Yeasts

Biochem Biophys Res Commun, 2001 Mar, 281(5), 1349 - 55
Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43; Westlind A et al.; On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily . The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71% . Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined . Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression . The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced . This indicates a nonfunctional protein or specific conditions required for proper folding . It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism .

Biochem Biophys Res Commun, 2001 Mar, 281(5), 1141 - 53
The small GTPase Rab4A interacts with the central region of cytoplasmic dynein light intermediate chain-1; Bielli A et al.; Rab4 belongs to the Rab family of small GTPases involved in the regulation of intracellular transport, and has been localized to early endosomes . We have employed the yeast two-hybrid system to identify proteins that specifically interact with Rab4AQ67L, a GTPase-deficient mutant form of Rab4A . Screening a mouse embryo cDNA library identified a clone (M449) that interacted with Rab4A in a nucleotide-dependent fashion . Data base searches identified this clone as the mouse cytoplasmic dynein light intermediate chain-1 (LIC-1) . Based on this finding, the full-length equivalent human cytoplasmic dynein LIC-1 was isolated by PCR . When Rab4A was overexpressed together with either M449 or dynein LIC-1 in HeLa cells, the proteins were found to colocalize in the perinuclear region . We characterize the localization of both overexpressed human dynein LIC-1 and the endogenous protein with respect to microtubules and show that it concentrates to the microtubule-organizing center and mitotic spindle . Additionally, GFPRab4A endosomes localize to microtubules and are redistributed by nocodazole treatment . This is the first described interaction between cytoplasmic dynein, a retrograde motor protein, and a Rab protein .

Biochem Biophys Res Commun, 2001 Mar, 281(5), 1065 - 9
Hrs and hbp: possible regulators of endocytosis and exocytosis; Komada M et al.; The molecular mechanisms of endocytosis and exocytosis are not yet fully understood . Hrs and Hbp, two tightly associated proteins in eukaryotic cells, have been implicated in these cellular processes . Hrs is homologous to Vps27p, an endosomal protein required for vacuolar and endocytic trafficking in yeast . Hrs is localized to early endosomes and is required for the normal morphology of early endosomes in mammalian cells . Hrs also associates with proteins implicated in endocytosis and exocytosis such as SNAP-25 and Eps15 . Hrs treatment inhibits neurotransmitter release in permeabilized neuronal cells and its overexpression inhibits internalization of transferrin . Overexpression of dominant-negative Hbp mutants inhibits ligand-induced downregulation of growth factor/receptor complexes and immunoglobulin E receptor-triggered degranulation of secretory granules in mast cells . These observations suggest an important role for the Hrs/Hbp protein complex in vesicular trafficking during endocytosis and exocytosis .

Methods, 2001 Mar, 23(3), 276 - 86
Probing RNA in vivo with methylation guide small nucleolar RNAs; Liu B et al.; Most box C/D small nucleolar RNAs (snoRNAs) direct the formation of 2'-O-methylated nucleotides in ribosomal RNA and, apparently, other RNAs present in the nucleolar complex . Sites to be modified are selected by a long (>10-nt) antisense guide sequence in the snoRNA and a distance measurement from a box D or D' element that follows the snoRNA guide sequence . Modification of the substrate occurs in the region of complementarity, at a position five nucleotides upstream from box D/D' . Methylation can be targeted to novel sites by expressing a snoRNA with a new guide sequence . In some cases methylation impairs the growth rate of the cell, indicating that a functionally important nucleotide has been altered . With a view to harnessing snoRNA-directed methylation for functional mapping, we have developed a method for constructing libraries of snoRNA genes that, in principle, can introduce methylation point mutations into any rRNA segment of interest . The strategy and procedures are described here, and preliminary results are presented that show the feasibility of using this technology to probe a region of the yeast large subunit rRNA that includes the core of the peptidyltransferase center.

J Mol Biol, 2001 Mar 2, 306(4), 717 - 26
Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain; Centner T et al.; The giant myofibrillar protein titin contains within its C-terminal region a serine-threonine kinase of unknown function . We have identified a novel muscle specific RING finger protein, referred to as MURF-1, that binds in vitro to the titin repeats A168/A169 adjacent to the titin kinase domain . In myofibrils, MURF-1 is present within the periphery of the M-line lattice in close proximity to titin's catalytic kinase domain, within the Z-line lattice, and also in soluble form within the cytoplasm . Yeast two-hybrid screens with MURF-1 as a bait identified two other highly homologous MURF proteins, MURF-2 and MURF-3 . MURF-1,2,3 proteins are encoded by distinct genes, share highly conserved N-terminal RING domains and in vitro form dimers/heterodimers by shared coiled-coil motifs . Of the MURF family, only MURF-1 interacts with titin repeats A168/A169, whereas MURF-3 has been reported to affect microtubule stability . Association of MURF-1 with M-line titin may potentially modulate titin's kinase activity similar to other known kinase-associated proteins, whereas differential expression and heterodimerization of MURF1, 2 and 3 may link together titin kinase and microtubule-dependent signal pathways in striated muscles .

Gene Expr, 2000, 9(3), 123 - 32
The Drosophila TATA binding protein contains a strong but masked activation domain; Um M et al.; TATA binding protein (TBP) is a critical transcription factor involved in transcription by all three RNA polymerases (RNAPs) . Studies using in vitro systems and yeast have shown that the C-terminal core domain (CTD) of TBP is necessary and sufficient for many TBP functions, but the significance of the N-terminal domain (NTD) of TBP is still obscure . Here, using transient expression assays in Drosophila Schneider cells, we show that the NTD of Drosophila TBP (dTBP) strongly activates transcription when fused to the GAL4 DNA binding domain (DBD) . Strikingly, the activity of the NTD is completely repressed in the context of full-length dTBP . In contrast to the much weaker activation obtained by either full-length dTBP or the dTBP CTD fused to the GAL4 DBD, activation by the NTD is dependent on the presence of GAL4 binding sites and is susceptible to the effects of a dominant negative TFIIB mutant, TFIIB deltaC202, a property observed previously with certain authentic activation domains . Activation by the NTD, but not full-length dTBP or the CTD, seems to be mediated by the action of a strong activation domain, likely a glutamine-rich region . In conclusion, the dTBP NTD can behave as a very strong activator that is masked in the full-length protein, suggesting possible roles for the dTBP NTD in RNAP II-mediated transcription.

BMC Mol Biol . 2001;2(1):3 . Epub 2001 Feb 27.
UAG readthrough in mammalian cells: effect of upstream and downstream stop codon contexts reveal different signals; Cassan M et al.; BACKGROUND: Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context . Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved . RESULTS: We have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10(-4) . We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon . There was no effect of amino acid identity or codon frequency . However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels . This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA . We then examined the importance of the downstream context using eight other constructs . No direct correlation between the +6 nucleotide and readthrough efficiency was observed . CONCLUSIONS: We conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery . Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.

Nature, 2001 Mar 1, 410(6824), 89 - 93
Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins; Jackson M et al.; Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters . To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1-5) . GLAST and GLT-1 are localized primarily in astrocytes, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9-11) and EAAT5 (ref 5) are neuronal . Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites . This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals . We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4--the glutamate transporter expressed predominately in the cerebellum--or in targeting and/or anchoring or clustering the transporter to the target site . Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.

Insect Mol Biol, 2001 Feb, 10(1), 77 - 86
The Drosophila gene Yippee reveals a novel family of putative zinc binding proteins highly conserved among eukaryotes; Roxstrom-Lindquist K et al.; An intracellular Drosophila protein, Yippee, was identified in a yeast interaction trap screen as physically interacting with Hyalophora cecropia Hemolin . The Yippee gene was isolated, structurally characterized, and mapped to the region 12A on the X-chromosome . Yippee contains a putative zinc-finger-like metal binding domain . It is the first characterized member of a conserved gene family of proteins present in diverse eukaryotic organisms, ranging from cellular slime mould to humans . A human cDNA clone was isolated and shown to be 76% identical to Drosophila Yippee . Yippee is ubiquitously expressed in different developmental stages of Drosophila and in different fetal tissues from human . Although the Hemolin-Yippee interaction remains to be further elucidated, the high degree of Yippee sequence conservation between a wide range of species suggests that this protein is of general importance in eukaryotes.

Enzyme Microb Technol, 2001 Mar 8, 28(4-5), 314 - 321
Factorial designs combined with the steepest ascent method to optimize serum-free media for CHO cells; Liu C et al.; A serum free medium for recombinant CHO NTHU 108 cell growth and fusion protein (CD20 linked to a human IgG-Fc gamma4 fragment) synthesis were systematically developed using factorial designs combined with the steepest ascent method . Experimental results indicate that the optimal composition of serum replacement for specific fusion protein production was 1% SITE (selenium, insulin, transferrin, ethanolamine), 0.3 g/L yeast extract, and 0.09% linoleic acid-BSA . Cell growth and fusion protein production of the adapted CHO NTHU 108 cultured in Iscove's modified Dulbecco's medium supplemented with these serum substitutes were comparable to those in the Ex-Cell 301 commercial serum-free medium . These serum substitutes can also promote CHO cell growth and fusion protein production in nine kinds of commercial media . The low protein content of the developed medium facilitates downstream processing and product purification.

FEBS Lett, 2001 Mar 2, 491(3), 193 - 9
Identification and kinetic analysis of the interaction between Nck-2 and DOCK180; Tu Y et al.; Nck-2 is a newly identified adapter protein comprising three N-terminal SH3 domains and one C-terminal SH2 domain . We have identified in a yeast two-hybrid screen DOCK180, a signaling protein implicated in the regulation of membrane ruffling and migration, as a binding protein for Nck-2 . Surface plasmon resonance analyses reveal that the second and the third SH3 domains interact with the C-terminal region of DOCK180 . The interactions mediated by the individual SH3 domains, however, are much weaker than that of the full length Nck-2 . Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduced the interaction of Nck-2 with DOCK180, suggesting that both SH3 domains contribute to the DOCK180 binding . A major Nck-2 binding site, which is recognized primarily by the third SH3 domain, has been mapped to residues 1819-1836 of DOCK180 . Two additional, albeit much weaker, Nck-2 SH3 binding sites are located to DOCK180 residues 1793-1810 and 1835-1852 respectively . Consistent with the mutational studies, kinetic analyses by surface plasmon resonance suggest that two binding events with equilibrium dissociation constants of 4.15+/-1.9x10(-7) M and 3.24+/-1.9x10(-9) M mediate the binding of GST-Nck-2 to GST fusion protein containing the C-terminal region of DOCK180 . These studies identify a novel interaction between Nck-2 and DOCK180 . Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain.

Trends Plant Sci, 2001 Mar, 6(3), 100 - 4
Vacuolar H+/Ca2+ transport: who's directing the traffic?
Hirschi K.
Physiological studies have established the role of plant high-capacity vacuolar H+/Ca2+ exchange activity in ion homeostasis and signal transduction . The molecular characterization and structure-function analyses of these transporters are just beginning to emerge . In yeast, Ca2+ signaling molecules regulate vacuolar H+/Ca2+ exchange . Recently, some of the Ca2+ dependent "molecular relay" molecules have been characterized in plants; however, the regulation of plant vacuolar H+/Ca2+ exchange remains an open question.

Mol Cell Biol, 2001 Mar, 21(6), 2154 - 64
Interaction with protein phosphatase 1 Is essential for bifocal function during the morphogenesis of the Drosophila compound eye; Helps NR et al.; The gene bifocal (bif), required for photoreceptor morphogenesis in the Drosophila compound eye, encodes a protein that is shown to interact with protein phosphatase 1 (PP1) using the yeast two-hybrid system . Complex formation between Bif and PP1 is supported by coprecipitation of the two proteins . Residues 992 to 995 (RVQF) in the carboxy-terminal region of Bif, which conform to the consensus PP1-binding motif, are shown to be essential for the interaction of Bif with PP1 . The interaction of PP1 with bacterially expressed and endogenous Bif can be disrupted by a synthetic peptide known to block interaction of other regulatory subunits with PP1 . Null bif mutants exhibit a rough eye phenotype, disorganized rhabdomeres (light-gathering rhodopsin-rich microvillar membrane structures in the photoreceptor cells) and alterations in the actin cytoskeleton . Expression of wild-type bif transgenes resulted in significant rescue of these abnormalities . In contrast, expression of transgenes encoding the Bif F995A mutant, which disrupts binding to PP1, was unable to rescue any aspect of the bif phenotype . The results indicate that the PP1-Bif interaction is critical for the rescue and that a major function of Bif is to target PP1c to a specific subcellular location . The role of the PP1-Bif complex in modulating the organization of the actin cytoskeleton underlying the rhabdomeres is discussed.

Genes Dev, 2001 Mar 1, 15(5), 522 - 34
C . elegans mre-11 is required for meiotic recombination and DNA repair but is dispensable for the meiotic G(2) DNA damage checkpoint; Chin GM et al.; We investigated the roles of Caenorhabditis elegans MRE-11 in multiple cellular processes required to maintain genome integrity . Although yeast Mre11 is known to promote genome stability through several diverse pathways, inviability of vertebrate cells that lack Mre11 has hindered elucidation of the in vivo roles of this conserved protein in metazoan biology . Worms homozygous for an mre-11 null mutation are viable, allowing us to demonstrate in vivo requirements for MRE-11 in meiotic recombination and DNA repair . In mre-11 mutants, meiotic crossovers are not detected, and oocyte chromosomes lack chiasmata but appear otherwise intact . gamma-irradiation of mre-11 mutant germ cells during meiotic prophase eliminates progeny survivorship and induces chromosome fragmentation and other cytologically visible abnormalities, indicating a defect in repair of radiation-induced chromosome damage . Whereas mre-11 mutant germ cells are repair-deficient, they retain function of the meiotic G(2) DNA damage checkpoint that triggers germ cell apoptosis in response to ionizing radiation . Although mre-11/mre-11 animals derived from heterozygous parents are viable and produce many embryos, there is a marked drop both in the number and survivorship of embryos produced by succeeding generations . This progressive loss of fecundity and viability indicates that MRE-11 performs a function essential for maintaining reproductive capacity in the species.

Carcinogenesis, 2001 Mar, 22(3), 515 - 7
Inactivate the remaining p53 allele or the alternate p73? Preferential selection of the Arg72 polymorphism in cancers with recessive p53 mutants but not transdominant mutants; Tada M et al.; Several reports have noted epidemiological differences in the prevalence or prognostic significance of p53 mutants with arginine (R) or proline (P) at the codon 72 polymorphism (R72/P72) in certain cancer types, but the biological significance of these variants is unclear . The ability of p53 mutants to interact with and inactivate the p53 homolog p73 was recently reported to depend on the conformational state of the p53 protein and the residue at codon 72 . Since the conformation of p53 mutants may influence their ability to transdominantly inhibit wild-type p53, we tested whether there was a correlation between the amino acid at codon 72 and the transdominance of p53 alleles found in tumors . The transdominance test was performed using a simple yeast transcription assay, and the amino acid at codon 72 was determined by sequencing . A total of 100 p53 mutants were tested . Compared with the germline frequency (R:P = 427:297), an extreme bias in favor of the R72 allele was observed with recessive mutants (R:P = 50:7, P < 0.0002), whereas no selection for the R72 allele was seen with transdominant mutants (R:P = 23:20) . p53 and p73 are known to transactivate overlapping sets of target genes . We interpret the R72 bias with recessive mutants as evidence that decreased activation of p53 target genes provides a selective growth advantage to tumor cells during the stage of tumorigenesis in which a wild-type and mutant p53 allele coexist . We suggest that transdominant p53 mutants achieve this by inactivation of the remaining wild-type p53 allele, whereas recessive p53 mutants achieve it through inactivation of p73.

Biochem J, 2001 Mar 15, 354(Pt 3), 635 - 43
Mitogen-stimulated TIS21 protein interacts with a protein-kinase-Calpha-binding protein rPICK1; Lin WJ et al.; TIS21 is induced transiently by PMA and a number of extracellular stimuli . Yeast two-hybrid screening has identified three TIS21 interacting clones from a rat cDNA library {Lin, Gary, Yang, Clarke and Herschman (1996) J . Biol . Chem 271, 15034-15044} . The amino acid sequence deduced from clone 5A shows 96.9% identity with the murine PICK1, a protein kinase Calpha (PKCalpha)-binding protein postulated to act as an intracellular receptor for PKC . A fusion protein of glutathione S-transferase and rPICK1 associates with the TIS21 translated in vitro, suggesting a direct physical interaction between these two proteins . TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins . This indicates that the interaction also occurs in mammalian cells . Deletion of the PDZ domain at the N-terminus of rPICK1 abolishes its interaction with TIS21 . A putative carboxylate-binding loop required for PICK1 to bind PKCalpha {Staudinger, Lu and Olson (1997) J . Biol . Chem 272, 32019-32024} is within this deleted region . Our results suggest a potential competition between TIS21 and PKC for binding to PICK1 . We show that recombinant TIS21 is phosphorylated by PKC in vitro . The catalytic activity of PKC towards TIS21 is significantly decreased in the presence of rPICK1, whereas phosphorylation of histone by PKC is not affected . rPICK1 seems to modulate the phosphorylation of TIS21 through specific interactions between these two proteins . TIS21 might have a role in PKC-mediated extracellular signal transduction through its interaction with rPICK1.

Mol Ther, 2001 Feb, 3(2), 262 - 73
A versatile framework for the design of ligand-dependent, transgene-specific transcription factors; Xu L et al.; The ability to regulate transgene expression will be essential for the safety and efficacy of many gene therapies . Various ligand-dependent transcription factors, including steroid hormone receptors, have been modified to enable transgene-specific regulation . To minimize effects on cellular gene expression, chimeric steroid receptors have been constructed by replacing their native DNA binding domain (DBD) with a heterologous DBD, like that from the yeast transcription factor GAL4 . This approach has limitations for human gene therapy, including the potential immunogenicity of the GAL4 domain and the inability to discriminate between different GAL4-linked transgenes in the same cell . To address this, we have constructed chimeric regulators containing the human estrogen receptor (ER) ligand binding domain (LBD) and a Cys(2)-His(2)-type zinc finger DBD . Cys(2)-His(2) zinc finger domains are common among human DNA binding proteins and can be engineered to selectively bind different DNA sequences . We demonstrate over 500-fold drug-dependent transgene induction with these chimeric regulators in vitro and the ability to regulate an adenovirus-delivered transgene in mice . Two chimeras containing different Cys(2)-His(2) domains displayed highly sequence-specific binding and regulation . Incorporating a point mutation in the ER LBD that ablates estrogen binding enables selective in vivo regulation with the clinically useful anti-estrogen tamoxifen . These Cys(2)-His(2)-ER LBD chimeras represent a versatile framework for creating transgene-specific regulators potentially useful for human gene therapy applications.

J Mol Biol, 2001 Feb 16, 306(2), 137 - 43
NMR identification of the Tom20 binding segment in mitochondrial presequences; Muto T et al.; Many mitochondrial proteins are synthesized in the cytosol as precursors with N-terminal presequences, and are imported into mitochondria with the aid of translocator protein complexes containing presequence-binding proteins . Tom20, a receptor protein which functions in an early step of the mitochondrial protein import, recognizes presequences with divergent amino acid sequences . Here, we report the identification of the segments involved in binding to Tom20 in mitochondrial presequences . We monitored the chemical shift perturbation of the NMR signals of five different 15N-labeled presequence peptides by the addition of the cytosolic receptor domain of rat or yeast Tom20 . The perturbed segments occupy different positions, either near the N terminus or at the C terminus, in the presequences . Spin label experiments revealed that this is not due to different orientations of the presequence peptides bound to Tom20 . The results presented here will offer a starting point to perform detailed analyses of Tom20-binding elements by systematic amino acid replacements.

Free Radic Res, 2000 Dec, 33(6), 851 - 5
Redox regulation by thioredoxin superfamily; protection against oxidative stress and aging; Tanaka T et al.; Thioredoxin (TRX) is a 12 kD protein with redox-active dithiol in the active site; -Cys-Gly-Pro-Cys- . We originally cloned human TRX as adult T cell leukemia derived factor (ADF) produced by HTLV-I transformed cells . TRX and related molecules maintain a cellular reducing enviroment, working in concert with the glutathione system . Physiologically, TRX has cytoprotective effects against oxidative stress . TRX promotes DNA binding of transcription factors such as NF-kB, AP-1, p53, and PEBP-2 . The TRX superfamily, including thioredoxin-2 (mitochondrial thioredoxin) and glutaredoxin, are involved in biologically important phenomena via the redox-regulating system . Thioredoxin-binding protein-2, which we recently identified by a yeast two-hybrid system, is a type of endogenous modulator of TRX activity . TRX is secreted from the cells and exhibits cytokine-like and chemokine-like activities . Redox regulation by TRX plays a crucial role in biological responses against oxidative stress.

Nature, 2001 Feb 15, 409(6822), 948 - 51
Integration of telomere sequences with the draft human genome sequence; Riethman HC et al.; Telomeres are the ends of linear eukaryotic chromosomes . To ensure that no large stretches of uncharacterized DNA remain between the ends of the human working draft sequence and the ends of each chromosome, we would need to connect the sequences of the telomeres to the working draft sequence . But telomeres have an unusual DNA sequence composition and organization that makes them particularly difficult to isolate and analyse . Here we use specialized linear yeast artificial chromosome clones, each carrying a large telomere-terminal fragment of human DNA, to integrate most human telomeres with the working draft sequence . Subtelomeric sequence structure appears to vary widely, mainly as a result of large differences in subtelomeric repeat sequence abundance and organization at individual telomeres . Many subtelomeric regions appear to be gene-rich, matching both known and unknown expressed genes . This indicates that human subtelomeric regions are not simply buffers of nonfunctional 'junk DNA' next to the molecular telomere, but are instead functional parts of the expressed genome.

Biochem Cell Biol, 2001, 79(1), 21 - 32
Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins; Hemming R et al.; To identify proteins interacting in the insulin-signaling pathway that might define new pathways or regulate existing ones, we have employed the yeast two-hybrid system . In a two-hybrid screen of a human liver cDNA library, we identified the human growth factor receptor bound 14 (hGrb14) adaptor protein as a partner of the activated insulin receptor . Additional analysis of the insulin receptor--hGrb14 interaction in the yeast two-hybrid system revealed that the SH2 domain of hGrb14 was not the sole region involved in binding the activated insulin receptor . The insulin-stimulated interaction between hGrb14 and the insulin receptor was also observed in different mammalian cultured cell lines . This association was detected at 1 min of insulin stimulation and was maximal at 10 nM and greater concentrations of insulin . Chinese hamster ovary cells stably expressing the insulin receptor (CHO-IR) and hGrb14 were used to examine the effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation of proteins; in general, increasing levels of hGrb14 expression resulted in a reduction in tyrosine phosphorylation . This decrease was demonstrated for the specific proteins src homology-containing and collagen-related protein (Shc), insulin receptor substrate-1 (IRS-1), and Downstream of tyrosine Kinase (Dok) . The broad effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation suggest that it acts early in the insulin-signaling pathway.

Biotechniques, 2001 Feb, 30(2), 380 - 6
The RITE assay: identifying effectors that target the transcription machinery using phage display technology; Mazzarelli JM et al.; We describe an approach using phage display to identify effectors (activators and repressors) of transcription based on the particular component of the general transcription machinery that they target . We refer to this approach as the reverse identification of transcriptional effectors (RITE) assay . A library of phages containing cDNA-encoded peptides displayed on their surfaces is screened using as the target a specific region of one of the general transcription factors (e.g., the C terminus of hTAFII135) . The amino acid sequence encoded by the cDNA of an interacting phage is determined and analyzed in a database homology search to identify known or novel factors that may interact with the target protein . Candidate effectors from the homology search are synthesized from recombinant clones and tested for their abilities to bind to the target protein and to functionally modulate transcription in vivo when co-expressed with the transcriptional target protein . Because the RITE assay is a direct measure of the interactions between general transcription proteins and their effectors, it has an advantage over the well-known yeast two-hybrid system, which is not amenable to identifying transcription factor interactions.

Biotechniques, 2001 Feb, 30(2), 296 - 8, 300, 302
Two-hybrid selection assay to identify proteins interacting with polymerase II transcription factors and regulators; Petrascheck M et al.; The RNA polymerase III-based two-hybrid system has been developed to detect interactions between proteins such as RNA polymerase II transcription factors and regulators that cannot be studied by the original RNA polymerase II two-hybrid system . This novel method appears to be most useful for a refined analysis of already known protein-protein interactions . However, the application of this system in library screenings has been impaired by the lack of a suitable assay for the selection of the activated pol III reporter gene in yeast . Here, we describe a novel selection assay for the pol III-based two-hybrid system that makes it readily usable for screening expression libraries to search for interacting partners . Our system utilizes a temperature-sensitive (ts) U6 snRNA, which is synthesized by RNA polymerase III from a mutated SNR6 gene in yeast . In this ts strain, interactions between hybrid proteins activate an artificial pol III reporter construct (UASG-SNR6), which controls expression of wild-type U6 snRNA . This wild-type U6 snRNA can suppress the ts phenotype and allow growth at the nonpermissive temperature of 37 degrees C, thus providing a positive selection system for interacting proteins.

Eur J Biochem, 2001 Mar, 268(5), 1340 - 51
Identification and characterization of SEB, a novel protein that binds to the acute undifferentiated leukemia-associated protein SET; Minakuchi M et al.; SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is a 39-kDa nuclear phosphoprotein and has an inhibitory activity for protein phosphatase 2A (PP2A) . SET is fused to a putative oncoprotein, CAN/NUP214, in AUL and is thought to play a key role in leukemogenesis by its nuclear localization, protein-protein interactions and PP2A inhibitory activity . Here, we describe the isolation and characterization of a novel cDNA encoding a protein with 1542 amino-acid residues that specifically interacts in a yeast two-hybrid system as well as in human cells with SET . This new protein, which we name SEB (SET-binding protein), is identified as a 170-kDa protein by immunoprecipitation with a specific antibody and is localized predominantly in the nucleus . SEB1238--1434 is determined as a SET-binding region that specifically binds to SET182--223 . SEB also has an oncoprotein Ski homologous region (amino acids 654--858), six PEST sequences and three sequential PPLPPPPP repeats at the C-terminus . SEB mRNA is expressed ubiquitously in all human adult tissues and cells examined . The SEB gene locus is assigned to the chromosome 18q21.1 that contains candidate tumor suppressor genes associated with deletions in cancer and leukemia . Although the function of SEB is not known, we propose that SEB plays a key role in the mechanism of SET-related leukemogenesis and tumorigenesis, perhaps by suppressing SET function or by regulating the transforming activity of Ski in the nucleus.

Curr Biol, 2001 Feb 6, 11(3), R112 - 4
Arabidopsis genome: life without notch; Wigge PA et al.; The complete genome sequence of the flowering plant Arabidopsis thaliana has been determined . New insights have come from comparisons between this sequence and genome sequences of other species, including those of cyanobacteria, yeast, worms and flies.

Curr Biol, 2001 Feb 6, 11(3), R106 - 8
Evolution: the evolvability enigma; Brookfield JF; A report that a switch of a yeast protein to a 'prion' state triggers diverse phenotypic changes has prompted re-examination of the processes of evolution . To what extent should processes of gene expression and control be interpreted in terms of their capacity to allow future evolution as well as present adaptation?

Curr Biol, 2001 Feb 6, 11(3), 151 - 60
The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila; Foley E et al.; Background: Exit from mitosis is a tightly regulated event . This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis . Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1 . In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction.Results: We now present evidence that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis . Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos . In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase . Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A . Rux has numerous functional similarities with Sic1 . While these proteins share no sequence similarity, we show that Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo.Conclusions: Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis . To our knowledge, this is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development.

Curr Biol, 2001 Feb 6, 11(3), 141 - 50
Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation; Schwab MS et al.; BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate . The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins . This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle . In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint . In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint . In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC . However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint . RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis . In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme . The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK . In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form . Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126 . Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity . CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner . Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo . These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).

Mech Dev, 2001 Mar, 101(1-2), 21 - 33
NudE-L, a novel Lis1-interacting protein, belongs to a family of vertebrate coiled-coil proteins; Sweeney KJ et al.; The LIS1-encoded protein (Lis1) plays a role in brain development because a hemizygous deletion or mutation of the human gene causes neuronal migration disorders, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) . Using a yeast two-hybrid screen, we have isolated a novel protein that interacts with mouse Lis1 (mLis1) which is termed mouse NudE-like protein (mNudE-L) because of its 49% amino acid conservation with NudE, a protein involved in nuclear migration in Aspergillus nidulans . GST pull-down assays and co-immunoprecipitation of fusion proteins expressed in mammalian cells confirmed the interaction of mLis1 and mNudE-L . mNudE-L gives rise to a approximately 2.3 kb mRNA and encodes an ORF corresponding to approximately 38 kDa protein . The overall amino acid sequence of mNudE-L is 49-95% identical to proteins found in a variety of organisms, thus establishing mNudE-L as a new member of a protein family . The hallmark of this family is an N-terminal region predicted to form a coiled-coil domain . We show that mNudE-L and mLis1 are coexpressed in the postnatal and adult cerebral cortices and in the Purkinje neurons of the cerebellum . In contrast to mLis1, mNudE-L transcripts are absent in the mitral cell layer of the olfactory bulb and in the inward migrating granular neurons of the developing cerebellum . Mutant mLis1 proteins modelling mutations found in human lissencephaly patients fail to interact with mNudE-L, raising the possibility that phenotypic changes result, in part, from the inability of mutant Lis1 proteins to interact with the human NudE-L polypeptide.

FEBS Lett, 2001 Jan 26, 489(1), 34 - 41
Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-2; Jin C et al.; A mouse cDNA that encodes a DNA-binding protein was identified by yeast two-hybrid screening, using activating transcription factor-2 (ATF-2) as the bait . The protein contained a bZIP (basic amino acid-leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2) . Mouse JDP2 interacted with ATF-2 both in vitro and in vivo via its bZIP domain . It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues . Furthermore, mouse JDP2 bound to the cAMP-response element (CRE) as a homodimer or as a heterodimer with ATF-2, and repressed CRE-dependent transcription that was mediated by ATF-2 . JDP2 was identified as a novel repressor protein that affects ATF-2-mediated transcription.

Acta Trop, 2001 Feb 23, 78(2), 147 - 54
Differences between coding and non-coding regions in the Trichomonas vaginalis genome: an actin gene as a locus model(1); Espinosa N et al.; The sequence of a cloned genomic fragment of Trichomonas vaginalis containing a complete actin gene was determined . An uninterrupted open reading frame of 1128 nucleotides was found that codes for an actin gene . Two overlapped consensus promoter sequences for T . vaginalis were found 12 nucleotides upstream the actin initiation codon . In addition to actin, two incomplete open reading frames were found at the 5' and 3' ends of the clone . These two sequences are expressed and showed similarity to adenylate cyclase genes and a yeast hypothetical protein . The overall sequence showed a higher G+C content and a lower frequency of repeated sequences in the coding regions when compared with the non-coding regions . A similar unequal nucleotide distribution was found in various T . vaginalis genes retrieved from data bases.

Mol Biol Evol, 2001 Mar, 18(3), 322 - 9
The Drosophila alcohol dehydrogenase gene may have evolved independently of the functionally homologous medfly, olive fly, and flesh fly genes; Brogna S et al.; cDNAs for alcohol dehydrogenase (ADH) isozymes were cloned and sequenced from two tephritid fruit flies, the medfly Ceratitis capitata and the olive fly Bactrocera oleae . Because of the high sequence divergence compared with the Drosophila sequences, the medfly cDNAs were cloned using sequence information from the purified proteins, and the olive fly cDNAs were cloned by functional complementation in yeast . The medfly peptide sequences are about 83% identical to each other, and the corresponding mRNAs have the tissue distribution shown by the corresponding isozymes, ADH-1 and ADH-2 . The olive fly peptide sequence is more closely related to medfly ADH-2 . The tephritid ADHs share less than 40% sequence identity with Drosophila ADH and ADH-related genes but are >57% identical to the ADH of the flesh fly Sarcophaga peregrina, a more distantly related species . To explain this unexpected finding, it is proposed that the ADH: genes of the family Drosophilidae may not be orthologous to the ADH: genes of the other two families, Tephritidae and Sarcophagidae.

