Cytokine, 2001 Aug 7, 15(3), 122 - 37 Evidence that HAX-1 is an interleukin-1 alpha N-terminal binding protein; Yin H et al.; During studies aimed at understanding the function of the N-terminal peptide of interleukin-1 alpha (IL-1 NTP, amino acids 1-112), which is liberated from the remainder of IL-1 alpha during intracellular processing, we identified by yeast two-hybrid analysis a putative interacting protein previously designated as HAX-1 . In vitro binding studies and transient transfection experiments confirmed that HAX-1 can associate with the IL-1 NTP . HAX-1 was first identified as a protein that associates with HS1, a target of non-receptor protein tyrosine kinases within haematopoietic cells . Recent data have also revealed interactions between HAX-1 and three disparate proteins, polycystin-2 (derived from the PKD2 gene), a protein linked to polycystic kidney disease, cortactin, and Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) . Sequence analysis of different HAX-1 binding domains revealed a putative consensus binding motif that is present in various intracellular proteins . Overlapping peptides comprising the IL-1 NTP were synthesized, and binding experiments revealed that discrete peptides were capable of interacting with HAX-1 . HAX-1 may serve to retain the IL-1 NTP in the cytoplasm, and complex formation between the IL-1 NTP and HAX-1 may play a role in motility and/or adhesion of cells . Biochem Biophys Res Commun, 2001 Sep 21, 287(2), 574 - 82 p38: A novel protein that associates with the vesicular stomatitis virus glycoprotein; Sevier CS et al.; The vesicular stomatitis virus glycoprotein (VSV G) is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway . The cytoplasmic domain of VSV G contains information for several intracellular sorting steps including efficient export from the ER, basolateral delivery, and endocytosis . In order to identify proteins that potentially interact with the polypeptide sorting motifs in the VSV G tail, the carboxy-terminal 27 amino acids of VSV G were used as bait in a yeast two-hybrid system . The protein identified most frequently in the screen is a novel protein of 38 kDa, p38 . In the present work, the initial molecular and biochemical characterization of p38 is described . Preliminary evidence suggests that p38 may interact transiently with endoplasmic reticulum (ER) membranes, and thus may affect VSV G and other cargo movement at the step of ER to Golgi traffic . Antioxid Redox Signal, 2001 Aug, 3(4), 635 - 50 SAG/ROC/Rbx/Hrt, a zinc RING finger gene family: molecular cloning, biochemical properties, and biological functions; Sun Y et al.; The RING (really interesting new gene) finger proteins containing a characteristic C3HC4 or C3H2C3 motif appear to act as E3 ubiquitin ligase and play important roles in many processes, including cell-cycle progression, oncogenesis, signal transduction, and development . This review is focused on SAG/ROC/Rbx/Hrt (sensitive to apoptosis gene/regulator of cullins/RING box protein), an evolutionarily conserved RING finger family of proteins that were cloned recently by several independent laboratories through differential display, yeast two-hybrid screening, or biochemical purification . SAG/ROC2/Rbx2/Hrt2 is expressed in multiple mouse adult tissues, as well as early embryos . In humans, both SAG and ROC1 are ubiquitously expressed at a very high level in heart, skeletal muscle, and testis . Expression of both SAG and ROC1 is induced by mitogenic stimulation . SAG is also induced by a redox agent in cultured cells, as well as in in vivo mouse brain upon ischemia/reperfusion . Structurally, SAG consists of four exons and three introns with at least one splicing variant and two pseudogenes . The SAG gene promoter is enriched with multiple transcription factor binding sites . Biochemically, SAG binds to RNA, has metal-ion binding/free radical scavenging activity, and is redox-sensitive . Most importantly, like ROC1, SAG/ROC2 binds to cullins and acts as an essential component of E3 ubiquitin ligase . Biologically, SAG is a growth-essential gene in yeast . In mammalian cells, SAG protects apoptosis mainly through inhibition of cytochrome c release/caspase activation, and promotes growth under serum deprivation at least in part by inhibiting p27 accumulation . Blocking SAG expression via antisense transfection inhibits tumor cell growth . Thus, SAG appears to be a valid drug target for anticancer therapy. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11539 - 44 Epub 2001 Sep 11. Oral immunization with hepatitis B surface antigen expressed in transgenic plants; Kong Q et al.; Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared . An oral adjuvant, cholera toxin, was used to increase immune responses . Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg . Electron microscopy of transgenic plant samples revealed evidence that the HBsAg accumulated intracellularly; we conclude that natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut . The correlate of protection from hepatitis B virus infection is serum antibody titers induced by vaccination; the protective level in humans is 10 milliunits/ml or greater . Mice fed HBsAg-transgenic potatoes produced HBsAg-specific serum antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long-lasting secondary antibody response . We have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule . The demonstrated success of oral immunization for hepatitis B virus with an "edible vaccine" provides a strategy for contributing a means to achieve global immunization for hepatitis B prevention and eradication. Mol Biol Cell, 2001 Sep, 12(9), 2688 - 98 Evidence for import of a lysyl-tRNA into marsupial mitochondria; Dorner M et al.; The mitochondrial tRNA gene for lysine was analyzed in 11 different marsupial mammals . Whereas its location is conserved when compared with other vertebrate mitochondrial genomes, its primary sequence and inferred secondary structure are highly unusual and variable . For example, eight species lack the expected anticodon . Because the corresponding transcripts are not altered by any RNA-editing mechanism, the lysyl-tRNA gene seems to represent a mitochondrial pseudogene . Purification of marsupial mitochondria and in vitro aminoacylation of isolated tRNAs with lysine, followed by analysis of aminoacylated tRNAs, show that a nuclear-encoded tRNA(Lys) is associated with marsupial mitochondria . We conclude that a functional tRNA(Lys) encoded in the nuclear genome is imported into mitochondria in marsupials . Thus, tRNA import is not restricted to plant, yeast, and protozoan mitochondria but also occurs also in mammals. J Biol Chem, 2001 Nov 16, 276(46), 42632 - 8 Epub 2001 Sep 11. Involvement of a novel zinc finger protein, MIZF, in transcriptional repression by interacting with a methyl-CpG-binding protein, MBD2; Sekimata M et al.; MBD2, a methyl-CpG-binding protein, is a component of the MeCP1 histone deacetylase (HDAC) complex and plays a critical role in DNA methylation-mediated transcriptional repression . To understand the molecular basis of the methylation-associated repression, we attempted to identify MBD2-interacting proteins by a yeast two-hybrid system . Using MBD2 as bait, we isolated a novel zinc finger protein, referred to as MIZF . A direct interaction between MBD2 and MIZF was confirmed by in vitro binding assays and immunoprecipitation experiments . Four of seven zinc fingers present in the C-terminal region of MIZF are required for binding with MBD2 . The MIZF mRNA is expressed in all human tissues and cell lines examined . The subcellular localization of MIZF is distinct from that of MBD2, although both proteins co-localize in some areas of the nuclei; MIZF localizes diffusely in the nucleoplasmic region, whereas MBD2 preferentially localizes in major satellites . A reporter assay demonstrated that MIZF significantly abrogates transcriptional activities . This repression is attenuated by an HDAC inhibitor, trichostatin A, and is completely dependent on the interaction with MBD2 . These results suggest that MIZF is abundantly present in cells and functions as a negative regulator of transcription by binding to MBD2 and recruiting HDAC-containing complexes. J Am Chem Soc, 2001 Sep 19, 123(37), 8902 - 13 Selective interactions of cationic porphyrins with G-quadruplex structures; Han H et al.; G-quadruplex DNA presents a potential target for the design and development of novel anticancer drugs . Because G-quadruplex DNA exhibits structural polymorphism, different G-quadruplex typologies may be associated with different cellular processes . Therefore, to achieve therapeutic selectivity using G-quadruplexes as targets for drug design, it will be necessary to differentiate between different types of G-quadruplexes using G-quadruplex-interactive agents . In this study, we compare the interactions of three cationic porphyrins, TMPyP2, TMPyP3, and TMPyP4, with parallel and antiparallel types of G-quadruplexes using gel mobility shift experiments and a helicase assay . Gel mobility shift experiments indicate that TMPyP3 specifically promotes the formation of parallel G-quadruplex structures . A G-quadruplex helicase unwinding assay reveals that the three porphyrins vary dramatically in their abilities to prevent the unwinding of both the parallel tetrameric G-quadruplex and the antiparallel hairpin dimer G-quadruplex DNA by yeast Sgs1 helicase (Sgs1p) . For the parallel G-quadruplex, TMPyP3 has the strongest inhibitory effect on Sgs1p, followed by TMPyP4, but the reverse is true for the antiparallel G-quadruplex . TMPyP2 does not appear to have any effect on the helicase-catalyzed unwinding of either type of G-quadruplex . Photocleavage experiments were carried out to investigate the binding modes of all three porphyrins with parallel G-quadruplexes . The results reveal that TMPyP3 and TMPyP4 appear to bind to parallel G-quadruplex structures through external stacking at the ends rather than through intercalation between the G-tetrads . Since intercalation between G-tetrads has been previously proposed as an alternative binding mode for TMPyP4 to G-quadruplexes, this mode of binding, versus that determined by a photocleavage assay described here (external stacking), was subjected to molecular dynamics calculations to identify the relative stabilities of the complexes and the factors that contribute to these differences . The DeltaG(o) for the external binding mode was found to be driven by DeltaH(o) with a small unfavorable TDeltaS(o) term . The DeltaG(o) for the intercalation binding model was driven by a large TDeltaS(o) term and complemented by a small DeltaH(o) term . One of the main stabilizing components of the external binding model is the energy of solvation, which favors the external model over the intercalation model by -67.94 kcal/mol . Finally, we propose that intercalative binding, although less favored than external binding, may occur, but because of the nature of the intercalative binding, it is invisible to the photocleavage assay . This study provides the first experimental insight into how selectivity might be achieved for different G-quadruplexes by using structural variants within a single group of G-quadruplex-interactive drugs. J Cell Biol, 2001 Sep 17, 154(6), 1161 - 71 Epub 2001 Sep 10. HES6 acts as a transcriptional repressor in myoblasts and can induce the myogenic differentiation program; Gao X et al.; HES6 is a novel member of the family of basic helix-loop-helix mammalian homologues of Drosophila Hairy and Enhancer of split . We have analyzed the biochemical and functional roles of HES6 in myoblasts . HES6 interacted with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif . HES6 repressed transcription from an N box-containing template and also when tethered to DNA through the GAL4 DNA binding domain . On N box-containing promoters, HES6 cooperated with HES1 to achieve maximal repression . An HES6-VP16 activation domain fusion protein activated the N box-containing reporter, confirming that HES6 bound the N box in muscle cells . The expression of HES6 was induced when myoblasts fused to become differentiated myotubes . Constitutive expression of HES6 in myoblasts inhibited expression of MyoR, a repressor of myogenesis, and induced differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain . Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant led to increased expression of MyoR and completely blocked the muscle development program . Our results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression. J Biol Chem, 2001 Nov 16, 276(46), 43074 - 82 Epub 2001 Sep 10. Functional interaction of Jun and homeodomain proteins; Schaefer LK et al.; We have used the yeast two-hybrid system to identify proteins that interact with the N-terminal region of c-Jun, which is known to be involved in regulatory interactions . One of the proteins identified is the homeodomain-containing protein Hex . The Hex homeodomain is sufficient for interaction; moreover, the homeodomains of several other transcription factors also interact . Mutations within helix III of the Hex homeodomain greatly reduce the interaction . In vitro, c-Jun/c-Fos, JunB/c-Fos, and JunD/c-Fos all interact with the Hex homeodomain more strongly than the respective Jun proteins (or c-Fos) alone, suggesting that heterodimerization exposes reactive regions in the N termini of the Jun proteins . In transfected cells, Hex expression inhibits Jun- or Jun/c-Fos-dependent transcription of a reporter gene; the presence of Hex-binding sites in the promoter enhances the inhibitory effect . Jun-dependent activation of transcription from the basic fibroblast growth factor gene, previously shown to be regulated by both Jun and homeodomain proteins, was also dramatically reduced by Hex expression . Furthermore, in contrast to the reduction of Jun-mediated transcription by Hex, we found that expression of the Drosophila ultrabithorax gene enhanced c-Jun-dependent transcription . We conclude that the functional interaction between members of the Jun and homeodomain families of transcription factors could play a critical role in regulating developmental and differentiation programs. Lancet, 2001 Sep 1, 358(9283), 696 - 701 Effect of preoperative oral immune-enhancing nutritional supplement on patients at high risk of infection after cardiac surgery: a randomised placebo-controlled trial; Tepaske R et al.; BACKGROUND: Elderly patients and those with poor ventricular function have increased morbidity and mortality rates when undergoing surgery . We aimed to ascertain whether an oral immune-enhancing nutritional supplement could improve preoperative host defence, and subsequently lower postoperative infections and organ dysfunction in patients undergoing elective cardiac surgery who are at high risk of infection . METHODS: In this prospective, randomised, double-blind, placebo-controlled study, we randomly assigned 50 patients who were scheduled to undergo coronary artery bypass to receive either an oral immune-enhancing nutritional supplement containing L-arginine, omega3 polyunsaturated fatty acids, and yeast RNA (n=25), or a control (n=25) for a minimum of 5 days . Patients were included if they were aged 70 years or older, or had an ejection fraction of less than 0.4, or were scheduled to undergo mitral valve replacement . The main outcome was preoperative host defence (delayed-type hypersensitivity response to recall antigens, expression of HLA-DR epitopes on monocytes, and concentration of interleukin 6 in plasma) . Analysis was per protocol . FINDINGS: Five patients (two in the treatment group) were excluded because they did not take the minimum dose . Preoperative expression of HLA-DR epitopes on monocytes was significantly higher in patients given the study treatment (109% {95% CI 92-128}) than those given the control (69% {58-82}) compared with baseline (100%) (p=0.02, repeated measures ANOVA) . However, concentration of interleukin 6 was significantly lower in the treatment group (0.90 pg/L {0.69-1.18}) than in the control group (1.94 pg/L {1.45-2.59}) (p=0.032, repeated measures ANOVA) . Additionally, delayed-type hypersensitivity response to recall antigens improved preoperatively and remained better until hospital discharge . INTERPRETATION: Intake of an oral immune-enhancing nutritional supplement for a minimum of 5 days before surgery can improve outlook in high-risk patients who are undergoing elective cardiac surgery. Phytochemistry, 2001 Sep, 58(2), 227 - 32 Modification of fatty acids changes the flavor volatiles in tomato leaves; Wang C et al.; Expression of the yeast Delta9 desaturase gene in tomato (Lycopersicon esculentum Mill.) resulted in changes in the profiles of fatty acids in tomato leaves . Transgenic leaves displayed a dramatic increase in cis-Delta9 16:1, which only existed in a small quantity in control leaves . Also higher, but not as dramatic, were 18:1 and 16:3 fatty acids . Several fatty acids, viz . 16:0, 18:0, and 18:3 declined in transgenic leaves . Changes in fatty acids were accompanied by changes in certain volatile compounds derived from fatty acids . On a percentage basis, most notable increases (>3-fold) were 1-hydroxy-2-butanone, 1-penten-3-ol, heptanal, 3-hexen-1-ol, 2-octanol,cis-3-hexenal, hexanal and 2-nonenal . Several flavor compounds not known to be biochemically derived from fatty acids, viz . 2-ethyl-furan, 5-ethyl-2-{5H}-furanone, eugenol, and 2-ethylthiophene also showed sharp increases in transgenic leaves. Mol Plant Microbe Interact, 2001 Sep, 14(9), 1105 - 13 Local modulation of host pH by Colletotrichum species as a mechanism to increase virulence; Prusky D et al.; The phytopathogenic fungus Colletotrichum gloeosporioides produces one pectate lyase (PL) that is a key virulence factor in disease development . During growth of C . gloeosporioides, Colletotrichum acutatum, and Colletotrichum coccodes in acidified yeast extract medium, the fungus secreted ammonia and increased the medium pH . Ammonia accumulation and the consequent pH change increased as a function of initial pH and buffer capacity of the medium . PL secretion by C . gloeosporioides correspondingly increased as the pH of the medium increased . The C . gloeosporioides pelB gene-disrupted mutant was able to increase ammonia accumulation and pH of the media similarly to the wild-type isolate . C . gloeosporioides in avocado, C . coccodes in tomato, and C . acutatum in apple showed ammonia accumulation in the infected area where pH increased to 7.5 to 8 and PL activity is optima . In nonhost interactions where C . gloeosporioides was inoculated in apples, the addition of ammonia-releasing compounds significantly enhanced pathogenicity to levels similar to those caused by the compatible C . acutatum-apple interaction . The results therefore suggest the importance of ammonia secretion as a virulence factor, enhancing environmental pH and pathogenicity of the Colletotrichum species. Plant Cell, 2001 Sep, 13(9), 2021 - 32 A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum; Tormakangas K et al.; Protein sorting to plant vacuoles is known to be dependent on a considerable variety of protein motifs recognized by a family of sorting receptors . This can involve either traffic from the endoplasmic reticulum (ER) through the Golgi apparatus or direct ER-to-vacuole transport . Barley aspartic protease (Phytepsin) was shown previously to reach the vacuole via trafficking through the Golgi apparatus . Here we show that Phytepsin normally exits the ER in a COPII-mediated manner, because the Phytepsin precursor accumulates in the ER upon specific inhibition of the formation of COPII vesicles in vivo . Phytepsin differs from its yeast and mammalian counterparts by the presence of a saposin-like plant-specific insert (PSI) . Deletion of this domain comprising 104 amino acids causes efficient secretion of the truncated molecule (Phytepsin Delta PSI) without affecting the enzymatic activity of the enzyme . Interestingly, deletion of the PSI also changes the way in which Phytepsin exits the ER . Inhibition of COPII vesicle formation causes accumulation of the Phytepsin precursor in the ER but has no effect on the secretion of Phytepsin Delta PSI . This suggests either that vacuolar sorting commences at the ER export step and involves recruitment into COPII vesicles or that the PSI domain carries two signals, one for COPII-dependent export from the ER and one for vacuolar delivery from the Golgi . The relevance of these observations with respect to the bulk flow model of secretory protein synthesis is discussed. Genomics, 2001 Aug, 76(1-3), 58 - 65 Molecular and functional analyses of the human and mouse genes encoding AFG3L1, a mitochondrial metalloprotease homologous to the human spastic paraplegia protein; Kremmidiotis G et al.; The identification of SPG7 as the gene defective in a recessive form of spastic paraplegia has drawn attention to the yeast protein family of ATP-dependent zinc metalloproteases . The protein encoded by SPG7, paraplegin, shows high homology to members of this protein family . Recently, many mammalian ATP-dependent zinc metalloproteases have been identified and considered as possible candidates for defects in other forms of hereditary spastic paraplegia and possibly other neurodegenerative disorders . So far only a partial sequence has been available for one of those genes, ATPase family gene-3, yeast-like-1 (AFG3L1) . We have carried out detailed molecular analysis of this gene and identified and characterized its mouse orthologue, Afg3l1 . Our data indicate that AFG3L1 is transcribed into four mRNA isoforms that are not translated in humans . Afg3l1 encodes a protein with high homology to paraplegin and the other members of the ATP-dependent zinc metalloprotease family . Like the other ATP-dependent zinc metalloproteases, Afg3l1 localizes to the mitochondria. Genomics, 2001 Aug, 76(1-3), 45 - 57 A high-resolution 6.0-megabase transcript map of the type 2 diabetes susceptibility region on human chromosome 20; Fossey SC et al.; Recent linkage studies and association analyses indicate the presence of at least one type 2 diabetes susceptibility gene in human chromosome region 20q12-q13.1 . We have constructed a high-resolution 6.0-megabase (Mb) transcript map of this interval using two parallel, complementary strategies to construct the map . We assembled a series of bacterial artificial chromosome (BAC) contigs from 56 overlapping BAC clones, using STS/marker screening of 42 genes, 43 ESTs, 38 STSs, 22 polymorphic, and 3 BAC end sequence markers . We performed map assembly with GraphMap, a software program that uses a greedy path searching algorithm, supplemented with local heuristics . We anchored the resulting BAC contigs and oriented them within a yeast artificial chromosome (YAC) scaffold by observing the retention patterns of shared markers in a panel of 21 YAC clones . Concurrently, we assembled a sequence-based map from genomic sequence data released by the Human Genome Project, using a seed-and-walk approach . The map currently provides near-continuous coverage between SGC32867 and WI-17676 ( approximately 6.0 Mb) . EST database searches and genomic sequence alignments of ESTs, mRNAs, and UniGene clusters enabled the annotation of the sequence interval with experimentally confirmed and putative transcripts . We have begun to systematically evaluate candidate genes and novel ESTs within the transcript map framework . So far, however, we have found no statistically significant evidence of functional allelic variants associated with type 2 diabetes . The combination of the BAC transcript map, YAC-to-BAC scaffold, and reference Human Genome Project sequence provides a powerful integrated resource for future genomic analysis of this region. Biochem Biophys Res Commun, 2001 Sep 14, 287(1), 282 - 7 M phase-specific association of human topoisomerase IIIbeta with chromosomes; Kobayashi M et al.; Two isoforms, 1 and 2, of human DNA topoisomerase IIIbeta were expressed in HeLa cells as a fusion protein to the C-terminus of green fluorescent protein (GFP) . The fusion protein of the isoform 1 was found to be localized to the nucleus, and to be associated with chromosomes during metaphase and anaphase . As yeast top3 mutants are known to exhibit phenotypes indicative of defective chromosome segregation, the result suggests that the isoform 1 of the human enzyme may also be involved in chromosome segregation . Two-hybrid screening for interaction partners of the isoform identified three candidate genes: CENP-F, a gene encoding a centromere protein and two genes of no known function, one of which was novel . The GFP fusion of the isoform 2 was found in the cytoplasm, indicating the nuclear localization signal sequence in the isoform 1 is in the C-terminal part that is different between the two isoforms . Biochem Biophys Res Commun, 2001 Sep 14, 287(1), 116 - 21 Cloning and characterization of hMAP126, a new member of mitotic spindle-associated proteins; Chang MS et al.; One novel gene product, hMAP126, was demonstrated to interact with p29 in the yeast two-hybrid assay . The full-length cDNA of hMAP126 has been obtained and encodes a protein of 1120 amino acids . Multiple tissue Northern blot analysis showed that hMAP126 was abundantly expressed in the testis . Polyclonal antiserum against hMAP126 was raised and affinity-purification of anti-hMAP126 antibodies was performed . The subcellular distribution of hMAP126 was localized to the mitotic spindle . Furthermore, hMAP126 was identified to be post-translationally modified and phosphorylated by p34(cdc2) kinase in vitro . Taken together, we have isolated a novel protein, hMAP126, which may be involved in the functional and dynamic regulation of mitotic spindles . Semin Thromb Hemost, 2001 Aug, 27(4), 357 - 72 Production and clinical development of a Hansenula polymorpha-derived PEGylated hirudin; Avgerinos GC et al.; This article describes the expression of the hirudin gene heterologously in the methylotrophic yeast Hansenula polymorpha, the establishment of an industrial-scale production process and the subsequent clinical development of polyethylene glycol (PEG)-hirudin . PEGylation increases the molecular weight of hirudin, thereby reducing its kidney filtration rate and immunogenicity and increasing its half-life in the circulation. J Membr Biol, 2001 Sep 1, 183(1), 39 - 50 Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions; Zvyagilskaya R et al.; In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying H(+)- and Na(+)-coupled phosphate transport systems . Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y . lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation . This was demonstrated for the first time for cells grown at pH 9.5-10.0 . In cells grown at pH 4.5, inorganic phosphate (P(i)) was accumulated by two kinetically discrete H(+)/P(i)-cotransport systems . The low-affinity system is most likely constitutively expressed and operates at high P(i) concentrations . The high-affinity system, subjected to regulation by both extracellular P(i) availability and intracellular polyphosphate stores, is mobilized during P(i)-starvation . In cells grown at pH 9.5-10, P(i) uptake is mediated by several kinetically discrete Na(+)-dependent systems that are specifically activated by Na(+) ions and insensitive to the protonophore CCCP . One of these, a low-affinity transporter operative at high P(i) concentrations is kinetically characterized here for the first time . The other two, high-affinity, high-capacity systems, are derepressible and functional during P(i)-starvation and appear to be controlled by extracellular P(i) . They represent the first examples of high-capacity, Na(+)-driven P(i) transport systems in an organism belonging to neither the animal nor bacterial kingdoms . The contribution of the H(+)- and Na(+)-coupled P(i) transport systems in Y . lipolytica cells grown at different pH values was quantified . In cells grown at pH values of 4.5 and 6.0, the H(+)-coupled P(i) transport systems are predominant . The contribution of the Na(+)/P(i) cotransport systems to the total cellular P(i) uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher. J Biol Chem, 2001 Nov 2, 276(44), 41049 - 58 Epub 2001 Sep 06. Phosphorylation of the human ubiquitin-conjugating enzyme, CDC34, by casein kinase 2; Block K et al.; The ubiquitin-conjugating enzyme, CDC34, has been implicated in the ubiquitination of a number of vertebrate substrates, including p27(Kip1), IkappaBalpha, Wee1, and MyoD . We show that mammalian CDC34 is a phosphoprotein that is phosphorylated in proliferating cells . By yeast two-hybrid screening, we identified the regulatory (beta) subunit of human casein kinase 2 (CK2) as a CDC34-interacting protein and show that human CDC34 interacts in vivo with CK2beta in transfected cells . CDC34 is specifically phosphorylated in vitro by recombinant CK2 and HeLa nuclear extract at five sites within the carboxyl-terminal 36 amino acids of CDC34 . Importantly, this phosphorylation is inhibited by heparin, a substrate-specific inhibitor of CK2 . We have also identified a kinase activity associated with CDC34 in proliferating cells, and we show that this kinase is sensitive to heparin and can utilize GTP, strongly suggesting it is CK2 . Phosphorylation of CDC34 by the associated kinase maps predominantly to residues 203 and 222 . Mutation of CDC34 at CK2-targeted residues, Ser-203, Ser-222, Ser-231, Thr-233, and Ser-236, abolishes the phosphorylation of CDC34 observed in vivo and markedly shifts nuclearly localized CDC34 to the cytoplasm . These results suggest a potential role for CK2-mediated phosphorylation in the regulation of CDC34 cell localization and function. J Biol Chem, 2001 Nov 23, 276(47), 44003 - 11 Epub 2001 Sep 06. Cloning and characterization of a p53-related protein kinase expressed in interleukin-2-activated cytotoxic T-cells, epithelial tumor cell lines, and the testes; Abe Y et al.; A human protein kinase, p53-related protein kinase (PRPK), was cloned from an interleukin-2-activated cytotoxic T-cell subtraction library . PRPK appears to be a homologue of a growth-related yeast serine/threonine protein kinase, YGR262c . However, a complementation assay using YGR262c-disrupted yeast indicated that PRPK is not functionally identical to the yeast enzyme . PRPK expression was observed in interleukin-2-activated cytotoxic T-cells, some human epithelial tumor cell lines, and the testes . The intrinsic transcriptional activity of p53 was up-regulated by a transient transfection of PRPK to COS-7 cells . PRPK was shown to bind to p53 and to phosphorylate p53 at Ser-15 . These results indicate that PRPK may play an important role in the cell cycle and cell apoptosis through phosphorylation of p53. J Biol Chem, 2001 Nov 2, 276(44), 41518 - 25 Epub 2001 Sep 06. Mammalian plasma membrane ecto-nucleoside triphosphate diphosphohydrolase 1, CD39, is not active intracellularly . The N-glycosylation state of CD39 correlates with surface activity and localization; Zhong X et al.; CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosphohydrolase family . The active site for native CD39 is located on the outer surface of the cellular plasma membrane; however, it is not yet known at what stage this enzyme becomes active along the secretory pathway to the plasma membrane . In this study, sucrose density fractionations performed on CD39-transfected COS-7 cell membranes suggest that CD39 activity resides primarily in the plasma membrane . Furthermore, we have created recombinant, soluble versions of CD39, one that is secreted and others that are retained in the endoplasmic reticulum, to demonstrate that CD39 is not active until it reaches the plasma membrane both in yeast and COS-7 cells . Moreover, the secreted active soluble CD39 in COS-7 cells is found to receive a higher degree of N-glycan addition than the inactive form retained intracellularly . When COS-7 cells were treated with tunicamycin to prevent N-glycosylation, soluble CD39 was not detected in the extracellular medium and remained inactive intracellularly . Surface biotinylation analysis also revealed that surface-expressed wild type CD39 receives a higher degree of N-glycosylation than intracellular forms and that inhibition of N-glycosylation prevents its plasma membrane localization . In addition, both intact and digitonin-permeablized COS-7 cells transfected with CD39 possess similar ecto-ATPase activities, further supporting the conclusion that only surface-expressed CD39 is enzymatically active . All of these data suggest that intracellular CD39 is inactive and that only a fully glycosylated CD39 has apyrase activity and is localized at the cell surface. J Biol Chem, 2001 Nov 9, 276(45), 42108 - 15 Epub 2001 Aug 23. Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications; Ghosh SS et al.; Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively . Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme . To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA . Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution . Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9 . Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain . Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain . A C127Y substitution abolished UGT1A1 activity, but not its dimerization . Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1 . Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme. J Biol Chem, 2001 Oct 26, 276(43), 39911 - 8 Epub 2001 Aug 23. Identification and characterization of hic-5/ARA55 as an hsp27 binding protein; Jia Y et al.; hsp27 has been reported to participate in a wide variety of activities, including resistance to thermal and metabolic stress, regulation of growth and differentiation, and acting as a molecular chaperone or a regulator of actin polymerization . We hypothesized that these diverse functions are regulated in a cell- or tissue-specific manner via interaction with various binding proteins . To investigate this hypothesis, we used hsp27 as a "bait" to screen a yeast two-hybrid cDNA library from rat kidney glomeruli and identified a novel hsp27 binding protein, hic-5 (also known as ARA55), a focal adhesion protein and steroid receptor co-activator . Biochemical interaction between hsp27 and hic-5 was confirmed by co-immunoprecipitation, and critical protein.protein interaction regions were mapped to the hic-5 LIM domains and the hsp27 C-terminal domain . Initial analysis of the functional role of hsp27.hic-5 interaction revealed that hic-5 significantly inhibited the protection against heat-induced cell death conferred by hsp27 overexpression in co-transfected 293T cells . In contrast, when a non-hsp27-interacting hic-5 truncation mutant (hic-5/DeltaLIM4) was co-expressed with hsp27, the hic-5 inhibition of hsp27 protection was absent . We conclude that hic-5 is a true hsp27 binding protein and inhibits the ability of hsp27 to provide protection against heat shock in an interaction-dependent manner. J Biol Chem, 2001 Oct 26, 276(43), 39903 - 10 Epub 2001 Aug 23. Protein kinase Calpha plays a critical role in mannosylerythritol lipid-induced differentiation of melanoma B16 cells; Zhao X et al.; Mannosylerythritol lipid (MEL), a novel extracellular glycolipid from yeast, was found to inhibit the proliferation of mouse melanoma B16 cells in a dose-dependent manner and to induce the apoptosis of B16 cells at concentrations higher than 10 microm (Zhao, X., Wakamatsu, Y., Shibahara, M., Nomura, N., Geltinger, C., Nakahara, T., Murata, T., and Yokoyama, K . K . (1999) Cancer Res . 59, 482-486) . We show here that exposure of B16 cells to MEL (5 microm) for 2 days resulted in an increase of the levels of differentiation-associated markers of melanoma cells such as melanogenesis and tyrosinase activity, which were accompanied by morphological changes . The MEL-induced differentiation of B16 cells at this concentration was closely associated with arrest of the cell cycle at G(1) phase, but no significant population of apoptotic cells was identified . Expression of protein kinase Calpha (PKCalpha) was enhanced after exposure of B16 cells to MEL for 48 h . Antisense oligodeoxynucleotides against the mouse gene for PKCalpha prevented MEL-induced melanogenesis in B16 cells . Conversely, the effects of the expression of a constitutively active form of PKCalpha mimicked the effects of MEL on B16 cells . These data suggest that MEL, a yeast-derived glycolipid, triggers the differentiation of B16 melanoma cells through a signaling pathway that involves PKCalpha. Development, 2001 Sep, 128(17), 3349 - 58 Developmental regulation of the heat shock response by nuclear transport factor karyopherin-alpha3; Fang X et al.; During early stages of Drosophila development the heat-shock response cannot be induced . It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo . Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo it does not enter the nucleus in response to heat shock . In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity . The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF . Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a Drosophila nuclear transport protein karyopherin-alpha3 (dKap-alpha3) . Biochemical methods demonstrate that the dKap-alpha3 protein binds specifically to the dHSF's nuclear localization sequence (NLS) . Furthermore, the dKap-alpha3 protein does not associate with NLSs that contain point mutations, which are not transported in vivo . Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-alpha3 . Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-alpha3 protein through cycle 12 . From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock. Development, 2001 Sep, 128(17), 3221 - 32 cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C . elegans; Navarro RE et al.; A high frequency of apoptosis is a conserved hallmark of oocyte development . In C . elegans, about half of all developing oocytes are normally killed by a physiological germline-specific apoptosis pathway, apparently so that they donate cytoplasm to the survivors . We have investigated the functions of CGH-1, the C . elegans ortholog of the predicted RNA helicase ste13/ME31B/RCK/p54, which is germline-associated in metazoans and required for sexual reproduction in yeast . We show that CGH-1 is expressed specifically in the germline and early embryo, and is localized to P granules and other possible mRNA-protein particles . cgh-1 is required for oocyte and sperm function . It is also needed to prevent the physiological germline apoptosis mechanism killing essentially all developing oocytes, making lack of cgh-1 function the first stimulus identified that can trigger this mechanism . We conclude that cgh-1 and its orthologs may perform conserved functions during gametogenesis, that in C . elegans certain aspects of oocyte development are monitored by the physiological germline apoptosis pathway, and that similar surveillance mechanisms may contribute to germline apoptosis in other species. Mol Cell, 2001 Aug, 8(2), 351 - 61 Structure of the TRFH dimerization domain of the human telomeric proteins TRF1 and TRF2; Fairall L et al.; TRF1 and TRF2 are key components of vertebrate telomeres . They bind to double-stranded telomeric DNA as homodimers . Dimerization involves the TRF homology (TRFH) domain, which also mediates interactions with other telomeric proteins . The crystal structures of the dimerization domains from human TRF1 and TRF2 were determined at 2.9 and 2.2 A resolution, respectively . Despite a modest sequence identity, the two TRFH domains have the same entirely alpha-helical architecture, resembling a twisted horseshoe . The dimerization interfaces feature unique interactions that prevent heterodimerization . Mutational analysis of TRF1 corroborates the structural data and underscores the importance of the TRFH domain in dimerization, DNA binding, and telomere localization . A possible structural homology between the TRFH domain of fission yeast telomeric protein Taz1 with those of the vertebrate TRFs is suggested. J Mol Biol, 2001 Sep 7, 312(1), 95 - 106 Probing the structure of F-actin: cross-links constrain atomic models and modify actin dynamics; Orlova A et al.; Cross-links between protomers in F-actin can be used as a very sensitive probe of both the dynamics and structure of F-actin . We have characterized filaments formed from a previously described yeast actin Q41C mutant, where disulfide bonds can be formed between the Cys41 that is introduced into subdomain-2 and Cys374 on an adjacent protomer . We find that the distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability cross-linking reaction . The random distribution suggests that disulfide formation does not cause a significant perturbation of the F-actin structure . Consistent with this lack of perturbation, three-dimensional reconstructions of extensively cross-linked filaments, using a new approach to helical image analysis, show very small structural changes with respect to uncross-linked filaments . This finding is in conflict with refined models but in agreement with the original Holmes et al . model for F-actin . Under conditions where 94 % of the protomers are linked by disulfide bonds, the distribution of filament twist becomes more heterogeneous with respect to control filaments . A molecular model suggests that strain, introduced by the disulfide, is relieved by increasing the twist of the long-pitch actin helices . Disulfide formation makes yeast actin filaments approximately three times less flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin . These observations support previous reports that the rigidity of F-actin can be controlled by the position of subdomain-2, and that this region is more flexible in yeast F-actin than in skeletal muscle F-actin . J Mol Biol, 2001 Sep 7, 312(1), 17 - 26 The 5'HS4 core element of the human beta-globin locus control region is required for high-level globin gene expression in definitive but not in primitive erythropoiesis; Navas PA et al.; To assess the contribution of DNase I-hypersensitive site 4 (HS4) of the beta-globin locus control region (LCR) to overall LCR function we deleted a 280 bp fragment encompassing the core element of 5'HS4 from a 248 kb beta-globin locus yeast artificial chromosome (beta-YAC) and analyzed globin gene expression during development in beta-YAC transgenic mice . Four transgenic lines were established; each contained at least one intact copy of the beta-globin locus . The deletion of the 5'HS4 core element had no effect on globin gene expression during embryonic erythropoiesis . In contrast, deletion of the 5'HS4 core resulted in a significant decrease of gamma and beta-globin gene expression during definitive erythropoiesis in the fetal liver and a decrease of beta-globin gene expression in adult blood . We conclude that the core element of 5'HS4 is required for globin gene expression only in definitive erythropoiesis . Absence of the core element of HS4 may limit the ability of the LCR to provide an open chromatin domain and/or enhance gamma and beta-globin gene expression in the adult erythroid cells . Electrophoresis, 2001 Aug, 22(13), 2812 - 23 Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis; Bruneau JM et al.; Previous studies in Aspergillus fumigatus (Mouyna I., Fontaine T., Vai M., Monod M., Fonzi W . A., Diaquin M., Popolo L., Hartland R . P., Latge J.-P, J . Biol . Chem . 2000, 275, 14882-14889) have shown that a glucanosyltransferase playing an important role in fungal cell wall biosynthesis is glycosylphosphatidylinositol (GPI) anchored to the membrane . To identify other GPI-anchored proteins putatively involved in cell wall biogenesis, a proteomic analysis has been undertaken in A . fumigatus and the protein data were matched with the yeast genomic data . GPI-anchored proteins of A . fumigatus were released from membrane preparation by an endogenous GPI-phospholipase C, purified by liquid chromatography and separated by two-dimensional electrophoresis . They were characterized by their peptide mass fingerprint through matrix-assisted laser desorption/ionization-time of flight-(MALDI-TOF)-mass spectrometry and by internal amino acid sequencing . Nine GPI-anchored proteins were identified in A . fumigatus . Five of them were homologs of putatively GPI-anchored yeast proteins (Csa1p, Crh1p, Crh2p, Ecm33p, Gas1p) of unknown function but shown by gene disruption analysis to play a role in cell wall morphogenesis . In addition, a comparative study performed with chitin synthase and glucanosyl transferase mutants of A . fumigatus showed that a modification of the growth phenotype seen in these mutants was associated to an alteration of the pattern of GPI-anchored proteins . These results suggest that GPI-anchored proteins identified in this study are involved in A . fumigatus cell wall organization. Nat Genet, 2001 Oct, 29(2), 206 - 11 Disruption of Trrap causes early embryonic lethality and defects in cell cycle progression; Herceg Z et al.; The transactivation/transformation-domain associated protein (TRRAP) belongs to the Ataxia-telangiectasia mutated (ATM) super-family and has been identified as a cofactor for c-MYC-mediated oncogenic transformation . TRRAP and its yeast homolog (Tra1p) are components of histone acetyltransferase (HAT) complexes, SAGA (refs . 2,4,5), PCAF (ref . 3) and NuA4 (ref . 6), which are important for the regulation of transcription and cell cycle progression and also have a role in cell viability . Yet the biological function of this molecule and how it controls proliferation are still unclear . Here we show that null mutation of Trrap in mice results in peri-implantation lethality due to a blocked proliferation of blastocysts . We use an inducible Cre-loxP system to show that loss of Trrap blocks cell proliferation because of aberrant mitotic exit accompanied by cytokinesis failure and endoreduplication . Trrap-deficient cells fail to sustain mitotic arrest despite chromosome missegregation and disrupted spindles, and display compromised cdk1 activity . Trrap is therefore essential for early development and required for the mitotic checkpoint and normal cell cycle progression. J Biol Chem, 2001 Nov 9, 276(45), 42370 - 81 Epub 2001 Sep 05. A direct interaction between the carboxyl-terminal region of CDC5L and the WD40 domain of PLRG1 is essential for pre-mRNA splicing; Ajuh P et al.; The human proteins CDC5L (hCDC5) and PLRG1 are both highly conserved components of a multiprotein complex that is a subunit of the spliceosome . The respective homologues in yeast of both proteins are also associated with a sub-spliceosomal multiprotein complex that has been shown to be important for pre-mRNA splicing . We show that these two human proteins are associated in vivo and will interact directly in vitro . The regions containing the interacting domains in both proteins have been identified . Our results indicate that the carboxyl-terminal region of CDC5L and the WD40 domain of PLRG1 are essential for direct interaction between both proteins . By using a bacterially expressed mutant protein, containing the PLRG1 interacting domain in CDC5L, we show that the CDC5L-PLRG1 interaction in HeLa nuclear extract can be disrupted causing pre-mRNA splicing to be inhibited . Thus, a direct interaction between the CDC5L protein and PLRG1 in the CDC5L complex is essential for pre-mRNA splicing progression. Adv Space Res, 1999, 24(3), 361 - 6 Experimental and mathematical models for small aqueous closed ecosystems with spatially separated components; Pisman TI et al.; Experimental and theoretical models of closed "autotroph-heterotroph" (chlorella-yeast, chlorella-protozoa) ecosystems with spatially separated components have been created and studied . The chart of flows and interaction of components of gas-closed "chlorella-yeast" system have formed the basis describe mathematically the functioning of the given system, experimental results have been found to agree with computer solution of the problem in terms of quality . Investigation of the experimental model of the "producer-consumer" trophic chain demonstrated the role of protozoa in nitrogen turnover . "Production-decomposition" and "production-grazing-decomposition" cycle models has been theoretically analyzed and compared . The predator has been shown to be a more intensive mineralizer than the reducer component. Plant Physiol Biochem, 1992, 30(2), 163 - 72 Characterization of GTP binding and hydrolysis in plasma membranes of zucchini; Perdue DO et al.; We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins . The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins . Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM . This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions . The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins . Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM . Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins. Life Support Biosph Sci, 1999, 6(2), 133 - 9 Experimental models of small closed systems with spatially separated unicellular organism-based components; Pis'man TI et al.; Experimental models of small biotic cycles of different degree of closure and complexity with spatially separated components based on unicellular organisms have been studied . Gas closure of components looped into "autotroph-heterotroph" (chlorella-yeast) system doubled the lifetime of the system (as opposed to individually cultivated components) . Higher complexity of the heterotroph component consisting of two yeast species also increased the lifetime of the system through more complete utilization of the substrate by competing yeast species . The lifetime of gas and substrate closed "producer-consumer" trophic chain (chlorella-paramecia) increased to 7 months . In 60 days the components' numbers reached their steady state followed by more than 40 cycles of the medium . The role of a predator organism (protozoan) in nitrogen cycling was demonstrated; reproduction of protozoa correlated directly with their emission of nitrogen in the ammonia form that is most optimum for growth of chlorella. Adv Space Res, 1994 Oct, 14(10), 979 - 88 Issues and problems for radiobiological research in space; Kiefer J; The uniqueness of the space radiation field creates specific problems in the evaluation of hazards to men and materials . Comprehensive measurements of all physical parameters are necessary but not sufficient . Particular attention has to be paid to variables like solar flares by applying fast-responding active dosimetry . The assessment of biological consequences poses even more problems . There are no human data for the kinds of particles seen in space and they will presumably never be available . The only reasonable approach is therefore to use the information obtained for other radiations and check their applicability for the space situation . This involves both the study of fundamental processes