Yeasts

Pigment Cell Res, 2000 Feb, 13(1), 15 - 20
Hermansky-Pudlak syndrome and pale ear: melanosome-making for the millennium; Spritz RA; Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid-lipofuscin lysosomal storage disease . These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles--melanosomes, platelet granules, and lysosomes--suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles . The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified . Functional studies indicate that the HPS protein is part of a high-molecular weight complex involved in the biogenesis of early melanosomes . Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.

Jpn J Cancer Res, 2000 Mar, 91(3), 293 - 300
Mapping of target regions of allelic loss in primary breast cancers to 1-cM intervals on genomic contigs at 6q21 and 6q25.3; Utada Y et al.; Allelic losses on the long arm of human chromosome 6 are frequently observed in cancers of the ovary, prostate, and breast . To identify the locations of putative tumor suppressor genes on 6q, we examined 192 primary breast cancers for patterns of allelic loss at 16 polymorphic microsatellite loci distributed along this chromosome arm . Allelic losses at one or more loci were observed in 105 (55%) of the tumors examined . Detailed deletion mapping with appropriate yeast artificial chromosome (YAC) contigs identified two distinct commonly deleted regions; one was confined to a 1-cM interval at 6q21 flanked by D6S1040 and D6S262 and the other to a 1-cM interval at 6q25.3 flanked by D6S305 and D6S411 . Allelic losses at 6q21 were more frequent in invasive solid tubular and scirrhous carcinomas than in tumors of less aggressive histologic types (P = 0.0006) . Allelic loss at 6q25.3 was associated with loss of progesterone receptor (P = 0.0256) . Our results suggest the presence of two tumor suppressor genes for breast cancer on 6q that are likely to be associated with tumor progression and / or loss of hormonal dependency.

FEBS Lett, 2000 Apr 7, 471(1), 17 - 22
An ARID family protein binds to the African swine fever virus encoded ubiquitin conjugating enzyme, UBCv1; Bulimo WD et al.; The NH(2)-terminal end of a protein, named SMCp, which contains an ARID (A/T rich interaction domain) DNA binding domain and is similar to the mammalian SMCY/SMCX proteins and retinoblastoma binding protein 2, was shown to bind the African swine fever virus encoded ubiquitin conjugating enzyme (UBCv1) using the yeast two hybrid system and in in vitro binding assays . Antisera raised against the SMCp protein were used to show that the protein is present in the cell nucleus . Immunofluorescence showed that although UBCv1 is present in the nucleus in most cells, in some cells it is in the cytoplasm, suggesting that it shuttles between the nucleus and cytoplasm . The interaction and co-localisation of UBCv1 with SMCp suggest that SMCp may be a substrate in vivo for the enzyme.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4233 - 8
Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2; Wu X et al.; PTEN is a tumor suppressor gene mutated in human cancers . Although many mutations target the phosphatase domain, others create a truncated protein lacking the C-terminal PDZ-binding motif or a protein that extends beyond the PDZ-binding motif . Using the yeast two-hybrid system, we isolated a membrane-associated guanylate kinase family protein with multiple PDZ domains {AIP-1 (atrophin interacting protein 1), renamed MAGI-2 (membrane associated guanylate kinase inverted-2)} . MAGI-2 contains eight potential protein-protein interaction domains and is localized to tight junctions in the membrane of epithelial cells . PTEN binds to MAGI-2 through an interaction between the PDZ-binding motif of PTEN and the second PDZ domain of MAGI-2 . MAGI-2 enhances the ability of PTEN to suppress Akt activation . Furthermore, certain PTEN mutants have reduced stability, which is restored by adding the minimal PDZ-binding motif back to the truncated protein . We propose that MAGI-2 improves the efficiency of PTEN signaling through assembly of a multiprotein complex at the cell membrane.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4023 - 8
Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression; da Silva Xavier G et al.; Elevated glucose concentrations stimulate the transcription of the pre-proinsulin (PPI), L-type pyruvate kinase (L-PK), and other genes in islet beta cells . In liver cells, pharmacological activation by 5-amino-4-imidazolecarboxamide riboside (AICAR) of AMP-activated protein kinase (AMPK), the mammalian homologue of the yeast SNF1 kinase complex, inhibits the effects of glucose, suggesting a key signaling role for this kinase . Here, we demonstrate that AMPK activity is inhibited by elevated glucose concentrations in MIN6 beta cells and that activation of the enzyme with AICAR prevents the activation of the L-PK gene by elevated glucose . Furthermore, microinjection of antibodies to the alpha2- (catalytic) or beta2-subunits of AMPK complex, but not to the alpha1-subunit or extracellular stimulus-regulated kinase, mimics the effects of elevated glucose on the L-PK and PPI promoter activities as assessed by single-cell imaging of promoter luciferase constructs . In each case, injection of antibodies into the nucleus and cytosol, but not the nucleus alone, was necessary, indicating the importance of either a cytosolic phosphorylation event or the subcellular localization of the alpha2-subunits . Incubation with AICAR diminished, but did not abolish, the effect of glucose on PPI transcription . These data suggest that glucose-induced changes in AMPK activity are necessary and sufficient for the regulation of the L-PK gene by the sugar and also play an important role in the regulation of the PPI promoter.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4017 - 22
The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton; Gallagher AR et al.; Despite the recent positional cloning of the PKD1 and PKD2 genes, which are mutated in the great majority of patients with autosomal-dominant polycystic kidney disease (PKD), the pathogenic mechanism for cyst formation is still unclear . The finding, that the PKD1 and PKD2 proteins interact with each other through their COOH termini, suggests that both proteins are part of the same protein complex or signal transduction pathway . Using a yeast two-hybrid screen with the PKD2 protein, we isolated the PKD2-interacting protein Hax-1 . The specificity of the interaction was demonstrated by the fact that PKD2L, a protein closely related to PKD2, failed to interact with Hax-1 . Immunofluorescence experiments showed that in most cells PKD2 and Hax-1 colocalized in the cell body, but in some cells PKD2 and Hax-1 also were sorted into cellular processes and lamellipodia . Furthermore we demonstrated an association between Hax-1 and the F-actin-binding protein cortactin, which suggests a link between PKD2 and the actin cytoskeleton . We speculate that PKD2 is involved in the formation of cell-matrix contacts, which are dysfunctional without a wild-type PKD2 protein, thus leading to cystic enlargement of tubular structures in the kidney, liver, and pancreas.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3999 - 4004
MIR16, a putative membrane glycerophosphodiester phosphodiesterase, interacts with RGS16; Zheng B et al.; We have identified the protein MIR16 (for Membrane Interacting protein of RGS16) from a yeast two-hybrid screen by using RGS16 as bait . MIR16 shares strong homology with bacterial glycerophosphodiester phosphodiesterases . It interacts with RGS16 and, more weakly, with several other selected RGS proteins . Analysis of deletion mutants showed that the N-terminal region of the RGS domain in RGS16 is required for its interaction with MIR16 . MIR16 is an integral membrane glycoprotein, because it remained associated with membrane fractions after alkaline treatment and because, in some cells, it is sensitive to digestion with endoglycosidase H . By immunofluorescence and immunoelectron microscopy, MIR16 was localized on the plasma membrane in liver and kidney and on intracellular membranes in rat pituitary and cultured pituitary cells . MIR16 represents the only integral membrane protein identified thus far to interact with an RGS domain and, to our knowledge, is the only mammalian glycerophosphodiester phosphodiesterase that has been cloned . The putative enzymatic activity of MIR16 and its interaction with RGS16 suggest that it may play important roles in lipid metabolism and in G protein signaling.

Genes Cells, 2000 Mar, 5(3), 191 - 202
NF-kappaB activation through IKK-i-dependent I-TRAF/TANK phosphorylation; Nomura F et al.; BACKGROUND: NF-kappaB is an ubiquitously expressed transcription factor that plays an important role in the immune, anti-apoptotic and inflammatory responses . NF-kappaB is normally sequestered in the cytoplasm by interacting with inhibitory IkappaB molecules . Upon stimulation, IkappaB is phosphorylated and subsequently degraded by the proteasome, allowing NF-kappaB to translocate into the nucleus where they regulate target gene expression . Two kinases, IKK-alpha and IKK-beta, which are responsible for IkappaB phosphorylation were recently identified . We have recently identified a cytokine inducible IKK-i, a kinase related to IKK-alpha and -beta . IKK-i significantly induced NF-kappaB activation upon over-expression, as did IKK-alpha and IKK-beta . Unlike IKK-alpha and IKK-beta, IKK-i phosphorylated Ser36 but not Ser32 in vitro, suggesting that IKK-i activates NF-kappaB by distinct mechanisms from the conventional IKKs . RESULTS: I-TRAF/TANK was isolated as a molecule that interacts specifically with inducible IkappaB kinase (IKK-i) by the yeast two-hybrid screening procedure . The association of IKK-i and I-TRAF is mediated via the interaction between the N-terminal domain of I-TRAF and the C-terminal portion of IKK-i . In vitro kinase assays demonstrate that IKK-i phosphorylates I-TRAF in the middle portion that associates with TRAF2 . Interestingly, TRAF2 is freed from the I-TRAF/TRAF2 complex after I-TRAF phosphorylation . NF-kappaB activation by IKK-i is significantly blocked by coexpression of the N-terminal domain of I-TRAF, dominant negative TRAF2, and dominant negative NIK and IKK-beta . IKK-i over-expression also induced c-Jun N-terminal kinase . These results show that I-TRAF is a substrate of IKK-i . NF-kappaB activation by IKK-i may be mediated through phosphorylation of I-TRAF by IKK-i and subsequent liberation of TRAF2 . CONCLUSION: These results indicate that NF-kappaB activation by IKK-i is mediated through phosphorylation of I-TRAF/TANK by IKK-i and subsequent liberation of TRAF2.

Eur J Biochem, 2000 Apr, 267(8), 2156 - 65
A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily; Castillo J et al.; The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described . A cDNA clone of its gene, which was designated psp54, was also isolated . The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54) . p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known . The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins . p16 is also 30% homologous with Nhp2p, a yeast nuclear protein . The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation . Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation) . p16 can be recovered from nuclei cross-linked to histone H3, when the disulfide bridges that occur in vivo are preserved . On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation . We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles . The function of p16 may be related to the protection of chromatin structure against desiccation during seed development.

Eur J Biochem, 2000 Apr, 267(8), 2135 - 49
Overexpression of enzymes that repair endogenous damage to DNA; Frosina G; A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin . Efficient repair mechanisms have evolved to cope with this . The main repair pathway involved in repair of endogenous damage is DNA base excision repair . In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine . In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities . In some cases (e.g . O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g . alkyl N-purine glycosylase or DNA polymerase beta) were detrimental and even produced a genome instability phenotype . The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3913 - 8
Identification and characterization of a host protein required for efficient template selection in viral RNA replication; Diez J et al.; Biochemical studies suggest that positive-strand RNA virus replication involves host as well as viral functions . Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of animal and plant positive-strand RNA viruses . Yeast expressing the BMV RNA replication proteins 1a and 2a supports BMV RNA replication and mRNA synthesis . Using the ability of BMV to replicate in yeast, we show that efficient BMV RNA replication requires Lsm1p, a yeast protein related to core RNA splicing factors but shown herein to be cytoplasmic . Haploid yeast with an Lsm1p mutation was defective in an early template selection step in BMV RNA replication, involving the helicase-like replication protein 1a and an internal viral RNA element conserved with tRNAs . Lsm1p dependence of this interaction was suppressed by adding 3' poly(A) to the normally unpolyadenylated BMV RNA . Our results show Lsm1p involvement in a specific step of BMV RNA replication and connections between Lsm1p and poly(A) function, possibly through interaction with factors binding mRNA 5' ends.

Plant Physiol, 2000 Apr, 122(4), 1301 - 10
ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation; Huang Y et al.; The modulation of mitogen-activated protein kinase (MAPK) activity regulates many intracellular signaling processes . In animal and yeast cells, MAP kinases are activated via phosphorylation by the dual-specificity kinase MEK (MAP kinase kinase) . Several plant homologs of MEK and MAPK have been identified, but the biochemical events underlying the activation of plant MAPKs remain unknown . We describe the in vitro activation of an Arabidopsis homolog of MAP kinase, ATMPK4 . ATMPK4 was phosphorylated in vitro by an Arabidopsis MEK homolog, AtMEK1 . This phosphorylation occurred principally on threonine (Thr) residues and resulted in elevated ATMPK4 kinase activity . A second Arabidopsis MEK isoform, ATMAP2Kalpha, failed to phosphorylate ATMPK4 in vitro . Tyr dephosphorylation by the Arabidopsis Tyr-specific phosphatase AtPTP1 resulted in an almost complete loss of ATMPK4 activity . Immunoprecipitates of Arabidopsis extracts with anti-ATMPK4 antibodies displayed myelin basic protein kinase activity that was sensitive to treatment with AtPTP1 . These results demonstrate that a plant MEK can phosphorylate and activate MAPK, and that Tyr phosphorylation is critical for the catalytic activity of MAPK in plants . Surprisingly, in contrast to the animal enzymes, AtMEK1 may not be a dual-specificity kinase but, rather, the required Tyr phosphorylation on ATMPK4 may result from autophosphorylation.

J Mol Med, 2000, 78(1), 36 - 46
A 500-kb region on chromosome 16p13.1 contains the pseudoxanthoma elasticum locus: high-resolution mapping and genomic structure; Cai L et al.; We have recently mapped the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and cardiovascular system, to chromosome 16p 13.1 . Here we report further data on the fine-mapping and genomic structure of this locus . Haplotype analysis of informative PXE families narrowed the locus to an interval of less than 500 kb located between markers D16B9621 and D16S764 . Three overlapping YAC clones were found to cover this region through YAC-STS content mapping . An overlapping BAC contig was then constructed to cover this interval and the surrounding region . About 80% of this chromosomal region has been fully sequenced using the BAC shotgun technique . Gene content and sequence analysis predicted four genes (MRP1, MRP6, PM5, and a novel transcript) and two pseudogenes (ARA and PKDI) within this interval . By screening a somatic cell hybrid panel we were able to precision-map the breakpoint of Cy185 and the starting point of a chromosomal duplication within 20 kb of BAC A962B4 . The present data further refine the localization of PXE, provide additional physical cloning resources, and will aid in the eventual identification of the genetic defect causing PXE.

Trends Biotechnol, 2000 May, 18(5), 218 - 23
Artificial chromosomes: ideal vectors?
Brown WR, Mee PJ, Hong Shen M.
Artificial chromosomes are DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes . Artificial chromosomes were first assembled in budding yeast and have since been useful in many aspects of yeast genetics . Several attempts have been made at building artificial chromosomes in mammals, although these have been met with limited success . Consequently, mini-chromosomes of defined structure have been developed to address questions regarding mammalian chromosome function and for biotechnological applications . Here we review progress in these areas and consider how it influences plans to build artificial chromosomes in plants and parasites.

Plant J, 2000 Feb, 21(3), 311 - 6
Induction of competence for elicitation of defense responses in cucumber hypocotyls requires proteasome activity; Becker J et al.; The epidermal cells of hypocotyls from etiolated cucumber seedlings are not constitutively competent for elicitation of the rapid H2O2 defense response . However, elicitor competence developed while conditioning the surface-abraded seedlings by rotating them in buffer for 4 h . Competence development was greatly potentiated by inducers of systemic acquired resistance and suppressed by specific inhibitors of proteasome activity, clastolactacystin beta-lactone (LAC) and carboxybenzoyl-L-leucyl-L-leucyl-L-leucinal (LLL) . In the freshly abraded seedlings, chitinase gene activation became evident approximately 4 h after elicitor addition . Accumulation of chitinase mRNA was enhanced upon conditioning prior to elicitation and was inhibited by LAC and LLL, indicating that the process which leads to H2O2 elicitation competence is also superimposed on the elicitation of chitinase mRNA . LAC and LLL caused an accumulation of ubiquitin-conjugated proteins and enhanced the expression of a proteasome alpha-subunit, suggesting that proteasome activity was specifically inhibited and that the effect observed on gene expression was not due to impaired gene induction in general . Together, our results suggest that the ubiquitin-proteasome system may play a crucial role in a process which switches the signaling pathway for diverse plant defense responses into a functional state, as is known for many basic cellular processes in both animals and yeast.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 489 - 500
Palaeococcus ferrophilus gen . nov., sp . nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney; Takai K et al.; A novel barophilic, hyperthermophilic archaeon was isolated from a deep-sea hydrothermal vent chimney at the Myojin Knoll in the Ogasawara-Bonin Arc, Japan . The cells were found to be irregular cocci and motile with multiple polar flagella . Growth was observed between 60 and 88 degrees C (opt . 83 degrees C; 30 min doubling time), pH 4.0 and 8.0 (opt . pH 6.0), 20 and 73 g sea salts l-1 (opt . 47 g l-1) and 0.1 and 60 MPa (opt . 30 MPa) . The isolate was a strictly anaerobic chemoorganotroph capable of utilizing proteinaceous substrates such as yeast extract, peptone, tryptone and casein in the presence of elemental sulfur or ferrous iron . The G + C content of the genomic DNA was 53.5 mol% . Phylogenetic analysis based on 16S rDNA sequences indicated that the isolate was a member of an ancient lineage of the Thermococcales that diverged prior to the formation of the two genera Thermococcus and Pyrococcus . On the basis of the physiological and molecular properties of the new isolate, the name Palaeococcus ferrophilus gen . nov., sp . nov . is proposed . The type strain is strain DMJT (= JCM 10417) {corrected}.

Mol Cell Biol, 2000 May, 20(9), 3137 - 46
The human SWI-SNF complex protein p270 is an ARID family member with non-sequence-specific DNA binding activity; Dallas PB et al.; p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein . The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain) . The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1 . The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression . Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties . Each binds preferentially to AT-rich sites . In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.

J Nat Prod, 2000 Mar, 63(3), 332 - 8
Antiyeast steroidal saponins from Yucca schidigera (Mohave yucca), a new anti-food-deteriorating agent; Miyakoshi M et al.; A saponin fraction from the stems of Yucca schidigera (Mohave yucca) exhibited potent growth-inhibitory activities against certain food-deteriorating yeasts, film-forming yeasts, and dermatophytic yeasts and fungi . From this fraction, a number of new anti-yeast monodesmosidic spirostanol saponins, named schidigera-saponins A1 (1), A2 (2), A3 (3), B1 (4), C1 (5),C2 (6); 25(R and S) schidigera-saponins D1 (7), D2 (8), E1 (12), F1 (13); and 25(S) schidigera-saponins D3 (9), D4 (10), D5 (11), and F2 (14) were isolated, together with several related known saponins, and the structures were elucidated by spectroscopic methods (see Chart 1) . The relationship between the antiyeast activities and the structures of these saponins is described.

Rev Med Chir Soc Med Nat Iasi, 1997 Jul-Dec, 101(3-4), 197 - 201
{Morphological diagnostic problems in a case of peritoneal blastomycosis}; Munteanu G et al.; Blastomycosis is a mycotic disease, caused by a fungal infection . It has a wide spectrum of clinical presentations, and, particularly, can mimic neoplastic disease . Correct diagnosis of the illness requires fungal culture and biopsy . In Romania, mycotic histopathology is insufficiently developed, and morphological tests are recommended to very few people who present this type of pathology . The paper discusses a case of peritoneal blastomycosis found at a patient with an abdominal pseudotumoral mass . The microscopic exam revealed the characteristic histologic features and budding yeast, in specific dyes, typical to the Blastomyces dermatidis (PAS, silver-methenamin) . Authors of this paper hereby intend to draw pathologists' attention on the existence and diagnosis of mycotic lesions, whose number is continuously increasing nowadays.

Genomics, 2000 Mar 15, 64(3), 264 - 76
An integrated map of human 6q22.3-q24 including a 3-Mb high-resolution BAC/PAC contig encompassing a QTL for fetal hemoglobin; Game L et al.; Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23 . An initial yeast artificial chromosome contig of 13 clones spanning this region was generated . Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb . We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region . The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC . A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb . About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms . Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig .

Genomics, 2000 Mar 15, 64(3), 221 - 9
Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia; McDonell N et al.; The chromosomal abnormality represented by an isodicentric X chromosome {idic(X)(q13)} is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females . A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene . Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region . In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs . Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations . FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML . Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region .

Mol Plant Microbe Interact, 2000 Apr, 13(4), 374 - 83
The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis; Takano Y et al.; The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants . Here we report that the C . lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps . CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea . Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant . Surprisingly, in contrast to M . grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination . However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination . Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant . This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi . Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

J Biomed Sci, 2000 Mar-Apr, 7(2), 160 - 8
Hepatitis B virus core protein interacts with the C-terminal region of actin-binding protein; Huang CJ et al.; Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes . There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis . To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis . A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified . This interaction was further confirmed both in vitro and in vivo . In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein . Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278 . The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs . The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed.

J Biomed Sci, 2000 Mar-Apr, 7(2), 152 - 9
Identification of okadaic-acid-induced genes by mRNA differential display in glioma cells; Chin LS et al.; To identify novel genes associated with apoptosis in glioma cells, we treated T98G glioma cells with okadaic acid (OA) . Differential display using 15 random primers was performed on RNA extracted from these cells . Upregulated bands were excised from polyacrylamide gels and cloned . Northern blots were used to confirm RNA expression in T98G cells . 18 RNA fragments corresponding to the untranslated region of genes were identified and sequenced . Three unknown gene fragments were used to screen a fetal brain cDNA library resulting in three complete cDNA sequences . The three sequences corresponded to a human gene homologous to the yeast translation initiation factor Sui-1, a cAMP-regulated phosphoprotein, ARPP-16/19, and a novel gene designated O48 . Transcription of Sui-1 increased in response to all stress factors tested, whereas ARPP only responded to OA . 2-kb and 4-kb O48 RNA species were identified . OA and stress factors increased 2-kb expression while K252a (protein kinase inhibitor) increased 4-kb expression . Differential display is effective for identifying genes associated with apoptosis . Novel genes may be identified by further analysis of the gene fragments identified in this study . The function of O48 is unknown.

Curr Opin Genet Dev, 2000 Apr, 10(2), 178 - 86
Think global, act local--how to regulate S phase from individual replication origins; Pasero P et al.; All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved . Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins . Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases . The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins . Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states.

Curr Opin Genet Dev, 2000 Apr, 10(2), 193 - 8
mRNA stability in eukaryotes; Mitchell P et al.; During the past two years, the role of the proteins HuR and hnRNP D in regulated mRNA degradation in humans has become clearer, and a putative mRNA deadenylase, DAN or PARN, has been identified . In yeast, the relationship between translation and mRNA turnover is clearer, but the mRNA decapping process has turned out to be unexpectedly complex.

Curr Opin Genet Dev, 2000 Apr, 10(2), 151 - 6
Replication and recombination intersect; Marians KJ; A bacterial housekeeping function, which requires both recombination and replication enzymes, has been identified that re-establishes inactivated replication forks under normal growth conditions . Some long-tract gene-conversion events initiated by double-strand breaks in yeast and mammalian cells can be attributed to recombination-directed DNA replication . Double-strand break repair in yeast has been shown to require both leading- and lagging-strand DNA synthesis . These observations suggest that the recombination and replication machinery cooperate to maintain genomic integrity.

Carcinogenesis, 2000 Apr, 21(4), 563 - 5
High frequency in esophageal cancers of p53 alterations inactivating the regulation of genes involved in cell cycle and apoptosis; Robert V et al.; Somatic mutations of the tumor suppressor gene p53 have been frequently detected in esophagal cancers, but their biological significance remains to be established . The tumor suppressor activity of p53 results in part from its ability to transactivate genes involved in the cell cycle and apoptosis, such as p21, bax and PIG3, and some p53 mutations may have a differential effect on the transactivation of these target genes . We developed yeast strains in which the activation by wild-type p53 of reporter plasmids containing p53 binding sites present within these target genes induces a change in the color of the colonies (red/white) . Using these strains, we analyzed 56 esophageal cancers from patients residing in Normandy, France, a high incidence geographic area . Forty-seven tumors (84%), scored as mutant with the p21, bax and PIG3 reporter strains and in most of the cases (76%), the percentage of red colonies suggested that both p53 alleles were inactivated . Sequencing analysis allowed the identification of a p53 mutation in each positive sample, and the spectrum of mutations was in agreement with the etiological role of tobacco and alcohol . These results confirm the high frequency of biallelic p53 mutations in esophageal carcinoma and strongly suggest that their biological consequence is the complete alteration of the transactivation of genes involved in the cell cycle and apoptosis, which indicates that p53 alteration is a key event in esophagus carcinogenesis.

Xenobiotica, 2000 Mar, 30(3), 219 - 33
Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling; Lightfoot T et al.; 1 . Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme . Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear . 2 . As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6 . 3 . Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position . The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems . 4 . A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable . An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine . 5 . This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6.

Protein Sci, 2000 Mar, 9(3), 536 - 43
Cytochrome c folds through a smooth funnel; Panda M et al.; A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding . The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state . A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2 . The results show that the thermodynamic properties of the transition states are very similar . A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues . At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms) . T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein . Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u) . The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic . In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.

J Biol Chem, 2000 Jun 16, 275(24), 18153 - 9
Mammalian mitochondrial ribosomal proteins (4) . Amino acid sequencing, characterization, and identification of corresponding gene sequences; O'Brien TW et al.; Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans . It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations . Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized . MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology . Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins . Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner . Three of them represent "new," i.e . formerly unknown mammalian mitochondrial ribosomal protein classes . Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins . In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.

J Biol Chem, 2000 Jun 23, 275(25), 18724 - 31
Identification and characterization of a novel protein from Sertoli cells, PASS1, that associates with mammalian small stress protein hsp27; Liu C et al.; hsp27 is involved in development of tolerance to stress, possibly by its involvement in molecular chaperoning, maintenance of glutathione status, and/or modulation of microfilament structure and function . We hypothesize that hsp27 function depends on specific association with other proteins . To discover proteins that associate with hsp27, we made a differentiated rat Sertoli cell cDNA expression library and screened it using the yeast two-hybrid system . We obtained a cDNA coding for a novel protein of 428 amino acids that we have named PASS1 (protein associated with small stress proteins 1) . BLAST searches did not reveal major similarity of PASS1 to any known protein, but the cDNA sequence matched several mouse EST clones and shares 34% homology with a Caenorhabditis elegans genomic sequence . In vitro, bacterially expressed glutathione S-transferase-PASS1 fusion protein bound to hsp27, and hsp27 was co-immunoprecipitated with c-Myc-tagged PASS1 overexpressed in several cell lines . The region of PASS1 responsible for association with hsp27 was identified as existing predominantly between amino acids 108 and 208 of PASS1 . Northern hybridization and Western blot analysis demonstrated that PASS1 is expressed in several tissues, with the highest expression occurring in testis, primarily in Sertoli cells . The presence of a 1.4-kilobase PASS1 mRNA in kidney as well as the 1 . 8-kilobase mRNA seen in other tissues suggests that alternate splicing may occur in this organ . Ectopic expression of PASS1 in two cultured cell lines was observed to inhibit the ability of hsp27 to protect cells against heat shock, indicating that PASS1 does interact with hsp27 in the live cell.

J Cell Sci, 2000 May, 113 ( Pt 9), 1553 - 64
The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin; Flick MJ et al.; Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus . The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments . Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined . To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners . From this screen, the cytoskeletal protein betaI-spectrin was isolated . Protein interaction assays demonstrate that MLP and betaI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions . betaI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins . The MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin . Confocal microscopy studies also reveal that MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue . Given that beta-spectrin is a known costamere protein, we propose that sarcolemma-associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the beta-spectrin network to the alpha-actinin crosslinked actin filaments of the myofibril.

J Cell Sci, 2000 May, 113 ( Pt 9), 1515 - 24
Lysosome-endosome fusion and lysosome biogenesis; Luzio JP et al.; Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle . This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created . Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents . In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation . We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Planta, 2000 Feb, 210(3), 371 - 82
Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.) . I . Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator; Hausler RE et al.; The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco . For this purpose, tobacco lines with an antisense repression of the endogenous TPT (alphaTPT) and tobacco lines overexpressing the TPT gene isolated from the C4 plant Flaveria trinervia (FtTPT) were used . The F . trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C3 plants . Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air . Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod . The FtTPT overexpressors incorporated more 14CO2 in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type . There were only small effects on labelling of amino acids and organic acids . The mobilisation of starch was enhanced in alphaTPT lines but decreased in FtTPT overexpressors compared to the wild type . Enzymes involved in starch mobilisation or utilisation, such as alpha-amylase or hexokinase were increased in alphaTPT plants and, in the case of amylases, decreased in FtTPT overexpressors . Moreover, alpha-amylase activity exhibited a pronounced diurnal variation in alphaTPT lines with a maximum activity after 8 h in the light . These changes in starch hydrolytic activities were confirmed by activity staining of native gels . Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity . There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate . In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO2 assimilation . However, in elevated CO2 (1500 microl x l(-1)) and low O2 (2%) the rate of CO2 assimilation was decreased in the alphaTPT lines and was slightly higher in FtTPT lines . This shows that the TPT limits maximum rates of photosynthesis in the wild type.

J Eukaryot Microbiol, 2000 Mar-Apr, 47(2), 129 - 38
Centrin protein and genes in Trichomonas vaginalis and close relatives; Brugerolle G et al.; Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina . Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina . Centrin was not demonstrated in the dividing spindle and paradesmosis . Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane . Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules . There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T . vaginalis . Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined . By screening a T . vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found . The sequence comprises the 4 typical EF-hand Ca++-binding domains present in every known centrin . Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).