J Clin Microbiol, 2001 Mar, 39(3), 1200 - 1
Recurrent self-limited fungemia caused by Yarrowia lipolytica in a patient with acute myelogenous leukemia; Chang CL et al.; Yarrowia lipolytica is a weakly pathogenic yeast that is rarely isolated from the blood . We observed transient recurrent catheter-related fungemia attributable to this organism in a leukemic patient . The fungemia and accompanying fever subsided spontaneously . The data suggest that it might be possible to withhold specific treatment for Y . lipolytica fungemia even in an immunocompromised patient.

Genome Res, 2001 Mar, 11(3), 341 - 55
Identification of human epidermal differentiation complex (EDC)-encoded genes by subtractive hybridization of entire YACs to a gridded keratinocyte cDNA library; Marenholz I et al.; The epidermal differentiation complex (EDC) comprises a large number of genes that are of crucial importance for the maturation of the human epidermis . So far, 27 genes of 3 related families encoding structural as well as regulatory proteins have been mapped within a 2-Mb region on chromosome 1q21 . Here we report on the identification of 10 additional EDC genes by a powerful subtractive hybridization method using entire YACs (950_e_2 and 986_e_10) to screen a gridded human keratinocyte cDNA library . Localization of the detected cDNA clones has been established on a long-range restriction map covering more than 5 Mb of this genomic region . The genes encode cytoskeletal tropomyosin TM30nm (TPM3), HS1-binding protein Hax-1 (HAX1), RNA-specific adenosine deaminase (ADAR1), the 34/67-kD laminin receptor (LAMRL6), and the 26S proteasome subunit p31 (PSMD8L), as well as five hitherto uncharacterized proteins (NICE-1, NICE-2, NICE-3, NICE-4, and NICE-5) . The nucleotide sequences and putative ORFs of the EDC genes identified here revealed no homology with any of the established EDC gene families . Whereas database searches revealed that NICE-3, NICE-4, and NICE-5 were expressed in many tissues, no EST or gene-specific sequence was found for NICE-2 . Expression of NICE-1 was up-regulated in differentiated keratinocytes, pointing to its relevance for the terminal differentiation of the epidermis . The newly identified EDC genes are likely to provide further insights into epidermal differentiation and they are potential candidates to be involved in skin diseases and carcinogenesis that are associated with this region of chromosome 1 . Moreover, the extended integrated map of the EDC, including the polymorphic sequence tag site (STS) markers D1S1664, D1S2346, and D1S305, will serve as a valuable tool for linkage analyses.

Genes Dev, 2001 Feb 15, 15(4), 404 - 14
Cdc13 both positively and negatively regulates telomere replication; Chandra A et al.; Cdc13 is a single-strand telomeric DNA-binding protein that positively regulates yeast telomere replication by recruiting telomerase to chromosome termini through a site on Cdc13 that is eliminated by the cdc13-2 mutation . Here we show that Cdc13 has a separate role in negative regulation of telomere replication, based on analysis of a new mutation, cdc13-5 . Loss of this second regulatory activity results in extensive elongation of the G strand of the telomere by telomerase, accompanied by a reduced ability to coordinate synthesis of the C strand . Both the cdc13-5 mutation and DNA polymerase alpha mutations (which also exhibit elongated telomeres) are suppressed by increased expression of the Cdc13-interacting protein Stn1, indicating that Stn1 coordinates action of the lagging strand replication complex with the regulatory activity of CDC13 . However, the association between Cdc13 and Stn1 is abolished by cdc13-2, the same mutation that eliminates the interaction between Cdc13 and telomerase . We propose that Cdc13 participates in two regulatory steps-first positive, then negative-as a result of successive binding of telomerase and the negative regulator Stn1 to overlapping sites on Cdc13 . Thus, Cdc13 coordinates synthesis of both strands of the telomere by first recruiting telomerase and subsequently limiting G-strand synthesis by telomerase in response to C-strand replication.

EMBO J, 2001 Mar 1, 20(5), 1051 - 63
The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases; Albrecht V et al.; The Arabidopsis calcineurin B-like calcium sensor proteins (AtCBLs) interact with a group of serine-threonine protein kinases (AtCIPKs) in a calcium-dependent manner . Here we identify a 24 amino acid domain (NAF domain) unique to these kinases as being required and sufficient for interaction with all known AtCBLs . Mutation of conserved residues either abolished or significantly diminished the affinity of AtCIPK1 for AtCBL2 . Comprehensive two-hybrid screens with various AtCBLs identified 15 CIPKs as potential targets of CBL proteins . Database analyses revealed additional kinases from Arabidopsis and other plant species harbouring the NAF interaction module . Several of these kinases have been implicated in various signalling pathways mediating responses to stress, hormones and environmental cues . Full-length CIPKs show preferential interaction with distinct CBLs in yeast and in vitro assays . Our findings suggest differential interaction affinity as one of the mechanisms generating the temporal and spatial specificity of calcium signals within plant cells and that different combinations of CBL-CIPK proteins contribute to the complex network that connects various extracellular signals to defined cellular responses.

EMBO J, 2001 Mar 1, 20(5), 1010 - 9
Arabidopsis glucosidase I mutants reveal a critical role of N-glycan trimming in seed development; Boisson M et al.; Glycoproteins with asparagine-linked (N-linked) glycans occur in all eukaryotic cells . The function of their glycan moieties is one of the central problems in contemporary cell biology . N-glycosylation may modify physicochemical and biological protein properties such as conformation, degradation, intracellular sorting or secretion . We have isolated and characterized two allelic Arabidopsis mutants, gcs1-1 and gcs1-2, which produce abnormal shrunken seeds, blocked at the heart stage of development . The mutant seeds accumulate a low level of storage proteins, have no typical protein bodies, display abnormal cell enlargement and show occasional cell wall disruptions . The mutated gene has been cloned by T-DNA tagging . It codes for a protein homologous to animal and yeast alpha-glucosidase I, an enzyme that controls the first committed step for N-glycan trimming . Biochemical analyses have confirmed that trimming of the alpha1,2- linked glucosyl residue constitutive of the N-glycan precursor is blocked in this mutant . These results demonstrate the importance of N-glycan trimming for the accumulation of seed storage proteins, the formation of protein bodies, cell differentiation and embryo development.

Appl Environ Microbiol, 2001 Mar, 67(3), 1268 - 73
Characteristics of a Streptomyces coelicolor A3(2) extracellular protein targeting chitin and chitosan; Saito A et al.; Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis . The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain . The CHB3 protein (14.9 kDa) was secreted by the S . lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin . The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration . In contrast to CHB1, CHB3 targets alpha-chitin, beta-chitin, and chitosan at pH 6.0 but does so relatively loosely . The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.

J Mol Diagn, 2000 Aug, 2(3), 139 - 44
Improving the detection of p53 mutations in breast cancer by use of the FASAY, a functional assay; Duddy PM et al.; The aim of this investigation was to examine the ability of the yeast-based functional assay, the functional analysis for the separation of alleles in yeast (FASAY), to detect p53 mutations in breast cancers when compared with immunohistochemistry and automated sequencing of the whole p53 gene (exons 1-11) . To achieve this, all three methods were carried out on a cohort of aggressive breast tumors . In those tumors, in which the FASAY analysis indicated the presence of a mutation, cDNA was extracted from red yeast colonies and was sequenced to identify the base change in the p53 gene . The FASAY detected all 24 mutations found in the series of 48 tumors, whereas initial automated sequencing of genomic DNA detected 18/24 mutations . A second round of automated sequencing carried out using an independent source of genomic DNA detected mutations in 3 of the 6 tumors that originally appeared to lack a mutation in genomic DNA . All but 1 of the mutations originally missed by sequencing of genomic DNA were point mutations . Five mutations in this series (21%) were outside the commonly investigated exons 5-8, reinforcing the need to extend sequencing beyond this region . Of 24 tumors, 14 had strong immunohistochemical staining, and all 14 had p53 mutations; the majority of mutations missed by immunohistochemistry produced a truncated protein . Strong staining was not seen in tumors lacking a p53 mutation . The FASAY proved to be a rapid, reliable, and effective method for identifying those breast tumors harboring p53 mutations.

Antioxid Redox Signal, 2000 Fall, 2(3), 461 - 5
Sensitivity of FRDA lymphoblasts to salts of transition metal ions; Wong A et al.; Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease resulting from decreased expression of the nuclear-encoded mitochondrial protein, frataxin . FRDA patients have characteristic iron deposits and dysfunction of mitochondrial enzymes in the heart . Inactivation of the frataxin homologue in yeast causes dysregulation of both mitochondrial iron levels and iron export . Previously, we have observed sensitivity of FRDA fibroblasts to FeCl3 and hydrogen peroxide, results consistent with the hypothesis that FRDA cells may experience increased Fenton chemistry . To determine whether the sensitivity of FRDA cells to transition metal ions is a general or specific property, we have compared the sensitivity of lymphoblasts from FRDA patients and healthy controls to the transition metal salts CoCl2, CuSO4 FeCl3 FeSO4, MnCl2, and ZnCl2 . FRDA lymphoblasts were significantly more sensitive to FeCl3 and MnCl2 than control cells . However, there were no significant differences observed in sensitivity to CoCl2, CuSO4, FeSO4 and ZnCl2 in the concentration ranges studied . Thus, the sensitivity of FRDA lymphoblasts exposed to transition metals appears to be specific, and could be relevant to the pathophysiological mechanism, which is discussed.

Curr Opin Plant Biol, 2001 Apr, 4(2), 136 - 42
Genome-wide expression analysis of plant cell cycle modulated genes; Breyne P et al.; Genome-wide expression analysis is rapidly becoming an essential tool for identifying and analysing genes involved in, or controlling, various biological processes ranging from development to responses to environmental cues . The control of cell division involves the temporal expression of different sets of genes, allowing the dividing cell to progress through the different phases of the cell cycle . A landmark study using DNA microarrays to follow the patterns of gene expression in synchronously dividing yeast cells has allowed the identification of several hundreds of genes that are involved in the cell cycle . Although DNA microarrays provide a convenient tool for genome-wide expression analysis, their use is limited to organisms for which the complete genome sequence or a large cDNA collection is available . For other organisms, including most plant species, DNA fragment analysis based methods, such as cDNA-AFLP, provide a more appropriate tool for genome-wide expression analysis . Furthermore, cDNA-AFLP exhibits properties that complement DNA microarrays and, hence, constitutes a useful tool for gene discovery.

Curr Opin Plant Biol, 2001 Apr, 4(2), 123 - 9
Gene silencing and DNA methylation processes; Paszkowski J et al.; Epigenetic gene silencing results from the inhibition of transcription or from posttranscriptional RNA degradation . DNA methylation is one of the most central and frequently discussed elements of gene silencing in both plants and mammals . Because DNA methylation has not been detected in yeast, Drosophila or Caenorhabditis elegans, the standard genetic workhorses, plants are important models for revealing the role of DNA methylation in the epigenetic regulation of genes in vivo.

Curr Opin Immunol, 2001 Apr, 13(2), 186 - 94
How to make ends meet in V(D)J recombination; Grawunder U et al.; In most vertebrate species analyzed so far, the diversity of soluble or membrane-bound antigen-receptors expressed by B and T lymphocytes is generated by V(D)J recombination . During this process, the coding regions for the variable domains of antigen-receptors are created by the joining of subexons that are randomly selected from arrays of tandemly repeated V, D (sometimes) and J gene segments . This involves the site-specific cleavage of chromosomal DNA by the lymphocyte-specific recombination-activating gene (RAG)-1/2 proteins, which appear to have originated from an ancient transposable element . The DNA double-strand breaks created by RAG proteins are subsequently processed and rejoined by components of the nonhomologous DNA end-joining pathway, which is conserved in all eukaryotic organisms - from unicellular yeast up to highly complex mammalian species.

FEBS Lett, 2001 Feb 23, 491(1-2), 148 - 53
GPI-anchored proteins and glycoconjugates segregate into lipid rafts in Kinetoplastida; Denny PW et al.; The plasma membranes of the divergent eukaryotic parasites, Leishmania and Trypanosoma, are highly specialised, with a thick coat of glycoconjugates and glycoproteins playing a central role in virulence . Unusually, the majority of these surface macro-molecules are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor . In mammalian cells and yeast, many GPI-anchored molecules associate with sphingolipid and cholesterol-rich detergent-resistant membranes, known as lipid rafts . Here we show that GPI-anchored parasite macro-molecules (but not the dual acylated Leishmania surface protein (hydrophilic acylated surface protein) or a subset of the GPI-anchored glycoinositol phospholipid glycolipids) are enriched in a sphingolipid/sterol-rich fraction resistant to cold detergent extraction . This observation is consistent with the presence of functional lipid rafts in these ancient, highly polarised organisms.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2413 - 8
ARF-GEP(100), a guanine nucleotide-exchange protein for ADP-ribosylation factor 6; Someya A et al.; A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP(100), which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified . On Northern blot analysis of human tissues, a approximately 8-kb mRNA that hybridized with an ARF-GEP(100) cDNA was abundant in peripheral blood leukocytes, brain, and spleen . ARF-GEP(100) accelerated {(35)S}GTPgammaS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca . 12-fold . The ARF-GEP(100) Sec7 domain contains Asp(543) and Met(555), corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe(535) and Ala(536), associated with BFA-insensitivity . The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding . Consistent with its structure, ARF-GEP(100) activity was not affected by BFA or phospholipids . After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic . On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas . The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP(100) in those areas . No similar coincidence of ARF-GEP(100) with AP-1, AP-2, catenin, LAMP-1, or 58K was observed . The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

Apoptosis, 2000 Jun, 5(3), 217 - 20
The Daxx enigma; Michaelson JS; Several reports describing Daxx and its putative role have emerged without a unifying theme . While Daxx has been implicated in apoptosis, it remains unclear whether Daxx is pro- or anti-apoptotic, and whether its role in apoptosis is direct or indirect . Moreover, whether Daxx plays alternative or additional roles in regulating transcription, centromere binding or any number of other activities within the cell, is uncertain . The ability of Daxx to interact with a wide variety of molecules in yeast-interaction trap systems (Table 1) has allowed for this range of speculation . The fact that Daxx contains no significant homology to other known proteins has rendered its study all the more challenging.

Fresenius J Anal Chem, 2000 Mar, 366(5), 504 - 7
Cationic cyanine as a near-infrared fluorescent probe for the determination of nucleic acids; Zheng H et al.; A new method with a cationic near-IR cyanine as fluorescent probe was developed for the determination of nucleic acids . The near-IR cyanine shows maximum excitation and emission wavelengths at 765 and 790 nm, respectively, in aqueous solution . The method is based on the fluorescence decrease of near-IR cyanine in the presence of nucleic acids . Under optimal conditions, the ratio of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.10-1.2 microg/mL for CT (calf thymus) DNA or SM (salmon sperm) DNA, and 0.10-1.6 microg/mL for yeast RNA . The detection limits were 30 ng/mL for CT DNA, 25 ng/mL for SM DNA and 70 ng/mL for yeast RNA . The relative standard deviation (n = 6) was 2.1% for 500 ng/mL CT DNA, 2.4% for 500 ng/mL SM DNA and 2.7% for 500 ng/mL yeast RNA, respectively.

Curr Opin Gastroenterol, 2001 Mar, 17(2), 177 - 183
Intestinal metal ion absorption: an update; Rolfs A et al.; Recent progress in the field of metal ion transport has significantly advanced our understanding of the mechanisms of intestinal metal ion absorption under normal and pathological conditions . In this brief review, we focus on the key proteins involved in intestinal absorption of iron, zinc, and copper . Following the initial description of the apical iron transporter, DCT1, the basolateral transporter complex has been identified, which consists of the metal transporter IREG1/MTP1 and the multicopper oxidase, hephaestin . Novel zinc and copper transporters have been identified as well, mostly based on their homology to yeast and plants transporters . The identification of a variety of copper and zinc transporters is consistent with the importance of copper and zinc in a wide variety of enzymatic reactions, free radical scavenging, and transcriptional control.

Mutat Res, 2001 Mar, 488(1), 39 - 64
Mutation processes at the protein level: is Lamarck back?
Chernoff YO.
The experimental evidence accumulated for the last half of the century clearly suggests that inherited variation is not restricted to the changes in genomic sequences . The prion model, originally based on unusual transmission of certain neurodegenerative diseases in mammals, provides a molecular mechanism for the template-like reproduction of alternative protein conformations . Recent data extend this model to protein-based genetic elements in yeast and other fungi . Reproduction and transmission of yeast protein-based genetic elements is controlled by the "prion replication" machinery of the cell, composed of the protein helpers responsible for the processes of assembly and disassembly of protein structures and multiprotein complexes . Among these, the stress-related chaperones of Hsp100 and Hsp70 groups play an important role . Alterations of levels or activity of these proteins result in "mutator" or "antimutator" affects in regard to protein-based genetic elements . "Protein mutagens" have also been identified that affect formation and/or propagation of the alternative protein conformations . Prion-forming abilities appear to be conserved in evolution, despite the divergence of the corresponding amino acid sequences . Moreover, a wide variety of proteins of different origins appear to possess the ability to form amyloid-like aggregates, that in certain conditions might potentially result in prion-like switches . This suggests a possible mechanism for the inheritance of acquired traits, postulated in the Lamarckian theory of evolution . The prion model also puts in doubt the notion that cloned animals are genetically identical to their genome donors, and suggests that genome sequence would not provide a complete information about the genetic makeup of an organism.

Gene, 2001 Jan 24, 263(1-2), 85 - 92
Identification and characterization of a novel human cDNA encoding a 21 kDa pRb-associated protein; Wen H et al.; The retinoblastoma protein (pRb) functions as a critical master regulator in cell cycle regulation, which is an important cell-regulatory process, through its interaction with various cellular proteins . Using the C-terminus of human pRb and the yeast two-hybrid system, a novel protein named RBP21 that contains 187 amino acid residues with a calculated molecular weight of 21 kDa was identified as a pRb-binding protein . Sequence analysis indicates that RBP21 shares homology with other retinoblastoma-binding proteins in the pRb-binding motif LxCxE at the C-terminal region . In vitro specific interaction between pRb and RBP21 was confirmed using in vitro translation products . When overexpressed in COS-7 cells, RBP21 could co-immunoprecipitate with pRb . This interaction requires the LxCxE motif of RBP21 and the entire pocket region of pRb . Each point mutation of the conserved amino acid residues in pRb-binding motif of RBP21 abolished its specific interaction with pRb . RH mapping result showed that this novel gene was mapped to chromosome region 15q21.1-21.3 . Northern blot analysis suggested that RBP21 was widely expressed in various human tissues and cancer cell lines . When expressed in HeLa cells as a green fluorescent protein fusion, RBP21 was distributed throughout the cell.

Mol Biochem Parasitol, 2001 Feb, 112(2), 229 - 37
Anopheles gambiae laminin interacts with the P25 surface protein of Plasmodium berghei ookinetes; Vlachou D et al.; Laminin is a major constituent of the basal lamina surrounding the midgut of the malaria vectors that has been implicated in the development of the Plasmodium oocyst . In this report we describe the cloning of the Anopheles gambiae gene encoding the laminin gamma 1 polypeptide and follow its expression during mosquito development . To further investigate the putative role of laminin in the transmission of the malaria parasite we studied the potential binding of the P25 surface protein of Plasmodium berghei using a yeast two-hybrid system . Heterodimer formation was observed and does not require any additional protein factors since purified fusion proteins can also bind each other in vitro . Laminin gamma 1 also interacts with the paralogue of P25, namely P28, albeit more weakly, possibly explaining why the two parasite proteins can substitute for each other in deletion mutants . This represents the first direct evidence for molecular interactions between a surface protein of the Plasmodium parasite with an Anopheles protein; the strong interplay between laminin gamma 1 and P25 suggests that this pair of proteins may function as a receptor/ligand complex regulating parasite development in the mosquito vector.

J Immunol Methods, 2001 Feb 1, 248(1-2), 77 - 90
Development and applications of surface-linked single chain antibodies against T-cell antigens; Griffin MD et al.; In this report the use of surface-linkage to expand the potential experimental and therapeutic applications of single chain antibody (scFv) constructs is reviewed . A strategy for the generation and functional characterization of surface-linked scFvs that bind selectively to the T-cell proteins CD3epsilon, CD28, and CD152 (CTLA-4) is described in detail . Experimental examples are provided of the use of these constructs to study the positive and negative regulation of T-cell activation and to manipulate the in vivo immunogenicity of tumor cells . In addition, a novel system for Simultaneous T-cell Activation and Retroviral Transduction (START) is described in which retroviral packaging cells are rendered mitogenic for T lymphocytes by combined expression of surface-linked scFvs . Finally, the use of random mutagenesis and yeast surface display to increase the affinity and functional efficacy of scFv constructs is demonstrated.

FEBS Lett, 2001 Feb 16, 490(3), 142 - 52
Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases; Waterman H et al.; The major process that regulates the amplitude and kinetics of signal transduction by tyrosine kinase receptors is endocytic removal of active ligand-receptor complexes from the cell surface, and their subsequent sorting to degradation or to recycling . Using the ErbB family of receptor tyrosine kinases we exemplify the diversity of the down regulation process, and concentrate on two sorting steps whose molecular details are emerging . These are the Eps15-mediated sorting to clathrin-coated regions of the plasma membrane and the c-Cbl-mediated targeting of receptors to lysosomal degradation . Like in yeast cells, sorting involves not only protein phosphorylation but also conjugation of ubiquitin molecules . The involvement of other molecules is reviewed and recent observations that challenge the negative regulatory role of endocytosis are described . Finally, we discuss the relevance of receptor down regulation to cancer therapy.

J Virol, 2001 Mar, 75(6), 2526 - 34
Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC; Hong S et al.; The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein . The p4 domain has no known role in M-PMV replication . We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids . Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex) . TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins . TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6 . Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis . In the p4 truncation mutants, the Gag-TRiC association was significantly reduced . These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly . We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding . Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.

Ecotoxicol Environ Saf, 2001 Mar, 48(3), 275 - 86
Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test; Fountain MT et al.; Current recommended ecotoxicological tests with the parthenogenetic springtail Folsomia candida using standard OECD soil do not allow for continuous monitoring during the exposure period . Effects of chemicals cannot be determined until the end of the experiment (typically after 4 weeks), since the animals stay below the soil surface . In this study, F . candida were maintained on a plaster of Paris/graphite substrate for 7 weeks and were supplied with an aqueous suspension of yeast contaminated with Cd, Cu, Pb, and Zn as nitrate salts . Growth rate, time to first batch of eggs, quantity of food consumed, and the presence of graphite in the gut (a sign of avoidance of yeast) were all affected by metal contaminated diets . The relative toxicities of Cd:Cu:Pb:Zn in the yeast were 1.0:1.07:12.0:4.3, respectively (on a weight basis) with Cd being the most toxic . Internal body concentrations increased, and the concentration factor (metal concentration in F . candida/metal concentration in yeast) decreased with increasing metal exposure . In general, metals are much less toxic when added to the food of F . candida than when incorporated into soil in standard tests . It is suggested that Collembola have a greater tolerance of metals in the diet since they avoid contaminated food, and are able to excrete assimilated metals at moulting via exfoliation of the midgut epithelium where the elements are retained as part of a storage--detoxification system . The methodology described in this article allows effects on growth to be observed as early as 7 days after the beginning of the experiment .

Dent Mater J, 2000 Sep, 19(3), 245 - 62
Estrogenic activity of dental materials and bisphenol-A related chemicals in vitro; Hashimoto Y et al.; Twenty-eight chemicals used as dental materials and bisphenol-A related chemicals were diluted with DMSO to concentrations ranging from 10(-7) to 10(-3) M and tested for estrogenicity . Bisphenol-A (BPA), bisphenol-F (BPF) and bisphenol-A-bischloroformate (BPACF) showed estrogenic activity using the yeast two-hybrid system, and BPA, BPF, BPACF and bisphenol-S (BPS) showed estrogenic activity using the fluorescence polarization system . However, none of the remaining chemicals and none of the dental materials showed any activity at concentrations between 10(-7) and 10(-3) M . Although BPA, BPF, BPACF, bisphenol-A-dimethacrylate and BPS showed estrogenic activity in the E-screen test, the remaining chemicals did not . Thus, most of the chemicals showed consistent results, either positive or negative, by the three testing methods, while two chemicals showed conflicting results . Further studies, together with in vivo and epidemiological examinations, are required . Elucidation of the structure-activity relationships of these chemicals is also needed to estimate the estrogenicity of a chemical from its structure.

Anal Chem, 2001 Feb 1, 73(3), 393 - 404
A multidimensional electrospray MS-based approach to phosphopeptide mapping; Annan RS et al.; A new, multidimensional electrospray MS-based strategy for phosphopeptide mapping is described which eliminates the need to radiolabel protein with 32P or 33P . The approach utilizes two orthogonal MS scanning techniques, both of which are based on the production of phosphopeptide-specific marker ions at m/z 63 and/or 79 in the negative ion mode . These scan methods are combined with liquid chromatography-electrospray mass spectrometry and nanoelectrospray MS/MS to selectively detect and identify phosphopeptides in complex proteolytic digests . Low-abundance, low-stoichiometry phosphorylation sites can be selectively determined in the presence of an excess of nonphosphorylated peptides, even in cases where the signal from the phosphopeptide is indistinguishable from background in the conventional MS scan . The strategy, which has been developed and refined in our laboratory over the past few years, is particularly well suited to phosphoproteins that are phosphorylated to varying degrees of stoichiometry on multiple sites . Sensitivity and selectivity of the method are demonstrated here using model peptides and a commercially available phosphoprotein standard . In addition, the strategy is illustrated by the complete in vitro and in vivo phosphopeptide mapping of Sic1p, a regulator of the G1/S transition in budding yeast.

Indian J Pathol Microbiol, 2000 Apr, 43(2), 165 - 8
Histoplasma capsulatum in adrenal gland aspirate--a case report; Mahajan R et al.; We report a case of disseminated histoplasmosis in a 60-year-old non-immunocompromised patient who presented to us with fever and hepatosplenomegaly . Sonographic & CT examination of the abdomen showed bilateral adrenal masses . Cytological examination of the aspirated material from the mass showed yeast forms of H . capsulatum.

Eur J Pediatr, 2000 Dec, 159 Suppl 3, S236 - 9
Organelle disease: peroxisomal disorders; Gartner J; Peroxisomes are virtually ubiquitous organelles involved in numerous catabolic and anabolic pathways . Interest in peroxisomes stems from an expanding group of genetic diseases in which there is either deficiency of a specific peroxisomal function (single protein defects) or failure to assemble the organelle resulting in defects of multiple peroxisome functions (peroxisome biogenesis disorders) . The paradigm for the former is X-linked adrenoleukodystrophy caused by mutations in the adrenoleukodystrophy gene and, for the latter, Zellweger syndrome caused by mutations in peroxin genes . CONCLUSION: The identification and functional characterisation of the peroxisomal disease genes is proceeding at rapid pace helped immeasurably by work in various yeast model systems . The ultimate goal is to elucidate how the encoded proteins produce normal appearing and functioning peroxisomes . The achievement of this goal will lead to a better understanding of peroxisomal disorders, their pathogenesis and treatment.

Planta, 2001 Jan, 212(2), 264 - 9
Cytosolic glutamine synthetase and not nitrate reductase from the green alga Chlamydomonas reinhardtii is phosphorylated and binds 14-3-3 proteins; Pozuelo M et al.; The nitrate reductase activity from Chlamydomonas reinhardtii was not altered when extracts were incubated with yeast 14-3-3 proteins in the presence of Mg-ATP . However, the C . reinhardtii extracts contained 14-3-3 proteins capable of inhibiting the spinach nitrate reductase, raising the question of their physiological substrates . Two C . reinhardtii proteins of about 48 and 35 kDa were eluted from 14-3-3 affinity chromatography columns and bound to 14-3-3s in overlay assays . The 48-kDa protein corresponded to the cytosolic isoform of glutamine synthetase (GS1) . The GSI was phosphorylated by a Ca2+-and calmodulin-dependent protein kinase partially purified from the alga . However, neither phosphorylation nor 14-3-3 binding seemed to change GS catalytic activity.

Nature, 2001 Feb 1, 409(6820), 581 - 8
Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion; Peters C et al.; SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles . But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear . Ca2+/calmodulin controls this terminal process in many intracellular fusion events . Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles . Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes . V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin . The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs . Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion . V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site . We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.

Antioxid Redox Signal, 2000 Winter, 2(4), 753 - 65
Cell-surface events for metallothionein-1 and heme oxygenase-1 regulation by the hemopexin-heme transport system; Sung L et al.; A model has been developed for the hemopexin receptor-mediated heme transport system based on iron uptake in yeast . Two steps are required: reduction followed by oxidation by a multi-copper-oxidase . Furthermore, in the hemopexin system, the surface redox events have been linked with gene regulation . The impermeable Cu(I) chelator bathocuproinedisulfonate (BCDS) is shown here to abrogate heme oxygenase-1 (HO-1) mRNA induction by heme-hemopexin . A role for Cu(I) in the regulation of HO-1 and MT-1 (Sung et al., 1999) by hemopexin supports the participation of electron transport processes at the cell surface as does competition by the reductase activator, ferric citrate, which inhibits the induction of MT-1 and HO-1 mRNA by heme-hemopexin . There is a key role for the hemopexin receptor because neither ferric citrate nor iron-transferrin alone regulates MT-1 or HO-1 . Cell-surface copper is the first molecule to link the concomitant regulation of HO-1 and MT-1 by the hemopexin receptor . In addition, cytochrome b5 and cytochrome b5 reductase are implicated here in the response of cells to heme-hemopexin . Reduction of one or more electron donors of the reductase and oxidation of the electron acceptor, b5 heme, leads to gene regulation, but only when heme-hemopexin is bound to its receptor . Protein kinase cascades, including JNK, are activated by the hemopexin receptor itself upon ligand binding but are modulated by a Cu(I)-dependent process likely to be heme uptake.

Cell Mol Life Sci, 1999 Oct 1, 56(1-2), 22 - 31
Small nucleolar RNAs; Eliceiri GL; Many small RNA species associate with the nucleolar structure . Some of these small nucleolar RNAs (snoRNAs) are required for cleavage processing of ribosomal RNA precursors . There are many pseudouridine residues and methylated riboses in mature ribosomal RNA . For most, if not all, of these modifications, each site is selected by base pairing with a specific snoRNA species . Some snoRNAs are needed for the 2'-O-ribose methylation of at least one spliceosomal small nuclear RNA . Many snoRNAs, particularly in yeast, are generated from independent transcription units . Most vertebrate snoRNAs are produced by processing of introns from protein-coding transcripts . Some snoRNAs are made by processing of introns from non-protein-coding transcripts.

J Hepatol, 2001 Jan, 34(1), 123 - 7
Rapid seroprotection against hepatitis B following the first dose of a Pre-S1/Pre-S2/S vaccine; Shapira MY et al.; BACKGROUND/AIMS: Will immunization with an experimental Pre-S1/Pre-S2/S hepatitis B vaccine (Bio-Hep-B) induce faster seroprotection using fewer doses as compared with a yeast derived S vaccine (Engerix B) . METHODS: Healthy volunteers, n = 36, mean age 23 y, randomized to receive 2 or 3 doses of both vaccines given months 0 and 6, or 0, 1 and 6 . RESULTS: Following primary immunization, seroprotection occurred in 6, 39, 53 and 60% in the Bio-Hep-B group at weeks 1, 2, 3 and 4, compared with 0, 12, 18 and 12.5% in the Engerix-B vaccinees, respectively . Six months following injection of the first dose, seroprotection was 70 and 25% in Pre-S/S and S vaccinees respectively . Area under the curve in vaccinees of Bio-Hep-B; versus Engerix-B showed mean anti-HBs level of 365 +/- 166 and 85 +/- 48 mIU/ml x day respectively (P = 0.012) . At month 7, 100% seroprotection was achieved in both groups while anti-HBs rose from 81 to 28,800 mIU/ml and from 12 to 923 mIU/ml in recipients of Bio-Hep-B and Engerix-B respectively (P < 0.025) . CONCLUSIONS: Bio-Hep-B induces rapid seroprotection against hepatitis B in 60-70% of vaccinees, within 4-24 weeks after the first dose . Two instead of the conventional three doses of the Pre-S/S vaccine may be sufficient to induce adequate seroprotection.