in ground experiments as well as their verification in space missions . Special emphasis has to be laid on the modification of radiation effects by flight-dynamic factors and microgravity . Radiation protection guidelines for space flights cannot simply be transformed from existent regulations designed for radiation workers on earth but have to be tailored to the specific situation in space. Adv Space Res, 1994 Oct, 14(10), 257 - 65 Mutation induction by heavy ions; Kiefer J et al.; Mutation induction by heavy ions is compared in yeast and mammalian cells . Since mutants can only be recovered in survivors the influence of inactivation cross sections has to be taken into account . It is shown that both the size of the sensitive cellular site as well as track structure play an important role . Another parameter which influences the probability of mutation induction is repair: Contrary to naive assumptions primary radiation damage does not directly lead to mutations but requires modification to reconstitute the genetic machinery so that mutants can survive . The molecular structure of mutations was analyzed after exposure to deuterons by amplification with the aid of polymerase chain reaction . The results--although preliminary--demonstrate that even with densely ionizing particles a large fraction does not carry big deletions which suggests that point mutations may also be induced by heavy ions. Adv Space Res, 1986, 6(11), 169 - 78 Quantitative interpretation of heavy ion effects: comparison of different systems and endpoints; Kiefer J; For a quantitative interpretation of biological heavy ion action the following parameters have to be taken into account: variations of energy depositions in microscopical sites, the dependence of primary lesion formation on local energy density and changes in reparability . They can be studied in objects of different size and with different sensitivities . Results on survival and mutation induction in yeast and in mammalian cells will be compared with theoretical predictions . It is shown that shouldered survival curves of diploid yeast can be adequately described if the final slope is adjusted according to the varying production of primary lesions . This is not the case for mammalian cells where the experiments show a rapid loss of the shoulder with LET, contrary to theoretical expectations . This behaviour is interpreted to mean that the reparability of heavy ion lesions is different in the two systems . Mutation induction is theoretically expected to decrease with higher LET . This is found in yeast but not in mammalian cells where it actually increases . These results suggest a higher rate of misrepair in mammalian cells. Bioessays, 2001 Sep, 23(9), 767 - 71 Decisive factors: a transcription activator can overcome heterochromatin silencing; Eissenberg JC; Eukaryotes organize certain chromosomal intervals into domains capable of silencing most genes . Examples of silencing domains include the HML/HMR loci and subtelomeric chromatin in yeast, the Barr body X chromosome in mammals, and the pericentric heterochromatin of Drosophila . Silencing chromatin is often correlated with more regularized nucleosomal array than that found in active chromatin, and transcriptional activators appear to be missing from their target sites in silent chromatin . In Drosophila, gene silencing by heterochromatin is often variegated, indicating that a gene may escape silencing in some cells . In a recent study, Ahmad and Henikoff(1) show that a yeast activator can compete successfully with Drosophila heterochromatic silencing factors for target sites in DNA . This competition, together with developmental change in the stability of heterochromatin itself, decides the transcriptional state for a gene subject to heterochromatin repression . Bioelectromagnetics, 2001 Sep, 22(6), 371 - 83 Mechanism of the fluorescent light induced suppression of Curly phenotype in Drosophila melanogaster; Pavelka J et al.; A dominant mutation Curly (Cy), frequently used as a marker on the second chromosome in Drosophila melanogaster, was previously shown to be suppressed by several factors, including larval crowding, low temperature, and fluorescent light . While the first two factors affect this mutation only partially, fluorescent tube exposed flies exhibit an almost completely suppressed (wild type) phenotype . This suppressive effect is the result of a combination of the electric field and light, both factors being produced by common fluorescent tubes . In this study, experiments were carried out to clarify the basic mechanism of this unique phenomenon . Two fluorescent tube sensitive stages of Drosophila development were found in the second half of embryonic development and first half of the pupal stage . Riboflavin, which is administered to Drosophila larvae with yeast, and decomposed by light, seems to play a key role in this phenomenon . In a medium lacking riboflavin caused by light exposure, Cy expression is inhibited by the action of electric field . Positive results of experiments with lithium ions, which block the opening of Ca(2+) channels, support the hypothesis that electromagnetic fields may alter ion currents during ontogenic development of Drosophila, and thus influence, expression of the Cy gene . Also, fluorescent light induces an overexpression of a specific protein in the imaginal wing disc of Cy pupae . J Neurobiol, 2001 Oct, 49(1), 62 - 78 Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation; Wolf RM et al.; During development of the central nervous system, oligodendrocyte progenitor cells differentiate into mature myelinating cells . The molecular signals that promote this process, however, are not well defined . One molecule that has been implicated in oligodendrocyte differentiation is the Src family kinase Fyn . In order to probe the function of Fyn in this system, a yeast two hybrid screen was performed . Using Fyn as bait, p190 RhoGAP was isolated in the screen of an oligodendrocyte cDNA library . Coimmunoprecipitation and in vitro binding assays verified that p190 RhoGAP bound to the Fyn SH2 domain . Phosphorylation of p190 required active Fyn tyrosine kinase and was increased threefold upon differentiation of primary oligodendrocytes . Moreover, complex formation between p190 and p120 RasGAP occurred in differentiated oligodendrocytes . p190 RhoGAP activity is known to regulate the RhoGDP:RhoGTP ratio . Indeed, expression of dominant negative Rho in primary oligodendrocytes caused a hyperextension of processes . Conversely, constitutively activated Rho caused reduced process formation . These findings define a pathway in which Fyn activity regulates the phosphorylation of p190, leading to an increase in RhoGAP activity with a subsequent increase in RhoGDP, which in turn, regulates the morphological changes that accompany oligodendrocyte differentiation . J Biol Chem, 2001 Nov 9, 276(45), 42339 - 46 Epub 2001 Sep 04. Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells; Weinman EJ et al.; Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC) . The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC . In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed . mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC . By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity . NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins . NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro . Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC . Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets. J Biol Chem, 2001 Dec 7, 276(49), 46237 - 42 Epub 2001 Sep 04. Proteome analysis and morphological studies reveal multiple effects of the immunosuppressive drug mycophenolic acid specifically resulting from guanylic nucleotide depletion; Escobar-Henriques M et al.; Mycophenolic acid (MPA), one of the most promising immunosuppressive drugs recently developed, is a potent inhibitor of IMP dehydrogenase, the first committed step toward GMP synthesis . We found that all the drug effects on yeast cells were prevented by bypassing GMP synthesis, thus confirming the high specificity of MPA . Although the primary target of MPA is clearly identified, we aimed to further understand how GTP depletion leads to growth arrest and developed a new approach based on proteome analysis combined with overexpression studies . Essential proteins down-expressed in the presence of MPA were identified by protein two-dimensional gel analysis and subsequently overexpressed in yeast . Two such proteins, Cdc37p and Sup45p, when overexpressed allowed partial relief of MPA toxicity, strongly suggesting that their lower amount after MPA treatment significantly contributed to the MPA effect . These conserved proteins involved in cell cycle progression and translation are therefore important secondary targets for MPA . Our data establish that MPA effects occur through inhibition of a unique primary target resulting in guanine nucleotides depletion, thereby affecting multiple cellular processes. Chromosoma, 2001 Aug, 110(4), 284 - 91 The centromere composition of multiple repetitive sequences on rice chromosome 5; Nonomura K et al.; The large-scale primary structure of the centromeric region of rice chromosome 5 was analyzed, the first example in a cereal species . The yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) contigs aligned on the centromere of rice chromosome 5 (CEN5) covered a distance of more than 670 kb . Strong suppression of genetic recombination, one of the features of a functional centromere, occurred along the contig region . The most remarkable feature of CEN5 is the composition of the multiple repetitive elements . Oryza-specific RCS2 short tandem repeats were clustered along less than 100 kb at one end of the contig . At least 15 copies of the conserved domain of the 1.9 kb RCE1 centromeric repeats, which are similar to the long terminal repeats (LTRs) of gypsy-type retrotransposon RIRE7, were dispersed mainly in 320 kb stretches next to RCS2 tandem clusters . Many copies of the LTR-like sequences of RIRE3 and RIRE8, another gypsy-type retrotransposon, were also found throughout the contig . On the other hand, the gagpol region was less conserved in the contig . These results indicate that the rice centromere is composed of multiple repetitive sequences with the RCS2 tandem cluster probably being situated as the core of a functional centromere of some hundreds of kilobases to megabases in length. Analyst, 2001 Aug, 126(8), 1367 - 71 Interaction of morin-cetyltrimethylammonium bromide with nucleic acids and determination of nucleic acids at nanograms per milliliter levels based on the enhancement of preresonance light scattering; Liu R et al.; A new preresonance light scattering (PRLS) assay of nucleic acids is presented . At pH 7.30, the weak PRLS of morin-cetyltrimethylammonium bromide (CTMAB) can be greatly enhanced by the addition of nucleic acids, owing to the interaction between the nucleic acid and morin-CTMAB . After the addition of morin and CTMAB to DNA, the zeta potential of DNA decreases and changes from negative to positive, which is due to the formation of an associate, the aggregation of morin on nucleic acids and the electric neutralization between DNA and the cationic surfactant CTMAB . Mechanism studies showed that the enhanced PRLS comes from the aggregation of morin in the presence of nucleic acids and CTMAB . The enhanced intensity of PRLS is in proportion to the concentration of nucleic acids in the range 7.5 x 10(-9)-1.0 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-1.0 x 10(-6) g ml(-1) for salmon sperm DNA and 1.0 x 10(-8)-1.0 x 10(-6) g ml(-1) for yeast RNA . The detection limits are 3.4, 6.2 and 4.1 ng ml(-1) for calf thymus DNA, salmon sperm DNA and yeast RNA, respectively . Synthetic samples were analyzed satisfactorily. Nat Cell Biol, 2001 Sep, 3(9), E201 - 4 A new view of mRNA export: separating the wheat from the chaff; Reed R et al.; Current models for the export of messenger RNA share the notion that the highly abundant class of nuclear RNA-binding proteins--the hnRNP proteins--have a key role in exporting RNA . But recent studies have led to a new understanding of several non-hnRNP proteins, including SR proteins and the conserved mRNA export factor ALY, which are recruited to the mRNA during pre-mRNA splicing . These studies, together with older work on hnRNP particles and assembly of the spliceosome, lead us to a new view of mRNA export . In our model, the non-hnRNP factors form a splicing-dependent mRNP complex that specifically targets mature mRNA for export, while hnRNP proteins retain introns in the nucleus . A machinery that is conserved between yeast and higher eukaryotes functions to export the mRNA. J Nutr, 2001 Sep, 