Curr Opin Oncol, 2000 Mar, 12(2), 143 - 8
Early detection and prevention of lung cancer; Wright GS et al.; Lung cancer remains the leading cause of cancer death in the United States and is one of the world's leading causes of preventable death . Technologic advances have brought new modalities that may be useful for the early detection of lung cancer . However, because of the large number of persons at increased risk for lung cancer, screening is a formidable task . There are several risk factors that can be identified, including potential susceptibility factors, which may aid in pinpointing individuals who need to participate in regular screening programs . Aside from recognized environmental exposures including cigarette smoking, there are a number of genetic and metabolic susceptibility factors that have been examined . These include polymorphisms in the cytochrome p450 enzymes and the metabolizing capability of glutathione s-transferase or acetylation . Additionally, defects in DNA repair and in bleomycin sensitivity assays may also aid in identifying individuals who are at an increased risk for lung cancer . Additional work has been done in the area of characterizing the molecular alterations in the bronchial epithelium in high-risk smokers . This manuscript addresses only selected molecular alterations that have been examined in preneoplastic bronchial epithelium . In addition to mutations in the k-ras oncogene and the p53 gene, which are frequently seen in malignancy, alterations in the p16 gene, microsatellite instability and loss of heterozygocity are also promising potential markers of preneoplasia . The hnRNP A2/B1 gene also shows some promising increased expression in preneoplasia . Lung cancer prevention has made some strides . A number of trials with molecular and morphologic intermediate endpoints have been conducted and have suggested that some of the molecular alterations and morphologic alterations are reversible . However, the rate of spontaneous regression of these lesions is, as yet, uncharacterized . Two recent large studies, the beta-carotene and retinol efficacy trial (CARET) trial conducted in the United States and the Alpha-Tocopherol Beta Carotene (ATBC) trial conducted in Finland, both demonstrated an unexpected increased risk for lung cancer associated with beta-carotene supplementation . The EUROSCAN trial evaluation of vitamin A and N-acetylcystine also showed no benefit to supplementation in reducing risk for lung cancer . Results from the Intergroup study of 1 3-cis-retinoic acid are pending, and plans are underway for an Intergroup trial studying high selenium yeast to reduce lung cancer risk . Hopefully, the combination of identifying markers of increased risk among the numerous current and former smokers will identify high-risk populations to participate in future trials of promising agents that may lead to reduction in incidence and mortality of the leading cause of cancer death.

J Biol Chem, 2000 Jun 30, 275(26), 20052 - 60
Crystal structure of a conformation-selective casein kinase-1 inhibitor; Mashhoon N et al.; Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis . Unlike most protein kinases, they appear to function as constitutively active enzymes . As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes . To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening . Here we report the crystal structure of 3-{(2,4,6-trimethoxyphenyl) methylidenyl}-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution . The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations . We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.

J Biol Chem, 2000 Jun 30, 275(26), 19866 - 76
Protein-arginine methyltransferase I, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3; Tang J et al.; Arginine methylation is a common post-translation modification found in many proteins . Protein-arginine methyltransferase I (PRMT1) contributes >90% of type I protein-arginine methyltransferase activity in cells and tissues . To expand our knowledge on the regulation and role of PRMT1 in cells, we used the yeast two-hybrid system to identify proteins that interact with PRMT1 . One of the interacting proteins we cloned is interleukin enhancer-binding factor 3 (ILF3), also known as M phase phosphoprotein 4 . ILF3 is closely related to nuclear factor 90 (NF90) . Using an immunofluorescence analysis, we determined that ILF3 and PRMT1 co-localize in the nucleus . Moreover, PRMT1 and ILF3 co-precipitate in immunoprecipitation assays and can be isolated together in "pull-down" experiments using recombinant fusion proteins . ILF3 is a robust substrate for methylation by PRMT1 and can modulate PRMT1 activity in in vitro methylation assays . Deletion studies demonstrated that the COOH-terminal region of ILF3, which is rich in arginine, glycine, and serine, is responsible for the strong interaction between PRMT1 and ILF3 and is the site of ILF3 methylation by PRMT1 . Although ILF3 and NF90 are highly similar, they differ in their carboxyl-terminal regions . Because of this difference, NF90 does not interact with PRMT1, is a much poorer substrate than ILF3 for PRMT1-dependent methylation, and does not modulate PRMT1 enzyme activity.

Biochem J, 2000 Apr 15, 347(Pt 2), 501 - 9
The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells; Warner AJ et al.; Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined . Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously . It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells . In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR . Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR . We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src-homology 2 (SH2) domain as tyrosine-1175 of KDR . We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells . Furthermore, we demonstrate that in the two-hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1.

Cancer Res, 2000 Mar 15, 60(6), 1690 - 7
A 700-kb physical map of a region of 16q23.2 homozygously deleted in multiple cancers and spanning the common fragile site FRA16D; Paige AJ et al.; We have identified a >600-kb region at 16q23.2 that is homozygously deleted from malignant ovarian ascites using representational difference analysis . Overlapping homozygous deletions were also observed in the colon carcinoma cell line HCT116 and a xenograft established from the small cell lung cancer cell line WX330 . This region coincides with that described previously by others as showing loss of heterozygosity in prostate and breast cancers (C . Li et al., Genes Chromosomes Cancer, 24: 175-182, 1999; A . Latil et al., Cancer Res., 57: 1058-1062, 1997; K . Driouch et al., Genes Chromosomes Cancer, 19: 185-191, 1997; A . Iida et al., Br . J . Cancer, 75: 264-267, 1997) . In addition, the minimally deleted region spans the common fragile site FRA16D . We have constructed a 700-kb physical map encompassing the deleted region . By fluorescence in situ hybridization of aphidicolin-induced metaphase chromosomes, we have preliminary data to suggest that P1-derived bacterial artificial chromosome clones from the contig lie on both sides of FRA16D . This is confirmed by extensive fluorescence in situ hybridization analysis of the region reported in the accompanying article (M . Mangelsdorf et al., Cancer Res., 60: 1683-1689, 2000) and is consistent with an involvement of this common fragile site in the loss of 16q23.2 material in various cancer types . The minimally deleted region of approximately 210 kb has been characterized using our own markers and public domain markers . Eleven distinct expressed sequences mapped to the region, providing a basis for identifying the predicted tumor suppressor gene in this region.

Cancer Res, 2000 Mar 15, 60(6), 1683 - 9
Chromosomal fragile site FRA16D and DNA instability in cancer; Mangelsdorf M et al.; It has been proposed that common aphidicolin-inducible fragile sites, in general, predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer . Although this appears to be the case for the fragile site FRA3B and may be the case for FRA7G, it is not yet clear whether this association is a general property of this class of fragile site . The major aim of the present study was to determine whether the FRA16D chromosomal fragile site locus has a role to play in predisposing DNA sequences within and adjacent to the fragile site to DNA instability (such as deletion or translocation), which could lead to or be associated with neoplasia . We report the localization of FRA16D within a contig of cloned DNA and demonstrate that this fragile site coincides with a region of homozygous deletion in a gastric adenocarcinoma cell line and is bracketed by translocation breakpoints in multiple myeloma, as reported previously (Chesi, M., et al., Blood, 91: 4457-4463, 1998) . Therefore, given similar findings at the FRA3B and FRA7G fragile sites, it is likely that common aphidicolin-inducible fragile sites exhibit the general property of localized DNA instability in cancer cells.

Cancer Res, 2000 Mar 15, 60(6), 1531 - 5
Potential role of BRCA2 in a mitotic checkpoint after phosphorylation by hBUBR1; Futamura M et al.; BRCA2, a gene responsible for inherited susceptibility to breast cancer in a number of families, is thought to be critical for replication and repair of DNA during S-phase . To elucidate the physiological functions of BRCA2, we used a yeast two-hybrid system to screen for proteins that could associate with BRCA2 . Here we report interaction of BRCA2 with a mitotic checkpoint protein, hBUBR1, and its phosphorylation by hBUBR1 in vitro . After cotransfection of BRCA2 and hBUBR1 expression vectors into the COS7 cell line, both proteins were stained together in the nuclei of cells whose spindle fibers were disrupted, but not in undamaged cells . Treatment with vincristine, which disrupts microtubules, significantly increased expression of both hBUBR1 and BRCA2 in the MCF7 cells . The results suggest that BRCA2 protein might be involved in a mitotic checkpoint in vivo after it has been phosphorylated by hBUBR1.

Nat Biotechnol, 2000 Apr, 18(4), 393 - 7
Large-scale functional analysis using peptide or protein arrays; Emili AQ et al.; The array format for analyzing peptide and protein function offers an attractive experimental alternative to traditional library screens . Powerful new approaches have recently been described, ranging from synthetic peptide arrays to whole proteins expressed in living cells . Comprehensive sets of purified peptides and proteins permit high-throughput screening for discrete biochemical properties, whereas formats involving living cells facilitate large-scale genetic screening for novel biological activities . In the past year, three major genome-scale studies using yeast as a model organism have investigated different aspects of protein function, including biochemical activities, gene disruption phenotypes, and protein-protein interactions . Such studies show that protein arrays can be used to examine in parallel the functions of thousands of proteins previously known only by their DNA sequence.

Biochim Biophys Acta, 2000 May 1, 1465(1-2), 1 - 16
The plant plasma membrane H(+)-ATPase: structure, function and regulation; Morsomme P et al.; The proton-pumping ATPase (H(+)-ATPase) of the plant plasma membrane generates the proton motive force across the plasma membrane that is necessary to activate most of the ion and metabolite transport . In recent years, important progress has been made concerning the identification and organization of H(+)-ATPase genes, their expression, and also the kinetics and regulation of individual H(+)-ATPase isoforms . At the gene level, it is now clear that H(+)-ATPase is encoded by a family of approximately 10 genes . Expression, monitored by in situ techniques, has revealed a specific distribution pattern for each gene; however, this seems to differ between species . In the near future, we can expect regulatory aspects of gene expression to be elucidated . Already the expression of individual plant H(+)-ATPases in yeast has shown them to have distinct enzymatic properties . It has also allowed regulatory aspects of this enzyme to be studied through random and site-directed mutagenesis, notably its carboxy-terminal region . Studies performed with both plant and yeast material have converged towards deciphering the way phosphorylation and binding of regulatory 14-3-3 proteins intervene in the modification of H(+)-ATPase activity . The production of high quantities of individual functional H(+)-ATPases in yeast constitutes an important step towards crystallization studies to derive structural information . Understanding the specific roles of H(+)-ATPase isoforms in whole plant physiology is another challenge that has been approached recently through the phenotypic analysis of the first transgenic plants in which the expression of single H(+)-ATPases has been up- or down-regulated . In conclusion, the progress made recently concerning the H(+)-ATPase family, at both the gene and protein level, has come to a point where we can now expect a more integrated investigation of the expression, function and regulation of individual H(+)-ATPases in the whole plant context.

J Biol Chem, 2000 May 26, 275(21), 16167 - 73
The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits; Hironaka K et al.; Glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays a crucial role in cerebellum-dependent motor learning . Although GluRdelta2 belongs to an ionotropic GluR family, little is known about its pharmacological features and downstream signaling cascade . To study molecular mechanisms underlying GluRdelta2-dependent motor learning, we employed yeast two-hybrid screening to isolate GluRdelta2-interacting molecules and identified protein-tyrosine phosphatase PTPMEG . PTPMEG is a family member of band 4.1 domain-containing protein-tyrosine phosphatases and is expressed prominently in brain . Here, we showed by in situ hybridization analysis that the PTPMEG mRNA was enriched in mouse thalamus and Purkinje cells . We also showed that PTPMEG interacted with GluRdelta2 as well as with N-methyl-d-aspartate receptor GluRepsilon1 in cultured cells and in brain . PTPMEG bound to the putative C-terminal PDZ target sequence of GluRdelta2 and GluRepsilon1 via its PDZ domain . Examination of the effect of PTPMEG on tyrosine phosphorylation of GluRepsilon1 unexpectedly revealed that PTPMEG enhanced Fyn-mediated tyrosine phosphorylation of GluRepsilon1 in its PTPase activity-dependent manner . Thus, we conclude that PTPMEG associates directly with GluRdelta2 and GluRepsilon1 . Moreover, our data suggest that PTPMEG plays a role in signaling downstream of the GluRs and/or in regulation of their activities through tyrosine dephosphorylation.

J Biol Chem, 2000 Jun 30, 275(26), 20033 - 44
The RIM/NIM family of neuronal C2 domain proteins . Interactions with Rab3 and a new class of Src homology 3 domain proteins; Wang Y et al.; RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins . RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles . We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain . Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C-terminal C(2) domains that are separated by long alternatively spliced sequences . Unexpectedly, the 3'-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2 . NIM2 is composed of a unique N-terminal sequence followed by the C-terminal part of RIM2 . Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected . To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium-triggered exocytosis in PC12 cells . NIM3 induced a dramatic increase in calcium-evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s . To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1 . Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified . RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction . In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C(2) domains, N-terminal to the beginning of NIMs . RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs . With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3.

J Biol Chem, 2000 Jun 2, 275(22), 16802 - 9
The autoimmune regulator protein has transcriptional transactivating properties and interacts with the common coactivator CREB-binding protein; Pitkanen J et al.; Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, caused by mutations in the autoimmune regulator (AIRE) gene, is an autosomal recessive autoimmune disease characterized by the breakdown of tolerance to organ-specific antigens . The 545 amino acid protein encoded by AIRE contains several structural motifs suggestive of a transcriptional regulator and bears similarity to cellular proteins involved in transcriptional control . We show here that AIRE fused to a heterologous DNA binding domain activates transcription from a reporter promoter, and the activation seen requires the full-length protein or more than one activation domain . At the structural level AIRE forms homodimers through the NH(2)-terminal domain, and molecular modeling for this domain suggests a four-helix bundle structure . In agreement, we show that the common transcriptional coactivator CREB-binding protein (CBP) interacts with AIRE in vitro and in yeast nuclei through the CH1 and CH3 conserved domains . We suggest that the transcriptional transactivation properties of AIRE together with its interaction with CBP might be important in its function as disease-causing mutations almost totally abolish the activation effect.

J Biol Chem, 2000 Jun 16, 275(24), 18225 - 33
Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA; MacDonald JI et al.; We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA . Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays . Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent . By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684) . By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding . Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.

J Biol Chem, 2000 Jun 2, 275(22), 16752 - 7
BSAP (Pax5)-importin alpha 1 (Rch1) interaction identifies a nuclear localization sequence; Kovac CR et al.; BSAP (Pax5) is an essential transcription factor for early B cell and central nervous system development . In later B cell development, BSAP has been implicated in the regulation of 3' Ig enhancers and a number of B cell-specific genes . Previous studies have suggested a role for BSAP-interacting proteins in the regulation of the function of BSAP . Using the yeast two-hybrid system, we identified importin alpha1 (Rch1) as a BSAP-interacting protein . Importin alpha proteins have been shown to escort proteins into the nucleus through interaction with a nuclear localization signal (NLS), composed of short stretches of basic amino acids . A predicted NLS in BSAP (NKRKRDE, located at amino acids 195-201 in the central domain) was confirmed to be essential for interaction with importin alpha1 by the yeast two-hybrid assay . Physical interaction between BSAP and importin alpha1 was detected in vitro by a glutathione S-transferase (GST) pulldown assay . The NLS sequence in BSAP conferred nuclear localization to green fluorescent protein (GFP)-BSAP fusion proteins . Although the N-terminal paired (DNA-binding) domain of BSAP also conferred nuclear localization when coupled to green fluorescent protein, this domain did not bind to importin alpha1 in the yeast two-hybrid assay . The NLS sequence in the central domain of BSAP binds to the C-terminal 98-amino acid fragment of importin alpha1.

J Biol Chem, 2000 May 12, 275(19), 14767 - 76
Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei . Sequence conservation and implications for 2'-O-ribose methylation of rRNA; Dunbar DA et al.; We report the identification of 17 box C/D fibrillarin-associated small nucleolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei . To systematically isolate and characterize these snoRNAs, the T . brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was cloned and polyclonal antibodies to the recombinant fibrillarin protein were generated in rabbits . Immunoprecipitations from T . brucei extracts with the anti-fibrillarin antibodies indicated that this trypanosomatid has at least 30 fibrillarin-associated snoRNAs . We have sequenced seventeen of them and designated them TBR for T . brucei RNA 1-17 . All of them bear conserved box C, D, C', and D' elements, a hallmark of fibrillarin-associated snoRNAs in eukaryotes . Fourteen of them are novel T . brucei snoRNAs . Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2'-O-ribose methylation . Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences . Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and vertebrate methylation guide snoRNAs . Our results indicate that T . brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2'-O-ribose methylation of rRNA and that the mechanism for targeting the nucleotide to be methylated at the fifth nucleotide upstream of box D or D' originated in early eukaryotes.

J Biol Chem, 2000 May 26, 275(21), 15851 - 60
Gamma1- and gamma2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells; Piluso G et al.; Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies . At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound . These are a family of cytoplasmic peripheral membrane proteins . Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa . Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins . The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids . The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues . We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25 . Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins . We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections . Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.

J Biol Chem, 2000 Jun 2, 275(22), 17173 - 9
The SCAN domain mediates selective oligomerization; Schumacher C et al.; The SCAN domain is described as a highly conserved, leucine-rich motif of approximately 60 amino acids found at the amino-terminal end of zinc finger transcription factors . Although no specific biological function has been attributed to the SCAN domain, its predicted amphipathic secondary structure led to the suggestion that this domain may mediate protein-protein associations . A yeast two-hybrid screen identified members of two SCAN domain protein families that interact with the SCAN domain of the zinc finger protein ZNF202 . The interacting ZNF191 protein represents the family of SCAN domain-containing zinc finger proteins, whereas the novel SDP1 protein establishes a new family of genes that encode an isolated SCAN domain . Isolated SCAN domain proteins may form asymmetric homodimers in solution . Biochemical binding studies confirmed the associations of ZNF191 and SDP1 with ZNF202 and established the SCAN domain as a selective hetero- and homotypic oligomerization domain . SCAN mediated protein associations might therefore represent a new regulatory mechanism of transcriptional activity.

J Biol Chem, 2000 May 26, 275(21), 15645 - 51
p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting directly with the N-terminal A/B domains; Kobayashi Y et al.; Estrogen receptor (ER) alpha and beta mediate estrogen actions in target cells through transcriptional control of target gene expression . For 17beta-estradiol-induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required . Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown . To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/CREB binding protein, steroid receptor coactivator-1/transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein- 205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function . We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ERalpha and human ERbeta . Direct interactions of p300 with the A/B domains of ERalpha and ERbeta were observed in an in vitro glutathione S-transferase pull-down assay in accordance with the interactions in yeast and mammalian two-hybrid assays . Furthermore, mutations in the p300 binding sites (56-72 amino acids in ERalpha and 62-72 amino acids in ERbeta) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ERalpha and ERbeta . Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ERalpha and ERbeta.

J Biol Chem, 2000 May 19, 275(20), 14791 - 4
Insertional mutation of the murine kisimo locus caused a defect in spermatogenesis; Yanaka N et al.; Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products . An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules . We cloned the disrupted locus of kisimo and identified a novel testis-specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse . The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-1epsilon, suggesting that THEG protein functions as a regulatory factor in protein assembly . Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility.

J Biol Chem, 2000 May 5, 275(18), 13167 - 70
Physical and functional interaction of rabphilin-11 with mammalian Sec13 protein . Implication in vesicle trafficking; Mammoto A et al.; Rab11a small G protein (Rab11p) is implicated in vesicle trafficking, especially vesicle recycling . We have previously isolated a downstream effector of Rab11p, named rabphilin-11 . We found here that rabphilin-11 directly bound the mammalian counterpart of yeast Sec13 protein (mSec13p) in cell-free and intact cell systems . Yeast Sec13p is involved as a component of coat proteins II in the Sar1p-induced vesicle formation from the endoplasmic reticulum, but the precise role of mSec13p is unknown . The interaction of rabphilin-11 with mSec13p was enhanced by GTP-Rab11p . Rabphilin-11 localized on the vesicles in perinuclear regions and along microtubules oriented toward the plasma membrane, whereas mSec13p partly colocalized with rabphilin-11 in the perinuclear regions, most presumably the Golgi complex . Disruption of the rabphilin-11-mSec13p interaction by overexpression of the mSec13p-binding region of rabphilin-11 impaired vesicle trafficking . These results indicate that the rabphilin-11-mSec13p interaction is implicated in vesicle trafficking.

Lett Appl Microbiol, 2000 Mar, 30(3), 183 - 7
Medium-size droplets of methyl ricinoleate are reduced by cell-surface activity in the gamma-decalactone production by Yarrowia lipolytica; Wache Y et al.; Size of methyl ricinoleate droplets during biotransformation into gamma-decalactone by Yarrowia lipolytica was measured in both homogenized and non-homogenized media . In non-homogenized but shaken medium, droplets had an average volume surface diameter d32 of 2.5 microm whereas it was 0.7 microm in homogenized and shaken medium . But as soon as yeast cells were inoculated, both diameters became similar at about 0.7 microm and did not vary significantly until the end of the culture . The growth of Y . lipolytica in both media was very similar except for the lag phase which was lowered in homogenized medium conditions.

Physiol Rev, 2000 Apr, 80(2), 593 - 614
Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm; Hsu SY et al.; Apoptosis is an essential physiological process by which multicellular organisms eliminate superfluous cells . An expanding family of Bcl-2 proteins plays a pivotal role in the decision step of apoptosis, and the differential expression of Bcl-2 members and their binding proteins allows the regulation of apoptosis in a tissue-specific manner mediated by diverse extra- and intracellular signals . The Bcl-2 proteins can be divided into three subgroups: 1) antiapoptotic proteins with multiple Bcl-2 homology (BH) domains and a transmembrane region, 2) proapoptotic proteins with the same structure but missing the BH4 domain, and 3) proapoptotic ligands with only the BH3 domain . In the mammalian ovary, a high rate of follicular cell apoptosis continues during reproductive life . With the use of the yeast two-hybrid system, the characterization of ovarian Bcl-2 genes serves as a paradigm to understand apoptosis regulation in a tissue-specific manner . We identified Mcl-1 as the main ovarian antiapoptotic Bcl-2 protein, the novel Bok (Bcl-2-related ovarian killer) as the proapoptotic protein, as well as BOD (Bcl-2-related ovarian death agonist) and BAD as the proapoptotic ligands . The activity of the proapoptotic ligand BAD is regulated by upstream follicle survival factors through its binding to constitutively expressed 14-3-3 or hormone-induced P11 . In contrast, the channel-forming Mcl-1 and Bok regulate cytochrome c release and, together with the recently discovered Diva/Boo, control downstream apoptosis-activating factor (Apaf)-1 homologs and caspases . Elucidation of the role of Bcl-2 members and their interacting proteins in the tissue-specific regulation of apoptosis could facilitate an understanding of normal physiology and allow the development of new therapeutic approaches for pathological states.

J Clin Microbiol, 2000 Apr, 38(4), 1599 - 608
Evaluation of phenotypic markers for selection and identification of Candida dubliniensis; Tintelnot K et al.; Candida dubliniensis is often associated with C . albicans in cultures . Easy-to-perform selective isolation procedures for these closely related species do not exist . Therefore, we evaluated previously described discriminatory phenotypic markers for C . dubliniensis . A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida . Dark green colonies described as being indicative of C . dubliniensis and other green colonies, 170 in total, were isolated . Chlamydospore formation, intracellular beta-D-glucosidase activity, ability to grow at 42 degrees C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization . Sequencing of the 5' end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for C . dubliniensis . C . dubliniensis was found in 34% of yeast-colonized HIV-infected patients . The color of the colonies on CHROMagar Candida proved to be insufficient for selecting C . dubliniensis, since only 30 of 53 proven C . dubliniensis isolates showed a dark green color in primary cultures . The described typical chlamydospore formation can give only some indication of C . dubliniensis . The assimilation pattern proved to be insufficient to discriminate C . dubliniensis from C . albicans . All C . dubliniensis strains showed no or highly restricted growth at 42 degrees C and a lack of beta-D-glucosidase activity . Unfortunately, atypical C . albicans strains can also exhibit these phenotypic traits . FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species.

J Cell Biol, 2000 Apr 3, 149(1), 209 - 22
The function of plakophilin 1 in desmosome assembly and actin filament organization; Hatzfeld M et al.; Plakophilin 1, a member of the armadillo multigene family, is a protein with dual localization in the nucleus and in desmosomes . To elucidate its role in desmosome assembly and regulation, we have analyzed its localization and binding partners in vivo . When overexpressed in HaCaT keratinocytes, plakophilin 1 localized to the nucleus and to desmosomes, and dramatically enhanced the recruitment of desmosomal proteins to the plasma membrane . This effect was mediated by plakophilin 1's head domain, which interacted with desmoglein 1, desmoplakin, and keratins in the yeast two-hybrid system . Overexpression of the armadillo repeat domain induced a striking dominant negative phenotype with the formation of filopodia and long cellular protrusions, where plakophilin 1 colocalized with actin filaments . This phenotype was strictly dependent on a conserved motif in the center of the armadillo repeat domain . Our results demonstrate that plakophilin 1 contains two functionally distinct domains: the head domain, which could play a role in organizing the desmosomal plaque in suprabasal cells, and the armadillo repeat domain, which might be involved in regulating the dynamics of the actin cytoskeleton.

Microbiology, 2000 Mar, 146 ( Pt 3), 695 - 9
L-{U-14C} lactate binding to a 43 kDa protein in plasma membranes of Candida utilis; Geros H et al.; To identify the putative lactate transporter protein of Candida utilis, plasma membranes from cells grown either on lactic acid (presence of lactate proton symport) or glucose (absence of lactate proton symport) were incubated with L-{U-14C}lactic acid and the membrane proteins were then separated by SDS-PAGE . A well-defined peak of radioactivity occurred in the lane of the gel containing plasma membrane proteins from lactic-acid-grown cells but not from glucose-grown cells . Binding was inhibited by unlabelled pyruvate and lactate, whereas succinate and citrate were not inhibitory . The monocarboxylate transporter inhibitor of animal cells, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, competitively inhibited the lactate proton symport in the whole yeast and also inhibited lactate binding to proteins of isolated plasma membranes . The polypeptide pattern of plasma membranes from lactic-acid-grown cells revealed a 43 kDa polypeptide associated with the peak of labelled lactate . Altogether the results suggest that this polypeptide is either the lactate transporter or a component of it.

Br Med Bull, 1999, 55(3), 544 - 55
Disorders of copper transport; Cox DW; Copper is an essential component of a number of important enzymes . Efficient systems have developed for providing sufficient copper for essential functions, while eliminating excess to avoid tissue toxicity . Copper transport is disrupted in two human diseases: Wilson disease and Menkes disease . Both have defects in copper transporting membrane proteins . Many other proteins are involved in copper transport . Some of these proteins have been identified through a study of the similar copper pathway in yeast . This suggests other copper transport diseases are yet to be discovered . Molecular diagnosis holds promise for reliable diagnosis of patients . Testing of flanking markers is a reliable way to detect presymptomatic sibs of a definite patient.

Med Mycol, 2000 Feb, 38(1), 81 - 3
Initial case of Candida dubliniensis infection from Asia: non-mucosal infection; Kamei K et al.; A yeast, repeatedly isolated from a post-surgical abdominal infection of a 75-year-old Japanese man, was genotyped as Candida dubliniensis . This is the first reported case in Asia of this recently described fungus.

Med Mycol, 2000 Feb, 38(1), 27 - 30
Isolation of Trichosporon asahii from environmental materials; Sugita T et al.; Trichosporon asahii is the most clinically important pathogenic yeast in the genus Trichosporon, as this species causes both deep-seated infection and summer-type hypersensitivity pneumonitis . We isolated 29 T . asahii colonies from environmental samples using the polymerase chain reaction (PCR) and dichloran rose bengal chloramphenicol (DRBC) medium . Our results suggest that T . asahii is common in nature.

Med Mycol, 2000 Feb, 38(1), 15 - 22
Monitoring internalization of Histoplasma capsulatum by mammalian cell lines using a fluorometric microplate assay; Scott AJ et al.; We have developed a fluorometric microtiter plate assay to quantify the internalization of Histoplasma capsulatum yeasts by macrophages . The assay utilizes the fluorescent dye Calcofluor White to label the yeast cell wall and the vital dye trypan blue, which does not enter viable macrophages, to quench fluorescence of extracellular labeled yeasts . Murine RAW 264.7 cells showed more efficient internalization of strain G217B yeasts than human U937 cells . Both cell lines exhibited a dependence upon actin, and, to a lesser degree, microtubules, in G217B uptake.

J Med Virol, 2000 May, 61(1), 29 - 36
Lack of clinical evidence for involvement of hepatitis C virus interferon-alpha sensitivity-determining region variability in RNA-dependent protein kinase-mediated cellular antiviral responses; Mihm S et al.; The hepatitis C virus (HCV) interferon-alpha (IFN-alpha) sensitivity-determining region (ISDR) has been shown to suppress double-stranded RNA-dependent protein kinase (PKR) activity in vitro in a yeast PKR expression system . Since variability of ISDR was shown to correlate with nonresponsiveness to IFN-alpha therapy in chronically HCV-infected patients, it has been suggested that prototype ISDR might be a viral inhibitor of cellular PKR . The present study evaluates the biological significance of ISDR variability in situ, relating it to PKR-mediated cellular antiviral responses within the liver . ISDR variability was determined in patients chronically infected with HCV genotypes 1a, 1b, and 3a by direct sequencing using liver-derived RNA preparations as starting material . As surrogate parameters for PKR-mediated cellular responses, hepatic endogenous IFN-alpha gene expression as well as MxA expression were analysed by a competitive, quantitative reverse transcription-polymerase chain reaction technique . Irrespectively of intra- or intergenotypic ISDR amino acid substitutions, ISDR variability was found not to correlate with endogenous hepatic IFN-alpha or with hepatic MxA gene expression . The data suggest that at least two prominent PKR-mediated cellular responses might be largely unaffected by HCV ISDR variability .

FEBS Lett, 2000 Mar 31, 470(3), 360 - 4
Isolation of Ich-1S (caspase-2S)-binding protein that partially inhibits caspase activity; Ito A et al.; Members of the caspase family are essential executors of apoptosis . Caspase-2 has two messenger RNAs generated by alternative splicing, which encode caspase-2L and caspase-2S . Although caspase-2L induces apoptosis, caspase-2S also has the ability to antagonize cell death . Experiments in caspase-2-deficient mice showed that caspase-2 functions to delay cell death in some neuronal populations, suggesting that caspase-2S dominantly acts for cell survival in the brain . However, the mechanism of caspase-2S-mediated anti-apoptotic effect is still unclear . Here, we isolated a protein that interacts with caspase-2S, designated as Ich-1S (caspase-2S)-binding protein (ISBP), by yeast two-hybrid screening using full-length caspase-2S cDNA as a bait . ISBP is identical to the recently isolated calcium and integrin-binding protein, and a small molecule calcium-binding protein with two EF-hand motifs of its C-terminus . In vitro transcribed and translated ISBP interacts specifically with glutathione-S-transferase-fused caspase-2S . Moreover, the interaction between ISBP and caspase-2S was observed in cultured cells . Northern blot analysis indicated that ISBP may be a ubiquitous protein . Interestingly, ISBP can partially inhibit the processing of pro-caspase-2L induced by anti-Fas antibody-treated Jurkat cytosolic lysates . These results suggested that ISBP may be the mediator for the survival function of caspase-2S.