Mol Cells, 2000 Dec 31, 10(6), 626 - 32
Interaction of PRK1 receptor-like kinase with a putative elF2B beta-subunit in tobacco; Park SW et al.; PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata . We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins . The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast beta-subunit of translation initiation factor 2B (eIF2B-beta), which was designated NeIF2Bbeta . eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells . Deletion mutants of NeIF2Bbeta were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2Bbeta, especially the region between residue 103 and 235, is important for the interaction . This protein association was confirmed by in vitro binding assay of the recombinant NeIF2Bbeta and PRK1 proteins . Despite high sequence homology between NeIF2Bbeta and its yeast counterpart, the NeIF2Bbeta cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-beta, when transferred into the mutant strain.

J Mol Neurosci, 2000 Aug, 15(1), 19 - 29
Phosphorylation of the common neurotrophin receptor p75 by p38beta2 kinase affects NF-kappaB and AP-1 activities; Wang JJ et al.; The signaling pathways invoked by ligand binding to the common neurotrophin receptor p75NTR are incompletely understood . Using the yeast two-hybrid system, we identified the mitogen-activated protein (MAP) kinase p38beta2 as a specific interactor with the 5th and 6th alpha helices of the p75NTR intracytoplasmic region . The consequences of this interaction were studied, using primary cultures of Schwann cells and the 293T cell line . Phosphorylation of p75NTR by p38beta2 was induced in vitro and in vivo by MAP kinase kinases (MKK) 6 activation . This pathway demonstrated feedback in that nerve growth factor (NGF) binding increased p38beta2 activity, causing an increase of nuclear factor-kappaB (NF-kappaB) activation and a decrease of AP-1 activation . The mechanisms described explain at least in part why NGF binding to p75NTR increases cell survival in certain circumstances.

Mamm Genome, 2001 Feb, 12(2), 150 - 6
Isolation and molecular characterization of rasfadin, a novel gene in the vicinity of the bovine prion gene; Comincini S et al.; A novel gene, rasfadin (RASSF2) was identified close to the bovine prion gene, and its genomic structure was derived with a combination of exon trapping and RACE . The gene covers at least 28 kb and maps to the same chromosomal region as the prion gene in cattle, sheep, and human . The RASSF2 ORF is composed of 987 base pairs divided into nine exons and shows a high nucleotide (88%) and amino acid similarity (95%) with a previously described human cDNA, KIAA0168 . The bovine 3'UTR region is significantly shorter than the human counterpart, but shares with it two highly conserved nucleotide blocks . The expression of the gene was investigated in brain, liver, and spleen . Alternative splicing yields a shorter product in the liver composed of only four exons . Computer analysis showed a highly significant similarity of the rasfadin protein with the Ras association (Ral-GDS/AF-6) domain family 2 and with the afadin family, respectively, for the longer brain/spleen and the shorter liver variants.

Mamm Genome, 2001 Feb, 12(2), 112 - 6
Smcy transgene does not rescue spermatogenesis in sex-reversed mice; Agulnik AI et al.; In mouse, the Sxr(b) deletion interval (delta Sxr(b)) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia . This deletion interval is approximately 1-2 Mb and contains eight known genes . In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr(b) interval including Zfy1, Ube1y, Smcy, and Eif2s3 . Two male and one female founder mice, transgenic for all four genes, were sterile . However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified . Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y . The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers . Introduction of the transgene into an X Sxr(b)/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males . These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy.

Neuroreport, 2001 Feb 12, 12(2), 233 - 5
An actin-binding protein, CAP, is expressed in a subset of rat taste bud cells; Ishimaru Y et al.; Single cell cDNA libraries were constructed from taste bud cells of rat circumvallate papillae . Using three steps of screening, including differential hybridization, sequence analyses and in situ hybridization, a clone encoding a rat homolog of yeast adenylyl cyclase-associated protein (CAP) was identified to be highly expressed in a subset of taste bud cells.

Hereditas, 2000, 133(1), 59 - 63
Half-sib analysis of three morphological traits in Drosophila melanogaster under poor nutrition; Bubliy OA et al.; Variation in thorax length, wing length and sternopleural bristle number was examined in Drosophila melanogaster reared in stressful and nonstressful environments using paternal half-sib design . Low concentration of yeast in the medium was used as a stress factor . Phenotypic variation of thorax length and wing length was higher under poor nutrition than in the control; in bristle number, phenotypic variation was relatively stable regardless of the environment . Heritability of all the traits analyzed was generally lower under nutritional stress . Heritability changes in thorax length and wing length were mainly due to an increase in the environmental variance under stress, whereas in bristle number, stress resulted in a decrease in genetic variation . Genetic variance in thorax length was higher under poor nutrition; in wing length, no difference in genetic variance between environments was found.

Protein Sci, 2000 Dec, 9(12), 2567 - 72
A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica; Smooker PM et al.; The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite . To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified . Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen . In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2 . Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position . Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite . Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed . Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F . hepatica cathepsin L proteases.

Life Sci, 2001 Jan 5, 68(7), 739 - 49
Panaxagin, a new protein from Chinese ginseng possesses anti-fungal, anti-viral, translation-inhibiting and ribonuclease activities; Ng TB et al.; From the roots of the Chinese ginseng Panax ginseng a protein designated panaxagin with ribonuclease activity, but possessing a sequence distinct from ribonucleases previously reported from ginseng calluses, was isolated . The purification protocol employed comprised extraction with cold saline, (NH4)2SO4 precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration on Superdex 75 by fast protein liquid chromatography . The purified protein was composed of two identical subunits each with a molecular weight of 26 kDa . Its N-terminal amino acid sequence exhibits sites of similarity with the sequences of plant ribosome inactivating proteins and fungal ribonucleases . The spectrum of biological activities of panaxagin encompassed ribonuclease activity toward yeast transfer RNA, translation-inhibitory activity in a rabbit reticulocyte lysate system, and antifungal activity against fungi including Coprinus comatus and Fusarium oxysporum, but not against Rhizoctonia solani . In addition it displayed an inhibitory activity against human immunodeficiency virus reverse transcriptase and succinylation augmented this activity.

Med Mycol, 2000 Dec, 38(6), 399 - 406
Host response and Histoplasma capsulatum/macrophage molecular interactions; Porta A et al.; Histoplasma capsulatum is the etiological agent of histoplasmosis, a chronic respiratory infection that is generally asymptomatic in healthy individuals, but severe or fatal in patients who are immunosuppressed or otherwise debilitated . H . capsulatum is found as a mould in soil and becomes a pathogenic yeast in the mammalian host . The first line of defense that H . capsulatum faces during host invasion is the attack of polymorphonuclear neutrophils and resident macrophages . In animal models, once phagocytosed, H . capsulatum is not killed by fusion of the phago-lysosomes, instead it multiplies within non-activated macrophages and destroys them . Upon induction of cell-mediated immunity, cytokines activate macrophages and destroy the yeast cells . Some aspects of the fungus-macrophage interaction have been elucidated, and it is clear that some of the mechanisms by which H . capsulatum escapes the lethal effects of this very hostile environment, involve the regulation of specific genes . Recently, using the differential display reverse transcriptase polymerase chain reaction technique, a number of H . capsulatum genes that are induced after the yeasts are ingested by macrophages have been identified . However, the mechanisms that underlie the capacity of H . capsulatum to adapt to the new environmental conditions present in macrophages remain to be clarified.

Med Mycol, 2000, 38 Suppl 1, 59 - 65
Natural pathogens of laboratory animals and their effects on research; Connole MD et al.; The natural fungal pathogens of laboratory animals such as rabbits and guinea pigs are mainly dermatophyte species, most commonly Trichophyton mentagrophytes and also, less frequently Microsporum gypseum and M . canis . However, the incidences of infection and clinical disease are low in well-managed animal facilities . Young or immunocompromised rabbits are thought to be most susceptible . Dermatophytes infect the epidermis and adnexal structures, including hair follicles and shafts, usually on or around the head, and cause pruritis, patchy alopecia, erythema and crusting . Histopathological changes in the underlying skin occur and these changes could confound histological studies involving the skin . Yeast infections usually due to Candida spp . have been reported occasionally in laboratory animals . In this paper, the role of rodents in the evaluation of topical antifungal agents, dermatophytosis and two species of Candida, which are natural pathogens of laboratory animals, are discussed in relation to their effects on research . Pneumocystis carinii, an inhabitant of the respiratory tract of laboratory mice and rats, is a pathogen only under conditions of induced or inherent immunodeficiency . Infected mice and rats are likely to develop severe pneumocystosis following immunosuppression and will be rendered unsuitable for most experimental purposes.

Med Mycol, 2000, 38 Suppl 1, 243 - 50
Black fungi: clinical and pathogenic approaches; De Hoog GS et al.; Data are presented on the clinically relevant black yeasts and their relatives, i.e., members of the Ascomycete order Chaetothyriales . In order to understand the pathology of these fungi it is essential to know their natural ecological niche . From a relatively low degree of molecular variability of the black yeast Exophiala dermatitidis, potential agent of brain infections in patients from East Asia, it is concluded that this species is an emerging pathogen, currently going through a process of active speciation . It is found to be an oligotrophic fungus in hot, moist environments, such as steambaths . Cladophialophora-, Fonsecaea- and Ramichloridium-like strains, known in humans as agents of chromoblastomycosis, are frequently found on rotten plant material, but the fungal molecular diversity in the environment is much higher than that on the human patient, so that it is difficult to trace the etiological agents of the disease with precision . This approach has been successful with Cladophialophora carrionii, of which cells resembling muriform cells, the tissue form of chromoblastomycosis, were found to occur in drying spines of cacti . Phagocytosis assays provide a method to distinguish between pathogens and non-pathogens, as the killing rates of strict saprobes proved to be consistently higher than of those species frequently known as agents of disease . The therapeutic possibilities for patients with chromoblastomycosis are reviewed.

Dev Biol, 2001 Jan 15, 229(2), 480 - 93
The Caenorhabditis elegans peb-1 gene encodes a novel DNA-binding protein involved in morphogenesis of the pharynx, vulva, and hindgut; Thatcher JD et al.; Gene expression in the Caenorhabditis elegans pharynx is regulated in part by organ-specific signals, which in the myo-2 gene target a regulatory sequence called the C sub-element . C sub-element activity requires the organ specification factor PHA-4, a winged-helix transcription factor expressed in all pharyngeal cells . To identify additional factors involved in pharyngeal organogenesis, we performed a yeast one-hybrid screen for C sub-element binding proteins . Here we describe the novel factor PEB-1, which is coexpressed with PHA-4 in many pharyngeal cell types, including muscles, epithelial cells, marginal cells, and glands, but is undetectable in the pharyngeal nervous system . PEB-1 is also detected outside the pharynx in cells surrounding the rectum and vulva, as well as in the germ line . Reduction of peb-1 function using RNAi results in morphological defects in the somatic tissues in which peb-1 is expressed . We have mapped the PEB-1 DNA-binding domain to a 158-residue region, which is unrelated to known DNA-binding proteins but shares some sequence similarity to the Drosophila Mod(mdg4) proteins . PEB-1 specifically recognizes a site in the C subelement that partially overlaps the PHA-4 binding site . Both the PEB-1 and the PHA-4 binding sites are necessary for strong C sub-element enhancer activity in some cells in which these factors are coexpressed . In contrast the PEB-1 site is dispensable for C sub-element activity in pharyngeal neurons . We propose that PEB-1 functions with PHA-4 to activate target gene expression in cells in which they are coexpressed.

Nature, 2001 Jan 18, 409(6818), 359 - 63
Pre-meiotic S phase is linked to reductional chromosome segregation and recombination; Watanabe Y et al.; Meiosis is initiated from G1 of the cell cycle and is characterized by a pre-meiotic S phase followed by two successive nuclear divisions . The first of these, meiosis I, differs from mitosis in having a reductional pattern of chromosome segregation . Here we show that meiosis can be initiated from G2 in fission yeast cells by ectopically activating the meiosis-inducing network . The subsequent meiosis I occurs without a pre-meiotic S phase and with decreased recombination, and exhibits a mitotic pattern of equational chromosome segregation . The subsequent meiosis II results in random chromosome segregation . This behaviour is similar to that observed in cells lacking the meiotic cohesin Rec8 (refs 3, 4), which becomes associated with chromosomes at G1/S phase, including the inner centromere, a region that is probably critical for sister-centromere orientation . If the expression of Rec8 is delayed to S phase/G2, then the centromeres behave equationally . We propose that the presence of Rec8 in chromatin is required at the pre-meiotic S phase to construct centromeres that behave reductionally and chromosome arms capable of a high level of recombination, and that this explains why meiosis is initiated from G1 of the cell cycle.

Ukr Biokhim Zh, 2000 Jul-Oct, 72(4-5), 169 - 74
{Division of Regulatory Systems of Cells of the Institute of Biochemistry of the Ukrainian National Academy of Sciences (L'viv) . History, achievements and perspectives}; Stoika RS; A short review presented deals with the history of biochemistry development in the western regions of Ukraine . Two principal biochemical schools were founded here by J . Parnas (1884-1949) and S . Gzhytskiy (1900-1976) . While most of the students and collaborators of Prof . J . Parnas left for Poland and other western states, those ones of Prof . S . Gzhytskiy stayed in Lviv and other scientific centers of Ukraine . In 1979 Prof . S . Kusen (one of Gzhytskiy's former students and collaborators) and Prof . G . Shavlovsky headed two scientific departments founded in Lviv at O . V . Palladin Institute of Biochemistry . This event could be considered as the beginning of modern biochemistry development in the western regions of Ukraine . Since 1992 in Lviv there exists the Division of Regulatory Cell Systems of O . V . Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine headed since 1995 by Prof . R . Stoika . Four Departments work in the structure of this Division: 1) the Department of Biochemistry of Cell Differentiation headed in 1979-1997 by S . Kusen and since 1997 by L . Drobot; 2) the Department of Regulation of Cell Proliferation created in 1993 and headed by R . Stoika; 3) the Department of Biochemical Genetics created in 1988 and headed by A . Sibirny; 4) the Department of Regulation of Synthesis of Low Molecular Compounds headed in 1979-1996 by G . Shavlovsky and since 1996 by D . Fedorovych . Division of Regulatory Cell Systems is presently the leading scientific center in Ukraine in the study of the biochemical mechanisms of proliferation, differentiation and apoptosis of normal and tumour cells and in the development of effective biotechnological processes for obtaining the biologically active substances using yeast . Numerous publications of its collaborators in the high impact factor scientific magazines as well as the realisation of the international grants confirm this statement . Taking into account the high level of scientific research and availability of highly skilled scientists at the Division in 1999 the Presidium of the National Academy of Sciences of Ukraine took a resolution to transform the Division into the Institute of Cell Biology of the National Academy of Sciences of Ukraine, which was founded in 2000 on the basis of the Division.

Phytochemistry, 2001 Jan, 56(1), 77 - 85
Profiling changes in metabolism of isoflavonoids and their conjugates in Lupinus albus treated with biotic elicitor; Bednarek P et al.; Liquid chromatography with ultraviolet and mass spectrometric detection was applied to monitor changes in profiles of isoflavonoid glycosides and free isoflavonoid aglycones in Lupinus albus L . Four isoflavonoid aglycones, fourteen isoflavonoid glycosides, four flavonol glycosides and flavone glycoside were identified in lupin tissue after LC/ESI/MS analyses . An elicitor preparation from purified yeast cell wall was used to inject the shoots of 3-week old seedlings or to infiltrate the cut lupin leaves . Qualitative and quantitative changes of isoflavonoids were measured at different time points after elicitation . In elicited lupin seedlings increased amounts of prenylated isoflavone aglycones were identified . The concentrations of glycosidic conjugates of isoflavones present in plant tissue were less affected.

Nippon Rinsho, 2001 Jan, 59(1), 147 - 51
{Possible pathogenesis of rheumatoid arthritis}; Nakazawa M et al.; Rheumatoid fibroblast-like synoviocytes characteristically proliferate in an anchorage-independent manner, and are deeply implicated in cartilage destruction . Our previous results showed that rheumatoid synoviocytes expressed Fas ligand and were induced apoptosis by anti-Fas antibody treatment . In addition, several transcriptional factors, such as NF kappa B and AP-1 significantly activated in rheumatoid synoviocytes . We are now clarifying the pathogenesis of RA with the method, yeast two-hybrid system as 'post genome' strategy . We recently found that several factors that related with cell differentiation highly expressed in rheumatoid synoviocytes . In this report we discuss possible pathogenesis of RA based on our recent data and application to the therapy tool against RA.

Plant Mol Biol, 2000 Nov, 44(4), 451 - 61
The role of hexokinase in plant sugar signal transduction and growth and development; Xiao W et al.; Previous studies have revealed a central role of Arabidopsis thaliana hexokinases (AtHXK1 and AtHXK2) in the glucose repression of photosynthetic genes and early seedling development . However, it remains unclear whether HXK can modulate the expression of diverse sugar-regulated genes . On the basis of the results of analyses of gene expression in HXK transgenic plants, we suggest that three distinct glucose signal transduction pathways exist in plants . The first is an AtHXK1-dependent pathway in which gene expression is correlated with the AtHXK1-mediated signaling function . The second is a glycolysis-dependent pathway that is influenced by the catalytic activity of both AtHXK1 and the heterologous yeast Hxk2 . The last is an AtHXK1-independent pathway in which gene expression is independent of AtHXK1 . Further investigation of HXK transgenic Arabidopsis discloses a role of HXK in glucose-dependent growth and senescence . In the absence of exogenous glucose, plant growth is limited to the seedling stage with restricted true leaf development even after a 3-week culture on MS medium . In the presence of glucose, however, over-expressing Arabidopsis or yeast HXK in plants results in the repression of growth and true leaf development, and early senescence, while under-expressing AtHXK1 delays the senescence process . These studies reveal multiple glucose signal transduction pathways that control diverse genes and processes that are intimately linked to developmental stages and environmental conditions.

Cancer Res, 2001 Jan 1, 61(1), 14 - 8
Usefulness of repeated direct intratumoral gene transfer using hemagglutinating virus of Japan-liposome method for cytosine deaminase suicide gene therapy; Kanyama H et al.; To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method . The in vivo growth of the s.c . transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p . injection of 5-FC when compared with tumors treated with the control vector . Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection . These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.

Chromosome Res, 2000, 8(8), 727 - 35
Comparative FISH mapping of the ancestral fusion point of human chromosome 2; Kasai F et al.; It is known that human chromosome 2 originated from the fusion of two ancestral primate chromosomes . This has been confirmed by chromosome banding and fluorescence in-situ hybridization (FISH) with human chromosome-2-specific DNA libraries . In this study, the order of 38 cosmid clones derived from the human chromosome region 2q12-q14 was exactly determined by high-resolution FISH in human chromosome 2 and its homologous chromosomes in chimpanzees (Pan trogrodydes, 2n=48) and cynomolgus monkeys (Macacafascicularis, 2n = 42) . This region includes the telomere-to-telomere fusion point of two ancestral ape-type chromosomes . As a result of comparative mapping, human chromosome region 2q12-q14 was found to correspond to the short arms of chimpanzee chromosomes 12 and 13 and cynomolgus monkey chromosomes 9 and 15 . It is noted that no difference was detected in the relative order of the cosmid clones between human and chimpanzee chromosomes . This suggests that two ancestral ape-type chromosomes fused tandemly at telomeres to form human chromosome 2, and the genomic organization of this region is thought to be considerably conserved . In the cynomolgus monkey, however, the order of clones in each homologue was inverted . In addition to cosmid mapping, two chromosome-2-specific yeast artificial chromosome (YAC) clones containing the fusion point were identified by FISH.

J Am Vet Med Assoc, 2001 Jan 15, 218(2), 238 - 42
Outbreaks of clinical mastitis caused by Trichosporon beigelii in dairy herds; Gonzalez RN et al.; Trichosporon beigelii is widely distributed in nature and is classically associated with white piedra, a mycosis that may involve the hair of the human body . Intramammary infections caused by T beigelii may be fatal in cows; the prevalence in affected dairy herds may be high . Affected cows may have hyperthermia, swelling of the udder, and substantially decreased milk production or agalactia . Intramammary infections caused by yeast, including T beigelii, may also be associated with high bacterial counts in bulk-tank milk.

Ann N Y Acad Sci, 2000, 922, 46 - 55
Mechanisms of resistance to camptothecins; Saleem A et al.; Camptothecins are broad-spectrum anticancer drugs that specifically target DNA topoisomerase I . Although the availability of camptothecins has had a significant impact on cancer therapeutics, de novo or acquired clinical resistance to camptothecins is common . Studies of camptothecin resistance using yeast and mammalian cell culture models suggest three general mechanisms of resistance: (1) reduced cellular accumulation of camptothecins, (2) alteration in the structure or location of topoisomerase I, and (3) alterations in the cellular response to camptothecin-DNA-ternary complex formation . The relevance of these mechanisms to clinical drug resistance is not yet known, but evaluation of these models in clinical specimens should enhance the use of camptothecins both as single agents and in combination with other anticancer drugs.

Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2328 - 35
Molecular cloning and sequence analysis of an endoinulinase gene from Penicillium sp . strain TN-88; Akimoto H et al.; A genomic DNA segment and cDNAs encoding an extracellular endoinulinase of Penicillium sp . strain TN-88 were cloned and sequenced . Southern blot analysis indicated that the endoinulinase gene (inuC) was present as a single copy in the genome . An open reading frame, consisting of 1,545 bp, was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein . The mature protein contained three Cys residues and ten potential N-linked glycosylation sites . Three distinct transcriptional start points were observed at positions -242 (A), -215 (A), and -75 (C) from the start codon . The 5'-noncoding region had a putative TATA box at position -120 (TATATATA) and two contiguous CAAT sequences at -159 to -151 . The deduced amino acid sequence showed 72 and 85% identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively . A neighbor-joining tree showed that fungal endoinulinases form a distinct cluster from other members of the beta-fructofuranosidase superfamily and that they are more closely related to bacterial levanases than to a fungal fructosyltransferase, yeast invertases, or a yeast exoinulinase.

Pharmacogenetics, 2000 Dec, 10(9), 799 - 807
Compound heterozygosity for missense mutations in the flavin-containing monooxygenase 3 (FM03) gene in patients with fish-odour syndrome; Dolphin CT et al.; Fish-odour syndrome is a highly unpleasant disorder of hepatic trimethylamine (TMA) metabolism characterized by a body odour reminiscent of rotting fish, due to excessive excretion of the malodorous free amine . Although fish-odour syndrome may exhibit as sequelae with other conditions (e.g . liver dysfunction), many patients exhibit an inherited, more persistent form of the disease . Ordinarily, dietary-derived TMA is oxidized to the nonodorous N-oxide by hepatic flavin-containing monooxygenase 3 (FMO3) . Our previous demonstration that a mutation, P153L (C to T), in the FMO3 gene segregated with the disorder and inactivated the enzyme confirmed that defects in FMO3 underlie the inherited form of fish-odour syndrome . We have investigated the genetic basis of the disorder in two further affected pedigrees and report that the three propositi are all compound heterozygotes for causative mutations of FMO3 . Two of these individuals possess the P153L (C to T) mutation and a novel mutation, N61S (A to G) . The third is heterozygous for novel, M4341 (G to A), and previously reported, R492W (C to T), mutations . Functional characterization of the S61, 1434 and W492 variants, via baculovirus-mediated expression in insect cells, confirmed that all three mutations either abolished, or severely attenuated, the capacity of the enzyme to catalyse TMA N-oxidation . Although 1434 and W492 were also incapable of catalysing the S-oxidation of methimazole, S61 was fully active with this sulphur-containing substrate . Since an asparagine is conserved at the equivalent position to N61 of FMO3 in mammalian, yeast and Caenorhabditis elegans FMOs, the characterization of the naturally occurring N61S (A to G) mutation may have identified this asparagine as playing a critical role specifically in FMO-catalysed N-oxidation.

Rev Neurol, 2000 Dec 1-15, 31(11), 1054 - 65
{Aging and neurodegeneration: molecular and cellular bases}; Peinado MA et al.; OBJECTIVE: A review about the possible cellular and molecular mechanisms of aging and related neurodegenerative diseases . DEVELOPMENT: The mechanisms involved in neuronal decrease, connectivity losses and glial reactivity, detected both in neurodegenerative (Alzheimer's disease) and physiological aging, are analyzed from the morphological and histological point of view to provide the morphofunctional base of the cognitive and intellectual alterations characterizing the senescence process . Taken together, these data are correlated to the possible genetical aspects implied in this process, reviewing the most relevant results on senescence and cellular death obtained from yeast, fruit fly and nematodes; besides this, a brief review of the molecular biology of gerontogenes was carried out, and the possible mechanisms inducing aging and neurodegenerative processes are analyzed according to the state-of-the-art related theories . Finally, cellular, biochemical and genetical data are correlated in the signal transduction way implied in the increase of the intracellular calcium level as the starting point of cell death . CONCLUSIONS: The main process implied in the neuronal cell death responsible for aging and the related neurodegenerative diseases are started by different agents such as the lacking of neurotrophic factors, hypoxia, hypoglycemia, excitotoxicity, and oxygen and nitrogen free radicals.

Wei Sheng Wu Xue Bao, 1997 Oct, 37(5), 374 - 7
{Studies on arachidonic acid production by Mortierella}; Bao S et al.; The effects of the incubation temperature, initial pH of the medium, carbon source and nitrogen source on the production of arachidonic acid by Mortierella sp . M10 were studied . Thought orthogonal experiments, the optimum culture medium was obtained (g/L): glucose, 100; yeast extract, 10; KNO3, 4.0; KH2PO4, 2.0; CaCl2.2H2O, 0.1; MgSO4.7H2O, 0.5; FeCl3.6H2O, 0.015; ZnSO4.7H2O, 0.0075; CuSO4.5H2O, 0.0005 . Under the optimum culture conditions, the dry cell weight and arachidonic acid was 33.51 g/L and 0.827 g/L, respectively . The flask culture process was analysed.

Fish Shellfish Immunol, 2000 Nov, 10(8), 677 - 93
Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation; Cima F et al.; Tapes philippinarum is a bivalve mollusc of the Pacific Ocean, successfully imported for human consumption into the northern Adriatic Sea (Europe) . For better knowledge of its considerable adaptive ability in comparison with similar autochthonous species, a morpho-functional characterisation of its haemocytes was carried out with the establishment of short-term cell cultures (60 min at 25 degrees C) . Various methods of cytochemical staining identified four cell types in the haemolymph: granulocytes (48.05% +/- 1.43), hyalinocytes (32.18% +/- 0.99), haemoblasts (18.97% +/- 0.63) and serous cells (0.8% +/- 0.19) . The granulocytes, possessing cytoplasmic granules with differing dye affinity, included basophils, neutrophils and acidophils . Such granules stained vitally with Neutral Red, and correspond to lysosomes . Hydrolytic and oxidative enzymes were mainly detectable after stimulation in the presence of yeast cells . Both granulocytes and hyalinocytes were positive for alkaline phosphatase, non-specific esterase, peroxidase, and cytochrome C oxidase, whereas only granulocytes were positive for beta-glucuronidase, acid esterase, and arylsulphatase . Both cell types were competent phagocytes towards yeast and plasma had an opsonising effect . Moreover, the respiratory burst accompanied phagocytosis with superoxide anion production, recognisable through cytoplasmic deposits of formazan after treatment with nitro blue tetrazolium . Haemoblasts were small undifferentiated cells which, due to their morphology and positivity to the anti-CD34 antibody, show the typical features of stem cells . Serous cells, probably arising from Keber's gland and belonging to another differentiation pathway, contained non-sulphate acid mucopolysaccharides and play an important role in early defence mechanisms, taking part in the formation of clots.

Phytomedicine, 2000 Jun, 7(3), 231 - 4
Evaluation of anti-pyretic potential of Jussiaea suffruticosa L . extract in rats; Murugesan T et al.; A study was carried out to evaluate the anti-pyretic potential of the methanol extract of the aerial part of Jussiaea suffruticosa Linn . (MEJS) on normal body temperature and yeast-induced pyrexia in albino rats . Yeast suspension (10 ml/kg body wt.) increased rectal temperature after 19 hours of subcutaneous injection . The MEJS, at doses of 100, 200 and 300 mg/kg body wt . p.o., showed significant reduction in normal body temperature and yeast-provoked elevated temperature in a dose-dependent manner . The effect also extended up to 5 hours after the drug administration . The anti-pyretic effect of MEJS was comparable to that of paracetamol (150 mg/kg body wt, p.o.), a standard anti-pyretic agent.

Arch Biochem Biophys, 2000 Nov 15, 383(2), 225 - 32
Expression and catalytic activity of mouse leukotriene B4 omega-hydroxylase, CYP4F14; Kikuta Y et al.; We have isolated a cDNA for a mouse leukotriene B4 omega-hydroxylase, CYP4F14 . The cDNA encoded a protein with 524 amino acids, whose sequence similarity is 95% that of rat CYP4F1 . The microsomes from yeast cells transfected with CYP4F14 expression vector showed 0.1 nmol P450/mg protein and catalyzed omega-hydroxylations of leukotriene B4, 6-trans-leukotriene B4, lipoxin A4, prostaglandin A1, and several hydroxyeicosatetraeonic acids (HETEs), with 8-HETE being the most active substrate . In contrast, no activity was detected toward lipoxin B4, laurate, and arachidonate . The mRNA for CYP4F14 had three different 5' untranslated sequences . Analysis of the CYP4F14 gene showed that two exon I sequences with different transcription start sites are located in the gene, and two splicing signals on the 3' end of intron I are alternatively used . The mRNA for this P450 was detected only in the liver by Northern blot analysis, whereas a small amount of the mRNA was detected in the brain using RT-PCR . Administration of clofibrate had no effect on microsomal 6-trans-leukotriene B4 omega-hydroxylase activity, but resulted in a marked reduction in the content of mRNA for this P450 in the liver . These findings indicate that CYP4F14 is very similar to CYP4F1 except for its expression in the brain and 5' untranslated sequences.

Biochem Biophys Res Commun, 2000 Nov 11, 278(1), 192 - 6
Isolation and characterization of the gene encoding glyceraldehyde-3-phosphate dehydrogenase; Jeong MJ et al.; A 1.2-kb full-length cDNA sequence of a glyceraldehyde-3-phosphate dehydrogenase (GPD) gene was isolated from the mushroom, Pleurotus sajor-caju . The full-length cDNA of the GPD gene consists of 1248 nucleotides, predicted to encode a 36-kDa polypeptide consisting of 335 amino acid residues . Sequence analysis revealed that the GPD gene has more than 72-78% amino acid sequence homology with those of other Basidiomycetes . Expression of the GPD gene increased when P . sajor-caju was treated with various abiotic stresses, such as salt, cold, heat, and drought . There was an eightfold induction by drought treatment . Salt and cold stress induced four- and twofold induction of GPD gene expression, respectively . There was also a fivefold induction by heat stress . The GPD gene exhibits different expression patterns under different stress conditions . It reached its maximum expression level within two hours under cold or heat treatment . The mRNA levels of this gene increased proportionally to increasing treatment time under salt or dry conditions . Because the expression of GPD was significantly increased, we tested whether GPD could confer abiotic stress resistance when it was introduced into yeast cells . For this, a transgenic yeast harboring P . sajor-caju GPD was generated under the control of a constitutively expressed GAL promoter . The results from biofunctional analyses with GPD yeast transformants showed that GPD yeast transformants had significantly higher resistance to cold, salt, heat, and drought stresses.

