cell transformation.

Mol Cell Biol, 2000 Jun, 20(12), 4350 - 8
Artificial recruitment of TFIID, but not RNA polymerase II holoenzyme, activates transcription in mammalian cells; Dorris DR et al.; In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain . Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells . Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters . In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains . In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter . Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters . Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.

J Biol Chem, 2000 Sep 15, 275(37), 28785 - 92
HPC3 is a new human polycomb orthologue that interacts and associates with RING1 and Bmi1 and has transcriptional repression properties; Bardos JI et al.; Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maintaining the transcriptionally repressed state of Hox/homeotic genes in a stable and heritable manner throughout development . A growing number of vertebrate genes related to the Drosophila PcG proteins have recently been identified, including two Polycomb orthologues, Pc2 and M33 . PcG proteins form multiprotein complexes, termed PcG bodies, that are thought to repress transcription by altering chromatin structure . Here we report the identification and characterization of HPC3 (human Polycomb 3), a novel PcG protein isolated in a yeast two-hybrid screen using human RING1 as bait . HPC3 shows strong sequence similarity to Drosophila Pc and also to vertebrate Pc2 and M33, particularly within the chromodomain and C-box . Previous studies indicate that M33 and human Pc2 (HPC2) can interact with RING1, and we show here that HPC3 also binds to RING1 . This interaction is dependent upon the HPC3 C-box but, only partially on the RING finger of RING1 . In contrast to HPC2, HPC3 interactions with RING1 are only observed in vivo with covalently modified forms of RING1 . HPC3 also colocalizes with other PcG proteins in human PcG bodies . Consistent with its role as a PcG member, HPC3 is able to act as a long range transcriptional silencer when targeted to a reporter gene by a heterologous DNA-binding domain . Taken together, these data suggest that HPC3 is part of a large multiprotein complex that also contains other PcG proteins and is involved in repression of transcriptional activity.

J Biol Chem, 2000 Aug 4, 275(31), 23666 - 73
Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice; Oshel KM et al.; We have previously demonstrated that the important cis-acting elements regulating transcription of the human GLUT4 gene reside within 895 base pairs (bp) upstream of the transcription initiation site (Thai, M . V., Guruswamy, S., Cao, K . T., Pessin, J . E., and Olson, A . L . (1998) J . Biol . Chem . 273, 14285-14292) . Our studies demonstrated that an MEF2 binding site within this region was necessary, but not sufficient, for GLUT4 promoter function in transgenic mice . We have identified a second regulatory element (Domain I) that functions cooperatively with the MEF2 domain in regulating GLUT4 transcription . Using a yeast-one hybrid screen, we obtained a partial cDNA and generated an antibody directed against a protein binding specifically to Domain I . Sequence analysis of the partial cDNA indicates that the protein binding to Domain I is a novel protein . The antibody specifically labels two proteins of approximately 70 and 50 kDa in Western blot analysis . These molecular masses correspond to Domain I binding proteins identified by UV-cross-linking nuclear extracts to a Domain I probe . The antibody raised against the Domain I binding protein inhibited formation of a Domain I-protein complex in electrophoretic mobility shift assays . We conclude that we have identified an authentic, novel, Domain I binding protein required for transcriptional regulation of the human GLUT4 promoter.

J Biol Chem, 2000 Aug 25, 275(34), 26172 - 7
Functional cloning and characterization of a novel nonhomeodomain protein that inhibits the binding of PBX1-HOX complexes to DNA; Abramovich C et al.; PBX1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during developmental and/or differentiation processes . A yeast two-hybrid screen of a fetal liver-hematopoietic cDNA library using PBX1a as bait led to the discovery of a novel non-homeodomain-containing protein that interacts with PBX1 as well as PBX2 and PBX3 . RNA analysis revealed it to be expressed in CD34(+) hematopoietic cell populations enriched in primitive progenitors, as is PBX1; search of the expressed sequence tag data base indicated that it is also expressed in other early embryonic as well as adult tissues . The full-length cDNA encodes a 731-amino acid protein that has no significant homology to known proteins . This protein that we have termed hematopoietic PBX-interacting protein (HPIP) is mainly localized in the cytosol and in small amounts in the nucleus . The region of PBX that interacts with HPIP includes both the homeodomain and immediate N-terminal flanking sequences . Strikingly, electrophoretic mobility shift assays revealed that HPIP inhibits the ability of PBX-HOX heterodimers to bind to target sequences . Moreover, HPIP strongly inhibits the transactivation activity of E2A-PBX . Together these findings suggest that HPIP is a new regulator of PBX function.

J Biol Chem, 2000 Sep 1, 275(35), 27084 - 93
The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM; Hawkins CJ et al.; The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death . All caspases studied to date cleave substrates COOH-terminal to an aspartate . Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate . DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate . DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate . Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death . Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM . These observations suggest that DRONC acts to promote cell death . However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35 . We discuss possible models for DRONC function as a cell death inhibitor.

Genes Chromosomes Cancer, 2000 Jun, 28(2), 153 - 63
A consistent pattern of RIN1 rearrangements in oral squamous cell carcinoma cell lines supports a breakage-fusion-bridge cycle model for 11q13 amplification; Shuster MI et al.; Gene amplification is a common feature of tumors . Overexpression of some amplified genes plays a role in tumor progression . Gene amplification can occur either extrachromosomally as double-minute chromosomes (dmin) or intrachromosomally in the form of homogeneously staining regions (hsrs) . Approximately one-half of our oral squamous cell carcinomas (OSCCs) are characterized by amplification of band 11q13, usually as an hsr located entopically (occurring or situated at the normal chromosomal site, as opposed to ectopically) . Using chromosomal fluorescence in situ hybridization (FISH), we confirmed the amplification of the cyclin D1 (CCND1/PRAD1) and fibroblast growth factor types 3 and 4 (FGF3/INT2 and FGF4/HSTF1) genes within the 11q13 amplicon in our series of primary OSCCs and derived cell lines . The human RIN1 gene was isolated as an RAS interaction/interference protein in a genetic selection in yeast and has been described as a putative effector of both the RAS and ABL oncogenes . We mapped RIN1 to 11q13.2 . FISH analysis of 10 11q13-amplified OSCC cell lines revealed high-level RIN1 amplification in two cell lines . Three additional cell lines have what appear to be duplications and/or low-level amplification of RIN1, visible in both interphase and metaphase cells . The hybridization pattern of RIN1 on the metaphase chromosomes is particularly revealing; RIN1 signals flank the 11q13 hsr, possibly as a result of an inverted duplication . The gene amplification model of Coquelle et al . (1997) predicted that gene amplification occurs by breakage-fusion-bridge (BFB) cycles involving fragile sites . Our data suggest that the pattern of gene amplification at 11q13 in OSCC cell lines is consistent with a BFB model . RIN1 appears to be a valuable probe for investigating the process of gene amplification in general and, specifically, 11q13 amplification in oral cancer.

Genes Chromosomes Cancer, 2000 Jun, 28(2), 138 - 44
MOZ is fused to p300 in an acute monocytic leukemia with t(8;22); Chaffanet M et al.; We report on the fusion of the monocytic leukemia zinc finger protein (MOZ) gene to the adenoviral E1A-associated protein p300 (p300) gene in acute monocytic leukemia M5 associated with a t(8;22)(p11;q13) translocation . We studied two patients with double-color fluorescence in situ hybridization (FISH) using the yeast artificial chromosome 176C9 and the bacterial artificial chromosome clone H59D10 specific to the MOZ and p300 genes, respectively . Both probes were split in the patients' chromosome metaphase cells, and the two derivative chromosomes were each labeled with both probes . We showed by Southern blot the rearrangement of the MOZ gene, and cloned the fusion transcripts in one patient carrying the t(8;22) by reverse transcription-polymerase chain reaction using MOZ- and p300-specific primers . Both fusion transcripts were expressed . This result defines a novel reciprocal translocation involving two acetyltransferases, MOZ and p300, resulting in an abnormal transcriptional co-activator that could play a critical role in leukemogenesis.

Biosens Bioelectron, 2000 Mar, 15(1-2), 77 - 83
Development of highly selective and stable potentiometric sensors for formaldehyde determination; Korpan YI et al.; Two types of biosensors selective to formaldehyde have been developed on the basis of pH-sensitive field effect transistor as a transducer . Highly or partially purified alcohol oxidase (AOX) and the permeabilised cells of methylotrophic yeast Hansenula polymorpha (as a source of AOX) have been used as sensitive elements . The response time in steady-state measurement mode is in the range of 10-60 s for the enzyme-based sensors and 60-120 s for the cell-based sensor . When measured in kinetic mode the response time of all biosensors developed was less than 5 s . The linear dynamic range of the sensor output signals corresponds to 5-200 mM formaldehyde for highly and partially purified alcohol oxidase, and 5-50 mM formaldehyde for the cells . The operational stability of the biosensors is not less than 7 h, and the relative standard deviation of intra-sensor response is approximately 2 and 5% for the enzyme- and cell-based sensors, respectively . When stored at 4 degrees C, the enzyme and cell sensor responses have been found stable for more than 60 and 30 days, respectively . Both types of biosensors demonstrate a high selectivity to formaldehyde with no potentiometric response to primary alcohols, including methanol, or glycerol and glucose . The possible reasons of such unexpected high selectivity of AOX-based FET-sensors to formaldehyde are discussed . The influence of the biomembrane composition and the effect of different buffers on the sensor response to formaldehyde are also discussed.

Proc Natl Acad Sci U S A, 2000 May 23, 97(11), 6224 - 9
A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis; Hwang I et al.; The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux . In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases . ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum . Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast . A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1 . In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities . An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition . Interestingly, prior binding of calmodulin blocked phosphorylation . This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition . These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

Proc Natl Acad Sci U S A, 2000 May 23, 97(11), 6212 - 7
Thyroid hormone receptor-binding protein, an LXXLL motif-containing protein, functions as a general coactivator; Ko L et al.; Nuclear hormone receptors activate gene transcription through ligand-dependent association with coactivators . Specific LXXLL sequence motifs present in these cofactors are sufficient to mediate these ligand-induced interactions . A thyroid hormone receptor (TR)-binding protein (TRBP) was cloned by a Sos-Ras yeast two-hybrid system using TRbeta1-ligand binding domain as bait . TRBP contains 2063 amino acid residues, associates with TR through a LXXLL motif, and is ubiquitously expressed in a variety of tissues and cells . TRBP strongly transactivates through TRbeta1 and estrogen receptor in a dose-related and ligand-dependent manner, and also exhibits coactivation through AP-1, CRE, and NFkappaB-response elements, similar to the general coactivator CBP/p300 . The C terminus of TRBP binds to CBP/p300 and DRIP130, a component of the DRIP/TRAP/ARC complex, which suggests that TRBP may activate transcription by means of such interactions . Further, the association of TRBP with the DNA-dependent protein kinase (DNA-PK) complex and DNA-independent phosphorylation of TRBP C terminus by DNA-PK point to a potential connection between transcriptional control and chromatin architecture regulation.

J Virol, 2000 Jun, 74(12), 5569 - 76
Rubella virus capsid associates with host cell protein p32 and localizes to mitochondria; Beatch MD et al.; Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA . The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes . The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein . Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system . One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins . The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria . Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region . The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid . Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.

J Biol Chem, 2000 Aug 11, 275(32), 24255 - 63
The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes; Lin Y et al.; The central regulatory role of the adipocyte in whole body energy homeostasis is well established . However, recent findings suggest that preadipocytes and adipocytes may play an important physiological role in the regulation of both the innate and adaptive immune response . To systematically characterize the molecular machinery of the adipocyte that mediates the recognition of pathogens, we have focused our analysis on the recently identified Toll-like receptors (TLRs) . These receptors have been implicated as mediators of the cellular response to bacterial lipopolysacharides (LPSs) . Here, we report the cloning and functional characterization of mouse TLR-2 from 3T3-L1 adipocytes . TLR-2 synthesis is strongly induced in the adipocyte by LPS, TNFalpha, and the yeast cell wall extract zymosan . TLR-2 undergoes a lengthy intracellular maturation process with a half-life of exit from the ER of approximately 3 h . Furthermore, LPS treatment of adipocytes results in dramatic changes at the level of gene expression, including the synthesis of a distinct set of secretory proteins such as interleukin-6 . Our studies demonstrate the presence of a fully intact pathway of innate immunity in the adipocyte that can be activated by LPS binding to the cell surface and results in the secretion of immunomodulatory molecules.

Oncogene, 2000 Apr 27, 19(18), 2186 - 93
Induction of human Cdc37 in prostate cancer correlates with the ability of targeted Cdc37 expression to promote prostatic hyperplasia; Stepanova L et al.; The Cdc37 gene encodes a 50 kDa protein which targets intrinsically unstable oncoprotein kinases such as Cdk4, Raf-1, and src to the molecular chaperone Hsp90 . This activity is thought to play an important role in the establishment of signaling pathways controlling cell proliferation . The budding yeast Cdc37 homolog is required for cell division and mammalian Cdc37 is expressed in proliferative zones during embryonic development and in adult tissues, consistent with a positive role in proliferation . Here we report that human prostatic tumors, neoplasias and certain pre-malignant lesions display increased Cdc37 expression, suggesting an important and early role for Cdc37 in prostatic transformation . To test the consequences of increased Cdc37 levels, transgenic mice expressing Cdc37 in the prostate were generated . These mice displayed a wide range of growth-related abnormalities including prostatic epithelial cell hyperplasia and dysplasia . These data suggest that the expression of Cdc37 may promote inappropriate proliferation and may be an important early step in the development of human prostate cancer.

J Biol Chem, 2000 May 26, 275(21), 15985 - 91
SOCS-3 is an insulin-induced negative regulator of insulin signaling; Emanuelli B et al.; The SOCS proteins are induced by several cytokines and are involved in negative feedback loops . Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2 . Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression . Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter . Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity . Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif . First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds . Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor . This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960 . Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3 . We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation . By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.

J Infect Dis, 2000 May, 181(5), 1720 - 8 Epub 2000 May 15.
Investigation of anti-WI-1 adhesin antibody-mediated protection in experimental pulmonary blastomycosis; Wuthrich M et al.; Infection with Blastomyces dermatitidis elicits strong antibody responses to the surface adhesin WI-1 . The antibodies are directed chiefly against the adhesive domain, a 25-amino-acid repeat . Tandem-repeat-specific monoclonal antibodies (mAbs) were studied for their opsonic activity in vitro and their capacity to adoptively transfer protection in murine experimental blastomycosis . mAbs to WI-1 enhanced binding and entry of B . dermatitidis yeasts into J774 . 16 cells but did not enhance killing or growth inhibition of the yeast . Passive transfer of 8 mAbs to WI-1 into 3 different inbred strains of mice also did not improve the course of experimental infection and sometimes worsened it . mu-deficient mice were more resistant to experimental blastomycosis than were intact littermates, and passive transfer of the mAbs into these mice did not protect them against experimental infection . Thus, antibody to WI-1 does not appear to improve the outcome of murine blastomycosis and may enhance the infection.

Dev Dyn, 2000 May, 218(1), 102 - 11
Rnf4, a RING protein expressed in the developing nervous and reproductive systems, interacts with Gscl, a gene within the DiGeorge critical region; Galili N et al.; A yeast 2-hybrid screen was performed to identify possible transcriptional modulators interactive with goosecoid-like (gscl), a transcription factor with suppressive activity, expressed during early brain and gonad development . The screen resulted in the identification of a RING protein known as rnf4 or snrf . Gscl/rnf4 interactions were confirmed by affinity chromatography and by immunoprecipitation . Northern analysis confirmed earlier reports of ubiquitous rnf4 expression in adult tissues . Immunohistochemical analysis of mouse embryos revealed expression primarily in the developing nervous system, with strong expression in the dorsal root ganglia and developing gonads . In contrast to previous reports, both cytoplasmic and nuclear expression of rnf4 was documented . The results reported here confirm and extend earlier reports of rnf4 expression . They suggest for the first time, that in addition to acting as a modulator of transcriptional activity, rnf4 may function, as do other RING proteins, to promote the formation of intracytoplasmic complexes involved in shuttling information between the cytoplasm and the nucleus.

Biochemistry, 2000 May 23, 39(20), 6207 - 18
Selection of viral RNA-derived tRNA-like structures with improved valylation activities; Wientges J et al.; The tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) RNA was previously shown to be efficiently charged by yeast valyl-tRNA synthetase (ValRS) . This RNA has a noncanonical structure at its 3'-terminus but mimics a tRNA L-shaped fold, including an anticodon loop containing the major identity nucleotides for valylation, and a pseudoknotted amino acid accepting domain . Here we describe an in vitro selection experiment aimed (i) to verify the completeness of the valine identity set, (ii) to elucidate the impact of the pseudoknot on valylation, and (iii) to investigate whether functional communication exists between the two distal anticodon and amino acid accepting domains . Valylatable variants were selected from a pool of 2 x 10(13) RNA molecules derived from the TYMV TLS randomized in the anticodon loop nucleotides and in the length (1-6 nucleotides) and sequence of the pseudoknot loop L1 . After nine rounds of selection by aminoacylation, 42 have been isolated . Among them, 17 RNAs could be efficiently charged by yeast ValRS . Their sequence revealed strong conservation of the second and the third anticodon triplet positions (A(56), C(55)) and the very 3'-end loop nucleotide C(53) . A large variability of the other nucleotides of the loop was observed and no wild-type sequence was recovered . The selected molecules presented pseudoknot domains with loop L1 varying in size from 3-6 nucleotides and some sequence conservation, but did neither reveal the wild-type combination . All selected variants are 5-50 times more efficiently valylated than the wild-type TLS, suggesting that the natural viral sequence has emerged from a combination of evolutionary pressures among which aminoacylation was not predominant . This is in line with the role of the TLS in viral replication.

Mol Cell Biochem, 2000 Feb, 205(1-2), 141 - 7
Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin; Miyagishi M et al.; A family of basic-helix-loop-helix (bHLH) nuclear factors play important roles in controlling cell growth and differentiation as critical regulatory components in transcription . Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin . Transactivation property of POD-1/Capsulin was analyzed by the Gal4 fusion system in six mammalian cell lines . The results indicated that an activation property was shown in HT1080 and HeLa cells, but a repression activity in HepG2 cells . Mapping analysis for the transactivation and repression activities revealed that the C-terminal domain of POD-1/Capsulin is essential for the transactivation and both the N-terminal and C-terminal domains are contributed to the repression activities . Furthermore, in order to identify possible interactants of the POD-1/Capsulin, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified a class A bHLH protein, ITF-2 as potential heterodimeric partner of the bHLH protein.

J Immunol, 2000 Jun 1, 164(11), 5805 - 14
GRID: a novel Grb-2-related adapter protein that interacts with the activated T cell costimulatory receptor CD28; Ellis JH et al.; Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners . These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs . Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28 . The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure . A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains . We term this protein GRID for Grb2-related protein with insert domain . GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28 . Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain . In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1) . These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.

J Mammary Gland Biol Neoplasia, 1998 Oct, 3(4), 413 - 21
Functional characterization of BRCA1 and BRCA2: clues from their interacting proteins; Sharan SK et al.; The familial breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2 have been the subject of extensive functional analysis studies since their cloning . Clues to their biological role in maintaining the genomic integrity were provided by studies that revealed their interaction with the recombination repair protein HsRad51 . The first clue of an interaction between HsRad51 and BRCA1 came from the colocalization of the characteristic nuclear foci formed by these two proteins during S phase of the cell cycle . An interaction between murine Brca2 and MmRad51 was detected by the yeast two hybrid system . Utilizing the yeast two hybrid system and other techniques several other Brca1 and Brca2 interacting proteins have been identified like, BARD1, importin-alpha, BIPs, RNA polymerase II holoenzyme, BRAP2 etc . Recently, mutations suggesting a role as a tumor suppressor have been identified in the BARD1 gene in primary human tumors . The identification of molecules that interact with Brca1 and Brca2 has greatly enhanced our knowledge of how BRCA1 and BRCA2 may function as tumor suppressors.

Ann N Y Acad Sci, 2000 Apr, 905, 54 - 60
Sphingosine 1-phosphate: a ligand for the EDG-1 family of G-protein-coupled receptors; Spiegel S; Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, cell growth, and survival . Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide, and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies . This ceramide/SPP rheostat is an evolutionarily conserved stress regulatory mechanism influencing growth and survival of yeast . In addition, SPP also has been identified as the ligand for the G-protein-coupled receptors EDG-1, -3, -5, and -6 . Binding of SPP to EDG-1 regulates chemotaxis and in vitro angiogenesis of endothelial cells, whereas EDG-5, and possibly EDG-3, are likely the cell surface receptors responsible for cell rounding and neurite retractions induced by SPP . Hence, the studies identify a family of highly specific SPP receptors that are capable of mediating different biological responses . Thus, it is suggested that SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers . Recently, sphingosine kinase was purified to homogeneity and the first mammalian sphingosine kinase, the enzyme responsible for the formation of SPP, was cloned . The studies should provide the necessary tools to develop insight into the biological roles of this important bioactive sphingolipid.

Ann Genet, 2000 Jan-Mar, 43(1), 5 - 9
Determination of the gene structure of human oligophrenin-1 and identification of three novel polymorphisms by screening of DNA from 164 patients with non-specific X-linked mental retardation; Billuart P et al.; We have recently shown that mutations in oligophrenin-1 (OPHN1) are responsible for non-specific X-linked mental retardation (MRX) . The structure of the gene encoding the OPHN1 protein was determined by isolation of genomic DNA clones from the human cosmid library . Genomic fragments containing exons were sequenced, and the sequences of the exons and flanking introns were defined . Knowledge of the genomic structure of the OPHN1 gene, which spans at least 500 kb and consists of 25 exons, will facilitate the search for additional mutations in OPHN1 . OPHN1 was screened for mutations in 164 subjects with non-specific mental retardation . Three nucleotide substitutions were identified, one of which was a silent mutation in the codon threonine 301 at position 903 (G-->C) . The other substitutions were located in exon 2, a G-->A substitution at position 133 (A45T), and in exon 10, a C-->T substitution at position 902 (T301M), but these are common polymorphisms rather than disease-causing mutations.

J Neurosci, 2000 Jun 1, 20(11), 4069 - 80
Identification of proteins in the postsynaptic density fraction by mass spectrometry; Walikonis RS et al.; Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs . In this study, we have completed the identification of most major proteins in the PSD fraction with the use of an analytical method based on mass spectrometry coupled with searching of the protein sequence databases . At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified . We found 7 proteins not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods . The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog of the yeast septin protein cdc10, which is important for bud formation in yeast . Both myosin-Va and cdc10 are threefold to fivefold enriched in the PSD fraction over brain homogenates . Immunocytochemical localization of myosin-Va in cultured hippocampal neurons shows that it partially colocalizes with PSD-95 at synapses and is also diffusely localized in cell bodies, dendrites, and axons . Cdc10 has a punctate distribution in cell bodies and dendrites, with some of the puncta colocalizing with PSD-95 . The results support a role for myosin-Va in transport of materials into spines and for septins in the formation or maintenance of spines.

J Biol Chem, 2000 Aug 25, 275(34), 26213 - 9
A Di-leucine signal in the ubiquitin moiety . Possible involvement in ubiquitination-mediated endocytosis; Nakatsu F et al.; Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes upon ligand-dependent ubiquitination . However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial . In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins . The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently . Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis . We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu(43)-Ile(44) . The Leu(43)-Ile(44) sequence mediated endocytosis and lysosomal sorting in a Leu(43)-dependent manner . These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.

J Biol Chem, 2000 Aug 4, 275(31), 23685 - 92
Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response; Pagani M et al.; Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins . A family of conserved genes, termed ERO for ER oxidoreductins, plays a key role in this process . We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo-sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M . R., Bulleid, N . J., and Sitia, R . (2000) J . Biol . Chem . 275, 4827-4833) . Here, we report the cloning and characterization of a novel human member of this family, ERO1-Lbeta . Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-Lbeta gene are primarily localized in the ER of mammalian cells . The ability to allow growth at 37 degrees C and to alleviate the "unfolded protein response" when expressed in ero1-1 cells indicates that ERO1-Lbeta is involved also in generating oxidative conditions in the ER . ERO1-L and ERO1-Lbeta display different tissue distributions . Furthermore, only ERO1-Lbeta transcripts are induced in the course of the unfolded protein response . Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.

Science, 2000 May 19, 288(5469), 1248 - 51
A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle; Milan D et al.; A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle . The mutation has beneficial effects on meat content but detrimental effects on processing yield . Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK) . Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage . Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.

Br J Cancer, 2000 May, 82(10), 1671 - 6
The expression profile for the tumour suppressor gene PTEN and associated polymorphic markers; Hamilton JA et al.; PTEN, a putative tumour suppressor gene associated with prostate and other cancers, is known to be located within the chromosomal region 10q23.3 . Transcription of the PTEN gives rise to multiple mRNA species . Analyses by Northern blots, using cell lines which express PTEN together with cell lines which have lost the PTEN or carry a truncated version of the gene, has allowed us to demonstrate that the pseudogene is not transcribed . In addition, 3' RACE studies confirmed that the multiple mRNA species arising from the gene probably result from the use of alternative polyadenylation sites . No evidence for tissue- or cell-specific patterns of transcription was found . Analysis by 5' RACE placed the putative site for the start of transcription around 830 bp upstream of the start codon . A map of the location of the PTEN gene with a series of overlapping YAC, BAC and PACs has been constructed and the relative position of eight microsatellite markers sited . Two known and one novel marker have been positioned within the gene, the others are in flanking regions . The more accurate location of these markers should help in future studies of the extent of gene loss . Several polymorphisms were also identified, all were within introns . Four of the common polymorphisms appear to be linked . In blood, DNA from 200 individuals, including normal, BPH and prostate cancer patients, confirmed this link . Only two samples of 200 did not carry the linked haplotype, both were patients with advanced prostate cancer . It is possible that such rearrangements within PTEN could be evidence of predisposition to prostate cancer in this small number of cases.