FEBS Lett, 2000 Mar 31, 470(3), 239 - 43
The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome; Savino C et al.; The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented . The fold typical of other plant toxins is conserved, despite some differences in the loop regions . The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate . Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications . A contact surface inside the C-terminal region of saporin has been identified . Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.

FEBS Lett, 2000 Mar 31, 470(3), 227 - 31
Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner; Menon RP et al.; We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble beta-galactoside-binding protein, galectin-3 . We utilised as bait murine full-length galectin-3 to screen a murine 3T3 cDNA library . Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology . In various assays in vitro the novel protein was shown to bind galectin-3 in a carbohydrate-independent manner . The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins . Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein.

J Med Genet, 2000 Apr, 37(4), 250 - 5
Mutation screening in Rett syndrome patients; Xiang F et al.; Rett syndrome (RTT) was first described in 1966 . Its biological and genetic foundations were not clear until recently when Amir et al reported that mutations in the MECP2 gene were detected in around 50% of RTT patients . In this study, we have screened the MECP2 gene for mutations in our RTT material, including nine familial cases (19 Rett girls) and 59 sporadic cases . A total of 27 sporadic RTT patients were found to have mutations in the MECP2 gene, but no mutations were identified in our RTT families . In order to address the possibility of further X chromosomal or autosomal genetic factors in RTT, we evaluated six candidate genes for RTT selected on clinical, pathological, and genetic grounds: UBE1 (human ubiquitin activating enzyme E1, located in chromosome Xp11.23), UBE2I (ubiquitin conjugating enzyme E2I, homologous to yeast UBC9, chromosome 16p13.3), GdX (ubiquitin-like protein, chromosome Xq28), SOX3 (SRY related HMG box gene 3, chromosome Xq26-q27), GABRA3 (gamma-aminobutyric acid type A receptor alpha3 subunit, chromosome Xq28), and CDR2 (cerebellar degeneration related autoantigen 2, chromosome 16p12-p13.1) . No mutations were detected in the coding regions of these six genes in 10 affected subjects and, therefore, alterations in the amino acid sequences of the encoded proteins can be excluded as having a causative role in RTT . Furthermore, gene expression of MECP2, GdX, GABRA3, and L1CAM (L1 cell adhesion molecule) was also investigated by in situ hybridisation . No gross differences were observed in neurones of several brain regions between normal controls and Rett patients.

Structure Fold Des, 2000 Mar 15, 8(3), 329 - 38
Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha; Conti E et al.; BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs) . The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif . Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor . RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution . Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide . The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues . CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them . The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS . In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region . Versatility in the specific recognition of NLSs relies on the modular.

Anal Chem, 2000 Mar 15, 72(6), 1334 - 41
DNA sensing on a DNA probe-modified electrode using ferrocenylnaphthalene diimide as the electrochemically active ligand; Takenaka S et al.; Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA . On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing . A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode . Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s . The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode . Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level . The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol.

Anal Chem, 2000 Mar 15, 72(6), 1163 - 8
Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer; Borchers C et al.; The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction . It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags) . Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity . Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search . This uncertainty, however, is overcome by MS/MS analysis of the peptides . Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein . This procedure is thus, highly sensitive, accurate, and rapid . In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling . Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.

Anal Chem, 2000 Mar 15, 72(6), 1112 - 8
Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching; Goodlett DR et al.; A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described . Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm . The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits . Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues . Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis . The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra . The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database . As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.

Prog Cell Cycle Res, 2000, 4, 107 - 14
Cell cycle regulation by the Cdc25 phosphatase family; Nilsson I et al.; Activation of cyclin-dependent kinases in higher eukaryotic cells can be achieved through dephosphorylation by members of the Cdc25 phosphatase family, Cdc25A, Cdc25B and Cdc25C . Cdc25A plays an important role at the G1/S-phase transition . Cdc25B undergoes activation during S-phase and plays a role in activating the mitotic kinase Cdk1/cyclin B in the cytoplasm . Active Cdk1/cyclin B then phosphorylates and activates Cdc25C leading to a positive feedback mechanism and to entry into mitosis . Cdc25A and B are potential human oncogenes . In addition, Cdc25 is a main player of the G2 arrest caused by DNA damage or in the presence of unreplicated DNA.

Trends Plant Sci, 2000 Apr, 5(4), 160 - 7
Pre-mRNA splicing in higher plants; Lorkovic ZJ et al.; Most plant mRNAs are synthesized as precursors containing one or more intervening sequences (introns) that are removed during the process of splicing . The basic mechanism of spliceosome assembly and intron excision is similar in all eukaryotes . However, the recognition of introns in plants has some unique features, which distinguishes it from the reactions in vertebrates and yeast . Recent progress has occurred in characterizing the splicing signals in plant pre-mRNAs, in identifying the mutants affected in splicing and in discovering new examples of alternatively spliced mRNAs . In combination with information provided by the Arabidopsis genome-sequencing project, these studies are contributing to a better understanding of the splicing process and its role in the regulation of gene expression in plants.

Exp Cell Res, 2000 Apr 10, 256(1), 168 - 78
Crk-associated substrate p130(Cas) interacts with nephrocystin and both proteins localize to cell-cell contacts of polarized epithelial cells; Donaldson JC et al.; Crk-associated substrate (p130(Cas), Cas) is a docking protein first recognized as having elevated phosphotyrosine content in mammalian cells transformed by v-Src and v-Crk oncoproteins . Subsequent studies have implicated Cas in the control of normal cell behavior through its roles in integrin-mediated signal transduction and organization of the actin cytoskeleton at sites of cell adhesion . In this study, we sought to gain new insight into normal Cas function by identifying previously unrecognized interacting proteins . A yeast two-hybrid screen using the C-terminal region of Cas as a bait identified the Src homology 3 (SH3) domain of the mouse "nephrocystin" protein-orthologous to a human protein whose loss of function leads to the cystic kidney disease familial juvenile nephronophthisis . The putative full-length mouse and partial canine nephrocystin sequences were deduced from cDNA clones . Additional studies using epitope-tagged mouse nephrocystin indicated that nephrocystin and Cas can interact in mammalian cells and revealed that both proteins prominently localize at or near sites of cell-cell contact in polarized Madin-Darby canine kidney epithelial cells . Our findings provide novel insight into the normal cellular activities regulated by both Cas and nephrocystin, and raise the possibility that these proteins have a related function in polarized epithelial cells .

Protein Sci, 2000 Jan, 9(1), 129 - 37
Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases; Raman CS et al.; The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39 . The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early . The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)) . N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation . A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter . Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c . This study illustrates that surface formation can be coupled to early events in protein folding . Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.

Protein Sci, 2000 Jan, 9(1), 83 - 94
Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB; Bateman KS et al.; Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases . Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme . All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases . To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB) . Four high resolution complex structures are being reported in this paper; the three beta-branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 A resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 A resolution . Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed . The beta-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type gamma-branched P1 residue for OMTKY3, Leu18I . Chi1 angles of approximately 40 degrees are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket . Dihedral angles of +60 degrees, -60 degrees, or 180 degrees are more commonly observed but 40 degrees is not unfavorable for the beta-branched side chains . Thr18I Ogamma1 also forms a hydrogen bond with Ser195 Ogamma in this orientation . The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.

Genes Chromosomes Cancer, 2000 May, 28(1), 121 - 5
Characterization of chromosome 1 abnormalities in malignant melanomas; Smedley D et al.; Chromosome 1 abnormalities are the most commonly detected aberrations in many cancers including malignant melanomas . Specific breakpoints are reported for malignant melanomas throughout the chromosome but especially at 1p36 and at several sites throughout 1p22-q21 . In addition, partial deletions and loss of heterozygosity have been found on 1p indicating the possible location of tumor suppressor genes . Here we have characterized the involvement of chromosome 1 in a series of seven malignant melanoma cell lines . Initial chromosome painting studies revealed that six of the cell lines had chromosome 1 rearrangements . Deletions involving 1p10-32, 1q11-44, and 1q25-44 were observed . The other rearrangement breakpoints included three in the 1q10-p11 region with the rest at 1p36, 1p34, 1p32, 1p31, 1p12-13, 1q21, and 1q23 . The breaks at 1q10-p11 were investigated further using an alpha-satellite 1 centromere probe and yeast artificial chromosomes (YACs) from the region . Two of the 1q10-p11 breaks mapped in the centromeric region, while the others mapped to variable sites . This suggests that the role of these rearrangements in the pathogenesis of melanomas does not involve the alteration of specific oncogenes in the breakpoint region . During the YAC mapping a previously undetected, small (<1 Mbp) del(1)(p10p11) was identified . This deletion lies within minimal overlapping deleted regions reported in head and neck as well as breast carcinomas and it could therefore facilitate the isolation of a carcinoma-associated tumor suppressor gene .

Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4144 - 9
Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis; Detter JC et al.; Few molecular events important to platelet biogenesis have been identified . Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents . Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins . Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon . Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets . Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets . These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis . Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.

Life Sci, 2000 Feb 18, 66(13), 1177 - 86
Purification and characterization of glycolactin, a novel glycoprotein from bovine milk; Ye XY et al.; A novel glycoprotein designated glycolactin, with a molecular weight of 64 kDa, a sequence hitherto unknown in the literature and capable of inhibiting the hemagglutinating activities of soybean lectin and Ricinus communis agglutinin 120, was isolated from bovine milk . Its lectin-inhibiting activity differed from that of lactoferrin, another milk protein . Like other milk proteins, glycolactin inhibited superoxide formation in vitro . Glycolactin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of about 31 nM . It exhibited ribonucleolytic (RNase) activity towards yeast transfer RNA with a pH optimum of 7.5, and specific RNase activity towards poly C . The purification protocol of glycolactin involved removal of globulin from the acid whey fraction of bovine milk by precipitation with 1.8 M (NH4)2SO4, and adsorption on the ion exchangers CM-Sepharose and Mono S . Deglycosylation of glycolactin using glycopeptidase F produced only a slight decrease of 4 kDa in the molecular weight of glycolactin.

Biosci Biotechnol Biochem, 2000 Feb, 64(2), 378 - 85
Identification of alpha-D-glucosylglycerol in sake; Takenaka F et al.; alpha-D-Glucosylglycerol (GG) was found for the first time in sake (Japanese rice wine) in an amount of about 0.5% . GG was also found in miso and mirin which had been brewed by using koji . GG was hydrolyzed into glucose and glycerol in an equimolar ratio with maltase (EC 3.2.1.20, alpha-glucosidase from yeast), but not with emulsin (EC 3.2.1.21, beta-glucosidase from almond) . The retention times and mass spectra of trimethylsilyl derivatives by a GC-MS analysis of GG in sake were comparable to those of various GG samples synthesized by glycol cleavage . It was proven that GG in sake consisted of three components, viz., 2-O-alpha-D-glucosyl-glycerol (GG-II), (2R)-1-O-alpha-D-glucosylglycerol (R-GG-I) and (2S)-1-O-alpha-D-glucosylglycerol (S-GG-I) . The ratio of the three components in GG was 6:66:28 for sake . It is considered that GG was formed by transglucosylation of the glucosyl groups to glycerol by alpha-glucosidase from koji in the sake mash.

Neurogenetics, 1998 Mar, 1(3), 189 - 96
Genetic fine mapping of the Miyoshi myopathy locus and exclusion of eight candidate genes; Bejaoui K et al.; Miyoshi myopathy (MM) is an early adult-onset, autosomal recessive disorder characterized by weakness and muscular atrophy starting in the distal muscles . The disease locus has been previously mapped by linkage analysis to chromosome 2p using the microsatellite marker D2S291 . Initial haplotype analysis of markers in families from three different origins (North American, Japanese, and Tunisian) suggested that the MM gene is located in a 4-cM region flanked by markers D2S292 on the telomeric side and D2S286 on the centromeric side . To delineate critical recombination events revealing a more refined localization of the MM gene, we have determined the pattern of segregation of 12 marker loci in two consanguineous families of Tunisian origin . In this study we have: (1) detected recombination events with the disease locus in one family, placing the MM gene most likely between markers D2S443 (CHLC.GGAA4D07.1876) and D2S2109; (2) generated a yeast artificial chromosome contig that spans approximately 3.8 megabases and extends from marker D2S358 to marker D2S286; (3) physically mapped 21 polymorphic markers, 5 genes, 3 STSs, and 1 EST within this contig; (4) detected and mapped a new polymorphism within this interval, allowing us to further reduce the MM locus to a 360-kilobase segment; (5) mapped the gene for the cytoskeletal protein beta-adducin within the MM candidate region, failing to find a consistent pattern of mutation of this gene in our MM patients; (6) excluded seven other candidate myopathy genes from the Miyoshi locus.

Bioseparation, 1999, 8(1-5), 77 - 83
Evaluation of the effect of in-bed sampling on expanded bed adsorption; Bruce LJ et al.; An expanded bed adsorption (EBA) column (5 cm diameter) has been modified to allow the abstraction of liquid samples from various positions along the height of an expanded bed . As the adsorbent particles were fluidized, in-bed monitoring of key component concentrations during feedstock application, washing and elution was achieved by the withdrawal of liquid samples from the voids within the expanded bed through ports along the wall of the column . Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line . On-line monitoring can be used to control the duration of the loading stage and as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption . Studies of residence time distributions indicated that the modifications to the column do not significantly affect liquid dispersion . Using the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on Streamline DEAE as a model system, comparison of breakthrough curves for runs when in-bed monitoring was and was not performed also suggested that separation efficiency is not appreciably affected by in-bed sampling.

Bioseparation, 1999, 8(1-5), 69 - 75
The effect of column verticality on separation efficiency in expanded bed adsorption; Bruce LJ et al.; The effect of column verticality on liquid dispersion and separation efficiency in expanded bed adsorption columns was investigated using 1 and 5 cm diameter columns . Column misalignment of only 0.15 degree resulted in the reduction of the Bodenstein number from 140 to 50 for the 1 cm dia . column and from 75 to 45 for the 5 cm dia . column . This degree of misalignment was not detectable by visual assessment of adsorbent particle movement within the column . Depending on the relative importance of transport limitations, kinetic limitations and dispersion to any specific separation, this increase in dispersion with column alignment can significantly affect separation efficiency . Pure protein breakthrough profiles resulting from the application of bovine serum albumin onto STREAMLINE Q XL demonstrated that, at 10% breakthrough, 7.8% more protein could be applied to a vertical 1 cm dia . column compared to the same column misaligned by 0.15 degree . When an unclarified yeast homogenate was applied to a 1 cm dia . vertical column packed with STREAMLINE DEAE, 10% breakthrough of glucose-6-phosphate dehydrogenase (G6PDH) corresponded to a load 55% greater compared to the same column aligned 0.185 degree off-vertical . The G6PDH breakthrough curves for vertical and 0.15 degree off-vertical runs performed using a 5 cm column were essentially indistinguishable.

FEBS Lett, 2000 Mar 24, 470(2), 207 - 10
The survival motor neuron protein interacts with the transactivator FUSE binding protein from human fetal brain; Williams BY et al.; To identify interacting proteins of survival motor neuron (SMN) in neurons, a fetal human brain cDNA library was screened using the yeast two-hybrid system . One identified group of SMN interacting clones encoded the DNA transactivator FUSE binding protein (FBP) . FBP overexpressed in HEK293 cells or endogenously expressed in fetal and adult mouse brain bound specifically in vitro to recombinant SMN protein . Furthermore, an anti-FBP antibody specifically co-immunoprecipitated SMN when both proteins were overexpressed in HEK293 cells . These results demonstrate that FBP is a novel interacting partner of SMN and suggests a possible role for SMN in neuronal gene expression.

FEBS Lett, 2000 Mar 24, 470(2), 147 - 50
Evidence for the existence of rhodanese (thiosulfate:cyanide sulfurtransferase) in plants: preliminary characterization of two rhodanese cDNAs from Arabidopsis thaliana; Hatzfeld Y et al.; The existence of rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) in plants has been highly controversial . We have isolated and characterized for the first time in plants two cDNAs encoding rhodanese isoforms in Arabidopsis thaliana, AtRDH1 and AtRDH2 . Both cDNAs contained a full-length open reading frame, the expression of which increased the rhodanese activity of transgenic yeast . AtRDH1 protein was mitochondrial, while AtRDH2 was cytosolic . AtRDH1 and AtRDH2 genes originated from the duplication of a large genomic region in chromosome 1 which took place before the appearance of the Arabidopsis genus . Our results confirm the existence of rhodanese in plants.

FEBS Lett, 2000 Mar 24, 470(2), 107 - 12
Arabidopsis phytochromes C and E have different spectral characteristics from those of phytochromes A and B; Eichenberg K et al.; The red/far-red light absorbing phytochromes play a major role as sensor proteins in photomorphogenesis of plants . In Arabidopsis the phytochromes belong to a small gene family of five members, phytochrome A (phyA) to E (phyE) . Knowledge of the dynamic properties of the phytochrome molecules is the basis of phytochrome signal transduction research . Beside photoconversion and destruction, dark reversion is a molecular property of some phytochromes . A possible role of dark reversion is the termination of signal transduction . Since Arabidopsis is a model plant for biological and genetic research, we focussed on spectroscopic characterization of Arabidopsis phytochromes, expressed in yeast . For the first time, we were able to determine the relative absorption maxima and minima for a phytochrome C (phyC) as 661/725 nm and for a phyE as 670/724 nm . The spectral characteristics of phyC and E are strictly different from those of phyA and B . Furthermore, we show that both phyC and phyE apoprotein chromophore adducts undergo a strong dark reversion . Difference spectra, monitored with phycocyanobilin and phytochromobilin as the apoprotein's chromophore, and in vivo dark reversion of the Arabidopsis phytochrome apoprotein phycocyanobilin adducts are discussed with respect to their physiological function.

J Biol Chem, 2000 Mar 31, 275(13), 9542 - 9
Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells; Su HP et al.; Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms . The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated . Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans . CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain . Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment . In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain . Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo . Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C . elegans, rodent, and human CED-6 proteins . We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

J Biol Chem, 2000 Mar 31, 275(13), 9461 - 7
The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex; Grimes JA et al.; The repressor REST/NRSF restricts expression of a large set of genes to neurons by suppressing their expression in non-neural tissues . We find that REST repression involves two distinct repressor proteins . One of these, CoREST, interacts with the COOH-terminal repressor domain of REST (Andres, M . E., Burger, C., Peral-Rubio, M . J., Battaglioli, E., Anderson, M . E., Grimes, J., Dallmanm J., Ballas, N . , and Mandel, G . (1999) Proc . Natl . Acad . Sci . U . S . A . 96, 9873-9878) . Here we show that the co-repressor mSin3A also interacts with REST . The REST-mSin3A association involves the NH(2)-terminal repressor domain of REST and the paired amphipathic helix 2 domain of mSin3A . REST forms complexes with endogenous mSin3A in mammalian cells, and both mSin3A and CoREST interact with REST in intact mammalian cells . REST repression is blocked in yeast lacking Sin3 and rescued in its presence . In mammalian cells, repression by REST is reduced when binding to mSin3A is inhibited . In mouse embryos, the distribution of mSin3A and REST transcripts is largely coincident . The pattern of CoREST gene expression is more restricted, suggesting that mSin3A is required constitutively for REST repression, whereas CoREST is recruited for more specialized repressor functions.

Biochem Biophys Res Commun, 2000 Apr 2, 270(1), 34 - 9
Molecular cloning and characterization of Xenopus RGS5; Saitoh O et al.; We identified six genes that encode putative RGS proteins (XRGSI-VI) in developing Xenopus embryos using PCR amplification with degenerate primers corresponding to the conserved region (RGS domain) of known RGS proteins . RT-PCR analysis revealed that mRNAs of these XRGSs are differentially expressed during embryogenesis . At stage 1, only XRGSII mRNA was detected . On the other hand, expression of XRGSVI mRNA increased apparently at stage 14 and expression of three of other XRGS (III, IV, V) elevated between stage 25 and 40 . To further characterize XRGS proteins expressed in Xenopus embryos, we isolated a cDNA clone for XRGSIII . Based on determined nucleotide sequence, XRGSIII was considered as a Xenopus homologue of mammalian RGS5 (XRGS5) . Genetic analysis using the pheromone response halo assay showed that expression of XRGS5 inhibits yeast response to alpha-factor, suggesting that XRGS5 negatively regulates the G-protein-mediated signaling pathway in developing Xenopus embryos .

Nucleic Acids Res . 2000 Apr 15;28(8):E30.
Rational design of landmark probes for quantitative DNA fiber mapping (QDFM); Hsieh HB et al.; Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs . We recently developed a procedure termed 'quantitative DNA fiber mapping' (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs . QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to approximately 2.3 kb/microm . In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping . Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs . As demonstrated in representative hybridizations, vector-specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences.

Nucleic Acids Res, 2000 Apr 15, 28(8), 1692 - 9
An ATM homologue from Arabidopsis thaliana: complete genomic organisation and expression analysis; Garcia V et al.; ATM is a gene mutated in the human disease ataxia telangiectasia with reported homologues in yeast, Drosophila, Xenopus and mouse . Whenever mutants are available they all indicate a role of this gene family in the cellular response to DNA damage . Here, we present the identification and molecular characterisation of the first plant homologue of ATM . The genomic locus of AtATM ( Arabidopsis thaliana homologue of ATM ) spans over 30 kb and is transcribed into a 12 kb mRNA resulting from the splicing of 79 exons . It is a single copy gene and maps to the long arm of chromosome 3 . Transcription of AtATM is ubiquitous and not induced by ionising radiation . The putative protein encoded by AtATM is 3856 amino acids long and contains a phosphatidyl inositol-3 kinase-like (Pi3k-l) domain and a rad3 domain, features shared by other members of the ATM family . The AtAtm protein is highly similar to Atm, with 67 and 45% similarity in the Pi3k-l and rad3 domains respectively . Interestingly, the N-terminal portion of the protein harbours a PWWP domain, which is also present in other proteins involved in DNA metabolism such as human mismatch repair enzyme Msh6 and the mammalian de novo methyl transferases, Dnmt3a/b.

Blood, 2000 Apr 1, 95(7), 2297 - 303
The alpha(E)C domain of human fibrinogen-420 is a stable and early plasmin cleavage product; Applegate D et al.; Human fibrinogen-420, (alpha(E)betagamma)(2), was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis . Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (alphabetagamma)(2), were found to be similar, suggesting little impact of the unique alpha(E)C domains of fibrinogen-420 on coagulation . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the alpha(E)C domain . One was a stable fragment (alpha(E)CX) comigrating with a 34-kd yeast recombinant alpha(E)C domain, and the other was an apparent precursor . Their release occurred early, before that of fragments D and E . Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

J Biochem (Tokyo), 2000 Jan, 127(1), 85 - 94
Cloning and sequencing of the gene for a Tetrahymena fimbrin-like protein; Watanabe A et al.; Tetrahymena F-actin-binding protein, which induces bundling of Tetrahymena F-actin, was localized to a division furrow during cytokinesis . We report here the cloning and characterization of the gene and cDNA of a Tetrahymena F-actin-binding protein . The cDNA encodes a protein comprising 579 deduced amino acids with a calculated molecular mass of 65.9 kDa . The predicted amino acid sequence shares 37.7, 41.8, and 39% identity with the sequences of yeast fimbrin, Arabidopsis thaliana fimbrin, and Dictyostelium discoideum plastin, respectively . The Tetrahymena F-actin-binding protein also shares two actin-binding domains previously identified in the fimbrin/plastin family, but lacks the EF-hand Ca2+-binding motif, suggesting that this protein is a novel-fimbrin-like protein in Tetrahymena . Moreover, we cloned a genomic DNA encoding the Tetrahymena fimbrin-like protein and performed Southern and Northern hybridizations . The results indicate that the genomic DNA possesses 9 introns and that both the gene and transcript of Tetrahymena fimbrin-like protein are single . Thus, we suggest that Tetrahymena fimbrin-like protein localizes to the division furrow and probably cross-links actin filaments in a Ca(2+)-insensitive manner during cytokinesis.

J Steroid Biochem Mol Biol, 2000 Jan-Feb, 72(1-2), 35 - 46
Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1a depends on residues flanking the motif; Needham M et al.; Steroid hormones induce the transcriptional activity of their cognate receptors by recruiting a variety of cofactors . One of these, steroid receptor co-activator-1 (SRC-1) interacts with the ligand binding domains of a number of different receptors by means of LXXLL motifs . We have investigated the relative interaction of four such motifs in SRC-1a using a yeast two-hybrid assay . We demonstrate that ERalpha, ERbeta and ERbeta2 preferentially interact with motif 2 while GR, AR, PPARalpha and PPARgamma preferentially interact with motif 4 . We show that the interactions depend not only on the LXXLL motif itself but also on residues flanking the motif.

Cancer Genet Cytogenet, 2000 Apr 1, 118(1), 28 - 34
Microdissection and FISH investigations in acute myeloid leukemia: a step forward to full identification of complex karyotypic changes; Falzetti D et al.; Complex chromosomal rearrangements in malignant hemopathies frequently remain unclarified because of paucity of material for further fluorescence in situ hybridization analyses and/or lack of suitable probes . Chromosome microdissection (MD) can be an adequate approach to elucidate chromosome aberrations unrecognizable by conventional karyotyping . We applied MD in two patients with acute myeloid leukemia (AML) and unidentified chromosome changes at karyotype . Microdissection of a ring chromosome in an AML-M5 case revealed 21q polysomy . In an AML-M4 case, MD of an add(15p) disclosed a t(8;15) with over-representation of both 8q22 and 8q24 bands . YAC probes were helpful in showing duplication of the ETO gene at 8q22, and amplification of C-MYC, at 8q24.

Neoplasma, 1999, 46(6), 384 - 9
p53 status in breast carcinomas revealed by FASAY correlates well with p53 protein accumulation determined by immunohistochemistry; Smardova J et al.; The prognostic and predictive value of p53 mutation in breast cancer is still conflicting . The choice of the p53 status detection method may account for some discrepancies . In this pilot study we compared two differently-based methods for detection of p53 alteration in 32 breast carcinoma samples: the immunohistochemical method using Bp53, DO1 and DO11 monoclonal antibodies for analysis of the p53 protein accumulation in cell nuclei and the functional method FASAY . FASAY - functional analysis of the separated alleles in yeast - tests the capability of the human p53 to transactivate a reporter with a p53 binding site RGC driving the ADE2 gene in yeast . In our group the percentage of breast cancers with accumulated p53 protein was 50%, as well as percentage of mutant p53 scored by FASAY was 50% . Although the agreement of both methods, when comparing the results of individual patients was high (94%), our results show that immunohistochemistry does not reflect the p53 status quite exactly.

Endocr Relat Cancer, 1999 Jun, 6(2), 149 - 56
Breast tumor aromatase: functional role and transcriptional regulation; Chen S et al.; Aromatase has been shown to be expressed at a higher level in human breast cancer tissue than in normal breast tissue, by means of enzyme activity measurement, immunocytochemistry, and RT-PCR analysis . Cell culture including MCF-7 breast cancer cells, animal experiments using aromatase-transfected breast cancer cells, and transgenic mouse studies have demonstrated that estrogen production in situ plays a more important role than circulating estrogens in breast tumor promotion . In addition, tumor aromatase is believed to be able to stimulate breast cancer growth through both autocrine and paracrine pathways, as demonstrated by a three-dimensional cell culture study . RT-PCR and gene transcriptional studies have revealed that the aromatase promoter is switched from a glucocorticoid-stimulated promoter, I.4, in normal tissue to cAMP-stimulated promoters, I.3 and II, in cancerous tissue . Recently, we identified and characterized a cAMP-responsive element (CREaro) upstream from promoter I.3 by DNA deletion and mutational analyses . Our results from promoter functional analysis also demonstrated an interaction between the CREaro and the silencer element (S1) that was identified previously in our laboratory . In the presence of cAMP, the positive regulatory CREaro can overcome the action of the silencer on the function of promoter I.3 . On the basis of results generated from our own and other laboratories, we propose that, in normal breast adipose stromal cells and fibroblasts, aromatase expression is driven by promoter I.4 (glucocorticoid dependent), and that the action of promoters I.3 and II is suppressed by the silencer negative regulatory element . However, in cancer cells and surrounding adipose stromal cells, the cAMP level increases, and aromatase promoters are switched to cAMP-dependent promoters - I.3 and II . Furthermore, we applied the yeast one-hybrid screening method to search for proteins interacting with the silencer element, S1 . The major protein identified was ERRalpha-1; however, SF-1, which is present in the ovary, is not detected in breast cancer tissue . Using a reporter plasmid with the aromatase genomic fragment containing promoter I.3 and S1, in breast cancer SK-BR-3 cells, ERRalpha-1 was found to have a positive regulatory function . It is believed that the silencer element in the human aromatase gene may function differently in different tissues, as a result of distinct expression patterns of transcription factors.

Br J Dermatol, 1999 Nov, 141 Suppl 56, 33 - 5
Resistance of Candida species to antifungal agents used in the treatment of onychomycosis: a review of current problems; Evans EG; Treatment of Candida infections with fluconazole has resulted in the emergence of drug resistance, a problem particularly apparent in HIV-infected patients . Frequently, the yeast is also cross-resistant to itraconazole and other azoles . In neutropenic patients fluconazole therapy or prophylaxis has caused overgrowth and infection by inherently less susceptible species of Candida, principally C . glabrata and C . krusei . Consequently, the use of intermittent long-term azole therapy to treat onychomycosis could result in changes in the commensal yeast flora of patients--either resistance or pathogen shift . An 'off-study' investigation undertaken in patients receiving either continuous terbinafine or intermittent itraconazole for toenail onychomycosis (L.I.ON . study) showed no evidence of changes in the yeast species present, nor in their sensitivity to itraconazole or fluconazole . Although intermittent itraconazole seems unlikely to cause problems in this respect, the situation with regard to intermittent fluconazole therapy of onychomycosis needs further study.