Traffic, 2000 Sep, 1(9), 724 - 37
Procathepsin L self-association as a mechanism for selective secretion; Yeyeodu S et al.; The lysosomal cysteine pro-protease procathepsin L was enriched in dense vesicles detectable when microsomes prepared from wild-type or transformed mouse fibroblasts were resolved on sucrose gradients . These dense vesicles did not comigrate with proteins characteristic of the endoplasmic reticulum, Golgi, endosomes or lysosomes . When gradient fraction vesicles were lysed at acidic pH in the presence of excess mannose 6-phosphate to prevent binding to mannose phosphate receptors, the majority of the procathepsin L was associated with the membrane, not the soluble, fraction . Immunogold labeling of procathepsin L in thin sections of cells or gradient fractions, using antibodies directed against the propeptide to avoid detection of the mature enzyme in dense lysosomes, revealed that the proenzyme was concentrated in dense cores localized in small vesicles near the plasma membrane and in multivesicular bodies . Consistent with the density of the gradient fraction and the electron density of the cores, yeast two-hybrid assays indicated the proenzyme could bind itself but could not interact with the aspartic proprotease procathepsin D . The data suggest that in mouse fibroblasts procathepsin L may self-associate into aggregates, initiating the formation of dense vesicles that could mediate the selective secretion of procathepsin L independent of mannose phosphate receptors.

Traffic, 2000 Nov, 1(11), 904 - 16
Overexpression of a novel sorting nexin, SNX15, affects endosome morphology and protein trafficking; Barr VA et al.; Sorting nexin (SNX) 15 is a novel member of the SNX family of proteins . Although the functions of most SNXs have not yet been determined, several family members (e.g., SNX1, SNX2, SNX3, and SNX8) are orthologs of yeast proteins involved in protein trafficking . Overexpression of myc-tagged SNX15 in COS-7 cells altered the morphology of several endosomal compartments . In transient transfection experiments, myc-SNX15 was first seen in small punctate spots and small ring structures . Later, myc-SNX15 was found in larger rings . Finally, myc-SNX15 was observed in large, amorphous membrane-limited structures . These structures contained proteins from lysosomes, late endosomes, early endosomes, and the trans-Golgi network . However, the morphology of the endoplasmic reticulum and Golgi was not affected by overexpression of myc-SNX15 . In myc-SNX15-overexpressing cells, the endocytosis of transferrin was severely inhibited and endocytosis of tac-trans-Golgi network (TGN) 38 and tac-furin was slowed . In addition, the recycling of internalized tac-TGN38 and tac-furin was also inhibited . Both the morphological and biochemical data indicate that SNX15 plays a crucial role in trafficking through the endocytic pathway . This is the first demonstration that a mammalian SNX protein is involved in protein trafficking.

Plant J, 2001 Feb, 25(3), 247 - 59
A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants; Kim JC et al.; Cold stress on plants induces changes in the transcription of cold response genes . A cDNA clone encoding C2H2-type zinc finger protein, SCOF-1, was isolated from soybean . The transcription of SCOF-1 is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity . Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants . SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters . However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro . SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system . The SGBF-1 transactivated the beta-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts . Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1 . These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein-protein interaction, which in turn enhances cold tolerance of plants.

Cell Microbiol, 2000 Dec, 2(6), 537 - 47
Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids; Kugler S et al.; Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase . Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures . In this study, the green fluorescent protein (GFP) was established as a reporter in H . capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages . One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells . By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated . Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome . Broth cultures of Histoplasma yeasts carrying a CBP-GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages . Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.

J Immunol, 2001 Mar 1, 166(5), 3158 - 66
The identification and characterization of a ligand for bovine CD5; Haas KM et al.; CD5, a type I glycoprotein expressed by T cells and a subset of B cells, is thought to play a significant role in modulating Ag receptor signaling . Previously, our laboratory has shown that bovine B cells are induced to express this key regulatory molecule upon Ag receptor cross-linking . To date, a ligand has not been described for bovine CD5 . Given the importance ligand binding presumably plays in the functioning of CD5 on this B cell subset and on T cells, we sought to characterize the ligand for this protein using a bovine CD5-human IgG1 (CD5Ig) fusion protein produced by both mammalian and yeast cells . As determined by CD5Ig binding, expression of this ligand is negative to low on freshly isolated lymphocytes, with low-density expression being limited to activated B cells . Activation with LPS, PMA, and calcium ionophore, or ligation of CD40 alone or in combination with anti-IgM, resulted in B cell-specific expression of this ligand . Interestingly, activation through B cell Ag receptor cross-linking alone, although able to induce CD5 expression, did not result in expression of CD5 ligand (CD5L) . In addition, we demonstrate a functional role for CD5L as a costimulatory molecule that augments CD40L-stimulated B cell proliferation . Finally, immunoprecipitation with CD5Ig suggests that the ligand characterized in this study has a molecular mass of approximately 200 kDa . The data reported herein, as well as future studies aimed at further characterizing this newly identified bovine CD5L, will undoubtedly aid in understanding the role that the CD5-CD5L interaction plays in immune responses.

Genome Biol . 2000;1(5):REVIEWS3002 . Epub 2000 Nov 10.
The F-box protein family; Kipreos ET et al.; SUMMARY: The F-box is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction . F-box proteins were first characterized as components of SCF ubiquitin-ligase complexes (named after their main components, Skp I, Cullin, and an F-box protein), in which they bind substrates for ubiquitin-mediated proteolysis . The F-box motif links the F-box protein to other components of the SCF complex by binding the core SCF component Skp I . F-box proteins have more recently been discovered to function in non-SCF protein complexes in a variety of cellular functions . There are 11 F-box proteins in budding yeast, 326 predicted in Caenorhabditis elegans, 22 in Drosophila, and at least 38 in humans . F-box proteins often include additional carboxy-terminal motifs capable of protein-protein interaction; the most common secondary motifs in yeast and human F-box proteins are WD repeats and leucine-rich repeats, both of which have been found to bind phosphorylated substrates to the SCF complex . The majority of F-box proteins have other associated motifs, and the functions of most of these proteins have not yet been defined.

Neuroscience, 2001, 102(4), 767 - 77
Calcium/calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain; Chang BH et al.; A second isoform of Ca2+/calmodulin-dependent-kinase II inhibitor protein (CaM-KIIN) has been identified using the yeast two-hybrid screen . The 1.8kb message encodes a 78 residue CaM-KIINalpha that is 65% identical in its putative open-reading frame and 95% identical in its inhibitory domain to the previously characterized CaM-KIINbeta . CaM-KIINalpha exhibits inhibitory properties towards recombinant mouse CaM-kinase IIalpha indistinguishable from CaM-KIINbeta . The 27 amino acid inhibitory peptide (CaM-KIINtide) derived from CaM-KIIN has the ability to inhibit brain CaM-kinase II activity from multiple organisms including rat, Drosophila and goldfish . Northern analysis of various rat tissues indicates that CaM-KIINalpha is specific to brain whereas CaM-KIINbeta message is also present in testis . In situ hybridization shows a general distribution of both isoforms in rat brain with stronger localization of CaM-KIINbeta in cerebellum and hindbrain and CaM-KIINalpha in frontal cortex, hippocampus and inferior colliculus . An antibody that recognizes both isoforms shows a distribution of CaM-KIIN in rat brain that correlates with immunoreactivity of CaM-kinase II . In cultured mature hippocampal neurons, CaM-KIIN is present in cell bodies and dendrites but, unlike CaM-kinase II, does not display punctate staining at synapses . These results suggest a localized function for CaM-KIIN in inhibiting specialized pools of CaM-kinase II.

J Lipid Res, 2001 Feb, 42(2), 159 - 69
Fine-mapping, mutation analyses, and structural mapping of cerebrotendinous xanthomatosis in U.S . pedigrees; Lee MH et al.; Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of bile acid biosynthesis . Clinically, CTX patients present with tendon xanthomas, juvenile cataracts, and progressive neurological dysfunction and can be diagnosed by the detection of elevated plasma cholestanol levels . CTX is caused by mutations affecting the sterol 27-hydroxylase gene (CYP27 ) . CTX has been identified in a number of populations, but seems to have a higher prevalence in the Japanese, Sephardic Jewish, and Italian populations . We have assembled 12 previously unreported pedigrees from the United States . The CYP27 locus had been previously mapped to chromosome 2q33-qter . We performed linkage analyses and found no evidence of genetic heterogeneity . All CTX patients showed segregation with the CYP27 locus, and haplotype analysis and recombinant events allowed us to precisely map CYP27 to chromosome 2q35, between markers D2S1371 and D2S424 . Twenty-three mutations were identified from 13 probands analyzed thus far; 11 were compound heterozygotes and 2 had homozygous mutations . Of these, five are novel mutations {Trp100Stop, Pro408Ser, Gln428Stop, a 10-base pair (bp) deletion in exon 1, and a 2-bp deletion in exon 6 of the CYP27 gene} . Three-dimensional structural modeling of sterol 27-hydroxylase showed that, while the majority of the missense mutations disrupt the heme-binding and adrenodoxin-binding domains critical for enzyme activity, two missense mutations (Arg94Trp/Gln and Lys226Arg) are clearly located outside these sites and may identify a potential substrate-binding or other protein contact site.

Hum Mol Genet, 2001 Mar 1, 10(5), 519 - 28
Extra-chromosomal telomeric DNA in cells from Atm(-/-) mice and patients with ataxia-telangiectasia; Hande MP et al.; Ataxia-telangiectasia (AT) is an autosomally recessive human genetic disease with pleiotropic defects such as neurological degeneration, immunodeficiency, chromosomal instability, cancer susceptibility and premature aging . Cells derived from AT patients and ataxia-telangiectasia mutated (ATM)-deficient mice show slow growth in culture and premature senescence . ATM, which belongs to the PI3 kinase family along with DNA-PK, plays a major role in signaling the p53 response to DNA strand breaks . Telomere maintenance is perturbed in yeast strains lacking genes homologous to ATM and cells from patients with AT have short telomeres . We examined the length of individual telomeres in cells from ATM(-/-) mice by fluorescence in situ hybridization . Telomeres were extensively shortened in multiple tissues of ATM(-/-) mice . More than the expected number of telomere signals was observed in interphase nuclei of ATM(-/-) mouse fibroblasts . Signals corresponding to 5-25 kb of telomeric DNA that were not associated with chromosomes were also noticed in ATM(-/-) metaphase spreads . Extrachromosomal telomeric DNA was also detected in fibroblasts from AT patients and may represent fragmented telomeres or by-products of defective replication of telomeric DNA . These results suggest a role of ATM in telomere maintenance and replication, which may contribute to the poor growth of ATM(-/-) cells and increased tumor incidence in both AT patients and ATM(-/-) mice.

Glycobiology, 2001 Jan, 11(1), 37 - 45
A new beta-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans; Takahashi S et al.; Recent studies have shown that O-mannosyl glycans are present in several mammalian glycoproteins . Although knowledge on the functional roles of these glycans is accumulating, their biosynthetic pathways are poorly understood . Here we report the identification and initial characterization of a novel enzyme capable of forming GlcNAc beta 1-2Man linkage, namely UDP-N-acetylglucosamine: O-linked mannose beta-1,2-N-acetylglucosaminyltransferase in the microsome fraction of newborn rat brains . The enzyme transfers GlcNAc to beta-linked mannose residues, and the formed linkage was confirmed to be beta 1-2 on the basis of diplococcal beta-N-acetylhexosaminidase susceptibility and by high-pH anion-exchange chromatography . Its activity is linearly dependent on time, protein concentration, and substrate concentration and is enhanced in the presence of manganese ion . Its activity is not due to UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,2-D-acetylglucosaminyltransferase II (GnT-II), which acts on the early steps of N-glycan biosynthesis, because GnT-I or GnT-II expressed in yeast cells did not show any GlcNAc transfer activity against a synthetic mannosyl peptide . Taken together, the results suggest that the GlcNAc transferase activity described here is relevant to the O-mannosyl glycan pathway in mammals.

J Cell Sci, 2001 Mar, 114(Pt 5), 953 - 63
Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase; Campbell MS et al.; Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset . Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells . HsMad1 is found localized at kinetochores in mitosis . The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase . In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled . We find HsMad1 associated with HsMad2 . Association with p55CDC, a protein previously shown to bind HsMad2, was not detected . Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase . This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions . HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins . The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.

J Cell Sci, 2001 Mar, 114(Pt 5), 867 - 74
Control of mitochondrial morphology by a human mitofusin; Santel A et al.; Although changes in mitochondrial size and arrangement accompany both cellular differentiation and human disease, the mechanisms that mediate mitochondrial fusion, fission and morphogenesis in mammalian cells are not understood . We have identified two human genes encoding potential mediators of mitochondrial fusion . The mitofusins (Mfn1 and Mfn2) are homologs of the Drosophila protein fuzzy onion (Fzo) that associate with mitochondria and alter mitochondrial morphology when expressed by transient transfection in tissue culture cells . An internal region including a predicted bipartite transmembrane domain (TM) is sufficient to target Mfn2 to mitochondria and requires hydrophobic residues within the TM . Co-expression of Mfn2 with a dominant interfering mutant dynamin-related protein (Drp1(K38A)) proposed to block mitochondrial fission resulted in long mitochondrial filaments and networks . Formation of mitochondrial filaments and networks required a wild-type Mfn2 GTPase domain, suggesting that the Mfn2 GTPase regulates or mediates mitochondrial fusion and that mitofusins and dynamin related GTPases play opposing roles in mitochondrial fusion and fission in mammals, as in yeast.

J Cell Sci, 2001 Mar, 114(Pt 5), 839 - 44
Novel roles for mammalian septins: from vesicle trafficking to oncogenesis; Kartmann B et al.; In recent years a convergence of various aspects of cell biology has become apparent, and yet investigators are only beginning to grasp the underlying unifying mechanisms . Among the proteins that participate in diverse aspects of cell biology are the septins . These are a group of novel GTPase proteins that are broadly distributed in many eukaryotes except plants . Although septins were originally identified as a protein family involved in cytokinesis in yeast, recent advances in the field have now ascribed additional functions to these proteins . In particular, the number of known mammalian septin family members has increased dramatically as more data has become available through genome analyses . We suggest a classification for the mammalian septins based on the sequence homologies in their highly divergent N- and C-termini . Recent work suggests novel functions for septins in vesicle trafficking, oncogenesis and compartmentalization of the plasma membrane . Given the ability of the septins to bind GTP and phosphatidylinositol 4,5-bisphosphate in a mutually exclusive manner, these proteins might be crucial elements for the spatial and/or temporal control of diverse cellular functions . As the functions of the septins become unraveled, our understanding of seemingly different cellular processes may move a step further.

BMC Genet . 2001;2(1):3 . Epub 2001 Feb 06.
Analysis of human sarcospan as a candidate gene for CFEOM1; O'Brien KF et al.; BACKGROUND: Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is an autosomal dominant eye movement disorder linked to the pericentromere of chromosome 12 (12p11.2 - q12) . Sarcospan is a member of the dystrophin associated protein complex in skeletal and extraocular muscle and maps to human chromosome 12p11.2 . Mutations in the genes encoding each of the other components of the skeletal muscle sarcospan-sarcoglycan complex (alpha - delta sarcoglycan) have been shown to cause limb girdle muscular dystrophy (LGMD2C-F) . To determine whether mutations in the sarcospan gene are responsible for CFEOM1 we: (1) attempted to map sarcospan to the CFEOM1 critical region; (2) developed a genomic primer set to directly sequence the sarcospan gene in CFEOM1 patients; and (3) generated an anti-sarcospan antibody to examine extraocular muscle biopsies from CFEOM1 patients . RESULTS: When tested by polymerase chain reaction, sarcospan sequence was not detected on yeast or bacterial artificial chromosomes from the CFEOM1 critical region . Sequencing of the sarcospan gene in CFEOM1 patients from 6 families revealed no mutations . Immunohistochemical studies of CFEOM1 extraocular muscles showed normal levels of sarcospan at the membrane . Finally, sarcospan was electronically mapped to bacterial artificial chromosomes that are considered to be outside of the CFEOM1 critical region . CONCLUSIONS: In this report we evaluate sarcospan as a candidate gene for CFEOM1 . We have found that it is highly unlikely that sarcospan is involved in the pathogenesis of this disease . As of yet no sarcospan gene mutations have been found to cause muscular abnormalities.

J Steroid Biochem Mol Biol, 2000 Dec 1, 75(1), 21 - 31
Identification and characterization of estrogen receptor variants in prostate cancer cell lines; Ye Q et al.; A sensitive semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was performed to evaluate estrogen receptor-alpha (ER-alpha) mRNA expression in prostate cancer cell lines . We demonstrated the presence of wild-type ER-alpha (wt ER-alpha) and five ER-alpha variants, designated ER-alphaA, B, C, D, and E . Unlike ER-alphaA and D, ER-alphaB, C, and E were not previously reported in normal or cancerous mammalian cells . DNA sequencing analysis of these ER-alpha variants revealed the genetic changes to be either in-frame or out-of-frame deletions . The expression of each ER-alpha variant differs significantly depending on the androgen responsiveness, tumorigenic and metastatic potentials of each prostate cancer cell line . The potential functional significance of ER-alpha variants was assessed in yeast two-hybrid and ERE promoter-reporter mammalian transcription assay systems . The results of these studies indicated that none of the ER-alpha variants can form homo- or heterodimers either with wt ER-alpha or among themselves in vivo, and that these ER-alpha variants have no demonstrable transcriptional or dominant-negative activity, as assessed in vitro.

Steroids, 2001 Mar-May, 66(3-5), 293 - 300
The role of vitamin D in prostate cancer; Zhao XY et al.; Prostate cancer is the second leading cause of cancer deaths in men in the United States . Developing new treatment strategies is critical to improving the health of men . This article will be a general review of the field with a focus on research from our laboratory . Our research has focused on four areas in which we have pursued the possible use of 1alpha,25(OH)(2)D(3) and its analogs to treat prostate cancer: 1) The ability of 1alpha,25(OH)(2)D(3) to up-regulate androgen receptors in LNCaP human prostate cancer cells . The implications of this finding on 1alpha,25(OH)(2)D(3)'s ability to inhibit cell growth in vivo are unclear at present.2) The reasons for an inability of 1alpha,25(OH)(2)D(3) to inhibit DU 145 prostate cancer cell growth were explored . We found that combination of an imidazole drug, Liarozole, with 1alpha,25(OH)(2)D(3) was capable of inhibiting DU 145 cell growth.3) A number of low-calcemic vitamin D analogs exhibit potent anti-proliferative activity on prostate cancer cells . We have developed a novel approach using the yeast two-hybrid system to screen for potent analogs.4) The results of a clinical trial of 1alpha,25(OH)(2)D(3) treatment of patients with early recurrent prostate cancer . We provide preliminary evidence that 1alpha,25(OH)(2)D(3) may be effective in slowing the rate of PSA rise in selected cases of prostate cancer.In conclusion, we believe that 1alpha,25(OH)(2)D(3) has a role in the treatment and/or prevention strategies being developed for prostate cancer . However, to increase antiproliferative potency without increasing side-effects, the use of less calcemic analogs appears to be the most reasonable approach.

Gene, 2001 Jan 10, 262(1-2), 267 - 73
Characterization of promoter region and genomic structure of the murine and human genes encoding Src like adapter protein; Kratchmarova I et al.; Src-like adapter protein (SLAP) was identified as a signaling molecule in a yeast two-hybrid system using the cytoplasmic domain of EphA2, a receptor protein tyrosine kinase (Pandey et al., 1995 . Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase . J . Biol . Chem . 270, 19201-19204) . It is very similar to members of the Src family of cytoplasmic tyrosine kinases in that it contains very homologous SH3 and SH2 domains (Abram and Courtneidge, 2000 . Src family tyrosine kinases and growth factor signaling . Exp . Cell . Res . 254, 1-13.) . However, instead of a kinase domain at the C-terminus, it contains a unique C-terminal region . In order to exclude the possibility that an alternative form exists, we have isolated genomic clones containing the murine Slap gene as well as the human SLA gene . The coding regions of murine Slap and human SLA genes contain seven exons and six introns . Absence of any kinase domain in the genomic region confirm its designation as an adapter protein . Additionally, we have cloned and sequenced approximately 2.6 kb of the region 5' to the initiator methionine of the murine Slap gene . When subcloned upstream of a luciferase gene, this fragment increased the transcriptional activity about 6-fold in a human Jurkat T cell line and approximately 52-fold in a murine T cell line indicating that this region contains promoter elements that dictate SLAP expression . We have also cloned the promoter region of the human SLA gene . Since SLAP is transcriptionally regulated by retinoic acid and by activation of B cells, the cloning of its promoter region will permit a detailed analysis of the elements required for its transcriptional regulation.

Gene, 2001 Jan 10, 262(1-2), 221 - 30
Receptor protein-tyrosine phosphatases: origin of domains (catalytic domain, Ig-related domain, fibronectin type III module) based on the sequence of the sponge Geodia cydonium; Muller CI et al.; Reversible tyrosine phosphorylation of proteins is one of the major regulatory physiological events in response to cell-cell- and cell-matrix contact in Metazoa . Previously it was documented that the tyrosine phosphorylating enzymes, the tyrosine kinases (TKs), are autapomorphic characters of Metazoa, including sponges . In this paper the tyrosine dephosphorylating enzymes, the protein-tyrosine phosphatases (PTPs), are studied which can be grouped into two subfamilies, the soluble PTPs and the receptor PTPs (RPTPs) . PTPs are characterized by one PTPase domain which interestingly comprises sequence similarity to yeast PTPs . In contrast to the PTPs, the RPTPs - which have been found only in Metazoa - are provided with two PTPase domains . To study the evolution of the RPTPs the full-length size RPTP was cloned from the marine demosponge Geodia cydonium, the phylogenetic oldest metazoan taxon . The 3253 bp long sequence has a putative open reading frame coding for a 999 aa long RPTP which is characterized by two fibronectin (type III; FN-III) domains in the extracellular portion, one intracellular immunoglobulin (Ig)-related domain, and two PTPase domains . Phylogenetic analysis revealed that the sponge FN-III domains form the basis of the metazoan FN-III domain with the common metazoan ancestor . The Ig-related, typical metazoan, module is classified to the disulphide lacking Ig members and represents the phylogenetic earliest member of this group . The beta-sheet propensity was calculated and the characteristic amino acids are present in the seven beta-sheets . The analysis of the two PTPase domains of the sponge RPTP demonstrates that the first domain is closely related to the PTPase domains present in the soluble PTPs, while the second PTPase domain is only distantly related to them . By constructing a rooted phylogenetic cladogram it became overt that the duplication of the PTPase domains must have occurred already in yeast . This interesting finding indicates that two conserved PTPase domains originated from a common ancestor in yeast while the evolutionary novelties, the FN-III domains and the Ig-related module, were added during the transition to the Metazoa . Hence, the tyrosine dephosphorylating enzyme, RPTP, is an example for a modular protein which is composed of ancient modules (PTPase domain{s}) and two metazoan novelties, while the tyrosine phosphorylating enzymes, the TKs, evolved only in Metazoa.

Gene, 2001 Jan 10, 262(1-2), 15 - 22
The black-pearl gene of Drosophila defines a novel conserved protein family and is required for larval growth and survival; Becker S et al.; Using a transposon insertion line of the Drosophila Genome Project we have cloned the black-pearl gene (blp), analyzed cDNA clones, generated various mutants, and characterized their phenotypes . The blp gene codes for a protein of 15.7 kDa calculated molecular weight that has been conserved from yeast to plants and mammals with high homology . A domain of these new proteins shows distant similarity to DnaJ domains indicating a functionally relevant interaction with other proteins . The P element insertion in line P1539 lies within the 5' untranslated leader of the black-pearl gene . Flies homozygous for this insertion are semi-lethal, escapers produce very few offspring and show melanotic inclusions in the hemocoel ('black pearls') similar to various melanotic 'tumor' mutants . Two small deletions confined to the blp gene and two EMS-induced mutations are homozygous lethal . These null mutants appear normal up to a prolonged first instar larval stage but fail to grow and die . Thus in Drosophila the blp gene is specifically required for larval growth . The evolutionary conservation in both unicellular and multicellular organisms suggests for the new protein family described here a fundamental role in cell growth.

EMBO J, 2001 Feb 15, 20(4), 880 - 90
The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts; Wang W et al.; The yeast UPF1, UPF2 and UPF3 genes encode trans-acting factors of the nonsense-mediated mRNA decay pathway . In addition, the upf1Delta strain demonstrates a nonsense suppression phenotype and Upf1p has been shown to interact with the release factors eRF1 and eRF3 . In this report, we show that both upf2Delta and upf3Delta strains demonstrate a nonsense suppression phenotype independent of their effect on mRNA turnover . We also demonstrate that Upf2p and Upf3p interact with eRF3, and that their ability to bind eRF3 correlates with their ability to complement the nonsense suppression phenotype . In vitro experiments demonstrate that Upf2p, Upf3p and eRF1 compete with each other for interacting with eRF3 . Con versely, Upf1p binds to a different region of eRF3 and can form a complex with these factors . These results suggest a sequential surveillance complex assembly pathway, which occurs during the premature translation termination process . We propose that the observed nonsense suppression phenotype in the upfDelta strains can be attributed to a defect in the surveillance complex assembly.

EMBO J, 2001 Feb 15, 20(4), 792 - 801
Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis; Zur A et al.; We have studied the ubiquitination and degradation patterns of the human securin/PTTG protein . We show that, in contrast to budding yeast pds1, securin degradation is catalyzed by both fzy (fizzy/cdc20) and fzr (fizzy-related/cdh1/hct1) . Both fzy and fzr also induce the APC/C to ubiquitinate securin in vitro . Securin degradation is mediated by an RXXL destruction box and a KEN box, and is inhibited only when both sequences are mutated . Interestingly, the non-degradable securin mutant is also partially ubiquitinated by fzy and fzr in vitro . Expressing the non-degradable securin mutant in cells frequently resulted in incomplete chromatid separation and gave rise to daughter cells connected by a thin chromatin fiber, presumably of chromosomes that failed to split completely . Strikingly, the mutant securin did not prevent the majority of sister chromatids from separating completely, nor did it prevent mitotic cyclin degradation and cytokinesis . This phenotype, reminiscent of the fission yeast cut (cells untimely torn) phenotype, is reported here for the first time in mammals.

Mol Cell Neurosci, 2001 Feb, 17(2), 317 - 28
Association of GABA(B) receptors and members of the 14-3-3 family of signaling proteins; Couve A et al.; Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently . Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression . We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1 . We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta . We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1 . Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3 . The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer .

AIDS Res Hum Retroviruses, 2001 Jan 20, 17(2), 105 - 11
HIV type 2 Vpx interaction with Gag and incorporation into virus-like particles; Jin L et al.; The domain of HIV-2 Vpx previously shown to be important for virion incorporation has been mapped to residues 73--89 . Mutational analysis of this domain was employed to further define the sequences important for incorporation into virus-like particles, using a vaccinia virus expression system . Deletion of residues 73--89 did not abrogate Vpx packaging, but substitution with alanines markedly reduced incorporation into virus-like particles . Moreover, alanine substitution also disrupted Vpx interaction with Gag, as demonstrated with glutathione S-transferase fusion proteins and the yeast two-hybrid system.

Bull Exp Biol Med, 2000 Oct, 130(10), 948 - 50
Chitotriosidase as a marker of macrophage stimulation; Korolenko TA et al.; Serum chitotriosidase activity was determined in different conditions accompanied by macrophage stimulation . Stimulation of macrophages with zymosan, yeast polysaccharide carboxymethylglucan (fraction II), and lysosomotropic preparation Triton WR-1339 1.5-2.0-fold increased enzyme activity . Chitotriosidase activity in intact Wistar rats was similar to that in humans, while in CBA and A/Sn mice this parameter was 5-fold higher . Sharp increase in chitotriosidase activity in the serum from patients with type I Gaucher's disease was probably related to intense secretion of the enzyme by macrophages . Under experimental conditions, stimulation of rat and mouse macrophages (mainly liver cells) caused no increase in chitotriosidase activity typical of patients with Gaucher's disease.

J Acquir Immune Defic Syndr, 2001 Jan 1, 26(1), 21 - 7
Phase 1 trial of the topical microbicide BufferGel: safety results from four international sites; van De Wijgert J et al.; AIM: To evaluate the safety of BufferGel (ReProtect LLC, Baltimore, MD), a spermicidal microbicide that acidifies semen and maintains the protective acidity of the vagina, in a high-dose tolerance trial . METHODS: HIV/STD negative, sexually abstinent, and sexually active women in India, Thailand, Malawi, and Zimbabwe were asked to insert one applicator ( approximately 5 ml) of BufferGel vaginally twice per day for 14 days . Sexually active women agreed to have sex (while using BufferGel and nonlubricated condoms) at least twice per week . RESULTS: In total, 98 women (30 sexually abstinent and 68 sexually active) were enrolled . Overall compliance with product use was 93% . Epithelial abnormalities detected by pelvic examination or colposcopy were uncommon (8 cases in 271 examinations) . Irritation was reported by approximately one quarter of the women (0.58 events per woman-week) but was generally mild and of short duration . The prevalence of bacterial vaginosis (BV) fell significantly, from 30% at enrollment to 6% at one week, and 7% at two weeks of BufferGel use . Thirty-two women acquired microscopically detectable yeast during BufferGel exposure, but only 3 developed symptomatic vaginitis . CONCLUSION: BufferGel appears to be safe and well tolerated by the cervicovaginal epithelium . Its effect on BV and yeasts merits further study.

Nat Genet, 2001 Feb, 27(2), 181 - 6
Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits; Puccio H et al.; Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence in diabetes . FRDA is caused by severely reduced levels of frataxin, a mitochondrial protein of unknown function . Yeast knockout models as well as histological and biochemical data from heart biopsies or autopsies of FRDA patients have shown that frataxin defects cause a specific iron-sulfur protein deficiency and intramitochondrial iron accumulation . We have recently shown that complete absence of frataxin in the mouse leads to early embryonic lethality, demonstrating an important role for frataxin during mouse development . Through a conditional gene-targeting approach, we have generated in parallel a striated muscle frataxin-deficient line and a neuron/cardiac muscle frataxin-deficient line, which together reproduce important progressive pathophysiological and biochemical features of the human disease: cardiac hypertrophy without skeletal muscle involvement, large sensory neuron dysfunction without alteration of the small sensory and motor neurons, and deficient activities of complexes I-III of the respiratory chain and of the aconitases . Our models demonstrate time-dependent intramitochondrial iron accumulation in a frataxin-deficient mammal, which occurs after onset of the pathology and after inactivation of the Fe-S-dependent enzymes . These mutant mice represent the first mammalian models to evaluate treatment strategies for the human disease.

Oncogene, 2000 Dec 14, 19(54), 6240 - 50
Colocalization and heteromerization between the two human oncogene POZ/zinc finger proteins, LAZ3 (BCL6) and PLZF; Dhordain P et al.; Most acute promyelocytic leukemia (APL) cases are associated with recurrent translocations between the gene of retinoic receptor alpha and that of PML (t(15;17)) or PLZF (t(11;17)) . PML localizes onto discrete intranuclear domains, the PML-nuclear bodies, and displays anti-oncogenic and pro-apoptotic properties . PLZF encodes a transcription factor belonging to the POZ/domain and Kruppel zinc finger (POK) family which interacts directly with PML . PLZF is related to another POK protein, LAZ3(BCL6), which is structurally altered, and presumably misexpressed, in many non-Hodgkin lymphoma (NHL) cases . PLZF and LAZ3 share many functional properties: both inhibit cell growth, concentrate into punctated nuclear subdomains and are sequence-specific transcriptional repressors recruiting a histone deacetylase-repressing complex . Given these similarities, we tested whether both proteins could be targeted by each other . Here, LAZ3 and PLZF are shown to colocalize onto nuclear dots . Moreover, truncated derivatives of one protein, which display a diffuse nuclear localization, are recruited onto nuclear dots by the full-length other . The colocalization and the reciprocal 'rescue' is the result of a direct interaction between LAZ3 and PLZF, as indicated by yeast two hybrid assays, in vitro immunoprecipitations, and GST pull down experiments . In contrast to LAZ3 homomerization, LAZ3/PLZF heteromerization in yeast does not solely depend on POZ/POZ contacts but rather also relies on interactions between the two zinc finger regions and 'cross' contacts between the zinc finger region and the POZ domain of each partner . Likewise, LAZ3 shows some colocalization with the PLZF partner PML upon stable overexpression of both proteins in CHO cells and interacts with PML in yeast . Finally, endogenous LAZ3 and PLZF are co-induced and partially colocalized in myeloid MDS cells . These data indicate that a physical interaction between LAZ3 and PLZF underlies their simultaneous recruitment onto multiproteic nuclear complexes, presumably involved in transcriptional silencing and whose integrity (for APL) and/or function (for APL and NHL) may be altered in oncogenesis.