Med Mycol, 2000 Apr, 38(2), 109 - 21
Presence of a pertussis toxin-sensitive G protein alpha subunit in Sporothrix schenckii; Delgado N et al.; As an initial step in the study of the role of G proteins in signal transduction in Sporothrix schenckii, we identified a Galphai subunit using different experimental approaches . Western blots of fungal membrane preparations using anti-Galphacommon and anti-Galphai1-Galphai2 antibodies identified a band of approximately 41 kDa . Pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation of these membrane fractions confirmed the presence of a protein substrate of 41 kDa . A 357 bp polymerase chain reaction (PCR) product obtained using fungal DNA as template and primers targeted to conserved Galphai sequences, was used as a probe to isolate a clone from an S . schenckii genomic library . A partial sequence for a Galphai subunit was obtained from this clone . The sequence was completed using the rapid amplification of cDNA ends (RACE) technique with mycelium and yeast cDNA . The cDNA sequence revealed a 1059 bp open reading frame encoding a 353 amino acid Galphai subunit of 41 kDa, more than 90% identical to the CPG-1 of Cryphonectria parasitica, and GNA-1 of Neurospora crassa . The genomic sequence was obtained by PCR using fungal DNA, and revealed a 1250 bp sequence and the presence of three introns . These results provide evidence for the first time of the presence and expression of a Galphai homolog in a pathogenic dimorphic fungus.

J Biol Chem, 2000 Jul 28, 275(30), 22650 - 6
SPECs, small binding proteins for Cdc42; Pirone DM et al.; The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling . We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities . The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively . Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain . Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA . Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain . Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity . Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain . These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities.

J Biol Chem, 2000 Aug 25, 275(34), 26206 - 12
Conformational activation of radixin by G13 protein alpha subunit; Vaiskunaite R et al.; G(13) protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface presumably involving more than one pathway . Yeast two-hybrid screening of a mouse brain cDNA library identified radixin, a member of the ERM family of three closely related proteins (ezrin, radixin, and moesin), as a protein that interacted with Galpha(13) . Interaction between radixin and Galpha(13) was confirmed by in vitro binding assay and by co-immunoprecipitation technique . Activated Galpha(13) induced conformational activation of radixin, as determined by binding of radixin to polymerized F-actin and by immunofluorescence in intact cells . Finally, two dominant negative mutants of radixin inhibited Galpha(13)-induced focus formation of Rat-1 fibroblasts but did not affect Ras-induced focus formation . Our results identifying a new signaling pathway for Galpha(13) indicate that ERM proteins can be activated by and serve as effectors of heterotrimeric G proteins.

J Biol Chem, 2000 Aug 4, 275(31), 23841 - 6
The survival motor neuron protein of Schizosacharomyces pombe . Conservation of survival motor neuron interaction domains in divergent organisms; Paushkin S et al.; Spinal muscular atrophy is a common often lethal neurodegenerative disease resulting from deletions or mutations in the survival motor neuron gene (SMN) . SMN is ubiquitously expressed in metazoan cells and plays a role in small nuclear ribonucleoprotein assembly and pre-mRNA splicing . Here we characterize the Schizosacharomyces pombe orthologue of SMN (yeast SMN (ySMN)) . We report that the ySMN protein is essential for viability and localizes in both the cytoplasm and the nucleus . Like human SMN, we show that ySMN can oligomerize . Remarkably, ySMN interacts directly with human SMN and Sm proteins . The highly conserved carboxyl-terminal domain of ySMN is necessary for the evolutionarily conserved interactions of SMN and required for cell viability . We also demonstrate that the conserved amino-terminal region of ySMN is not required for SMN and Sm binding but is critical for the housekeeping function of SMN.

Microsc Res Tech, 2000 Apr 15, 49(2), 168 - 72
Involvement of PCH family proteins in cytokinesis and actin distribution; Lippincott J et al.; Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms . These proteins are characterized by the presence of several conserved sequence and structural motifs . Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis . Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members . We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis .

Anal Chem, 2000 May 1, 72(9), 2154 - 9
High-efficiency capillary isoelectric focusing of peptides; Shen Y et al.; Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins . Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry . In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point . Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ microL were detectable using conventional UV detection . CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels . Very small differences in peptide isoelectric points (deltapI approximately 0.01) could be resolved, High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.

Toxicol Lett, 2000 Jun 5, 115(3), 231 - 8
Re-evaluation of the first synthetic estrogen, 1-keto-1,2,3, 4-tetrahydrophenanthrene, and bisphenol A, using both the ovariectomised rat model used in 1933 and additional assays; Ashby J et al.; 1-Keto-1,2,3,4-tetrahydrophenanthrene (THP-1) was reported by Cook et al . in 1933 as the first synthetic estrogen . Estrogenic activity was assessed by the induction of vaginal cornification in ovariectomised rats . The corresponding 4-isomer (THP-4) was shown to be inactive . Both chemicals have been re-synthesised and assessed for hormonal activity . Each chemical bound weakly and to the same extent to isolated estrogen receptors, but only at high concentrations . However, they each lacked estrogenic or anti-estrogenic activity when evaluated in vitro using a yeast hER assay, and both failed to induce vaginal cornification or uterotrophic effects in ovariectomised rats . THP-1, and to a lesser extent THP-4, were shown to possess weak androgenic and anti-androgenic activity in vitro when evaluated using an hAR yeast assay . Estrogenic activity for bisphenol A (BPA) was subsequently demonstrated by {Dodds and Lawson, Synthetic, oestrogenic agents without the phenanthrene nucleus, Nature 137, (1936)} using the same ovariectomised rat protocol, and this activity has been confirmed and supplemented by positive uterotrophic effects for BPA in the same bioassays . The present results illustrate the complexity of deriving conclusions regarding the hormonal activities of chemicals . First, some activities observed in isolated hormonal receptor binding assays may not be expressed in functional hormonal assays . This indicates the need for functional hormonal assays in any screening programme . Second, that activities observed for a chemical in one hormonal assay may not be reflected in related hormonal assays . This indicates the need to define assay protocols with some precision when incorporating them into screening batteries . Finally, that some literature reports of hormonal activity for chemicals may not be capable of independent confirmation under apparently identical conditions of test . This illustrates the need to use lists of hormonally active chemicals with care.

Hum Mol Genet, 2000 May 22, 9(9), 1303 - 13
A novel protein with RNA-binding motifs interacts with ataxin-2; Shibata H et al.; Spinocerebellar ataxia type 2 (SCA2) is caused by expansion of a polyglutamine tract in ataxin-2, a protein of unknown function . Using the yeast two-hybrid system, we identified a novel protein, A2BP1 (ataxin-2 binding protein 1) which binds to the C-terminus of ataxin-2 . Northern blot analysis showed that A2BP1 was predominantly expressed in muscle and brain . By immunocfluorescent staining, A2BP1 and ataxin-2 were both localized to the trans -Golgi network . Immunocytochemistry showed that A2BP1 was expressed in the cytoplasm of Purkinje cells and dentate neurons in a pattern similar to that seen for ataxin-2 labeling . Western blot analysis of subcellular fractions indicated enrichment of A2BP1 in the same fractions as ataxin-2 . Sequence analysis of the A2BP1 cDNA revealed an RNP motif that is highly conserved among RNA-binding proteins . A2BP1 had striking homology with a human cDNA clone, P83A20, of unknown function and at least two copies of A2BP1 homologs are found in the Caenorhabditis elegans genome database . A2BP1 and related proteins appear to form a novel gene family sharing RNA-binding motifs.

Virology, 2000 May 25, 271(1), 9 - 17
Interactions of HIV-1 nef with the mu subunits of adaptor protein complexes 1, 2, and 3: role of the dileucine-based sorting motif; Craig HM et al.; HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits . However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized . Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3 . The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2 . Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1 . Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits . However, leucine residues of the sorting motif were not required for this subcellular localization of Nef . These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein .

Biochem Biophys Res Commun, 2000 May 19, 271(3), 719 - 25
Adaptor gamma ear homology domain conserved in gamma-adaptin and GGA proteins that interact with gamma-synergin; Takatsu H et al.; We identified a novel family of proteins that have a VHS domain and an AGEH (adaptor gamma ear homology) domain that is homologous to the ear domain of the gamma-adaptin subunit of the AP-1 clathrin adaptor . When overexpressed, the proteins, called GGA1, GGA2, and GGA3, localized to the trans-Golgi network (TGN) and often caused fragmentation and vacuolation of the compartment . Yeast two-hybrid analysis showed that the AGEH domains of the GGA proteins as well as those of gamma-adaptins are able to interact with gamma-synergin, which was previously shown to localized in the TGN region and interact with gamma-adaptin . Furthermore, gamma-synergin and either of the GGA proteins coexpressed were colocalized in the TGN region . These results suggest that the GGA proteins regulate the function of the TGN or membrane trafficking from this compartment and that the AGEH domains of GGAs and gamma-adaptins, like the ear domain of alpha-adaptin, are involved in interaction with molecules that modulate their functions .

Proc Natl Acad Sci U S A, 2000 May 23, 97(11), 6114 - 9
Squamous epithelial proliferation induced by walleye dermal sarcoma retrovirus cyclin in transgenic mice; Lairmore MD et al.; Walleye dermal sarcoma (WDS) is a common disease of walleye fish in the United States and Canada . These proliferative lesions are present autumn through winter and regress in the spring . Walleye dermal sarcoma virus (WDSV), a retrovirus distantly related to other members of the family Retroviridae, has been etiologically linked to the development of WDS . We have reported that the D-cyclin homologue {retroviral (rv) cyclin} encoded by WDSV rescues yeast conditionally deficient for cyclin synthesis from growth arrest and that WDSV-cyclin mRNA is present in developing tumors . These data strongly suggest that the rv-cyclin plays a central role in the development of WDS . To test the ability of the WDSV rv-cyclin to induce cell proliferation, we have generated transgenic mice expressing the rv-cyclin in squamous epithelia from the bovine keratin-5 promoter . The transgenic animals were smaller than littermates, had reduced numbers of hair follicles, and transgenic females did not lactate properly . Following injury the transgenic animals developed severe squamous epithelial hyperplasia and dysplasia with ultrastructural characteristics of neoplastic squamous epithelium . Immunocytochemistry studies demonstrated that the hyperplastic epithelium stained positive for cytokeratin and were abnormally differentiated . Furthermore, the rv-cyclin protein was detected in the thickened basal cell layers of the proliferating lesions . These data are the first to indicate that the highly divergent WDSV rv-cyclin is a very potent stimulator of eukaryotic cell proliferation and to demonstrate the potential of a cyclin homologue encoded by a retrovirus to induce hyperplastic skin lesions.

Proc Natl Acad Sci U S A, 2000 May 23, 97(11), 5924 - 9
Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor; Xu W et al.; The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins . To identify co-binding proteins, we performed a yeast two-hybrid screen . The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins . In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta . DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE) . Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element . In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins . Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4 . Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor . The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.

J Biol Chem, 2000 Oct 6, 275(40), 31266 - 8
Structure of a conserved domain common to the transcription factors TFIIS, elongin A, and CRSP70; Booth V et al.; TFIIS is a transcription elongation factor that consists of three domains . We have previously solved the structures of domains II and III, which stimulate arrested polymerase II elongation complexes in order to resume transcription . Domain I is conserved in evolution from yeast to human species and is homologous to the transcription factors elongin A and CRSP70 . Domain I also interacts with the transcriptionally active RNA polymerase II holoenzyme and therefore, may have a function unrelated to the previously described transcription elongation activity of TFIIS . We have solved the structure of domain I of yeast TFIIS using NMR spectroscopy . Domain I is a compact four-helix bundle that is structurally independent of domains II and III of the TFIIS . Using the yeast structure as a template, we have modeled the homologous domains from elongin A and CRSP70 and identified a conserved positively charged patch on the surface of all three proteins, which may be involved in conserved functional interactions with the transcriptional machinery.

J Biol Chem, 2000 Jul 28, 275(30), 22847 - 53
Functional consequence of substitutions at residue 171 in human galactose-1-phosphate uridylyltransferase; Crews C et al.; Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (hGALT) results in the potentially lethal disorder classic galactosemia . Although a variety of naturally occurring mutations have been identified in patient alleles, few have been well characterized . We have explored the functional significance of a common patient mutation, F171S, using a strategy of conservative substitution at the defined residue followed by expression of the wild-type and, alternatively, substituted proteins in a null-background strain of yeast . As expected from patient studies, the F171S-hGALT protein demonstrated <0.1% wild-type levels of activity, although two of three conservatively substituted moieties, F171L- and F171Y-hGALT, demonstrated approximately 10% and approximately 4% activity, respectively . The third protein, F171W, demonstrated severely reduced abundance, precluding further study . Detailed kinetic analyses of purified wild-type, F171L- and F171Y-hGALT enzymes, coupled with homology modeling of these proteins, enabled us to suggest that the effects of these substitutions resulted largely from altering the position of a catalytically important residue, Gln-188, and secondarily, by altering the subunit interface and perturbing hexose binding to the uridylylated enzyme . These results not only provide insight into the functional impact of a single common patient allele and offer a paradigm for similar studies of other clinically or biochemically important residues, but they further help to elucidate activity of the wild-type human GALT enzyme.

EMBO J, 2000 May 15, 19(10), 2292 - 303
Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family; Eberhard D et al.; Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and lymphomagenesis . Here we demonstrate that Pax5 exerts its repression function by recruiting members of the Groucho corepressor family . In a yeast two-hybrid screen, the groucho-related gene product Grg4 was identified as a Pax5 partner protein . Both proteins interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and C-terminal transactivation domain of Pax5 . The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding . Moreover, Grg4 efficiently represses the transcriptional activity of Pax5 in an octapeptide-dependent manner . Similar protein interactions resulting in transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families . In agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos . These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein family.

EMBO J, 2000 May 15, 19(10), 2237 - 46
Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis; Ayllon V et al.; Growth factor deprivation is a physiological mechanism to regulate cell death . We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation . Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1alpha (PP1alpha) . Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis . IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase . A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation . This enzymatic activity also dephosphorylates in vivo (32)P-labeled Bad . Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death . Finally, Ras activation controls the catalytic activity of PP1alpha . These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1alpha phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1alpha phosphatase, whose enzymatic activity is regulated by Ras.

Cancer Res, 2000 May 1, 60(9), 2411 - 8
Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation; Hayes F et al.; The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor, but its precise biochemical function remains unknown . The BRCA1 COOH terminus acts as a transcription activation domain, and germ-line cancer- predisposing mutations in this region abolish transcription activation, whereas benign polymorphisms do not . These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis . Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment . The BRCA1 COOH terminus (amino acids 1560-1863) consists of two BRCTs preceded by a region likely to be nonglobular . We combined site-directed and random mutagenesis, followed by a functional transcription assay in yeast: (a) error-prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat, and human BRCA1; (b) random insertion of a variable pentapeptide cassette generated 21 insertion mutants . All pentapeptide insertions NH2-terminal to the BRCTs retained wild-type activity, whereas insertions in the BRCTs were, with few exceptions, deleterious; and (c) site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the COOH terminus . Deletion analysis revealed that the integrity of the most COOH-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity . We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function . Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay.

Exp Nephrol, 2000 May-Jun, 8(3), 152 - 60
Cloning of rabbit Cct6 and the distribution of the Cct complex in mammalian tissues; Schwartz GJ et al.; Cct6 protein is one of the subunits of the Cct complex involved in ATP-dependent folding of cellular proteins . We used the cDNA of the human CCT6 subunit to obtain a full-length cDNA from a rabbit kidney cortex library . Two transcripts of 2 and 2.5 kb were detected in rabbit kidney and liver by Northern analysis . The rabbit CCT6 was 93% identical to the human gene; the deduced amino acid sequence was 97% identical . A phylogenetic analysis of Cct6 proteins from mouse, rabbit, human, and yeast showed greater similarities of Cct6 protein among the species than among other Cct subunits . The ATP-binding sites were perfectly conserved among mammals and yeast, supporting the role of Cct complex in ATP-dependent protein folding . Using a polyclonal antibody to human Cct6 protein and Western analysis, we found expression of this subunit in a variety of rabbit organs and tissues, as well as in bovine testes, human lymphocytes, human and rabbit reticulocytes, and in two cultured kidney cell lines . We also found Cct1 protein by Western analysis in several rabbit organs as well as in bovine testes . These data characterize the rabbit Cct6 subunit and compare it to its homologues . The Western analyses support the concept that Cct complex is widely distributed among tissues and highly conserved among eukaryotes .

Protein Eng, 2000 Apr, 13(4), 239 - 45
Evaluation of protein-protein association energies by free energy perturbation calculations; Brandsdal BO et al.; The association energy upon binding of different amino acids in the specificity pocket of trypsin was evaluated by free energy perturbation calculations on complexes between bovine trypsin (BT) and bovine pancreatic trypsin inhibitor (BPTI) . Three simulations of mutations of the primary binding residue (P(1)) were performed (P(1)-Ala to Gly, P(1)-Met to Gly and P(1)-Met to Ala) and the resulting differences in association energy (DeltaDeltaG(a)) are 2 . 28, 5.08 and 2.93 kcal/mol for P(1)-Ala to Gly, P(1)-Met to Gly and to Ala with experimental values of 1.71, 4.62 and 2.91 kcal/mol, respectively . The calculated binding free energy differences are hence in excellent agreement with the experimental binding free energies . The binding free energies, however, were shown to be highly dependent on water molecules at the protein-protein interface and could only be quantitatively estimated if the correct number of such water molecules was included . Furthermore, the cavities that were formed when a large amino acid side-chain is perturbed to a smaller one seem to create instabilities in the systems and had to be refilled with water molecules in order to obtain reliable results . In addition, if the protein atoms that were perturbed away were not replaced by water molecules, the simulations dramatically overestimated the initial state of the free energy perturbations.

Genome Res, 2000 May, 10(5), 679 - 90
Analysis of 5S rDNA arrays in Arabidopsis thaliana: physical mapping and chromosome-specific polymorphisms; Cloix C et al.; A physical map of a pericentromeric region of chromosome 5 containing a 5S rDNA locus and spanning approximately 1000 kb was established using the CIC YAC clones . Three 5S rDNA arrays were resolved in this YAC contig by PFGE analysis and we have mapped different types of sequences between these three blocks . 5S rDNA units from each of these three arrays of chromosome 5, and from chromosomes 3 and 4, were isolated by PCR . A total of 38 new DNA sequences were obtained . Two types of 5S rDNA repeated units exist: the major variant with 0.5-kb repeats and one with short repeats (251 bp) only detected on YAC 11A3 from chromosome 3 . Although the 38 sequences displayed noticeable heterogeneity, we were able to group them according to their 5S array origin . The presence of 5S array-specific variants was confirmed with the restriction polymorphism study of all the YACs carrying 5S units.

Int J Dermatol, 2000 Apr, 39(4), 266 - 9
Onychomycosis and Trichosporon beigelii in Korea; Han MH et al.; BACKGROUND: Onychomycosis is a common superficial fungal infection . Causative organisms in onychomycosis have been extensively studied, but the role of nondermatophytes is controversial . Trichosporon beigelii is a soil and water inhabiting yeast and is occasionally found in the flora normally associated with human skin, mouth, and nails . Several reports in the literature have suggested that T . beigelii is one of the pathogens in onychomycosis . METHODS: We performed a survey of the mycologic laboratory records of patients clinically suspected of having onychomycosis from July 1996 to December 1998 . RESULTS: Out of a total of 2591 nail samples examined, 1222 (47.2%) were culture positive, including 262 cases (10.1%) with T . beigelii . The overall positive rate for the KOH mount examination was 58.8%, and in the cases with T . beigelii was 89.1% . Among the age groups, the incidence rate was highest in the fifth decade (26.6%) . The most common causative organism of microscopy-positive onychomycosis was Trichophyton rubrum (61.4%); the others in decreasing frequency were T . beigelii (20.4%), Candida spp . (7.3%), Trichophyton mentagrophytes (4.1%), and mixed infection (2.9%) . T . beigelii was repeatedly isolated in successive nail cultures from 10 of 20 patients selected from those with T . beigelii nail infection . CONCLUSIONS: T . beigelii was the second most commonly isolated fungus in onychomycosis and had a high positive rate on KOH mount examination of the nails and successive repeated cultures . We suggest that T . beigelii might be a common pathogen of onychomycosis in Korea.

Spectrochim Acta A Mol Biomol Spectrosc, 2000 Apr, 56(5), 1013 - 20
Application of aluminium(III) complex with salicylidene-o-aminophenol to the fluorometric determination of nucleic acids; Hao YM et al.; In buffer medium of hexamethylene tetraamine-HCl at pH 5.9 the aluminium(III) complex with salicylidene-o-aminophenol (SAP) has a fluorescence peak at 508 nm with excitation at 410 nm . When nucleic acid coexists, it reacts with the complex within 8 min at room temperature to produce a non-fluorescent product, resulting in the decrease of fluorescence intensity of the aluminium complex . On basis of this, a new fluorometric method for nucleic acids determination is proposed . The calibration graphs for calf thymus DNA, fish sperm DNA and yeast RNA are linear up to 5.0, 4.0 and 3.0 microg ml(-1), respectively, and corresponding detection limits are 49, 52 and 62 ng ml(-1) . The synthetic samples are analyzed with relative standard deviation of five measurements of 3.9-6.0% . DNA in an extraction product from human blood is determined using the calibration graph for calf thymus DNA, and the result is very close to that by the ethidium bromide assay . Compared with some established fluorometric methods, this procedure is sensitive, selective, reliable, reproducible and practical . The association constant of calf thymus DNA with the complex is estimated by two graphic methods . It is suggested that the binding reaction between nucleic acids with the complex proceeds in an intercalation way.

J Biol Chem, 2000 Aug 11, 275(32), 24595 - 600
Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes . Role of conserved residues; Gaullier JM et al.; FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P) . Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes . The binding of the FYVE finger to PtdIns(3)P is Zn(2+)-dependent, and Zn(2+) could not be replaced by any other bivalent cations tested . By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K(D) of about 50 nm and a 1:1 stoichiometry . Mutagenesis of cysteines involved in Zn(2+) coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6- to >100-fold decreased affinity for PtdIns(3)P . A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy . Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic . Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect . These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.

J Biol Chem, 2000 Jul 28, 275(30), 23097 - 105
The human thioesterase II protein binds to a site on HIV-1 Nef critical for CD4 down-regulation; Cohen GB et al.; A HIV-1 Nef affinity column was used to purify a 35-kDa Nef-interacting protein from T-cell lysates . The 35-kDa protein was identified by peptide microsequence analysis as the human thioesterase II (hTE) enzyme, an enzyme previously identified in a yeast two-hybrid screen as a potential Nef-interacting protein . Immunofluorescence studies showed that hTE localizes to peroxisomes and that coexpression of Nef and hTE leads to relocalization of Nef to peroxisomes . Interaction of Nef and hTE was abolished by point mutations in Nef at residues Asp(108), Leu(112), Phe(121), Pro(122), and Asp(123) . All of these mutations also abrogated the ability of Nef to down-regulate CD4 from the surface of HIV-infected cells . Based on the x-ray and NMR structures of Nef, these residues define a surface on Nef critical for CD4 down-regulation . A subset of these mutations also affected the ability of Nef to down-regulate major histocompatibility complex class I . These results, taken together with previous studies, identify a region on Nef critical for most of its known functions . However, not all Nef alleles bind to hTE with high affinity, so the role of hTE during HIV infection remains uncertain.

J Hum Genet, 2000, 45(3), 188 - 91
Molecular cloning and expression analysis of the human DA41 gene and its mapping to chromosome 9q21.2-q21.3; Hanaoka E et al.; DA41 was previously identified as one of the DAN-binding proteins, via a yeast-based two-hybrid screening strategy . In the present study, we cloned a human homolog of DA41 cDNA . Structural analysis revealed that human DA41 cDNA consisted of 2,861 nucleotides in length and encoded a protein of 589 amino acids, with a predicted molecular mass of 62.4kDa . Human DA41 exhibited an 86% amino acid sequence identity to rat DA41, indicating the evolutionarily conserved structure and function of DA41 . A database search for DA41-related protein(s) identified mouse PLIC-1, PLIC-2, frog XDRP1, and yeast DSK2 . DA41 and each DA41-related protein contain a ubiquitin-like domain in their amino-terminal regions . DA41 was expressed ubiquitously in adult human tissues, with relatively higher levels in pituitary gland, adrenal gland, kidney, thymus, and placenta . Fluorescence in situ hybridization (FISH) revealed that DA41 was mapped to human chromosome 9q21.2-q21.3, a position overlapping the candidate tumor suppressor locus for bladder cancer.