Community Dent Oral Epidemiol, 2000 Apr, 28(2), 141 - 9
Effects of an oral health program on the occurrence of oral candidosis in a long-term care facility; Budtz-Jorgensen E et al.; OBJECTIVES: The aim was to evaluate the effectiveness of a preventive oral health program on the prevalence of oral candidosis in 237 frail or dependent residents in a long-term care facility . Half of the residents were included in an experimental group which benefited from a preventive oral hygiene program including instruction of the carers and implementation of a recall program for professional oral hygiene care . METHODS: Intraoral examinations and yeast cultures from the oral mucosa and the fitting denture surface were carried out at baseline and 18 months later . The outgrowth of yeast was estimated on Oricult-N dip slides using the scale: no growth; 1-20 colonies; 21-100 colonies; >100 colonies . RESULTS: At baseline (n = 237) and at 18 months (n = 159) the experimental and the control groups were similar with regard to the residents' distribution by age, sex, dental and prosthetic status and prevalence of denture stomatitis . The 78 residents lost had the same baseline characteristics as the survivors, except for being older . In the experimental group the severity of the inflammation of the palate decreased (P = 0.005) as well as the prevalence of glossitis (P = 0.005) . At baseline high yeast scores from the mucosa (>20) were observed in about 50% of the residents in the experimental as well as the control group . At 18 months this figure was 23.4% for the experimental and 48.7% for the control group (P = 0.001) . There was also a reduction of the number of residents with positive cultures and the denture yeast scores at 18 months in the experimental group (P = 0.05) . CONCLUSIONS: This study has shown that the preventive program was effective in reducing the colonization of the oral mucosa and dentures by Candida and thereby improving the health of the oral mucosa.

Genomics, 2000 Mar 1, 64(2), 195 - 202
Identification and characterization of novel genes located at the t(1;15)(p36.2;q24) translocation breakpoint in the neuroblastoma cell line NGP; Amler LC et al.; The distal portion of chromosome 1p is frequently deleted in several human cancers, suggesting the presence of one or more putative tumor suppressor genes on this chromosomal arm . In human neuroblastoma, a consistently deleted region at 1p36.1-p36.2 has been defined by comparison of molecular loss of heterozygosity (LOH) analyses . Recently we described the identification of a yeast artificial chromosome, YAC 927G4, that spans a translocation/duplication breakpoint within the minimally defined LOH region at 1p36.1-p36.2 in the neuroblastoma cell line NGP . Here we describe the identification of two overlapping P1 artificial chromosomes comprising 220 kb at the distal end of YAC 927G4, which we have used as hybridization probes under modified conditions to screen a composite, normalized cDNA library (IMAGE cDNA library) . Hybridization screening resulted in the rapid and comprehensive identification of partial cDNAs of which a portion comprised two novel candidate genes, termed DNB1/ARPh and DNB5, which encode putative proteins of 1011 and 447 amino acids, respectively . The DNB1/ARPh gene, which was found to be ubiquitously expressed in human adult and fetal tissues, is highly related to the DRPLA gene, in which expansion of a CAG triplet appears to be causal in the dentatorubral and pallidolysian atrophy disease phenotype . The DNB5 sequence, in contrast, which is predominantly expressed in brain tissues and fetal kidney, failed to show any similarity to sequences in the public domain . A preliminary assessment of transcription and sequence of both genes in several neuroblastoma cell lines does not, thus far, support a causal role in neuroblastoma . However, further analyses are required to confirm these results .

Biochem J, 2000 Mar 1, 346 Pt 2, 281 - 93
Pancreatic eukaryotic initiation factor-2alpha kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress; Sood R et al.; In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha) . Recently, we identified a new family member, pancreatic eIF-2alpha kinase (PEK) from rat pancreas . PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER . In this study, we identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans . Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues . In mammalian cells subjected to ER stress, we found that elevated eIF-2alpha phosphorylation was coincident with increased PEK autophosphorylation and eIF-2alpha kinase activity . Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen . To address the role of C . elegans PEK in translational control, we expressed this kinase in yeast and found that it inhibits growth by hyperphosphorylation of eIF-2alpha and inhibition of eIF-2B . Furthermore, we found that vaccinia virus K3L protein, an inhibitor of the eIF-2alpha kinase PKR involved in an anti-viral defence pathway, also reduced PEK activity . These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway.

Eur J Biochem, 2000 Apr, 267(7), 2113 - 21
Identification of tudor repeat associator with PCTAIRE 2 (Trap) . A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain; Hirose T et al.; PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron . To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system . A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2) . The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila tudor protein . Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains . PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap . Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2 . Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells . Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum . These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons.

Eur J Biochem, 2000 Apr, 267(7), 2046 - 53
Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella; Frobius AC et al.; Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level . These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation . ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae . Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples . The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3 . The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.

Biochim Biophys Acta, 2000 Apr 15, 1501(1), 63 - 9
Identification of a novel human member of the DEAD box protein family; Suk K et al.; The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM) . From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family . Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family . DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division . RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family . Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets . In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.

Biochimie, 2000 Feb, 82(2), 117 - 22
The selenocysteine insertion sequence binding protein SBP is different from the Y-box protein dbpB; Fagegaltier D et al.; In eukaryotes, translation of internal UGA selenocysteine codons requires the SECIS stem-loop structure in the 3'UTR of selenoprotein mRNAs . In an earlier work, we identified SBP as a selenocysteine insertion sequence (SECIS)-binding protein . Here, the yeast three-hybrid screen was employed to capture the cDNA of SBP . One candidate, satisfying the genetic screens, was identified as the already known dbpB protein . Although it was also found by another group, but with a different strategy, to carry SECIS-binding activity, further experiments enabled us to show that dbpB was unable to bind the SECIS element in vitro . Altogether, our findings led us to conclude that, under our conditions, dbpB and SBP are two distinct proteins.

Neuropharmacology, 2000 Apr 3, 39(6), 919 - 30
Interactions between AMPA receptors and intracellular proteins; Braithwaite SP et al.; alpha-Amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors mediate most fast excitatory synaptic transmission in the mammalian CNS . They play a central role in synapse stabilisation and plasticity and their prolonged activation is potently neurotoxic . Developmental and activity-dependent changes in the functional synaptic expression of these receptors are subject to tight cellular regulation . The molecular and cellular mechanisms which control the postsynaptic insertion and arrangement of individual AMPA receptor variants are therefore the subject of intense investigation and in the last two years there has been significant progress towards elucidating some of the processes involved . Much of the new information has come from the application of the yeast two-hybrid assay, which has led to the discovery of a hitherto unexpected complexity of proteins which selectively interact with individual AMPA receptor subunits . These proteins have been implicated in the regulation of AMPA receptor post-translational modification, targeting and trafficking, surface expression and anchoring . The aim of this article is to present an overview of the major interacting proteins described so far and to place these in the context of how they may participate in the well ordered series of events controlling the cell biology of AMPA receptors.

J Clin Invest, 2000 Mar, 105(6), 757 - 64
Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia; Furuyama K et al.; The first and the rate-limiting enzyme of heme biosynthesis is delta-aminolevulinate synthase (ALAS), which is localized in mitochondria . There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N) . To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system . Our screening led to the isolation of the beta subunit of human ATP-specific succinyl CoA synthetase (SCS-betaA) . Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-betaA associates specifically with ALAS-E and not with ALAS-N . Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-betaA, but the D190V mutant did not . Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-betaA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria.

Biochem J, 2000 Apr 1, 347 Pt 1, 55 - 9
PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor which binds to the two mouse type-I collagen gene promoters; Hasegawa T et al.; We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes . We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain . One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction . Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene . In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines . Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proalpha2(I) collagen proximal promoter in vitro . The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proalpha2(I) collagen proximal promoter activity . We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

Biochemistry, 2000 Mar 28, 39(12), 3472 - 9
Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme; Arai M et al.; Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain . A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast . Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique . Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type . Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding . In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism . Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate . The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.

Biochemistry, 2000 Mar 28, 39(12), 3206 - 15
Fetal Alz-50 clone 1 (FAC1) protein interacts with the Myc-associated zinc finger protein (ZF87/MAZ) and alters its transcriptional activity; Jordan-Sciutto KL et al.; Transcription factors mediate their regulatory effects through interaction with DNA and numerous nuclear proteins . The fetal Alz-50 clone 1 (FAC1) protein, a novel DNA-binding protein with the capacity to repress transcription, is likely to function through a similar mechanism (1) . Using the two-hybrid yeast screen, we have shown that FAC1 interacts with the myc-associated zinc finger protein (ZF87/MAZ) . This association was confirmed in vitro with recombinant protein . The ZF87/MAZ interaction domain was mapped to the region containing a putative nuclear localization signal (NLS) and nuclear export sequence (NES) of FAC1, using deletion mutants of the FAC1 protein . FAC1, on the other hand, recognizes a conformational interface that includes the proline/alanine-rich domain of ZF87/MAZ and the first zinc finger . Cotransfection of NIH3T3 cells with ZF87/MAZ and a luciferase reporter containing the SV40 promoter and enhancer results in an increase in transcriptional activation, suggesting ZF87/MAZ is able to recognize its consensus binding site present in the SV40 promoter . Cotransfection with FAC1 reduces the level of ZF87/MAZ-induced activation of the SV40 promoter in a dose dependent manner . A mutant FAC1, lacking the ZF87/MAZ interaction domain, does not alter ZF87/MAZ activation of the SV40 promoter . These data demonstrate that interaction between FAC1 and ZF87/MAZ alters the transactivation capacity of ZF87/MAZ . By immunoblot analysis, FAC1 and ZF87/MAZ exhibit similar tissue distribution and co-localize to pathologic structures in Alzheimer's disease brain . Coexpression of FAC1 and ZF87/MAZ suggest that interaction of these two proteins will have biological implications for gene regulation in neurodegeneration.

DNA Seq, 1999, 10(4-5), 263 - 99
A 356-Kb sequence of the subtelomeric part of the MHC Class I region; Hampe A et al.; The subtelomeric part of the MHC Class I region contains 11 of the 21 genes described on chromosome 6 at position 6p21.3 . The general organization of those and other genes resident in the region was revealed by determining a 356,376 bp sequence . Potential exons for new genes were identified by computer analysis and a large number of ESTs were selected by testing the sequence by the BLAST algorithm against the GenBank nonredundant and EST databases . Most of the ESTs are clustered in two regions . In contrast, the whole HLA-gene region is crammed with LINE and SINE repeats, fragments of genes and microsatellites, which tends to hinder the identification of new genes.

Eur J Cell Biol, 2000 Feb, 79(2), 92 - 103
Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants; Lee SS et al.; Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology . By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively . W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding . The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged . Binding to phospholipids was indistinguishable from the wild-type profilin . The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D . discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994) . Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development . Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations . Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles . These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.

Mutat Res, 2000 Mar 20, 459(2), 123 - 33
Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle; Pichierri P et al.; Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood . The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases . The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III . In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III . The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II . This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor . Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle . No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage . These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.

J Gen Virol, 2000 Apr, 81(Pt 4), 939 - 47
A short leucine-rich sequence in the Borna disease virus p10 protein mediates association with the viral phospho- and nucleoproteins; Wolff T et al.; Borna disease virus (BDV) is unique among the non-segmented negative-strand RNA viruses of animals and man because it transcribes and replicates its genome in the nucleus of the infected cell . It has recently been discovered that BDV expresses a gene product of 87 amino acids, the p10 protein, from an open reading frame that overlaps with the gene encoding the viral p24 phosphoprotein . In addition, the p10 protein has been localized to intranuclear BDV-specific clusters containing viral antigens . Here, characterization of p10 interactions with the viral nucleoprotein p38/p39 and the p24 phosphoprotein is reported . Immunoaffinity chromatography demonstrated the presence of high-salt stable complexes of p10 containing the p24 and p38/p39 proteins in extracts of BDV-infected cells . Analyses in the yeast two-hybrid system and biochemical co-precipitation experiments suggested that the p10 protein binds directly to the p24 phosphoprotein and indirectly to the viral nucleoprotein . Mutational analysis demonstrated that a leucine-rich stretch of amino acids at positions 8-15 within the p10 protein is critical for interaction with p24 . Furthermore, binding of p10 to the viral phosphoprotein was shown to be important for association with the BDV-specific intranuclear clusters that may represent the sites of virus replication and transcription in infected cells . These findings are discussed with respect to possible roles for the p10 protein in viral RNA synthesis or ribonucleoprotein transport.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3596 - 601
The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses; Garcia RA et al.; Neuregulins regulate the expression of ligand- and voltage-gated channels in neurons and skeletal muscle by the activation of their cognate tyrosine kinase receptors, ErbB 1-4 . The subcellular distribution and mechanisms that regulate the localization of ErbB receptors are unknown . We have found that ErbB receptors are present in brain subcellular fractions enriched for postsynaptic densities (PSD) . The ErbB-4 receptor is unique among the ErbB proteins because its C-terminal tail (T-V-V) conforms to a sequence that binds to a protein motif known as the PDZ domain . Using the yeast two-hybrid system, we found that the C-terminal region of ErbB-4 interacts with the three related membrane-associated guanylate kinases (MAGUKs) PSD-95/SAP90, PSD-93/chapsyn-110, and SAP 102, which harbor three PDZ domains, as well as with beta(2)-syntrophin, which has a single PDZ domain . As with N-methyl-D-aspartate (NMDA) receptors, ErbB4 interacts with the first two PDZ domains of PSD-95 . Using coimmunoprecipitation assays, we confirmed the direct interactions between ErbB-4 and PSD-95 in transfected heterologous cells, as well as in vivo, where both proteins are coimmunoprecipitated from brain lysates . Moreover, evidence for colocalization of these proteins was also observed by immunofluorescence in cultured hippocampal neurons . ErbB-4 colocalizes with PSD-95 and NMDA receptors at a subset of excitatory synapses apposed to synaptophysin-positive presynaptic terminals . The capacity of ErbB receptors to interact with PDZ-domain proteins at cell junctions is conserved from invertebrates to mammals . As discussed, the interactions found between receptor tyrosine kinases and MAGUKs at neuronal synapses may have important implications for activity-dependent plasticity.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3730 - 4
The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance; Liu J et al.; In Arabidopsis thaliana, the Salt Overly Sensitive 2 (SOS2) gene is required for intracellular Na(+) and K(+) homeostasis . Mutations in SOS2 cause Na(+) and K(+) imbalance and render plants more sensitive toward growth inhibition by high Na(+) and low K(+) environments . We isolated the SOS2 gene through positional cloning . SOS2 is predicted to encode a serine/threonine type protein kinase with an N-terminal catalytic domain similar to that of the yeast SNF1 kinase . Sequence analyses of sos2 mutant alleles reveal that both the N-terminal catalytic domain and the C-terminal regulatory domain of SOS2 are functionally essential . The steady-state level of SOS2 transcript is up-regulated by salt stress in the root . Autophosphorylation assays show that SOS2 is an active protein kinase . In the recessive sos2-5 allele, a conserved glycine residue in the kinase catalytic domain is changed to glutamate . This mutation abolishes SOS2 autophosphorylation, indicating that SOS2 protein kinase activity is required for salt tolerance.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3735 - 40
The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3; Halfter U et al.; The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na(+) and K(+) homeostasis and plant tolerance to high Na(+) and low K(+) environments . SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B . SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family . We report here that SOS3 physically interacts with and activates SOS2 protein kinase . Genetically, sos2sos3 double mutant analysis indicates that SOS2 and SOS3 function in the same pathway . Biochemically, SOS2 interacts with SOS3 in the yeast two-hybrid system and in vitro binding assays . The interaction is mediated by the C-terminal regulatory domain of SOS2 . SOS3 activates SOS2 protein kinase activity in a Ca(2+)-dependent manner . Therefore, SOS3 and SOS2 define a novel regulatory pathway important for the control of intracellular ion homeostasis and salt tolerance in plants.

J Cell Biol, 2000 Mar 20, 148(6), 1255 - 65
Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building; Takeda S et al.; Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins . The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells . To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out . They significantly blocked fast axonal transport and brought about inhibition of neurite extension . A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3 . This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations . Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles . In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components . Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.

Development, 2000 Apr, 127(8), 1651 - 60
A S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes; Corellou F et al.; S/M checkpoints prevent various aspects of cell division when DNA has not been replicated . Such checkpoints are stringent in yeast and animal somatic cells but are usually partial or not present in animal embryos . Because little is known about S/M checkpoints in plant cells and embryos, we have investigated the effect of aphidicolin, a specific inhibitor of DNA polymerases (alpha) and (delta), on cell division and morphogenesis in Fucus and Pelvetia zygotes . Both DNA replication and cell division were inhibited by aphidicolin, indicating the presence, in fucoid zygotes, of a S/M checkpoint . This checkpoint prevents chromatin condensation, spindle formation, centrosomal alignment with the growth axis and cytokinesis but has no effect on germination or rhizoid elongation . This S/M checkpoint also prevents tyrosine dephosphorylation of cyclin-dependent kinase-like proteins at the onset of mitosis . The kinase activity is restored in extracts upon incubation with cdc25A phosphatase . When added in S phase, olomoucine, a specific inhibitor of cyclin-dependent kinases, has similar effects as aphidicolin on cell division although alignment of the centrosomal axis still occurs . We propose a model involving the inactivation of CDK-like proteins to account for the S/M DNA replication checkpoint in fucoid zygotes and embryos.

J Cell Sci, 2000 Apr, 113 ( Pt 8), 1355 - 64
Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro; Laoukili J et al.; Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins . It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca(2+)-sensitive structures . Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells . Three centrin genes have been cloned in human cells . In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process . RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation . Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression . Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies . It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3 . This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.

J Agric Food Chem, 2000 Mar, 48(3), 849 - 52
Insulin-like biological activity of culinary and medicinal plant aqueous extracts in vitro; Broadhurst CL et al.; To evaluate the possible effects on insulin function, 49 herb, spice, and medicinal plant extracts were tested in the insulin-dependent utilization of glucose using a rat epididymal adipocyte assay . Cinnamon was the most bioactive product followed by witch hazel, green and black teas, allspice, bay leaves, nutmeg, cloves, mushrooms, and brewer's yeast . The glucose oxidation enhancing bioactivity was lost from cinnamon, tea, witch hazel, cloves, bay leaf and allspice by poly(vinylpyrrolidone) (PVP) treatment, indicating that the active phytochemicals are likely to be phenolic in nature . The activity of sage, mushrooms, and brewers's yeast was not removed by PVP . Some products such as Korean ginseng, flaxseed meal, and basil have been reported to be effective antidiabetic agents; however, they were only marginally active in our assay . Our technique measures direct stimulation of cellular glucose metabolism, so it may be that the active phytochemicals in these plants improve glucose metabolism via other mechanisms or that this in vitro screening is not a reliable predictor of hypoglycemic effects in vivo for some products . In summary, the positive effects of specific plant extracts on insulin activity suggest a possible role of these plants in improving glucose and insulin metabolism.

Allergy, 2000 Feb, 55(2), 163 - 70
Indoor air pollutants in schools: nasal patency and biomarkers in nasal lavage; Norback D et al.; BACKGROUND: There is growing concern about the respiratory health aspects of the indoor air quality in schools . METHODS: A standardized investigation, including nasal lavage (NAL), measurement of the nasal cavity by acoustic rhinometry, and hygienic measurements of airborne pollutants, was performed in classrooms, outside the pollen season . All 279 school personnel working in the main buildings of 12 randomly selected primary schools in an urban community in central Sweden (Uppsala) were invited to enroll in the study; 234 (84%) participated . Eosinophil cationic protein (ECP), myeloperoxidase (MPO), lysozyme, and albumin were analyzed in NAL fluid . Crude statistical analysis, as well as multiple regression analysis, was performed, controlling for room temperature, age, sex, current smoking, and a history of atopy . RESULTS: Most classrooms (83%) did not meet the Swedish ventilation standards . A lower degree of nasal patency was found at higher concentrations of respirable dust, nitrogen dioxide (NO2), formaldehyde, and total molds, and in the presence of Aspergillus spp . in the classroom air . The most consistent findings were observed for formaldehyde, NO2, and Aspergillus spp., related to both decreased nasal patency and increase of ECP and lysozyme in NAL . The presence of yeast was associated with an increase of ECP and lysozyme in NAL, but was not related to nasal patency . CONCLUSIONS: Ventilation flow was below current hygienic standards in the classrooms . Air pollutants in the classroom air may influence nasal patency and inflammatory response in the nasal mucosa.

Philos Trans R Soc Lond B Biol Sci, 2000 Feb 29, 355(1394), 191 - 8
The breast cancer susceptibility gene, BRCA2: at the crossroads between DNA replication and recombination?
Venkitaraman AR.
The identification and cloning of the familial breast cancer susceptibility gene, BRCA2, has excited much interest in its biological functions . Here, evidence is reviewed that the protein encoded by BRCA2 has an essential role in DNA repair through its association with mRad51, a mammalian homologue of bacterial and yeast proteins involved in homologous recombination . A model is proposed that the critical requirement for BRCA2 in cell division and the maintenance of chromosome stability stems from its participation in recombinational processes essential for DNA replication.

Nature, 2000 Mar 9, 404(6774), 201 - 4
hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response; Lee JS et al.; Mutations in the BRCA1 (ref . 1) tumour suppressor gene are found in almost all of the families with inherited breast and ovarian cancers and about half of the families with only breast cancer . Although the biochemical function of BRCA1 is not well understood, it is important for DNA damage repair and cell-cycle checkpoint . BRCA1 exists in nuclear foci but is hyperphosphorylated and disperses after DNA damage . It is not known whether BRCA1 phosphorylation and dispersion and its function in DNA damage response are related . In yeast the DNA damage response and the replication-block checkpoint are mediated partly through the Cds1 kinase family . Here we report that the human Cds1 kinase (hCds1/Chk2) regulates BRCA1 function after DNA damage by phosphorylating serine 988 of BRCA1 . We show that hCds1 and BRCA1 interact and co-localize within discrete nuclear foci but separate after gamma irradiation . Phosphorylation of BRCA1 at serine 988 is required for the release of BRCA1 from hCds1 . This phosphorylation is also important for the ability of BRCA1 to restore survival after DNA damage in the BRCA1-mutated cell line HCC1937.

Bioessays, 2000 Apr, 22(4), 351 - 63
Checkpoints controlling mitosis; Clarke DJ et al.; Each year many reviews deal with checkpoint control.((1-5)) Here we discuss checkpoint pathways that control mitosis . We address four checkpoint systems in depth: budding yeast DNA damage, the DNA replication checkpoint, the spindle assembly checkpoint and the mammalian G2 topoisomerase II-dependent checkpoint . A main focus of the review is the organization of these checkpoint pathways . Recent work has elucidated the order-of-function of several checkpoint components, and has revealed that the S phase, DNA damage and spindle assembly checkpoints each have at least two parallel branches . These steps forward have largely come from kinetic studies of checkpoint-defective mutants .

Bioessays, 2000 Apr, 22(4), 327 - 36
Transcription elongation factor SII; Wind M et al.; RNA chain elongation by RNA polymerase II (pol II) is a complex and regulated process which is coordinated with capping, splicing, and polyadenylation of the primary transcript . Numerous elongation factors that enable pol II to transcribe faster and/or more efficiently have been purified . SII is one such factor . It helps pol II bypass specific blocks to elongation that are encountered during transcript elongation . SII was first identified biochemically on the basis of its ability to enable pol II to synthesize long transcripts . ((1)) Both the high resolution structure of SII and the details of its novel mechanism of action have been refined through mutagenesis and sophisticated in vitro assays . SII engages transcribing pol II and assists it in bypassing blocks to elongation by stimulating a cryptic, nascent RNA cleavage activity intrinsic to RNA polymerase . The nuclease activity can also result in removal of misincorporated bases from RNA . Molecular genetic experiments in yeast suggest that SII is generally involved in mRNA synthesis in vivo and that it is one type of a growing collection of elongation factors that regulate pol II . In vertebrates, a family of related SII genes has been identified; some of its members are expressed in a tissue-specific manner . The principal challenge now is to understand the isoform-specific functional differences and the biology of regulation exerted by the SII family of proteins on target genes, particularly in multicellular organisms .

Biochim Biophys Acta, 2000 Mar 17, 1496(1), 23 - 35
Regulation of the enzymatic and motor activities of myosin I; Barylko B et al.; Myosins I were the first unconventional myosins to be purified and they remain the best characterized . They have been implicated in various motile processes, including organelle translocation, ion channel gating and cytoskeletal reorganization but their exact cellular functions are still unclear . All members of the myosin I family, from yeast to man, have three structural domains: a catalytic head domain that binds ATP and actin; a tail domain believed to be involved in targeting the myosins to specific subcellular locations and a junction or neck domain that connects them and interacts with light chains . In this review we discuss how each of these three domains contributes to the regulation of myosin I enzymatic activity, motor activity and subcellular localization.

FEBS Lett, 2000 Mar 17, 470(1), 70 - 6
A novel C-terminal kinesin subfamily may be involved in chromosomal movement in caenorhabditis elegans; Ali MY et al.; C-terminal kinesin motor proteins, such as the Drosophila NCD and yeast KAR3, are involved in chromosomal segregation . Previously we have described two orthologs of NCD in Caenorhabditis elegans, KLP-3 and KLP-17, which also participate in chromosome movement . Here we report cDNA cloning of klp-15 and klp-16, and the expression pattern of the genes encoding C-terminal motor kinesins including klp-15 and klp-16 . Interestingly KLP-15 and KLP-16 form a unique class of C-terminal kinesins, distinct from the previously known C-terminal motors in other organisms . Using in situ hybridization and RNA interference assay, we show that although all of these motors mediate chromosome segregation, they do so in a combination of unique and overlapping manners, suggesting a complex hierarchy of kinesin motor function in metazoans.

Genesis, 2000 Mar, 26(3), 189 - 97
Pc-G/trx-G and the SWI/SNF connection: developmental gene regulation through chromatin remodeling; Gebuhr TC et al.; Pc-G and trx-G genes are responsible for maintenance of transcriptional regulation and provide a cellular memory mechanism throughout development . Studies in fly, yeast, mouse, and human have implicated modulation of higher-order chromatin structure as an important component in this process . Specifically, connections between SWI/SNF complexes and trx-G genes have provided a mechanistic link between chromatin remodeling and transcriptional regulation . Here we discuss recent genetic and biochemical data that has shed light on the molecular mechanisms and pathways associated with Pc-G and trx-G function in developmental processes such as cell cycle control and hematopoiesis . genesis 26:189-197, 2000 .

Stud Health Technol Inform, 1999, 68, 400 - 5
A new way for the analysis of the exocytosis; Sanchez JL et al.; Exocytosis is the most common way for cells to secrete substances . This is a wide distributed phenomenon that involves all cell types and all animal species from yeast to human . Catecholamine (CA) release from adrenal chromaffin cells occurs by the exocytosis of secretory vesicles also called chromaffin granules . Individual secretory events can be easily monitored by the use of carbon fibers encased in glass microelectrodes . Catecholamines oxidized at the electrode tip produce a transient electrical current wave called secretory spike . The typical secretory spike consists in a sharp elevation of current followed by a rapid exponential decrease to the basal level . A 35-50% of the spikes showed a pre-spike wave called "foot" which represents the CA release through the fusion pore . The time course of secretory spikes observed is altered by extracellular phenomena's like diffusion . It has been suggested that amperometric spikes can be described by the convolution of a Gaussian with a decreasing exponential . The exponential function is partially governed by the diffusion of the secreted substances towards the electrode . In this article a method to deconvolve both functions is proposed . This mathematical approach allows the observation of the original secretory profile--the Gaussian--without the distortion caused by the diffusional broadening of catecholamines.

J Biol Chem, 2000 Mar 24, 275(12), 8540 - 8
JAB1 interacts with both the progesterone receptor and SRC-1; Chauchereau A et al.; JAB1 (Jun activation domain-binding protein-1) has previously been described as a coactivator of AP1 transcription factor . We show here, by yeast and mammalian two-hybrid analyses and by pull-down experiments, that JAB1 also interacts with both the progesterone receptor (PR) and the steroid receptor coactivator 1 (SRC-1) and that it stabilizes PR-SRC-1 complexes . We also show that JAB1 potentiates the activity of a variety of transcription factors known to associate with SRC-1 (nuclear receptors, activator protein-1, and nuclear factor kappaB) . This occurs without any modification of PR or SRC-1 concentration . JAB1 is a subunit of a large multiprotein complex that has been called the COP9 signalosome . The latter is present in plant and animal cells and has been shown to be involved in a variety of cellular mechanisms including transcription regulation, cell cycle control, and phosphorylation cascades . We now show that it is also involved in the mechanisms of action of nuclear receptors and of their coactivators.

J Biol Chem, 2000 Mar 24, 275(12), 8267 - 70
Smad6 as a transcriptional corepressor; Bai S et al.; Smad6 and Smad7, a subgroup of Smad proteins, antagonize the signals elicited by transforming growth factor-beta . These two Smads, induced by transforming growth factor-beta or bone morphogenetic protein (BMP) stimulation, form stable associations with their activated type I receptors, blocking phosphorylation of receptor-regulated Smads in the cytoplasm . Here we show that Smad6 interacts with homeobox (Hox) c-8 as a transcriptional corepressor, inhibiting BMP signaling in the nucleus . The interaction between Smad6 and Hoxc-8 was identified by a yeast two-hybrid approach and further demonstrated by co-immunoprecipitation assays in cells . Gel shift assays show that Smad6, but not Smad7, interacts with both Hoxc-8 and Hoxa-9 as a heterodimer when binding to DNA . More importantly, the Smad6-Hoxc-8 complex inhibits interaction of Smad1 with Hoxc-8- and Smad1-induced transcription activity . These data indicate that Smad6 interacts with Hox transcription factors as part of the negative feedback circuit in the BMP signaling pathway.