Eur J Hum Genet, 2001 Jan, 9(1), 34 - 8
Linkage disequilibrium narrows locus for venous malformation with glomus cells (VMGLOM) to a single 1.48 Mbp YAC; Irrthum A et al.; Venous malformations with glomus cells are localised cutaneous lesions of vascular dysmorphogenesis . They are usually sporadic, but sometimes familial . Using five families, we mapped the locus, VMGLOM, to chromosome 1p21-p22 . In order to refine this locus, spanning 4-6 Mbp, we then studied seven additional families . They exhibited linkage to VMGLOM and the combined lod score for all 12 families was 18.41 at theta = 0.0 for marker D1S188 . We found a distinct haplotype shared by seven families, comprising seven alleles which are rare in the general population (P < 0.01) . This indicates that the haplotype is identical by descent in all seven families, and hence the locus can be refined by inferring ancestral crossovers . Using this approach, we position the causative gene between two markers on the same non-chimeric YAC of 1.48 Mbp, a feasible size for positional cloning . As there is no known gene involved in vasculogenesis and/or angiogenesis in this YAC, the identification of the causative gene is likely to reveal a novel regulator or vascular development.

Dev Neurosci, 2001, 23(1), 17 - 24
The orphan nuclear receptor NOR-1 interacts with the homeobox containing protein Six3; Ohkura N et al.; Neuron-derived orphan receptor 1 (NOR-1) is a member of the NGFI-B subfamily within the nuclear receptor superfamily . In order to identify cofactors that associate with NOR-1 in the fetal forebrain, we tested a yeast two-hybrid system with the NOR-1 cDNA fragment lacking a transactivating domain as a bait . By screening of the rat fetal brain embryonic day 17 library, a rat homologue of Six3 was identified as an associated protein . We demonstrated that NOR-1 interacted with Six3 in yeast and in vitro, and the association was required for the DNA binding and AF2 domains of NOR-1 . Regarding the other members of the family (NGFI-B and RNR-1), association with Six3 was not observed in yeast . In addition, cotransfection experiments with Six3 and NOR-1 indicated that Six3 had a negative activity against the transactivation by NOR-1 through the NBRE response element in a dose-dependent manner . The overlap in expression of NOR-1 and Six3 was mainly detected in the rat fetal forebrain on embryonic day 18 . Thereafter, the expression of both genes diminished rapidly . These results suggest that a dimer consisting of a homeobox containing protein Six3 and transcriptional factor NOR-1 might regulate gene expression during the late stage of the fetal forebrain development . This study provides, after the association of Ftz and Ftz-F1 in Drosophila, another example of a dimer formation of a homeobox protein and an orphan nuclear receptor .

Cytogenet Cell Genet, 2000, 91(1-4), 141 - 7
Cloning and characterization of the breakpoint regions of a chromosome 11;18 translocation in a patient with hamartoma of the retinal pigment epithelium; Kutsche K et al.; Mutations of various tumor suppressor genes, e.g., PTEN, TSC1, and TSC2, are known to be responsible for different inherited diseases presenting with multiple hamartomas, a benign tumor resembling neoplasia that results from faulty organ development . Combined hamartoma of the retinal pigment epithelium (RPE) and retina is a rare, congenital, focal malformation of the fundus . So far, no disease gene has been associated with this disorder . By molecular analysis of an apparently balanced and reciprocal translocation between the short arms of chromosomes 11 and 18, t(11;18)(p13;p11.31), in a patient with hamartoma of the RPE and retina, we selected PAC clones crossing the breakpoints on both derivative chromosomes 11 and 18 . For the overlapping chromosome 11 clone, two EST clusters were identified, suggesting the existence of at least two genes in the breakpoint region . We constructed a PAC contig and showed that at least three exons of a novel gene map to the breakpoint region on chromosome 18 . Based on the results of FISH analysis with the PAC clones of this contig, we suggest the occurrence of a complex rearrangement .

Cytogenet Cell Genet, 2000, 91(1-4), 67 - 71
TSPY variants in six loci on the human Y chromosome; Dechend F et al.; We have studied the structure, organization, and evolution of the human TSPY gene family by mapping three sequence variants identified through RT-PCR analysis onto genomic clones derived from two different YAC contigs . TSPY gene family members occur in at least six locations on the human Y chromosome, and each cluster contains a unique combination of variants . Our data further suggest that an 18-bp tandem duplication found in TSPY exon 1 originated from an unequal sister chromatid exchange between two tandemly arranged TSPY clusters .

J Biochem (Tokyo), 2001 Feb, 129(2), 321 - 7
Interaction between emerin and nuclear lamins; Sakaki M et al.; Emerin is an inner nuclear membrane protein that is involved in X-linked recessive Emery-Dreifuss muscular dystrophy (X-EDMD) . Although the function of this protein is still unknown, we revealed that C-terminus transmembrane domain-truncated emerin (amino acid 1-225) binds to lamin A with higher affinity than lamin C . Screening for the emerin binding protein and immunoprecipitation analysis showed that lamin A binds to emerin specifically . We also used the yeast two-hybrid system to clarify that this interaction requires the top half of the tail domain (amino acid 384-566) of lamin A . Lamin A and lamin C are alternative splicing products of the lamin A/C gene that is responsible for autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) . These results indicate that the emerin-lamin interaction requires the tail domains of lamin A and lamin C . The data also suggest that the lamin A-specific region (amino acids 567-664) plays some indirect role in the difference in emerin-binding capacity between lamin A and lamin C . This is the first report that refers the difference between lamin A and lamin C in the interaction with emerin . These data also suggest that lamin A is important for nuclear membrane integrity.

Virus Res, 2001 Mar, 73(2), 103 - 12
Identification of the genome-linked protein in virions of Potato virus A, with comparison to other members in genus Potyvirus; Oruetxebarria I et al.; Viruses of the genus Potyvirus, the largest genus of plant-infecting viruses, have a messenger-polarity ssRNA genome encapsidated by approximately 2000 units of the viral coat protein (CP), resulting in filamentous virions . Only few studies have examined potyvirus virions for the presence of other structural proteins . A protein linked covalently to the 5'-end of the genome has been identified in Tobacco vein mottling virus (TVMV) and Tobacco etch virus (TEV) . In TEV, it is either the viral NIa protein or only its N-terminal domain (VPg) separated autocatalytically from the C-terminal proteinase domain (NIa-Pro) . Virions of TVMV carry only the VPg . We examined virions of Potato virus A (PVA) for the genome-linked protein using immunoblotting or iodination and immunoprecipitation . The VPg ( approximately 25 kDa) only, and not the unprocessed NIa, was detected . Another signal corresponding to approximately 49 kDa was detected in disrupted, RNase-treated virions with anti-VPg antibodies but not with antibodies to NIa-Pro . Since it possibly represented a dimeric form of the VPg, self-interaction of the VPg was tested using the yeast two-hybrid system, which showed that the VPg self-interacts in the absence of viral RNA.

FEBS Lett, 2001 Feb 9, 490(1-2), 65 - 9
Conserved role for 14-3-3epsilon downstream of type I TGFbeta receptors; McGonigle S et al.; Schistosoma mansoni receptor kinase-1 (SmRK1) is a divergent type I transforming growth factor beta (TGFbeta) receptor on the surface of adult parasites . Using the intracellular domain of SmRK1 as bait in a yeast two-hybrid screen we identified an interaction with S . mansoni 14-3-3epsilon . The interaction which is phosphorylation-dependent is not specific to schistosomes since 14-3-3epsilon also binds to TbetaRI, the human type I TGFbeta receptor . 14-3-3epsilon enhances TGFbeta-mediated signaling by TbetaRI and is the first TbetaRI-interacting non-Smad protein identified that positively regulates this receptor . The interaction of 14-3-3epsilon with schistosome and human TbetaRI suggests a conserved, but previously unappreciated, role for this protein in TGFbeta signaling pathways.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 2065 - 70 Epub 2001 Feb 06.
The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway; Helliwell CA et al.; We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA(12) . A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains . Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein . Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified . Arabidopsis thaliana has two CYP88A genes, both of which are expressed . Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA(12) . Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1877 - 82 Epub 2001 Feb 06.
Herpes simplex virus 1 alpha regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1; Kawaguchi Y et al.; The infected cell protein no . 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a promiscuous transactivator shown to enhance the expression of gene introduced into cells by infection or transfection . At the molecular level, ICP0 is a 775-aa ring finger protein localized initially in the nucleus and late in infection in the cytoplasm and mediates the degradation of several proteins and stabilization of others . None of the known functions at the molecular level account for the apparent activity of ICP0 as a transactivator . Here we report that ICP0 functionally interacts with cellular transcription factor BMAL1, a member of the basic helix-loop-helix PER-ARNT-SIM (PAS) super family of transcriptional regulators . Specifically, sequences mapped to the exon II of ICP0 interacted with BMAL1 in the yeast two-hybrid system and in reciprocal pull-down experiments in vitro . Moreover, the enhancement of transcription of a luciferase reporter construct whose promoter contained multiple BMAL1-binding sites by ICP0 and BMAL1 was significantly greater than that observed by ICP0 or BMAL1 alone . Although the level of BMAL1 present in nuclei of infected cells remained unchanged between 3 and 8 h after infection, the level of cytoplasmic BMAL1 was reduced at 8 h after infection . The reduction of cytoplasmic BMAL1 was significantly greater in cells infected with the ICP0-null mutant than in the wild-type virus-infected cells, suggesting that ICP0 mediates partial stabilization of the protein . These results indicate that ICP0 interacts physically and functionally with at least one cellular transcription-regulatory factor.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1811 - 6 Epub 2001 Jan 30.
The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis; Holbert S et al.; Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the protein huntingtin (htt) . Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably, transcription factors) . To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library . We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests . A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an imperfect (Gln-Ala)(38) tract encoded by a polymorphic repeat DNA . CA150 interacted in vitro with full-length htt from lymphoblastoid cells . The expression of CA150, measured immunohistochemically, was markedly increased in human HD brain tissue compared with normal age-matched human brain tissue, and CA150 showed aggregate formation with partial colocalization to ubiquitin-positive aggregates . In 432 HD patients, the CA150 repeat length explains a small, but statistically significant, amount of the variability in the onset age . Our data suggest that abnormal expression of CA150, mediated by interaction with polyglutamine-expanded htt, may alter transcription and have a role in HD pathogenesis.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1637 - 42 Epub 2001 Jan 30.
A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic; Cormont M et al.; The small GTPase Rab4 is implicated in endocytosis in all cell types, but also plays a specific role in some regulated processes . To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for an effector(s) that specifically recognizes its GTP-bound form . We cloned a ubiquitous 69-kDa protein, Rabip4, that behaves as a Rab4 effector in the yeast two-hybrid system and in the mammalian cell . Rabip4 contains two coiled-coil domains and a FYVE-finger domain . When expressed in CHO cells, Rabip4 is present in early endosomes, because it is colocated with endogenous Early Endosome Antigen 1, although it is absent from Rab11-positive recycling endosomes and Rab-7 positive late endosomes . The coexpression of Rabip4 with active Rab4, but not with inactive Rab4, leads to an enlargement of early endosomes . It strongly increases the degree of colocalization of markers of sorting (Rab5) and recycling (Rab11) endosomes with Rab4 . Furthermore, the expression of Rabip4 leads to the intracellular retention of a recycling molecule, the glucose transporter Glut 1 . We propose that Rabip4, an effector of Rab4, controls early endosomal traffic possibly by activating a backward transport step from recycling to sorting endosomes.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1595 - 600 Epub 2001 Feb 06.
Myozenin: an alpha-actinin- and gamma-filamin-binding protein of skeletal muscle Z lines; Takada F et al.; To better understand the structure and function of Z lines, we used sarcomeric isoforms of alpha-actinin and gamma-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system . Here we describe myozenin (MYOZ), an alpha-actinin- and gamma-filamin-binding Z line protein expressed predominantly in skeletal muscle . Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by alpha-helical regions with no strong homologies to any known genes . The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2 . Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues . Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for alpha-actinin, and immunogold electron microscopy confirms localization specifically to Z lines . Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.

Development, 2001 Feb, 128(4), 603 - 15
Drosophila homologues of the transcriptional coactivation complex subunits TRAP240 and TRAP230 are required for identical processes in eye-antennal disc development; Treisman J; We have identified mutations in two genes, blind spot and kohtalo, that encode Drosophila homologues of human TRAP240 and TRAP230, components of a large transcriptional coactivation complex homologous to the yeast Mediator complex . Loss of either blind spot or kohtalo has identical effects on the development of the eye-antennal disc . Eye disc cells mutant for either gene can express decapentaplegic and atonal in response to Hedgehog signaling, but they maintain inappropriate expression of these genes and fail to differentiate further . Mutant cells in the antennal disc lose expression of Distal-less and misexpress eyeless, suggesting a partial transformation towards the eye fate . blind spot and kohtalo are not required for cell proliferation or survival, and their absence cannot be rescued by activation of the Hedgehog or Notch signaling pathways . These novel and specific phenotypes suggest that TRAP240 and TRAP230 act in concert to mediate an unknown developmental signal or a combination of signals.

J Cell Sci, 2001 Feb, 114(Pt 3), 609 - 22
Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity; Steinberg G et al.; Many fungal pathogens undergo a yeast-hyphal transition during their pathogenic development that requires rearrangement of the cytoskeleton, followed by directed membrane traffic towards the growth region . The role of microtubules and their dynamic behavior during this process is not well understood . Here we set out to elucidate the organization, cellular role and in vivo dynamics of microtubules in the dimorphic phytopathogen Ustilago maydis . Hyphae and unbudded yeast-like cells of U . maydis contain bundles of spindle pole body-independent microtubules . At the onset of bud formation two spherical tubulin structures focus microtubules towards the growth region, suggesting that they support polar growth in G(2), while spindle pole body-nucleated astral microtubules participate in nuclear migration in M and early G(1) . Conditional mutants of an essential alpha-tubulin gene from U . maydis, tub1, confirmed a role for interphase microtubules in determination of cell polarity and growth . Observation of GFP-Tub1 fusion protein revealed that spindle pole body-independent and astral microtubules are dynamic, with elongation and shrinkage rates comparable to those found in vertebrate systems . In addition, very fast depolymerization was measured within microtubule bundles . Unexpectedly, interphase microtubules underwent bending and rapid translocations within the cell, suggesting that unknown motor activities participate in microtubule organization in U . maydis . Movies available on-line: http://www.biologists.com/JCS/movies/jcs1792.html

J Cell Sci, 2001 Feb, 114(Pt 3), 525 - 38
Parvin, a 42 kDa focal adhesion protein, related to the alpha-actinin superfamily; Olski TM et al.; We have identified and cloned a novel 42-kDa protein termed alpha-parvin, which has a single alpha-actinin-like actin-binding domain . Unlike other members of the alpha-actinin superfamily, which are large multidomain proteins, alpha-parvin lacks a rod domain or any other C-terminal structural modules and therefore represents the smallest known protein of the superfamily . We demonstrate that mouse alpha-parvin is widely expressed as two mRNA species generated by alternative use of two polyadenylation signals . We analyzed the actin-binding properties of mouse alpha-parvin and determined the K(d) with muscle F-actin to be 8.4+/-2.1 microM . The GFP-tagged alpha-parvin co-localizes with actin filaments at membrane ruffles, focal contacts and tensin-rich fibers in the central area of fibroblasts . Domain analysis identifies the second calponin homology domain of parvin as a module sufficient for targeting the focal contacts . In man and mouse, a closely related paralogue beta-parvin and a more distant relative gamma-parvin have also been identified and cloned . The availability of the genomic sequences of different organisms enabled us to recognize closely related parvin-like proteins in flies and worms, but not in yeast and Dictyostelium . Phylogenetic analysis of alpha-parvin and its para- and orthologues suggests, that the parvins represent a new family of alpha-actinin-related proteins that mediate cell-matrix adhesion.

Biochem Soc Trans, 2000 Dec, 28(6), 947 - 50
Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana; Smith M et al.; Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil . By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively . In Arabidopsis plants with an active ER Delta12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels . Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.

Biochem Soc Trans, 2000 Dec, 28(6), 910 - 2
Effect of copper and lead on lipid metabolism in bryophytes and lichens; Guschina IA et al.; Bryophytes and lichens have a widespread occurrence and can survive under extreme environmental conditions, such as drought, low temperatures, continuous light or prolonged darkness . It has been shown that lipid metabolism is sensitive to both metal response and metal resistance mechanisms in many organisms, including yeast, Silene cucubalus, and in the marine brown algae Fucus spp . and Ascophyllum nodosum . In the present study, the effects of lead and copper on lipid metabolism have been studied in two moss species, Rhytidiadelphus squarrosus and Dicranum scoparium, and also in the lichen Peltigera horizontalis with a cyanobacterial Nostoc photobiont.

Biochem Soc Trans, 2000 Dec, 28(6), 867 - 70
omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence; Pinot F et al.; The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa . The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH . After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer . Both the 9S,10R and 9R,10S enantiomers were incubated separately . We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer . This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V . sativa microsomes . Cutin analysis of V . sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid . Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence . Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.

Biochem J, 2001 Feb 1, 353(Pt 3), 655 - 61
Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation; Row PE et al.; Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic . The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole . A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved . To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells . Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor . In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor . We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes . We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.

Biochem J, 2001 Feb 1, 353(Pt 3), 483 - 92
mSiglec-E, a novel mouse CD33-related siglec (sialic acid-binding immunoglobulin-like lectin) that recruits Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2; Yu Z et al.; The sialic acid-binding immunoglobulin-like lectins (siglecs) represent a recently defined distinct subset of the immunoglobulin superfamily . By using the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatase SHP-1 as bait in a yeast two-hybrid screen, we have identified a new member of the mouse siglec family, mSiglec-E . The mSiglec-E cDNA encodes a protein of 467 amino acids that contains three extracellular immunoglobulin-like domains, a transmembrane region and a cytoplasmic tail bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) . mSiglec-E is highly expressed in mouse spleen, a tissue rich in leucocytes . The ITIMs of mSiglec-E can recruit SHP-1 and SHP-2, two inhibitory regulators of immunoreceptor signal transduction . This suggests that the function of mSiglec-E is probably an involvement in haematopoietic cells and the immune system as an inhibitory receptor . When expressed in COS-7 cells, mSiglec-E was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that mSiglec-E may function through cell-cell interactions . In comparison with the known members of the siglec family, mSiglec-E exhibits a high degree of sequence similarity to both human siglec-7 and siglec-9 . The gene encoding mSiglec-E is localized in the same chromosome as that encoding mouse CD33 . Phylogenetic analysis reveals that neither mouse mSiglec-E nor CD33 shows a clear relationship with any human siglecs so far identified.

Biochem J, 2001 Feb 1, 353(Pt 3), 417 - 39
Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling; Janssens V et al.; Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated . Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes . Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices . The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs . The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development . PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases . Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity . Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.

Genomics, 2001 Feb 1, 71(3), 307 - 14
Characterization of a widely expressed gene (LUC7-LIKE; LUC7L) defining the centromeric boundary of the human alpha-globin domain; Tufarelli C et al.; We have identified the first gene lying on the centromeric side of the alpha-globin gene cluster on human 16p13.3 . The gene, called 16pHQG;16 (HGMW-approved symbol LUC7L), is widely transcribed and lies in the opposite orientation with respect to the alpha-globin genes . This gene may represent a mammalian heterochromatic gene, encoding a putative RNA-binding protein similar to the yeast Luc7p subunit of the U1 snRNP splicing complex that is normally required for 5' splice site selection . To examine the role of the 16pHQG;16 gene in delimiting the extent of the alpha-globin regulatory domain, we mapped its mouse orthologue, which we found to lie on mouse chromosome 17, separated from the mouse alpha-cluster on chromosome 11 . Establishing the full extent of the human 16pHQG;16 gene has allowed us to define the centromeric limit of the region of conserved synteny around the human alpha-globin cluster to within an 8-kb segment of chromosome 16 .

Biotechnol Prog, 2001 Jan-Feb, 17(1), 134 - 9
A novel magnetic affinity support for protein adsorption and purification; Tong XD et al.; A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material . Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA . The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field . These facts indicated that the particles were superparamagnetic . Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption . Lysozyme was used as a model protein to test the adsorption properties of the MAS . The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm . The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3-5 micromol/g) . In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme . Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates . Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity.

Biochemistry, 2001 Jan 30, 40(4), 1053 - 62
The ubiquitin-proteasome pathway plays an essential role in proteolysis during Trypanosoma cruzi remodeling; de Diego JL et al.; Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage . The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc . This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels . About 30 proteins in a range of 25-110 kDa were detected in the purified T . cruzi 26S proteasome . Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T . cruzi 20S core specifically recognized components of T . cruzi 26S . To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling . Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes . If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites . As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.

Biochemistry, 2001 Jan 23, 40(3), 847 - 51
Effect of pressure on deuterium isotope effects of formate dehydrogenase; Quirk DJ et al.; High pressure causes biphasic effects on the oxidation of formate by yeast formate dehydrogenase as expressed on the kinetic parameter V/K, which measures substrate capture . Moderate pressure increases capture by accelerating hydride transfer . The transition state for hydride transfer has a smaller volume than the free formate plus the capturing form of enzyme, with DeltaV(double dagger) = -9.7 +/- 1.0 mL/mol . Pressures above 1.5 kbar decrease capture, reminiscent of effects on the conformational change associated with the binding of nicotinamide adenine dinucleotide (NAD(+)) to yeast alcohol dehydrogenase {Northrop, D . B., and Y . K . Cho (2000) Biochemistry 39, 2406-2412} . The collision complex, E-NAD(+), has a smaller volume than the more tightly bound reactant-state complex, E-NAD(+), with DeltaV = +83.4 +/- 5.2 mL/mol . A comparison of the effects of pressure on the oxidation of normal and deuteroformate shows that the entire isotope effect on hydride transfer, 2.73 +/- 0.20, arises solely from transition-state phenomena, as was also observed previously with yeast alcohol dehydrogense . In contrast, normal primary isotope effects arise solely from different zero-point energies in reactant states, and those that express hydrogen tunneling arise from a mixture of both reactant-state and transition-state phenomena . Moreover, pressure increases the primary intrinsic deuterium isotope effect, the opposite of what was observed with yeast alcohol dehydrogense . The lack of a decrease in the isotope effect is also contrary to empirical precedents from chemical reactions suspected of tunneling and to theoretical constructs of vibrationally enhanced tunneling in enzymatic reactions . Hence, this new experimental design penetrates transition states of enzymatic catalysis as never before, reveals the presence of phenomena foreign to chemical kinetics, and calls for explanations of how enzymes work beyond the tenants of physical organic chemistry.

J Cell Biochem, 2001, 80(4), 483 - 90
Molecular interaction between human tumor marker protein p150, the largest subunit of eIF3, and intermediate filament protein K7; Lin L et al.; The human tumor marker protein p150 was identified as the largest subunit of eukaryotic translation initiation factor 3 (eIF3) (also known as p170/p180) . Its expression level is not only upregulated in many transformed cell lines, but also in several human cancers including breast, cervical, esophageal, and stomach carcinomas . The function of p150 in cancer and initiation of translation are not well understood . Using the yeast two-hybrid genetic screen, we found that a portion of p150 interacts with hPrt1, another subunit of eIF3, and cytokeratin 7, an intermediate filament protein . The interactions between p150 and hPrt1, and between p150 and cytokeratin 7 were verified both in vivo and in vitro . The interaction site for hPrt1 was mapped to the carboxyl half of the coiled-coil region of the p150 protein between amino acids 664-835 . The expression of hPrt1 was clearly upregulated in cancer tissue, similarly to that of p150 . By contrast, no substantial difference in the expression level of cytokeratin 7 was observed between cancer and normal breast tissue, suggesting that cytokeratin 7 expression is not co-regulated with p150 . Taken together, our studies suggest a new role for p150 in translation initiation, possibly by acting as an adapter molecule between the translation initiation apparatus and the cytoskeleton structure in the cell .

J Pathol, 2001 Jan, 193(1), 55 - 65
Association of allelic loss at the FHIT locus and p53 alterations with tumour kinetics and chromosomal instability in non-small cell lung carcinomas (NSCLCs); Garinis GA et al.; The FHIT gene, located at the FRA3B fragile site of chromosome 3p14.2, encodes a 16.8 kD homologue of the yeast enzyme diadenosine tetraphosphate (Ap(4)A) hydrolase . Frequent allelic losses at this region in various malignancies, including non-small cell lung carcinomas (NSCLCs), imply that FHIT may represent a tumour suppressor gene (TSG) . Increasing evidence suggests that multiple TSG impairment has a synergistic effect on tumour growth . The present study of 67 NSCLCs investigated the allelic imbalance (AIm) within the FHIT locus and its relationship with p53 abnormalities, kinetic parameters {proliferative activity or proliferation index (PI) and apoptotic index (AI)}, and ploidy status of the carcinomas . Allelic imbalance at FHIT was observed in 35 out of 55 informative (heterozygous: H) cases (64%) . Similar frequencies of loss of heterozygosity (LOH) were noticed among squamous cell lung carcinomas and adenocarcinomas . The high percentage of AIm in stage I tumours (71%) is indicative of its relatively early involvement in NSCL carcinogenesis . No association was found between LOH at FHIT, kinetic parameters, and ploidy status of the tumours . Concurrent loss at FHIT and p53 overexpression {FHIT(LOH)/p53(P)} was the most frequent pattern and was observed in 39% of the informative cases . The latter pattern was not associated with smoking, supporting the hypothesis that in patients with a history of tobacco exposure, FHIT allelic loss may not be a consequence of p53 checkpoint defects, but the outcome of tobacco-induced mutagenesis . Statistically significant differences in the presence of FHIT(LOH)/p53(P) and FHIT(LOH)/p53(N) patterns were noted at the proliferative and apoptotic level, whereas ploidy was similar amongst all groups, implying that wild-type (wt) p53 may play a safeguard role against altered FHIT function . However, the possibility of a masking effect from wt p53 cannot be excluded, since the FHIT(LOH)/p53(P) profile demonstrated a higher growth index (GI=PI/AI mean value ratio) than FHIT(H)/p53(P) (32 vs . 8), although this was not significant . Further studies are needed in order to elucidate the role of FHIT and its relationships with other cell-cycle regulatory molecules involved in NSCL carcinogenesis.

Eur J Neurosci, 2001 Jan, 13(2), 241 - 7
p35/cdk5 binds and phosphorylates beta-catenin and regulates beta-catenin/presenilin-1 interaction; Kesavapany S et al.; The neuronal cyclin-dependent kinase p35/cdk5 comprises a catalytic subunit (cdk5) and an activator subunit (p35) . To identify novel p35/cdk5 substrates, we utilized the yeast two-hybrid system to screen for human p35 binding partners . From one such screen, we identified beta-catenin as an interacting protein . Confirmation that p35 binds to beta-catenin was obtained by using glutathione S-transferase (GST)-beta-catenin fusion proteins that interacted with both endogenous and transfected p35, and by showing that beta-catenin was present in p35 immunoprecipitates . p35 and beta-catenin also displayed overlapping subcellular distribution patterns in cells including neurons . Finally, we demonstrated that p35/cdk5 phosphorylates beta-catenin . beta-catenin also binds to presenilin-1 and altered beta-catenin/presenilin-1 interactions may be mechanistic in Alzheimer's disease (AD) . Abnormal p35/cdk5 activity has also been suggested to contribute to AD . We therefore investigated how modulation of p35/cdk5 activity influenced beta-catenin/presenilin-1 interactions . Inhibition of p35/cdk5 with roscovitine did not alter the steady state levels of either beta-catenin or presenilin-1 but reduced the amount of presenilin-1 bound to beta-catenin . Thus, p35/cdk5 binds and phosphorylates beta-catenin and regulates its binding to presenilin-1 . The findings reported here therefore provide a novel molecular framework to connect p35/cdk5 with beta-catenin and presenilin-1 in AD.

Int J Biochem Cell Biol, 2001 Jan, 33(1), 1 - 10
Frataxin: its role in iron metabolism and the pathogenesis of Friedreich's ataxia; Becker E et al.; Friedreich's ataxia (FA) is a severe neurodegenerative condition with an incidence of 1:50000 in the European population . In 97% of patients this disease is due to an intronic GAA triplet repeat expansion in the FRDA gene resulting in a marked decrease in its expression . The protein encoded by this gene is known as frataxin which is found within the mitochondrion . Upon deletion of the homologous gene (YFH1) in the yeast, there was an accumulation of iron (Fe) within the mitochondrion . When the YFH1 gene was reintroduced back into the yeast cell Fe was exported out of the mitochondrion and into the cytosol . Evidence that human frataxin is also involved in mitochondrial Fe-overload comes from studies in FA patients that have shown an accumulation of Fe within the heart . While the precise role of human frataxin remains to be determined, the molecule appears to be involved indirectly in regulating the export and/or import of mitochondrial Fe . The finding of mitochondrial Fe-overload suggests that the use of specific Fe chelators which can permeate the mitochondrion may have potential in the treatment of this disease.

Curr Opin Biotechnol, 2001 Feb, 12(1), 41 - 7
New developments in microarray technology; Blohm DH et al.; Microarrays have emerged as indispensable research tools for gene expression profiling and mutation analysis . New classification of cancer subtypes, dissecting the yeast metabolism and large-scale genotyping of human single nucleotide polymorphisms are important results being obtained with this technique . Realizing the microsphere-based massively parallel signature sequencing technique as fluid microarrays, building new types of protein arrays and constructing miniaturized flow-through systems, which can potentially take this technology from the research bench into industrial, clinical and other routine applications, exemplify the intense developments that are now ongoing in this field.

Gene, 2000 Dec 31, 261(2), 197 - 203
Characterization and expression analysis of two human septin genes, PNUTL1 and PNUTL2; Zieger B et al.; The presence and role of septin proteins in yeast is well documented, but there is a growing appreciation for this family of proteins beyond yeast and extending to human cells . In this report we present the characterization and comparison of two highly similar human septin genes, PNUTL1 and PNUTL2 . We compare the exon/intron structure of both genes, the steady-state mRNA levels in tumor cell lines and adult organs, the conceptual translation products from alternatively processed mRNAs and the development of specific immunologic reagents distinguishing either PNUTL1 or PNUTL2 . The results illustrate a remarkable similarity between the two genes and their protein products while identifying specific differences in mRNA expression patterns . A summary of the described functional roles for mammalian septins is discussed along with an attempt to assimilate the alternative nomenclature existing for the same human septins, such as references to PNUTL1 and PNUTL2 as hCDCrel-1 and hCDCrel-2, respectively . The characterization of PNUTL1 and PNUTL2 represents a fundamental step in completing the characterization of the entire family of human septin genes.

Curr Opin Chem Biol, 2001 Feb, 5(1), 34 - 9
Genomic analysis of biochemical function; Grayhack EJ et al.; Establishing the linkage between an individual biochemical activity and the gene(s) specifying that activity has been facilitated by advances in mass spectrometry and affinity purification methods . In addition, a genomic protein array has been produced in yeast by fusing each yeast open reading frame to glutathione-S-transferase, thus linking each protein with its cognate gene . Purification and biochemical assay of pools of glutathione-S-transferase-open-reading-frame proteins allows analysis of the entire proteome for biochemical activities, followed by simple deconvolution to identify the responsible open reading frame . An alternative method to analyze large sets of proteins is the use of protein microarrays in which over 10,000 individual proteins can be immobilized and assayed on a single slide.