Neurosurgery, 2000 May, 46(5), 1034 - 51
Molecular biology and neurosurgery in the third millennium; Rutka JT et al.; The application of techniques in molecular biology to human neurosurgical conditions has led to an increased understanding of disease processes that affect the brain and to novel forms of therapy that favorably modify the natural history of many of these conditions . Molecular strategies are currently being either used or sought for brain tumors, stroke, neurodegenerative diseases, vascular malformations, spinal degenerative diseases, and congenital malformations of the central nervous system . Considering that the structure of deoxyribonucleic acid was ascertained by Watson and Crick as recently as 1953, the progress that has been made to implement molecular medicine in clinical practice has been meteoric . More than 2000 patients have been treated in approved gene therapy trials throughout the world . Many of these patients have been treated for neurological diseases for which conventional medical therapies have been of limited utility . As part of this continuing series on advances in neurosurgery in the third millennium, we first reflect on the history of the nascent field of molecular biology . We then describe the powerful techniques that have evolved from knowledge in this field and have been used in many publications in Neurosurgery, particularly within the past decade . These methods include commonly used techniques such as advanced cytogenetics, differential display, microarray technology, molecular cell imaging, yeast two-hybrid assays, gene therapy, and stem cell utilization . We conclude with a description of the rapidly growing field of bioinformatics . Because the Human Genome Project will be completed within 5 years, providing a virtual blueprint of the human race, the next frontier (and perhaps our greatest challenge) will involve the development of the field of "proteomics," in which protein structure and function are determined from the deoxyribonucleic acid blueprint . It is our conviction that neurosurgeons will continue to be at the forefront of the treatment of patients with neurological diseases using molecular strategies, by performing essential research leading to increased understanding of diseases, by conducting carefully controlled studies to test the effects of treatments on disease processes, and by directly administering (by neurosurgical, endovascular, endoscopic, or stereotactic means) the treatments to patients.

Gene, 2000 May 2, 248(1-2), 77 - 87
Structure of the mouse metalloprotease meprin beta gene (Mep1b): alternative splicing in cancer cells; Jiang W et al.; The mouse meprin beta gene encodes an integral membrane protease that is expressed in a tissue-specific manner in embryonic and adult epithelial cells, and in carcinoma cells . The meprin beta mRNA in the embryo, kidney and intestinal cells is 2.5kb, whereas the isoform in carcinoma cells (beta' mRNA) is 2.7kb . The work herein was initiated to explore the molecular mechanism responsible for the different isoforms . Overlapping fragments containing the Mep1b gene were obtained from a yeast artificial chromosome clone using polymerase chain reactions . The gene spans approximately 40kb and consists of 18 exons and 17 introns . The first three exons are unique to the 5' end of beta' mRNA; the next two exons correspond to the 5' end of beta mRNA . The rest of the exons (13 total) encode the regions common to both beta and beta' messages . In conjunction with the cDNA sequences, the gene structure establishes that alternative splicing of 5' exons is responsible for the generation of the mRNA isoforms . The DNA regions between beta'- and beta-specific exons and upstream of the first beta' exon have been completely sequenced to identify potential regulatory elements for beta and beta' transcription . There is significant homology between the two regions, indicating that a duplication event occurred during evolution of the Mep1b gene . Potential promoter elements and transcription factor-binding sites were identified from comparisons to sequences in the databanks . This is the first gene structure that has been completed for meprin subunits from all species . The work elucidates molecular mechanisms that regulate differential expression of the Mep1b gene.

Plant Physiol, 2000 May, 123(1), 297 - 306
High-affinity potassium transport in barley roots . Ammonium-sensitive and -insensitive pathways; Santa-Maria GE et al.; In an attempt to understand the process mediating K(+) transport into roots, we examined the contribution of the NH(4)(+)-sensitive and NH(4)(+)-insensitive components of Rb(+) transport to the uptake of Rb(+) in barley (Hordeum vulgare L.) plants grown in different ionic environments . We found that at low external Rb(+) concentrations, an NH(4)(+)-sensitive component dominates Rb(+) uptake in plants grown in the absence of NH(4)(+), while Rb(+) uptake preferentially occurs through an NH(4)(+)-insensitive pathway in plants grown at high external NH(4)(+) concentrations . A comparison of the Rb(+)-uptake properties observed in roots with those found in heterologous studies with yeast cells indicated that the recently cloned HvHAK1 K(+) transporter may provide a major route for the NH(4)(+)-sensitive component . HvHAK1 failed to complement the growth of a yeast strain defective in NH(4)(+) transport, suggesting that it could not act as an NH(4)(+) transporter . Heterologous studies also showed that the HKT1 K(+)/Na(+)-cotransporter may act as a pathway for high-affinity Rb(+) transport sensitive to NH(4)(+) . However, we found no evidence of an enhancement of Rb(+) uptake into roots due to Na(+) addition . The possible identity of the systems contributing to the NH(4)(+)-insensitive component in barley plants is discussed.

J Biol Chem, 2000 Jul 21, 275(29), 22284 - 92
A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts; Paine CT et al.; Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins . To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization . Polyclonal antibodies were prepared against two recombinant variants of this protein . Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39 . Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA . In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts . Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize . Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix . TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates . Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.

J Cell Sci, 2000 Jun, 113 ( Pt 11), 1939 - 50
HZwint-1, a novel human kinetochore component that interacts with HZW10; Starr DA et al.; HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10 . HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain . Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads . This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum . The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein . In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase . This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase . HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes . HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.

Clin Oral Investig, 1999 Sep, 3(3), 120 - 5
Time-related bisphenol-A content and estrogenic activity in saliva samples collected in relation to placement of fissure sealants; Arenholt-Bindslev D et al.; It was recently reported that estrogenic activity was detected in saliva samples collected during 1 h after placement of one fissure sealant (Delton) and this related to Bisphenol-A (BPA) content . The aim of the present study was to determine the time-related BPA content and estrogenic activity in saliva samples collected before and after placement of two fissure sealants each with a different monomer composition . Eight healthy male volunteers with no history of prior placement of fissure sealants or composite resin fillings had four molars sealed with either Delton LC (four people) or Visio-Seal (four people) . Base-line saliva samples were collected preexperimentally, in the morning when fasting . Fissure sealants were placed and saliva samples collected immediately, 1 h and 24 hs after placement of the fissure sealant . BPA was found in saliva samples collected immediately after placement of Delton LC (range 0.3-2.8 ppm) . No detectable amounts of BPA were determined 1 h and 24 h after Delton treatment (detection limit < or = 0.1 ppm) . In base-line samples and in all samples collected from Visio-Seal treated individuals, no BPA was detected . In a recombinant yeast cell assay, significantly increased estrogenic activity was found in saliva samples collected immediately after placement of Delton LC sealant (P < 0.05; ANOVA) whereas no statistically significant estrogenic activity was observed in the remaining groups . In conclusion, minute amounts of BPA, however considerably lower than previously reported, were detected in saliva samples collected immediately after but not 1 and 24 h(s) after placement of Delton LC fissure sealant . BPA was not detected after placement of Visio-Seal fissure sealant.

J Biol Chem, 2000 Jul 28, 275(30), 23169 - 74
A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor; Proenza C et al.; We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle . The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D) . Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D) . For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR . The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873 . Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube . The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression . Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.

J Biol Chem, 2000 Jul 7, 275(27), 20942 - 8
GCIP, a novel human grap2 and cyclin D interacting protein, regulates E2F-mediated transcriptional activity; Xia C et al.; Regulation of mammalian cell growth and proliferation is governed through receptor-mediated signaling networks that ultimately converge on the cell cycle machinery . Adaptor proteins play essential roles in the formation of intracellular signaling complexes, relaying extracellular signals from the plasma membrane to the nucleus of a cell . The leukocyte-specific adaptor protein Grap2 is a central linker protein in immune cell signaling and activation . Using Grap2 as bait protein, we identified a novel human protein, GCIP (Grap2 cyclin-D interacting protein) . We found that GCIP bound to Grap2 in both yeast two-hybrid assays and in mammalian cells through binding to the COOH-terminal unique domain and SH3 domain (designated QC domain) of Grap2 . GCIP also associated with cyclin D both in vitro and in vivo . The expression of GCIP was found in all human tissues examined with the highest level of expression in the heart, muscle, peripheral blood leukocytes, and brain . Furthermore, phosphorylation of retinoblastoma protein by cyclin D-dependent protein kinase was reduced and E2F1-mediated transcription activity was inhibited in cells transfected with GCIP . High level expression of GCIP in terminally differentiated tissues and the inhibition of E2F1 transcription activation suggest that GCIP could play an important role in controlling cell differentiation and proliferation.

Curr Biol, 2000 Apr 20, 10(8), 479 - 82
Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice; de Klein A et al.; Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase {1} {2} {3} . The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation {4} {5} {6} . The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) {7} {8} . ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis {8} {9} {10} {11}, and may phosphorylate p53 {12} {13}, suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage . Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5) . Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level . No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents . Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal . Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

J Mol Biol, 2000 May 19, 298(5), 817 - 32
The intermediate filament protein consensus motif of helix 2B: its atomic structure and contribution to assembly; Herrmann H et al.; Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central alpha-helical rod domain . We have analyzed its contribution to the various stages of assembly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them with the wild-type and tailless proteins . It is surprising that in buffers of low ionic strength and high pH where the full-length proteins form tetramers, both VimIAT and DesIAT associated into various high molecular weight complexes . After initiation of assembly, both VimIAT and DesIAT aggregated into unit-length-type filaments, which rapidly longitudinally annealed to yield filaments of around 20 nm in diameter . Mass measurements by scanning transmission electron microscopy revealed that both VimIAT and DesIAT filaments contained considerably more subunits per cross-section than standard intermediate filaments . This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control of filament width, rather than elongation, during assembly . To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amino-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation . The atomic structure shows that starting from Tyr400 the two helices gradually separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis .

J Oral Pathol Med, 2000 May, 29(5), 200 - 5
Effects of date extract on adhesion of Candida species to human buccal epithelial cells in vitro; Abu-Elteen KH; The adherence of three Candida species to human buccal epithelial cells (BEC) following treatment with different concentrations of date extract was investigated in vitro, as well as the effect of a mouth rinse with date extract on the adhesion of yeast to BEC . Adhesion of C . albicans, C . tropicalis and C . kefyr to BEC was significantly reduced after both short- and long-term periods of yeast exposure to various concentrations of date extract (reduction between 25% and 52% of the control value) . A similar inhibition of adherence was observed upon pre-incubation of BEC with date extract . There was a significant reduction (P<0.001) in the adherence of yeast to BEC collected immediately or 5-20 min after an oral rinse with 10% date extract . No statistically significant difference was observed in the adhesion of BEC collected 30 min after an oral rinse with date extract and control BEC . In addition, pre-treatment of either Candida or BEC, or both, with date extract resulted in reduced adherence, the magnitude of which was largest when both types of cells were pre-treated . Date extract also inhibited germ-tube formation of C . albicans (56-85% inhibition), which might contribute to the effects on adherence.

J Immunol, 2000 May 15, 164(10), 5296 - 305
A contig map of the Mhc class I genomic region in the zebrafish reveals ancient synteny; Michalova V et al.; In contrast to the human and mouse Mhc, in which the clusters of class I and class II loci reside in close vicinity to one another, in the zebrafish, Danio rerio, they are found in different linkage groups . Chromosome walking using BAC (bacterial artificial chromosome) and PAC (P1 artificial chromosome) clones reveals the zebrafish class I region to occupy a segment of approximately 450 kb and to encompass at least 19 loci . These include three class I (Dare-UDA, -UEA, -UFA), five proteasome subunit beta (PSMB8, -9A, -9C, -11, -12), two TAPs (TAP2A, TAP2B), and one TAP binding protein (TAPBP) . This arrangement contrasts with the arrangements found in human and mouse Mhc, in which the orthologues of the PSMB, TAP, and TAPBP loci reside within the class II region . In addition to this main zebrafish class I contig, a shorter contig of about 150 kb contains two additional class I (UBA, UCA) and at least five other loci . It probably represents a different haplotype of part of the class I region . The previously identified UAA gene shares an identical 5' part with UEA, but the two genes differ in their 3' parts . One of them is probably the result of an unequal crossing over . The described organization has implications for the persistence of syntenic relationships, coevolution of loci, and interpretation of the origin of the human/mouse Mhc organization.

Vet Microbiol, 2000 May 22, 74(1-2), 87 - 100
The influenza A virus M1 protein interacts with the cellular receptor of activated C kinase (RACK) 1 and can be phosphorylated by protein kinase C; Reinhardt J et al.; The M1 protein of influenza A virus has multiple regulatory functions during the infectious cycle, which include mediation of nuclear export of viral ribonucleoproteins, inhibition of viral transcription and a crucial role in virus assembly and budding . The only known modification of the M1 protein is by phosphorylation through yet-to-be-identified kinases . We postulated that at least some of the M1 functions are exerted or regulated through interactions with cellular components . In a screen for such cellular mediators, the protein receptor of the activated C-kinase (RACK 1) was identified by its interaction with the viral M1 protein in the yeast two hybrid system . The physical M1-RACK 1 interaction was confirmed in glutathione-S-transferase-based coprecipitation assays for the diverged M1 proteins of avian, swine and human influenza A virus strains . This conservation suggests that the M1-RACK 1 interaction is of general importance during influenza A virus infections . RACK 1 has previously been identified to specifically bind the activated form of protein kinase C (PKC) and is assumed to anchor the kinase at membranes in the vicinity of its substrates . Since the M1 protein becomes phosphorylated during influenza virus infection, we examined if PKC could catalyze the phosphate transfer . We demonstrate that virion-derived and recombinant M1 protein can indeed be efficiently phosphorylated by purified PKC . Moreover, in cell extracts, we detected M1 phosphorylation activity that was strongly reduced in the presence of the PKC-specific inhibitor compound GF109203X . These data suggest that PKC is the main M1-phosphorylating activity in the cell . Since both, the M1 protein and PKC have been shown to interact with RACK 1, we suggest that the M1-RACK 1 interaction is involved in M1 phosphorylation.

J Virol, 2000 Jun, 74(11), 5190 - 7
Involvement of the mannose receptor in infection of macrophages by influenza virus; Reading PC et al.; Influenza viruses A/PR/8/34 (PR8; H1N1), A/Aichi/68 X-31 (HKx31; H3N2), and A/Beijing/89 X-109 (BJx109; H3N2) show marked differences in their ability to infect murine macrophages, including resident alveolar and peritoneal macrophages as well as the macrophage-derived cell line J774 . The hierarchy in infectivity of the viruses (PR8 < HKx31 < BJx109) resembles that of their reactivity with mannose-binding lectins of the collectin family . Since the macrophage mannose receptor recognizes the same spectrum of monosaccharides as the collectins do, we investigated the possible involvement of this receptor in infection of macrophages by influenza virus . In competitive binding studies, the binding of (125)I-labeled mannosylated bovine serum albumin to macrophages was inhibited by the purified hemagglutinin and neuraminidase (HANA) glycoproteins of influenza virus but not by HANA that had been treated with periodate to oxidize its oligosaccharide side chains . The inhibitory activity of HANA from the three strains of virus differed markedly and correlated with the infectivity of each virus for macrophages . Infection of macrophages, but not MDCK cells, by influenza virus was inhibited by yeast mannan . A variant line of J774 cells, J774E, which expresses elevated levels of the mannose receptor, was more readily infected than J774, and the sensitivity of J774E cells to infection was greatly reduced by culture in the presence of D-mannose, which down-modulated mannose receptor expression . Together, the data implicate the mannose receptor as a major endocytic receptor in the infectious entry of influenza virus, and perhaps other enveloped viruses, into murine macrophages.

J Biol Chem, 2000 May 12, 275(19), 14295 - 306
Identification and characterization of CKIP-1, a novel pleckstrin homology domain-containing protein that interacts with protein kinase CK2; Bosc DG et al.; The catalytic subunits of protein kinase CK2, CK2alpha and CK2alpha', are closely related to each other but exhibit functional specialization . To test the hypothesis that specific functions of CK2alpha and CK2alpha' are mediated by specific interaction partners, we used the yeast two-hybrid system to identify CK2alpha- or CK2alpha'-binding proteins . We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2alpha, but not CK2alpha', in the yeast two-hybrid system . CKIP-1 also interacts with CK2alpha in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2alpha and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e . EGFP-CKIP-1) when they are co-expressed . CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions . The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000 . EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane . The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain . We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e . CK2alpha) with a potential role in targeting CK2alpha to a particular cellular location.

J Biol Chem, 2000 May 12, 275(19), 14167 - 72
Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity; Moskovitz J et al.; Peptide methionine sulfoxide reductases (MsrA) from many different organisms share a consensus amino acid sequence (GCFWG) that could play an important role in their active site . Site-directed single substitution of each of these amino acids except glycines in the yeast MsrA resulted in total loss of enzyme activity . Nevertheless, all the recombinant MsrA mutants and native proteins had a very similar circular dichroism spectrum . The demonstration that either treatment with iodoacetamide or replacement of the motif cysteine with serine leads to inactivation of the enzyme underscores the singular importance of cysteine residues in the activity of MsrA . The recombinant yeast MsrA was used for general characterization of the enzyme . Its K(m) value was similar to the bovine MsrA and appreciably lower than the K(m) of the bacterial enzyme . Also, it was shown that the enzymatic activity increased dramatically with increasing ionic strength . The recombinant yeast MsrA activity and the reduction activity of free methionine sulfoxide(s) were stereoselective toward the L-methionine S-sulfoxide and S-methyl p-tolyl sulfoxide . It was established that a methionine auxotroph yeast strain could grow on either form of L-methionine sulfoxide.

Hum Genet, 2000 Mar, 106(3), 269 - 76
SOX14 is a candidate gene for limb defects associated with BPES and Möbius syndrome; Wilmore HP et al.; Members of the SOX gene family encode proteins with homology to the HMG box DNA-binding domain of SRY, the Y-linked testis-determining gene . SOX genes are expressed during embryogenesis and are involved in the development of a wide range of different tissues . Mutations in SRY, SOX9 and SOX10 have been shown to be responsible for XY sex reversal, campomelic dysplasia and Waardenburg-Hirschsprung disease, respectively . It is likely that mutations in other SOX genes are responsible for a variety of human genetic diseases . SOX14 has been identified from a human genomic library and the mouse and chicken sequences obtained by polymerase chain reaction amplification . The SOX14 amino acid sequence is highly conserved across these species, suggesting an important role for this protein in vertebrate development . SOX14 is expressed in the neural tube and apical ectodermal ridge of the developing chicken limb . This is the only SOX gene known to be expressed in the apical ectodermal ridge, a structure that directs outgrowth of the embryonic limb bud . Human SOX14 is localised to a 1.15-Mb yeast artificial chromosome on chromosome 3q23, close to loci for BPES (blepharophimosis, ptosis, epicanthus inversus syndrome) and Mobius syndrome . Although SOX14 maps outside these loci, its expression pattern and chromosomal localisation suggest that it is a candidate gene for the limb defects frequently associated with these syndromes.

Cochrane Database Syst Rev . 2000;(2):CD000100.
Vaccines for preventing hepatitis B in health-care workers; Jefferson T et al.; BACKGROUND: Hepatitis B causes acute and chronic liver disease and may be prevented by vaccination . OBJECTIVES: To assess the effectiveness and safety of plasma-derived vaccines against acute and chronic hepatitis B in health-care workers in protecting them from hepatitis B infection and its consequences . SEARCH STRATEGY: MEDLINE and Excerpta Medica Database (EMBASE) search using standard Cochrane strategy, Cochrane Library, full text searching of the journal "Vaccine", bibliography of retrieved studies and correspondence with authors, researchers and manufacturers . SELECTION CRITERIA: All original prospective randomised comparisons of yeast-derived vaccines and plasma-derived vaccines against no intervention, placebo, or vaccines against other disease (control vaccines) . Assessment of trial quality was made according to: 1 . generation of allocation schedule 2 . measure(s) taken to conceal treatment allocation 3 . drop-out of allocated health-care worker participants from the analysis of trial results 4 . measures taken to implement double blinding Trial reports were blinded by removal of authors and their affiliation, journal reference, introduction, results, and discussion . DATA COLLECTION AND ANALYSIS: To assess efficacy the incidence rates of acute hepatitis B were observed in the surveillance of the vaccinated and control groups of the trials included in the review . Safety was assessed from side-effect rates, classified as systemic (malaise, nausea, fever, arthralgias, rash, headache) or local (induration and soreness at the site of the inoculation) . MAIN RESULTS: Four trials fulfilling the criteria were identified and the data synthesised . All trials compared plasma-derived vaccines versus placebo . Differences in the settings (and level of incidence) between three of the trial settings and Dienstag's led us to stratify our comparison grouping the three trials performed in dialysis units together . After our stratification, the Desmyter, Smuzness and Crosnier group appears to be homogeneous (Chi-square = 0.11, degrees of freedom = 2) . Our estimates of effectiveness and safety in the high risk group favour treatment, the OR for cases of HB being 0.34; 95% CI (0.21, 0.55) . The analysis also revealed a non-significant trend towards benefit in the lower risk health-care workers (Dienstag trial, OR 0.26 (0.05, 1.30) . Overall the evidence strongly favours vaccination (OR=0.33; 95% CI (0.21, 0.53)) . There was no difference in the incidence and severity of side-effects between the two arms of the trials . We calculated that it was necessary to vaccinate between 145 (assuming a baseline rate of 10 cases/1000/year) and 7 (for a baseline rate of 200/1000/year) health-care workers with plasma-derived vaccines to avoid one case of hepatitis B . Completeness of trial reporting was not good with all four trials failing to report titre results on antibodies against hepatitis B surface antigen and hepatitis B core antigen in the placebo arms (correspondence with two of the four authors failed to shed light on the reasons for such an omission) . All four trials achieved low scores in the four quality dimensions assessed (generation of allocation schedule, measure(s) taken to conceal treatment allocation, exclusion of allocated participants from the analysis of the trial and measures taken to implement and protect double blinding) . Mean length of follow-up was 14.5 months . REVIEWER'S CONCLUSIONS: Plasma-derived vaccines appear to be efficacious and safe for use in high risk health-care workers, such as staff of renal dialysis and transplant units . There is some uncertainty concerning the effectiveness of the vaccine in lower risk health-care workers, although the trend is towards benefit . We found no evidence of a long-term protective effect due to the short follow-up time of the four trials included in this review . We found relatively poor standard of trial reporting, possibly related to the age of the trials.

Comp Biochem Physiol A Mol Integr Physiol, 2000 Mar, 125(3), 351 - 7
Involvement of tyrosine kinase and phosphatidylinositol 3-kinase in phagocytosis by ascidian hemocytes; Ishikawa G et al.; It has been proposed that protein tyrosine phosphorylation plays important roles in signal transduction in mammalian T- and B-cells and monocytes . During our investigations on the ascidian host defense system, we have shown that the monoclonal antibody A74 strongly inhibits both phagocytosis of sheep red blood cells (SRBCs) by hemocytes and hemocyte aggregation, and that the A74 antigen protein has two immunoreceptor tyrosine-based activation motifs and several other motifs that are thought to function in signal transduction in mammals . In this study, we found that the A74 antibody strongly inhibited phagocytosis by ascidian hemocytes of yeast cells, as strongly as that of SRBCs, but not that of latex beads . We also found that herbimycin A and an erbstatin analog, tyrosine kinase inhibitors, and wortmannin, a specific inhibitor for phosphatidylinositol 3-kinase (PI3-kinase), inhibited the phagocytosis of yeast cells . We investigated which hemocyte proteins were specifically tyrosine-phosphorylated during phagocytosis by ascidian hemocytes and found that a protein with a molecular mass of 100 kDa was specifically tyrosine-phosphorylated upon phagocytosis; its tyrosine phosphorylation was inhibited by the A74 antibody . These results strongly suggest that both tyrosine kinase and PI3-kinase play important roles in phagocytosis by ascidian hemocytes.

Aquat Toxicol, 2000 Apr 1, 48(4), 419 - 429
Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin; Cima F et al.; In the colonial ascidian Botryllus schlosseri, tributyltin (TBT), a powerful antifouling biocide, acts as immunotoxic xenobiotic since, at a sublethal concentration (10 microM), it causes an irreversible and significant decrease in in vitro yeast phagocytosis, associated with considerable changes in the shape of phagocytes, which withdraw their pseudopodia and become spherical, due to structural damage of cytoskeletal components . The addition of TBT to the culture medium causes a significant decrease in the amoebocytic index, i.e . the percentage of amoeboid-shaped haemocytes, and prolonged washing in sea water never succeeds in restoring amoeboid shape . In these cytoskeletal alterations, F-actin undergoes extensive depolymerisation, resulting in the absence of FITC-phalloidin fluorescence . Microtubules are not recognisable as single filaments with anti-alpha-tubulin immunofluorescence, although the centrosome is not affected . The addition of increasing exogenous calmodulin (CaM) concentrations (from 20 to 120 microM) after incubation in TBT determines a significant increase in the amoebocytic index, although it is not able to bring it to that of controls, suggesting that CaM in the medium in any case externally exerts an influence on haemocytes pretreated with TBT . The copresence of TBT and exogenous CaM at concentrations higher than 80 microg/ml restores the amoebocytic index and cytoskeletal morphology . The latter appears complete for microtubules and partial for microfilaments . Experiments with isodynamic mixtures of TBT and specific CaM inhibitors, i.e . chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloronaphtalene-1-sulfonamide (W-7), reveal the synergistic effect of antagonism, indicating competition for the same site - a Ca(2+)-CaM hydrophobic region - by both interacting substances and, therefore, the formation of a TBT-CaM complex . Instead, isodynamic mixtures with thapsigargin, an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, have an effect of potentiation, suggesting that TBT indirectly interacts with this Ca(2+)-ATPase activity . We hypothesise that the main mechanism of action of TBT in B . schlosseri phagocytes is alteration of Ca(2+) homeostasis by means of direct interaction with endogenous CaM, which induces a conformational change preventing the regulative activity of CaM on Ca(2+)-ATPase . Consequently, an excess of cytosolic Ca(2+) accumulates which, together with the inhibition of CaM-dependent kinases and Ca(2+)-regulated proteins, produces extensive cytoskeletal disorganisation.