131(9), 2343 - 50 Selenium from selenium-rich Spirulina is less bioavailable than selenium from sodium selenite and selenomethionine in selenium-deficient rats; Cases J et al.; The bioavailabilty of selenium (Se) from selenium-rich Spirulina (SeSp) was assessed in Se-deficient rats by measuring tissue Se accumulation and glutathione peroxidase (GSH-Px) activity . For 42 d, rats were subjected to dietary Se depletion by consumption of a Torula yeast (TY)-based diet with no Se; controls were fed the same diet supplemented with 75 microg Se/kg diet as sodium selenite . Se-deficient rats were then repleted with Se (75 microg/kg) by the addition of sodium selenite, selenomethionine (SeMet) or SeSp to the TY basal diet . Selenium speciation in SeSp emphasized the quasi-absence of selenite (2% of total Se); organic Se comprised SeMet (approximately 18%), with the majority present in the form of two selenoproteins (20-30 kDa and 80 kDa) . Gross absorption of Se from SeSp was significantly lower than from free SeMet and sodium selenite . SeMet was less effective than sodium selenite in restoring Se concentration in the liver but not in kidney . SeSp was always much less effective . Similarly, Se from SeSp was less effective than the other forms of Se in restoring GSH-Px activity, except in plasma and red blood cells where no differences were noted among the three sources . This was confirmed by measuring the bioavailability of Se by slope-ratio analysis using selenite as the reference form of Se . Although Se from SeSp did not replenish Se concentration and GSH-Px activity in most tissues to the same degree as the other forms of Se, we conclude that it is biologically useful and differently metabolized due to its chemical form. J Virol, 2001 Oct, 75(19), 9393 - 406 Interactions and nuclear import of the N and P proteins of sonchus yellow net virus, a plant nucleorhabdovirus; Goodin MM et al.; We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus . Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import . Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124 . The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST) . Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain . Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region . Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains . N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS . Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization. J Biol Chem, 2001 Nov 2, 276(44), 41318 - 24 Epub 2001 Aug 30. Identification of a novel interaction of 14-3-3 with p190RhoGEF; Zhai J et al.; Activation of Rho GTPases by guanine nucleotide exchange factors (GEFs) mediates a broad range of cytoskeletal alterations that determine cell shape . In the nervous system, Rho GTPases are essential for establishing highly asymmetrical neuronal forms and may fine-tune the shape of dendrites in differentiated neurons . p190RhoGEF is a brain-enriched, RhoA-specific GEF whose highly interactive C-terminal domain provides potential linkage to multiple pathways in the cell . In the present study, a yeast two-hybrid screen was used to identify 14-3-3eta and 14-3-3epsilon as additional binding partners of p190RhoGEF . Interactions between p190RhoGEF and 14-3-3eta were confirmed biochemically and by colocalization of the respective proteins when fused to fluorescent markers and transfected in neuronal cells . We also mapped a unique phosphorylation-independent binding site (I(1370)QAIQNL) in p190RhoGEF . Deletion of the binding site abolished interactions in vitro as well as the ability of 14-3-3eta to alter the cytoplasmic aggregation of p190RhoGEF in cotransfected cells . The findings suggest a potential role for 14-3-3 in modulating p190RhoGEF activity or in linking p190RhoGEF to the activities of other pathways in the neuron. Hum Mol Genet, 2001 Aug 15, 10(17), 1767 - 73 The mutational spectrum of human malignant autosomal recessive osteopetrosis; Sobacchi C et al.; Human malignant infantile osteopetrosis (arOP; MIM 259700) is a genetically heterogeneous autosomal recessive disorder of bone metabolism, which, if untreated, has a fatal outcome . Our group, as well as others, have recently identified mutations in the ATP6i (TCIRG1) gene, encoding the a3 subunit of the vacuolar proton pump, which mediates the acidification of the bone/osteoclast interface, are responsible for a subset of this condition . By sequencing the ATP6i gene in arOP patients from 44 unrelated families with a worldwide distribution we have now established that ATP6i mutations are responsible for approximately 50% of patients affected by this disease . The vast majority of these mutations (40 out of 42 alleles, including seven deletions, two insertions, 10 nonsense substitutions and 21 mutations in splice sites) are predicted to cause severe abnormalities in the protein product and are likely to represent null alleles . In addition, we have also analysed nine unrelated arOP patients from Costa Rica, where this disease is apparently much more frequent than elsewhere . All nine Costa Rican patients bore either or both of two missense mutations (G405R and R444L) in amino acid residues which are evolutionarily conserved from yeast to humans . The identification of ATP6i gene mutations in two families allowed us for the first time to perform prenatal diagnosis: both fetuses were predicted not to be affected and two healthy babies were born . This study contributes to the determination of genetic heterogeneity of arOP and allows further delineation of the other genetic defects causing this severe condition. Carcinogenesis, 2001 Sep, 22(9), 1335 - 41 Counteracting spontaneous transformation via overexpression of rate-limiting DNA base excision repair enzymes; Frosina G; DNA damage of endogenous origin may significantly contribute to human cancer . A major pathway involved in DNA repair of endogenous damage is DNA base excision repair (BER) . BER is rather efficient in human cells but a certain amount of endogenous damage inevitably escapes mending and likely contributes to human carcinogenesis . Apart from some glycosylases that are particularly sluggish (e.g . 8-oxoG DNA glycosylase), recent work suggests that the general rate-limiting steps of BER may be trimming of 2-deoxyribose 5-phosphate in case the process is started by a monofunctional glycosylase or trimming of a 3'-blocking fragment, in case BER is started by a bifunctional glycosylase or in the case of single-strand breaks produced by free radical attack . Overexpression of the 5'-deoxyribophosphodiesterase (dRPase) domain of DNA polymerase beta, on the one hand, and of yeast APN1 protein, containing an efficient 3' repair activity, on the other, may lead to improved BER in mammals . The recently characterized S3 protein of Drosophila, containing both dRPase and 3'-trimming activities, could also be considered for overexpression studies . The possible protecting role of enhanced BER could be investigated in cultured rodent embryonic fibroblasts undergoing spontaneous transformation, a most interesting system that merits rediscovery. Genome Biol . 2001;2(8):SOFTWARE0001 . Epub 2001 Aug 06. AFM 4.0: a toolbox for DNA microarray analysis; Breitkreutz BJ et al.; We have developed a series of programs, collectively packaged as Array File Maker 4.0 (AFM), that manipulate and manage DNA microarray data . AFM 4.0 is simple to use, applicable to any organism or microarray, and operates within the familiar confines of Microsoft Excel . Given a database of expression ratios, AFM 4.0 generates input files for clustering, helps prepare colored figures and Venn diagrams, and can uncover aneuploidy in yeast microarray data . AFM 4.0 should be especially useful to laboratories that do not have access to specialized commercial or in-house software. Biochem Genet, 2001 Jun, 39(5-6), 201 - 11 Phylogenetic relationships of the seven coat protein subunits of the coatomer complex, and comparative sequence analysis of murine xenin and proxenin; Chow VT et al.; The coatomer complex is involved in intracellular protein transport and comprises an assembly of seven polypeptide subunits designated alpha, beta, beta', gamma, delta, epsilon, and zeta COP . Rooted phylogenetic trees constructed from the full-length cDNA and amino acid sequences of 49 COP entities in different eukaryotes from yeast to man generally revealed striking conservation of each subunit through evolution . Both nucleotide and protein trees displayed close relationships between alpha and beta' subunits, between beta and gamma subunits, and between delta and zeta subunits, implying evolution from common ancestors as well as functional similarity . Interestingly, although 6 out of 7 epsilon-COP genes appeared to be grouped and related to the beta-COP genes, 4 out of 7 epsilon Biochimie, 2001 Aug, 83(8), 791 - 9 Balancing N-linked glycosylation to avoid disease; Freeze HH et al.; Complete loss of N-glycosylation is lethal in both yeast and mammals . Substantial deficiencies in some rate-limiting biosynthetic steps cause human congenital disorders of glycosylation (CDG) . Patients have a range of clinical problems including variable degrees of mental retardation, liver dysfunction, and intestinal disorders . Over 60 mutations in phosphomannomutase (encoded by PMM2) diminish activity and cause CDG-Ia . The severe mutation R141H in PMM2 is lethal when homozygous, but heterozygous in about 1/70 Northern Europeans . Another disorder, CDG-Ic, is caused by mutations in ALG6, an alpha 1,3glucosyl transferase used for lipid-linked precursor synthesis, yet some function-compromising mutations occur at a high frequency in this gene also . Maintenance of seemingly deleterious mutations implies a selective advantage or positive heterosis . One possible explanation for this is that production of infective viruses such as hepatitis virus B and C, or others that rely heavily on host N-glycosylation, is substantially inhibited when only a tiny fraction of their coat proteins is misglycosylated . In contrast, this reduced glycosylation does not affect the host . Prevalent functional mutations in rate-limiting glycosylation steps could provide some resistance to viral infections, but the cost of this insurance is CDG . A balanced glycosylation level attempts to accommodate these competing agendas . By assessing the occurrence of a series of N-glycosylation-compromising alleles in multi-genic diseases, it may be possible to determine whether impaired glycosylation is a risk factor or a major determinant underlying their pathology. Cell Mol Life Sci, 2001 Jul, 58(8), 1067 - 84 Trans-Golgi network sorting; Gu F et al.; The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations . The TGN also receives extracellular materials and recycled molecules from endocytic compartments . In this review, we summarize recent progress on understanding TGN structure and the dynamics of trafficking to and from this compartment . Protein sorting into different transport vesicles requires specific interactions between sorting motifs on the cargo molecules and vesicle coat components that recognize these motifs . Current understanding of the various targeting signals and vesicle coat components that are involved in TGN sorting are discussed, as well as the molecules that participate in retrieval to this compartment in both yeast and mammalian cells . Besides proteins, lipids and lipid-modifying enzymes also participate actively in the formation of secretory vesicles . The possible mechanisms of action of these lipid hydrolases and lipid kinases are discussed . Finally, we summarize the fundamentally different apical and basolateral cell surface delivery mechanisms and the current facts and hypotheses on protein sorting from the TGN into the regulated secretory pathway in neuroendocrine cells. Nat Genet, 2001 Sep, 29(1), 57 - 60 A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure; de Lonlay P et al.; Complex III (CIII; ubiquinol cytochrome c reductase of the mitochondrial respiratory chain) catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-linked dehydrogenases to cytochrome c . CIII is made up of 11 subunits, of which all but one (cytochrome b) are encoded by nuclear DNA . CIII deficiencies are rare and manifest heterogeneous clinical presentations . Although pathogenic mutations in the gene encoding mitochondrial cytochrome b have been described, mutations in the nuclear-DNA-encoded subunits have not been reported . Involvement of various genes has been indicated in assembly of yeast CIII (refs . 