Curr Biol, 2001 Jan 9, 11(1), 8 - 17
DNA damage leads to a Cyclin A-dependent delay in metaphase-anaphase transition in the Drosophila gastrula; Su TT et al.; BACKGROUND: In response to DNA damage, fission yeast, mammalian cells, and cells of the Drosophila gastrula inhibit Cdk1 to delay the entry into mitosis . In contrast, budding yeast delays metaphase-anaphase transition by stabilization of an anaphase inhibitor, Pds1p . A variation of the second response is seen in Drosophila cleavage embryos; when nuclei enter mitosis with damaged DNA, centrosomes lose gamma-tubulin, spindles lose astral microtubules, chromosomes fail to reach a metaphase configuration, and interphase resumes without an intervening anaphase . The resulting polyploid nuclei are eliminated . RESULTS: The cells of the Drosophila gastrula can also delay metaphase-anaphase transition in response to DNA damage . This delay accompanies the stabilization of Cyclin A, a known inhibitor of sister chromosome separation in Drosophila . Unlike in cleavage embryos, gamma-tubulin remains at the spindle poles, and anaphase always occurs after the delay . Cyclin A mutants fail to delay metaphase-anaphase transition after irradiation and show an increased frequency of chromosome breakage in the subsequent anaphase . CONCLUSIONS: DNA damage delays metaphase-anaphase transition in Drosophila by stabilizing Cyclin A . This delay may normally serve to preserve chromosomal integrity during segregation . To our knowledge this is the first report of a metazoan metaphase-anaphase transition being delayed in response to DNA damage . Though mitotic progression is modulated in response to DNA damage in both cleaving and gastruating embryos of Drosophila, different mechanisms operate . These differences are discussed in the context of differential cell cycle regulation in cleavage and gastrula stages.

Mech Dev, 2001 Feb, 100(2), 327 - 30
Bambi is coexpressed with Bmp-4 during mouse embryogenesis; Grotewold L et al.; Signaling of TGF-beta superfamily members is tightly controlled by an elaborate network of regulators (for recent review see Trends Genet . 15 (1999) 3; Genes Dev . 14 (2000) 627) . Recently, the transmembrane protein BAMBI (BMP and activin membrane-bound inhibitor) has been shown to interfere with Bmp and activin-like signaling by inhibiting Tgf-beta type I receptor activation (Nature 401 (1999) 480) . In striking contrast to other Bmp antagonists like noggin (Cell 86 (1996) 599) or chordin (Cell 86 (1996) 589), BAMBI is strictly coexpressed with Bmp-4 during early Xenopus embryogenesis . The grouping of genes according to their shared complex spatial expression pattern and their involvement in the same biological signaling pathway has been referred to as synexpression group . This concept facilitates prognoses about the roles of a group member with unknown function . Apparently, only a minority of genes is organized in synexpression groups and up to now they have mainly been described in yeast and Xenopus (for review see Nature 402 (1999) 483) . In the frog, BAMBI is a member of the Bmp-4 synexpression group (Nature 401 (1999) 480) . We identified two murine homologues of BAMBI one of which, named Bambi-psi, is a pseudogene . We show that the spatiotemporal expression pattern of Bambi closely matches that of Bmp-4 during mouse embryonic development . Moreover, we show that Bambi expression is induced in mouse embryonic fibroblasts by Bmp-4 . Hence, we provide first evidence for the existence of an evolutionarily conserved Bmp-4 synexpression group in mammals.

Mol Cell Endocrinol, 2001 Feb 14, 172(1-2), 47 - 56
The expression of pregnane X receptor and its target gene, cytochrome P450 3A1, in perinatal mouse; Masuyama H et al.; Recently, pregnane X receptor (PXR) has been described to mediate the genomic effects of several steroid hormones, such as progesterone (P), glucocorticoid (Dex), pregnenolone (Preg), and xenobiotics through the cytochrome P-450 3A gene family (CYP3A), which are monooxygenases, responsible for the oxidative metabolism of some endogenous substrates and xenobiotics . In the present study, we used a transient transfection reporter gene expression assay of COS-7 cells to demonstrate that P, Dex and Preg significantly stimulate PXR-mediated transcription at relatively high concentration comparable with that of progesterone near term pregnancy . In yeast two-hybrid protein interaction assay, PXR interacted with nuclear receptor coactivator proteins, SRC1, RIP140, and SUG1 in a ligand-dependent manner . The expression of PXR mRNA was observed in the liver, intestine, uterus, ovary and placenta . The expressions of PXR mRNA in the liver and ovary increased towards term about fifty-fold compared with that of non-pregnancy and decreased postpartum . Its expression in the placenta was not drastically changed towards term . CYP3A, a target gene of PXR, was also expressed in the liver, ovary, and placenta . The expressions of CYP3A mRNA as well as PXR in the liver and ovary increased about 20-fold during prenatal period . These data suggest that PXR may play certain roles in perinatal period, possibly in the protection of the feto-maternal system from the toxic effect of endogenous steroids and foreign substrates.

Mol Cell Endocrinol, 2001 Jan 22, 171(1-2), 199 - 204
17beta-hydroxysteroid dehydrogenase type 7--an ancient 3-ketosteroid reductase of cholesterogenesis; Breitling R et al.; 17beta-hydroxysteroid dehydrogenase type 7 (17beta-HSD7) is a novel estrogenic hydroxysteroid dehydrogenase from mammals . We modeled the three-dimensional structure of human 17beta-HSD7, analyzed the phylogeny of 17beta-HSD7 homologues and determined its expression pattern by in silico Northern blotting . Predominant expression is found not only in reproductive tissues (breast, ovary, placenta) but also in liver and developing brain, principal sites of cholesterol synthesis . The substrate binding pocket is opening towards a conserved membrane-associated helix, which is indicative for a conversion of a membrane component . 17beta-HSD7 shows significant homology to a yeast 3-ketosteroid reductase (ERG27) involved in ergosterol biosynthesis . Our results lead to the conclusion that 17beta-HSD7 is not only involved in estradiol production but plays another (and possibly more important) role as a 3-ketosteroid reductase in cholesterogenesis . This agrees with the striking absence of 17beta-HSD7 homologues in the complete genomes of Drosophila and C . elegans, which are both auxotrophic for cholesterol.

Regul Pept, 2001 Mar 2, 97(2-3), 147 - 52
Mutation of FKBP associated protein 48 (FAP48) at proline 219 disrupts the interaction with FKBP12 and FKBP52; Neye H; Immunophilins are known as intracellular receptors for the immunosuppressant drugs, cyclosporin A, FK506, and rapamycin . They can be divided into two groups, cyclophilins that bind cyclosporin A and FK506 binding proteins (FKBPs) that bind FK506 and rapamycin . Many efforts were made to elucidate the physiological role of the immunophilins . Many of them are involved in intracellular signalling as they bind to calcium channels or to steroid receptor complexes . A yeast two-hybrid screen was used to identify further target proteins that interact with known proteins . Recently, a 48-kDa FKBP associated protein (FAP48) was isolated that binds to FKBP12 and FKBP52 . Binding of FAP48 to FKBPs is inhibited by the macrolide FK506 indicating that the binding sites on the immunophilins coincide with the binding site for FK506 . A peptidyl-prolyl motif on FAP48 should be responsible for the binding of the protein to FKBPs . We sequentially point mutated proline sites on FAP48 and checked the mutant proteins for interaction with FKBP12 and FKBP52 . Mutation of proline 219 to alanine leads to a loss of interaction indicating that a cysteinyl prolyl site might be responsible for the binding of FAP48 to FKBPs . Thus we identified proline 219 being essential for the interaction.

Plant Sci, 2000 Dec 7, 160(1), 37 - 47
Induction of isoflavonoid pathway in the model legume Lotus japonicus: molecular characterization of enzymes involved in phytoalexin biosynthesis; Shimada N et al.; Treatment of the seedlings of Lotus japonicus, a model legume for molecular genetic studies, with reduced glutathione (GSH) resulted in the accumulation of an isoflavan phytoalexin, vestitol . Using PCR strategies based on the conserved amino acid sequences, full length P450 cDNAs were obtained from GSH-treated seedling roots . When the clones, LjCYP-1 (CYP93C family) and LjCYP-2 (CYP81E family), were heterologously expressed in yeast, the proteins exhibited 2-hydroxyisoflavanone synthase (IFS) and isoflavone 2'-hydroxylase (I2'H) activities, respectively . The transcription levels of LjCYP-1, LjCYP-2 and isoflavone reductase, which are all involved in vestitol biosynthesis, coordinately increased upon elicitation . Genomic Southern blot analysis indicated that the IFS gene forms a small gene family and a single copy of the I2'H gene is present in the L . japonicus genome . Molecular biological aspects of P450s involved in the isoflavonoid pathway and the genomic approach to flavonoid metabolism in this unique plant are discussed.

FEBS Lett, 2001 Jan 19, 488(3), 201 - 5
Identification of two proteins, S14 and UIP1, that interact with UCH37; Li T et al.; By the use of the yeast two-hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1) . The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co-immunoprecipitation analysis . The C-terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two-hybrid assay and the in vitro binding assay . Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.

FEBS Lett, 2001 Jan 19, 488(3), 116 - 22
The C-terminus of human glutaminase L mediates association with PDZ domain-containing proteins; Olalla L et al.; The enzyme glutaminase in brain is responsible for the synthesis of neurotransmitter glutamate . We used the two-hybrid genetic selection system in yeast to look for interactors of glutaminase in human brain . We have identified two proteins containing PDZ domains, alpha1-syntrophin and a glutaminase-interacting protein, named GIP, that showed association with human glutaminase L, as deduced from specificity test of the two-hybrid system . The complete GIP cDNA clone has 1315 nucleotides with a 372-base open reading frame encoding a 124-amino acids protein . Glutaminase associates with both PDZ proteins through its C-terminal end; mutagenesis of single amino acids revealed the sequence -ESXV as essential for the interaction . These data suggest the possibility that PDZ domain-containing proteins are involved in the regulation of glutaminase in brain.

Mol Biochem Parasitol, 2000 Dec, 111(2), 415 - 24
Mitochondrial genetic code in cestodes; Nakao M et al.; The flatworm mitochondrial genetic code, which has been used for all species of the Platyhelminthes, is mainly characterized by AUA codon for isoleucine, AAA codon for asparagine and UAA codon for tyrosine . In eight species of cestodes (Echinococcus multilocularis, Echinococcus granlosus, Taenia solium Taenia saginata, Taenia hydatigena, Taenia crassiceps, Hymenolepis nama and Mesocestoides corti), the cytochrome c oxidase subunit I (COI) genes were partially sequenced to verify this genetic code . Comparison of the COI-encoding nucleotide sequences with those of human, sea urchin, fruit fly, nematode and yeast indicated that the assignments of AUA and AAA codons are adequate for cestodes . In addition, the nucleotide sequences of ATPase subunit 6 (ATP6) gene and its flanking region were compared to examine initiation and stop codons . In the related species of T . solium and T . saginata, the deduced amino acid sequences of ATP6 were homogeneous; however, the conversion of initiation codon AUG into GUG was observed in T . saginata . We also found the similar conversion in T . crassiceps . The C-terminal sequences of putative ATP6 proteins were highly conserved among the eight species and the stop codon UAG was altered to UAA in all Taenia species . The features of the gene-junctional region between NADH dehydrogenase subunit 4 (ND4) and glutamine tRNA (tRNAGln) genes also supported that UAA serves as a stop codon . Based on these results, we propose that the flatworm mitochondrial code should be modified for cestodes, particularly, in an initiating methionine codon (GUG) and a terminating codon (UAA).

Mol Biochem Parasitol, 2000 Dec, 111(2), 401 - 14
Chromosome structure and sequence organization between pathogenic and non-pathogenic Leishmania spp; Tamar S et al.; We have used a chromosome fragmentation strategy based on systematic genomic insertions of the rare cutting yeast I-SceI endonuclease to assess structure and sequence organization of homologous chromosomes between evolutionary divergent pathogenic and non-pathogenic Leishmania species . This method was combined to physical mapping and hybridization studies using a number of specific chromosomal markers as probes . Our studies have concentrated on two different chromosomes of Leishmania major (L . major), L . donovani and L . infantum and of the non-pathogenic species L . tarentolae . Specific chromosome fragmentation events at the level of multiple I-SccI genomic integrations indicated that very similar distances separated internal genomic sequences between homologous chromosomes and that distances from chromosome ends were more variable . The order and orientation of genes along the homologous chromosomes were also conserved between species . With only few exceptions, genome organization between pathogenic and non-pathogenic Leishmania species was found to be highly conserved . Genomic comparison of pathogenic and non-pathogenic species may be useful for depicting regions involved in species-specific related pathologies.

Mol Biochem Parasitol, 2000 Dec, 111(2), 261 - 73
The molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427; Melville SE et al.; We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427, clone 221a . This cloned stock is most commonly used in research laboratories in genetic manipulation experiments and in studies of antigenic variation . Using 116 previously characterised chromosome-specific markers, we identify 11 diploid pairs of megabase chromosomes and detect no loss of synteny in EST and gene marker distribution between this stock and the genome project reference stock TREU 927/4 . Nevertheless, the chromosomes of 427 are all larger than their homologues in 927, except chromosomes IIa and IXa . The greatest size variation is seen in chromosome I, the smallest of which is 1.1 Mb (927-Ia) and the largest 3.6 Mb (427-Ib) . The total nuclear DNA content of both stocks has been estimated by comparison of the mobility of T . brucei and yeast chromosomes . Trypanosomes of stock 427 contain approximately 16.5 Mb more megabase chromosomal DNA than those of stock 927 . We have detected the presence of bloodstream-form expression-site-associated sequences on eight or more megabase chromosomes . These sequences are not found on the same chromosomes in each stock . We have determined the chromosomal band location of nine characterised variant surface glycoprotein genes, including the currently expressed VSG 221 . Our results demonstrate both the stability of the T . brucei genome, as illustrated by the conservation of syntenic groups of genes in the two stocks, and the polymorphic nature of the genomic regions involved in antigenic variation . We propose that the chromosomes of stock 427 be numbered to correspond to their homologues in the genome project reference stock TREU 927/4.

Mol Immunol, 2000 Aug, 37(10), 603 - 12
Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein; He X et al.; Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling . In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait . A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein . The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively . Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells . In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological . The interacting domains of both proteins were mapped using yeast two-hybrid assays . Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280 . The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains . These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

Mol Immunol, 2000 Aug, 37(10), 591 - 602
Autoantigen Ro52 directly interacts with human IgG heavy chain in vivo in mammalian cells; Yang YS et al.; Previously, when we used in vivo yeast two-hybrid and in vitro protein-protein interaction analyses, we demonstrated a direct interaction between autoantigen Ro52 and the human IgG heavy chain . This interaction occurred in the absence of antibody-antigen specific interaction . Here, by employing a novel strategy, we further demonstrated that Ro52 co-localized with IgG in transfected mammalian cells . The co-localization was specific to IgG1 but not IgG3 . Co-immunoprecipitating IgG with Ro52 from transfected cell lysates suggested that protein complex containing Ro52 and IgG contributed to the in vivo co-localization . In addition, IgG from normal human serum was shown to bind to the surface of apoptotic keratinocytes and the binding could be competitively blocked by 50-fold excesses of IgG1, not IgG3 . With a direct binding study, we also demonstrated that IgG1 could bind to the surface of apoptotic cells while IgG3 bound barely . This binding was not competed by Fcgamma fragments indicating a non-Fcgamma receptor mediated interaction . Finally, in a competition analysis the addition of GST-RFP could reduce the IgG binding to the cell surface . Thus, we suggested that the binding of IgG to the apoptotic keratinocytes might be mediated through the interactions with the surface exposed Ro52 . The potential role of forming this protein complex on the apoptotic cells will be discussed.

FEBS Lett, 2001 Jan 5, 487(3), 377 - 83
Alternative splice variants of hTrp4 differentially interact with the C-terminal portion of the inositol 1,4,5-trisphosphate receptors; Mery L et al.; The molecular basis of capacitative (or store-operated) Ca2+ entry is still subject to debate . The transient receptor potential proteins have been hypothesized to be structural components of store-operated Ca2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N-terminal region of the inositol 1,4,5-triphosphate receptors (InsP3R) . In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as alpha-hTrp4 and beta-hTrp4 . The shorter variant beta-hTrp4 is generated through alternative splicing and lacks the C-terminal amino acids G785-S868 . Using a yeast two-hybrid assay and glutathione-S-transferase-pulldown experiments, we found that the C-terminus of alpha-hTrp4, but not of beta-hTrp4, associates in vitro with the C-terminal domain of the InsP(3) receptors type 1, 2 and 3 . Thus, we describe a novel interaction between Trp proteins and InsP3R and we provide evidence suggesting that the formation of hTrp4-InsP3R complexes may be regulated by alternative splicing.

Cell, 2000 Dec 22, 103(7), 1133 - 42
Generation of superhelical torsion by ATP-dependent chromatin remodeling activities; Havas K et al.; ATP-dependent chromatin remodeling activities participate in the alteration of chromatin structure during gene regulation . All have DNA- or chromatin-stimulated ATPase activity and many can alter the structure of chromatin; however, the means by which they do this have remained unclear . Here we describe a novel activity for ATP-dependent chromatin remodeling activities, the ability to generate unconstrained negative superhelical torsion in DNA and chromatin . We find that the ability to distort DNA is shared by the yeast SWI/SNF complex, Xenopus Mi-2 complex, recombinant ISWI, and recombinant BRG1, suggesting that the generation of superhelical torsion represents a primary biomechanical activity shared by all Snf2p-related ATPase motors . The generation of superhelical torque provides a potent means by which ATP-dependent chromatin remodeling activities can manipulate chromatin structure.

Curr Opin Genet Dev, 2001 Feb, 11(1), 78 - 82
DNA damage: Chk1 and Cdc25, more than meets the eye; Walworth NC; Control of mitotic entry is a component of the checkpoint response that contributes to cell survival following DNA damage . In some eukaryotic cells, mitotic entry relies heavily on regulation of the state of tyrosine phosphorylation of the cyclin-dependent kinase Cdc2 . Evidence that checkpoint regulation of cell-cycle progression operates through controlling the state of Cdc2 tyrosine phosphorylation exists . Whether other targets of the checkpoint pathway could play important roles in the response to DNA damage is a subject of ongoing investigations.

Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 892 - 7
cDna cloning and expression of CYP4F12, a novel human cytochrome P450; Bylund J et al.; cDNA of a novel human cytochrome P450 was cloned from human liver by reverse transcription-polymerase chain reaction and designated CYP4F12 . The open reading frame coded for 524 amino acids, and the sequence could be aligned with 78-83% amino acid identity to the four human CYP4F enzymes (CYP4F2, CYP4F3, CYP4F8 and CYP4F11) . Northern blot analysis suggested three major transcripts of CYP4F12, which were detected in liver, kidney, colon, small intestine and heart . The CYP4F12 gene contained 13 exons and was located at chromosome 19p13.1 . CYP4F12, expressed in yeast, oxidized arachidonic acid to 18-hydroxyarachidonic acid, and the omega-side chain of two stable prostaglandin (PG) H(2) analogs (11,9-epoxymethano-PGH(2) and 9,11-diazo-15-deoxy-PGH(2)) . CYP4F12 oxidized the omega-side chain of leukotriene B(4), PGE(2), PGF(2 alpha), PGH(2), and 9,11-epoxymethano-PGH(2) poorly . Several CYP4F enzymes are important omega 1- and omega 2-hydroxylases of eicosanoids . The physiological function of CYP4F12 merits further investigation .

Biochem Biophys Res Commun, 2001 Jan 19, 280(2), 535 - 40
Molecular cloning of a novel ubiquitin-like protein, UBIN, that binds to ER targeting signal sequences; Matsuda M et al.; To identify proteins that interact with HSP47, an endoplasmic reticulum (ER)-resident molecular chaperone, a yeast two-hybrid screening was performed using mouse full-length HSP47 including an N-terminal signal sequence as a bait . Analysis of several positive clones led to the identification and cloning of a novel gene, ubin, encoding a ubiquitin-like protein . Unlike other ubiquitin-like proteins, UBIN was shown to interact with signal sequences of various secretory and ER-luminal proteins, including HSP47, but not interact with signal sequences of mitochondrial targeting in two-hybrid system . The possible function of UBIN will be discussed with regards to novel characteristics of binding to signal sequences for ER targeting .

Biochem Biophys Res Commun, 2001 Jan 19, 280(2), 513 - 7
Interactions among subunits of human Arp2/3 complex: p20-Arc as the hub; Zhao X et al.; The Arp2/3 complex is critical for nucleation and crosslinking of actin filaments . To gain insight into its subunit topology and assembly pathway, we systematically examined interactions among subunits of human Arp2/3 complex by yeast two-hybrid assays . It was shown that p20-Arc was able to interact with p21-Arc, p34-Arc, and p16-Arc, respectively . In contrast, p41-Arc only interacted with p20-Arc/p16-Arc heterodimer . In addition, we found that structural integrity was important for association between p20-Arc and p21-Arc, while the N-terminal half of p34-Arc was dispensable for its binding to p20-Arc . Our data suggest a key role of p20-Arc and a multistep pathway for the complex formation .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 293 - 300
The double-stranded RNA-activated protein kinase PKR is dispensable for regulation of translation initiation in response to either calcium mobilization from the endoplasmic reticulum or essential amino acid starvation; Kimball SR et al.; The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR . Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation . Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR) . However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation . Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation . However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported . In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity . Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha . Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells) . In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells . Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation .

Biochem Biophys Res Commun, 2000 Dec 29, 279(3), 989 - 95
Genomic organization and molecular characterization of the human ninein gene; Hong YR et al.; The centrosome plays a key role in the formation of the mitotic spindle, cell polarity, and cell locomotion . Previously we identified a novel centrosomal associated protein hNinein using GSK-3beta as a bait in the yeast two-hybrid assay . In this report, the hNinein genome was found to correspond to 29 exons of genomic sequence on human chromosome 14q22 . Promoter analysis predicts that hNinein contains a TATA, two CCAAT, and three GC boxes . The promoter exhibits the following potential transcription factor binding sites: Sp1, p300, and AP-1 . In addition, an alternatively spliced isoform, encoded a 2041-amino-acid protein of 237,900 Da, which was designated hNinein-Lm (GenBank AF302773) . The hNinein-Lm genome was found to correspond to 28 exons (2'-29) . Amino acid sequence comparison with hNinein showed that hNinein-Lm exhibited an EF-hand Ca2+ binding domain in the N-terminus which similar to mouse ninein . Northern blot showed that this hNinein-Lm isoform was expressed more than hNinein in tissues examined . Differential RT-PCR combining Southern blotting also showed that hNinein-Lm is much more abundant compared to hNinein . Two forms of ninein may also imply the status of ninein associated with a pair of the centrioles in the centrosome structure . Furthermore, molecular characterization shows that human ninein is oligomerized at the C-terminal end which overlapped with GSK-3beta binding site, suggesting that oligomerization of ninein may be regulated by GSK-3beta phosphorylation.

Blood Cells Mol Dis, 2001 Jan-Feb, 27(1), 1 - 15
Cloning and characterization of a potential transcriptional activator of human gamma-globin genes; Yang Y et al.; Hybrids produced by fusing human fetal erythroblasts (HFE) with mouse erythroleukemia (MEL) cells initially produce predominantly or exclusively human gamma-globin and switch to human beta globin expression as time in culture advances . One explanation for the initially predominant expression of gamma-globin gene in these hybrids is the presence of trans-acting factors that activate gamma-globin gene transcription . We used differential display of hybrids before and after the gamma to beta switch as well as fetal liver and adult erythroblasts to identify cDNAs that could be candidates for potential gamma gene activators . Identically sized amplicons which were present in fetal liver erythroblasts and in the hybrids expressing only gamma-globin but were absent in the adult erythroblasts and in the same hybrids after they had switched to beta globin expression were cloned and sequenced . Fifty pairs of cDNAs fitting these criteria were chosen for further analysis . The sequences of the two members of 48 pairs differed from each other, revealing the low efficiency of this experimental approach . One clone pair coded for human proteosome subunit X . The second pair coded for a protein containing an acidic domain in the N-terminus and three consecutive CDC10/SW16/ankyrin repeats in the C-terminus . Transactivation assays in the yeast hybrid system and transient transfection assays in COS cells showed that a potent trans-activating domain resides in the N-terminus of this protein . Northern blot and RT-PCR assays showed that this gene is expressed in several fetal tissues but not in adult tissues . Stable transfection assays provided evidence that the product of this gene may increase the level of gamma mRNA in HFE x MEL cell hybrids that undergo the gamma to beta switch, suggesting that this new gene encodes a protein that may function as gamma gene activator .

J Mol Biol, 2001 Jan 26, 305(4), 839 - 49
Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi{COO}-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi{CH2NH2+}-Asp19I; Bateman KS et al.; X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3) . The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi{COO}-Leu18I) . Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors . The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I . The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond . In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi{COO}-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group . Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding . A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi{CH2NH2+}-Asp19I) has also been determined by X-ray crystallography . This variant has very weak association equilibrium constants with SGPB and with chymotrypsin . The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor . Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole .

Genomics, 2001 Jan 15, 71(2), 222 - 34
SH3GLB, a new endophilin-related protein family featuring an SH3 domain; Pierrat B et al.; A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax . SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C . elegans genome (GenBank Accession No . U46675) . Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1 . The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions . Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action . Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus . SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells .

Genomics, 2001 Jan 15, 71(2), 174 - 81
Construction of a detailed physical and transcript map of the candidate region for Russell-Silver syndrome on chromosome 17q23-q24; Dorr S et al.; Russell-Silver syndrome (RSS) is a heterogeneous disorder characterized mainly by pre- and postnatal growth retardation and characteristic dysmorphic features . The genetic cause of this syndrome is unknown . However, two autosomal translocations involving chromosome 17q25 were reported in association with RSS . Molecular analysis of the breakpoint on chromosome 17 of the de novo translocation previously described as t(1;17)(q31;q25) enabled us to refine the localization of the chromosome 17 breakpoint to 17q23-q24 . Since no detailed mapping data were available for this region, we established a contig of yeast artificial chromosomes, P1 artificial chromosomes, bacterial artificial chromosomes, and cosmid clones for a 17q segment flanked by the sequence-tagged site (STS) markers D17S1557 and D17S940 . This contig covers a physical distance of 4-5 Mb encompassing several novel markers . A transcript map was constructed by assigning genes and expressed sequence tags to the clone contig, and altogether 74 STS markers were mapped . Furthermore, the locus order and content provide insight into several duplication events that have occurred in the chromosomal region 17q23-q24 . On the basis of our refined map, we have reduced the translocation breakpoint region to 65 kb between the newly derived markers 58T7 and CF20b . These data provide the molecular tools for the final identification of the RSS gene in 17q23-q24 .

Genomics, 2001 Jan 15, 71(2), 142 - 9
Characterization of human MAPRE genes and their proteins; Su LK et al.; The MAPRE genes encode the EB1 family proteins . The yeast EB1 protein had been shown to play important roles in microtubule dynamic regulation, cytokinesis, mitotic spindle positioning, and episome segregation . To facilitate functional studies of mammalian EB1 family proteins, we characterized the human MAPRE genes (MAPRE1, MAPRE2, and MAPRE3) and their proteins (EB1, RP1, and EBF3) . We found that the three MAPRE genes had similar genomic structures but were on different chromosomes . We showed that EB1 family proteins appeared to be expressed ubiquitously . We identified two EBF3 proteins, which were encoded by alternatively spliced MAPRE3 mRNAs . We demonstrated that there were also two RP1 proteins, which were products of translation from different initiation codons . We showed that the three EB1 family proteins had different abilities to interact with APC in vitro, and we provided the first direct evidence for the association between endogenous EB1 and APC .

Genomics, 2000 Dec 15, 70(3), 292 - 9
Radial transformation-associated recombination cloning from the mouse genome: isolation of Tg.AC transgene with flanking DNAs; Humble MC et al.; Transformation-associated recombination (TAR) cloning allows entire genes and large chromosomal regions to be specifically, accurately, and quickly isolated from total genomic DNA . We report the first example of radial TAR cloning from the mouse genome . Tg.AC mice carry a zeta-globin promoter/v-Ha-ras transgene . Fluorescence in situ hybridization localized the transgene integrant as a single site proximal to the centromere of chromosome 11 . Radial TAR cloning in yeast was utilized to create orientation-specific yeast artificial chromosomes (YACs) to explore the possibility that cis-flanking regions were involved in transgene expression . YACs containing variable lengths of 5' or 3' flanking chromosome 11 DNA and the Tg.AC transgene were specifically chosen, converted to bacterial artificial chromosomes (BACs), and assayed for their ability to promote transcription of the transgene following transfection into an FVB/N carcinoma cell line . A transgene-specific reverse transcription-polymerase chain reaction assay was utilized to examine RNA transcripts from stably transfected clones . All Tg.AC BACs expressed the transgene in this in vitro system . This report describes the cloning of the v-Ha-ras transgene and suggests that transcriptional activity may not require cis elements flanking the transgene's integration site .

Exp Cell Res, 2001 Feb 1, 263(1), 131 - 44
An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles; Dye BT et al.; Using fusions with green fluorescent protein (GFP), we have identified sequences in the polypyrimidine tract binding protein-associated splicing factor (PSF) that are involved in nuclear and subnuclear localization . Like other splicing factors, PSF localizes to the nucleus, is absent from nucleoli, and accumulates in punctate structures within the nucleus referred to as speckles . However, PSF lacks the known speckle localization domains that have been identified in other proteins . Instead, the localization of PSF to speckles is dependent on an RNA recognition motif (RRM) . PSF comprises an N-terminal proline- and glutamine-rich domain, two RRMs (RRM1 and RRM2), and a C-terminal region that contains two nuclear localization signals, both of which are required for complete nuclear localization . Deletion of RRM2 led to a complete loss of speckle localization and resulted in diffuse accumulation of PSF in the nucleus, indicating that RRM2 is required for subnuclear localization . Thus, PSF appears to localize to speckles through a novel pathway that is dependent on its second RRM . Consistent with the use of a novel subnuclear targeting pathway, PSF redistributes to perinucleolar clusters upon the addition of a transcription inhibitor whereas other splicing factors display increased localization to speckles in the absence of transcription . A yeast two-hybrid screen identified four-and-a-half LIM-only protein 2 (FHL2) as a potential RRM2 interaction partner, indicating a possible role for zinc-finger or LIM domains in the localization of splicing factors to subnuclear speckles.

Mol Cell Neurosci, 2001 Jan, 17(1), 97 - 106
Functional characterization of two splice variants of rat bad and their interaction with Bcl-w in sympathetic neurons; Hamner S et al.; Neuronal cell death is in many cases regulated by competitive interactions between pro- and antiapoptotic proteins of the Bcl-2 family . In this study we have identified two splice variants of the rat proapoptotic molecule Bad, which differ in their carboxy-terminal regions . Both splice variants of Bad interacted with the antiapoptotic molecule Bcl-w as shown by yeast two-hybrid assay and by co-immunoprecipitation experiments from transfected cells . mRNA expression for the two variants of bad were detected in all neonatal and adult rat tissues tested . Overexpression of either of the two isoforms of Bad in nerve growth factor (NGF)-maintained sympathetic neurons by microinjection induced the cell death of these neurons, which was neutralized by co-expression of Bcl-w . Overexpression of Bcl-w in sympathetic neurons also counteracted death induced by NGF deprivation, which was not reduced by co-expression of either of the two Bad variants . The results suggest that Bcl-w, Bad-alpha, and Bad-beta may participate in the regulation of apoptosis in the sympathetic nervous system.

Nucleic Acids Res, 2001 Feb 1, 29(3), 767 - 73
Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription; Chao SH et al.; The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing . Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD . Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo . However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II . Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation . Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone . The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA . Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.

J Virol, 2001 Mar, 75(5), 2493 - 8
Self-association and mapping of the interaction domain of hepatitis E virus ORF3 protein; Tyagi S et al.; Hepatitis E virus (HEV) is a major human pathogen in the developing world . In the absence of an in vitro culture system, very little information on the basic biology of the virus exists . A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV . The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains . The C-terminal half of pORF3 is rich in proline residues and contains a putative src homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site . In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system . We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro . The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.