IUBMB Life, 1999 Aug, 48(2), 169 - 74
Expression of a human serum albumin fragment (consisting of subdomains IA, IB, and IIA) and a study of its properties; Park DS et al.; Site-directed mutagenesis and a yeast expression system were used to synthesize a human serum albumin (HSA) fragment (amino acids 1-297) . The HSA fragment (half HSA) was evaluated with a number of biophysical techniques and found to be similar to the corresponding region in wild-type HSA . Specifically, the circular dichroism spectra of half HSA and wild-type HSA were superimposable, indicating that the highly alpha-helical secondary structure of wild-type HSA is preserved in half HSA . Additionally, half HSA was partially reactive with a polyclonal antibody against authentic HSA . Half HSA, which contains subdomain IIA, had an affinity for thyroxine and several thyroxine analogs, similar to that observed previously for wild-type HSA . This study suggests that the production of recombinant HSA fragments will be useful for the study of HSA ligand interactions.

Gene Expr, 1999, 8(4), 207 - 17
LIM domain-containing protein trip6 can act as a coactivator for the v-Rel transcription factor; Zhao MK et al.; The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-kappaB family of eukaryotic transcription factors . v-Rel malignantly transforms a variety of cell types in vitro and in vivo, and its transforming activity is dependent on the ability of v-Rel to bind to DNA and activate transcription . In this report, we used the yeast two-hybrid assay to identify proteins that interact with C-terminal sequences of v-Rel that are needed for transcriptional activation and transformation . One protein, Trip6, that we identified in this screen was previously identified as a thyroid hormone receptor-interacting protein . Trip6 is a member of a subfamily of LIM domain-containing proteins that are thought to transport intracellular signals from the cell surface to the nucleus . By several criteria, we show that sequences from Trip6, which include the LIM domains, behave as a coactivator for transcriptional activation by v-Rel . That is, a GAL4-Trip6 fusion protein can activate transcription in yeast and chicken cells, Trip6 can enable C-terminal sequences of v-Rel to activate transcription in yeast, and Trip6 can enhance activation by v-Rel from a kappaB site reporter plasmid in yeast . Although full-length Trip6 localizes to adhesion plaques, deletion of N-terminal sequences allows human Trip6 to enter the nucleus of chicken cells . Lastly, Northern blotting shows that Trip6 mRNA is expressed in many human tissues . Coexpression of Trip6 does not affect the transforming activity of v-Rel . Taken together, our results indicate that Trip6 may be a protein that is important for the ability of v-Rel to activate transcription and transform cells, and may represent a potential target for blocking Rel-mediated oncogenesis and transcriptional activation.

Mol Biol Cell, 2000 May, 11(5), 1535 - 46
Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends; Guo Z et al.; Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely . Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases . We report that Xenopus Cds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-free Xenopus egg extracts . Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks . In contrast, Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends . Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends . These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.

Virology, 2000 May 10, 270(2), 291 - 8
HTLV-1 tax oncoprotein binds to DNA topoisomerase I and inhibits its catalytic activity; Suzuki T et al.; HTLV-1 Tax oncoprotein exerts pleiotropic effects on cellular regulatory systems, such as transcription and the cell cycle, through the interaction with various cellular factors . During our search for additional cellular targets of Tax using a yeast two-hybrid screening system, we isolated a cDNA encoding human DNA topoisomerase I . Tax was demonstrated to bind to topoisomerase I in vitro, and the Tax-topoisomerase I complex was also detected in HTLV-1-infected T-cells by immunoprecipitation . Furthermore, Tax inhibited the catalytic activity of topoisomerase I as measured by relaxation of supercoiled DNA, although complete inhibition was not observed under the conditions used . The binding of topoisomerase I to DNA was inhibited by the addition of the wild type of Tax but not by a mutant of Tax that cannot bind to topoisomerase I . Consistent with these observations, expression of Tax induced an in vivo reduction of the covalent association of topoisomerase I with chromosomal DNA, which accumulates in the presence of camptothecin . These results suggest that Tax has a novel potential to affect various cellular processes such as transcription and maintenance of genomic stability, in which DNA topoisomerase I is involved .

Plant J, 2000 Apr, 22(1), 1 - 8
Transactivation properties of parsley proline-rich bZIP transcription factors; Sprenger-Haussels M et al.; Light-responsive chalcone synthase (CHS) gene activation requires LRUCHS, a light regulatory promoter unit including the MYB recognition element MRECHS and the ACGT-containing element ACECHS . ACECHS is bound by the parsley basic region/leucine zipper (bZIP) factors CPRF1 and 4 . Factors containing the bZIP domain exist in animals, plants and yeast, and recognize DNA sequence-specifically after formation of homo- or heterodimers . To determine the potential role of CPRFs in the regulation of CHS promoter activity, we investigated the functions of distinct CPRF domains in a homologous co-transfection system . The proline-rich domains of CPRF1 and CPRF4 activate transcription, indicating that CPRF1 and CPRF4 have transactivating properties . Over-expression of the CPRF1 bZIP domain caused a reduction of LRUCHS-mediated light inducibility, and point mutations throughout ACECHS affected both responsiveness to UV-containing white light and transactivation by CPRF1:VP16 . The data suggest that a CPRF1-containing bZIP heterodimer interacts with ACECHS in vivo . We discuss regulatory steps in light-induced CHS transcription that may be influenced by CPRF1 and/or related bZIP factors.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5089 - 94
Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton; Dong LQ et al.; Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells . However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown . Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components . PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN . In addition to a direct interaction, PDK1 also phosphorylated Thr(774) in the activation loop and activated PKN . Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Czeta, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR) . However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN . Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

J Cell Biol, 2000 May 1, 149(3), 707 - 18
Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids; Tvrdik P et al.; Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood . Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids . The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern . Their translation products are all integral membrane proteins with five putative transmembrane domains . By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)) . Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range . Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity . Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity . Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.

J Biol Chem, 2000 Jul 21, 275(29), 21844 - 9
Identification of CYP4F8 in human seminal vesicles as a prominent 19-hydroxylase of prostaglandin endoperoxides; Bylund J et al.; A novel cytochrome P450, CYP4F8, was recently cloned from human seminal vesicles . CYP4F8 was expressed in yeast . Recombinant CYP4F8 oxygenated arachidonic acid to (18R)-hydroxyarachidonate, whereas prostaglandin (PG) D(2), PGE(1), PGE(2), PGF(2alpha), and leukotriene B(4) appeared to be poor substrates . Three stable PGH(2) analogues, 9,11-epoxymethano-PGH(2) (U-44069), 11, 9-epoxymethano-PGH(2) (U-46619), and 9,11-diazo-15-deoxy-PGH(2) (U-51605) were rapidly metabolized by omega2- and omega3-hydroxylation . U-44069 was oxygenated with a V(max) of approximately 260 pmol min(-)(1) pmol P450(-1) and a K(m) of approximately 7 micrometer . PGH(2) decomposes mainly to PGE(2) in buffer and to PGF(2alpha) by reduction with SnCl(2) . CYP4F8 metabolized PGH(2) to 19-hydroxy-PGH(2), which decomposed to 19-hydroxy-PGE(2) in buffer and could be reduced to 19-hydroxy-PGF(2alpha) with SnCl(2) . 18-Hydroxy metabolites were also formed (approximately 17%) . PGH(1) was metabolized to 19- and 18-hydroxy-PGH(1) in the same way . Microsomes of human seminal vesicles oxygenated arachidonate, U-44069, U-46619, U-51605, and PGH(2), similar to CYP4F8 . (19R)-Hydroxy-PGE(1) and (19R)-hydroxy-PGE(2) are the main prostaglandins of human seminal fluid . We propose that they are formed by CYP4F8-catalyzed omega2-hydroxylation of PGH(1) and PGH(2) in the seminal vesicles and isomerization to (19R)-hydroxy-PGE by PGE synthase . CYP4F8 is the first described hydroxylase with specificity and catalytic competence for prostaglandin endoperoxides.

Genome, 2000 Apr, 43(2), 412 - 5
Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR; Yang ZN et al.; Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed . Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR . BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini . After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+) . PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11 . We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones . This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used . With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes.

Genetics, 2000 May, 155(1), 213 - 23
Defective pigment granule biogenesis and aberrant behavior caused by mutations in the Drosophila AP-3beta adaptin gene ruby; Kretzschmar D et al.; Lysosomal protein trafficking is a fundamental process conserved from yeast to humans . This conservation extends to lysosome-like organelles such as mammalian melanosomes and insect eye pigment granules . Recently, eye and coat color mutations in mouse (mocha and pearl) and Drosophila (garnet and carmine) were shown to affect subunits of the heterotetrameric adaptor protein complex AP-3 involved in vesicle trafficking . Here we demonstrate that the Drosophila eye color mutant ruby is defective in the AP-3beta subunit gene . ruby expression was found in retinal pigment and photoreceptor cells and in the developing central nervous system . ruby mutations lead to a decreased number and altered size of pigment granules in various cell types in and adjacent to the retina . Humans with lesions in the related AP-3betaA gene suffer from Hermansky-Pudlak syndrome, which is caused by defects in a number of lysosome-related organelles . Hermansky-Pudlak patients have a reduced skin pigmentation and suffer from internal bleeding, pulmonary fibrosis, and visual system malfunction . The Drosophila AP-3beta adaptin also appears to be involved in processes other than eye pigment granule biogenesis because all ruby allele combinations tested exhibited defective behavior in a visual fixation paradigm.

Genetics, 2000 May, 155(1), 159 - 66
Drosophila wee1 has an essential role in the nuclear divisions of early embryogenesis; Price D et al.; In Drosophila, the maternally expressed mei-41 and grp genes are required for successful execution of the nuclear division cycles of early embryogenesis . In fission yeast, genes encoding similar kinases (rad3 and chk1, respectively) are components of a cell cycle checkpoint that delays mitosis by inhibitory phosphorylation of Cdk1 . We have identified mutations in a gene encoding a Cdk1 inhibitory kinase, Drosophila wee1 (Dwee1) . Like mei-41 and grp, Dwee1 is zygotically dispensable but is required maternally for completing the embryonic nuclear cycles . The arrest phenotype of Dwee1 mutants, as well as genetic interactions between Dwee1, grp, and mei-41 mutations, suggest that Dwee1 is functioning in the same regulatory pathway as these genes . These findings imply that inhibitory phosphorylation of Cdk1 by Dwee1 is required for proper regulation of the early syncytial cycles of embryogenesis.

J Clin Microbiol, 2000 May, 38(5), 1965 - 6
Acute cerebral phaeohyphomycosis due to Wangiella dermatitidis accompanied by cerebrospinal fluid eosinophilia; Chang CL et al.; We report a case of cerebral phaeohyphomycosis due to Wangiella dermaitidis in an immunocompetent adult man . His cerebrospinal fluid (CSF) showed pleocytosis with a high eosinophil count but without peripheral blood eosinophilia . The present case suggested that this black yeast-like fungus should be included when the causes of CSF eosinophilia are considered, even though it is an extremely rare pathogen.

Food Addit Contam, 1999 Dec, 16(12), 533 - 42
Screening of selected pesticides for oestrogen receptor activation in vitro; Vinggaard AM et al.; Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an MCF7 cell proliferation assay and a Yeast Oestrogen Screen . The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 microM induces a 2.0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone) . The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor . Several pesticides did not have any effect on the proliferation response after 6 days of exposure, including: chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon . Some pesticides resulted in a negligible proliferation response, which was not statistically significant under the present experimental conditions . These were: bromopropylate, chlorfenvinphos, chlorobenzilate, dicofol, heptachlor, and imazalil . Fenarimol and dicofol also gave rise to a positive oestrogenic response in yeast cells transfected with the oestrogen receptor alpha, whereas the remaining compounds resulted in a negative response due either to biological inactivity or cytotoxocity to the yeast cells . The EC50 for fenarimol was estimated to be 13 microM in the yeast cells, compared with an EC50 of 3 microM in the MCF7 cells, indicating higher sensitivity of the latter assay . No in vivo data for fenarimol, triadimefon or triadimenol have previously been published that support oestrogenic activity in the intact animal . Thus, from the present results we suggest that oestrogen receptor activation may not be an important mode of action for these compounds . The need to include at least two bioassays in a screening procedure and for combining in vitro and in vivo data is emphasized.

J Biol Chem, 2000 May 5, 275(18), 13933 - 9
Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion; Galliano MF et al.; ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family . ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown . Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo . In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation . Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12 . In vitro binding assays with GST fusion proteins confirmed the specific interaction . The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membrane-distal region . Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2 . Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion . Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.

J Biol Chem, 2000 May 5, 275(18), 13737 - 45
Interaction of a kinesin-like calmodulin-binding protein with a protein kinase; Day IS et al.; Kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that is involved in cell division and trichome morphogenesis . KCBP is unique among all known kinesins in having a myosin tail homology-4 region in the N-terminal tail and a calmodulin-binding region following the motor domain . Calcium, through calmodulin, has been shown to negatively regulate the interaction of KCBP with microtubules . Here we have used the yeast two-hybrid system to identify the proteins that interact with the tail region of KCBP . A protein kinase (KCBP-interacting protein kinase (KIPK)) was found to interact specifically with the tail region of KCBP . KIPK is related to a group of protein kinases specific to plants that has an additional sequence between subdomains VII and VIII of the conserved C-terminal catalytic domain and an extensive N-terminal region . The catalytic domain alone of KIPK interacted weakly with the N-terminal KCBP protein but strongly with full-length KCBP, whereas the noncatalytic region did not interact with either protein . The interaction of KCBP with KIPK was confirmed using coprecipitation assays . Using bacterially expressed full-length and truncated proteins, we have shown that the catalytic domain is capable of phosphorylating itself . The association of KIPK with KCBP suggests regulation of KCBP or KCBP-associated proteins by phosphorylation and/or that KCBP is involved in targeting KIPK to its proper cellular location.

J Biol Chem, 2000 May 5, 275(18), 13713 - 20
Syntaxin 18, a SNAP receptor that functions in the endoplasmic reticulum, intermediate compartment, and cis-Golgi vesicle trafficking; Hatsuzawa K et al.; Members of the syntaxin family are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors involved in vesicle docking and/or fusion within the exocytic and endocytotic pathways . By using the yeast two-hybrid system, we have identified a novel member of the syntaxin family, syntaxin 18, that binds to alpha-soluble N-ethylmaleimide-sensitive factor-attachment protein . Subcellular fractionation and immunocytochemical analysis revealed that syntaxin 18 is principally located in the endoplasmic reticulum . We examined the effect of overexpression of FLAG-tagged syntaxin 18 and a mutant lacking the N-terminal 81 amino acid residues on protein transport and organelles in the early secretory pathway . Both expressed proteins localized to the endoplasmic reticulum, and the expressed FLAG-syntaxin 18 caused remarkable aggregation of endoplasmic reticulum membranes . Although expression of the FLAG-syntaxin 18 lacking the N-terminal region produced less effect on the morphology of the endoplasmic reticulum, dispersion of the endoplasmic reticulum-Golgi intermediate compartment and cis-Golgi was elicited . Moreover, overexpression of the FLAG-syntaxin 18 mutant inhibited protein export from the endoplasmic reticulum . These results taken together suggest that syntaxin 18 functions in transport between the endoplasmic reticulum and Golgi.

J Biol Chem, 2000 May 5, 275(18), 13510 - 6
Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR; Zhu Y et al.; We previously isolated and identified steroid receptor coactivator-1 (SRC-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/PPARBP) as coactivators for PPAR, using the ligand-binding domain of PPARgamma as bait in a yeast two-hybrid screening . As part of our continuing effort to identify cofactors that influence the transcriptional activity of PPARs, we now report the isolation of a novel coactivator from mouse, designated PRIP (peroxisome proliferator-activated receptor interacting protein), a nuclear protein with 2068 amino acids and encoded by 13 exons . Northern analysis showed that PRIP mRNA is ubiquitously expressed in many tissues of adult mice . PRIP contains two LXXLL signature motifs . The amino-terminal LXXLL motif (amino acid position 892 to 896) of PRIP was found to be necessary for nuclear receptor interaction, but the second LXXLL motif (amino acid position 1496 to 1500) appeared unable to bind PPARgamma . Deletion of the last 12 amino acids from the carboxyl terminus of PPARgamma resulted in the abolition of the interaction between PRIP and PPARgamma . PRIP also binds to PPARalpha, RARalpha, RXRalpha, ER, and TRbeta1, and this binding is increased in the presence of specific ligands . PRIP acts as a strong coactivator for PPARgamma in the yeast and also potentiates the transcriptional activities of PPARgamma and RXRalpha in mammalian cells . A truncated form of PRIP (amino acids 786-1132) acts as a dominant-negative repressor, suggesting that PRIP is a genuine coactivator.

J Biol Chem, 2000 May 5, 275(18), 13386 - 93
p38JAB1 binds to the intracellular precursor of the lutropin/choriogonadotropin receptor and promotes its degradation; Li S et al.; Using the C-terminal tail of the rat lutropin/choriogonadotropin receptor (rLHR) as "bait" in a yeast two-hybrid screen resulted in the identification of p38(JAB1) (a protein initially identified as a co-activator of c-Jun) as a putative rLHR binding partner . More recently p38(JAB1) has been shown to promote the degradation of a cyclin-dependent kinase inhibitor and to be a component of the COP9 signalosome . Microscopic localization of an epitope-tagged p38(JAB1) expressed in 293 cells revealed a punctuated perinuclear and cytosolic localization, while cell fractionation studies showed that most of the p38(JAB1) was in a high speed supernatant . Co-transfection of 293 cells revealed that p38(JAB1) binds to the immature 68-kDa precursor of the rLHR that resides in the endoplasmic reticulum and promotes its degradation . It does not appear to interact with the cell surface rLHR, however, and it does not affect its expression . When transfected into HeLa cells, p38(JAB1) potentiates the transcriptional activity of c-Jun, but co-transfection with rLHR prevents this effect . We conclude that p38(JAB1) interacts with the rLHR precursor and promotes its degradation . These results reveal a novel protein binding partner of the rLHR and are consistent with current views of the functions of p38(JAB1).

J Lipid Res, 2000 May, 41(5), 797 - 806
Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii; Toledo MS et al.; Major neutral glycosphingolipid components were extracted from Sporothrix schenckii, a dimorphic fungus exhibiting a hyphal saprophytic phase and a yeast parasitic phase responsible for chronic mycotic infections in mammalian hosts . These components, one from the mycelial form and two from the yeast form, were purified and their structures were elucidated by (1)H nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and tandem ESI-MS/MS . All three were characterized as cerebrosides (monohexosylceramides) containing (4E, 8E)-9-methyl-4,8-sphingadienine as the long-chain base attached to N-2'-hydroxyoctadecanoate and N-2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty acyl components . However, while the mycelial form expressed only beta-glucopyranosylceramide, the yeast form expressed both beta-gluco- and beta-galactopyranosylceramides in approximately equal amounts . In addition, while the glucosylceramides of both mycelial and yeast forms had similar proportions of saturated and (E)-Delta(3) unsaturated 2-hydroxy fatty acid, the galactocerebroside of the yeast form had significantly higher levels of (E)-Delta(3) unsaturation.The differences in cerebroside hexose structure represent a novel type of glycosphingolipid dimorphism not previously reported in fungi . Possible implications of these findings with respect to regulation of morphological transitions in S . schenckii and other dimorphic fungi are discussed.

Biol Bull, 2000 Apr, 198(2), 272 - 83
Vacuolar-type ATPase in the accessory boring organ of Nucella lamellosa (Gmelin) (Mollusca : Gastropoda): role in shell penetration; Clelland ES et al.; The structure and function of the accessory boring organ (ABO) of muricid gastropods has been described in numerous studies, and the ABO of Nucella lamellosa was found to be similar to those of other muricid species . The active cap region of the ABO is composed of tall, mitochondria-rich cells with distinct brush borders at their apicies, surrounding a hemolymph-containing central sinus . Using antibodies specific for vacuolar-type ATPase (V-ATPase), enzyme immunoreactivity was found to be limited to the brush border of the epithelial cells . Electron immunohistochemistry revealed that V-ATPase immunoreactivity resides in the plasma membranes of the microvilli . Immunodot blotting using yeast V-ATPase as a positive control confirmed the specificity of the reactions . SDS-PAGE of membrane suspensions from the ABO revealed protein bands of the requisite molecular weight for V-ATPase subunits . Western blots suggest that antibodies raised against mammalian V-ATPase subunits recognize subunits of the molluscan V-ATPase . The molecular weights of these identified subunits are similar to those in mammals . The V-ATPase-specific inhibitor bafilomycin A1 inhibited ATPase activity in samples of ABO homogenate by about 10% relative to control, providing further evidence for the presence of V-ATPase . Specific V-ATPase activity was about 67 picomoles of inorganic phosphate per microgram of protein per minute in the homogenate . Collectively this evidence strongly suggests that a vacuolar-type proton transporting ATPase is present in the brush border of the accessory boring organ of Nucella lamellosa, and is responsible for acidifying secretions from this gland . Similarities between the ABO, osteoclasts, and the mantle of freshwater bivalves also suggest that the mechanism for decalcification of calcareous substrates is conserved.

Mod Pathol, 2000 Apr, 13(4), 433 - 7
FGF4 and INT2 oncogenes are amplified and expressed in Kaposi's sarcoma; Kiuru-Kuhlefelt S et al.; Kaposi's sarcoma (KS) is a vascular tumor, the pathogenesis of which has been suggested to include human herpesvirus 8 (HHV-8) as well as various cytokines and growth factors . Very little is known about cytogenetic and molecular genetic changes in KS . We studied DNA copy number changes in KS and found a recurrent gain at 11q13 . We then analyzed the amplification and expression status of two known oncogenes, FGF4 and INT2, residing at 11q13 . Comparative genomic hybridization, interphase fluorescence in situ hybridization with yeast artificial chromosome probes containing FGF4 and INT2, and immunoperoxidase immunostaining with anti-FGF4 and -INT2 antibodies were used on 12 KS samples . All samples tested were shown by polymerase chain reaction to be HHV-8 positive . A recurrent gain at 11q13 was shown by comparative genomic hybridization in 4 of 10 cases studied . Of six cases studied by interphase fluorescence in situ hybridization, four showed a 3- to 4-fold amplification with the probes containing FGF4 and INT2 . Expression of FGF4 and INT2 was found in nine and three cases, respectively, of nine studied . Amplification and expression of these genes is particularly interesting in the context of oncovirus involvement, because INT2 is a homolog of mouse int2 which causes mammary carcinoma in mice when activated by integration of retrovirus mouse mammary tumor virus . This raises the question of whether HHV-8 represents an integrating oncovirus that causes amplification and activation of genomic oncogenes in humans.

Cancer Res, 2000 Apr 15, 60(8), 2225 - 31
HMSH6 alterations in patients with microsatellite instability-low colorectal cancer; Parc YR et al.; Two microsatellite instability (MSI) phenotypes have been described in colorectal cancer (CRC): MSI-H (instability at >30% of the loci examined) and MSI-L (MSI at 1-30% of the loci examined) . The MSI-H phenotype, observed in both hereditary nonpolyposis colon cancer-associated CRC and approximately 15% of sporadic CRC, generally results from mutational or epigenetic inactivation of the DNA mismatch repair (MMR) genes hMSH2 or hMLH1 . The genetic basis for the MSI-L phenotype, however, is unknown . Several other proteins, including hMSH3 and hMSH6, also participate in DNA MMR . Inactivating mutations of MSH6 in yeast and human tumor cell lines are associated with an impaired ability to repair single-base mispairs and small insertion-deletion loops but not large insertion-deletion loops . This suggests that hMSH6 mutations are more likely to be associated with a MSI-L phenotype than a MSI-H phenotype in CRC . To explore this possibility, we screened tumors from 41 patients with MSI-L CRC for hMSH6 mutations with conformation-sensitive gel electrophoresis (CSGE) and for hMSH6 protein expression by immunohistochemistry . Alterations found with CSGE were confirmed by DNA sequencing of normal and tumor tissue . One somatic (Asp389Asn) and 15 germ-line changes were found . Of the 15 germ-line changes, 9 were found in an intron (none involving splice junctions), and 6 were found in an exon (Gly39Glu, Leu395Val, and 4 silent alterations) . Immunohistochemical staining for hMSH6 performed on 34 of the 41 tumors revealed strong nuclear hMSH6 expression in all of the cases . Overall, our results suggest that hMSH6 mutations do not play a major role in the development of sporadic CRC with a MSI-L phenotype.