Gene, 2000 Jan 25, 242(1-2), 285 - 94
Cloning of three novel neuronal Cdk5 activator binding proteins; Ching YP et al.; Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics . The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a) . As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998 . Association of neurofilament proteins with neuronal Cdk5 activator . J . Biol . Chem . 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen . The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively . Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas . The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate . Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk . Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

Kidney Int, 2000 Mar, 57(3), 787 - 93
Pathogenesis of Dent's disease and related syndromes of X-linked nephrolithiasis; Thakker RV; Renal stone disease, which affects 12% of males and 5% of females by the seventh decade, occurs as an inherited disorder in 45% of patients and is most commonly associated with hypercalciuria . The biochemical basis for hereditary nephrolithiasis and hypercalciuria is unknown, and this has therefore been investigated by a "positional cloning" approach . As a first step in this approach, the chromosomal locations of two disorders referred to as Dent's disease and X-linked recessive nephrolithiasis (XRN) were determined . These two disorders, which represent unusual forms of the renal Fanconi syndrome, are characterized by a low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and renal failure . An X-linked inheritance for XRN was established by studies of a North American kindred, and a similar inheritance for Dent's disease was indicated by the observation of a greater disease severity in males and an absence of male-to-male transmission in five British families . X-linked polymorphic genetic markers were used in linkage studies of these families, and the genes causing Dent's disease and XRN were mapped to Xp11 . In addition, in one family with Dent's disease, a microdeletion involving the DNA probe M27 beta was identified . This microdeletion was further characterized by using yeast artificial chromosomes (YACs) and its size was estimated to be 515 Kb . A search for renal-expressed genes from this region identified a novel gene encoding a chloride channel (CLCN5) with similarities to a family of voltage-gated chloride channels . Molecular genetic studies of CLCN5 demonstrated that mutations, which resulted in a functional loss, were associated with Dent's disease and XRN . In addition, such CLCN5 mutations that would result in a functional loss have also been demonstrated in Japanese children with idiopathic low molecular weight proteinuria, hypercalciuria and nephrocalcinosis, and an Italian kindred with X-linked recessive hypophosphatemic rickets (XLRH) and hypercalciuria . Thus, four hereditary disorders of nephrolithiasis are due to mutations of the novel chloride channel, CLCN5.

Methods, 2000 Apr, 20(4), 465 - 73
Analysis of phosphoinositides in protein trafficking; Hama H et al.; Phosphoinositides are key regulators of vesicle-mediated protein trafficking . Their roles include recruiting vesicle coat and effector proteins to the site of budding and promoting vesicle fusion . The intracellular levels of phosphoinositides and their localization to intracellular membranes are critical to their functions . An analytical procedure was developed that optimizes the recovery of radiolabeled cellular phosphoinositides . Quantitative analyses of yeast cellular phosphoinositides indicated that this approach is useful for examining the intracellular membrane phosphoinositide compositions related to trafficking phenomena . The approach will also enable investigators to determine whole-plant phosphoinositide compositions that have been difficult to achieve in the past . These analytical advances should be generally applicable to studies of phosphoinositide dynamics related to membrane trafficking in yeast, plant, and animal cells .

Methods, 2000 Apr, 20(4), 399 - 402
Use of the two-hybrid system to identify Rab-interacting proteins; Janoueix-Lerosey I et al.; The yeast two-hybrid system has been useful for identifying many partners and effectors of small GTPases of the Rab family . We describe here such a screen using Rab6, a protein involved in the regulation of intracellular transport at the level of the Golgi apparatus, as bait .

Pharmacol Toxicol, 2000 Jan, 86(1), 24 - 9
Endocrine-disrupting agents on healthy human tissues; Paganetto G et al.; A vast number of substances have been suggested as possibly contributing to perturbation of the endocrine system . Several have been tested with different approaches ranging from yeast expression system of human oestrogenic receptors to human breast cancer cells assays . Surprisingly, no inhibition-binding experiments to steroid receptors on healthy human tissue have been performed so far . Our study provides inhibition binding experiments to oestrogens, progesterone, testosterone and retinoic acid receptors in prostate and uterine human tissue of organochlorine pesticides, phthalate esters, oestrogenic constituents derived from plants and phenol derivates . Affinities of significant extent of phthalates on oestrogenic, progestinic and androgenic receptors have not been detected . As for retinoic acid receptors, mono(2-ethylexyl)phthalate provokes a notable reduction of the binding of the tritiated retinoic acid, phtalic acid ethyl-n-butyl ester and 4-octylphenol show an affinity comparable to that of isoflavonoid genistein, whereas 4-nonylphenol reduces the binding of retinoic acid in prostate.

Int J Cancer, 2000 Jan 20, 89(1), 92 - 9
Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers; Takahashi M et al.; A total of 76 primary breast cancers were screened for p53 mutations using the yeast p53 functional assay, and the mutations were determined by DNA sequencing . Clonal mutations of p53 were detected in 30 tumors (39%) . Immunohistochemical staining for nuclear p53 accumulation performed on the yeast assay-positive cases clearly differentiated missense mutations in the DNA binding domain (contact mutant; 17 cases) as positive stain and nonsense-type mutations or missense mutations that may affect 3D-structure of p53 protein (structural mutant; 13 cases) as negative stain . Enzyme immunoassay revealed loss of estrogen receptor in 36 tumors (50%) . Prognostic values of p53 mutation and loss of estrogen receptor were evaluated after a median follow-up period of 44 months . p53 mutations were associated with a short overall survival (log rank test, p = 0.0319), whereas it was not related to disease-free (recurrence-free) survival . Contact mutants were associated with slightly shorter survival compared with structural mutants . Inversely, loss of estrogen receptor was associated with early recurrence (p = 0.0461) but not with short overall survival . The patients with tumors harboring both p53 mutation and loss of estrogen receptor had the poorest outcome (p = 0.0019 and 0.0075 for overall and disease-free survivals, respectively), suggesting independent and additive effects of the 2 factors . The independent role of the 2 factors was confirmed by a multivariate analysis using the Cox proportional hazard model stratified according to clinical tumor stages . Although preliminary, due to the small number of patients studied and the relatively short follow-up time, our results suggest that p53 mutations and loss of estrogen receptor cooperatively affect the prognosis of primary breast cancer patients.

Genes Chromosomes Cancer, 2000 Apr, 27(4), 373 - 9
Cervical cancer suppressor gene is within 1 cM on 6p23; Rader JS et al.; We previously showed loss of heterozygosity at 6p to be a common genetic alteration in cervical cancer and cervical intraepithelial neoplasia . To characterize this critical area of deletion in chromosome 6, we evaluated 107 invasive cervical cancers, using 23 polymorphic markers . Genomic DNA from microdissected frozen or paraffin-embedded cervical tumors and corresponding normal tissue was analyzed . Fifty-three percent (57/107) of the cervical tumors showed loss in 6p . Deletions were found in all stages and histologic types . Ninety-one percent (52/57) of these tumors had a loss at 6p23 . One tumor defined the distal area of deletion at marker D6S429 . Two tumors defined the proximal area of deletion at marker D6S1578 . Genotyping of parental DNA was done on 16 cases to evaluate the origin of chromosomal loss . The deletion occurred in the paternal chromosome in 10 tumors and in the maternal in six . Within each tumor, the same parental chromosome was lost at all tested heterozygous 6p markers . The order of the polymorphic markers and estimate of distances in the critical region were confirmed by generation of a yeast artificial chromosome (YAC) contig and pulse-field gel electrophoresis . Our data strongly suggest that a gene important in cervical cancer tumorigenesis is located within a 1-cM region of 6p23, and it is not imprinted .

Genes Chromosomes Cancer, 2000 Apr, 27(4), 358 - 61
Patients with both pancreatic adenocarcinoma and melanoma may harbor germline CDKN2A mutations; Lal G et al.; Germline mutations of the CDKN2A tumor suppressor gene have been identified in melanoma kindreds linked to 9p21, and pancreatic adenocarcinoma is the second most common malignancy in some of these families . We hypothesized that unselected patients with both primary cancers, i.e., pancreatic cancer and malignant melanoma, have a genetic predisposition to tumor development, and that this susceptibility may be due to germline CDKN2A mutations . Fourteen patients, with both pathologically verified pancreatic adenocarcinoma and melanoma, were assessed for germline CDKN2A mutations by polymerase chain reaction amplification and sequencing of six overlapping fragments encompassing exons 1alpha and 2 . A yeast two-hybrid assay was used to assess the functional consequences of CDKN2A variants . Germline CDKN2A mutations were identified in 2/14 patients: I49S, a novel substitution in exon 1alpha, and M53I, a previously reported missense mutation in exon 2 . Both variants lead to compromised CDKN2A function . We conclude that the occurrence of both pancreatic cancer and melanoma, in the same patient, signals an inherited susceptibility to cancer, and that this predisposition is, in some cases, due to germline CDKN2A mutations . This finding has important implications not only for the proband, but also for other family members .

Mutagenesis, 2000 Mar, 15(2), 127 - 32
p53 mutations experimentally induced by 8-methoxypsoralen plus UVA (PUVA) differ from those found in human skin cancers in PUVA-treated patients; Monti P et al.; 8-Methoxypsoralen (8-MOP) plus UVA irradiation (PUVA therapy) has been used for the treatment of psoriasis . PUVA therapy has been associated with an increased risk of developing skin squamous cell carcinoma (SCC) . In order to determine the PUVA-induced p53 mutation spectrum, a yeast expression vector harbouring a human wild-type p53 cDNA was incubated with 8-MOP, and UVA irradiated in vitro . PUVA-damaged and undamaged DNA was transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter . An 8-MOP concentration-dependent decrease in survival and increase in mutant frequency were observed . At a fixed 8-MOP concentration, survival decreased and mutant frequency increased as UVA irradiation increased . Eleven mutant clones contained 11 mutations: 10 were single base pair substitutions, the remaining one being a complex mutation . All eight T:A-targeted mutations were at 5'-TpA sites, hallmark mutations of PUVA mutagenesis . Through a rigorous statistical test, the PUVA-induced p53 mutation spectrum appears to differ significantly (P < 0.0002) from that observed in SCC in PUVA-treated patients . The present work demonstrates that a specific PUVA-induced mutational fingerprint could be obtained and recognized on human p53 cDNA . This result may suggest that PUVA therapy can be a risk factor for the development of SCC in psoriasis patients through a mechanism not involving the induction of p53 mutations.

Science, 2000 Mar 17, 287(5460), 2023 - 6
Start sites of bidirectional DNA synthesis at the human lamin B2 origin; Abdurashidova G et al.; The initiation sites of bidirectional synthesis at the DNA replication origin located at the 3' end of the human lamin B2 gene were investigated . RNA-primed nascent DNA molecules were subjected to second-strand synthesis with appropriate primers, amplified by ligation-mediated polymerase chain reaction, and size fractionated . Evidence for precise start sites was obtained . Exploration of close to 1 kilobase, coupled to inhibition of Okazaki fragment synthesis, demonstrates that the leading strands initiate at precise nucleotides on either helix, overlapping by three base pairs, within the area bound to a protein complex possibly analogous to the prereplicative complex of yeast.

Leukemia, 2000 Mar, 14(3), 427 - 30
Frequent deletions at 11q23 and 13q14 in B cell prolymphocytic leukemia (B-PLL); Lens D et al.; Deletions of the long arm of chromosomes 11 and 13 are the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders . However, these regions have not been studied so far in B cell prolymphocytic leukemia (B-PLL) . We have investigated the incidence of 13q deletions in 18 B-PLL cases by fluorescence in situ hybridization (FISH), using molecular probes for the RB1 and D13S25 loci . Chromosome 11q deletions were evaluated by FISH using the yeast artificial chromosome (YAC) clone 755b11 from the chromosome 11q22.3-q23.1 region, which has been previously shown to be deleted in 20% of cases of chronic lymphocytic leukemia . Chromosome 11q23 deletions were found in 7/18 (39%) cases of B-PLL . Monoallelic loss of RB1, D13S25 and BRCA2 was present in 10/18 (55%), 6/18 (33%) and 3/18 (16%) of the cases, respectively . All the cases with D13S25 and BRCA2 deletion showed RB1 loss . Deletions of 13q14 and 11q23 are frequent chromosome aberrations in B-PLL and, in contrast to CLL, there is a preferential loss of RB1 with respect to the D13S25 locus suggesting that allelic loss of the RB1 gene may play a role in the pathogenesis of B-PLL.

Int J Mol Med, 2000 Apr, 5(4), 327 - 30
Lack of chemopreventive efficacy of DL-selenomethionine in colon carcinogenesis; Reddy BS et al.; Epidemiologic observations and laboratory research have suggested that dietary selenium reduces the risk of colon cancer . Selenium-enriched brewer's yeast as a dietary supplement reduces the incidence of and mortality from cancer of the colon in humans . It is not clear whether the observed inhibitory effect is due to selenomethionine, or to other forms of selenium, or to a mixture of the selenium compounds present in selenium-enriched brewer's yeast . Therefore, bioassay described in this study examined the chemopreventive efficacy of 10 and 15 ppm selenomethionine, equivalent to 3.6 and 5.4 ppm as selenium, against azoxymethane (AOM)-induced colon carcinogenesis . At five weeks of age, groups of male F344 rats were fed diets containing 0 (control diet), 10 or 15 ppm selenomethionine . At seven and eight weeks of age, all rats except those in vehicle-treated groups received s.c . injections of AOM at a dose rate of 15 mg/kg body wt . The rats were maintained on their respective diets for 52 weeks and were then sacrificed . Colon tumors were processed and evaluated histopathologically . Colon tumor incidence and multiplicity were analyzed statistically . No obvious toxic effects were observed following dietary administration of 10 or 15 ppm selenomethionine as indicated by body weight gain . Administration of 10 or 15 ppm selenomethionine had no significant effect on colon tumor incidence and multiplicity . This study suggests that i) selenomethionine lacks chemopreventive efficacy against AOM-induced colon carcinogenesis and ii) other forms of selenium or a mixture of selenium compounds present in selenium-enriched brewer's yeast need to be evaluated for their chemopreventive efficacy.

DNA Res, 2000 Feb 28, 7(1), 9 - 17
Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis; Nishiyama T et al.; The moss, Physcomitrella patens has been used as a useful material in many fields, because of its simple body plan, ease of gene targeting, and other reasons . Although many mutants have been reported, no method to isolate the corresponding genes was reported . We developed a gene tagging and gene-trap system in P . patens by using the shuttle mutagenesis technique, which has been used in the budding yeast . In 5264 tagged lines, 203 mutants with altered developmental or morphological phenotypes were obtained . In 129 of 4757 gene-trap lines, beta-glucuronidase (GUS) activity was detected in some tissue . Although multiple copies of a tag were detected in many tagged lines by Southern analyses, most copies are likely integrated at the same locus according to PCR analyses.

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2597 - 602
BAR: An apoptosis regulator at the intersection of caspases and Bcl-2 family proteins; Zhang H et al.; Two major pathways for induction of apoptosis have been identified-intrinsic and extrinsic . The extrinsic pathway is represented by tumor necrosis factor family receptors, which utilize protein interaction modules known as death domains and death effector domains (DEDs) to assemble receptor signaling complexes that recruit and activate certain caspase-family cell death proteases, namely procaspases-8 and -10 . The intrinsic pathway for apoptosis involves the participation of mitochondria, which release caspase-activating proteins . Bcl-2 family proteins govern this mitochondria-dependent apoptosis pathway, with proteins such as Bax functioning as inducers and proteins such as Bcl-2 and Bcl-X(L) serving as suppressors of cell death . An apoptosis regulator, BAR, was identified by using a yeast-based screen for inhibitors of Bax-induced cell death . The BAR protein contains a SAM domain, which is required for its interactions with Bcl-2 and Bcl-X(L) and for suppression of Bax-induced cell death in both mammalian cells and yeast . In addition, BAR contains a DED-like domain responsible for its interaction with DED-containing procaspases and suppression of Fas-induced apoptosis . Furthermore, BAR can bridge procaspase-8 and Bcl-2 into a protein complex . The BAR protein is anchored in intracellular membranes where Bcl-2 resides . BAR therefore may represent a scaffold protein capable of bridging two major apoptosis pathways.

EMBO J, 2000 Mar 15, 19(6), 1378 - 88
Cyclin F regulates the nuclear localization of cyclin B1 through a cyclin-cyclin interaction; Kong M et al.; The key regulator of G(2)-M transition of the cell cycle is M-phase promoting factor (MPF), a complex composed of cdc2 and a B-type cyclin . Cyclin B1 nuclear localization involves phosphorylation within a region called the cytoplasmic retention signal, which also contains a nuclear export signal . The mechanism of MPF nuclear localization remains unclear since it contains no functional nuclear localization signal (NLS) . We exploited the yeast two-hybrid screen to find protein(s) potentially mediating localization of cyclin B1 and identified a novel interaction between cyclin B1 and cyclin F . We found that cdc2, cyclin B1 and cyclin F form a complex that exhibits histone H1 kinase activity . Cyclin B1 and cyclin F also colocalize through immunofluorescence studies . Additionally, deletion analysis revealed that each putative NLS of cyclin F is functional . Taken together, the data suggest that the NLS regions of cyclin F regulate cyclin B1 localization to the nucleus . The interaction between cyclin B1 and cyclin F represents the first example of direct cyclin-cyclin binding, and elucidates a novel mechanism that regulates MPF localization and function.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3084 - 8
Unambiguous demonstration of triple-helix-directed gene modification; Barre FX et al.; Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies . However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated . To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region . The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified . Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site . Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Semin Neurol, 1999, 19(4), 385 - 95
Recent insights into the molecular pathogenesis of Huntington disease; Leavitt BR et al.; Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the HD gene resulting in expression of an uninterrupted polyglutamine stretch within the N-terminus of its protein product huntingtin (htt) . In this article we review the clinical, genetic, and neuropathological features of HD and discuss recent insights into the pathogenesis of HD . Examining the role of CAG repeat size on age of onset and penetrance in HD using a refined database of human HD patients has provided further support for the importance of the CAG repeat in the pathogenesis of HD and information leading to a predictive model for the likelihood of being affected by a specific age for a particular CAG expansion . In a YAC transgenic mouse model that replicates key elements of the HD phenotype, the development of selective striatal neurodegeneration is coincident with cleavage of htt and translocation of the N-terminal htt fragment into the nucleus . We also review in vitro evidence that htt is a substrate for cleavage by a group of cysteine proteases involved in apoptotic death-the caspases, and that caspase cleavage of htt results in the generation of a toxic N-terminal fragment . Inhibiting caspase cleavage of huntingtin eliminates the toxicity of the mutant htt protein . These results suggest that cleavage of huntingtin resulting in production of a truncated N-terminal fragment may be a crucial step in the pathogenesis of Huntington disease and that inhibition of this process may be a potential therapeutic strategy for this currently untreatable disorder.

Int J Radiat Biol, 2000 Feb, 76(2), 189 - 98
Identification of genes regulated by UV/salicylic acid; Paunesku T et al.; PURPOSE: Previous work from the authors' group and others has demonstrated that some of the effects of UV irradiation on gene expression are modulated in response to the addition of salicylic acid to irradiated cells . The presumed effector molecule responsible for this modulation is NF-kappaB . In the experiments described here, differential-display RT-PCR was used to identify those cDNAs that are differentially modulated by UV radiation with and without the addition of salicylic acid . MATERIALS AND METHODS: Differential-display RT-PCR was used to identify differentially expressed genes . RESULTS: Eight such cDNAs are presented: lactate dehydrogenase (LDH-beta), nuclear encoded mitochondrial NADH ubiquinone reductase 24 kDa (NDUFV2), elongation initiation factor 4B (eIF4B), nuclear dots protein SP100, nuclear encoded mitochondrial ATPase inhibitor (IF1), a cDNA similar to a subunit of yeast CCAAT transcription factor HAP5, and two expressed sequence tags (AA187906 and AA513156) . CONCLUSIONS: Sequences of four of these genes contained NF-kappaB DNA binding sites of the type that may attract transrepressor p55/p55 NF-kappaB homodimers . Down-regulation of these genes upon UV irradiation may contribute to increased cell survival via suppression of p53 independent apoptosis.

Int J Biochem Cell Biol, 2000 Mar, 32(3), 303 - 7
How do Rab proteins function in membrane traffic?
Armstrong J.
The Rabs are a group of GTP-binding proteins implicated for some time in the targeting of different transport vesicles within the cell, but it has been unclear how they function, or how they relate to a second group of targeting proteins, the SNAREs . Recent work, discussed in this review, has used biophysical, biochemical and genetic approaches to begin to answer these questions for Rab3, Rab5 and the yeast protein Sec4p . However, the results from these three Rabs lead to a surprising conclusion: the different Rabs seem to function via highly diverse target proteins.

Plant Cell, 2000 Mar, 12(3), 443 - 55
A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype; Woeste KE et al.; A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype . Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding . In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype . The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking . Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family . However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion . The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors . Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

Plant Cell, 2000 Mar, 12(3), 343 - 56
A conserved domain of the arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding; Grebe M et al.; The Arabidopsis GNOM protein, a guanine nucleotide exchange factor (GEF) that acts on ADP ribosylation factor (ARF)-type G proteins, is required for coordination of cell polarity along the apical-basal embryo axis . Interallelic complementation of gnom mutants suggested that dimerization is involved in GNOM function . Here, direct interaction between GNOM molecules is demonstrated in vitro and by using a yeast two-hybrid system . Interaction was confined to an N-terminal domain conserved within a subgroup of large ARF GEFs . The same domain mediated in vitro binding to cyclophilin 5 (Cyp5), which was identified as a GNOM interactor in two-hybrid screening . Cyp5 displayed peptidylprolyl cis/trans-isomerase and protein refolding activities that were sensitive to cyclosporin A . Cyp5 protein accumulated in several plant organs and, like GNOM, was partitioned between cytosolic and membrane fractions . Cyp5 protein was also expressed in the developing embryo . Our results suggest that Cyp5 may regulate the ARF GEF function of the GNOM protein during embryogenesis.

J Mol Biol, 2000 Mar 24, 297(2), 301 - 8
Transposition and exon shuffling by group II intron RNA molecules in pieces; Hiller R et al.; In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules . Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNA-containing viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking . Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria . We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuffling . The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions .

Eur J Hum Genet, 2000 Jan, 8(1), 75 - 8
Refined mapping of the human serotonin transporter (SLC6A4) gene within 17q11 adjacent to the CPD and NF1 genes; Shen S et al.; The SLC6A4 gene encodes the serotonin transporter, the target of an important class of antidepressant drugs (serotonin selective reuptake inhibitors) . Polymorphisms in the SLC6A4 gene have been reported to be associated with susceptibility to depression and other psychiatric disorders . We have constructed a 1 Mb YAC and PAC contig which harbours both the SLC6A4 and the carboxypeptidase D (CPD) genes . The order of loci within the contig was cen-D17S975-D17S1549-24R-D17S1294-SLC6A4-28L+ ++-(CPD, D17S2009, D17S2004)-D17S2120-ter . Both genes were deleted in one of 17 neurofibromatosis type 1 (NF1) patients carrying submicroscopic NF1 contiguous gene deletions.

Eur J Hum Genet, 2000 Jan, 8(1), 63 - 70
Opposite deletions/duplications of the X chromosome: two novel reciprocal rearrangements; Giglio S et al.; Paralogous sequences on the same chromosome allow refolding of the chromosome into itself and homologous recombination . Recombinant chromosomes have microscopic or submicroscopic rearrangements according to the distance between repeats . Examples are the submicroscopic inversions of factor VIII, of the IDS gene and of the FLN1/emerin region, all resulting from misalignment of inverted repeats, and double recombination . Most of these inversions are of paternal origin possibly because the X chromosome at male meiosis is free to refold into itself for most of its length . We report on two de novo rearrangements of the X chromosome found in four hypogonadic females . Two of them had an X chromosome deleted for most of Xp and duplicated for a portion of Xq and two had the opposite rearrangement (class I and class II rearrangements, respectively) . The breakpoints were defined at the level of contiguous YACs . The same Xp 11.23 breakpoint was found in the four cases . That of the long arm coincided in three cases (Xq21.3) and was more proximal in case 4 (Xq21.1) . Thus class I rearrangements (cases 1 and 2) are reciprocal to that of case 3, whilst that of case 4 shares only the Xp breakpoint . The abnormal X was paternal in the three cases investigated . Repeated inverted sequences located at the breakpoints of rearrangements are likely to favour the refolding of the paternal X chromosome and the recombination of the repeats . The repeat at the Xp11 may synapse with either that at Xq21.3 or that at Xq21.1 . These rearrangements seem to originate as the Xq28 submicroscopic inversions but they are identifiable at the microscopic level and result from a single recombination event.

J Biol Chem, 2000 Mar 17, 275(11), 7950 - 7
A human importin-beta family protein, transportin-SR2, interacts with the phosphorylated RS domain of SR proteins; Lai MC et al.; Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA . RS domains are also found in proteins that have influence on other aspects of gene expression . Proteins that contain an RS domain are often located in the speckled domains of the nucleus . Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles . By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-beta family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR . This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins . More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains . Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35 . Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins.

J Biol Chem, 2000 Mar 17, 275(11), 7771 - 8
Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14 . Characterization of a new receptor binding partner; Reilly JF et al.; Using the cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) as bait in a yeast two-hybrid screen, Grb14 was identified as a FGFR1 binding partner . A kinase-inactive mutant of FGFR1 failed to interact with Grb14, indicating that activation of FGFR1 is necessary for binding . Deletion of the C-tail or mutation of both C-tail tyrosine residues of FGFR1 to phenylalanine abolished binding, and deletion of the juxtamembrane domain of the receptor reduced binding, suggesting that Grb14 binds to FGFR1 at multiple sites . Co-immunoprecipitation and in vitro binding assays demonstrated that binding of Grb14 to FGFR1 in mammalian cells was dependent on receptor activation by fibroblast growth factor-2 (FGF-2) . Deletion of the Src homology 2 (SH2) domain of Grb14 reduced but did not block binding to FGFR1 and eliminated dependence on receptor activation . The SH2 domain alone bound both FGFR1 and platelet-derived growth factor receptor, whereas full-length Grb14 bound only FGFR1, suggesting that regions upstream of the SH2 domain confer specificity for FGFR1 . Grb14 was phosphorylated on serine and threonine residues in unstimulated cells, and treatment with FGF-2 enhanced this phosphorylation . Expression of exogenous Grb14 inhibited FGF-2-induced cell proliferation, whereas a point-mutated form of Grb14 incapable of binding to FGFR1 enhanced FGF-2-induced mitogenesis . These data demonstrate an interaction between activated FGFR1 and Grb14 and suggest a role for Grb14 in FGF signaling.

J Biol Chem, 2000 Mar 17, 275(11), 7447 - 50
Fidelity of human DNA polymerase eta; Johnson RE et al.; Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers . The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta) . Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer . Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer . Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA . The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.

Mol Cell Biol, 2000 Apr, 20(7), 2604 - 18
The orphan nuclear receptor Ear-2 is a negative coregulator for thyroid hormone nuclear receptor function; Zhu XG et al.; Thyroid hormone (T3) nuclear receptors (TR) are ligand-dependent transcription factors which regulate growth, differentiation, and development . One emerging hypothesis suggests that TR mediate these diverse effects via a large network of coregulators . Recently, we found that TR-mediated transcriptional responses varied in six cell lines derived from different tissues . We therefore used human TR subtype beta1 (TRbeta1) as bait to search for coregulators in human colon carcinoma RKO cells with a yeast two-hybrid system . RKO cells exhibited T3-dependent and -independent transcriptional activation . One of the three positive clones was identified as Ear-2, which is a distant member of the chick ovalbumin upstream promoter-transcription factors of the orphan nuclear receptor family . The physical interaction between Ear-2 and TRbeta1 was further confirmed by specific binding of Ear-2 to glutathione S-transferase-TRbeta1 . In addition, Ear-2 was found to associate with TRbeta1 in cells . As a result of this physical interaction, binding of TRbeta1 to the T3 response elements was inhibited . Using reporter systems, we found that both the basal activation and the T3-dependent activation mediated by TRbeta1 were repressed by Ear-2 in CV1 cells . In RKO cells, however, the T3-independent transcriptional activity was more sensitive to the repression effect of Ear-2 than the T3-dependent transcriptional activity . The repression effect of Ear-2 was reversed by steroid hormone receptor coactivator 1 . These results suggest that TR-mediated responses reflect a balance of corepressors and coactivators in cells . These findings further strengthen the hypothesis that the diverse activities of TR are achieved via a large network of coregulators that includes Ear-2.

Mol Cell Biol, 2000 Apr, 20(7), 2411 - 22
A tissue-specific coactivator of steroid receptors, identified in a functional genetic screen; Knutti D et al.; Steroid receptors mediate responses to lipophilic hormones in a tissue- and ligand-specific manner . To identify nonreceptor proteins that confer specificity or regulate steroid signaling, we screened a human cDNA library in a steroid-responsive yeast strain . One of the identified cDNAs, isolated in the screen as ligand effect modulator 6, showed no homology to yeast or Caenorhabditis elegans proteins but high similarity to the recently described mouse coactivator PGC-1 and was accordingly termed hPGC-1 . The hPGC-1 DNA encodes a nuclear protein that is expressed in a tissue-specific manner and carries novel motifs for transcriptional regulators . The expression of hPGC-1 in mammalian cells enhanced potently the transcriptional response to several steroids in a receptor-specific manner . hPGC-1-mediated enhancement required the receptor hormone-binding domain and was dependent on agonist ligands . Functional analysis of hPGC-1 revealed two domains that interact with steroid receptors in a hormone-dependent manner, a potent transcriptional activation function, and a putative dimerization domain . Our findings suggest a regulatory function for hPGC-1 as a tissue-specific coactivator for a subset of nuclear receptors.

Mol Cell Biol, 2000 Apr, 20(7), 2400 - 10
SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC To facilitate NotchIC function; Zhou S et al.; Notch proteins are transmembrane receptors that mediate intercell communication and direct individual cell fate decisions . The activated intracellular form of Notch, NotchIC, translocates to the nucleus, where it targets the DNA binding protein CBF1 . CBF1 mediates transcriptional repression through the recruitment of an SMRT-histone deacetylase-containing corepressor complex . We have examined the mechanism whereby NotchIC overcomes CBF1-mediated transcriptional repression . We identified SKIP (Ski-interacting protein) as a CBF1 binding protein in a yeast two-hybrid screen . Both CBF1 and SKIP are highly conserved evolutionarily, and the SKIP-CBF1 interaction is also conserved in assays using the Caenorhabditis elegans and Drosophila melanogaster SKIP homologs . Protein-protein interaction assays demonstrated interaction between SKIP and the corepressor SMRT . More surprisingly, SKIP also interacted with NotchIC . The SMRT and NotchIC interactions were mutually exclusive . In competition binding experiments SMRT displaced NotchIC from CBF1 and from SKIP . Contact with SKIP is required for biological activity of NotchIC . A mutation in the fourth ankyrin repeat that abolished Notch signal transduction did not affect interaction with CBF1 but abolished interaction with SKIP . Further, NotchIC was unable to block muscle cell differentiation in myoblasts expressing antisense SKIP . The results suggest a model in which NotchIC activates responsive promoters by competing with the SMRT-corepressor complex for contacts on both CBF1 and SKIP.