J Virol, 2001 Feb, 75(4), 2019 - 23
Hantavirus nucleocapsid protein oligomerization; Alfadhli A et al.; Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome . The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein . The N protein is a 420- to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood . We have undertaken a biochemical and genetic analysis of N protein oligomerization . Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers . Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer . We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions . Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion . These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils . Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates.

J Virol, 2001 Feb, 75(4), 1899 - 908
Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis; Momose F et al.; Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis . A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity . Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library . In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48-NP complex was dissociated following addition of free RNA . Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase . RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding . Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.

J Virol, 2001 Feb, 75(4), 1879 - 87
Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane; Van Der Heijden MW et al.; Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2 . P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain . Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity . A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction . In P2, the sequence of the N-terminal 241 aa was required for the interaction . In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies . While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell . As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M . A . Restrepo-Hartwig and P . Ahlquist, J . Virol . 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.

J Virol, 2001 Feb, 75(4), 1656 - 63
Role of vaccinia virus A20R protein in DNA replication: construction and characterization of temperature-sensitive mutants; Ishii K et al.; Previous analyses of randomly generated, temperature-sensitive vaccinia virus mutants led to the mapping of DNA synthesis negative complementation groups to the B1R, D4R, D5R, and E9L genes . Evidence from the yeast two-hybrid system that the D4R and D5R proteins can interact with the A20R protein suggested that A20R was also involved in DNA replication . We found that the A20R gene was transcribed early after infection, consistent with such a role . To investigate the function of the A20R protein, targeted mutations were made by substituting alanines for charged amino acids occurring in 11 different clusters . Four mutants were not isolated, suggesting that they were lethal, two mutants exhibited no temperature sensitivity, two mutants exhibited partial temperature sensitivity, and two mutants formed no plaques or infectious virus at 39 degrees C . The two mutants with stringent phenotypes were further characterized . Temperature shift-up experiments indicated that the crucial period was between 6 and 12 h after infection, making it unlikely that the defect was in virus entry, early gene expression, or a late stage of virus assembly . Similar patterns of metabolically labeled viral early proteins were detected at permissive and nonpermissive temperatures, but one mutant showed an absence of late proteins under the latter conditions . Moreover, no viral DNA synthesis was detected when cells were infected with either stringent mutant at 39 degrees C . The previous yeast two-hybrid analysis together with the present characterization of A20R temperature-sensitive mutants suggested that the A20R, D4R, and D5R proteins are components of a multiprotein DNA replication complex.

J Virol, 2001 Feb, 75(4), 1601 - 10
ATP-dependent simian virus 40 T-antigen-Hsc70 complex formation; Sullivan CS et al.; Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation . T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly . J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates . It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities . However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53 . In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70) . Furthermore, the T-antigen--Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 microM), but T-antigen--Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site . N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis . Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain . These results provide direct evidence that the T-antigen--Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70 . This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.

J Neurosci, 2001 Jan 15, 21(2), 423 - 33
Densin-180 forms a ternary complex with the (alpha)-subunit of Ca2+/calmodulin-dependent protein kinase II and (alpha)-actinin; Walikonis RS et al.; Densin-180 is a transmembrane protein that is tightly associated with the postsynaptic density in CNS neurons and is postulated to function as a synaptic adhesion molecule . Here we report the identification of the alpha-subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alpha-actinin-4 as potential binding partners for the densin-180 intracellular segment . We demonstrate by yeast two-hybrid and biochemical assays that the intracellular portion of densin-180, the alpha-subunit of CaMKII (CaMKIIalpha), and alpha-actinin interact with each other at distinct binding sites and can form a ternary complex stabilized by multiple interactions . Densin-180 binds specifically to the association domain of CaMKIIalpha and does not bind with high affinity to holoenzymes of CaMKII that contain beta-subunit . The PDZ (PSD-95, DIg, Z0-1) domain of densin contributes to its binding to alpha-actinin . A distinct domain of alpha-actinin interacts with the kinase domains of both alpha- and beta-subunits of CaMKII . Autophosphorylation of CaMKII increases its affinity for densin-180 from an EC(50) of >1 micrometer to an EC(50) of <75-150 nM . In contrast, phosphorylation of densin-180 by CaMKII at serine-1397 only slightly decreases its affinity for CaMKII . The specific interaction of densin-180 with holoenzymes of CaMKII containing only alpha-subunit and the increased affinity of CaMKII for densin-180 after autophosphorylation suggest that densin-180 may be involved in localization of activated CaMKII synthesized in dendrites.

J Lipid Res, 2001 Jan, 42(1), 128 - 36
Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis; Bellamine A et al.; Sterol 14 alpha-demethylase (14DM) is a cytochrome P-450 involved in sterol biosynthesis in eukaryotes . It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium tuberculosis (MT) infection, although the origin of the cholesterol is unknown . A protein product from MT having about 30% sequence identity with eukaryotic 14 alpha-demethylases has been found to convert sterols to their 14-demethyl products indicating that a sterol pathway might exist in MT . To determine the optimal sterol structure recognized by MT 14DM, binding of 28 sterol and sterol-like (triterpenoids) molecules to the purified recombinant 14 alpha-demethylase was examined . Like eukaryotic forms, a 3 beta-hydroxy group and a 14 alpha-methyl group are essential for substrate acceptability by the bacterial 14 alpha-demethylase . The high affinity binding of 31-norcycloartenol without detectable activity indicates that the Delta(8)-bond is required for activity but not for binding . As for plant 14 alpha-demethylases, 31-nor-sterols show a binding preference for MT 14DM . Similar to enzymes from mammals and yeast, a C24-alkyl group is not required for MT 14DM binding and activity, whereas it is for plant 14 alpha-demethylases.Thus, substrate binding to MT 14DM seems to share common features with all eukaryotic 14 alpha-demethylases, the MT form seemingly having the broadest substrate recognition of all forms of 14 alpha-demethylase studied so far . - Bellamine, A., A . T . Mangla, A . L . Dennis, W . D . Nes, and M . R . Waterman . Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis . J . Lipid Res . 2001 . 42: 128;-136.

J Lipid Res, 2001 Jan, 42(1), 60 - 9
Determinants of human apolipoprotein {a} secretion from mouse hepatocyte cultures; Wang J et al.; Efforts to develop an in vitro model system to analyze apolipoprotein {a} (apo{a}) gene transcription, mRNA translation, and protein secretion have been complicated by the limited tissue and species distribution of apo{a} and the presence of regulatory DNA sequences remote from the apo{a} transcription start site . In the current study we examined primary hepatocytes cultured from apo{a} transgenic mice as a model system for analyzing apo{a} biogenesis . Hepatocytes from mice transgenic for a yeast artificial chromosome (YAC) encoding the entire apo{a} gene in its own genomic context (YAC-apo{a} hepatocytes) were unable to maintain apo{a} expression beyond 48 h of culture . This suggests that the apo{a} promoter was not active in cultured YAC-apo{a} hepatocytes . In contrast, apo{a} expression was maintained for at least 7 days in hepatocytes cultured from mice transgenic for an apo{a} cDNA under control of the mouse transferrin promoter (transferrin-apo{a} hepatocytes) . Pulse-chase experiments established that more than 80% of apo{a} synthesized by both transferrin-apo{a} and YAC-apo{a} hepatocytes was degraded prior to secretion, independently of the coexpression of human apoB.Thus, low secretion efficiency appears to be a general characteristic of human apo{a} proteins in mouse liver . Apo{a} secretion was increased somewhat (from 18% to 32%) in the presence of lipoprotein-containing serum . Transformed cell lines derived from transferrin apo{a} hepatocytes retained characteristics of apo{a} secretion similar to those observed in primary cells . Primary and transformed apo{a} transgenic hepatocytes may provide valuable additional models with which to study posttranslational mechanisms regulating apo{a} secretion . - Wang, J., J . Boedeker, H . H . Hobbs, and A . L . White . Determinants of human apolipoprotein {a} secretion from mouse hepatocyte cultures . J . Lipid Res . 2001 . 42: 60;-69.

J Immunol, 2001 Feb 15, 166(4), 2665 - 73
The WI-1 adhesin blocks phagocyte TNF-alpha production, imparting pathogenicity on Blastomyces dermatitidis; Finkel-Jimenez B et al.; The WI-1 adhesin is indispensable for pathogenicity of Blastomyces dermatitidis and is thought to promote pulmonary infection by fixing yeast to lung tissue and cells . Recent findings suggest that WI-1 confers pathogenicity by mechanisms in addition to adherence . Here, we investigated whether WI-1 modulates host immunity by altering production of pro-inflammatory cytokines . Production of TNF-alpha in lung alveolar fluids of mice infected with B . dermatitidis was severalfold higher for WI-1 knockout yeast compared with wild-type yeast, and in vitro coculture of unseparated lung cells with these isogenic yeast disclosed similar differences . Upon coculture with purified macrophages and neutrophils, wild-type yeast blocked TNF-alpha production, yet WI-1 knockout yeast stimulated production . Coating knockout yeast with purified WI-1 converted them from stimulating TNF-alpha production to inhibiting production . Addition of purified WI-1 into stimulated phagocyte cultures led to concentration-dependent inhibition of TNF-alpha production . Neutralization of TNF-alpha in vivo exacerbated experimental pulmonary infection, particularly for the nonpathogenic WI-1 knockout yeast . Inducing increased TNF-alpha levels in the lung by adenovirus-vectored gene therapy controlled infection with wild-type yeast . Thus, the WI-1 adhesin on yeast modulates host immunity through blocking TNF-alpha production by phagocytes, which fosters progression of pulmonary infection.

J Immunol, 2001 Feb 1, 166(3), 1763 - 70
Constitutive association of SHP-1 with leukocyte-associated Ig-like receptor-1 in human T cells; Sathish JG et al.; The intracellular Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP-1) is a negative regulator of cell signaling and contributes to the establishment of TCR signaling thresholds in both developing and mature T lymphocytes . Although there is much functional data implicating SHP-1 as a regulator of TCR signaling, the molecular basis for SHP-1 activation in T lymphocytes is poorly defined . A modification of the yeast two-hybrid system was employed to identify in T cells phosphotyrosine-containing proteins capable of binding the SH2 domains of SHP-1 . From this yeast tri-hybrid screen, the p85beta subunit of phosphatidylinositol 3-kinase and the immunoreceptor tyrosine-based inhibitory motif-containing receptors, leukocyte-associated Ig-like receptor-1 (LAIR-1) and programmed death-1 (PD-1), were identified . Coimmunoprecipitation studies demonstrated that the exclusive phosphotyrosine-containing protein associated with SHP-1 in Jurkat T cells under physiological conditions is LAIR-1 . Significantly, this interaction is constitutive and was detected only in the membrane-enriched fraction of cell lysates . Ligand engagement of the SH2 domains of SHP-1 is a prerequisite to activation of the enzyme, and, consistent with an association with LAIR-1, SHP-1 was found to be constitutively active in unstimulated Jurkat T cells . Importantly, a constitutive interaction between LAIR-1 and SHP-1 was also detected in human primary T cells . These results illustrate the sustained recruitment and activation of SHP-1 at the plasma membrane of resting human T cells by an inhibitory receptor . We propose that this mechanism may exert a constitutive negative regulatory role upon T cell signaling.

J Immunol, 2001 Feb 1, 166(3), 1710 - 5
Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi; Miyazawa S et al.; Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B . To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals . Using degenerate primers, we isolated two integrin alpha subunits (alpha(Hr1) and alpha(Hr2)) from the hemocyte mRNA of H . roretzi, by RT-PCR, and the entire coding sequence of alpha(Hr1) was determined from cDNA clones . alpha(Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family . A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha(Hr1) in the monophyletic cluster of I domain-containing alpha integrins . The alpha(Hr1) gene shows hemocyte-specific expression on Northern blot analysis . Western blot analysis and immunocytochemical staining of the hemocytes of H . roretzi using anti-alpha(Hr1) Ab showed that alpha(Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes . Furthermore, anti-alpha(Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes . These observations strongly suggest that alpha(Hr1) constitutes an integrin molecule on the hemocytes of H . roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian.

Blood, 2001 Feb 15, 97(4), 901 - 10
mDYRK3 kinase is expressed selectively in late erythroid progenitor cells and attenuates colony-forming unit-erythroid development; Geiger JN et al.; DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1, an inhibitor of cell cycle progression in yeast . At present, mDYRK-3 and mDYRK-2 have been cloned, and mDYRK-3 has been characterized with respect to kinase activity, expression among tissues and hematopoietic cells, and possible function during erythropoiesis . In sequence, mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a, but is 91.3% identical overall to hDYRK-3 . Catalytically, mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro . Expression of mDYRK-1a, mDYRK-2, and mDYRK-3 was high in testes, but unlike mDYRK1a and mDYRK 2, mDYRK-3 was not expressed at appreciable levels in other tissues examined . Among hematopoietic cells, however, mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells . In developmentally synchronized erythroid progenitor cells, expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone), and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver . Interestingly, antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation . Thus, it is proposed that mDYRK-3 kinase functions as a lineage-restricted, stage-specific suppressor of red cell development . (Blood . 2001;97:901-910)

Am J Physiol Lung Cell Mol Physiol, 2001 Feb, 280(2), L369 - 75
Association between airway hyperreactivity and bronchial macrophage dysfunction in individuals with mild asthma; Alexis NE et al.; Little is known about the functional capabilities of bronchial macrophages (BMs) and their relationship to airway disease such as asthma . We hypothesize that BMs from asthmatics may be modulated in their function compared with similar cells from healthy individuals . BMs obtained by induced sputum from mild asthmatics (n = 20) and healthy individuals (n = 20) were analyzed using flow cytometry for CD16, CD64, CD11b, CD14, and human leukocyte antigen-DR expression, phagocytosis of IgG opsonized yeast, and oxidant production . Asthma status was assessed by lung function {percent predicted forced vital capacity and forced expiratory volume in 1 s (FEV(1))}, percent sputum eosinophils, and nonspecific airway responsiveness {provocative concentration that produces a 20% fall in FEV(1) (PC(20,FEV1))} . Asthmatics with >5% airway eosinophils (AEo+) had decreased BM CD64 expression and phagocytosis compared with asthmatics with <5% eosinophils (AEo-) . Among asthmatics, a significant correlation was found between CD64 expression and BM phagocytosis (R = 0.7, P < 0.009) . Phagocytosis was also correlated with PC(20,FEV1) (R = 0.6, P < 0.007), lung function (%predicted FEV(1), R = 0.7, P < 0.002) and percent eosinophils (R = -0.6, P < 0.01) . In conclusion, BM from asthmatics are functionally modulated, possibly by Th2 cytokines involved in asthma pathology.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 920 - 5 Epub 2001 Jan 16.
Protein-protein interactions with subunits of human nuclear RNase P; Jiang T et al.; A yeast two-hybrid system was used to analyze interactions among the protein subunits of human nuclear RNase P themselves and with other interacting partners encoded in a HeLa cell cDNA library . Subunits hpop1, Rpp21, Rpp29, Rpp30, Rpp38, and Rpp40 are involved in extensive, but weak, protein-protein interactions in the holoenzyme complex . Rpp14, Rpp20, and Rpp30 were found to have strong interactions with proteins encoded in the cDNA library . The small heat shock protein 27, which interacts with Rpp20 in the two-hybrid assay, binds to Rpp20 during affinity chromatography and can be found to be associated with, and enhances the activity of, highly purified RNase P . RNase P activity in HeLa cell nuclei also increases under the stress of heat shock.

Plant Cell, 2001 Jan, 13(1), 89 - 100
A mutation of the mitochondrial ABC transporter Sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik; Kushnir S et al.; A mutation in the Arabidopsis gene STARIK leads to dwarfism and chlorosis of plants with an altered morphology of leaf and cell nuclei . We show that the STARIK gene encodes the mitochondrial ABC transporter Sta1 that belongs to a subfamily of Arabidopsis half-ABC transporters . The severity of the starik phenotype is suppressed by the ectopic expression of the STA2 homolog; thus, Sta1 function is partially redundant . Sta1 supports the maturation of cytosolic Fe/S protein in Deltaatm1 yeast, substituting for the ABC transporter Atm1p . Similar to Atm1p-deficient yeast, mitochondria of the starik mutant accumulated more nonheme, nonprotein iron than did wild-type organelles . We further show that plant mitochondria contain a putative l-cysteine desulfurase . Taken together, our results suggest that plant mitochondria possess an evolutionarily conserved Fe/S cluster biosynthesis pathway, which is linked to the intracellular iron homeostasis by the function of Atm1p-like ABC transporters.

Mol Cell Biol, 2001 Feb, 21(4), 1077 - 88
SH2 domain-mediated interaction of inhibitory protein tyrosine kinase Csk with protein tyrosine phosphatase-HSCF; Wang B et al.; The protein tyrosine kinase (PTK) Csk is a potent negative regulator of several signal transduction processes, as a consequence of its exquisite ability to inactivate Src-related PTKs . This function requires not only the kinase domain of Csk, but also its Src homology 3 (SH3) and SH2 regions . We showed previously that the Csk SH3 domain mediates highly specific associations with two members of the PEP family of nonreceptor protein tyrosine phosphatases (PTPs), PEP and PTP-PEST . In comparison, the Csk SH2 domain interacts with several tyrosine phosphorylated molecules, presumed to allow targetting of Csk to sites of Src family kinase activation . Herein, we attempted to understand better the regulation of Csk by identifying ligands for its SH2 domain . Using a modified yeast two-hybrid screen, we uncovered the fact that Csk associates with PTP-HSCF, the third member of the PEP family of PTPs . This association was documented not only in yeast cells but also in a heterologous mammalian cell system and in cytokine-dependent hemopoietic cells . Surprisingly, the Csk-PTP-HSCF interaction was found to be mediated by the Csk SH2 domain and two putative sites of tyrosine phosphorylation in the noncatalytic portion of PTP-HSCF . Transfection experiments indicated that Csk and PTP-HSCF synergized to inhibit signal transduction by Src family kinases and that this cooperativity was dependent on the domains mediating their association . Finally, we obtained evidence that PTP-HSCF inactivated Src-related PTKs by selectively dephosphorylating the positive regulatory tyrosine in their kinase domain . Taken together, these results demonstrate that part of the function of the Csk SH2 domain is to mediate an inducible association with a PTP, thereby engineering a more efficient inhibitory mechanism for Src-related PTKs . Coupled with previously published observations, these data also establish that Csk forms complexes with all three known members of the PEP family.

Mol Cell Biol, 2001 Feb, 21(4), 1066 - 76
p53 down-regulates CHK1 through p21 and the retinoblastoma protein; Gottifredi V et al.; Both fission yeast and mammalian cells require the function of the checkpoint kinase CHK1 for G2 arrest after DNA damage . The tumor suppressor p53, a well-studied stress response factor, has also been shown to play a role in DNA damage G2 arrest, although in a manner that is probably independent of CHK1 . p53, however, can be phosphorylated and regulated by both CHK1 as well as another checkpoint kinase, hCds1 (also called CHK2) . It was therefore of interest to determine whether reciprocally, p53 affects either CHK1 or CHK2 . We found that induction of p53 either by diverse stress signals or ectopically using a tetracycline-regulated promoter causes a marked reduction in CHK1 protein levels . CHK1 downregulation by p53 occurs as a result of reduced CHK1 RNA accumulation, indicating that repression occurs at the level of transcription . Repression of CHK1 by p53 requires p21, since p21 alone is sufficient for this to occur and cells lacking p21 cannot downregulate CHK1 . Interestingly, pRB is also required for CHK1 downregulation, suggesting the possible involvement of E2F-dependent transcription in the regulation of CHK1 . Our results identify a new repression target of p53 and suggest that p53 and CHK1 play interdependent and complementary roles in regulating both the arrest and resumption of G2 after DNA damage.

J Cell Biol, 2001 Feb 5, 152(3), 579 - 94
Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin; Govind S et al.; Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells . To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay . Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target . IRS-58 is a brain-enriched protein comprising at least four protein-protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain . Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters . In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity . Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells . In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia . An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization . These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

J Cell Biol, 2001 Feb 5, 152(3), 503 - 18
Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex; Otte S et al.; Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching . We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles . Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles . Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species . Erv41p and Erv46p were further characterized . These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation . Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity . The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain . When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains . A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.

Hum Mol Genet, 2001 Feb 15, 10(4), 383 - 94
Large-scale evaluation of imprinting status in the Prader-Willi syndrome region: an imprinted direct repeat cluster resembling small nucleolar RNA genes; Meguro M et al.; Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader-Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive . Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established . Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS . Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15 . Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval . This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion . Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs) . Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, XENOPUS: and DROSOPHILA: These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.

Hum Mol Genet, 2001 Feb 15, 10(4), 353 - 60
Functional analysis of BRCA1 C-terminal missense mutations identified in breast and ovarian cancer families; Vallon-Christersson J et al.; Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1 are responsible for the majority of cases involving hereditary breast and ovarian cancer . Whereas all truncating mutations are considered as functionally deleterious, most of the missense variants identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms . The C-terminal domain of BRCA1 displays an intrinsic transactivation activity, and mutations linked to disease predisposition have been shown to confer loss of such activity in yeast and mammalian cells . In an attempt to clarify the functional importance of the BRCA1 C-terminus as a transcription activator in cancer predisposition, we have characterized the effect of C-terminal germline variants identified in Scandinavian breast and ovarian cancer families . Missense variants A1669S, C1697R, R1699W, R1699Q, A1708E, S1715R and G1738E and a truncating mutation, W1837X, were characterized using yeast- and mammalian-based transcription assays . In addition, four additional missense variants (V1665M, D1692N, S1715N and D1733G) and one in-frame deletion (V1688del) were included in the study . Our findings demonstrate that transactivation activity may reflect a tumor-suppressing function of BRCA1 and further support the role of BRCA1 missense mutations in disease predisposition . We also report a discrepancy between results from yeast- and mammalian-based assays, indicating that it may not be possible to unambiguously characterize variants with the yeast assay alone . We show that transcription-based assays can aid in the characterization of deleterious mutations in the C-terminal part of BRCA1 and may form the basis of a functional assay.

Genome Res, 2001 Feb, 11(2), 266 - 73
Establishment of a chemical synthetic lethality screen in cultured human cells; Simons A et al.; The synthetic lethality screen is a powerful genetic method for unraveling functional interactions between proteins in yeast . Here we demonstrate the feasibility of a chemical synthetic lethality screen in cultured human cells, based in part on the concept of the yeast method . The technology employs both an immortalized human cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the gene of interest, and the use of a novel double-label fluorescence system . Selective pressure imposed by any one of several synthetic lethal metabolic inhibitors prevented the spontaneous loss of the episomal survival plasmid . Retention or loss over time of this plasmid could be sensitively detected in a blind test, while cells were grown in microtiter plates . Application of this method should thus permit high throughput screening of drugs, which are synthetically lethal with any mutant human gene of interest, whose normal counterpart can be expressed . This usage is particularly attractive for the search of drugs, which kill malignant cells in a gene-specific manner, based on their predetermined cellular genotype . Moreover, by replacing the chemicals used in this example with a library of either DNA oligonucleotides or expressible dominant negative genetic elements, one should be able to identify synthetic lethal human genes.

Genes Dev, 2001 Jan 15, 15(2), 173 - 87
Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression; Sullivan E et al.; Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem-loop . Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3' end . Stem-loop-binding protein (SLBP) binds to the histone mRNA 3' end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation . To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP . dSLBP function is required both zygotically and maternally . Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells . These histone mRNAs are cytoplasmic and have polyadenylated 3' ends like other polymerase II transcripts . Hypomorphic dSLBP alleles support zygotic development but cause female sterility . Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles . This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase . These data demonstrate that dSLBP is required in vivo for 3' end processing of histone pre-mRNA, and that this is an essential function for development . Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle.

EMBO J, 2001 Feb 1, 20(3), 499 - 509
Modulation of Drosophila heat shock transcription factor activity by the molecular chaperone DROJ1; Marchler G et al.; Heat shock transcription factors (HSFs) play important roles in the cellular response to physiological stress signals . To examine the control of HSF activity, we undertook a yeast two-hybrid screen for proteins interacting with Drosophila HSF . DROJ1, the fly counterpart of the human heat shock protein HSP40/HDJ1, was identified as the dominant interacting protein (15 independent isolates from 58 candidates) . Overexpression of DROJ1 in Drosophila SL2 cells delays the onset of the heat shock response . Moreover, RNA interference involving transfection of SL2 cells with double-stranded droj1 RNA depletes the endogenous level of DROJ1 protein, leading to constitutive activation of endogenous heat shock genes . The induction level, modest when DROJ1 was depleted alone, reached maximal levels when DROJ1 and HSP70/HSC70, or DROJ1 and HSP90, were depleted concurrently . Chaperone co-depletion was also correlated with strong induction of the DNA binding activity of HSF . Our findings support a model in which synergistic interactions between DROJ1 and the HSP70/HSC70 and HSP90 chaperones modulate HSF activity by feedback repression.

Appl Environ Microbiol, 2001 Feb, 67(2), 938 - 41
Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes; Stender H et al.; A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described . The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis . The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy . The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D . bruxellensis and that the new method is able to identify Brettanomyces (D . bruxellensis) with 100% sensitivity and 100% specificity.

Genetics, 2001 Feb, 157(2), 765 - 75
Molecular Dissection of the 5' Region of no-on-transientA of Drosophila melanogaster Reveals cis-Regulation by Adjacent dGpi1 Sequences; Sandrelli F et al.; The nonA gene of Drosophila melanogaster is important for normal vision, courtship song, and viability and lies approximately 350 bp downstream of the dGpi1 gene . Full rescue of nonA mutant phenotypes can be achieved by transformation with a genomic clone that carries approximately 2 kb of 5' regulatory material and that encodes most of the coding sequence of dGpi1 . We have analyzed this 5' region by making a series of deleted fragments, fusing them to yeast GAL4 sequences, and driving UAS-nonA expression in a mutant nonA background . Regions that both silence and enhance developmental tissue-specific expression of nonA and that are necessary for generating optomotor visual responses are identified . Some of these overlap the dGpi1 sequences, revealing cis-regulation by neighboring gene sequences . The largest 5' fragment was unable to rescue the normal electroretinogram (ERG) consistently, and no rescue at all was observed for the courtship song phenotype . We suggest that sequences within the nonA introns that were missing in the UAS-nonA cDNA may carry enhancer elements for these two phenotypes . Finally, we speculate on the striking observation that some of the cis-regulatory regions of nonA appear to be embedded within the coding regions of dGpi1.

Sex Transm Infect, 1999 Dec, 75(6), 417 - 9
Microscopic features of vaginal candidiasis and their relation to symptomatology; Sonnex C et al.; OBJECTIVES: To document the microscopic features of vaginal candidiasis and to examine the relation between yeast morphology and patient symptomatology . METHOD: The study population comprised women undergoing screening for genital infection at a department of genitourinary medicine . RESULTS/CONCLUSION: Data were collected on 267 women of whom 234 were found to have vaginal candidiasis by vaginal culture . The remaining 33 patients had microscopic features of candidiasis (spores and/or hyphae) but were culture negative . Of the culture positive women, microscopy was positive in 182 (78%) . "Spores only" were identified in 65 (28%), "hyphae only" in 16 (7%), and both "spores and hyphae" in 101 (43%) . 68% of culture positive women were symptomatic, the commonest symptoms being irritation alone (27%) or irritation plus vaginal discharge (25%) . No association was found between yeast morphology (spores, budding/non-budding; hyphae, branching/non-branching) as identified on microscopy of vaginal secretions and symptomatology.

Int J Cancer, 2000 Mar 20, 89(2), 187 - 93
Clinical significance of p53 functional loss in squamous cell carcinoma of the oropharynx; Obata A et al.; We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations.We also explored the clinical importance of p53 mutations in oropharyngeal SCC . An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast-based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations . The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations . Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations . Subsequently, the relationship between p53 mutations and the clinico-pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay . Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation . Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption . Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy . The results showed that tumors with wild-type p53 decreased in size significantly compared to those with mutant p53 . In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild-type p53 tumor than in patients with a mutant p53 tumor . We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC .

J Biol Chem, 2000 Apr 14, 275(15), 10767 - 71
A G protein-coupled receptor for UDP-glucose; Chambers JK et al.; Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates . It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist . Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells . Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor . The receptor is expressed in a wide variety of human tissues, including many regions of the brain . These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

Curr Opin Biotechnol, 2000 Apr, 11(2), 162 - 7
Monitoring genome-wide expression in plants; Schaffer R et al.; When completed this year, the Arabidopsis genome will represent the first plant genome to be fully sequenced . This sequence information, together with the large collection of expressed sequence tags, has established the basics for new approaches to studying gene expression patterns in plants on a global scale . We can now look at biology from the perspective of the whole genome . This revolution in the study of how all genes in an organism respond to certain stimuli has encouraged us to think in new dimensions . Expression profiles can be determined over a range of experimental conditions and organized into patterns that are diagnostic for the biological state of the cell . The field of genome-wide expression in plants has yet to produce its fruit; however, the current application of microarrays in yeast and human research foreshadows the diverse applications this technology could have in plant biology and agriculture.

Biochem Biophys Res Commun, 2000 Apr 13, 270(2), 504 - 9
The carboxyl terminal domain of phosducin functions as a transcriptional activator; Zhu X et al.; In previous work, we identified a set of phosducin (Phd) isoforms with unknown function including the phosducin (Phd)-like orphan protein 1 (PhLOP1), an amino terminal truncated isoform of the retinal Phd lacking the Gbetagamma binding domain . To investigate the potential biological function of PhLOP1, PhLOP1 was fused at its amino terminus with the DNA binding domain (BD) of the yeast transcriptional factor, GAL4, and used as bait in a yeast two-hybrid screen . Two potential functional protein partners were identified during the screen: SUG1, a subunit of the 26S proteasome and a putative transcriptional mediator, and CRX, a retina- and pineal-specific transcription factor . Upon localizing the interacting domain of PhLOP1 with one of the new partners, SUG1, we found that a domain of 40 amino acids at the carboxyl terminus of Phd and PhLOP1 had intrinsic transcriptional activation activity in yeast . The transactivation activity was further confirmed in mammalian cells . This region contains an acidic domain that has been shown to be involved in the function of several transcriptional activators . In addition, we showed that Phd is cytoplasmic while PhLOP1 is localized predominantly to the nucleus when fused to an enhanced green fluorescent protein (EGFP) and transiently expressed in transfected cells, suggesting that PhLOP1 may play a distinct functional role in transcriptional regulation independent of the known Phd interaction/regulation of Gbetagamma transduction .

Allergy, 2000 Mar, 55(3), 274 - 80
Allergens of Epicoccum nigrum grown in different media for quality source material; Bisht V et al.; BACKGROUND: The Epicoccum nigrum (EN) extract used in allergy disorders exhibits batch-to-batch variations in protein composition and allergenic potency . In this study, the allergens of EN grown in different media were investigated . METHODS: EN was grown in five different nutrient media as stationary cultures at 25 degrees C for 5-23 days . The growth pattern was characterized by measuring dry weight, protein and carbohydrate content . The antigenic and allergenic content of EN extract was evaluated with EN-positive patients' sera and antibodies raised in rabbit . RESULTS: The growth of EN in Czapeck Dox medium yielded insufficient material, while Sabouraud's broth with yeast extract (SBY) gave maximum spore-mycelial mass and protein content . Potato dextrose broth (PDB) and potato dextrose agar (PDA) showed higher dry weight and protein in 7-9-day cultures . SDS-PAGE resolved 26, 22, and 21 protein bands in EN extracts from cultures of day-13 SBY, day-7 PDB, and day-9 PDA, respectively . IgE/IgG immunoblots showed more allergenic (25)/antigenic (25) bands in EN cultured in SBY than in the others . Specific IgE ELISA and intradermal tests showed EN extract from day-13 culture in SBY to be the most potent . CONCLUSIONS: The day-13 culture of EN in SBY was the most potent and may be selected for preparing EN extracts for diagnosis of allergy and future studies.