Cancer Res, 2000 Apr 15, 60(8), 2190 - 6
Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth; Yokoyama Y et al.; Ovarian cancer is the leading cause of fatality among gynecological malignancies . Ovarian cancer growth is angiogenesis-dependent, and an increased production of angiogenic growth factors such as vascular endothelial growth factor is prognostically significant even during early stages of the disease . Therefore, we investigated whether antiangiogenic treatment can be used to inhibit the growth of ovarian cancer in an experimental model system . Mouse angiostatin (kringle 1-4) and endostatin were expressed in yeast . Purified angiostatin and endostatin were then used to treat established ovarian cancers in athymic mice . These studies showed that both angiostatin and endostatin inhibited tumor growth . However, angiostatin treatment was more effective in inhibiting ovarian cancer growth when compared with endostatin in parallel experiments . Residual tumors obtained from angiostatin- and endostatin-treated animals showed decreased number of blood vessels and, as a consequence, increased apoptosis of tumor cells . Subsequently, the efficacy of a combined treatment with angiostatin and endostatin was investigated . In the presence of both angiostatic proteins, endothelial cell proliferation was synergistically inhibited . Similarly, a combination regimen using equal amounts of angiostatin and endostatin showed more than additive effect in tumor growth inhibition when compared with treatment with individual angiostatic protein . These studies demonstrate synergism between two angiostatic molecules and that antiangiogenic therapy can be used to inhibit ovarian cancer growth.

Biochim Biophys Acta, 2000 Jan 31, 1490(1-2), 189 - 97
Nuclear receptor binding factor-2 (NRBF-2), a possible gene activator protein interacting with nuclear hormone receptors; Yasumo H et al.; A protein named nuclear receptor binding factor-2 (NRBF-2) was identified by yeast two-hybrid screening, as an interaction partner of peroxisome proliferator-activated receptor alpha as well as several other nuclear receptors . NRBF-2 exhibited a gene activation function, when tethered to a heterologous DNA binding domain, in both mammalian cells and yeast.

Biochim Biophys Acta, 2000 Jan 31, 1490(1-2), 11 - 20
Identification of the copper chaperone SAH in Ovis aries: expression analysis and in vitro interaction of SAH with ATP7B; Lockhart PJ et al.; A clone encoding the putative copper chaperone protein Sheep Atx1 Homologue (SAH) was isolated from a sheep liver cDNA library . The 466-bp cDNA encoded a predicted protein of 68 amino acids, with 44 and 81% amino acid identity to the yeast Atx1 and human Atox1 copper chaperone proteins, respectively . The characteristic MTCxxC and KTGK motifs were conserved in SAH . Northern blot analysis revealed an abundant 0.5-kb mRNA in all tissues examined . Elevated hepatic copper content did not affect the level of SAH mRNA in the liver . Analysis of SAH mRNA in the developing liver revealed low levels of expression in the foetal period, with a steady increase to adult levels occurring during development . In vitro two-hybrid analysis demonstrated SAH interacted with the amino terminal portion of the sheep Wilson's disease protein (ATP7B) . The extent of this interaction was significantly reduced by the addition of the copper chelator bathocuproine disulfonic acid to the media . These results suggest SAH is a functional copper chaperone that is able to interact with ATP7B in a copper-dependent manner to facilitate copper transport into the secretory pathway.

J Nat Prod, 2000 Apr, 63(4), 457 - 60
Bioactive compounds from Combretum erythrophyllum; Schwikkard S et al.; A methanol extract of Combretum erythrophyllum showed inhibitory bioactivities in a yeast-based microtiter assay for DNA-damaging agents . Bioassay-guided fractionation of this extract yielded two known bioactive compounds, combretastatin A-1 and (-)-combretastatin, and two new bioactive glucosides, combretastatin A-1 2'-beta-D-glucoside (1) and combretastatin B-1 2'-beta-D-glucoside (2) . The structures of the new compounds were assigned by (1)H and (13)C NMR, DEPT, HMQC, and HMBC spectra.

Genomics, 2000 Apr 15, 65(2), 104 - 12
Functional and genomic analysis of the human mitochondrial intermediate peptidase, a putative protein partner of frataxin; Chew A et al.; We showed recently that the yeast mitochondrial intermediate peptidase (YMIP polypeptide; gene symbol, OCT1) promotes mitochondrial iron uptake by catalyzing the maturation of iron-utilizing proteins and exacerbates the mitochondrial iron accumulation that results from loss of yeast frataxin, a mitochondrial protein required for mitochondrial iron efflux . This suggests that the human MIP (HMIP polypeptide; gene symbol MIPEP) may be one of the loci predicted to influence the clinical manifestations of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease caused by lack of human frataxin . To begin to test this hypothesis, we have characterized HMIP at the functional and genomic levels . We show that HMIP can complement a yeast knock-out mutant lacking YMIP, demonstrating that HMIP and YMIP are functional homologues . The MIPEP gene spans 57 kb and consists of 19 exons that correlate with the functional domains of HMIP . Primer extension analysis has identified a major transcript of the MIPEP gene expressed differentially and predominantly in tissues with high oxygen consumption, while sequence analysis of approximately 2 kb of 5'-flanking DNA has revealed putative Mt1/3/4, NF-kappaB, and AP-1 elements that may regulate MIPEP expression in these tissues . Using a new polymorphic (CA)(n) repeat in intron 4, MIPEP has been genetically mapped within a 7-cM interval between markers D13S283 and D13S217 on 13q12 . This work provides the basis for molecular analysis of MIPEP in FRDA and possibly other neurodegenerative diseases .

Nat Cell Biol, 2000 Apr, 2(4), 226 - 31
A new self-assembled peroxisomal vesicle required for efficient resealing of the plasma membrane; Jedd G et al.; The Woronin body is a membrane-bound organelle that has been observed in over 50 species of filamentous fungi . However, neither the composition nor the precise function of the Woronin body has yet been determined . Here we purify the Woronin body from Neurospora crassa and isolate Hex1, a new protein containing a consensus sequence known as peroxisome-targeting signal-1 (PTS1) . We show that Hex1 is localized to the matrix of the Woronin body by immunoelectron microscopy, and that a green fluorescent protein- (GFP-)Hex1 fusion protein is targeted to yeast peroxisomes in a PTS1- and peroxin-dependent manner . The expression of the HEX1 gene in yeast generates hexagonal vesicles that are morphologically similar to the native Woronin body, implying a Hex1-encoded mechanism of Woronin-body assembly . Deletion of HEX1 in N . crassa eliminates Woronin bodies from the cytoplasm and results in hyphae that exhibit a cytoplasmic-bleeding phenotype in response to cell lysis . Our results show that the Woronin body represents a new category of peroxisome with a function in the maintenance of cellular integrity.

Virchows Arch, 2000 Mar, 436(3), 271 - 5
Feasibility of simultaneous fluorescence immunophenotyping and fluorescence in situ hybridization study for the detection of estrogen receptor expression and deletions of the estrogen receptor gene in breast carcinoma cell lines; Zhang Y et al.; For the first time, combined immunophenotyping and fluorescence in situ hybridization (FISH) technique according to the "fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms" (FICTION) technique have been successfully applied in solid tumors . Thus, we were able to visualize the antigen expression of cells with chromosomal deletions of a tumor suppressor region directly . In six breast carcinoma cell lines, we investigated the correlation between estrogen receptor (ER) expression status and deletions of the estrogen receptor gene (ESR) . To screen for deletions of the ESR gene, dual-color FISH was performed with a YAC (yeast artificial chromosome) probe containing the ESR gene and, as internal control, with a centromeric probe of chromosome 6 . Deletions of the ESR gene were detected in four of six cell lines . For direct comparison of ER expression with the copy number of the ESR gene at the single cell level, immunophenotyping with mouse anti-human ER antibody was combined with FISH with the YAC probe containing the ESR gene according to the FICTION technique . There was no correlation between lack of or reduced ER expression and deletions of the ESR gene . One cell line with deletions of the ESR gene did express ER on the protein level, while another cell line without a deletion did not . Cells with deletions of the ESR gene were either ER expression positive or negative . The staining intensity of ER expression was not associated with the copy number of the ESR gene . Thus, this FICTION study unequivocally shows that deletions of the ESR gene are not the major cause of absent or reduced ER expression in breast carcinoma cell lines.

Sex Transm Dis, 2000 Apr, 27(4), 230 - 5
Candida vaginitis: self-reported incidence and associated costs; Foxman B et al.; BACKGROUND AND OBJECTIVES: Incidence of Candida vaginitis by age and racial or ethnic group is poorly described . GOAL: Estimate incidence, cumulative probability of presumed C vaginitis by age, racial or ethnic group, and associated costs . STUDY DESIGN: Random digit-dialing survey of 2000 US women . RESULTS: A total of 6.5 percent (95% CI, 5.4-7.5%) of women older than 18 years reported a least one episode of presumed C vaginitis during the previous 2 months . Women reporting a 1-year period with four or more episodes comprised 8.0% of the sample but accounted for 37.2% of women reporting episodes . Black women reported approximately three times more yeast infections in the previous 2 months (17.4%; 95% CI, 11.2-23.5%) than white women (5.8%; 95% CI, 4.7-6.9%) . CONCLUSION: The high incidence and the propensity for recurrence underscore the need for a better understanding of the epidemiology and pathogenesis, and stress the need for the development of more accurate, rapid diagnostics and effective treatments.

Bioorg Med Chem Lett, 2000 Apr 17, 10(8), 839 - 41
Synthesis and antifungal activity of a ferrocene-fluconazole analogue; Biot C et al.; A novel ferrocene fluconazole analogue was synthesized and its antifungal properties investigated against yeast strains of medical importance, including those intrinsically resistant to fluconazole . In vitro tests revealed a slight increase in fungal growth and a reversal of the effect of fluconazole at minimal inhibitory concentrations.

Infect Control Hosp Epidemiol, 2000 Apr, 21(4), 264 - 9
Comparison of higher-dose intradermal hepatitis B vaccination to standard intramuscular vaccination of healthcare workers; Henderson EA et al.; OBJECTIVE: To compare the immunogenicity of hepatitis B vaccine administered via intradermal (ID) versus intramuscular (IM) route . METHODS: Subjects chose either to specify the route of immunization or to undergo random allocation to vaccination by the ID (0.15 mL) or the IM (1.0 mL) route . Yeast-derived recombinant hepatitis B vaccine was given at 0, 30, and 180 days . Hepatitis B surface antibody (HBsAb) and hepatitis B core antibody (HBcAb) were measured by microparticle enzyme immunoassay . RESULTS: 763 subjects were enrolled . Baseline screening identified 65 subjects (8%) who were positive for HBsAb or HBcAb . Vaccination was completed by 590 (85%) of 698 enrollees (370 ID, 220 IM) . Seroconversion rates (geometric mean titers {GMT}>0 IU/mL HBsAb) for those vaccinated ID were 99% and 96% for screening at 9 months and 1 year post-vaccination, respectively; subjects vaccinated intramuscularly had similar rates of 95% and 96% . Seropositivity rates (GMT > or = 10 IU/mL HBsAb) showed a similar pattern, with 95%, 92%, and 73% at 9 months and 1 and 2 years, respectively, for those vaccinated ID, and 94%, 93%, and 81% for those having IM vaccination . GMT for HBsAb was significantly higher for individuals vaccinated IM than for those vaccinated ID (P<.0001) . The GMT ratio for the IM and ID routes decreased over time, being 9.3 at 9 months, 7.8 at 1 year, and 5.9 at 2 years . An unanticipated side effect of intradermal vaccination was skin discoloration at injection sites, which persisted for at least 2 years postvaccination . Two thirds (112/166) of respondents reported that they would have selected the ID route despite the discoloration . CONCLUSIONS: Higher-dose ID vaccination (3 vs 1 microg per injection) uses one sixth of the dose required for standard IM vaccination . It is a cost-effective way to vaccinate populations against hepatitis B virus, but the long-term efficacy of the ID route must still be investigated.

Proc Soc Exp Biol Med, 2000 May, 224(1), 32 - 40
The familial mediterranean fever protein interacts and colocalizes with a putative Golgi transporter; Chen X et al.; The biological function of pyrin, the protein mutated in Familial Mediterranean Fever (FMF), has not been elucidated . Based on sequence homology, a transcription factor activity was proposed for this neutrophil-specific protein . In a yeast two-hybrid assay, neither transcription activation activity nor any self interaction was detected for pyrin . Screening of an expression cDNA library of peripheral blood leukocytes using as bait the carboxyl portion of pyrin (amino acids 557-781), which contains most of the FMF mutations, led to the identification of P/M-IP1 (pyrin/marenostrin interacting protein 1) . A splice variant of P/M-IP1, GTC-90, had previously been described as a component of the 13S hetero-oligomeric protein complex that stimulates in vitro Golgi transport . We have now shown that P/M-IP1 colocalizes with pyrin in the perinuclear cytoplasm of Cos-7 cells and that the interaction between these two proteins is impaired by FMF causing mutations in pyrin . These data suggest that, at some stage of its functional pathway, pyrin resides in the cytoplasm and might be involved in, or impacted by, cellular protein sorting by the Golgi apparatus . The data also imply that P/M-IP1 may be involved in the abnormal inflammatory response that occurs in patients with FMF.

Mech Dev, 2000 May, 93(1-2), 221 - 31
Formin-2, a novel formin homology protein of the cappuccino subfamily, is highly expressed in the developing and adult central nervous system; Leader B et al.; Formin-1 is the founding member of a family of genes of emerging biological and medical importance that share specific domains of homology, allowing them to be classified together as the formin homology proteins . Although deficiency mutations in formin-1 lead to profound developmental defects in limb and kidney formation, similar deficiency mutations in more distantly related members of this family (diaphanous and cappuccino in Drosophila and BNI1 in yeast) have ostensibly unrelated phenotypes . Here we describe murine and human formin-2 (Fmn2), a gene which bears a high degree of similarity to formin-1 and cappuccino . The mouse gene, which encodes a putative 1567-amino-acid open reading frame and maps to mouse Chromosome 1, is expressed almost exclusively in the developing and mature central nervous system . Expression begins at embryonic day 9 . 5 in the developing spinal cord and brain structures and continues in neonatal and adult brain structures including the olfactory bulb, cortex, thalamus, hypothalamus, hippocampus and cerebellum . Human formin-2 has a similar expression pattern.

Mol Immunol, 2000 Jan-Feb, 37(1-2), 73 - 84
TRAF-3 interacts with p62 nucleoporin, a component of the nuclear pore central plug that binds classical NLS-containing import complexes; Gamper C et al.; The TRAF-3 gene encodes a number of splice-variant isoforms that function as adapter molecules in NF-kappaB signaling, in part by associating with the cytoplasmic tails of CD40 or other TNF-receptor (TNF-R) family members . To identify downstream molecules in TRAF-3 signaling, a yeast two-hybrid library was screened with a full-length TRAF-3 construct . Nine independent TRAF-3 interacting clones encoded fragments of p62 Nucleoporin (p62), a 522 amino acid (aa) component of the nuclear pore central plug, that is known to bind karyopherin-beta/classical-NLS import factor complexes . The interaction of p62 with TRAF-3 was specific, since p62 failed to interact with TRAF-2, -4, -5, or -6 . Deletional analysis in yeast revealed that the p62:TRAF-3 interaction is mediated by a p62 carboxy (C)-terminal coiled-coil domain and TRAF-3's fifth zinc (Zn) finger and coiled-coil domain . In human 293 T cells, recombinant TRAF-3 or p62 specifically co-immunoprecipitates the other species . In addition, endogenous p62 co-precipitates over-expressed TRAF-3 . The functional effects of over-expressing a TRAF-3 binding fragment, p62(aa 336-522) were studied on NF-kappaB-dependent, or control STAT1-dependent reporter activity in 293 T cells, either resting or after stimulation by CD40 or IFN-gamma, respectively . Over-expression of p62(aa 336-522) induces NF-kappaB activation in resting cells and augments CD40-induced NF-kappaB activation, but has no effect on control STAT1 reporter activity, either at baseline or after IFN-gamma induction . The finding that TRAF-3 binds p62, suggests that TRAF-3 may serve as an adapter molecule at the nuclear membrane, in addition to its known adapter function at the plasma membrane.

J Biol Chem, 2000 Jun 23, 275(25), 19422 - 7
A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product; Chien W et al.; Pituitary tumor-transforming gene (PTTG) is a recently characterized oncogene whose expression product contains a transcriptional activation domain at the C terminus . To understand the mechanisms involved in PTTG biological functions, we used yeast two-hybrid screening to identify proteins that interact with PTTG . This study reports the isolation and characterization of a novel PTTG-binding factor (PBF) . PBF contains an open reading frame of 179 amino acids with a predicted molecular mass of 22 kDa . In Northern blot analyses, PBF mRNA was ubiquitously expressed in human tissues . Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that PBF interacts specifically with PTTG under both in vitro and in vivo conditions . The PTTG binding domain in PBF was located within the C-terminal 30-amino acid region that contain a nuclear localization signal . Immunofluorescence and subcellular fractionation studies showed that PTTG is predominantly expressed in the cytoplasm with partial nuclear localization, whereas PBF is localized both in the cytoplasm and the nucleus . The interaction between PBF and PTTG facilitated PTTG translocation from the cytoplasm to the nucleus . Furthermore, PBF is required for transcriptional activation of basic fibroblast growth factor by PTTG . In summary, we have characterized a novel PTTG-binding protein that facilitates PTTG nuclear translocation and potentiates its transcriptional activation function.

Proc Natl Acad Sci U S A, 2000 Apr 25, 97(9), 4879 - 84
Genome-wide analysis of vaccinia virus protein-protein interactions; McCraith S et al.; To detect interactions between proteins of vaccinia virus, we carried out a comprehensive two-hybrid analysis to assay every pairwise combination . We constructed an array of yeast transformants that contained each of the 266 predicted viral ORFs as Gal4 activation domain hybrid proteins . The array was individually mated to transformants containing each ORF as a Gal4-DNA-binding domain hybrid, and diploids expressing the two-hybrid reporter gene were identified . Of the approximately 70,000 combinations, we found 37 protein-protein interactions, including 28 that were previously unknown . In some cases, e.g., late transcription factors, both proteins were known to have related roles although there was no prior evidence of physical associations . For some other interactions, neither protein had a known role . In the majority of cases, however, one of the interacting proteins was known to be involved in DNA replication, transcription, virion structure, or host evasion, thereby providing a clue to the role of the other uncharacterized protein in a specific process.

Eur J Hum Genet, 2000 Mar, 8(3), 167 - 73
FISH mapping of the sex-reversal region on human chromosome 9p in two XY females and in primates; Shan Z et al.; Accumulating evidence suggests that haploinsufficiency of a dosage-sensitive gene(s) in human chromosome 9p24.3 is responsible for the failure of testicular development and feminisation in XY patients with monosomy for 9p . We have used molecular cytogenetic methods to characterise the sex-reversing 9p deletions in two XY females . Fluorescence in situ hybridisation (FISH) with YACs from the critical 9p region containing an evolutionarily conserved sex-determining gene, DMRT1, is a very fast and reliable assay for patient screening . Comparative YAC mapping on great ape and Old and New World monkey chromosomes demonstrated that the critical region was moved from an interstitial position on the ancestral primate chromosome to a very subtelomeric position in chimpanzee and humans by a pericentric inversion(s) . Pathological 9p rearrangements may be the consequence of an evolutionary chromosome breakpoint in close proximity to the sex-reversal region.

Nucleic Acids Symp Ser, 1999, (42), 65 - 6
Statistical analysis of DNA sequencing data (1): accuracy test of DNA data by partial re-sequencing; Yoshida K et al.; To qualify DNA data, we have developed a statistical method of deciding whether the DNA data has an acceptable accuracy in sequencing process . The method is to test the probability of sequencing errors, based on partial re-sequencing . The method was successfully applied to a yeast mitochondrial DNA which is previously sequenced (1) . The analysis indicates that the entire sequence is very accurate although we found one base change error on the ND1 gene sequence data by a partial re-sampling . This method is applicable to any DNA data.

Pharmacogenetics, 1999 Aug, 9(4), 421 - 34
Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping; Vasiliou V et al.; As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time . The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes . To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced . These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible . An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family . Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site . For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g . 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines be used for all species other than mouse and Drosophila . Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g . 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity' . If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out in chronological order as they are reported to us . In addition, 20 human ALDH variant alleles that have been reported to date are listed herein and are recommended to be given numbers (or a number plus a capital letter) following an asterisk (e.g . 'ALDH3A2*2, ALDH2*4C') . It is anticipated that this eukaryotic ALDH gene nomenclature system will be extended to include bacterial genes within the next 2 years and that this nomenclature system will require updating on a regular basis; an ALDH Web site has been established for this purpose and will serve as a medium for interaction amongst colleagues in this field.

J Immunol, 2000 May 1, 164(9), 4569 - 74
Faithful expression of the human 5q31 cytokine cluster in transgenic mice; Lacy DA et al.; Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31 . We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself . Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro . Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo . Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes . These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.

Mol Biochem Parasitol, 2000 Apr 15, 107(2), 227 - 40
Plasmodium falciparum phosphoenolpyruvate carboxykinase is developmentally regulated in gametocytes; Hayward RE; Plasmodium species have the capacity to fix carbon dioxide during intracellular development . This process contributes to the pool of free amino acids and metabolites, which are the end products of glucose metabolism in the malaria parasite . A gene encoding phosphoenolpyruvate carboxykinase (PEPCK), an enzyme known to catalyze CO(2) fixation was identified in the genome of the human parasite Plasmodium falciparum by DNA microarray analysis experiments and was cloned and characterized . PfPEPCK is a 66.2 kDa, ATP-dependent enzyme which is closely related to PEPCK from plants and yeast but markedly different from the host enzyme human PEPCK . PfPEPCK transcript and active enzyme levels are upregulated in the transmissible and zygote stages of parasite development relative to the asexual blood stages . Elevated expression of PfPEPCK during the extracellular zygote phase of P . falciparum development within the microenvironment of the mosquito midgut may reflect a glucose-rare medium and suggests a possible switch in carbohydrate metabolism to a gluconeogenesis pathway.

Genome Res, 2000 Apr, 10(4), 516 - 22
Ab initio gene finding in Drosophila genomic DNA; Salamov AA et al.; Ab initio gene identification in the genomic sequence of Drosophila melanogaster was obtained using (human gene predictor) and Fgenesh programs that have organism-specific parameters for human, Drosophila, plants, yeast, and nematode . We did not use information about cDNA/EST in most predictions to model a real situation for finding new genes because information about complete cDNA is often absent or based on very small partial fragments . We investigated the accuracy of gene prediction on different levels and designed several schemes to predict an unambiguous set of genes (annotation CGG1), a set of reliable exons (annotation CGG2), and the most complete set of exons (annotation CGG3) . For 49 genes, protein products of which have clear homologs in protein databases, predictions were recomputed by Fgenesh+ program . The first annotation serves as the optimal computational description of new sequence to be presented in a database . Reliable exons from the second annotation serve as good candidates for selecting the PCR primers for experimental work for gene structure verification . Our results shows that we can identify approximately 90% of coding nucleotides with 20% false positives . At the exon level we accurately predicted 65% of exons and 89% including overlapping exons with 49% false positives . Optimizing accuracy of prediction, we designed a gene identification scheme using Fgenesh, which provided sensitivity (Sn) = 98% and specificity (Sp) = 86% at the base level, Sn = 81% (97% including overlapping exons) and Sp = 58% at the exon level and Sn = 72% and Sp = 39% at the gene level (estimating sensitivity on std1 set and specificity on std3 set) . In general, these results showed that computational gene prediction can be a reliable tool for annotating new genomic sequences, giving accurate information on 90% of coding sequences with 14% false positives . However, exact gene prediction (especially at the gene level) needs additional improvement using gene prediction algorithms . The program was also tested for predicting genes of human Chromosome 22 (the last variant of Fgenesh can analyze the whole chromosome sequence) . This analysis has demonstrated that the 88% of manually annotated exons in Chromosome 22 were among the ab initio predicted exons . The suite of gene identification programs is available through the WWW server of Computational Genomics Group at html.