8-11) . So far only one such gene, BCS1L, has been identified in human . BCS1L represents, therefore, an obvious candidate gene in CIII deficiency . Here, we report BCS1L mutations in six patients, from four unrelated families and presenting neonatal proximal tubulopathy, hepatic involvement and encephalopathy . Complementation study in yeast confirmed the deleterious effect of these mutations . Mutation of BCS1L would seem to be a frequent cause of CIII deficiency, as one-third of our patients have BCS1L mutations. Cytogenet Cell Genet, 2001, 93(3-4), 188 - 94 Comparative genomic hybridization based strategy for the analysis of different chromosome imbalances detected in conventional cytogenetic diagnostics; Tonnies H et al.; Today, conventional cytogenetics (CC) is the main technique in routine genetic diagnostics for the analysis of genotype/phenotype correlations . Additionally, fluorescence in situ hybridization (FISH) has proven to be useful for the characterization of structural chromosome aberrations found in conventional cytogenetics . Comparative genomic hybridization (CGH) is a molecular cytogenetic FISH approach developed for the detection of genomic imbalances with cytogenetic resolution . CGH allows the genome-wide assessment of relative DNA copy number changes using extracted specimen DNA as a probe . We investigated the capacity of CGH in cases referred for conventional cytogenetic diagnostics for the detection of chromosome imbalances . Three different groups of conspicuous karyotypes after CC (intrachromosomal rearrangements, interchromosomal rearrangements, and additional marker chromosomes) in pre- and postnatal diagnostic cases were surveyed by CGH to characterize the underlying imbalances of chromosome segments . All together, we investigated more than 100 cases by CGH and validated the results with other molecular cytogenetic methods . Here we present eight of these cases in order to demonstrate our CGH based strategy to analyze chromosomal de novo rearrangements . CGH provided additional cytogenetic information to complement conventional cytogenetic investigations . Additionally, CGH refined the description of the aberrant chromosome segments allowing us to further characterize the underlying mechanisms involved . Biochem Biophys Res Commun, 2001 Sep 7, 286(5), 1087 - 97 Interaction of Myc-associated zinc finger protein with DCC, the product of a tumor-suppressor gene, during the neural differentiation of P19 EC cells; Ugai H et al.; Expression of the DCC (deleted in colorectal cancer) protein is strongly induced during the neural differentiation of mouse P19 embryonal carcinoma (EC) cells that occurs when these cells are treated with retinoic acid (RA) . Myc-associated zinc finger protein (MAZ) is a DNA-binding protein that is widely expressed and functions in human, mouse and hamster cells as an activator, an initiator or a terminator of transcription . However, the biological functions of MAZ remain elusive . We report here that MAZ associates with the cytoplasmic domain of the DCC protein in vivo and in vitro . Yeast two-hybrid assays confirmed this association . An immunofluorescence study demonstrated that DCC protein is expressed at elevated levels in neuron-like P19 EC cells, in particular in axons, in which the MAZ protein is also expressed . We found that MAZ was translocated from the nucleus to the cytoplasm during the RA-induced terminal differentiation of P19 EC cells with resultant loss of the ability of MAZ to bind to the ME1a1 site of the c-myc promoter . Taken together, our observations imply that the DCC protein might play a critical role as a signaling molecule in the regulation of the transcriptional activity of MAZ during the neural differentiation of P19 EC cells . Curr Genet, 2001 Jul, 39(5-6), 319 - 26 The grp78 promoter of Neurospora crassa: constitutive, stress and differentiation-dependent protein-binding patterns; Monnerjahn C et al.; In order to characterize the transcriptional regulation of the grp78 (glucose-regulated protein 78 kDa) gene of Neurospora crassa, a 1.65-kb genomic fragment upstream of the protein-coding region was sequenced and analysed . A single transcription start point was mapped 160 nt upstream of the first codon . Several distinct protein-DNA binding sites were identified in the promoter region by non-radioactive scanning electrophoretic mobility shift analysis during growth, stress treatment and differentiation of conidia . A protein DNA binding complex induced by tunicamycin was linked to a promoter motif similar to the unfolded protein response element consensus of yeast . Another binding complex in differentiating aerial hyphae was found, which differs from the known cis-elements involved in conidiation-dependent gene expression. J Cell Biol, 2001 Sep 3, 154(5), 973 - 82 Epub 2001 Aug 27. Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components; Toyooka K et al.; alpha-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG) . Immunocytochemical analysis of the cotyledon cells with anti-alpha-amylase antibody showed that alpha-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins . To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted . The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar alpha-amylase . The autophagy for SG was structurally similar to micropexophagy detected in yeast cells . In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells . When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms . The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon. Domest Anim Endocrinol, 2001 Jul, 21(1), 55 - 72 Structure of the genes for porcine endometrial secreted and membrane folate binding proteins; Vallet JL et al.; The endometrium of the pig produces two types of folate binding proteins (FBP) which, based on their sequences, are likely to be membrane (m) and secreted (s) forms . A clone containing both a gene coding for the sFBP cDNA and a gene coding for the mFBP was isolated from a yeast artificial chromosome (YAC) library . Each gene was subcloned and sequenced . The gene for sFBP spanned 4.4 kbp and included 5 exons . The mFBP gene spanned 7.0 kbp and also contained 5 exons . Structures of the genes were very similar for the last three exons, and this similarity was shared with other known FBP/folate receptor (FR) gene sequences . Unexpectedly, portions of introns 3 and 4 of both genes were highly homologous, suggesting the possibility that sequences within these introns served some as yet unknown function . In contrast, the structures of the 5' exons differed between the two genes and other known FBP/FR genes . Comparison of putative promoter regions for the two genes with promoter regions for human FBP/FR genes revealed significant sequence homology between sFBP and human gammaFBP and between mFBP and human alphaFR . These regions of homology may play a role in control of transcription of each gene. Biochemistry, 2001 Sep 4, 40(35), 10717 - 22 Quaternary and domain structure of glycoprotein processing glucosidase II; Trombetta ES et al.; Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system . We have recently proposed that glucosidase II consisted of two different subunits, alpha and beta . The alpha subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity . Deletion of the homologue of the noncatalytic beta subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the beta subunit in glucosidase II activity has not been established . Furthermore, a direct interaction between alpha and beta subunits has not been demonstrated . Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (alpha(1)beta(1)) with a molecular mass of 161 kDa . The complex had an s value of 6.3 S, indicative of a highly nonglobular shape . The asymmetric shape of the alpha(1)beta(1) complex was confirmed by its high susceptibility to proteases . The beta subunit could be proteolytically removed from the alpha(1)beta(1) complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro . Furthermore, we isolated a monomeric C-terminal fragment of the alpha subunit, which retained full glucosidase activity . We conclude that the catalytic core of glucosidase II resides in a globular domain of the alpha subunit, which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER) . Through its C-terminal HDEL signal, the beta subunit may retain the complete alpha(1)beta(1) complex in the ER. J Tongji Med Univ, 2001, 21(2), 168 - 70 Study on the role of interleukin-4 in experimental murine systemic candidiasis; Liu Z et al.; In order to investigate the role of interleukin-4 (IL-4) in experimental murine systemic Candidiasis, we created the intact and dexamethasone-induced immunosuppressed murine systemic Candidiasis models . In these models, two-site ELISA and RT-PCR were applied to determine the level of IL-4 protein and mRNA expression in spleens respectively, clone forming units (CFUs) of infected kidneys were determined with the plating dilution method, and mean survival time (MST) of the mice was recorded . The results showed that, when compared with the controls, protein level of IL-4 increased in both intact mice infected with lethal doses of yeast (day 3, P < 0.05; day 7, P < 0.001) and immunosuppressed mice infected with sublethal doses of yeast (day 3, P > 0.05; day 7, P < 0.05) . Furthermore, the level of IL-4 was higher on day 7 than on day 3 after infection (P < 0.001 and P < 0.05 respectively in two groups) . The tendency of IL-4 mRNA expression was similar with that of IL-4 protein . As for fungal loads in kidneys, CFUs were significantly higher on day 7 than on day 3 after infection (P < 0.001 in both groups) . Mice in both groups succumbed to infection within several days . It was suggested that IL-4 might play a promoting role in the development of murine systemic Candidiasis. J Biol Chem, 2001 Nov 23, 276(47), 43915 - 23 Epub 2001 Aug 24. Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library . Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner; Baltrusch S et al.; The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver . To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage . This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase . The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver . The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism . Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization . 5'-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2 . A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain . The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform . The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes . In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling . Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion. Mol Biochem Parasitol, 2001 Sep 3, 116(2), 209 - 18 A Schistosoma mansoni Pad1 homologue stabilizes c-Jun; Nabhan JF et al.; We report the cloning and functional analysis of a Pad1 homologue (SmPOH) from Schistosoma mansoni . SmPOH encodes a protein of approximately 35 kDa with high amino acid identities to yeast Pad1 (65%) and its human homologue, POH1 (78%) . Members of the Pad1 family are subunits of the 26S proteasome and have been implicated as positive modulators of transcription in yeast . Recombinant SmPOH expressed in COS7 cells exhibited a punctate pattern of distribution throughout the cytoplasm and nucleus, predominantly in the nuclear periphery, a distribution consistent with that of the cellular proteasome . Transient overexpression of SmPOH in COS7 cells caused a dose-dependent stimulation in AP-1 transcriptional activity, as determined by a reporter gene assay . This effect was associated with a pronounced increase in the levels of cellular c-Jun . In vitro degradation assays further demonstrated that SmPOH specifically decreased the rate of c-Jun degradation in a dose dependent manner . Taken together, these results suggest that SmPOH, and possibly other related Pad1 proteins, function as positive modulators of transcription by increasing the stability of cellular c-Jun, making elevated amounts of this protein available for transactivation of AP-1-responsive genes. Oncogene, 2001 Aug 9, 20(35), 4853 - 63 Expression, cellular distribution and protein binding of the glioma amplified sequence (GAS41), a highly conserved putative transcription factor; Munnia A et al.; The glioma amplified sequence 41 (GAS41) was previously isolated by microdissection mediated cDNA capture from the glioblastoma multiforme cell line TX3868 and shown to be frequently amplified in human gliomas . We determined the complete cDNA sequence of the GAS41 gene, demonstrated that the GAS41 protein is evolutionarily conserved, specifically at the N-terminus, and identified the yeast transcription factor tf2f domain within the GAS41 sequence . A human multiple-tissue Northern blot revealed ubiquitous expression of GAS41 with the highest expression in human brain . After generating polyclonal antibodies we found GAS41 protein expression in the