Plant Physiol, 2000 Mar, 122(3), 977 - 83
Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit; Hadfield KA et al.; Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening . Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process . Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein . The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins . The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues . When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed . One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene . A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues . The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene . Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development . The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.

Plant Physiol, 2000 Mar, 122(3), 695 - 704
A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth; van der Knaap E et al.; Os-GRF1 (Oryza sativa-GROWTH-REGULATING FACTOR1) was identified in a search for genes that are differentially expressed in the intercalary meristem of deepwater rice (Oryza sativa L.) internodes in response to gibberellin (GA) . Os-GRF1 displays general features of transcription factors, contains a functional nuclear localization signal, and has three regions with similarities to sequences in the database . One of these regions is similar to a protein interaction domain of SWI2/SNF2, which is a subunit of a chromatin-remodeling complex in yeast . The two other domains are novel and found only in plant proteins of unknown function . To study its role in plant growth, Os-GRF1 was expressed in Arabidopsis . Stem elongation of transformed plants was severely inhibited, and normal growth could not be recovered by the application of GA . Our results indicate that Os-GRF1 belongs to a novel class of plant proteins and may play a regulatory role in GA-induced stem elongation.

Bone, 2000 Mar, 26(3), 207 - 13
The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants; Scimeca JC et al.; Osteosclerosis (oc) is an autosomal recessive lethal mutation that impairs bone resorption by osteoclasts, and induces a general increase of bone density in affected mice . Genetic mapping of the oc mutation was used as a backbone in a positional cloning approach in the pericentromeric region of mouse chromosome 19 . Perfect cosegregation of the osteopetrotic phenotype with polymorphic markers enabled the construction of a sequence-ready bacterial artificial chromosome (BAC) contig of this region . Genomic sequencing of a 200-kb area revealed the presence of the mouse homologue to the human gene encoding the osteoclast-specific 116-kDa subunit of the vacuolar proton pump . This gene was located recently on human 11q13, a genomic region conserved with proximal mouse chromosome 19 . Sequencing of the 5' end of the gene in oc/oc mice showed a 1.6-kb deletion, including the translation start site, which impairs genuine transcription of this subunit . The inactivation of this osteoclast-specific vacuolar proton ATPase subunit could be responsible for the lack of this enzyme in the apical membranes of osteoclast cells in oc/oc mice, thereby preventing the resorption function of these cells, which leads to the osteopetrotic phenotype.

Int J Immunopharmacol, 2000 May, 22(5), 383 - 94
Immunopharmacological and immunotoxicological activities of a water-soluble (1-->3)-beta-D-glucan, CSBG from Candida spp; Tokunaka K et al.; We have established a convenient, two-step procedure to solubilize the yeast cell wall (1-->3)-beta-D-glucan using the combination of NaClO oxidation and DMSO extraction . Candida soluble beta-D-glucan (CSBG) was mainly composed of a linear beta-1,3 glucan with a linear beta-1,6-glucan moiety . In this study, we screened for several immunopharmacological activities of CSBG and found the following activities: (1) interleukin-6 synthesis of macrophages in vitro; (2) antagonistic effect for zymosan mediated-tumor necrosis factor synthesis of macrophages; (3) augmentation for lipopolysaccharide mediated tumor necrosis factor and nitrogen oxide syntheses of macrophages; (4) activation of alternative pathway of complement; (5) hematopoietic response on cyclophosphamide induced leukopenia; (6) the antitumor effect on ascites form tumor; (7) Enhanced vascular permeability; (8) priming effect on lipopolysaccharide triggered TNF-alpha synthesis; and (9) adjuvant effect on antibody production . These results strongly suggested that CSBG possessed various immunopharmacological activity.

FEBS Lett, 2000 Mar 3, 469(1), 72 - 6
Direct interaction of nerve growth factor receptor, TrkA, with non-receptor tyrosine kinase, c-Abl, through the activation loop; Koch A et al.; The nerve growth factor receptor, TrkA, is essential for the survival and differentiation of neurons in the central and peripheral nervous systems . To understand the molecular principles underlying this differentiation step, we employed a yeast two-hybrid screening protocol using human TrkA as bait . We isolated c-Abl as a TrkA-interacting protein, in addition to known proteins such as phospholipase Cgamma and SH2-B . This interaction was confirmed by an in vitro binding assay using glutathione S-tranferase-Abl fusion protein . Furthermore, we show here that c-Abl binds to phosphotyrosine residue(s) in the kinase activation loop of TrkA.

Genomics, 2000 Feb 15, 64(1), 1 - 14
A 6-Mb high-resolution physical and transcription map encompassing the hereditary prostate cancer 1 (HPC1) region; Carpten JD et al.; Several hereditary disease loci have been genetically mapped to the chromosome 1q24-q31 interval, including the hereditary prostate cancer 1 (HPC1) locus . Here, we report the construction of a 20-Mb yeast artificial chromosome contig and a high-resolution 6-Mb sequence-ready bacterial artificial chromosome (BAC)/P1-derived artificial chromosome (PAC) contig of 1q25 by sequence and computational analysis, STS content mapping, and chromosome walking . One hundred thirty-six new STSs, including 10 novel simple sequence repeat polymorphisms that are being used for genetic refinement of multiple disease loci, have been generated from this contig and are shown to map to the 1q25 interval . The integrity of the 6-Mb BAC/PAC contig has been confirmed by restriction fingerprinting, and this contig is being used as a template for human chromosome 1 genome sequencing . A transcription mapping effort has resulted in the precise localization of 18 known genes and 31 ESTs by database searching, exon trapping, direct cDNA hybridization, and sample sequencing of BACs from the 1q25 contig . An additional 11 known genes and ESTs have been placed within the larger 1q24-q31 interval . These transcription units represent candidate genes for multiple hereditary diseases, including HPC1 .

Nucleic Acids Res, 2000 Apr 1, 28(7), 1647 - 55
Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta; Shimamoto A et al.; The RecQ helicase superfamily has been implicated in DNA repair and recombination . At least five human RecQ-related genes exist: RecQ1, BLM, WRN, RecQ4 and RecQ5 . Mutations in BLM, WRN and RecQ4 are associated with Bloom, Werner and Rothmund-Thomson syndromes, respectively, involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability . RecQ5 is small, containing only a core part of the RecQ helicase, but three isomer transcripts code for small RecQ5alpha (corresponding to the original RecQ5 with 410 amino acids), new large RecQ5beta (991 amino acids) and small RecQ5gamma (435 amino acids) proteins that contain the core helicase motifs . By determining the genomic structure, we found that the three isoforms are generated by differential splicing from the RecQ5 gene that contains at least 19 exons . Northern blot analysis using a RecQ5beta-specific probe indicates that RecQ5beta mRNA is expressed strongly in the testis . Immunocytochemical staining of three N-terminally tagged RecQ5 isomers expressed in 293EBNA cells showed that RecQ5beta migrates to the nucleus and exists exclusively in the nucleoplasm, while the small RecQ5alpha and RecQ5gamma proteins stay in the cytoplasm . Immunoprecipitation and an extended cytochemical experiment suggested that the nucleoplasmic RecQ5beta, like yeast Sgs1 DNA helicase, binds to topoisomerases 3alpha and 3beta, but not to topoisomerase 1 . These results predict that RecQ5beta may have an important role in DNA metabolism and may also be related to a distinct genetic disease.

Nucleic Acids Res, 2000 Apr 1, 28(7), 1548 - 54
Molecular characterisation of two paralogous SPO11 homologues in Arabidopsis thaliana; Hartung F et al.; The Spo11 protein of yeast has been found to be covalently bound to double-strand breaks in meiosis, demonstrating a unique role of the protein in the formation of these breaks . Homologues of the SPO11 gene have been found in various eukaryotes, indicating that the machinery involved in meiotic recombination is conserved in eukaryotes . Here we report on SPO11 homologues in plants . In contrast to what is known from other eukaryotes, Arabidopsis thaliana carries in its genome at least two SPO11 homologues, AtSPO11-1 and AtSPO11-2 . Both genes are not more closely related to each other than to other eukaryotic SPO11 homologues, indicating that they did not arise via a recent duplication event during higher plant evolution . For both genes three different poly-adenylation sites were found . AtSPO11-1 is expressed not only in generative but also to a lesser extent in somatic tissues . We were able to detect in different organs various AtSPO11-1 cDNAs in which introns were differently spliced-a surprising phenomenon also reported for SPO11 homologues in mammals . In the case of AtSPO11-2 we found that the 3' end of the mRNA is overlapping with a mRNA produced by a gene located in inverse orientation next to it . This points to a possible antisense regulation mechanism . Our findings hint to the intriguing possibility that, at least for plants, Spo11-like proteins might have more and possibly other biological functions than originally anticipated for yeast.

Nucleic Acids Res, 2000 Apr 1, 28(7), 1506 - 13
The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity; Beamish HJ et al.; DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus . Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system . The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact . The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity . Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity . We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells . Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase . We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.

Mol Carcinog, 2000 Mar, 27(3), 219 - 28
Somatic cell hybrids for high-density mapping of chromosome 2 breakpoints in radiation-induced myeloid leukemia cell lines from inbred mice; Pazzaglia S et al.; Chromosome 2 (chr 2) deletions are recurrent abnormalities in acute myeloid leukemia (AML) induced by ionizing radiation in the mouse . The localization of deletion sites has proven extremely useful in providing information on the molecular mechanisms of leukemogenesis . The models available for the study of AML are mostly represented by inbred mouse strains, in which the molecular resolution of breakpoints is problematic . In this study, we have examined five leukemic cell lines exhibiting hemizygous chr 2 loss, derived from CBA, C3H, or (C57BLxCBA/H) F1 mice in which AML had been induced by a whole-body dose of radiation . By application of a somatic cell hybridization technique, we have generated interspecific cell hybrids retaining the deleted murine chr 2 homologue . This strategy permitted a very detailed genetic analysis allowing the utilization of any genetic marker on chr 2 without a requirement for polymorphism . Somatic cell hybrid clones were subjected to a high-density polymerase chain reaction-based microsatellite screening using 62-106 informative markers for each cell line . Detailed maps accurately defining chr 2 breakpoints were obtained . The identification of critical breakpoint markers allowed the construction of partial yeast artificial chromosome contigs across chr 2 breakpoints . These maps represent an essential resource for cloning of the breakpoint regions.

Ann Oncol, 2000, 11 Suppl 1, 127 - 30
The ATM gene in the pathogenesis of mantle-cell lymphoma; Stilgenbauer S et al.; BACKGROUND: Mantle-cell lymphoma (MCL) is genetically characterized by the translocation t(11;14)(q13;q32) leading to an overexpression of cyclin-D1, but additional chromosomal abnormalities appear to be required for MCL pathogenesis . PATIENTS AND METHODS: Deletions involving chromosome 11q, which were recently found as recurrent aberrations in MCL, were analyzed at the molecular level in a series of 81 MCL by fluorescence in situ hybridization (FISH) with probes from a contiguous set of yeast artificial chromosomes (YACs) spanning bands 11q14-q24 . RESULTS AND CONCLUSIONS: Loss of chromosome 11 material was observed in 37 of the 81 MCL cases (46%) . The consensus deletion comprised YAC 801e11 containing the ATM gene . The minimal region of loss was further narrowed with P1-derived artificial chromosome (PAC) probes . This allowed the identification of a deletion confined to the genomic region of ATM, which, together with intragenic mutations found in the coding sequence, suggests a role of ATM as a tumor suppressor gene in MCL.

Blood, 2000 Mar 15, 95(6), 2138 - 43
Deletions of chromosome 5q13.3 and 17p loci cooperate in myeloid neoplasms; Castro PD et al.; Nonrandom interstitial deletions and monosomy of chromosomes 5, 7, and 17 in refractory myelodysplasia (MDS) and acute myelogenous leukemia (AML) suggest a multistep pathway that culminates in aggressive clinical course . Because cytogenetic studies frequently identify chromosome 5 and 17 deletions within a single clone, we searched for allele loss for 5q loci and TP53 gene mutations in the same leukemic samples . Cosegregating deletions of chromosomes 5 and 17 were found to specifically include the 5q13.3 interval between the loci D5S672 and D5S620/D5S626, a locus hypothesized to harbor a tumor suppressor gene(1) and the TP53 gene on 17p . A rare patient with secondary refractory MDS and an unbalanced translocation {der(5;17)}, which resulted in deletions of the 5q13.3-qter and 17p loci, provided clues on the sequence of genetic alterations . Serial molecular analysis of this patient revealed a dysplastic clone with der(5;17), which gave rise to a leukemic clone on acquiring an inactivating mutation of TP53 . Our findings are consistent with functional cooperation between a putative tumor suppressor gene at 5q13.3 that contributes toward the progression of early stages of MDS, and the TP53 gene when mutated, causes transformation to AML . (Blood . 2000;95:2138-2143)

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2836 - 9
Specific association of estrogen receptor beta with the cell cycle spindle assembly checkpoint protein, MAD2; Poelzl G et al.; Estrogen receptors (ERs) are ligand-activated transcription factors that regulate gene expression and cell growth . Two ERs now have been identified: ERalpha and the more recently discovered ERbeta . The physiological function of ERbeta remains unclear, but evidence from vascular injury studies and from ERbeta knockout mice suggests that ERbeta may be involved in the regulation of cellular proliferation . Here we show a direct and specific interaction between ERbeta and the cell cycle mitotic spindle assembly checkpoint protein, MAD2 (mitosis arrest-deficient 2) . The ERbeta-MAD2 interaction was identified by screening of a yeast two-hybrid system vascular endothelial cell library with ERbeta and confirmed with glutathione S-transferase-fusion protein interaction studies . In contrast, ERalpha did not interact with MAD2 in either the two-hybrid system or in the protein-protein interaction experiments . Amino acids 173-208 in the hinge region of ERbeta were sufficient to mediate the interaction with MAD2 in the two-hybrid system and in glutathione S-transferase-fusion protein studies . These data identify a link between ERbeta and MAD2 of potential importance to regulation of the cell cycle and support a function of ERbeta distinct from the established role of ERs as transcription factors.

Biol Pharm Bull, 2000 Feb, 23(2), 231 - 4
Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase; Hayashi H et al.; A cDNA clone (GgCAS1) encoding cycloartenol synthase (CAS) has been isolated from Glycyrrhiza glabra (licorice) by cross-hybridization with that of Pisum sativum CAS as a probe . The deduced amino acid sequence of GgCAS1 exhibits 89%, 83% and 81% identity to those of Pisum sativum, Panax ginseng and Arabidopsis thaliana CASs, respectively . CAS activity has been detected in the homogenate of the yeast transformed with the expression vector containing the open reading frame of GgCAS1 . Southern blot analysis suggested that at least two CAS genes exist in the licorice genome . In Northern blot analysis, the strong signal for CAS mRNA is detected in the cultured licorice cells of all growth phases, but no significant increase of CAS mRNA expression was observed in the cells treated with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, pravastatin.

Dev Dyn, 2000 Feb, 217(2), 225 - 31
YAC transgene-mediated olfactory receptor gene choice; Ebrahimi FA et al.; In the mouse, individual olfactory neurons express one of a thousand distinct olfactory receptor genes . Furthermore, only one allele of the expressed gene is transcribed . This phenomenon, random allelic inactivation, along with the observation that the olfactory receptor genes reside in large chromosomal arrays, suggests a role for long-range gene regulation in olfactory receptor gene choice . We have constructed a 300-kb yeast artificial chromosome (YAC) transgene in which a single receptor gene is marked while maintaining its coding region . This 300-kb piece of DNA functions as an independent olfactory receptor gene locus in directing olfactory receptor gene choice in both the olfactory system and the accessory olfactory system (vomeronasal organ, VNO) . Furthermore, the transgene, like endogenous olfactory receptor loci, is subject to allelic inactivation.

J Formos Med Assoc, 1999 Nov, 98(11), 787 - 9
Mandibular osteomyelitis caused by Blastoschizomyces capitatus in a child with acute myelogenous leukemia; Cheung MY et al.; A 6-year-old girl with acute myelogenous leukemia (AML) developed fungal mandibular osteomyelitis during chemotherapy . Blastoschizomyces capitatus was recognized histologically by its yeast-like morphology and formation of annelloconidia, and was confirmed by culture . The fungal osteomyelitis of the mandible was treated successfully with prolonged antifungal medication, extensive surgical debridement and an oral care program, without interrupting leukemia chemotherapy . B . capitatus osteomyelitis of the mandible may occur during chemotherapy in AML patients with poor dental condition . Successful treatment can be achieved by careful management without interruption of antineoplastic chemotherapy.

Mech Dev, 2000 Mar 1, 91(1-2), 451 - 4
GFP-tagged balancer chromosomes for Drosophila melanogaster; Casso D et al.; We constructed green fluorescent protein (GFP)-expressing balancer chromosomes for each of the three major chromosomes of Drosophila melanogaster . Expression of GFP in these chromosomes is driven indirectly by a Kruppel (Kr) promoter, via the yeast GAL4-UAS regulatory system . GFP fluorescence can be seen in embryos as early as the germ band extension stage, and can also be seen in larvae, pupae, and adults . We show the patterns of GFP expression of these balancers and demonstrate the use of the balancers to identify homozygous progeny.

Cancer Lett, 2000 Mar 31, 150(2), 191 - 9
p53-dependent radioresistance in ovarian carcinoma cell lines; Concin N et al.; In the present study, we investigated the radiosensitivity profiles of three established human ovarian carcinoma cell lines, PA-1, Caov-3, and SK-OV-3, using the adenosine triphosphate-cell viability assay (ATP-CVA) . We have correlated radioresponsiveness with the p53 status and the p53 accumulation after irradiation as well as with the Bcl-2 expression and the growth rate of these cell lines . The p53 status was examined by immunocytochemistry and a functional assay (functional analysis of separated alleles in yeast, FASAY); the p53 accumulation was determined by immunocytochemistry and flow cytometry . Furthermore, the Bcl-2 expression before and after irradiation was examined by immunocytochemistry . PA-1, expressing wild-type p53, showed an unequivocal accumulation of p53 protein following exposure to irradiation . This cell line was found to be strongly sensitive to irradiation . The two p53 mutant cell lines Caov-3 and SK-OV-3 showed radioresistance at different degrees and irradiation did not result in p53 accumulation . None of the cell lines examined expressed Bcl-2 protein and no change was seen after irradiation . Furthermore, the most sensitive cell line to irradiation, PA-1, showed the highest proliferative activity, while Caov-3 and SK-OV-3, the more resistant cell lines, exhibited lower growth rates . Our findings indicate that the presence of p53 protein is a possible determinant for the cytotoxicity induced by irradiation in the investigated ovarian carcinoma cell lines . Bcl-2 expression does not seem to determine the response to irradiation in these cell lines . Additionally, an association between radioresponsiveness and the growth rate is suggested in PA-1, Caov-3, and SK-OV-3.

J Cell Sci, 2000 Apr, 113 ( Pt 7), 1127 - 38
Progesterone regulates the accumulation and the activation of Eg2 kinase in Xenopus oocytes; Frank-Vaillant M et al.; Xenopus prophase oocytes reenter meiotic division in response to progesterone . The signaling pathway leading to Cdc2 activation depends on neosynthesized proteins and a decrease in PKA activity . We demonstrate that Eg2 protein, a Xenopus member of the Aurora/Ipl1 family of protein kinases, accumulates in response to progesterone and is degraded after parthenogenetic activation . The polyadenylation and cap ribose methylation of Eg2 mRNA are not needed for the protein accumulation . Eg2 protein accumulation is induced by progesterone through a decrease in PKA activity, upstream of Cdc2 activation . Eg2 kinase activity is undetectable in prophase and is raised in parallel with Cdc2 activation . In contrast to Eg2 protein accumulation, Eg2 kinase activation is under Cdc2 control . Furthermore, by using an anti-sense strategy, we show that Eg2 accumulation is not required in the transduction pathway leading to Cdc2 activation . Altogether, our results strongly suggest that Eg2 is not necessary for Cdc2 activation, though it could participate in the organization of the meiotic spindles, in agreement with the well-conserved roles of the members of the Aurora family, from yeast to man.

Genomics, 2000 Feb 1, 63(3), 433 - 8
The human CYP2C locus: a prototype for intergenic and exon repetition splicing events; Finta C et al.; In human there are four known CYP2C genes that have been mapped to chromosome 10q24 with the order Cen-2C18-2C19-2C9-2C8-Tel . Previously we have shown that splicing events joining exons from the neighboring 2C18 and 2C19 genes occur in human liver and epidermis . Here evidence is presented that the terminal genes of this cluster, 2C18 and 2C8, are also involved in intergenic splicing . Most interestingly, several of these 2C18/2C8 RNAs were composed of all nine exons, thus conceivably having the potential for coding functional proteins . Moreover, chimeric RNA species consisting of exons originating not only from the CYP2C8 and CYP2C18 genes, but also from the CYP2C19 gene were detected . In all cases the exons from the different CYP2C genes were joined at the correct canonical splice sites . However, the closely linked RBP4 gene is not participating in intergenic splicing with the CYP2C genes . In addition, CYP2C8 gene expression was found to generate a variety of scrambled RNA molecules including species that contained repetitions of certain exons .

Genomics, 2000 Feb 1, 63(3), 307 - 13
Molecular cytogenetic resources for chromosome 4 and comparative analysis of phylogenetic chromosome IV in great apes; Marzella R et al.; We have generated a panel of 55 somatic cell hybrids retaining fragments of human chromosome 4 . Each hybrid has been characterized cytogenetically by FISH and molecularly by 37 STSs, evenly spaced along the chromosome . The panel can be exploited to map subregionally DNA sequences on chromosome 4 and to generate partial chromosome paints useful in the characterization of chromosomal rearrangements involving this chromosome . Furthermore, a panel of 84 YACs mapping on chromosome 4 has been characterized by FISH . A subset of this panel is recognized by STSs used in the somatic cell hybrid characterization . In this way a correlation between the genetic and the physical maps can be established . These resources have been used to investigate the conservation of the phylogenetic chromosome IV in great apes . The results indicate that all the pericentric inversions that differentiate chromosome IV in these species are distinct and that one of the breakpoints frequently lies very close to the centromere . In 4 instances, the YAC containing the breakpoint was identified . The breakpoint in IVq of PTR and MMU lies in the same YAC, suggesting that this breakpoint has been utilized twice in the evolutionary history of this chromosome .

Eur Urol, 2000 Feb, 37(2), 228 - 33
Identification of a novel microsatellite marker tightly linked to the KAI-1 gene for predicting prostate cancer progression; Maraj BH et al.; BACKGROUND: We have mapped the human prostate-specific membrane antigen (PSM) gene to the chromosome 11p11.2 region at 62.5 cM, a region which also contains the prostatic cancer metastasis suppressor gene KAI-1 . The genetic marker D11S1344 has been utilised for loss of heterozygosity (LOH) studies on the KAI-1 gene in a large series of prostate cancer specimens . The results were negative and it was concluded that deletions of the KAI-1 gene were not involved in the development of the metastatic phenotype in these tumours . One possible explanation for this result could be that D11S1344 is not sufficiently tightly linked to the KAI-1 gene to detect small deletions . OBJECTIVE: To attempt to identify a genetic marker more tightly linked to the KAI-1 gene than D11S1344 . METHODS: Yeast artificial chromosome (YAC) clones containing the KAI-1 gene and the neighbouring marker D11S1344 were analysed by the fluorescent in situ hybridisation technique . The human genomic inserts in these novel clones were sized by pulsed field gel electrophoresis . For more accurate mapping of the KAI-1 gene, YACs containing it were screened for polymorphic markers (including D11S1344) from the 11p11.2 region . RESULTS: The novel YAC clones localised exclusively to the 11p11.2 region, with single hybridisation signals compared to the dual signals consistently obtained with nearby PSM-containing YACs . All the KAI-1 clones found had small inserts (<300 kb) . The only known microsatellite which gave amplification products with these YACs was D11S986 which has been mapped at 61.3 cM on human chromosome 11 . CONCLUSIONS: We have precisely localised KAI-1 at 61.3 cM on human chromosome 11 . This is some 1.2 cM away from the previously utilised LOH microsatellite marker, D11S1344 . We suggest that the very tightly linked microsatellite D11S986 may be a more accurate marker to assess LOH of the KAI-1 gene and thus predict progression of prostate cancer . The region of genetic duplication around the PSM gene does not extend as far distally on 11p as KAI-1.

Phytochemistry, 2000 Feb, 53(3), 345 - 8
Stimulation of the production of hypericins by mannan in Hypericum perforatum shoot cultures; Kirakosyan A et al.; Shoot organ cultures were established from callus derived from anthers of Hypericum perforatum flowers and the effect of elicitors on hypericin and pseudohypericin production in shoot organ cultures was investigated . Mannan stimulated pseudohypericin production up to four fold (0.82 mg/g dry wt) and hypericin production up to two fold (0.04 mg/g dry wt.) beta-1,3-glucan and pectin slightly stimulated pseudohypericin production (ca . two fold), but had no effect on hypericin production . On the other hand, yeast extract showed no stimulatory effect, on either hypericin or pseudohypericin production.

Am J Pathol, 2000 Mar, 156(3), 781 - 9
ATIC-ALK: A novel variant ALK gene fusion in anaplastic large cell lymphoma resulting from the recurrent cryptic chromosomal inversion, inv(2)(p23q35); Colleoni GW et al.; The subset of CD30-positive anaplastic large cell lymphomas (ALCL) with the NPM-ALK gene fusion arising from the t(2;5)(p23;q35) forms a distinct clinical and prognostic entity . Recently, various cytogenetic, molecular, and protein studies have provided evidence for the existence of several types of variant ALK fusions in up to 20% of ALK+ ALCL, of which only one, a TPM3-ALK fusion resulting from a t(1;2)(q25;p23), has so far been cloned . A cryptic inv(2)(p23q35) has been described as another recurrent cytogenetic alteration involving ALK and an unidentified fusion partner in some ALCL . In a screen for variant ALK gene fusions, we identified two ALCL that were negative for NPM-ALK by reverse transcriptase-polymerase chain reaction, but were positive for cytoplasmic ALK with both polyclonal and monoclonal antibodies to the ALK tyrosine kinase domain, consistent with ALK deregulation by an alteration other than the t(2;5) Case 1 was a T-lineage nodal and cutaneous ALCL in a 52-year-old woman, and Case 2 was a T-lineage nodal ALCL in a 12-year-old girl . FISH analysis confirmed ALK rearrangement in both cases . An inverse polymerase chain reaction approach was then used to identify the ALK translocation partner in Case 1 . We found an in-frame fusion of ALK to ATIC, a gene previously mapped to 2q34-q35 . We then confirmed by DNA polymerase chain reaction the localization of ATIC to yeast artificial chromosome (YAC) 914E7 previously reported to span the 2q35 break in the inv(2)(p23q35) . FISH analysis in Case 1 confirmed rearrangement of YAC 914E7 and fusion to ALK . The ATIC-ALK fusion was confirmed in Case 1 and also identified in Case 2 by conventional reverse transcriptase-polymerase chain reaction using ATIC forward and ALK reverse primers . ATIC encodes an enzyme involved in purine biosynthesis which, like other fusion partners of ALK, is constitutively expressed and appears to contain a dimerization domain . ATIC-ALK fusion resulting from the inv(2)(p23q35) thus provides a third mechanism of ALK activation in ALK+ ALCL.

Cancer Genet Cytogenet, 2000 Feb, 117(1), 66 - 70
A broad amplification pattern at 3q in squamous cell lung cancer--a fluorescence in situ hybridization study; Kettunen E et al.; Frequent DNA copy number gain at 3q, with minimal overlapping area at 3q24-qter, has previously been reported in squamous cell carcinoma of the lung (SQCC), implicating the importance of genes at 3q in the tumorigenesis of SQCC . To further characterize the gain of DNA sequences at 3q, we performed interphase fluorescence in situ hybridization (FISH) analysis on 16 paraffin-embedded SQCC tumor samples that had previously been studied by comparative genomic hybridization (CGH) . Eleven yeast artificial chromosome (YAC) probes located at 3q25-q27 and a chromosome 3-specific centromeric probe were used in the analysis . All SQCC tumors showed increase in DNA sequence copy number with 9-11 probes . In 5 tumors (31%) the number of centromeric signals varied from 3 to 5 and the YAC/centromeric signal ratio was 1.0, suggesting that the increase in DNA sequence copy number at 3q in these cases resulted from polysomy of chromosome 3 . In 11 tumors (69%), the YAC/centromeric signal ratio varied between 1.5 and 4.7, indicating that the increase in DNA sequence copy number was due to intrachromosomal gain of DNA sequences at 3q . In each case, several YACs showed increased number of signals, demonstrating that the gained area was relatively large . Our findings therefore suggest that multiple genes located at 3q25-q27 are involved in the tumorigenesis of SQCC.

Cancer Genet Cytogenet, 2000 Feb, 117(1), 9 - 18
Molecular definition of a small amplification domain within 3q26 in tumors of cervix, ovary, and lung; Sugita M et al.; A common amplification target encompassing chromosome region 3q25 to q27 has been identified by comparative genomic hybridization analyses in tumors of the cervix, ovary, endometrium, lung, and head and neck . Because this segment spans at least 30 megabases, we undertook a molecular analysis of copy number to more precisely define the amplification domain . Our Southern blot and fluorescence in situ hybridization results with the use of 17 markers confirmed the presence of low-level 3q amplification events in cervical, ovarian, and variant SCLC tumors . Most of the tumor types studied appeared to have similar, broad amplification domains centered within 3q26.2, suggesting that the same target is being affected in all . The ovarian carcinoma cell line NIH:OVCAR3 had a highly restricted amplification domain spanned by four overlapping YAC clones, suggesting a small target . The region of highest amplification included the gene for the RNA component of telomerase (hTR), supporting it as a potential target . Although the importance of low-level amplification is unknown, the consistent and reproducible nature of this event in a variety of carcinomas suggests that 3q26.2 harbors an oncogene whose low-level amplification has a significant influence on tumor biology.