Blood, 2000 May 1, 95(9), 2838 - 46
REDK, a novel human regulatory erythroid kinase; Lord KA et al.; We have identified a novel regulatory erythroid kinase (REDK) that is homologous to a family of dual-specificity kinases . The yeast homolog of REDK negatively regulates cell division, suggesting a similar function for REDK, which is primarily localized in the nucleus . REDK is present in hematopoietic tissues, such as bone marrow and fetal liver, but the RNA is expressed at significant levels only in erythroid or erythropoietin (EPO)-responsive cells . Two novel forms of cDNA (long and short) for REDK have been isolated that appear to be alternative splice products and imply the presence of polypeptides with differing amino termini . The ratio of short-to-long forms of REDK increases dramatically in CD34(+) cells cultured with EPO, suggesting differing regulation and function for each form . REDK is predominantly found in nuclear, rather than cytoplasmic, protein extracts, and immunoprecipitated REDK is active in phosphorylating histones H2b, H3, myelin basic protein, and other coimmunoprecipitated proteins . Antisense REDK oligonucleotides promote erythroid colony formation by human bone marrow cells, without affecting colony-forming unit (CFU)-GM, CFU-G, or CFU-GEMM numbers . Maximal numbers of CFU-E and burst-forming unit-erythroid were increased, and CFU-E displayed increased sensitivity to suboptimal EPO concentrations . The data indicate that REDK acts as a brake to retard erythropoiesis . (Blood . 2000;95:2838-2846)

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5387 - 92
In vitro evolution of a T cell receptor with high affinity for peptide/MHC; Holler PD et al.; T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower . It has been suggested that TCRs undergo selection in vivo to maintain lower affinities . Here, we show that there is not an inherent genetic or structural limitation on higher affinity . Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using yeast display and selection from a library of Valpha CDR3 mutants . Selected mutants had greater than 100-fold higher affinity (K(D) approximately 9 nM) for the peptide/MHC ligand while retaining a high degree of peptide specificity . Among the high-affinity TCR mutants, a strong preference was found for CDR3alpha that contained Pro or Gly residues . Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide/MHC complexes on antigen-presenting cells . These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide/MHC complexes for diagnostic and therapeutic purposes.

Cell, 2000 Mar 31, 101(1), 103 - 13
Drosophila p53 binds a damage response element at the reaper locus; Brodsky MH et al.; The tumor suppressor gene p53 regulates multiple cellular responses to DNA damage, but the transcriptional targets that specify these responses are incompletely understood . We describe a Drosophila p53 homolog and demonstrate that it can activate transcription from a promoter containing binding sites for human p53 . Dominant-negative forms of Drosophila p53 inhibit both transactivation in cultured cells and radiation-induced apoptosis in developing tissues . The cis-regulatory region of the proapoptotic gene reaper contains a radiation-inducible enhancer that includes a consensus p53 binding site . Drosophila p53 can activate transcription from this site in yeast and a multimer of this site is sufficient for radiation induction in vivo . These results indicate that reaper is a direct transcriptional target of Drosophila p53 following DNA damage.

Nippon Ishinkin Gakkai Zasshi, 2000, 41(2), 89 - 95
{Phaeomycotic cyst caused by Phaeoacremonium parasiticum}; Kitamura K et al.; Phaeoacremonium parasiticum was identified as the causative agent of a phaeomycotic cyst seen just below the right knee of a 59-year-old healthy woman . She had no history of trauma . Direct KOH examination of the pus aspirated from the subcutaneous nodule revealed abundant mycelia, which were not too deeply brown in color . The nodule was surgically excised, and there was no recurrence during a half year of observation . Tissue section of the excised material revealed rather a large cavity extending from the cutis to the subcutis . The cavity had a thick wall composed of granulomatous tissues . Mycelial and yeast-like fungal elements were seen within the cavity and the granulomatous tissues . A dematiaceous fungus was cultured from both pus and the excised material . The isolates were characterized by a dark green to black colony, unbranched or infrequently branched, brownish conidiophores bearing an aculeate monophialide with a narrow funnel-shaped collarette, and slimy, hyaline, one-celled, ellipsoid to allantoid conidia.

Biochem Biophys Res Commun, 2000 Apr 29, 271(1), 144 - 50
Human adrenoleukodystrophy protein and related peroxisomal ABC transporters interact with the peroxisomal assembly protein PEX19p; Gloeckner CJ et al.; Four ABC half transporters (ALDP, ALDRP, PMP70, and PMP69) have been identified in the mammalian peroxisomal membrane but no function has been unambiguously assigned to any of them . To date X-linked adrenoleukodystrophy (X-ALD) is the only human disease known to result from a defect of one of these ABC transporters, ALDP . Using the yeast two-hybrid system and in vitro GST pull-down assays, we identified the peroxin PEX19p as a novel interactor of ALDP, ALDRP, and PMP70 . The cytosolic farnesylated protein PEX19p was previously shown to be involved in an early step of the peroxisomal biogenesis . The PEX19p interaction occurs in an internal N-terminal region of ALDP which we verified to be important for proper peroxisomal targeting of this protein . Farnesylated wild-type PEX19p and a farnesylation-deficient mutant PEX19p did not differ in their ability to bind to ALDP . Our data provide evidence that PEX19p is a cytosolic acceptor protein for the peroxisomal ABC transporters ALDP, PMP70, and ALDRP and might be involved in the intracellular sorting and trafficking of these proteins to the peroxisomal membrane .

Genomics, 2000 Apr 1, 65(1), 70 - 4
Cloning and expression analysis of a novel gene, RP42, mapping to an autism susceptibility locus on 6q16; Mas C et al.; We isolated a novel mouse gene, RP42, in a systematic search for genes expressed in proliferating neuroblasts whose human orthologs map to susceptibility loci for autism . This gene is intronless and encodes a putative 259-amino-acid protein that exhibits 30-36% overall sequence identity to a fission yeast and a nematode protein (GenPept Accession Nos . CAA17006 and CAB54261) . Nevertheless, no homology to any known gene was found . RP42 has developmentally regulated expression, particularly in proliferating neuroblasts from which neocortical neurons originate . Its human ortholog is located in a cluster of embryonic neuronally expressed genes on the 6q16 chromosome, making it a positional candidate susceptibility gene for autism .

Genomics, 2000 Apr 1, 65(1), 34 - 43
A clone contig of 12q24.3 encompassing the distal hereditary motor neuropathy type II gene; Irobi J et al.; We previously assigned the disease locus for autosomal dominant hereditary motor neuropathy type II (distal HMN II) within a 13-cM interval at chromosome 12q24.3 . We constructed a physical map of the distal HMN II region based on yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) using an STS content mapping approach . The contig contains 26 YAC, 15 PAC, and 60 BAC clones and covers a physical distance of approximately 5 Mb . A total of 99 STS markers, including 25 known STSs and STRs, 49 new STSs generated from clone end-fragments, 20 ESTs, and 5 known genes, were located on the contig . This physical map provides a valuable resource for mapping genes and markers located within the distal HMN II region and facilitates the positional cloning of the distal HMN II gene .

J Biol Chem, 2000 Apr 28, 275(17), 12896 - 9
The Elp2 subunit of elongator and elongating RNA polymerase II holoenzyme is a WD40 repeat protein; Fellows J et al.; A novel yeast gene, ELP2, is shown to encode the 90-kDa subunit of the Elongator complex and elongating RNA polymerase II holoenzyme . ELP2 encodes a protein with eight WD40 repeats, and cells lacking the gene display typical elp phenotypes, such as temperature and salt sensitivity . Generally, different combinations of double and triple ELP gene deletions cause the same phenotypes as single ELP1, ELP2, or ELP3 deletion, providing genetic evidence that the ELP gene products work together in a complex.

J Biol Chem, 2000 Apr 28, 275(17), 12857 - 67
Identification of a novel SCAN box-related protein that interacts with MZF1B . The leucine-rich SCAN box mediates hetero- and homoprotein associations; Sander TL et al.; The SCAN box or leucine-rich (LeR) domain is a conserved motif found within a subfamily of C(2)H(2) zinc finger proteins . The function of a SCAN box is unknown, but it is predicted to form alpha-helices that may be involved in protein-protein interactions . Myeloid zinc finger gene-1B (MZF1B) is an alternatively spliced human cDNA isoform of the zinc finger transcription factor, MZF1 . MZF1 and MZF1B contain 13 C(2)H(2) zinc finger motifs, but only MZF1B contains an amino-terminal SCAN box . A bone marrow cDNA library was screened for proteins interacting with the MZF1B SCAN box domain and RAZ1 (SCAN-related protein associated with MZF1B) was identified . RAZ1 is a novel cDNA that encodes a SCAN-related domain and arginine-rich region but no zinc finger motifs . Co-immunoprecipitation assays demonstrate that the SCAN box domain of MZF1B is necessary for association with RAZ1 . By yeast two-hybrid analysis, the carboxyl terminus of RAZ1 is sufficient for interaction with the MZF1B SCAN box . Furthermore, MZF1B and RAZ1 each self-associate in vitro via a SCAN box-dependent mechanism . These data provide evidence that the SCAN box is a protein interaction domain that mediates both hetero- and homoprotein associations.

J Biol Chem, 2000 Apr 28, 275(17), 12806 - 12
Human procathepsin B interacts with the annexin II tetramer on the surface of tumor cells; Mai J et al.; To study potential roles of plasma membrane-associated extracellular cathepsin B in tumor cell invasion and metastasis, we used the yeast two-hybrid system to screen for proteins that interact with human procathepsin B . The annexin II light chain (p11), one of the two subunits of the annexin II tetramer, was one of the proteins identified . We have confirmed that recombinant human procathepsin B interacts with p11 as well as with the annexin II tetramer in vitro . Furthermore, procathepsin B could interact with the annexin II tetramer in vivo as demonstrated by coimmunoprecipitation . Cathepsin B and the annexin II tetramer were shown by immunofluorescent staining to colocalize on the surface of human breast carcinoma and glioma cells . Taken together, our results indicate that the annexin II tetramer can serve as a binding protein for procathepsin B on the surface of tumor cells, an interaction that may facilitate tumor invasion and metastasis.

J Biol Chem, 2000 Apr 28, 275(17), 12470 - 4
Silencing mediator of retinoic acid and thyroid hormone receptors, as a novel transcriptional corepressor molecule of activating protein-1, nuclear factor-kappaB, and serum response factor; Lee SK et al.; Silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is known to interact with Sin3 and recruit the histone deacetylases (HDACs) that lead to hypoacetylation of histones and transrepression of target transcription factors . Herein, we found that coexpression of SMRT significantly repressed transactivations by activating protein-1 (AP-1), nuclear factor-kappaB (NFkappaB), and serum response factor (SRF) in a dose-dependent manner, but not in the presence of trichostatin A, a specific inhibitor of HDAC . Similarly, coexpression of HDAC1 and mSin3A also showed repressive effects . Consistent with these results, the C-terminal region of SMRT directly interacted with SRF, the AP-1 components c-Jun and c-Fos, and the NFkappaB components p50 and p65, as demonstrated by the yeast and mammalian two hybrid tests as well as the glutathione S-transferase pull down assays . Thus, we concluded that SMRT serves to recruit Sin3/HDACs to SRF, NFkappaB, and AP-1 in vivo and modulate their transactivation.

J Biol Chem, 2000 Apr 28, 275(17), 12446 - 52
Multiple interactions between receptor protein-tyrosine phosphatase (RPTP) alpha and membrane-distal protein-tyrosine phosphatase domains of various RPTPs; Blanchetot C et al.; Receptor protein-tyrosine phosphatase (RPTP) alpha belongs to the large family of receptor protein-tyrosine phosphatases containing two tandem phosphatase domains . Most of the catalytic activity is retained in the first, membrane-proximal domain (RPTPalpha-D1), and little is known about the function of the second, membrane-distal domain (RPTPalpha-D2) . We investigated whether proteins bound to RPTPalpha using the two-hybrid system and found that the second domain of RPTPsigma interacted with the juxtamembrane domain of RPTPalpha . We confirmed this interaction by co-immunoprecipitation experiments . Furthermore, RPTPalpha not only interacted with RPTPsigma-D2 but also with RPTPalpha-D2, LAR-D2, RPTPdelta-D2, and RPTPmu-D2, members of various RPTP subfamilies, although with different affinities . In the yeast two-hybrid system and in glutathione S-transferase pull-down assays, we show that the RPTP-D2s interacted directly with the wedge structure of RPTPalpha-D1 that has been demonstrated to be involved in inactivation of the RPTPalpha-D1/RPTPalpha-D1 homodimer . The interaction was specific because the equivalent wedge structure in LAR was unable to interact with RPTPalpha-D2 or LAR-D2 . In vivo, we show that other interaction sites exist as well, including the C terminus of RPTPalpha-D2 . The observation that RPTPalpha, but not LAR, bound to multiple RPTP-D2s with varying affinities suggests a specific mechanism of cross-talk between RPTPs that may regulate their biological function.

J Biol Chem, 2000 Apr 28, 275(17), 12363 - 6
ICln is essential for cellular and early embryonic viability; Pu WT et al.; pICln is a 26-kDa protein that is ubiquitously expressed and highly conserved from Xenopus laevis to Homo sapiens . The physiological functions of pICln remain to be established . To address this question, we disrupted the ICln gene in embryonic stem cells . We found that murine embryos lacking ICln die early in gestation (between stages E3.5 and E7.5) . Furthermore, we found that ICln is essential for embryonic stem cell viability . Previously, we showed that pICln interacts directly with a homolog of a yeast protein that binds a PAK-like kinase and participates in the regulation of cell morphology and cell cycling . pICln also forms a complex with several core spliceosomal proteins, and this interaction may play a role in the regulation of spliceosomal biogenesis . Collectively, these data strongly suggest that pICln participates in critical cellular pathways, including regulation of the cell cycle and RNA processing.

J Biol Chem, 2000 Jun 23, 275(25), 19401 - 8
Recruitment of nuclear receptor corepressor and coactivator to the retinoic acid receptor by retinoid ligands . Influence of DNA-heterodimer interactions; Klein ES et al.; Ligand activation of retinoic acid receptors (RARs) involves coordinated changes in their interaction with coregulatory molecules . Binding of the agonist all-trans-retinoic acid to the RAR results in increased interaction with coactivator molecules as well as a decreased interaction with corepressor molecules . Thus, an all-trans-retinoic acid antagonist might function either by preventing agonist induction of such events or, additionally, by actively increasing repression via corepressor recruitment . We demonstrate that the repression of the transcriptional activity of a constitutively active RARgamma-VP-16 chimeric receptor by the inverse agonist AGN193109 requires a functional Co-R box and that binding of this ligand to RARgamma leads to an increased interaction with the corepressor N-CoR both in glutathione S-transferase pull-down and yeast two-hybrid analyses . Detection of nuclear receptor corepressor (N-CoR) association with RARgamma was greatly facilitated by inclusion of a RARE oligonucleotide in coimmunoprecipitation analyses, a result of an increase in association of the ternary complex consisting of RAR, RXR, and DNA . Similarly, this DNA-dependent increase in heterodimer formation likewise resulted in an increase in agonist-mediated recruitment efficiency of the coactivator SRC-1 . Under conditions which favor ternary complex formation, a RAR neutral antagonist is distinguished from an inverse agonist with respect to corepressor recruitment as is a RAR partial agonist distinguished from an agonist with respect to coactivator recruitment . These results indicate that it is possible to design RAR ligands with distinct recruitment capabilities for coregulators, both coactivators as well as corepressors . In addition, using this recruitment assay, we show that SRC-1 and the related coactivator molecule ACTR associate with the ternary complex via utilization of different helical motifs within their conserved receptor interaction domains.

Oncogene, 2000 Mar 30, 19(14), 1807 - 19
A murine ATFa-associated factor with transcriptional repressing activity; De Graeve F et al.; The ATFa proteins, which are members of the CREB/ATF family of transcription factors, have previously been shown to interact with the adenovirus E1a oncoprotein and to mediate its transcriptional activity; they heterodimerize with Jun, Fos or related transcription factors, possibly altering their DNA-binding specificity; they also stably bind JNK2, a stress-induced protein kinase . Here we report the identification and characterization of a novel protein isolated in a yeast two-hybrid screen using the N-terminal half of ATFa as a bait . This 1306-residue protein (mAM, for mouse ATFa-associated Modulator) is rather acidic (pHi 4.5) and contains high proportions of Ser/Thr (21%) and Pro (11%) residues . It colocalizes and interacts with ATFa in mammalian cells, contains a bipartite nuclear localization signal and possesses an ATPase activity . Transfection experiments show that mAM is able to downregulate transcriptional activity, in an ATPase-independent manner . Our results indicate that mAM interacts with several components of the basal transcription machinery (TFIIE and TFIIH), including RNAPII itself . Together, these findings suggest that mAM may be involved in the fine-tuning of ATFa-regulated gene expression, by interfering with the assembly or stability of specific preinitiation transcription complexes.

Oncogene, 2000 Mar 30, 19(14), 1801 - 6
Epstein-Barr virus encoded nuclear protein EBNA-3 binds XAP-2, a protein associated with Hepatitis B virus X antigen; Kashuba E et al.; EBNA-3 (also called EBNA-3A) is one of the EBV encoded nuclear antigens that are necessary for B-cell transformation . EBNA-3 is known to target RBPs, nuclear proteins that also interacts with EBNA-2, EBNA-4 and EBNA-6 . In order to identify additional EBNA-3 targets, an EBV-transformed human lymphocyte cDNA library was screened in the yeast two-hybrid system with N-terminus truncated EBNA-3 that cannot interact with RBP-Jkappa . A clone, encoding Xap-2 protein, a cellular partner of Hepatitis B virus X-antigen was isolated . This protein is also known as the p38 subunit of the aryl hydrocarbon receptor complex (ARA9) . The specific binding to EBNA-3 was confirmed by showing that the GST-Xap-2 precipitated EBNA-3 from CV1 cells that were infected with recombinant vaccinia virus expressing EBNA-3 . Deletion of the C-terminus of Xap-2 eliminated the binding . Fusion with green fluorescent protein showed that Xap-2 is preferentially cytoplasmic but translocates to the nucleus upon expression of EBNA-3.

Oncogene, 2000 Mar 30, 19(14), 1752 - 63
B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos; Echlin DR et al.; B-ATF is a nuclear basic leucine zipper protein that belongs to the AP-1/ATF superfamily of transcription factors . Northern blot analysis reveals that the human B-ATF gene is expressed most highly in hematopoietic tissues . Interaction studies in vitro and in vivo show that the leucine zipper of B-ATF mediates dimerization with members of the Jun family of proteins . Chimeric proteins consisting of portions of B-ATF and the DNA binding domain of the yeast activator GAL4 do not stimulate reporter gene expression in mammalian cells, indicating that B-ATF does not contain a conventional transcription activation domain . Jun/B-ATF dimers display similar DNA binding profiles as Jun/Fos dimers, with a bias toward binding TRE (12-O-tetradecanolyphorbol-13-acetate-response element) over CRE (cyclic AMP-response element) DNA sites . B-ATF inhibits transcriptional activation of a reporter gene containing TRE sites in a dose-dependent manner, presumably by competing with Fos for Jun and forming transcriptionally inert Jun/B-ATF heterodimers . Stable expression of B-ATF in C3H10T1/2 cells does not reduce cell viability, but does result in a reduced cellular growth rate when compared to controls . This effect is dominant in the presence of the growth promoting effects of the H-Ras or the v-Fos oncoproteins, since expression of B-ATF restricts the efficiency of focus formation by these transforming agents . These findings demonstrate that B-ATF is a tissue-specific transcription factor with the potential to function as a dominant-negative to AP-1.

Oncogene, 2000 Mar 30, 19(14), 1744 - 51
ENL, the MLL fusion partner in t(11;19), binds to the c-Abl interactor protein 1 (ABI1) that is fused to MLL in t(10;11)+; Garcia-Cuellar MP et al.; Translocations of the chromosomal locus 11q23 that disrupt the MLL gene (alternatively ALL-1 or HRX) are frequently found in children's leukemias . These events fuse the MLL amino terminus in frame with a variety of unrelated proteins . Up to date, 16 different fusion partners have been characterized and more are likely to exist . No general unifying property could yet be detected amongst these proteins . We show here that the frequent MLL fusion partner ENL at 19p13.1 interacts with the human homologue of the mouse Abl-Interactor 1 (ABI1) protein . ABI1 in turn, is fused to MLL in the t(10;11)(p11.2;q23) translocation . ABI1 was identified as an ENL binding protein by a yeast two-hybrid screen . The interaction of ENL and ABI1 could be verified in vitro by far-Western blot assays and GST-pulldown studies as well as in vivo by co-immunoprecipitation experiments . A structure-function analysis identified an internal region of ENL and a composite motif of ABI1 including an SH3 domain as mutual binding partners . These data introduce novel aspects that might contribute to the understanding of the process of leukemogenesis by MLL fusion proteins.

J Virol, 2000 May, 74(10), 4705 - 9
An adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis complexes with dynein and a small GTPase; Lukashok SA et al.; Adenoviruses (Ad) code for immunoregulatory and cytokine regulatory proteins, one of which is the early region 3, 14.7-kDa protein (Ad E3-14.7K), which has been shown to inhibit tumor necrosis factor alpha-induced apoptosis . In an effort to understand the mechanism of action of Ad E3-14.7K, we previously searched for cell proteins with which it interacted . Three Ad E3-14.7K-interacting proteins (FIP-1, -2, and -3) were isolated . FIP-1 is a small GTPase which was used in this report as bait in the yeast two-hybrid system to find other interacting cell targets . The search resulted in the isolation of a protein, which we called GIP-1 (GTPase-interacting protein) that subsequently was shown to be identical to one of the light-chain components of human dynein (TCTEL1) . FIP-1 was able to bind both TCTEL1 and Ad E3-14.7K simultaneously and was necessary to form a complex in which the viral protein was associated with a microtubule-binding motor protein . The functional significance of these interactions is discussed with respect to the steps of the Ad life cycle which are microtubule associated.

Am J Hum Genet, 2000 Jun, 66(6), 1736 - 43 Epub 2000 Apr 20.
Identification of the alpha-aminoadipic semialdehyde synthase gene, which is defective in familial hyperlysinemia; Sacksteder KA et al.; The first two steps in the mammalian lysine-degradation pathway are catalyzed by lysine-ketoglutarate reductase and saccharopine dehydrogenase, respectively, resulting in the conversion of lysine to alpha-aminoadipic semialdehyde . Defects in one or both of these activities result in familial hyperlysinemia, an autosomal recessive condition characterized by hyperlysinemia, lysinuria, and variable saccharopinuria . In yeast, lysine-ketoglutarate reductase and saccharopine dehydrogenase are encoded by the LYS1 and LYS9 genes, respectively, and we searched the available sequence databases for their human homologues . We identified a single cDNA that encoded an apparently bifunctional protein, with the N-terminal half similar to that of yeast LYS1 and with the C-terminal half similar to that of yeast LYS9 . This bifunctional protein has previously been referred to as "alpha-aminoadipic semialdehyde synthase," and we have tentatively designated this gene "AASS." The AASS cDNA contains an open reading frame of 2,781 bp predicted to encode a 927-amino-acid-long protein . The gene has been sequenced and contains 24 exons scattered over 68 kb and maps to chromosome 7q31.3 . Northern blot analysis revealed the presence of several transcripts in all tissues examined, with the highest expression occurring in the liver . We sequenced the genomic DNA from a single patient with hyperlysinemia (JJa) . The patient is the product of a consanguineous mating and is homozygous for an out-of-frame 9-bp deletion in exon 15, which results in a premature stop codon at position 534 of the protein . On the basis of these and other results, we propose that AASS catalyzes the first two steps of the major lysine-degradation pathway in human cells and that inactivating mutations in the AASS gene are a cause of hyperlysinemia.

Toxicol Sci, 2000 Apr, 54(2), 338 - 54
Evaluation of a Tier I screening battery for detecting endocrine-active compounds (EACs) using the positive controls testosterone, coumestrol, progesterone, and RU486; O'Connor JC et al.; After previously examining 12 compounds with known endocrine activities, we have now evaluated 4 additional compounds in a Tier I screening battery for detecting endocrine-active compounds (EACs): a weak estrogen receptor (ER) agonist (coumestrol; COUM), an androgen receptor (AR) agonist (testosterone; TEST), a progesterone receptor (PR) agonist (progesterone; PROG), and a PR antagonist (mifepristone; RU486) . The Tier I battery incorporates 2 short-term in vivo tests (5-day ovariectomized female battery; 15-day intact male battery) and an in vitro yeast transactivation system (YTS) . The Tier I battery is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptors; steroid biosynthesis inhibitors (aromatase, 5alpha-reductase, and testosterone biosynthesis); or compounds that alter thyroid function . In addition to the Tier I battery, a 15-day dietary restriction experiment was performed using male rats to assess confounding due to treatment-related decreases in body weight . In the Tier I female battery, TEST administration increased uterine weight, uterine stromal cell proliferation, and altered hormonal concentrations (increased serum testosterone {T} and prolactin {PRL}; and decreased serum FSH and LH) . In the male battery, TEST increased accessory sex gland weights, altered hormonal concentrations (increased serum T, dihydrotestosterone {DHT}, estradiol {E2}, and PRL; decreased serum FSH and LH), and produced microscopic changes of the testis (Leydig cell atrophy and spermatid retention) . In the YTS, TEST activated gene transcription in the yeast containing the AR or PR . In the female battery, COUM administration increased uterine weight, uterine stromal cell proliferation, and uterine epithelial cell height, and increased serum PRL concentrations . In the male battery, COUM altered hormonal concentrations (decreased serum T, DHT, E2; increased serum PRL) and, in the YTS, COUM activated gene transcription in the yeast containing the ER . In the female battery, PROG administration increased uterine weight, uterine stromal cell proliferation, and uterine epithelial cell height and altered hormonal concentrations (increased serum progesterone and decreased serum FSH and LH) . In the male battery, PROG decreased epididymis and accessory sex gland weights, altered hormonal concentrations (decreased serum T, PRL, FSH, and LH; increased serum progesterone and E2), and produced microscopic changes of the testis (Leydig cell atrophy) . In the YTS, PROG activated gene transcription in the yeast containing the AR or PR . In the female battery, RU486 administration increased uterine weight and decreased uterine stromal cell proliferation . In the male battery, RU486 decreased epididymis and accessory sex gland weights and increased serum FSH and LH concentrations . In the YTS, RU486 activated gene transcription in the yeast containing the ER, AR, or PR . Dietary restriction data demonstrate that confounding due to decrements in body weight are not observed when body weight decrements are 10% or less in the Tier I male battery . In addition, minimal confounding is observed at body decrements of 15% (relative liver weight, T3, and T4) . Hence, compounds can be evaluated in this Tier I at levels that produce a 10% decrease in body weight without confounding of the selected endpoints . Using the responses obtained for all the endpoints in the Tier I battery, a distinct "fingerprint" was produced for each type of endocrine activity against which compounds with unknown activity can be compared . These data demonstrate that the described Tier I battery is useful for identifying EACs and they extend the compounds evaluated to 16.