nucleus of the TX3868 cell line by Western blot analysis and immunofluorescence microscopy . The nuclear localization was confirmed for several human tumors including gliomas of different grades of malignancy . In neuroblastoma however, GAS41 was found in the nucleoli but not in the nucleoplasm . Yeast two-hybrid screening of the TX3868 cell line identified the nuclear mitotic apparatus protein (NuMA), the KIAA1009 protein, and prefoldin subunit 1 (PFDN1) as potential interacting partners of GAS41 . We generated a polyclonal antibody against the KIAA1009 protein and we demonstrated that the KIAA1009 protein is a nuclear protein, which appears to be co-localized with the GAS41 protein and NuMA. Oncogene, 2001 Aug 9, 20(35), 4807 - 16 Independent control of cell survival by Raf-1 and Bcl-2 at the mitochondria; Zhong J et al.; Bcl-2 family proteins play a critical role in the regulation of cell survival by controlling the activation of the cell death executing caspase machinery . Recent work demonstrated that they also provide a link between growth factor signaling and cell survival control . Raf-1 has been identified initially as an essential component of the mitogenic Ras-Raf-MEK-ERK cascade . However, expression of oncogenic Raf-1 also efficiently suppresses apoptotic cell death . This process requires mitochondrial translocation of Raf-1 which can be achieved either by co-expression of the anti-apoptotic protein Bcl-2 or by fusion with the transmembrane domain of the yeast outer mitochondrial membrane protein Mas 70p . It is currently unclear how mitochondrial Raf-1 prevents apoptosis . One possible mechanism involves the phosphorylation of the pro-apoptotic protein Bad resulting in the restoration of Bcl-2 function . Alternatively, the role of Bcl-2 could be limited to the mitochondrial translocation of Raf-1 and survival signaling by Raf-1 is Bcl-2 independent . To test for the mutual requirement of Raf-1 and Bcl-2 in apoptosis suppression the individual proteins were singly tested for survival activity in a genetic background which precludes the expression of the other . The results obtained in these studies demonstrate that ablation of Raf-1 or Bcl-2 expression in fibroblast cells significantly increases the sensitivity towards doxorubicin induced cell death . Reversion of the mutant phenotype could be achieved in either case by introducing a functional bcl-2 gene or a mitochondria targeted version of oncogenic Raf-1, demonstrating that each protein by itself is sufficient to confer protection . Our data thus suggest the existence of two separate pathways of survival signaling at the mitochondria controlled either by Bcl-2 or by Raf-1. Cell Immunol, 2001 Jun 15, 210(2), 143 - 53 Intracellular transport of MHC class II and associated invariant chain in antigen presenting cells from AP-3-deficient mocha mice; Sevilla LM et al.; MHC class II-restricted antigen presentation requires trafficking of newly synthesized class II-invariant chain complexes from the trans-Golgi network to endosomal, peptide-loading compartments . This transport is mediated by dileucine-like motifs within the cytosolic tail of the invariant chain . Although these signals have been well characterized, the cytosolic proteins that interact with these dileucine signals and mediate Golgi sorting and endosomal transport have not been identified . Recently, an adaptor complex, AP-3, has been identified that interacts with dileucine motifs and mediates endosomal/lysosomal transport in yeast, Drosophila, and mammals . In this report, we have assessed class II-invariant chain trafficking in a strain of mice (mocha) which lacks expression of AP-3 . Our studies demonstrate that the lack of AP-3 does not affect the kinetics of invariant chain degradation, the route of class II-invariant chain transport, or the rate and extent of class II-peptide binding as assessed by the generation of SDS-stable dimers . The possible role of other known or unknown adaptor complexes in class II-invariant chain transport is discussed . J Biol Chem, 2001 Nov 9, 276(45), 42389 - 400 Epub 2001 Aug 22. The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK; Korkaya H et al.; The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis . The biology and pathogenesis of HEV remain poorly understood . We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins . This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the adaptor protein Grb2 . A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction . The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins . Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3.SH3 complex . In pORF3-expressing cells, pp60(src) was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells . However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells . These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling . The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection. Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 332 - 5 {The metabolism of trehalose and intracellular glycerol in Candida krusei responding to high osmosis}; Zhang Y et al.; The trehalose and intracellular glycerol contents of osmotolerant yeast Candida krusei cells rapidly increased in response to the medium containing NaCl . Addition of NaCl at early exponential phase or steady phase increased trehalose content only, but the content of extracellular glycerol increased gradually . Development of resistance was accompanied by accumulation of trehalose and was apparently unrelated to intracellular glycerol content which was always low under these conditions . There seems to be an overlap function between trehalose and glycerol in Candida krusei cells in response to the osmotic conditions. Proc Natl Acad Sci U S A, 2001 Aug 28, 98(18), 10172 - 7 Epub 2001 Aug 21. Interaction between growth arrest-DNA damage protein 34 and Src kinase Lyn negatively regulates genotoxic apoptosis; Grishin AV et al.; Genotoxic stresses activate intracellular signaling molecules, which lead to growth arrest, DNA repair, and/or apoptosis . Among these molecules are the growth arrest and DNA damage protein 34 (GADD34) and the Src-related protein tyrosine kinase Lyn . Here, we report that these two proteins physically and functionally interact to regulate DNA damage-induced apoptosis . Multiple isolates of GADD34 and the related murine protein MyD116 were identified as binding partners of Lyn in a yeast two-hybrid screen . The specific interaction was confirmed by in vitro association of GADD34 with glutathione S-transferase fusion proteins containing the Src Homology 3 (SH3) domain of Lyn, as well as coimmunoprecipitation of GADD34 and Lyn from mammalian cells . GADD34 was tyrosine-phosphorylated in vivo in a Lyn-dependent manner . Lyn efficiently phosphorylated affinity-purified GADD34 in vitro . Lyn negatively regulated the proapoptotic function of GADD34 in a kinase-dependent manner . Expression of wild-type, but not kinase-inactive, Lyn weakened promotion of apoptosis by GADD34 following treatment with methyl-methanesulfonate or ionizing radiation in HEK293 and HeLa cells . In contrast, pretreatment of cells with the Src-specific tyrosine kinase inhibitor PP1 strengthened promotion of apoptosis by GADD34 . We propose that Lyn regulates the proapoptotic function of GADD34 by binding and phosphorylating it. Proc Natl Acad Sci U S A, 2001 Aug 28, 98(18), 10380 - 5 Epub 2001 Aug 21. Cloning and characterization of PIMT, a protein with a methyltransferase domain, which interacts with and enhances nuclear receptor coactivator PRIP function; Zhu Y et al.; The nuclear receptor coactivators participate in the transcriptional activation of specific genes by nuclear receptors . In this study, we report the isolation of a nuclear receptor coactivator-interacting protein from a human liver cDNA library by using the coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) (ASC2/AIB3/RAP250/NRC/TRBP) as bait in a yeast two-hybrid screen . Human PRIP-interacting protein cDNA has an ORF of 2,556 nucleotides, encodes a protein with 852 amino acids, and contains a 9-aa VVDAFCGVG methyltransferase motif I and an invariant GXXGXXI segment found in K-homology motifs of many RNA-binding proteins . The gene encoding this protein, designated PRIP-interacting protein with methyltransferase domain (PIMT), is localized on chromosome 8q11 and spans more than 40 kb . PIMT mRNA is ubiquitously expressed, with a high level of expression in heart, skeletal muscle, kidney, liver, and placenta . Using the immunofluorescence localization method, we found that PIMT and PRIP proteins appear colocalized in the nucleus . PIMT strongly interacts with PRIP under in vitro and in vivo conditions, and the PIMT-binding site on PRIP is in the region encompassing amino acids 773-927 . PIMT binds S-adenosyl-l-methionine, the methyl donor for methyltransfer reaction, and it also binds RNA, suggesting that it is a putative RNA methyltransferase . PIMT enhances the transcriptional activity of peroxisome proliferator-activated receptor gamma and retinoid-X-receptor alpha, which is further stimulated by coexpression of PRIP, implying that PIMT is a component of nuclear receptor signal transduction apparatus acting through PRIP . Definitive identification of the specific substrate of PIMT and the role of this RNA-binding protein in transcriptional regulation remain to be determined. J Neurosci, 2001 Sep 1, 21(17), 6597 - 607 c-Jun N-terminal kinase (JNK)-interacting protein-1b/islet-brain-1 scaffolds Alzheimer's amyloid precursor protein with JNK; Matsuda S et al.; Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries . In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK . The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary . APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs . JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner . Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs . Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells . Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner . Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells . These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules. Curr Biol, 2001 Aug 7, 11(15), 1207 - 14 Analysis of an essential requirement for the poly(A) binding protein function using cross-species complementation; Chekanova JA et al.; Poly(A) binding protein (PABP) is an essential, well-conserved, multifunctional protein involved in translational initiation, mRNA biogenesis, and degradation {1--5} . We have used a cross-species complementation approach to address the nature of the essential requirement for PABP in yeast . The expression of Pab3p, a member of the Arabidopsis thaliana PABP multigene family, rescues the lethal phenotype associated with the loss of the yeast Pab1p . However, Pab3p neither protects the mRNA 5' cap from premature removal, nor does it support poly(A)-dependent translational initiation or the synergistic enhancement of translation by the poly(A) tail and 5' cap in yeast . However, Pab3p corrects the temporal lag prior to the entry of the mRNA into the degradation pathway characteristic of pab1 Delta yeast strains . Furthermore, this lag correction by Pab3p requires Pan3p, a subunit of poly(A) nuclease, an enzyme involved in the mRNA 3'-end processing . Importantly, the substitution of Pab3p for the yeast Pab1p is synthetically lethal with the PAN3 gene deletion . These results show that the function of PABP in mRNA biogenesis alone could be sufficient to support cell viability in yeast. Curr Biol, 2001 Aug 7, 11(15), 1197 - 201 Securin is not required for cellular viability, but is required for normal growth of mouse embryonic fibroblasts; Mei J et al.; Sister chromatid separation depends on the release of cohesion by the activity of Esp1, a member of the caspase family {1, 2} . In budding yeast, Esp1p is kept inactive by its association with Pds1p, until the onset of anaphase, when Pds1p is ubiquitinated by the APC/Cdc20 complex {3--5} and subsequently degraded by the 26S proteasome . Pds1 is not an essential gene in budding yeast, but is required for cell cycle arrest prior to anaphase in response to the disruption of spindle structures {6, 7} . Thus, Pds1 mutant yeast cells display precocious sister chromatid separation in the presence of nocodazole {6} . Mammalian orthologs of yeast Esp1 and Pds1, separin and securin, have been identified {8}, and, as anticipated, a nondegradable mutant form of securin inhibits sister separation when added to mitotic Xenopus egg extracts {8} . Securin was also independently identified as PTTG (pituitary tumor transforming gene), a gene overexpressed in pituitary tumors {9} . The relationship between its overexpression in tumors and its control of sister chro