Biochim Biophys Acta, 2000 Feb 28, 1495(3), 281 - 96
Phospholipase D-dependent and -independent mechanisms are involved in milk protein secretion in rabbit mammary epithelial cells; Boisgard R et al.; Phospholipase D has been implicated in membrane traffic in the secretory pathway of yeast and of some mammalian cell lines . Here we investigated the involvement of phospholipase D in protein transport at various steps of the secretory pathway of mammary epithelial cells . Treatment of rabbit mammary explants with butanol, which blocks the formation of phosphatidic acid, decreased the secretion of caseins and to a lesser extent that of whey acidic protein . Butanol interfered with both the endoplasmic reticulum to Golgi complex transport of the caseins and secretory vesicle formation from the trans-Golgi network . In contrast, the transport of whey acidic protein to the Golgi was less affected . Activation of protein kinase C enhanced the overall secretion of both markers and interestingly, this stimulation of secretion was maintained for whey acidic protein in the presence of butanol . Transphosphatidylation assays demonstrated the existence of a constitutive phospholipase D activity which was stimulated by the activation of protein kinase C . We conclude that phospholipase D plays a role in casein transport from the endoplasmic reticulum to the Golgi and in the secretory vesicle formation from the trans-Golgi network . Moreover, our results suggest a differential requirement for phospholipase D in the secretion of caseins and that of whey acidic protein.

Hum Mol Genet, 2000 Mar 1, 9(4), 631 - 6
Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice; Alami R et al.; Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells) . It is well established that locus control regions (LCRs) generally overcome position effects in transgenes . However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined . We report that single-copy yeast artificial chromosome transgenes containing an unmodified human beta -globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested . We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement . DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region . We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter-dependent mechanisms.

Hum Mol Genet, 2000 Mar 1, 9(4), 617 - 29
Expression of the human CFTR gene from episomal oriP-EBNA1-YACs in mouse cells; Huertas D et al.; Plasmids carrying the origin of plasmid replication ( oriP ) and expressing the EBNA-1 protein from the Epstein-Barr virus replicate and segregate in human cells and are thus potentially useful vectors for gene therapy . As very large circular molecules, up to 660 kb in size, can be maintained episomally using this system, it is possible to include intact human genes with all their long-range controlling elements which might give high levels of tissue-specific and controlled gene expression . We have shown previously that a 320 kb yeast artificial chromosome (YAC) carrying the intact human CFTR gene can complement the Cambridge null cystic fibrosis mice as a transgene . We have now modified this YAC to a circular molecule carrying both oriP and the EBNA-1 gene . We show that this oriP-EBNA1-YAC can be stably maintained as unrearranged episomes in mouse LA-9 cells, which do not express endogenous cftr, and in mouse CMT-93 cells, which do express endogenous cftr . The human CFTR gene is expressed in some of the cell lines, but the level of expression is very variable between cell lines and is not related to the copy number of the elements.

Hum Mol Genet, 2000 Mar 1, 9(4), 561 - 74
Developmentally distinct effects on human epsilon-, gamma- and delta-globin levels caused by the absence or altered position of the human beta-globin gene in YAC transgenic mice; Bauchwitz R et al.; The human beta-globin locus has been an important model system in the study of developmentally regulated transcription in multigene chromosomal domains . In this study, primer extension and sensitive real-time RT-PCR assays were used to quantify the effects of beta-globin sequence modifications on epsilon-, gamma- and delta-globin levels in transgenic mice . E11.5 primitive erythroid cells showed a surprisingly large increase in epsilon-globin in the absence of the beta-globin gene (beta- locus), which is weakly expressed at that stage of development . E17.5 fetal liver and adult erythroid cells, in which beta-globin expression approaches its maximum, showed an unexpectedly small, statistically insignificant stimulation of gamma- and delta-globin levels in the absence of beta-globin sequence . Analysis of erythroid colonies produced by in vitro differentiation of embryonic stem cells indicated that the absence of the human beta-globin gene had no effect on gamma-globin expression . These results suggest that competitive influences need not be linked directly to transcription level or distance from the locus control region (LCR), and that the large increases in gamma-globin levels seen in some human deletional beta-thalassemias and hereditary persistence of fetal hemoglobin conditions are most likely to be due to effects other than loss of beta-globin competition . In transgenic mice with beta-globin sequences inserted between epsilon and the LCR in a beta- locus (betaup), the expression of epsilon-, gamma- and delta-globins suggested that stage-specific sensitivity to loss of LCR activity may be a more important parameter than position relative to the LCR . The relationship of these measurements of transgenic globin expression to a possible binary model of globin LCR action and to mimicry from red blood cell loss due to transgenic globin imbalances are discussed.

Oncogene, 2000 Feb 10, 19(6), 754 - 61
Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell carcinomas: increased copy number and overexpression of the MET proto-oncogene; Glukhova L et al.; Molecular cytogenetic analysis of several sporadic papillary renal cell carcinomas and of their xenografts in immunodeficient mice had previously allowed us to delimit a minimal overrepresented region of chromosome 7 shared by all of them to band 7q31 . We have refined the location of the overlapping region to the junction of the subbands 7q31.2 and 7q31.3 by reverse painting with two differently labelled probes prepared from the small chromosome 7 derivatives microdissected from the cells of two distinct tumours . This small region was shown to contain the MET proto-oncogene, present at three to four copies per cell as determined by Southern blot analysis . The increased copy number of the MET gene was found to be associated with its overexpression at the mRNA level . However, no change in MET copy number or expression level was observed in the cells from two xenografted tumours serially transplanted into immunodeficient mice, as compared to those from the corresponding initial tumours . Our results indicate that expression of the MET proto-oncogene above a critical threshold is required for the maintenance of the tumorigenic phenotype of at least some papillary renal cell carcinomas, but does not further increase during tumour progression.

Oncogene, 2000 Feb 10, 19(6), 745 - 53
EAP1/Daxx interacts with ETS1 and represses transcriptional activation of ETS1 target genes; Li R et al.; ETS1 is a member of the evolutionarily conserved family of ets genes, which are transcription factors that bind to unique DNA sequences, either alone or by association with other proteins . In this study, we have used the yeast two-hybrid system to identify an ETS1 interacting protein . The ETS1 N-terminal amino acid region was used as bait and an interaction was identified with the Daxx protein, referred to as EAP1 (ETS1 Associated Protein 1)/Daxx . This interactin has been shown to exist in yeast and in vitro . EAP1/Daxx and ETS1 are co-localized in the nucleus of mammalian cells . The region in EAP1/Daxx which specifically binds to ETS1 is located within its carboxy terminal 173 amino acid region . The ETS1 interaction region is located within its N-terminal 139 amino acids and is referred as the Daxx Interaction Domain (DID) . The DID appears to be conserved in several other ets family members, as well as in other proteins known to interact with Daxx . The EAP1/Daxx interacts with both isoforms of ETS1, p51-ETS1 and p42-ETS1 . Interaction of EAP1/Daxx with ETS1 causes the repression of transcriptional activation of the MMP1 and BCL2 genes . The interaction domains of both ETS1 and EAP1/Daxx are required for this repression and deletion of either domain abolishes this activity.

Cancer J Sci Am, 2000 Jan-Feb, 6(1), 2 - 10
Translational research offers individually tailored treatments for cancer patients; Bartelink H et al.; The measurement of the effect of cisplatin on DNA has become feasible with the development of antibodies against DNA adducts . In a phase II dose escalation trial with concomitant radiotherapy and daily cisplatin in lung cancer, we found that patients with high DNA adduct levels measured in the buccal mucosa had a much higher survival rate than patients with a low or undetectable amount of cisplatin-DNA adducts . The use of this assay may therefore allow the selection of individual patients for concomitant treatment with cisplatin and radiotherapy, as has been shown to be effective in randomized trials in patients with lung, head and neck, and cervix malignancies . To predict the response to radiation treatment, assays have been developed for tumor growth potential by measuring the labeling index after intravenous injection of IdUrd or by estimating cyclin D1 expression . Intrinsic radiation sensitivity of human tumors can be estimated by conventional techniques, which are probably too slow or cumbersome for routine use, or with more rapid assays, such as those for chromosome damage with fluorescent probes . These assays should be able to guide us in the adaptation of the individual radiation doses that should be applied and to select patients for an accelerated or hyperfractionated regimen . Pretreatment levels of apoptosis may also be helpful in predicting treatment outcome, although the data so far show inconsistent results . A better understanding of the signal transduction pathways involved in radiation-induced apoptosis may help in the design of studies aimed at modulating the apoptotic response, thereby increasing cell kill . We have recently shown that alkyllysophospholipids, which inhibit mitogenic signaling, induce apoptosis in a variety of tumor cell lines . In combination with ionizing radiation, these compounds cause an enhancement of apoptotic cell kill . This type of a signaling-based intervention could form the basis for new therapeutic strategies . The role of hormonal therapy in breast cancer patients, both in an adjuvant setting and for the treatment of disseminated disease, is becoming increasingly important . The development of a functional assay for the estrogen receptor (ER-FASAY), based on a yeast growth assay, provides a better way than the classical immunohistochemistry assay of estimating abnormal function of the receptor in tumors . These assays are simply examples, illustrating how clinicians could improve the therapeutic outcome for their patients by implementing knowledge obtained in the laboratory in clinical decision making . With further optimization of these assays, this holds the promise for the future that the treatment for each patient can be tailored rationally to the biology of the individual.

J Biol Chem, 2000 Mar 10, 275(10), 7416 - 23
FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions; Peterson RT et al.; The FKBP12-rapamycin associated protein (FRAP, also RAFT, mTOR) belongs to a family of phosphatidylinositol kinase-related kinases . These kinases mediate cellular responses to stresses such as DNA damage and nutrient deprivation in a variety of eukaryotes from yeast to humans . FRAP regulates G(1) cell cycle progression and translation initiation in part by controlling the phosphorylation states of a number of translational and cell cycle regulators . Although FRAP is known to be phosphorylated in vivo and to phosphorylate several proteins (including itself) in vitro, FRAP's phosphorylation sites and substrate specificity are unknown . We report here the identification of a FRAP autophosphorylation site . This site, Ser-2481, is located in a hydrophobic region near the conserved carboxyl-terminal FRAP tail . We demonstrate that the COOH-terminal tail is required for FRAP kinase activity and for signaling to the translational regulator p70(s6k) (ribosomal subunit S6 kinase) . Phosphorylation of wild-type but not kinase-inactive FRAP occurs at Ser-2481 in vivo, suggesting that Ser-2481 phosphorylation is a marker of FRAP autokinase activity in cells . FRAP autophosphorylation is blocked completely by wortmannin treatment but not by rapamycin treatment, amino acid deprivation, or serum withdrawal, treatments that lead to acute dephosphorylation of eIF4E-binding protein (4E-BP1) and p70(s6k) . Ser-2481 phosphorylation increases slightly upon c-Akt/PKB activation and dramatically upon calyculin A treatment of T-cells . These results suggest that FRAP-responsive dephosphorylation of 4E-BP1 and p70(s6k) occurs through a mechanism other than inhibition of intrinsic FRAP kinase activity.

J Biol Chem, 2000 Mar 10, 275(10), 7373 - 7
Characterization of a novel serine/threonine kinase associated with nuclear bodies; Trost M et al.; A novel protein kinase, Mx-interacting protein kinase (PKM), has been identified in a yeast two-hybrid screen for interaction partners of human MxA, an interferon-induced GTPase with antiviral activity against several RNA viruses . A highly conserved protein kinase domain is present in the N-terminal moiety of PKM, whereas an Mx interaction domain overlaps with C-terminal PEST sequences . PKM has a molecular weight of about 127,000 and exhibits high sequence homology to members of a recently described family of homeodomain-interacting protein kinases . Recombinant PKM has serine/threonine kinase activity that is abolished by a single amino acid substitution in the ATP binding domain (K221W) . PKM catalyzes autophosphorylation and phosphorylation of various cellular and viral proteins . PKM is expressed constitutively and colocalizes with the interferon-inducible Sp100 protein and murine Mx1 in discrete nuclear structures known as nuclear bodies.

J Biol Chem, 2000 Mar 10, 275(10), 7212 - 23
Cloning and characterization of a novel Kruppel-associated box family transcriptional repressor that interacts with the retinoblastoma gene product, RB; Skapek SX et al.; The retinoblastoma gene product, RB, seems to function as a key tumor suppressor by repressing the expression of genes activated by members of the E2F family of transcription factors . In order to accomplish this, RB has been proposed to interact with a transcriptional repressor . However, no genuine transcriptional repressors have been identified by virtue of interaction with RB . By using the yeast two-hybrid system, we have identified a novel member of a known family of transcriptional repressors that contain zinc fingers of the Kruppel type and a portable transcriptional repressor motif known as the Kruppel-associated box (KRAB) . The mouse and human forms of the novel RB-associated KRAB protein (RBaK) are widely expressed . The amino acid motif that links the KRAB domain and zinc fingers appears to be required for interaction with RB in vitro . Human RBaK ectopically expressed in fibroblasts is an 80-kDa protein that is localized to the nucleus . The expression of either RB or RBaK in 10T1/2 fibroblasts represses the activation of an E2F-dependent promoter and decreases DNA synthesis to a similar degree . However, a mutant form of RBaK that cannot interact with RB in vitro is unable to prevent DNA synthesis . We present a model in which RB physically interacts with the novel transcriptional repressor RBaK to repress E2F-dependent genes and prevent DNA synthesis.

J Biol Chem, 2000 Mar 10, 275(10), 7184 - 8
Hepatitis C virus NS5A protein modulates transcription through a novel cellular transcription factor SRCAP; Ghosh AK et al.; Hepatitis C virus NS5A protein transcriptionally modulates cellular genes and promotes cell growth . NS5A is likely to exert its activity in concert with cellular factor(s) . Using a yeast two-hybrid screen, we have demonstrated that NS5A interacts with the C-terminal end of a newly identified cellular transcription factor, SRCAP . The authenticity of this interaction was verified by a mammalian two-hybrid assay, in vitro pull-down experiment, and an in vivo coimmunoprecipitation assay in human hepatoma (HepG2) cells . An in vitro transient transfection assay demonstrated that SRCAP can efficiently activate transcription when recruited by the Gal4 DNA-binding domain to the promoter . However, down-regulation of p21 promoter activity by NS5A was enhanced following ectopic expression of SRCAP . Together these results suggest that the interaction of NS5A and SRCAP may be one of the mechanisms by which NS5A exerts its effect on cell growth regulation contributing to hepatitis C virus-mediated pathogenesis.

J Biol Chem, 2000 Mar 10, 275(10), 7095 - 100
The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7; Mongiat M et al.; Perlecan is a multifaceted heparan sulfate proteoglycan that is expressed not only as an intrinsic constituent of basement membranes but also as a cell-surface and pericellular proteoglycan . Perlecan functions as a ligand reservoir for various growth factors that become stabilized against misfolding or proteolysis and acts as a co-receptor for basic fibroblast growth factor by augmenting high affinity binding and receptor activation . These biological properties are mediated by the heparan sulfate moiety . Rather little is known about the protein core's mediation of functions . We have recently discovered that fibroblast growth factor-7 (FGF7) binds to perlecan protein core and that exogenous perlecan efficiently reconstitutes FGF7 mitogenic activity in perlecan-deficient cells . In this report we examined the specific binding of FGF7 to various domains and subdomains of perlecan protein core . Using several experimental approaches including overlay protein assays, radioligand binding experiments, and the yeast two-hybrid system, we demonstrate that FGF7 binds specifically to the N-terminal half of domain III and to a lesser extent to domain V, with affinity constants in the range of 60 nM . Thus, perlecan protein core should be considered a novel biological ligand for FGF7, an interaction that could influence cancer growth and tissue remodeling.

J Biol Chem, 2000 Mar 10, 275(10), 6901 - 7
Cell surface monkey CD9 antigen is a coreceptor that increases diphtheria toxin sensitivity and diphtheria toxin receptor affinity; Cha JH et al.; Monkey (Mk) CD9 antigen has been shown previously to increase the diphtheria toxin (DT) sensitivity of cells when co-expressed with Mk proHB-EGF (DT receptor) . We have elucidated here the mechanism whereby Mk CD9 influences Mk proHB-EGF and present evidence that Mk CD9 is a coreceptor for DT . We observed that Mk CD9 not only increased the DT sensitivity but also increased the DT receptor affinity of cells . Furthermore, the higher the Mk CD9/Mk proHB-EGF ratio, the higher the affinity . In contrast, mouse (Ms) CD9 did not increase the toxin sensitivity or receptor affinity of cells when co-expressed with Mk proHB-EGF . Using Mk/Ms chimeric CD9 molecules, we determined that the second extracellular domain of Mk CD9 is responsible for both increased sensitivity and receptor affinity . This domain of Mk CD9 also interacts with Mk proHB-EGF in a yeast two-hybrid system . Our findings thus suggest that Mk CD9 has a direct physical interaction with Mk proHB-EGF to form a DT receptor complex and that this contact may change the conformation of the receptor to increase DT binding affinity and consequently increase toxin sensitivity . We thus propose that Mk CD9 is a coreceptor for DT.

Parassitologia, 1999 Sep, 41(1-3), 489 - 92
Malaria entomology: can genomics help?
Louis C.
The field of genomics has advanced over the last decades to the forefront of molecular genetic research . Many genomes, including that of yeast, have already been sequenced and the determination of the genome sequence of several higher organisms is now within sight, including the complete DNA sequence of Homo sapiens . Here I review the state of the Anopheles gambiae genomic research and I present the current plans for an 'Anopheles Genome Project' . An understanding of the structure and function of the vector's genome may ultimately provide better tools for the control of the malaria mosquito.

Lipids, 2000 Jan, 35(1), 55 - 9
Lipoproteins modify the macrophage uptake of triacylglycerol emulsion and of zymosan particles by similar mechanisms; Carvalho MD et al.; The uptake of lipids and formation of foam cells are key events in atherosclerosis and in eruptive xanthomata formation in primary hyperchylomicronemia . Here we have compared the influence of low density lipoprotein (LDL), oxidized LDL (oxLDL), high density lipoprotein (HDL), and delipidated HDL (apoHDL) on the uptake by macrophages of zymosan (an insoluble fraction of yeast cell walls) and of triglyceride-rich emulsion (EM) particles that resemble chylomicrons, but, like zymosan, are equally devoid of protein components . Zymosan internalization is known to occur through unspecific phagocytosis, whereas natural chylomicrons are taken up by several specific lipoprotein receptors . We found that phagocytosis is not promoted as much by oxLDL as by normal LDL . HDL-coated zymosan was found to be inert and apoHDL slightly enhanced phagocytosis . LDL and apoHDL promoted the uptake of EM while oxLDL and HDL significantly inhibited the uptake . Therefore, the data support that HDL, and not apoHDL, particles inhibit EM uptake . We concluded that by using lipoprotein-coated zymosan particles, we could demonstrate different biological effects of LDL, oxLDL, HDL, and apoHDL on macrophage phagocytosis and that this method could be useful to delineate components of the various lipoproteins important for the propagation or inhibition of the formation of foam cells.

Exp Cell Res, 2000 Mar 15, 255(2), 184 - 91
Cdk1 is essential for mammalian cyclosome/APC regulation; Listovsky T et al.; The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis . Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr) . This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro . Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint . We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1 . Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation . These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint . Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome .

Exp Cell Res, 2000 Mar 15, 255(2), 135 - 43
Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9; Xu W et al.; The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells . To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF . The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9 . The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay . Although hUBC9 is known to interact preferentially with SENTRIN/SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN . Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation . MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin-proteasome system in MITF degradation . Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation . Furthermore, we identified lysine 201 as a potential ubiquitination site . A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo . Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation .

Biochem Biophys Res Commun, 2000 Mar 5, 269(1), 203 - 8
Quinqueginsin, a novel protein with anti-human immunodeficiency virus, antifungal, ribonuclease and cell-free translation-inhibitory activities from American ginseng roots; Wang HX et al.; A homodimeric protein designated quinqueginsin, with a molecular weight of 53 kDa, has been isolated from the roots of American ginseng Panax quinquefolium . It was unadsorbed on DEAE cellulose in low ionic strength and neutral pH, and adsorbed on Affigel blue gel and SP-Sepharose under similar conditions . Its N-terminal sequence bore similarity to those of plant ribosome inactivating proteins and fungal ribonucleases . The protein displayed a variety of biological activities . It possessed ribonucleolytic activity toward yeast tRNA and specific activity toward poly C . It inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 0.26 nM, and exerted antifungal action against Fusarium oxysporum, Rhizoctonia solani, and Coprinus comatus . An inhibitory action was expressed toward human immunodeficiency virus-1 reverse transcriptase . This action was potentiated after chemical modification with succinic anhydride .

Mol Pharmacol, 2000 Mar, 57(3), 446 - 52
Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280); Li M et al.; Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling . In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors . The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding . The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280 . D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells . Maximal inhibitory responses of D(2) receptor activation were also reduced . Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358) . Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation . This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase . PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association . A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells . Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

J Biol Chem, 2000 Mar 3, 275(9), 6381 - 7
Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system; Chin MT et al.; We have cloned a cardiovascular-restricted basic helix-loop-helix factor that interacts with arylhydrocarbon receptor nuclear translocator (ARNT) in a yeast two-hybrid screen . Cardiovascular helix-loop-helix factor 1 (CHF1) is distantly related to the hairy family of transcriptional repressors . We analyzed its expression pattern during mouse embryo development . At day 8.5, the expression of CHF1 is first detected in the primitive ventricle of the primordial heart tube and persists throughout gestation . In rat hearts, this expression is down-regulated after birth, concurrent with terminal differentiation of cardiomyocytes . In the developing vasculature, CHF1 first appears in the dorsal aorta at day 9.0, which precedes the reported expression of smooth muscle cell markers, and persists into adulthood . In an in vitro system of smooth muscle cell differentiation, CHF1 mRNA was barely detectable in undifferentiated cells but was induced highly in differentiated smooth muscle cells . To determine whether CHF1 might affect the function of ARNT, we performed transfection studies . Co-transfection of CHF1 inhibited ARNT/EPAS1-dependent transcription by 85%, and this inhibition is dose-dependent . In electrophoretic mobility studies, CHF1 inhibited the binding of the ARNT/EPAS1 heterodimer to its target site . Our data suggest that CHF1 functions as a transcriptional repressor and may play an important role in cardiovascular development.

Genes Dev, 2000 Feb 15, 14(4), 475 - 82
A Krüppel-like zinc finger protein is involved in nitrogen-fixing root nodule organogenesis; Frugier F et al.; Mechanisms regulating plant host differentiation of the nitrogen-fixing root nodules remain mostly unknown . Sinorhizobium meliloti induces this process in Medicago sativa in which the Mszpt2-1 gene is expressed in vascular bundles of roots and nodules . This gene codes for a Kruppel-like zinc finger protein, a class of transcription factors involved in many animal developmental processes . Expression of Mszpt2-1 in yeast cells conferred osmotic tolerance . Antisense plants grew normally but developed nonfunctional nodules, in which differentiation of the nitrogen-fixing zone and bacterial invasion were arrested . Hence, a vascular bundle-associated Kruppel-like gene is required for the formation of the central nitrogen-fixing zone of the root nodule.

J Agric Food Chem, 2000 Feb, 48(2), 457 - 63
Purification and characterization of a mannan-binding lectin specifically expressed in corms of saffron plant (Crocus sativus L.); Escribano J et al.; Despite the economical interest of Crocus sativus, its biochemistry has been poorly studied . Herein, we have isolated a lectin present in saffron corm by gel-filtration, anion-exchange, and reversed-phase chromatography . One- and two-dimensional PAGE, MALDI-MS, and N-terminal amino acid sequence analyses indicated that the native protein forms noncovalently linked aggregates of about 80 kDa apparent molecular mass, mainly composed of two charged heterogeneous (pI's, 6.69-6.93) basic subunits of approximately 12 kDa . Their N-terminal sequences shared 25% similarity and were homologous to the N- and C-terminal domains of monocotyledonous mannose-binding lectins, respectively . An additional polypeptide of around 28 kDa apparent molecular mass was also detected, probably corresponding to a precursor processed into two mature subunits . In addition, the N-terminal domain subunit exhibited 56% similarity with curculin, a sweet protein with taste-modifying activity . The native lectin specifically interacts with a yeast mannan and is a major corm protein specifically expressed in this organ.

J Mol Neurosci, 1999 Aug-Oct, 13(1-2), 141 - 58
Activity-dependent interaction of the intracellular domain of rat trkA with intermediate filament proteins, the beta-6 proteasomal subunit, Ras-GRF1, and the p162 subunit of eIF3; MacDonald JI et al.; Many responses to nerve growth factor (NGF) are regulated through the receptor tyrosine kinase trkA . To understand more fully the functions of trkA in NGF responsive cells, we have expressed the intracellular domain of rat trkA as a fusion protein with the yeast gal4 transcription factor, and used the fusion protein to probe rat and mouse cDNA libraries by the yeast two-hybrid system . We have identified a direct interaction between the intracellular domain of trkA and two members of the intermediate filament (IF) family of proteins, the guanine-nucleotide exchange protein Ras-GRF1, the p162 subunit of eIF3, and the beta-6 proteasome subunit . The interactions are dependent on an active trkA kinase, and RasGRF1, the beta-6 proteasomal subunit, and peripherin are directly phosphorylated by trkA . The interaction with trkA is not affected by mutations at either Tyr499 or Tyr794, the two major phosphotyrosine residues essential to the activation and receptor binding of Shc, FRS-2/SNT, and phospholipase Cgamma-1, and it is highly specific in vitro for trkA, with little or no binding observed with trkB and/or trkC . The results show that trkA may play a regulatory role in a variety of cellular functions in addition to neuritogenesis, including regulated protein degradation and transcriptional activation.

DNA Res, 1999 Dec 31, 6(6), 401 - 5
Sequence-ready 1-Mb YAC, BAC and cosmid contigs covering the distal imprinted region of mouse chromosome 7; Kato R et al.; We have constructed approximately 1-Mb contigs of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones covering the imprinted region in mouse chromosome band 7F4/F5 . This region is syntenic to human chromosome 11p15.5, which is associated with Beckwith-Wiedemann syndrome (BWS) and certain childhood and adult tumors . These contigs provide the basis for genomic sequencing, identification of genes and their regulatory elements, and functional studies in transgenic and knockout mice, which should be of help to understand not only the mechanisms of imprinting but also the molecular events involved in the genesis of BWS and tumors.

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2686 - 91
Chip interacts with diverse homeodomain proteins and potentiates bicoid activity in vivo; Torigoi E et al.; The Drosophila protein Chip potentiates activation by several enhancers and is required for embryonic segmentation . Chip and its mammalian homologs interact with and promote dimerization of nuclear LIM proteins . No known Drosophila LIM proteins, however, are required for segmentation, nor for expression of most genes known to be regulated by Chip . Here we show that Chip also interacts with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and that Chip potentiates activity of one of these homeodomain proteins in Drosophila embryos and in yeast . These and other observations help explain the roles of Chip in segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers.

Biochemistry, 2000 Feb 22, 39(7), 1675 - 82
Active intracellular domain of Notch enhances transcriptional activation of CCAAT/enhancer binding protein beta on a rat pregnancy-specific glycoprotein gene; Chen H et al.; Pregnancy-specific glycoproteins (PSGs) are primarily expressed in the placenta and become the major glycoproteins at term . To understand the regulation of PSG expression, we characterized the promoter elements of a rodent PSG gene, rnCGM3, and delineated three nuclear protein binding sites: FPI, -II, and -III in the 5'-flanking region of the gene . The FPII-binding factor is shown to be C/EBPbeta, which positively regulates rnCGM3 expression {Chen, H., et al . (1995) DNA Cell Biol . 14, 681-688} . In the current study, we used the yeast one-hybrid system to isolate transcription factors binding to the FPIII site and demonstrated that a rodent J kappa recombination signal sequence binding protein, rRBPJ kappa-2N, bound to the FPIII site . Electrophoretic mobility shift assay with rat placental nuclear proteins revealed a constitutive occupancy of the FPIII site by RBPJ kappa . By transient expression analyses, we demonstrated that rRBPJ kappa-2N repressed the expression from an FPIII-driven SV40 promoter . However, this repression effect was counteracted by the active intracellular domain of Notch (NotchIC) . Using the native rnCGM3 promoter construct, we demonstrated that the promoter activity stimulated by C/EBP beta was also repressed by rRBPJ kappa-2N but enhanced by NotchIC . Additionally, we found that NotchIC can stimulate expression through another RBPJkappa site within the FPI site . A functional interaction between factors binding to the FPI, FPII, and FPIII sites is proposed.

Enzyme Microb Technol, 2000 Feb 1, 26(2-4), 252 - 258
The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida; Pitta-Alvarez SI et al.; Hairy root cultures of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine . In an attempt to increase productivity, several biotic and abiotic elicitors were tested . Salicylic acid increased significantly the release of both alkaloids (2- to 12-fold) and it also acted positively on specific production without altering the production profile . AgNO(3) increased significantly scopolamine release (3-fold) and both alkaloid's accumulation (5- to 8-fold) in the roots, thus favoring the production of scopolamine (up to 2-fold) . The inhibiting effects of AgNO(3) and salicylic acid on ethylene could be partly responsible for these responses . Yeast extract incremented the intracellular content of both alkaloids (ca . 3-fold), but particularly increased the release of scopolamine (7-fold) . CaCl(2) had little effect on accumulation or release of either alkaloid . CdCl(2) acted positively on the release of both alkaloids (3- to 24-fold), but was highly detrimental to growth . Hairy roots of B . candida are therefore susceptible to elicitation by biotic and abiotic elicitors, with variations in the kinetics of induction and the extent of release of each metabolite, thereby also exerting different effects on the alkaloid profile.

Enzyme Microb Technol, 2000 Feb 1, 26(2-4), 216 - 221
The kinetics of affinity-mediated cell-surface attachment; Ming F et al.; Data and a semi-empirical model are presented that describe the affinity interaction of yeast cells with a Concanavalin A derivatised surface . The model uses 3 parameters to describe the time course of cell attachment from a flowing suspension of yeast cells, over a range of flow rates, and gives an effective global fit to the data obtained . Further modifications allow the effects of a soluble competitor (glucose) on binding to be quantified in terms of a saturation effect, and an effective global fit is obtained . A comparison was made between the relationship between steady-state attached fraction and applied shear with similar data reported earlier (Ming, F . et al, 1998) for the detachment of pre-adsorbed cells . This shows that there is an order of magnitude difference between the forces required to effect complete detachment in the two systems, and that the nature of the relationship between shear and attached fraction is profoundly different . The magnitude of this time-dependent stabilization might be explained in terms of a progressive reorientation of cell relative to the surface such that the number of bonds is maximized.