Annu Rev Med, 2000, 51, 443 - 64
Genetic disorders affecting proteins of iron metabolism: clinical implications; Sheth S et al.; Remarkable progress is being made in understanding the molecular basis of disorders of human iron metabolism . Recent work has uncovered unanticipated relationships with the immune and nervous systems, intricate interconnections with copper metabolism, and striking homologies between yeast and human genes involved in the transport of transition metals . This review examines the clinical consequences of new insights into the pathophysiology of genetic abnormalities affecting iron metabolism . The proteins recently found to be involved in the absorption, transport, utilization, and storage of iron are briefly described, and the clinical manifestations of genetic disorders that affect these proteins are discussed . This chapter considers the most common inherited disorder in individuals of European ancestry (hereditary hemochromatosis), a widespread disease in sub-Saharan populations for which the genetic basis is still uncertain (African dietary iron overload), and several less frequent or rare disorders (juvenile hemochromatosis, atransferrinemia, aceruloplasminemia, hyperferritinemia with autosomal dominant congenital cataract, Friedreich's ataxia, and X-linked sideroblastic anemia with ataxia).

Cell Death Differ, 2000 Apr, 7(4), 374 - 83
Regions essential for the interaction between Bcl-2 and SMN, the spinal muscular atrophy disease gene product; Sato K et al.; The SMN gene is implicated in spinal muscular atrophy (SMA), and its product has been shown to interact with Bcl-2 protein to enhance its anti-apoptotic activity . In this study, we determined the regions that were essential for the interaction of Bcl-2 and SMN by co-immunoprecipitation of deletion mutants . Bcl-2 lacking its amino-terminal 20 amino acid residues or its carboxyl-terminal membrane-anchoring domain showed no or greatly reduced binding with SMN, respectively . However, Bcl-2 lacking other regions could still bind to SMN . Because Bcl-2 lacking the membrane-anchoring domain could bind to SMN in a yeast two-hybrid system, the amino-terminal region of Bcl-2 seems to be the most important domain for binding with SMN . A fragment of SMN encoded by exon 6 could bind to Bcl-2, but SMN lacking this region could not . From these results, we concluded that Bcl-2 and SMN proteins bound with each other at the amino-terminal region near the BH4 domain of Bcl-2 and the region encoded by exon 6 of SMN, both regions known to be important for their function.

Cytogenet Cell Genet, 2000, 88(1-2), 163 - 7
Mouse linkage cytogenetics (L-C) probes; Liechty MC et al.; We have identified 149 hybridization probes at 10-cM intervals in the mouse and have confirmed their order and linkage by fluorescence in situ hybridization . These probes represent a new resource for mapping in the mouse and can be used to correlate linkage and cytogenetic maps, to map novel sequences to within a few centimorgans, to relate cytogenetic abnormalities to the genetic map, and to make cross-species comparisons .

Cytogenet Cell Genet, 2000, 88(1-2), 159 - 62
Characterization of a new TSPY gene family member in Yq (TSPYq1); Ratti A et al.; We investigated subinterval 6E on the human Y chromosome, a region frequently deleted in infertile males . YAC yOX17, mapped within subinterval 6E by STS-PCR, was analyzed for the presence of new genes . TSPYq1, a member of the TSPY multi-copy gene family, was isolated and characterized from a yOX17 cosmid subclone . PCR and FISH analysis performed on normal subjects and on patients with microdeletions of Yq suggested the presence of multiple copies of TSPY in Yq .

Cytogenet Cell Genet, 2000, 88(1-2), 82 - 6
Eukaryotic translation termination factor gene (ETF1/eRF1) maps at D5S500 in a commonly deleted region of chromosome 5q31 in malignant myeloid diseases; Guenet L et al.; The human genome contains four ETF1 (eukaryotic translation termination factor 1) homologous sequences, localized on chromosomes 5, 6, 7 and X, and corresponding to a functional gene on chromosome 5 and three processed pseudogenes on the other chromosomes . ETF1 genomic or cDNA probes were mapped by fluorescence in situ hybridization to 5q31, 6p21, 7q11 and Xp11.4-->p11.1 . A microsatellite marker (D5S500) was identified in intron 7 of the functional ETF1 gene providing its exact position in the 5q31 band . Thus, the ETF1 gene is located in a 5q region which contains unidentified genes responsible for genetic or malignant disorders, and it might be considered as a candidate gene involved in the pathogenesis of these diseases .

Cytogenet Cell Genet, 2000, 88(1-2), 62 - 7
HMG20A and HMG20B map to human chromosomes 15q24 and 19p13.3 and constitute a distinct class of HMG-box genes with ubiquitous expression; Sumoy L et al.; The HMG box encodes a conserved DNA binding domain found in many proteins and is involved in the regulation of transcription and chromatin conformation . We describe HMG20A and HMG20B, two novel human HMG box-containing genes, discovered within the EURO-IMAGE Consortium full-length cDNA sequencing initiative . The predicted proteins encoded by these two genes are 48.4% identical (73.9% within the HMG domain) . The HMG domain of both HMG20 proteins is most similar to that of yeast NHP6A (38% to 42%) . Outside of this domain, HMG20 proteins lack any significant homology to other known proteins . We determined the genomic structure and expression pattern of HMG20A and HMG20B . Both genes have several alternative transcripts, expressed almost ubiquitously . HMG20A maps to chromosome 15q24 (near D15S1227) and HMG20B to 19p13.3 (between D19S209 and D19S216) . The HMG20 genes define a distinct class of mammalian HMG box genes .

Cytogenet Cell Genet, 2000, 88(1-2), 35 - 7
Cloning and mapping of murine superoxide dismutase copper chaperone (Ccsd) and mapping of the human ortholog; Moore SD et al.; Copper does not exist in a free state within cells but is found consistently bound to metalloproteins . Specific metallochaperones escort copper to numerous targets within the cell, providing protection from the toxic effects of intracellular free copper . Many metallochaperones have been characterized in yeast, mouse, and human . To further characterize mouse metallochaperones, we cloned murine Ccsd from an adult mouse cDNA brain library, including both the coding region and the 5' and 3' UTRs . We obtained a 1,174-bp cDNA with an 825-bp open reading frame, translating a 274 amino acid protein that is 86.9% identical to human CCS . Using a mouse x hamster radiation hybrid panel, we mapped Ccsd to a proximal position on mouse chromosome 19 . We mapped human CCS to 11q13 (homologous with mouse chromosome 19), utilizing a human x hamster radiation hybrid panel . The human and mouse metallochaperones are ubiquitously expressed in the major tissues of the body but seem to have different transcription products .

Gene, 2000 Apr 18, 247(1-2), 241 - 53
Structure and chromosomal localization of the human and mouse muscle fructose-1,6-bisphosphatase genes; Tillmann H et al.; Mammalian skeletal muscle contains fructose-1,6-bisphosphatase (Fru-1,6-P(2)ase), a key enzyme of glyconeogenesis . We have shown previously that muscle Fru-1,6-P(2)ase is encoded by a gene different from that coding for the liver isoenzyme . Starting with genomic YAC libraries and based on the cDNA sequences of human and mouse muscle Fru-1,6-P(2)ases together with the known gene structures of two mammalian liver fructose-1,6-bisphosphatases, we have PCR-amplified and sequenced all functional parts of the human and mouse muscle fructose-1,6-bisphosphatase genes and determined their chromosomal localization . The human gene (FBP2), localized at chromosome 1p36.1-2, spans about 30 kb, while the mouse gene (Fbp2) at chromosome 13B3-C1 is more compact (about 21 kb) . Intron lengths are only poorly conserved between the two genes, while intron number and positions are identical in all hitherto analyzed mammalian fructose-1,6-bisphosphatase isoenzyme genes . Transcriptional start sites were found to be located 97 and 95bp before the start codon in the human gene and 35 bp before the start codon in the mouse homolog . A comparison of the 5'-flanking sequences of the two genes revealed a 56% homology up to human bp -607 before the first transcriptional start point, while upstream of this region we found no similarity . The data presented in this paper provide a basis for further studies of the mechanism of expression regulation and the elucidation of the physiological role of the enzyme.

Gene, 2000 Apr 18, 247(1-2), 175 - 80
A satellite DNA containing CENP-B box-like motifs is present in the antarctic scallop Adamussium colbecki; Canapa A et al.; The DNA of the Antarctic scallop Adamussium colbecki was found to contain a highly repeated sequence identifiable upon restriction with endonuclease BglII . The monomeric unit - denominated pACS (about 170bp long) - was cloned . Southern blot hybridization yielded a ladder-like banding pattern, indicating that the repeated elements are tandemly arranged in the genome and therefore represent a sequence of satellite DNA.Sequence analysis of five different clones revealed the presence of various subfamilies, some of which showed a high degree of divergence . In each clone, regions homologous to the mammalian CENP-B box were observed . A region homologous to the CDEIII centromeric sequence of yeast was also found in one of the clones . These observations suggest a relationship of the pACS family to the centromeric area in A . colbecki.

Cancer Lett, 2000 May 1, 152(2), 193 - 9
Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines; Myrie KA et al.; Genetic instability is a hallmark feature of breast, colorectal and other types of cancers . One type characterized by chromosomal instability is thought to be important in the pathogenesis of many solid tumors displaying aneuploidy . Two related protein kinases and homologues of the yeast checkpoint genes, hBUB1 and hBUB1B, have been implicated in the pathogenesis of colorectal cancers . Mutations in hBUB1 have demonstrated a dominant negative effect by disrupting the mitotic checkpoint when transfected into euploid colon cancer cell lines . In Brca2 deficient murine cells, Bub1 mutants potentiate growth and cellular transformation . This would suggest that aneuploidy in solid tumors including breast, could be the result of defects in mitotic checkpoint genes and may be responsible for a chromosomal instability phenotype contributing to tumor progression . We conducted mutational analysis of 19 aneuploid breast cancer cell lines . No mutations were found but we identified nine sequence variations including five previously unreported sequence variants in hBUB1B, two of which affect restrictions sites . None of these nucleotide changes predict significant changes in the predicted protein structure . Expression analysis by Northern blot of breast cell lines showed variable expression of hBUB1 and hBUB1B genes . This suggest that while regulation of expression of these genes may be important in cancer, the lack of putative deleterious mutations in the coding sequence does not support a frequent role for mutant hBUB1 and hBUB1B alleles in the pathogenesis of breast cancer.

Biochem Biophys Res Commun, 2000 Apr 21, 270(3), 868 - 79
Characterization of Bax-sigma, a cell death-inducing isoform of Bax; Schmitt E et al.; The Ced-9/Bcl-like family of genes codes for proteins that have antiapoptotic and proapoptotic activity . Several Bax isoproteins have been detected by 2-D gel electrophoresis, and a novel human member, designated as Bax-sigma, has been identified and cloned from human cancer promyelocytic cells . Bax-sigma contains BH-3, BH-1, and BH-2 domains, putative alpha-5 and alpha-6 helices, and the carboxy-terminal hydrophobic transmembrane domain but lacks amino acids 159 to 171 compared to Bax-alpha . mRNA expression analysis by reverse transcription-polymerase chain reaction and RNase protection assays have revealed that Bax-sigma is expressed in a variety of human cancer cell lines and normal tissues . To investigate the potential role of Bax-sigma in apoptosis, first its effects were compared to those of Bax-alpha by transient expression in human B lymphoma Namalwa cells . Both Bax-sigma and Bax-alpha promoted apoptosis, as detected by DNA fragmentation and morphological analysis by electron microscopy . The apoptosis induced by Bax-sigma and Bax-alpha was correlated with their expression, cytochrome c release, and caspase activation . In a yeast two-hybrid system, Bax-sigma interacted with several Ced-9/Bcl family members but had no affinity for the human Egl-1 homologs Bik and Bad and the Ced-4 homolog Apaf-1 . In human cells, Bax-sigma function was counteracted by Bcl-xL overexpression, and co-immunoprecipitation experiments indicated that Bax-sigma was associated with Bcl-xL . Furthermore, Bax-sigma overexpression increased cell death induced by various concentrations of genotoxic agents with the most pronounced effect occurring at low camptothecin and vinblastine dose levels . Our results suggest that Bax-sigma, a novel variant of Bax, encodes a protein with a proapoptotic effect and mode of action similar to those of Bax-alpha .

J Mol Biol, 2000 May 5, 298(3), 417 - 29
An algorithm for the prediction of proteasomal cleavages; Kuttler C et al.; Proteasomes, major proteolytic sites in eukaryotic cells, play an important part in major histocompatibility class I (MHC I) ligand generation and thus in the regulation of specific immune responses . Their cleavage specificity is of outstanding interest for this process.In order to generalize previously determined cleavage motifs of 20 S proteasomes, we developed network-based model proteasomes trained by an evolutionary algorithm with experimental cleavage data of yeast and human 20 S proteasomes . A window of ten flanking amino acid residues proved sufficient for the model proteasomes to reproduce the experimental results with 98-100 % accuracy . Actual experimental data were reproduced significantly better than randomly selected cleavage sites, suggesting that our model proteasomes were able to extract rules inherent to proteasomal cleavage data . The affinity parameters of the model, which decide for or against cleavage, correspond with the cleavage motifs determined experimentally . The predictive power of the model was verified for unknown (to the program) test conditions: the prediction of cleavage numbers in proteins and the generation of MHC I ligands from short peptides.In summary, our model proteasomes reproduce and predict proteasomal cleavages with high degree of accuracy . They present a promising approach for predicting proteasomal cleavage products in future attempts and, in combination with existing algorithms for MHC I ligand prediction, will be tested to improve cytotoxic T lymphocyte epitope prediction .

J Mol Biol, 2000 May 5, 298(3), 395 - 405
Protein-protein interaction among hnRNPs shuttling between nucleus and cytoplasm; Kim JH et al.; Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in several RNA-related biological processes such as transcription, pre-mRNA processing, mature mRNA transport to the cytoplasm, and translation . About 20 major hnRNPs from A1 to U are known . Among them, hnRNP A, D, E, I, and K are known to shuttle between the nucleus and the cytoplasm . hnRNP E2 has been seen to stabilize alpha-globin mRNA and to enhance polioviral mRNA translation . hnRNP K modulates transcription and translation of some mRNAs . hnRNP I and its homologue hnRNP L have been suggested to enhance translation of some IRES-dependent mRNAs . In order to better understand the molecular mechanisms of the biological functions of hnRNPs, we investigated protein-protein interactions of six hnRNPs (hnRNP A1, C1, E2, I, K, and L) using the yeast two-hybrid system and in vitro co-precipitation assays . All of the hnRNPs tested exerted homomeric interactions, and hnRNP E2, I, K, and L interacted with each other . In the case of hnRNP E2 and hnRNP K, the N-terminal half of the proteins containing two KH (K homologous) domains were required for protein-protein interaction, and the second quarter of hnRNP I and hnRNP L containing RRM2 (RNA recognition motif 2) was essential for protein-protein interaction . hnRNP A1 and C1 did not form complexes with other hnRNPs in our assay systems . This suggests that the hnRNPs could fall into two groups: one group, including hnRNP A1 and C1, involved in hnRNP core complex formation and another group, including hnRNP E2, I, K, and L, involved in a variety of RNA-related biological processes . Different combinations of the proteins of the second group may facilitate different biological processes in conjunction with other factors .

Exp Cell Res, 2000 May 1, 256(2), 545 - 54
Retinoic acid and its receptors repress the expression and transactivation functions of Nur77: a possible mechanism for the inhibition of apoptosis by retinoic acid; Kang HJ et al.; Nur77 (NGFI-B) is an orphan nuclear receptor that has been implicated in activation-induced T-cell apoptosis . Retinoids, potent immune modulators, were shown to inhibit the activation-induced apoptosis of immature thymocytes and T-cell hybridomas . To illustrate the mechanism of the inhibition, we examined the effects of retinoic acid (RA) on the expression and transactivation functions of Nur77 in the human peripheral blood mononuclear cells and the human T-cell leukemia, Jurkat . All-trans-RA remarkably repressed the DNA binding and transcriptional induction of Nur77 . Among the two potential trans-acting factors that activate Nur77 gene promoter, i.e., AP-1 and related serum response factor (RSRF), all-trans-RA repressed DNA binding and reporter gene activity of AP-1 but not that of RSRF, suggesting that the inhibition may be mediated through AP-1 . We also demonstrated a posttranscriptional regulation of Nur77 function by retinoid receptors by showing that transactivation activity of Nur77 was significantly inhibited by cotransfection of RARalpha or RXRalpha . Nur77 bound RARalpha or RXRalpha in both yeast and mammalian two-hybrid tests, suggesting that direct protein-protein interaction between these receptors may mediate the inhibition . Taken all together, we demonstrated that RA repressed Nur77 function through multiple mechanisms that may provide the basis for RA inhibition on the apoptosis of activated T-lymphocytes .

Exp Cell Res, 2000 May 1, 256(2), 515 - 21
B2-1, a Sec7- and pleckstrin homology domain-containing protein, localizes to the Golgi complex; Lee SY et al.; B2-1 is a human protein that contains both a Sec7 and a pleckstrin homology domain . The yeast Sec7 protein was previously shown to be involved in vesicle formation in the Golgi and endoplasmic reticulum . Recently, several groups have shown that B2-1 and highly similar proteins (e.g., ARNO, ARNO3) have varied cellular functions and subcellular locations . One of these is an association of the B2-1 Sec7 domain with the plasma membrane, binding to the cytoplasmic portion of the integrin beta2 chain (CD18) and is postulated to be involved in inside-out signaling . Other groups have shown that B2-1 and these related proteins are guanine nucleotide-exchange factors that act upon ADP ribosylation factors (ARFs) and are localized to the Golgi or plasma membrane . Here we report the subcellular localization of B2-1 protein . Interestingly, B2-1 does not localize to the plasma membrane but rather associates with a distinct Golgi complex compartment . B2-1's distribution can be disrupted by brefeldin A, a drug that rapidly disrupts the Golgi apparatus by inhibiting ARF activity . Furthermore, transient transfection of GFP-tagged B2-1 shows Golgi complex targeting . Excessive overexpression of transfected B2-1 causes partial Golgi dispersion .

Dev Biol, 2000 May 1, 221(1), 87 - 100
A sequence-specific RNA binding complex expressed in murine germ cells contains MSY2 and MSY4; Davies HG et al.; The protamine mRNAs are stored for up to 8 days as translationally repressed ribonucleoprotein particles during murine spermatogenesis . Translational repression of the protamine 1, Prm1, mRNA is controlled by sequences in its 3'-untranslated region (UTR) . In this study we used the yeast three-hybrid system to clone Msy4, which encodes a novel member of the Y box family of nucleic acid binding proteins . MSY4 specifically binds to a site within the 5' most 37 nucleotides in the Prm1 3' UTR . Msy4 is highly expressed in the testis, and the protein is detected in the cytoplasm of germ cells in both the testis and the ovary, where repressed messages are stored . Analysis of a previously described 48/50-kDa binding activity in testis extracts by electrophoretic mobility shift assays and immunoprecipitation indicates the activity is composed of MSY4 and MSY2, another mouse Y box protein . Polysome analysis demonstrates MSY4 is associated with mRNPs, consistent with MSY4 having a role in storing repressed messages .

Dev Biol, 2000 May 1, 221(1), 23 - 40
The L63 gene is necessary for the ecdysone-induced 63E late puff and encodes CDK proteins required for Drosophila development; Stowers RS et al.; The pulse of ecdysone that triggers Drosophila metamorphosis activates six early genes in a primary response made visible by polytene chromosome puffs . The secondary response is detected by the induction of over 100 late puffs, only a few of which have been subject to molecular genetic analysis . We present a molecular and mutational analysis of the L63 gene responsible for the late puff at 63E . This gene contains overlapping L63A, B, and C transcription units of which the A unit encodes two isoforms and the B unit three . The C unit, which exhibits little activity, encodes one of the B isoforms . Evidence that L63B, but not L63A, transcription is ecdysone responsive derives from their developmental transcription profiles and from P-element mutagenesis showing that ecdysone induction of the 63E puff requires sequences adjacent to the 5' end of L63B but not those adjacent to the 5' end of L63A . L63-specific lethal mutations showed that L63 is required not only for metamorphosis, but also maternally and for embryonic and larval development . The L63 proteins contain a common C-terminal 294-aa sequence that is 71% identical to the CDK sequence of the murine PFTAIRE protein . In vivo tests of L63 proteins altered by site-directed mutagenesis showed that they exhibit CDK functions . L63 proteins are widely distributed among late larval and prepupal tissues and are unlikely to be involved in cell cycle functions .

Appl Microbiol Biotechnol, 2000 Mar, 53(3), 278 - 81
The influence of nitrogen sources on the alpha-amylase productivity of Aspergillus oryzae in continuous cultures; Pedersen H et al.; The influence of the nitrogen source on the alpha-amylase productivity of Aspergillus oryzae was quantified in continuous cultivations . Both inorganic and complex nitrogen sources were investigated and glucose was used as the carbon and energy sources . For production of alpha-amylase, nitrate was shown to be inferior to ammonia as a nitrogen source . A mixture of ammonia and complex nitrogen sources, such as yeast extract or casein hydrolysate, was better than with ammonia as the sole nitrogen source . Even a low concentration of casein hydrolysate (0.05 g l(-1)) resulted in a 35% increase in the alpha-amylase productivity . The higher alpha-amylase productivity during growth on casein hydrolysate was not caused by increased transcription of the alpha-amylase genes but was caused by a faster secretion of alpha-amylase or by a lower binding of alpha-amylase to the biomass.

IUBMB Life, 2000 Jan, 49(1), 63 - 70
Specific sequence of motifs of mitochondrial uncoupling proteins; Jezek P et al.; We have searched for the exclusivity of common sequence motifs of the mitochondrial uncoupling proteins (UCP1, UCP2, UCP3, UCP4, BMCP1, and plant UCP {PUMP}) within the gene family of mitochondrial anion carrier proteins . The UCP-specific sequences, "UCP signatures", were found in the first, second, and fourth alpha-helices . First: Ala/Ser-Cys/Thr/n-n/Phe-Ala/Gly-{negatively charged residue}-n/Phe-n/Cys-Thr-Phe/n; second: Gly/Ala-Ile/Leu-Gln/X-{positively charged residue}-NH-n/Cys-Ser/nphi/X-n/Ser-OH/Gly-n-{positively charged residue}-Ile/Met-Gly/Val-n/Thr; fourth: Pro-Asn/ Thr-n-X-{positively charged residue}-Asn/Ser/Ala-n-n-Ile/Leu-n-Asn/Val-Cys/n-n/Thr-{negatively charged residue}-n-n/Thr/Pro-OH/Val (n, nonpolar; phi, aromatic; (positively charged residue/negatively charged residue, charged residue) . The second and part of the third signature are also present in the yeast dicarboxylate transporter . The UCP signature excluding BMCP1 was also found in the second matrix segment: {positively charged residue}-(Pro/ del-Leu/del)-{positively charged residue}-phi-X-Gly/Ser-Thr/n-X-NH/{negatively charged residue}-Ala-phi . These UCP signatures are thought to be involved in fatty acid anion binding and translocation.

J Cell Biochem, 2000 Apr, 77(4), 596 - 603
Characterization of a murine gene encoding an acidic-basic dipeptide repeat that interacts with GADD34; Hasegawa T et al.; GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest . To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34 . We utilized as bait the partial product of GADD34 cDNA including the PEST region and the gamma(1)34.5 . One cDNA clone was almost the same as MuRED, which encodes an acidic-basic dipeptide repeat; we named it G34BP . The interaction between GADD34 and G34BP was also confirmed in the NIH3T3 cells by in vivo two-hybrid analysis . For the binding of two proteins, the PEST region was important, and the C-terminal of G34BP was necessary . G34BP was detected in all the mouse tissues examined . Although GADD34 was significantly elevated with methyl methanesulfonate treatment, G34BP expression was not induced . Overexpression of G34BP in the NIH3T3 cells inhibited the cell growth analyzed by WST1 assay .