Int J Mol Med, 2000 Mar, 5(3), 269 - 73
Molecular interactions between presenilin and calpain: inhibition of m-calpain protease activity by presenilin-1, 2 and cleavage of presenilin-1 by m-, mu-calpain; Maruyama K et al.; Several mutations of presenilin (PS)-1, 2 result in early onset Alzheimer disease . Using the yeast two-hybrid system, the interaction between PS2 loop domain and the C-terminal region of mu-calpain was previously identified . Calpain is a calcium dependent-protease and there are two isoforms, m-calpain and mu-calpain, which differ in the calcium concentration required for activation . m-Calpain needs about 10(-3) M calcium ions, whereas mu-calpain about 10(-5) M . When PS and calpain were separately expressed in COS cells by cDNA transfection and then combined in vitro, or both were co-transfected to be co-expressed in vivo in COS cells, PS1 and PS2 reduced the casein proteolysis activity of m-calpain but not that of mu-calpain . Some of the PS mutations related to Alzheimer disease decreased this inhibitory activity . On the other hand, PS1 was cleaved by m-calpain and mu-calpain at a different site from those already reported (constitutive cleavage or alternative cleavage) . These results suggest a regulatory function of presenilin on the calpain system.

Mol Cell Biol, 2000 Mar, 20(6), 2158 - 66
The branch point enzyme of the mevalonate pathway for protein prenylation is overexpressed in the ob/ob mouse and induced by adipogenesis; Vicent D et al.; We have recently reported that skeletal muscle of the ob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes . Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus . GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway . Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis . Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa . Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs . GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/ob mice by Northern blot analysis . Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA . Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold . Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate . Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation . Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

Int J Biochem Cell Biol, 2000 Feb, 32(2), 235 - 41
Ribonuclease, cell-free translation-inhibitory and superoxide radical scavenging activities of the iron-binding protein lactoferrin from bovine milk; Ye XY et al.; The purpose of this study was to characterize the ribonuclease (RNase) and cell-free translation-inhibitory activities of lactoferrin isolated from bovine milk . It was found that bovine lactoferrin exhibited ribonucleolytic activity toward yeast transfer RNA in a dose-dependent manner . The pH optimum for this RNase activity was in the vicinity of 7.5 . Lactoferrin exerted RNase activity on poly C with an activity of 2.15 U/mg . No activity was detected toward poly A, poly G, and poly U . The milk protein inhibited cell-free translation in rabbit reticulocyte lysate with an IC50 of 9.6 microM . The protein was devoid of N-glycosidase activity characteristic of ribosome inactivating proteins which also possess RNase and cell-free translation-inhibitory activities . It inhibited superoxide radical formation.

Hum Hered, 2000 May-Jun, 50(3), 151 - 7
Physical and cDNA mapping in the DBH region of human chromosome 9q34; Gilbert JR et al.; Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region . During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker . The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs . We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones . In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons . The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region .

Brain Res Mol Brain Res, 2000 Feb 22, 75(2), 237 - 47
Identification of a brain specific protein that associates with a refsum disease gene product, phytanoyl-CoA alpha-hydroxylase; Lee ZH et al.; Refsum disease is an autosomal recessive neurologic disorder of the lipid metabolism . Major diagnostic clinical findings include retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, increased cerebrospinal fluid protein without pleocytosis, nerve deafness, and cardiac involvement . We have identified a novel protein (PAHX-AP #1) associated with phytanoyl-CoA alpha-hydroxylase (PAHX), a Refsum disease gene product, using the yeast-based two-hybrid assay . The middle portion (amino acids 83-264) of PAHX was used as a bait and a mouse brain cDNA library was searched . The ability of PAHX-AP #1 to interact with PAHX was confirmed using immunoprecipitation and Western blot studies in NIH3T3 cells which stably expressed both PAHX and PAHX-AP #1 . Northern and Western blot analyses demonstrated a unique pattern of developmental PAHX-AP #1 expression which was targeted to the adult brain, but ubiquitous expressions of PAHX were observed in all examined tissues . In situ hybridization analyses of the brain showed specific localization of PAHX-AP #1 to the supragranular layer in the cerebral cortex, dentate gyrus, hippocampus, Purkinje cell layer, deep cerebellar nucleus, trigeminal nucleus, abducent nucleus, facial nucleus, cochlear and vestibular nucleus, ganglion cell and nuclear layer of the retina . These data indicate that localization of PAHX-AP #1 in the brain is correlated with central neurologic symptoms of Refsum disease such as retinitis pigmentosa, cerebellar ataxia, nerve deafness and suggest that PAHX-AP #1 may be involved in the development of the central neurologic deficits of Refsum disease.

Biol Reprod, 2000 Mar, 62(3), 789 - 96
Expression of deoxyribonucleic acid repair enzymes during spermatogenesis in mice; Richardson LL et al.; Meiotic recombination during gametogenesis is critical for proper chromosome segregation . However, the participating proteins and mechanics of recombination are not well understood in mammals . DNA repair enzymes play an essential role in both mitosis and meiosis in yeast . The mammalian mismatch repair system consists of homologues of the bacterial MutH, MutL, and MutS proteins . As part of our goal of understanding the function of enzymes that mediate meiotic recombination, we used a reverse transcription-polymerase chain reaction approach to identify germ cell transcripts for the MutL homologue, Pms2, and two members of the MutS family, Msh2 and Msh3 . Both the Pms2 and the Msh2 genes were highly expressed in mitotically proliferating spermatogonia, and early in meiotic prophase in the leptotene and zygotene spermatocytes . Thereafter, expression declined in early and mid pachytene spermatocytes, and was negligible in postmeiotic spermatids . In contrast, expression of Msh3 was at its highest level in pachytene spermatocytes . Protein levels were similar to gene expression patterns, and both PMS2 and MSH2 were localized in spermatogonia and spermatocytes . These patterns of expression for genes encoding mismatch repair enzymes are consistent with the proposed roles of the gene products in mismatch repair during both DNA replication and recombination.

Biochemistry, 2000 Feb 15, 39(6), 1316 - 23
Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity?
Mori M, Konno T, Ozawa T, Murata M, Imoto K, Nagayama K.
The voltage-dependent sodium channel (VDSC) interacts with intracellular molecules to modulate channel properties and localizations in neuronal cells . To study protein interactions, we applied yeast two-hybrid screening to the cytoplasmic C-terminal domain of the main pore-forming alpha-subunit . We found a novel interaction between the C-terminal domain and calmodulin (CaM) . By two-hybrid interaction assays, we specified the interaction site of VDSC in a C-terminal region, which is composed of 38 amino acid residues and contains both IQ-like and Baa motifs . Using a fusion protein of the C-terminal domain, we showed that interaction with CaM occurred in the presence and absence of Ca(2+) . Two synthetic peptides, each covering the IQ-like (NaIQ) or the Baa motifs (NaBaa), were used to examine the binding property by a gel mobility shift assay . Although the NaIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca(2+)-bound Ca(2+)CaM, whereas NaIQ binds to both Ca(2+)CaM and Ca(2+)-free apoCaM . Fluorescence spectroscopy of dansylated CaM showed Ca(2+)-dependent spectral changes not only for NaBaa.CaM but also for NaIQ.CaM . The results, taken together with other results, indicate that whereas the NaBaa.CaM complex is formed in a Ca(2+)-dependent manner, the NaIQ.CaM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca(2+) concentration . These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca(2+)-dependent conformational transition of the complex.

Nucleic Acids Res . 2000 Mar 15;28(6):E15.
A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells; Arndt GM et al.; We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (10(5)) for any target sequence for use in human cells . In this system, target sequences are fused to the 5' end of the lacZ reporter gene and expressed in yeast . Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype . We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors . GSCs specific for c-myc were shown to regulate expression of both a c-myc-lacZ fusion gene and the endogenous c-myc gene in human cells.

FEBS Lett, 2000 Feb 18, 468(1), 68 - 72
Intra- and intermolecular interactions of the catalytic domains of human CD45 protein tyrosine phosphatase; Hayami-Noumi K et al.; We have investigated protein-protein interaction between distinct domains of the human CD45 cytoplasmic region using a yeast two-hybrid system . Consequently, we have found that the spacer region between two tandem PTP domains specifically interacts with the membrane-distal PTP domain (D2) . This interaction is mediated by a stretch of amino acid residues in the carboxyl-terminal half of the spacer region . Although the membrane proximal region does not directly interact with either of the two PTP domains, it appears to function in stabilizing the interaction between the spacer region and D2 . We also demonstrate that the interaction between the spacer region and D2 might take place intramolecularly.

FEBS Lett, 2000 Feb 18, 468(1), 59 - 64
The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region; Acquati F et al.; We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so-called 'Down critical region' in 21q22.3 . Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones . One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family . In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved . Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules . We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane . Furthermore DRAP/BACE2 shares a high homology with a newly described beta-secretase enzyme (BACE-1) which is a transmembrane aspartic protease . The implications of this finding for Down syndrome are discussed.

J Virol, 2000 Mar, 74(6), 2840 - 6
Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential; Rahmani Z et al.; Understanding the mechanism(s) of action of the hepatitis B virus (HBV)-encoded protein HBx is fundamental to elucidating the underlying mechanisms of chronic liver disease and hepatocellular carcinoma caused by HBV infection . In our continued attempts to identify cellular targets of HBx, we have previously reported the identification of a novel cellular protein with the aid of a yeast two-hybrid assay . This cellular gene was identified as a third member of the family of human genes that encode the voltage-dependent anion channel (HVDAC3) . In the present study, physical interaction between HBx and HVDAC3 was established by standard in vitro and in vivo methods . Confocal laser microscopy of transfected cells with respective expression vectors colocalized HVDAC3 and HBx to mitochondria . This novel, heretofore unreported subcellular distribution of HBx in mitochondria implies a functional role of HBx in functions associated with mitochondria . Using a stable cationic fluorophore dye, CMXRos, we show that HBx expression in cultured human hepatoma cells leads to alteration of mitochondrial transmembrane potential . Such functional roles of HBx in affecting mitochondrial physiology have implications for HBV-induced liver injury and the development of hepatocellular carcinoma.

J Virol, 2000 Mar, 74(6), 2510 - 24
Covalent modification of the transactivator protein IE2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins SUMO-1 and hSMT3b; Hofmann H et al.; The 86-kDa IE2 protein (IE2-p86) of human cytomegalovirus (HCMV) is a potent transactivator of viral as well as cellular promoters . Several lines of evidence indicate that this broad transactivation spectrum is mediated by protein-protein interactions . To identify novel cellular binding partners, we performed a yeast two-hybrid screen using a N-terminal deletion mutant of IE2-p86 comprising amino acids 135 to 579 as a bait . Here, we report the isolation of two ubiquitin-homologous proteins, SUMO-1 and hSMT3b, as well as their conjugating activity hUBC9 (human ubiquitin-conjugating enzyme 9) as specific interaction partners of HCMV IE2 . The polypeptides SUMO-1 and hSMT3b have previously been shown to be covalently coupled to a subset of nuclear proteins such as the nuclear domain 10 (ND10) proteins PML and Sp100 in a manner analogous to ubiquitinylation, which we call SUMOylation . By Western blot analysis, we were able to show that the IE2-p86 protein can be partially converted to a 105-kDa isoform in a dose-dependent manner after cotransfection of an epitope-tagged SUMO-1 . Immunoprecipitation experiments of the conjugated isoforms using denaturing conditions further confirmed the covalent coupling of SUMO-1 or hSMT3b to IE2-p86 both after transient transfection and after lytic infection of human primary fibroblasts . Moreover, we defined two modification sites within IE2, located in an immediate vicinity at amino acid positions 175 and 180, which appear to be used alternatively for coupling . By using a SUMOylation-defective mutant, we showed that the targeting of IE2-p86 to ND10 occurs independent of this modification . However, a strong reduction of IE2-mediated transactivation of two viral early promoters and a heterologous promoter was observed in cotransfection analysis with the SUMOylation-defective mutant . This suggests a functional relevance of covalent modification by ubiquitin-homologous proteins for IE2-mediated transactivation, possibly by providing an additional interaction motif for cellular cofactors.

J Anim Sci, 2000 Jan, 78(1), 100 - 5
Effect of organic and inorganic selenium sources and levels on sow colostrum and milk selenium content; Mahan DC; A study was conducted to evaluate the short-term effects of feeding two dietary Se sources at various Se levels on the transfer of Se to the dam's milk and nursing pig . Six dietary treatments were arranged in a 2 x 2 factorial arrangement with two additional treatments in a randomized complete block designed experiment . Inorganic (sodium selenite) or organic (Se-enriched yeast) Se sources were added to the diet at .15 or .30 ppm Se . A non-Se-fortified corn-soybean meal basal diet served as a negative control, and a sixth group was fed .15 ppm Se from both inorganic and organic Se sources . A total of 43 sows were fed their treatment diets at 2.2 kg/d from 6 d prepartum to parturition and at full feed through a 14-d lactation period . Ten sows were initially bled at 6 d prepartum, and three sows and three pigs from their litters were bled at 7 and 14 d postpartum . Serum was analyzed for its Se concentration and glutathione peroxidase (GSH-Px) activity . Colostrum was collected within 12 h postpartum and milk at 7 and 14 d of lactation . When the basal diet was fed, sow serum GSH-Px activity declined from 6 d prepartum and remained low throughout lactation . When dietary Se levels increased, sow serum Se concentration and serum GSH-Px activity increased (P < .05) at both 7 and 14 d postpartum . The short-term feeding of either Se source at .15 or .30 ppm Se did not affect colostrum Se content when inorganic Se was fed, but it was increased when organic Se was provided . This resulted in a significant Se source x Se level interaction (P < .01) . Milk Se at 7 and 14 d postpartum was 2.5 to 3 times higher when the organic Se source was provided and resulted in a significant Se source x Se level interaction (P < .05) . When the combination of inorganic and organic Se was fed at .15 ppm Se, colostrum and milk Se contents were similar to those of sows fed .15 ppm Se from the organic Se source . Pig serum GSH-Px activity was not affected at 7 and 14 d of age by dietary Se level or Se source fed to the sow, but serum Se increased (P < .05) as dietary Se level increased, particularly when sows had been fed organic Se . The results demonstrated that organic Se increased milk Se content more than did inorganic Se and increased the nursing pig's serum Se . These results indicate that inorganic Se was more biologically available for sow serum GSH-Px activity, but organic Se was more effectively incorporated into milk.

J Biol Chem, 2000 Feb 25, 275(8), 5504 - 11
Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle; Huber A et al.; Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle . In the gastrointestinal tract substrates of cGKI have not been identified yet . In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system . The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T . (1993) FEBS Lett . 333, 51-55) . Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro . Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo . Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation . Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus . The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine.

J Biol Chem, 2000 Feb 25, 275(8), 5485 - 92
Identification and characterization of a PDZ protein that interacts with activin type II receptors; Shoji H et al.; We have identified a mouse PDZ protein that interacts with the activin type IIA receptor (ActRIIA), which we named activin receptor-interacting protein 1 (ARIP1) . By using yeast two-hybrid screening, we isolated a cDNA clone of ARIP1 from a mouse brain cDNA library . We detected two forms of ARIP1, ARIP1-long and ARIP1-short, which may be produced by alternative splicing . ARIP1-long had one guanylate kinase domain in the NH(2)-terminal region, followed by two WW domains and five PDZ domains (PDZ1-5) . ARIP1-short had a deletion in the NH(2)-terminal region and lacked the guanylate kinase domain . Both forms interacted with ActRIIA through PDZ5 . The COOH-terminal residues of ActRIIA (ESSL) agree with a PDZ-binding consensus motif, and ARIP1 recognized the consensus sequence . ARIP1 interacts specifically with ActRIIA among the receptors for the transforming growth factor beta family . Interestingly, ARIP1 also interacted with Smad3, which is an activin/transforming growth factor beta intracellular signaling molecule . The mRNA of ARIP1 was more abundant in the brain than in other tissues . Overexpression of ARIP1 controls activin-induced and Smad3-induced transcription in activin-responsive cell lines . These findings suggest that ARIP1 has a significant role in assembling activin signaling molecules at specific subcellular sites and in regulating signal transduction in neuronal cells.

Reprod Fertil Dev, 1999, 11(1), 17 - 23
Molecular cloning of translocation breakpoints in a case of constitutional translocation t(11;22)(q23;q11) and preparation of probes for preimplantation genetic diagnosis; Fung J et al.; In vitro fertilization (IVF) centres with preimplantation genetic diagnosis (PGD) programmes are often confronted with the problem of identifying chromosomal abnormalities in interphase cells biopsied from preimplantation embryos of carriers of a reciprocal translocation . The present authors have developed a DNA testing based approach to analyse embryos from translocation carriers, and this report describes breakpoint-spanning probes to detect abnormalities in cases of the most common human translocation (i.e . the t(11;22)(q23;q11)) . Screening a yeast artificial chromosome (YAC) library for probes covering the respective breakpoint regions in the patient lead to probes for the breakpoint on chromosome 11q23 . The physically mapped YAC and bacterial artificial chromosome (BAC) clones from chromosome 22 were then integrated with the cytogenetic map, which allowed localization of the breakpoint on chromosome 22q11 to an interval of less than 84 kb between markers D22S184 and KI457 and to prepare probes suitable for interphase cell analysis . In summary, breakpoint localization could be accomplished in about 4 weeks with additional time needed to optimize probes for use in PGD.

Mycoses, 1999, 42(11-12), 645 - 7
The influence of alcohol disinfection of nail samples in the laboratory; Willemsen R et al.; Samples of two hundred finger- and toenails cultured after a 70% alcohol disinfection for 2 min were compared with non-disinfected samples cultured on a classical way . Alcohol treatment caused a significant decrease in isolation of yeast and moulds . Both organisms were cultivated at 25.5 and 31%, respectively, when not treated with alcohol, whereas the isolation rate dropped to 10 and 17.5%, respectively, after alcohol treatment . Isolation of dermatophytes decreased from 27.5 to 23.5% when alcohol disinfection was used . In conclusion, although alcohol disinfection of nail samples in the laboratory effectively decreases the isolation rate of unwanted contaminants on Sabouraud glucose agar without cycloheximide, it hampers the isolation of dermatophytes and pathogenic yeasts on cycloheximide-containing media.

Curr Opin Cell Biol, 2000 Feb, 12(1), 126 - 32
Cytokinesis without myosin II; Gerisch G et al.; The ability of substrate-anchored Dictyostelium cells to divide without myosin II has opened the possibility of analysing the formation of cleavage furrows in the absence of a contractile ring made of filamentous myosin and actin . Similar possibilities exist in mutants of budding yeast and, less strictly, also in drug-treated mammalian cells . Myosin-II-independent activities in Dictyostelium include the microtubule-induced programming of the cell surface into ruffling areas and regions that are converted into a concave furrow, as well as the translocation of cortexillins and cross-linked membrane proteins towards the cleavage furrow . A centripetal flow of actin filaments followed by their disassembly in the cleavage furrow is proposed to underlie the translocation.

Curr Opin Cell Biol, 2000 Feb, 12(1), 113 - 8
gamma-tubulin complexes: binding to the centrosome, regulation and microtubule nucleation; Schiebel E; Microtubule assembly is initiated in vivo by gamma-tubulin complexes . Cytoplasmic gamma-tubulin complexes are targeted to centrosomes or to other microtubule organizing centers (MTOCs) via a set of so called gamma-tubulin complex binding proteins (GTBPs) that probably interact with the conserved Spc97p/Spc98p protein family of gamma-tubulin complexes . In other cell types, gamma-tubulin complexes may initiate microtubule formation near chromosomes in a MTOC-independent manner . Recently, major advances have been achieved through the finding that gamma-tubulin, Spc97p and Spc98p form a conserved core that is probably responsible for microtubule nucleation, and by the discovery that a yeast GTBP is regulated in a cell-cycle-dependent manner and in response to an external signal . Furthermore, it was found that the small GTPase Ran in its GDP-bound state may promote spindle assembly . In addition, an essential function of gamma-tubulin in basal body duplication has been demonstrated in Paramecium.

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 893 - 8
Three rat brain alternative splicing dynamin-like protein variants: interaction with the glycogen synthase kinase 3beta and action as a substrate; Chen CH et al.; Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have shown that it may be involved in the formation of coated vesicles . In this report, three different alternatively spliced dynamin-like protein variants (DLP1-WT, -11, and -37) from rat brain were identified by reverse transcription/polymerase chain reaction (RT-PCR) . One novel rat alternatively spliced variant (DLP1-37), not described previously, was identified . We examined the interaction of these three rat brain dynamin-like protein variants with glycogen synthase kinase 3beta (Gsk-3beta) using the yeast two-hybrid screening, in vitro binding assay, and immunoprecipitation analysis . It was found that all three examined rat brain dynamin-like protein variants can bind to Gsk-3beta . Moreover, in vitro kinase (phosphorylation) assay showed that mammalian dynamin-like protein acts as a substrate for glycogen synthase kinase 3beta . These data suggest that Gsk-3beta may participate in a functional role in dynamin-like proteins in vesicle trafficking .

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 697 - 703
Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains; Masuhara M et al.; To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1(+)c-kit(+)Lin(-)) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed . By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites . RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit . The highest expression was observed in the CD34(low/-)Sca-1(+)c-kit(+)Lin(-) stem cell-enriched fraction . The murine myeloid cell line, M1, expressed a high level of STAP-1 . However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type . A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2 . In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit . An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5 . These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells .

Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 345 - 53
Cloning and characterization of the cytochrome P450 oxidoreductase gene from the zygomycete fungus Cunninghamella; Yadav JS et al.; The filamentous fungus Cunninghamella utilizes cytochrome P450 system(s) in the metabolism of a broad range of polyaromatic and aliphatic pollutants and a variety of drugs, but prior attempts at isolation of P450 system components of this fungus have been generally unsuccessful . We report upon the cytochrome P450 oxidoreductase (CPR) gene from two widely studied species, C . elegans and C . echinulata . The C . elegans CPR gene was obtained by screening a genomic library using as probe a PCR amplicon obtained with degenerate primers based on known CPRs . The 2420 bp coding region contained two apparent introns (149 bp and 138 bp) . Northern blot analysis showed that the CPR gene is transcriptionally expressed in C . elegans and appears to be inducible by an alkane substrate, n-tetradecane . Phylogenetic comparison of the deduced C . elegans CPR (710 aa) suggested that it is more closely related to animal CPRs (41-42%) than to yeast (38-41%) and plant (35-36%) forms . A 2074 bp sequence containing most of the CPR gene homolog from C . echinulata was also isolated .

Cancer Res, 2000 Feb 1, 60(3), 530 - 3
Differences in estrogen receptor alpha variant messenger RNAs between normal human breast tissue and primary breast carcinomas; van Dijk MA et al.; We evaluated the differences in prevalence and functional activity of human estrogen receptor alpha (hER) variant mRNA between 21 normal breast tissues and 41 primary breast carcinomas using a functional assay in yeast for the hER First, we found that the presence of wild-type hER, relative to the total amount of hER, differs markedly (P < 0.0001) between normal breast tissue (median, 85% wild-type hER) and breast tumors (median, 74% wild-type hER) . Second, the hER variants with altered function that are present in normal breast tissue are mainly one-exon deleted splicing variants (median, 100%), whereas in breast tumors only half of all variants lack just one single exon (median, 50%; P < 0.0001) . Our results suggest that hER-dependent estrogen responsiveness of breast tissue may change during tumor outgrowth, indicating that specific hER variants may play a role in breast cancer development or progression.

Cancer Res, 2000 Feb 1, 60(3), 510 - 6
Expression of a highly conserved protein, p27BBP, during the progression of human colorectal cancer; Sanvito F et al.; The highly conserved protein p27BBP is a cytoplasmic interactor of integrin beta4 expressed in epithelia . p27BBP is found in two pools: one nuclear pool enriched in the perinucleolar region, and one cytoplasmic pool . Deletion of p27BBP in yeast is lethal as a result of loss of the ribosomal 60S subunit . The aim of this study was to investigate the distribution of p27BBP in gut epithelium and its behavior during progression of human colorectal carcinomas . Results indicated that p27BBP is high in rapidly cycling cells and decreased in villous cells committed to apoptotic cell death . In dysplastic adenomas and carcinomas, p27BBP displayed a large increase of its nucleolar component that was superimposable to argyrophylic nucleolar organizing region-associated proteins and was associated with the nuclear matrix . Western blotting confirmed increased p27BBP in dysplastic adenomas and in carcinomas . In particular, p27BBP increased progressively from adenomas to carcinomas and, in the latter, was related to the tumor stage . The overexpression of p27BBP corresponded to mRNA up-regulation in carcinomas, supporting the idea of transcriptional or post-transcriptional regulation of its expression . Results suggested that p27BBP alterations are an early event in the transition from benign to malignant colorectal phenotypes and provide a novel tool in surgical pathology.

J Gen Virol, 2000 Mar, 81(Pt 3), 821 - 30
The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6; Torres-Vega MA et al.; Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa . We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells . Here we have mapped the domains of NSP5 responsible for these associations . Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay . The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted alpha-helical structure . A deletion mutant lacking the 10 C-terminal aa (DeltaC10) failed to multimerize both in yeast cells and in an in vitro affinity assay . When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms) . In contrast, the DeltaC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein . The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5 . NSP6 was also found to interact with wild-type NSP5, but not with its mutant DeltaC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.

J Chromatogr B Biomed Sci Appl, 1999 Dec 24, 736(1-2), 153 - 66
Aggregation of recombinant hepatitis B surface antigen induced in vitro by oxidative stress; Tleugabulova D et al.; In order to examine whether oxygen radicals could be responsible for aggregation of recombinant hepatitis B surface antigen (HBsAg) during its assembly in yeast, purified HBsAg was oxidized with ammonium peroxodisulphate (AP) and analyzed by non-denaturing and denaturing size exclusion chromatography, immunoassay and immunoelectron microscopy . As a result, peroxodisulphate radicals induced a reproducible aggregation of HBsAg . At 44 mM AP, the aggregation process took a few hours and the resulting structures were large, branched and non-antigenic . During more gentle oxidation with 9 mM AP and 20-80 microM Cu2+, a continuous structural modification to HBsAg delaying for tens of hours preceded the aggregation event . During this pre-aggregation period, peroxidation of HBsAg lipids and covalent cross-linking of S protein chains occurred that led a complete loss of antigenicity of oxidized particles . In contrast, yeast-derived HBsAg aggregate is decomposed to S monomers under reducing conditions and recognized by anti-HBsAg polyclonal and monoclonal antibodies, suggesting that is has been assembled in vivo from antigenic and reversibly cross-linked particles . Based on these observations, we conclude that oxidation, at least with respect to the specific molecular sites oxidized by AP, is not a primary event in HBsAg aggregate formation in vivo . Since oxidized HBsAg was shown to be irreversibly cross-linked and non-antigenic, there are no suitable techniques for detection HBsAg oxidation in biological samples . Hence, at present, the magnitude of the in-vivo oxidative damage to HBsAg cannot be evaluated and thus, whether the plasma-derived HBsAg undergoes radical-induced oxidation in the course of viral hepatitis remains to be established . If this occurs, this process is expected to contribute to low HBsAg levels in chronic hepatitis B carriers, failure of the currently available immunoassays to identify HBsAg-positive blood donors and inconsistency in the results provided by HBsAg- and anti-HBsAg-based tests in several recent reports.

Gene, 2000 Feb 8, 243(1-2), 179 - 85
Characterization of a Rab11 homologue in Trypanosoma cruzi; Mauricio de Mendonca SM et al.; Vesicle trafficking between organelles occurs through fusion of donor and specific acceptor membranes . This process is highly regulated and ensures proper direction in sorting and packaging of a number of molecules in eukaryotic cells . Monomeric GTPases of the Rab family play a pivotal role in the control of membrane fusion and vesicle traffic . In this paper, we characterize a Trypanosoma cruzi Rab 11 homologue (TcRab11) that shares at, the amino acid level, 40% similarity with human rab11, Arabdopsis thaliana rab11 and yeast rab11 homologue genes . Western blot analysis, using a polyclonal rabbit antiserum raised against a synthetic peptide derived from the COOH-terminus of predicted the TcRab11 protein, reacted to a 26kDa protein . In immunofluorescence assays, TcRab 11, was shown to be expressed in epimastigote and amastigote forms, but it was absent in trypomastigotes . Interestingly, the TcRab11 product seems to be located at the reservosome complex, a site of active endocytosis and vesicle fusion present only in the epimastigote stage . Therefore, TcRab11 may represent the first molecular marker of this peculiar organelle.

FEMS Microbiol Lett, 2000 Feb 15, 183(2), 307 - 12
A non-stop antisense reading frame in the grp78 gene of Neurospora crassa is homologous to the Achlya klebsiana NAD-gdh gene but is not being transcribed; Monnerjahn C et al.; A long non-stop reading frame exists on the antisense strand of the grp78 gene (cDNA and genomic DNA) of Neurospora crassa . Computer analysis revealed a strong similarity of the putative antisense protein to the 10th exon of the NAD-dependent glutamate dehydrogenase gene (NAD-gdh) of Achlya klebsiana, which is itself located on the complementary strand of a transcribed hsc70 gene homologue . In Neurospora, no grp78 antisense mRNA was detected by Northern blot and reverse transcription-coupled polymerase chain reaction analyses, indicating that this long reading frame is not being transcribed . Hypotheses for the presence of such unexpressed non-stop reading frames are discussed.

FEBS Lett, 2000 Feb 11, 467(2-3), 316 - 20
Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system; De Jaeger G et al.; The coding sequences of three single-chain variable (scFv) fragments (A4, G4 and H3), which bind to dihydroflavonol-4-reductase (DFR) of Petunia hybrida, and the DFR-encoding sequence were cloned in two-hybrid vectors . The vectors were transformed in the yeast strain HF7c (his3-200, trp1-901, leu2-3) and the scFv-DFR interaction was analyzed by measuring yeast growth on medium without histidine . ScFv-G4 and, to a lesser extent, scFv-A4 could interact with DFR in the yeast nucleus . On the contrary, scFv-H3 showed no interaction with its antigen in yeast . The results of a previous expression analysis of the same scFv fragments in the plant cytosol correlate with those of the two-hybrid test . This suggests that it is possible to evaluate the antigen-scFv interaction in a reducing subcellular environment with the two-hybrid test . Therefore, the yeast two-hybrid system can be useful to identify candidate scFv fragments for intracellular antibody applications.

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