J Invest Dermatol, 2000 May, 114(5), 1050 - 6
A novel mouse gene, Sh3yl1, is expressed in the anagen hair follicle; Aoki N et al.; In an attempt to investigate the genes expressed in the anagen hair follicle, we differentially screened a mouse anagen skin cDNA library, and identified a cDNA encoding a novel protein containing one Src homology 3 domain at the carboxyl terminus . The predicted amino acid sequence revealed a significant homology with YHRO 16c/Ysc 84, the yeast Src homology 3 domain-containing protein, for which transcripts are expressed at high levels during meiosis . The sequence identity was remarkable at the amino terminus as well as the carboxyl-terminal Src homology 3 domain, suggesting that the novel protein is a mouse homolog of the yeast protein, and we have termed this protein Sh3yl1 . In northern blot study, the transcripts were detected not only in the skin but also in other tissues, especially the kidney, stomach, small intestine, and colon . Furthermore, in mouse skin, the expression of these transcripts basically followed the hair-growth cycle, increasing significantly during mid and late anagen phases, and decreasing during catagen, telogen, and early anagen phases . An in situ hybridization experiment showed that the Sh3yl1 transcripts are expressed predominantly in the hair bulb, the hair shaft, inner root sheath, and outer root sheath in the lower half of the follicle during mid and late anagen phases . These transcripts were not detected in catagen, telogen, and early anagen hair follicles, or in other skin components . Thus, these data suggest the possible involvement of Sh3yl1 in the development of hair follicles during the anagen phase.

Biochim Biophys Acta, 2000 Apr 17, 1496(2-3), 356 - 61
Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca(2+)-binding proteins in Dictyostelium discoideum; Dorywalska M et al.; During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca(2+)-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca(2+)-binding protein (termed CBP4a) . The protein is composed of 162 amino acids and contains four consensus EF-hands . PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca(2+)-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a . Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome . CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA . In addition, both novel proteins bind (45)Ca(2+) and interact with CBP1 in vitro in a Ca(2+)-dependent manner.

J Cell Sci, 2000 May, 113 ( Pt 10), 1651 - 9
The nuclear pore complex: mediator of translocation between nucleus and cytoplasm; Allen TD et al.; The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm . These sites, termed nuclear pore complexes (NPCs), number 100-200 in yeast, a few thousand in mammalian cells and approximately 50 million in the giant nuclei of amphibian oocytes . NPCs are large (125 MDa) macromolecular complexes that comprise 50-100 different proteins in vertebrates . In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division . Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass . Each pore can facilitate both import and export . The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms . Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export . The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.

Biochem J, 2000 May 1, 347 Pt 3, 741 - 7
Identification of the catalytic residues of the first family of beta(1-3)glucanosyltransferases identified in fungi; Mouyna I et al.; A new family of glycosylphosphatidylinositol-anchored beta(1-3)glucanosyltransferases (Gelp), recently identified and characterized in the filamentous fungus Aspergillus fumigatus, showed functional similarity to the Gas/Phr/Epd protein families, which are involved in yeast morphogenesis . Sequence comparisons and hydrophobic cluster analysis (HCA) showed that all the Gas/Phr/Epd/Gel proteins belong to a new family of glycosylhydrolases, family 72 . We confirmed by site-directed mutagenesis and biochemical analysis that the two conserved glutamate residues (the putative catalytic residues of this family, as determined by HCA) are involved in the active site of this family of glycosylhydrolases.

Biochem J, 2000 May 1, 347 Pt 3, 613 - 21
Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin; Oleinikov AV et al.; gp600/megalin, an endocytic receptor, belongs to the low-density lipoprotein receptor family . It is most abundant in the renal proximal tubular cells, where it is implicated in the reabsorption of a number of molecules filtered through the glomerulus . The cytoplasmic tail (CT) of gp600/megalin contains a number of sequence similarities, which indicate that gp600/megalin might be involved in signal transduction . To find intracellular proteins that would interact with the gp600/megalin CT, a human kidney cDNA library was screened by using the yeast two-hybrid system . The phosphotyrosine interaction domain (PID) of the Disabled protein 2 (Dab2), a mammalian structural analogue of Drosophila Disabled, was found to bind to the gp600/megalin CT in this system . The interaction between these two proteins was confirmed by a binding assay in vitro and by the co-immunoprecipitation of both proteins from renal cell lysates . The gp600/megalin CT contains three PsiXNPXY motifs (in which Psi represents a hydrophobic residue) that are potentially able to interact with PID . Analysis of the CT deletion and point-mutation variants of gp600/megalin by the two-hybrid system revealed that the third PsiXNPXY motif is most probably involved in this interaction . Dab2 is a mitogen-responsive phosphoprotein thought to be an adaptor molecule involved in signal transduction, and a suggested negative regulator of cell growth . Dab2 is the first intracellular ligand identified for gp600/megalin; gp600/megalin is the first known transmembrane receptor that interacts with the cytosolic protein Dab2 . We speculate that their interaction might involve gp600/megalin in signal transduction pathways or might mediate the intracellular trafficking of this receptor.

Biochemistry, 2000 Apr 25, 39(16), 4575 - 80
Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase eta; Vaisman A et al.; Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication . Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells . Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay . We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts . Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers . In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta . Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts . However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases . We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.

AIDS, 2000 Mar 10, 14(4), 387 - 95
Phase III study of granulocyte-macrophage colony-stimulating factor in advanced HIV disease: effect on infections, CD4 cell counts and HIV suppression . Leukine/HIV Study Group; Angel JB et al.; OBJECTIVE: To evaluate the effect of adjuvant granulocyte-macrophage colony-stimulating factor (GM-CSF) (sargramostim, yeast-derived recombinant human GM-CSF) on incidence and time to opportunistic infection or death, plasma HIV-RNA, and CD4 cell count in patients with advanced HIV disease . METHODS: This Phase III randomized, double-blind, placebo-controlled trial enrolled subjects with CD4 cell counts < or = 50 x 10(6)/l or < or = 100 x 10(6)/l with a prior AIDS-defining illness on stable antiretroviral therapy . Subjects were stratified by baseline HIV-RNA level (> or = or < 30,000 copies/ml) and randomized to receive subcutaneous injections of GM-CSF 250 microg or placebo three times per week for 24 weeks . Subjects were permitted to continue on blinded drug for up to 20 months . Subjects were evaluated for infections, plasma HIV-RNA, lymphocyte counts, changes in antiretroviral therapy, toxicity, and survival . RESULTS: Three-hundred and nine subjects received at least one dose of study drug, 70% completed 24 weeks of therapy . Groups were well matched at baseline . Significant increases in CD4 cell and neutrophil counts were observed at 1, 3, and 6 months in the GM-CSF group . GM-CSF significantly reduced the incidence of overall infections (78% placebo versus 67% GM-CSF; P = 0.03) and delayed time to first infection (56 days placebo versus 97 days GM-CSF; P = 0.04) . No statistical difference in cumulative opportunistic infections was observed between groups; however, among subjects without an opportunistic infection prior to study, the GM-CSF group demonstrated a trend towards fewer subjects with an opportunistic infection on study (26% placebo versus 8% GM-CSF; P = 0.08) . Change in HIV-RNA was not significantly different between groups, but significantly fewer GM-CSF subjects with baseline viral load < 30,000 copies/ml had changes in antiretroviral therapy for increased viral load (42% placebo versus 21% GM-CSF; P = 0.01) . In patients with HIV-RNA levels below the limit of detection at baseline, more GM-CSF patients maintained an undetectable viral load at 24 weeks (54% placebo versus 83% GM-CSF; P = 0.02) . GM-CSF was well tolerated . CONCLUSIONS: GM-CSF significantly increased CD4 cell count and decreased virological breakthrough and overall infection rate in subjects with advanced HIV disease.

J Am Acad Dermatol, 2000 May, 42(5 Pt 2), 929 - 31
Trichosporon cutaneum (Trichosporon asahii) infection mimicking hand eczema in a patient with leukemia; Nakagawa T et al.; Trichosporon Cutaneum is a yeast-like fungus that causes white piedra and onychomycosis . Recently, it has also been recognized as an opportunistic pathogen in immunocompromised hosts . We describe a 64-year-old woman who developed a superficial Trichosporon infection mimicking hand eczema during chemotherapy for her chronic myelocytic leukemia . To our knowledge, no cases of superficial infection like this one have previously been reported . This case suggests that careful examination is required in diagnosing Trichosporon infection in immunocompromised hosts, because the infection can be invasive or unusual in appearance.

Hum Mol Genet, 2000 May 1, 9(8), 1219 - 26
Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation; Cossee M et al.; Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is caused in almost all cases by homozygous intronic expansions resulting in the loss of frataxin, a mitochondrial protein conserved through evolution, and involved in mitochondrial iron homeostasis . Yeast knockout models, and histological and biochemical data from patient heart biopsies or autopsies indicate that the frataxin defect causes a specific iron-sulfur protein deficiency and mitochondrial iron accumulation leading to the pathological changes . Affected human tissues are rarely available to further examine this hypothesis . To study the mechanism of the disease, we generated a mouse model by deletion of exon 4 leading to inactivation of the Frda gene product . We show that homozygous deletions cause embryonic lethality a few days after implantation, demonstrating an important role for frataxin during early development . These results suggest that the milder phenotype in humans is due to residual frataxin expression associated with the expansion mutations . Surprisingly, in the frataxin knockout mouse, no iron accumulation was observed during embryonic resorption, suggesting that cell death could be due to a mechanism independent of iron accumulation.

Hum Mol Genet, 2000 May 1, 9(8), 1201 - 8
Co-localisation of CCG repeats and chromosome deletion breakpoints in Jacobsen syndrome: evidence for a common mechanism of chromosome breakage; Jones C et al.; Folate-sensitive fragile sites are associated with the expansion and hypermethylation of CCG-repeats . The fragile site in 11q23.3, FRA11B, has been shown to cause chromosome deletions in vivo, its expression being associated with Jacobsen (11q-) syndrome . However, the majority of Jacobsen deletions are distal to FRA11B and are not related to its expression . To test the hypothesis that other unidentified fragile sites might be located in 11q23.3-24 and may cause these deletions, we have identified and characterised CCG-trinucleotide repeats within a 40 Mb YAC contig spanning distal chromosome 11q . Only eight CCG-repeats were identified within the entire YAC contig (not including FRA11B ), six of which map to the region of 11q23.3-24 that includes Jacobsen deletions . We have previously collated the deletion mapping data of 24 Jacobsen patients with the physical map of chromosome 11q, and accurately localised six breakpoints to short intervals corresponding to individual YAC clones . We now show that in each of these cases, YAC clones found to contain a deletion breakpoint also contain a CCG-repeat . The improved analysis of one of these deletions, together with those of several new Jacobsen cases, further strengthens this association by localising five breakpoints to individual PAC clones containing CCG-repeats . These data provide strong evidence for the non-random clustering of chromosome deletion breakpoints with CCG-repeats, and suggests that they may play an important role in a common mechanism of chromosome breakage.

Hum Mol Genet, 2000 May 1, 9(8), 1145 - 59
(Over)correction of FMR1 deficiency with YAC transgenics: behavioral and physical features; Peier AM et al.; Fragile X syndrome is a common cause of mental retardation involving loss of expression of the FMR1 gene . The role of FMR1 remains undetermined but the protein appears to be involved in RNA metabolism . Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macroorchidism and behavioral abnormalities . As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued . Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence . We observed that the YAC transgene supported production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner . Macro-orchidism was absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein . Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, we performed a more thorough evaluation of the Fmr1 knockout phenotype using additional behavioral assays that had not previously been reported for this animal model . The mouse displayed reduced anxiety-related responses with increased exploratory behavior . FMR1 YAC transgenic mice overexpressing the human protein did produce opposing behavioral responses and additional abnormal behaviors were also observed . These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype.

Hum Mol Genet, 2000 Apr 12, 9(7), 1131 - 40
Hailey-Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca(2+) pump; Sudbrak R et al.; Hailey-Hailey disease (HHD) is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis . Previous genetic linkage studies localized the gene to a 5 cM interval on human chromosome 3q21 . After reducing the disease critical region to <1 cM, we used a positional cloning strategy to identify the gene ATP2C1, which is mutated in HHD . ATP2C1 encodes a new class of P-type Ca(2+)-transport ATPase, which is the homologue for the rat SPLA and the yeast PMR1 medial Golgi Ca(2+)pumps and is related to the sarco(endo)plasmic calcium ATPase (SERCA) and plasma membrane calcium ATPase (PCMA) families of Ca(2+)pumps . The predicted protein has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases . ATP2C1 produces two alternative splice variants of approximately 4.5 kb encoding predicted proteins of 903 and 923 amino acids . We identified 13 different mutations, including nonsense, frameshift insertion and deletions, splice-site mutations, and non-conservative missense mutations . This study demonstrates that defects in ATP2C1 cause HHD and together with the recent identification of ATP2A2 as the defective gene in Darier's disease, provide further evidence of the critical role of Ca(2+)signaling in maintaining epidermal integrity.

Hum Mol Genet, 2000 Apr 12, 9(6), 887 - 92
Recent advances in the molecular pathogenesis of Friedreich ataxia; Puccio H et al.; Friedreich ataxia, the most frequent cause of recessive ataxia, is due in most cases to a homozygous intronic expansion resulting in the loss of function of frataxin . Frataxin is a mitochondrial protein conserved through evolution . Yeast knock-out models and histological data from patient heart autopsies have shown that frataxin defect causes mitochondrial iron accumulation . Biochemical data from patient heart biopsies or autopsies have revealed a specific deficiency in the activities of aconitases and of mitochondrial iron-sulfur proteins . These results suggest that frataxin may play a role either in mitochondrial iron transport or in iron-sulfur cluster assembly or transport . Iron abnormalities suggest a pathogenic mechanism involving free radical production and oxidative stress, a process that might be sensitive to antioxidant therapies.

Mol Genet Metab, 2000 Mar, 69(3), 223 - 32
Screening human EST database for identification of candidate genes in respiratory chain deficiency; Rotig A et al.; Disorders of mitochondrial oxidative phosphorylation (OXPHOS) are now recognized as major causes of human metabolic diseases and several mutations of mitochondrial and nuclear genes encoding respiratory chain components have been reported . Interestingly, mutations of nuclear genes involved in mitochondrial respiratory chain assembly, protein trafficking, and iron metabolism are also known to alter oxidative phosphorylation . While several hundred of these genes have been described in yeast, only a few nuclear genes have been hitherto identified in humans . Yeast gene databases present therefore an invaluable tool for identification of human homologues that should be regarded as candidate genes in OXPHOS diseases . In an attempt to identify the human counterparts of yeast genes, we developed a systematic comparison of yeast protein sequences to the GenBank dbEST database . Starting from 340 yeast protein sequences as templates, we searched the human dbEST counterparts using the BLAST similarity searching program and identified 102 groups of human EST likely to represent orthologues of yeast genes because of significant homology . This collection of human genes possibly related to mitochondrial OXPHOS may help identify nuclear genes responsible of mitochondrial disorders .

Biopolymers, 2000, 57(2), 77 - 84
Circular dichroism and resonance raman comparative studies of wild type cytochrome c and F82H mutant; Zheng J et al.; The UV-visible, circular dichroism (CD), and resonance Raman (RR) spectra of the wild type yeast iso-1-cytochrome c (WT) and its mutant F82H in which phenylalanine-82 (Phe-82) is substituted with His are measured and compared for oxidized and reduced forms . The CD spectra in the intrinsic and Soret spectral region, as well as RR spectra in high, middle, and low frequency regions, are discussed . From the analysis of the spectra, it is determined that in the oxidized F82H the two axial ligands to the heme iron are His-18 and His-82 whereas in the reduced form the sixth ligand switches from His-82 to Met-80 providing the coordination geometry similar to that of WT . Based on the spectroscopic data, the conclusion is that the porphyrin macrocycle is less distorted in the oxidized F82H compared to the oxidized WT . Similar distortions are present in the reduced form of the proteins . Frequency shifts of Raman bands, as well as the decrease of the alpha-helix content in the CD spectra, indicate more open conformation of the protein around the heme.

J Biol Chem, 2000 Apr 21, 275(16), 12313 - 20
Antagonism between members of the CNC-bZIP family and the immediate-early protein IE2 of human cytomegalovirus; Huang CF et al.; The HCMV IE2 protein negatively autoregulates its own expression as well as represses the transactivation activity of p53 . Using the repression domain of IE2 as bait in the yeast two-hybrid system, Nrf1 and Nrf2, members of the CNC-bZIP family, were found to be IE2-interacting proteins . Residues 331-448 encompassing the DNA-binding and the dimerization domains of Nrf1 are sufficient for the interaction . The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation . In transient transfection studies, transcription driven by six copies of an NF-E2 site or by chimeric proteins between the DNA-binding domain of LexA and members of the CNC-bZIP family is repressed by IE2 . Importantly, the DNA binding activity of the Nrf1/MafK heterodimer is not impeded by IE2 . In a parallel study, CNC-bZIP factors attenuate the negative autoregulation of IE2 . The attenuation could be explained by the finding that Nrf1 functions alone and synergistically with its heterodimerization partner, MafK, in inhibiting the DNA binding activity of IE2 . Taken together, these results demonstrate the existence of antagonism between members of the CNC-bZIP family and IE2.

J Biol Chem, 2000 Apr 21, 275(16), 12108 - 18
Differential role of hepatocyte nuclear factor-1 in the regulation of glucose-6-phosphatase catalytic subunit gene transcription by cAMP in liver- and kidney-derived cell lines; Streeper RS et al.; In liver and kidney, the terminal step in gluconeogenesis is catalyzed by glucose-6-phosphatase . To examine the effect of the cAMP signal transduction pathway on transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into either the liver-derived HepG2 or kidney-derived LLC-PK cell line . Co-transfection of an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA) markedly stimulated G6Pase-CAT fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple regions are required for this PKA response in both the HepG2 and LLC-PK cell lines . A sequence in the G6Pase promoter that resembles a cAMP response element is required for the full PKA response in both HepG2 and LLC-PK cells . However, in LLC-PK cells, but not in HepG2 cells, a hepatocyte nuclear factor-1 (HNF-1) binding site was critical for the full induction of G6Pase-CAT expression by PKA . Changing this HNF-1 motif to that for the yeast transcription factor GAL4 reduces the PKA response in LLC-PK cells to the same degree as deleting the HNF-1 site . However, co-transfection of this mutated construct with chimeric proteins comprising the GAL4-DNA binding domain ligated to the coding sequence for HNF-1alpha, HNF-1beta, HNF-3, or HNF-4 completely restored the PKA response . Thus, we hypothesize that, in LLC-PK cells, HNF-1 is acting as an accessory factor to enhance PKA signaling through the cAMP response element by altering G6Pase promoter conformation or accessibility rather than specifically affecting some component of the PKA signal transduction pathway.

J Biol Chem, 2000 Apr 21, 275(16), 11545 - 52
Roles of the histone H2A-H2B dimers and the (H3-H4)(2) tetramer in nucleosome remodeling by the SWI-SNF complex; Boyer LA et al.; SWI-SNF is an ATP-dependent chromatin remodeling complex required for expression of a number of yeast genes . Previous studies have suggested that SWI-SNF action may remove or rearrange the histone H2A-H2B dimers or induce a novel alteration in the histone octamer . Here, we have directly tested these and other models by quantifying the remodeling activity of SWI-SNF on arrays of (H3-H4)(2) tetramers, on nucleosomal arrays reconstituted with disulfide-linked histone H3, and on arrays reconstituted with histone H3 derivatives site-specifically modified at residue 110 with the fluorescent probe acetylethylenediamine-(1,5)-naphthol sulfonate . We find that SWI-SNF can remodel (H3-H4)(2) tetramers, although tetramers are poor substrates for SWI-SNF remodeling compared with nucleosomal arrays . SWI-SNF can also remodel nucleosomal arrays that harbor disulfide-linked (H3-H4)(2) tetramers, indicating that SWI-SNF action does not involve an obligatory disruption of the tetramer . Finally, we find that although the fluorescence emission intensity of acetylethylenediamine-(1,5)-naphthol sulfonate-modified histone H3 is sensitive to octamer structure, SWI-SNF action does not alter fluorescence emission intensity . These data suggest that perturbation of the histone octamer is not a requirement or a consequence of ATP-dependent nucleosome remodeling by SWI-SNF.

J Biol Chem, 2000 Jun 16, 275(24), 18520 - 6
RGS4 binds to membranes through an amphipathic alpha -helix; Bernstein LS et al.; RGS4, a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S . P., Bernstein, L . S., Blumer, K . J., and Linder, M . E . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 5584-5589) . In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix . RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids . Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an alpha-helical conformation in the presence of anionic phospholipids . Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast . Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes . Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an alpha-helix as measured by circular dichroism spectroscopy . These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.

J Biol Chem, 2000 Jun 9, 275(23), 17566 - 70
NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR; Mukai J et al.; The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins . To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system . We identified one positive clone and named NADE (p75NTR-associated cell death executor) . Mouse NADE has marked homology to the human HGR74 protein . NADE specifically binds to the cell-death domain of p75NTR . Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells . However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis . Furthermore, p75NTR/NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent . We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes . Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.

J Mol Biol, 2000 Apr 14, 297(5), 1183 - 94
Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha; Fontes MR et al.; Importin-alpha is the nuclear import receptor that recognizes cargo proteins which contain classical monopartite and bipartite nuclear localization sequences (NLSs), and facilitates their transport into the nucleus . To determine the structural basis of the recognition of the two classes of NLSs by mammalian importin-alpha, we co-crystallized an N-terminally truncated mouse receptor protein with peptides corresponding to the monopartite NLS from the simian virus 40 (SV40) large T-antigen, and the bipartite NLS from nucleoplasmin . We show that the monopartite SV40 large T-antigen NLS binds to two binding sites on the receptor, similar to what was observed in yeast importin-alpha . The nucleoplasmin NLS-importin-alpha complex shows, for the first time, the mode of binding of bipartite NLSs to the receptor . The two basic clusters in the NLS occupy the two binding sites used by the monopartite NLS, while the sequence linking the two basic clusters is poorly ordered, consistent with its tolerance to mutations . The structures explain the structural basis for binding of diverse NLSs to the sole receptor protein .

Leukemia, 2000 Apr, 14(4), 594 - 601
The interaction between EEN and Abi-1, two MLL fusion partners, and synaptojanin and dynamin: implications for leukaemogenesis; So CW et al.; The mixed lineage leukaemia gene, MLL (also called HRX, ALL-1) in acute leukaemia is fused to at least 16 identified partner genes that display diverse structural and biochemical properties . Using GST pull down and the yeast two hybrid system, we show that two different MLL fusion partners with SH3 domains, EEN and Abi-1, interact with dynamin and synaptojanin, both of which are involved in endocytosis . Synaptojanin, a member of the inositol phosphatase family that has recently been shown to regulate cell proliferation and survival, is also known to bind to Eps15, the mouse homologue of AF1p, another fusion partner of MLL . Expression studies show that synaptojanin is strongly expressed in bone marrow and immature leukaemic cell lines, very weakly in peripheral blood leukocytes and absent in Raji, a mature B cell line . We found that the SH3 domains of EEN and Abi-1 interact with different proline-rich domains of synaptojanin while the EH domains of Eps15 interact with the NPF motifs of synaptojanin . In vitro competitive binding assays demonstrate that EEN displays stronger binding affinity than Abi-1 and may compete with it for synaptojanin . These findings suggest a potential link between MLL fusion-mediated leukaemogenesis and the inositol-signalling pathway.

Int J Mol Med, 2000 May, 5(5), 553 - 6
A new region of synteny between human chromosome 1p22 and mouse chromosome 5; Chelsea DM et al.; By constructing a physical map of the p22 region of human chromosome 1 we have been able to show the relative orientation of four genes; GFI1, NB4S/EVI5, RPL5 and DR1 . Analysis of the mouse physical map shows that the murine orthologs of these genes are located on mouse chromosome 5 . Through this analysis we have established a new region of synteny between mouse chromosome 5 and human chromosome region 1p22.

Pharmacogenetics, 2000 Mar, 10(2), 95 - 104
CYP2C9 Ile359 and Leu359 variants: enzyme kinetic study with seven substrates; Takanashi K et al.; To assess the effects of Ile359 to Leu359 change on CYP2C9-mediated metabolism, we performed site-directed mutagenesis and cDNA expression in yeast for CYP2C9 and examined in detail the kinetics of seven metabolic reactions by wild-type CYP2C9 (Ile359) and its Leu359 variant . For the metabolism of all the substrates studied, the Leu359 variant exhibited smaller Vmax/Km values than did the wild-type . The differences in the Vmax/Km values between the wild-type and the Leu359 variant varied from 3.4-fold to 26.9-fold . The Leu359 variant had higher Km values than did the wild-type for all the reactions studied . Among the seven reactions studied, the greatest difference in the Vmax values between the wild-type and the Leu359 variant was for piroxicam 5'-hydroxylation (408 versus 19 pmol/min/nmol P450), whereas there were no differences in the Vmax values between the wild-type and the Leu359 variant for diclofenac 4'-hydroxylation and tolbutamide methylhydroxylation . These results indicate that the Ile359 to Leu359 change significantly decreases the catalytic activity of all the CYP2C9-mediated metabolisms studied, whereas the extent of the reduction in activity and changes of the kinetic parameters varies between substrates . Moreover, the amino acid substitution decreased the enantiomeric excess in the formation of 5-(4-hydroxyphenyl)-5-phenylhydantoin from phenytoin.

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