Acta Oncol, 2000, 39(1), 71 - 5
Gynaecological infections as risk determinants of subsequent cervical neoplasia; Viikki M et al.; A longitudinal cohort study was carried out to determine whether gynaecological infections other than human papillomavirus (HPV) are also related to the subsequent increased risk of cervical neoplasia . The study comprised 19114 women attending the organized mass screening in Finland in 1985-1990 with cytologically detected HPV, Actinomyces, herpes simplex, Trichomonas vaginalis, or yeast . The women were followed-up for subsequent preinvasive lesions and invasive cancers until the end of 1994 by linkage to the nation-wide Cancer Registry . Standardized incidence ratios (SIR) with rates for the whole of Finland as reference and 95% confidence intervals (CI) were estimated . Trichomonas vaginalis and HPV were associated with a high relative risk of cervical cancer, SIR 6.4 (CI 3.7-10, preinvasive lesion and invasive cancer combined) and SIR 5.5 (CI 4.2 7.2, preinvasive lesion and invasive cancer combined), respectively . Herpes simplex was rarely detected, but the highest and statistically most significant point estimate was observed (SIR 12, CI 2.4-34, preinvasive lesion and invasive cancer combined) . Neither Actinomyces nor yeast was associated with a significantly increased risk of cervical cancer . None of these results could be accounted for by the confounding effect of the other infections . Our results, based on a prospective design, lead us to propose that Trichomonas vaginalis and herpes simplex virus are also predictors for cervical neoplasia.

J Biol Chem, 2001 Apr 20, 276(16), 12974 - 82 Epub 2001 Jan 09.
Interaction of serotonin 5-hydroxytryptamine type 2C receptors with PDZ10 of the multi-PDZ domain protein MUPP1; Becamel C et al.; By using the yeast two-hybrid system, we previously isolated a cDNA clone encoding a novel member of the multivalent PDZ protein family called MUPP1 containing 13 PDZ domains . Here we report that the C terminus of the 5-hydroxytryptamine type 2C (5-HT(2C)) receptor selectively interacts with the 10th PDZ domain of MUPP1 . Mutations in the extreme C-terminal SSV sequence of the 5-HT(2C) receptor confirmed that the SXV motif is critical for the interaction . Co-immunoprecipitations of MUPP1 and 5-HT(2C) receptors from transfected COS-7 cells and from rat choroid plexus verified this interaction in vivo . Immunocytochemistry revealed an SXV motif-dependent co-clustering of both proteins in transfected COS-7 cells as well as a colocalization in rat choroid plexus . A 5-HT(2C) receptor-dependent unmasking of a C-terminal vesicular stomatitis virus epitope of MUPP1 suggests that the interaction triggers a conformational change within the MUPP1 protein . Moreover, 5-HT(2A) and 5-HT(2B), sharing the C-terminal EX(V/I)SXV sequence with 5-HT(2C) receptors, also bind MUPP1 PDZ domains in vitro . The highest MUPP1 mRNA levels were found in all cerebral cortical layers, the hippocampus, the granular layer of the dentate gyrus, as well as the choroid plexus, where 5-HT(2C) receptors are highly enriched . We propose that MUPP1 may serve as a multivalent scaffold protein that selectively assembles and targets signaling complexes.

J Biol Chem, 2001 Mar 30, 276(13), 10263 - 71 Epub 2001 Jan 08.
Fibroblast growth factor-binding protein is a novel partner for perlecan protein core; Mongiat M et al.; Perlecan, a widespread heparan sulfate proteoglycan, functions as a bioactive reservoir for growth factors by stabilizing them against misfolding or proteolysis . These factors, chiefly members of the fibroblast growth factor (FGF) gene family, are coupled to the N-terminal heparan sulfate chains, which augment high affinity binding and receptor activation . However, rather little is known about biological partners of the protein core . The major goal of this study was to identify novel proteins that interact with the protein core of perlecan . Using the yeast two-hybrid system and domain III of perlecan as bait, we screened approximately 0.5 10(6) cDNA clones from a keratinocyte library and identified a strongly interactive clone . This cDNA corresponded to FGF-binding protein (FGF-BP), a secreted protein previously shown to bind acidic and basic FGF and to modulate their activities . Using a panel of deletion mutants, FGF-BP binding was localized to the second EGF repeat of domain III, a region very close to the binding site for FGF7 . FGF-BP could be coimmunoprecipitated with an antibody against perlecan and bound in solution to recombinant domain III-alkaline phosphatase fusion protein . Immunohistochemical analyses revealed colocalization of FGF-BP and perlecan in the pericellular stroma of various squamous cell carcinomas suggesting a potential in vivo interaction . Thus, FGF-BP should be considered a novel biological ligand for perlecan, an interaction that could influence cancer growth and tissue remodeling.

J Biol Chem, 2001 Apr 27, 276(17), 14059 - 66 Epub 2001 Jan 08.
The 8-kDa dynein light chain binds to its targets via a conserved (K/R)XTQT motif; Lo KW et al.; Cytoplasmic dynein is a large, multisubunit molecular motor that translocates cargoes toward the minus ends of microtubules . Proper functioning of the dynein motor requires precise assembly of its various subunits . Using purified recombinant proteins, we show that the highly conserved 8-kDa light chain (DLC8) binds to the intermediate chain of the dynein complex . The DLC8-binding region was mapped to a highly conserved 10-residue fragment (amino acid sequence SYSKETQTPL) C-terminal to the second alternative splicing site of dynein intermediate chain . Yeast two-hybrid screening using DLC8 as bait identified numerous additional DLC8-binding proteins . Biochemical and mutational analysis of selected DLC8-binding proteins revealed that DLC8 binds to a consensus sequence containing a (K/R)XTQT motif . The (K/R)XTQT motif interacts with the common target-accepting grooves of DLC8 dimer . The role of each conserved amino acid residue in this pentapeptide motif in supporting complex formation with DLC8 was systematically studied using site-directed mutagenesis.

J Biol Chem, 2001 Apr 6, 276(14), 10683 - 91 Epub 2000 Dec 29.
The Golgi PMR1 P-type ATPase of Caenorhabditis elegans . Identification of the gene and demonstration of calcium and manganese transport; Van Baelen K et al.; In recent years, it has been well established that the Ca(2+) concentration in the lumen of intracellular organelles is a key determinant of cell function . Despite the fact that essential functions of the Golgi apparatus depend on the Ca(2+) and Mn(2+) concentration in its lumen, little is known on the transport system responsible for ion accumulation . The Golgi ion pump PMR1 has been functionally studied only in yeast . In humans, mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease . We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells . The C . elegans genome is predicted to contain a single PMR1 orthologue on chromosome I . We found evidence for alternative splicing in the 5'-untranslated region, but no indication for the generation of different protein isoforms . C . elegans PMR1 overexpressed in COS-1 cells transports Ca(2+) and Mn(2+) with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner . Part of the accumulated Ca(2+) can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca(2+) store.

J Biol Chem, 2001 Mar 23, 276(12), 8793 - 7 Epub 2000 Dec 28.
Phosphorylation and Cu+ coordination-dependent DNA binding of the transcription factor Mac1p in the regulation of copper transport; Heredia J et al.; Copper ions are essential at a proper level yet toxic when present in excess . To maintain a proper intracellular level, cells must be able to sense the changes in copper ion concentrations . The yeast transcription factor Mac1p plays a critical role in the transcriptional regulation of CTR1 and CTR3, both encoding high affinity copper ion transporters . Here we report that the Mac1p binding of the copper ion-responsive elements (CuREs) in the promoters of CTR1 and CTR3 is affected by copper ions . On one hand, the Mac1p DNA binding is Cu(+) coordination-dependent, and on the other hand, exogenous Cu(+) and isoelectronic Ag(+) ions disrupt the DNA binding of Mac1p . These results suggest that the Mac1p is able to sense two different levels of copper ions . These two levels are probably the physiological and toxic copper levels in yeast cells . Furthermore, we found that Mac1p undergoes posttranslational phosphorylation modification in yeast and that the phosphorylation is required for the Mac1p to become DNA-binding active . Nonphosphorylated Mac1p is unable to bind the CTR1 promoter DNA . The data support the model of intradomain interactions and indicate further that the phosphorylation probably prevents the inhibition of DNA-binding domain activity by the activation domain of Mac1p . Taken together, these findings demonstrate that Mac1p functions critically in maintaining a proper intracellular concentration of copper ions.

J Biol Chem, 2001 Mar 30, 276(13), 9726 - 32 Epub 2000 Dec 27.
A direct inhibitory role for the Rab3-specific effector, Noc2, in Ca2+-regulated exocytosis in neuroendocrine cells; Haynes LP et al.; Rab proteins comprise a family of GTPases, conserved from yeast to mammals, which are integral components of membrane trafficking pathways . Rab3A is a neural/neuroendocrine-specific member of the Rab family involved in Ca(2+) -regulated exocytosis, where it functions in an inhibitory capacity controlling recruitment of secretory vesicles into a releasable pool at the plasma membrane . The effector by which Rab3A exerts its inhibitory effect is unclear as the Rab3A effectors Rabphilin and RIM have been excluded from for this role . One putative Rab3A effector in dense-core granule exocytosis is the cytosolic zinc finger protein, Noc2 . We have established that overexpression of Noc2 in PC12 cells has a direct inhibitory effect upon Ca(2+)-triggered exocytosis in permeabilized cells . We demonstrate specific nucleotide-dependent binding of Noc2 to Rab3A and show that the inhibition of exocytosis is dependent upon this interaction since Rab3A binding-deficient mutants of Noc2 do not inhibit exocytosis . We propose that Noc2 may be a negative effector for Rab3A in regulated exocytosis of dense-core granules from endocrine cells.

Genome Res, 2001 Jan, 11(1), 12 - 27
Biased distribution of inverted and direct Alus in the human genome: implications for insertion, exclusion, and genome stability; Stenger JE et al.; Alu sequences, the most abundant class of large dispersed DNA repeats in human chromosomes, contribute to human genome dynamics . Recently we reported that long inverted repeats, including human Alus, can be strong initiators of genetic change in yeast . We proposed that the potential for interactions between adjacent, closely related Alus would influence their stability and this would be reflected in their distribution . We have undertaken an extensive computational analysis of all Alus (the database is at to better understand their distribution and circumstances under which Alu sequences might affect genome stability . Alus separated by <650 bp were categorized according to orientation, length of regions sharing high sequence identity, distance between highly identical regions, and extent of sequence identity . Nearly 50% of all Alu pairs have long alignable regions (>275 bp), corresponding to nearly full-length Alus, regardless of orientation . There are dramatic differences in the distributions and character of Alu pairs with closely spaced, nearly identical regions . For Alu pairs that are directly repetitive, approximately 30% have highly identical regions separated by <20 bp, but only when the alignments correspond to near full-size or half-size Alus . The opposite is found for the distribution of inverted repeats: Alu pairs with aligned regions separated by <20 bp are rare . Furthermore, closely spaced direct and inverted Alus differ in their truncation patterns, suggesting differences in the mechanisms of insertion . At larger distances, the direct and inverted Alu pairs have similar distributions . We propose that sequence identity, orientation, and distance are important factors determining insertion of adjacent Alus, the frequency and spectrum of Alu-associated changes in the genome, and the contribution of Alu pairs to genome instability . Based on results in model systems and the present analysis, closely spaced inverted Alu pairs with long regions of alignment are likely at-risk motifs (ARMs) for genome instability.

Cancer Res, 2000 Dec 15, 60(24), 7039 - 47
Identification of a caspase-2 isoform that behaves as an endogenous inhibitor of the caspase cascade; Droin N et al.; Procaspase-2 is one of the aspartate-specific cysteine proteases that are activated in response to various apoptotic stimuli . Two isoforms of human procaspase-2 have been described initially . Overexpression of the long isoform (caspase-2L) promotes cell death whereas the short isoform (caspase-2S) antagonizes some apoptotic pathways . In the present study, we identified two additional CASP-2 mRNAs, designated CASP-2L-Pro and CASP-2s-Pro . The proteins encoded by these isoforms corresponded to the prodomain of procaspase-2L and -2S, in which the last alpha-helix of their caspase recruitment domains was deleted . Caspase-2L-Pro mRNA and protein were detected in a series of human tissues and cell lines . Yeast 2-hybrid assays and immunoprecipitation studies indicated that caspase-2L-Pro can interact with procaspase-2L and the adaptor protein RAIDD/CRADD, but not with FADD/MORT1 or APAF-1 adaptor proteins . The addition of recombinant caspase-2L-Pro negatively interfered with cytochrome c/dATP-mediated activation of the caspase cascade in a cell-free system . In transient expression studies of human B lymphoma Namalwa cells, overexpression of caspase-2L-Pro weakly induced apoptosis, which was prevented by a D83A/E87A double mutation . In stable selected CASP-2L-Pro-transfected Namalwa cells, overexpression of caspase-2L-Pro delayed apoptotic DNA fragmentation induced by death receptor agonists (anti-Fas antibodies, tumor necrosis factor-alpha) and DNA topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and prevented etoposide-induced activation of the caspase cascade . These inhibitory effects were not observed in stable transfected cells expressing the D83A/E87A double mutant . Altogether, these data indicated that the caspase-2L-Pro isoform functions as an endogenous apoptosis inhibitory protein that antagonizes caspase activation and cell death.

Plant Physiol, 2001 Jan, 125(1), 339 - 50
Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation; Reichler SA et al.; The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome . A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported . These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis . To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings . These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes . These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome . Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance . After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase . Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis . Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis . Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

Vitam Horm, 2001, 61, 241 - 66
L-ascorbic acid biosynthesis; Smirnoff N; Biosynthesis of L-ascorbate (vitamin C) occurs by different pathways in plants and mammals . Yeast contain D-erythroascorbate, a C5 analog of ascorbate . UDP-D-glucuronic acid is the precursor in mammals . Loss of UDP forms glucuronic acid/glucuronolactone . Reduction of these at C-1 then forms L-gulonic acid/L-gulono-1,4-lactone . The lactone is oxidized by a microsomal L-gulono-1,4-lactone oxidase to ascorbate . Only the L-gulono-1,4-lactone oxidase has been purified and cloned, and very little is known about the properties of the other enzymes . Plants form ascorbate from GDP-D-mannose via GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone . The final oxidation of L-galactono-1,4-lactone to ascorbate is catalyzed by a mitochondrial L-galactono-1,4-lactone dehydrogenase located on the inner membrane and using cytochrome c as electron acceptor . GDP-mannose pyrophosphorylase and L-galactono-1,4-lactone dehydrogenase have been cloned . Yeast synthesizes D-erythroascorbate from D-arabinose and D-arabinono-1,4-lactone in a pathway analogous to that in plants . The plant, mammalian, and yeast aldonolactone oxidase/dehydrogenases that catalyze the last step in each pathway have significant sequence homology . L-Gulono-1,4-lactone oxidase is mutated and not expressed in animals, such as primates, that have lost ascorbate biosynthesis capacity . Assessment of the literature reveals that little is known about many of the enzymes involved in ascorbate biosynthesis or about the factors controlling flux through the pathways . There is also a possibility that minor alternative pathways exist in plants and mammals.

Cell Signal, 2000 Dec, 12(11-12), 769 - 79
SETA is a multifunctional adapter protein with three SH3 domains that binds Grb2, Cbl, and the novel SB1 proteins; Borinstein SC et al.; Expression of the src homology 3 (SH3)-encoding, expressed in tumorigenic astrocytes (SETA) gene is associated with astrocyte transformation in culture and tumors in the adult brain . SETA binds to the apoptosis regulator apoptosis-linked gene 2 (ALG-2) interacting protein 1 (AIP1), and modulates apoptosis in astrocytes . The predicted protein structure of SETA revealed two SH3 domains, while related proteins were reported to have three . Here we report the identification of an additional SH3 domain N-terminal to the previously identified SETA sequence . Yeast two-hybrid screening of a p53(-/-) astrocyte cDNA library with this SH3 domain identified a novel gene, SETA binding protein 1 (SB1), with 55% amino acid identity to the renal tumor antigen, NY-REN-45 . In vitro confrontation and co-immunoprecipitation experiments confirmed the binding of SB1 to SETA . Evidence that SETA binds to the CD2 protein, the proto-oncogene c-Cbl, and the signal transduction molecule Grb2, and can dimerize via its C-terminal coiled coil (CC) domain is also presented.

Am J Respir Cell Mol Biol, 2001 Jan, 24(1), 30 - 37
BR22, a novel protein, interacts with thyroid transcription factor-1 and activates the human surfactant protein B promoter; Yang YS et al.; Surfactant protein (SP)-B expression is restricted to type II pneumocytes and Clara cells in the lung . Previously, a promoter region of human SP-B gene from -64 to -118 has been identified as critical for the tissue-specific expression of this gene . Two cis-elements for thyroid transcription factor (TTF)-1 and hepatocyte nuclear factor (HNF)-3alpha binding were found within this area . Using an oligonucleotide fragment, we incorporated this region sequence into the promoter of a HIS3 reporter gene in yeast . With this modified yeast a human lung complementary DNA (cDNA) library was screened for DNA-binding proteins, other than TTF-1 and HNF-3alpha, that interacted with this promoter segment . A cDNA clone encoding a novel polypeptide, BR22, was identified that activated the reporter gene expression in yeast . This gene is expressed in many tissues and encodes a protein with bipartite nuclear localization signals . Studies using in vivo yeast two-hybrid analysis, in vitro protein-protein interactions, and coimmunoprecipitation analyses demonstrated that BR22 formed a protein complex with TTF-1 . In vivo cotransfection studies further indicated that BR22 could act with TTF-1 to synergistically activate the SP-B promoter in mammalian cells . Our data suggest that BR22 is a TTF-1-associated protein . Through a protein-protein interaction with TTF-1, BR22 can form a complex and activate the human SP-B promoter in vivo.

Eur J Cell Biol, 2000 Dec, 79(12), 986 - 92
The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization; Becker B et al.; Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans . This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain . To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells . Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization . The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi . In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi . A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi . Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization . A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi . These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.

Eur J Cell Biol, 2000 Dec, 79(12), 924 - 35
Nerve growth factor- and epidermal growth factor-regulated gene transcription in PC12 pheochromocytoma and INS-1 insulinoma cells; Groot M et al.; PC12 and INS-1 cells both express the nerve growth factor (NGF) receptors trkA and p75NTR and the epidermal growth factor receptor (EGF) . In PC12 cells, NGF treatment initiates a signaling cascade that ultimately leads to a change of the genetic program of the cell . We have investigated the role of NGF in regulating gene transcription in PC12 and INS-1 cells, in order to define if there are NGF-regulated genes per se . Furthermore, to distinguish between growth factor stimulation via receptor tyrosine kinases in general and NGF-specific changes in gene transcription, we analyzed the effects of EGF on gene transcription . First, we tested the biological activities of fusion proteins consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the phosphorylation-dependent activation domains of the transcription factors Elk1, CREB, ATF2 and c-jun in NGF- or EGF-treated PC12 cells . We found a striking increase in the transcriptional activity of the GAL4-Elk1 fusion protein that is a major substrate for the extracellular signal-regulated protein kinase (ERK) . This effect was observed in NGF- as well as in EGF-treated PC12 cells . In INS-1 cells, however, the activity of the GAL4-Elk1 fusion protein was induced by NGF, but not by EGF . The effects of NGF and EGF on gene transcription were subsequently studied with plasmids containing reporter genes under control of the Egr-1, c-jun, HES-1 or Bc12 regulatory sequences . NGF stimulated Egr-1 promoter activities in PC12 and INS-1 cells, although the effect was much more pronounced in PC12 cells than in INS-1 cells . EGF also stimulated Egr-1 promoter activity in both PC12 and INS-1 cells . Stimulation of c-jun promoter activity by NGF was observed only in PC12 cells . Deletion mutagenesis demonstrated the importance of the 12-O-tetradecanoylphorbol-13-acetate response elements within the c-jun promoter for basal and NGF-mediated transcriptional induction . Likewise, NGF activated HES1 and Bcl2 P1 promoter activities in PC12 cells but not in INS-1 cells and EGF did not show any effects on these promoters . We conclude that in PC12 and INS-1 cells, NGF signaling leads to an activation of the ERK subtype of mitogen-activated protein kinases in the nucleus and a subsequent activation of Egr-1 gene transcription . The NGF-induced transcription of the c-jun, HES1 and Bc12 genes is, in contrast, cell type-specific, indicating that NGF can trigger different gene expression programs dependent on the signaling pathways present in a particular cell type . EGF is clearly able to activate gene transcription, suggesting that the differences in the biological activities of EGF and NGF cannot be explained by the inability of EGF to stimulate gene transcription.

Chromosoma, 2000 Nov, 109(7), 498 - 505
Isolation and molecular analysis of inv dup(15) and construction of a physical map of a common breakpoint in order to elucidate their mechanism of formation; Wandstrat AE et al.; An inverted duplication of chromosome 15 {inv dup(15)} is the most common supernumerary marker chromosome, comprising approximately 50% of all chromosomes in this class . Structurally, the inv dup(15) is a mirror image with the central axis defining a distal break within either the heterochromatic alpha-satellite array or along the euchromatin in the long (q) arm of the chromosome . There are several types of inv dup(15), classified by the amount of euchromatic material present . Generally, they are bisatellited, pseudodicentric and have a breakpoint in 15q11-q14 . A suggested mechanism of formation of inv dup(15) involves illegitimate recombination between homologous chromosomes followed by nondisjunction and centromere inactivation . The proximal portion of chromosome 15 contains several low-copy repeat sequence families and it has been hypothesized that errors in pairing among these repeats may result in structural rearrangements of this chromosome including the inv dup(15) . To test this hypothesis and to determine the mechanism of formation, the inv dup(15) from four cases was isolated in somatic cell hybrids and polymerase chain reaction microsatellite markers were used to determine the origin of exchange . Two appeared to result from interchromosomal and two from intrachromosomal exchange, one of which occurred post-recombination . In addition, a detailed physical map of the breakpoint region in the largest inv dup(15) was constructed placing eight new sequence-tagged sites and ten new bacterial artificial chromosome markers in the region.

Chromosoma, 2000 Nov, 109(7), 482 - 9
Evolutionary conservation of kinetochore protein sequences in plants; ten Hoopen R et al.; The evolutionary conservation of structural/functional kinetochore proteins has been studied on isolated nuclei and pro-/metaphase chromosomes of mono- and dicot plants . The cross-reactivities of antibodies against human CENPC, CENPE and CENPF, and against maize CENPCa with the centromeric regions of mitotic chromosomes of Vicia faba and/or Hordeum vulgare are shown . Putative homologs of the kinetochore protein SKP1 (suppressor of kinetochore protein 1p of yeast) were found in both species and of CBF5p (centromere binding factor 5 of yeast) in barley . Antibodies against synthetic peptides derived from partial sequences encoding these proteins were produced and recognized the centromeric regions on mitotic chromosomes as detected by indirect immunofluorescence.

Trends Cardiovasc Med, 2000 Jan, 10(1), 15 - 9
In vivo modulation of human beta-globin gene switching; Tanimoto K et al.; Continuing accumulation of information from the genome projects requires parallel development of technologies to assess the in vivo functions of conserved sequences . We can now manipulate huge DNA molecules (such as yeast artificial chromosomes; YACs) in vivo, permitting the analysis of very large single genes or multigene loci as they exist in the chromosome . However, since transgenes integrate randomly into different chromatin environments, accurate evaluation of gene function in such transgenic mice is still fraught with pitfalls . We recently developed the use of cre-mediated homologous recombination to manipulate YAC transgenic mice in vivo, and successfully applied this strategy to the analysis of human beta-globin locus regulation . Here we describe these results and discuss the application of this technology to the analysis of this and related problems.

J Cell Biol, 2001 Jan 8, 152(1), 157 - 64
The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle; Kickhoefer VA et al.; Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1 . Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay . An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice . Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice . Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault . Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA . Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.

FASEB J, 2001 Jan, 15(1), 79 - 89
NOSIP, a novel modulator of endothelial nitric oxide synthase activity; Dedio J et al.; Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments . Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown . Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain . Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain . NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP . Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS . Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP . Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments . We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.

Plant Cell, 2000 Dec, 12(12), 2441 - 2454
Chilling tolerance in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases; Gomes E et al.; The lipid composition of membranes is a key determinant for cold tolerance, and enzymes that modify membrane structure seem to be important for low-temperature acclimation . We have characterized ALA1 (for aminophospholipid ATPase1), a novel P-type ATPase in Arabidopsis that belongs to the gene family ALA1 to ALA11 . The deduced amino acid sequence of ALA1 is homologous with those of yeast DRS2 and bovine ATPase II, both of which are putative aminophospholipid translocases . ALA1 complements the deficiency in phosphatidylserine internalization into intact cells that is exhibited by the drs2 yeast mutant, and expression of ALA1 results in increased translocation of aminophospholipids in reconstituted yeast membrane vesicles . These lines of evidence suggest that ALA1 is involved in generating membrane lipid asymmetry and probably encodes an aminophospholipid translocase . ALA1 complements the cold sensitivity of the drs2 yeast mutant . Downregulation of ALA1 in Arabidopsis results in cold-affected plants that are much smaller than those of the wild type . These data suggest a link between regulation of transmembrane bilayer lipid asymmetry and the adaptation of plants to cold.

J Cell Sci, 2001 Jan, 114(Pt 2), 389 - 99
Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization; Seipel K et al.; Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis . This actin remodeling is regulated in part by Rho family GTPases . Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases . Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development . Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance . Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region . Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies . Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II . Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies . Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin . Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events . Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B . We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.

Biochemistry, 2001 Jan 16, 40(2), 524 - 30
Amino acid residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity; Numata T et al.; The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordica charantia), consists of 190 amino acids and is characterized by specific cleavage at the 5'-side of uridine . Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73, and Arg74, at the alpha4-alpha5 loop between alpha4 and alpha5 helices for recognition of uracil base by RNase MC1 . Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1 were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylic acid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect to enzymatic activity . Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutant N71T exhibited 7.0-fold increased K(m) and 2.3-fold decreased k(cat), while the mutant L73A had 14.4-fold increased K(m), although it did retain the k(cat) value comparable to that of the wild-type . In contrast, replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had little effect on the enzymatic activity . This observation is consistent with findings in the crystal structure analysis that Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1 . The role of Asn71 in enzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln, and Asp . Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogen bonding, and the other is to stabilize the conformation of the alpha4-alpha5 loop by hydrogen bonding to the peptide backbone, events which possibly result in an appropriate orientation of the alpha-helix (alpha5) containing active site residues . Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity, as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA and homopolynucleotides.

J Cell Physiol, 2001 Jan, 186(1), 35 - 46
IRS-1 and IRS-2 are recruited by TrkA receptor and oncogenic TRK-T1; Miranda C et al.; TRK-T1 oncogene is generated by the rearrangement of the NGF receptor TrkA with TPR . This gives rise to the constitutive tyrosine autophosphorylation and activation of the kinase . To study TRK-T1 oncogenic signaling and compare it to that induced by the genuine receptor TrkA, we investigated the involvement of IRS-1, a docking protein implicated in mitogenic signaling induced by several growth factors, in TRK-T1 and TrkA signaling . Here, we show that IRS-1 and IRS-2 are phosphorylated on tyrosine in presence of both TRK-T1 and the activated TrkA receptor . These tyrosine phosphorylations lead to IRS-1- and IRS-2-induced recruitment of p85PI3K, SHP-2, and Grb2 and increase in PI 3-kinase activity associated with IRS-1 . Furthermore, we found that TRK-T1 is able to activate c-fos serum responsive element in cooperation with IRS-1 and IRS-2 . We observed that TRK-T1 stimulates DNA synthesis in wild-type fibroblasts but not in IRS-1(-/-) mouse embryo fibroblasts . Yeast two-hybrid system experiments showed the occurrence of direct interaction between TRK and IRS molecules, which suggests involvement of different modes of interactions . On the whole, our results suggest that IRS-1 and IRS-2 could be substrates of TRK-T1 and TrkA, and hence could participate in their signal generation.

J Cell Physiol, 2001 Jan, 186(1), 136 - 45
Characterization of SWI/SNF protein expression in human breast cancer cell lines and other malignancies; Decristofaro MF et al.; Organization of genomic DNA into chromatin aids in the regulation of gene expression by limiting access to transcriptional machinery . The SWI/SNF family of complexes, which are conserved from yeast to humans, are ATP-dependent chromatin-remodeling enzymes required for the transcription of a number of genes in yeast . In humans, the gene encoding the BAF47/hSNF5 subunit of the complex, located at 22q11.2, has been found to be mutated in a number of human tumors including rhabdoid, rhabdomyosarcoma, chronic myeloid leukemia, and CNS tumors such as medulloblastomas and choroid plexus carcinomas . In addition, loss of heterozygosity (LOH) has been reported for the BAF47 region in breast and liver cancer . LOH has also been reported in breast and ovarian cancer within 17q12-25, a gene-rich area including BRCA1, BAF60B, and BAF57 . Interestingly, the gene encoding the BAF155/hSWI3 subunit of the complex maps to 3p21-p23, an area of chromosomal deletion seen in a number of human adenocarcinomas including breast, kidney, pancreas, and ovary . To look for abnormalities in these proteins as well as the SWI/SNF complex in general, we have determined the protein status of core human SWI/SNF components BAF170, BAF155, BAF57, BAF53a, and BAF47 in 21 breast cell lines . The complex status in other human tumor cell lines of various tissue types was also examined . We also determined the protein status of the human SWI2 homologues, hBRM/SWI2alpha and BRG1/SWI2beta as well as two other proteins found in human SWI/SNF complexes, BAF180 and BAF250 . In this study, we identified the first cell line negative for the BAF57 protein as well as a pancreatic carcinoma cell line negative for both the BRG-1 and hBRM proteins.

Nat Cell Biol, 2000 Dec, 2(12), 944 - 7
Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores; Chan GK et al.; Here we show that human Zeste White 10 (Zw10) and Rough deal (Rod) are new components of the mitotic checkpoint, as cells lacking these proteins at kinetochores fail to arrest in mitosis when exposed to microtubule inhibitors . Checkpoint failure and premature mitotic exit may explain why cells defective for hZw10 and hRod divide with lagging chromosomes . As Zw10 and Rod are not conserved in yeast, our data, combined with an accompanying study of Drosophila Zw10 and Rod, indicate that metazoans may require an elaborate spindle checkpoint to monitor complex kinetochore functions.

Nat Cell Biol, 2000 Dec, 2(12), 939 - 43
Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila; Basto R et al.; The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells . A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle . Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy . Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies . Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis . These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.

Nat Cell Biol, 2001 Jan, 3(1), E12 - 4
Together until separin do us part; Amon A; Loss of sister-chromatid cohesion triggers chromosome segregation . Several recent reports show that the protease Esp1 cleaves the cohesin subunit Scc1/Mcd1 to induce sister-chromatid segregation in yeast and vertebrates . This finding indicates that cohesin cleavage may control sister-chromatid separation in all vertebrates.

Am J Med Genet, 2000 Dec 18, 95(5), 467 - 72
Patient with large 17p11.2 deletion presenting with Smith-Magenis syndrome and Joubert syndrome phenotype; Natacci F et al.; We report on a 22-year-old woman carrying a del(17)(p11.2p12) and presenting with the clinical manifestations of both Smith-Magenis syndrome (SMS) and Joubert syndrome (JS) . Her facial anomalies, brachydactyly, severe mental retardation, and self-injuring behavior could be attributed to SMS, whereas the cerebellar vermis hypoplasia, hypotonia, ataxic gait, developmental delay, and abnormal respiratory pattern were suggestive of JS . By fluorescent in situ hybridization analyses with Yeast Artificial Chromosomes (YAC) mapping to the 17p11.2 region, as well as locus-specific probes generated through a novel procedure, we could establish that the deletion encompasses a 4-Mb interval with centromeric and telomeric breakpoints at loci D17S793 and D17S953, the latter close to the locus Charcot Marie Tooth 1A (CMT1A)-REP . The deletion differs from that commonly found in SMS in its telomeric boundary, which is more distal than usually observed . The presence of JS phenotype in our patient and the detection of an unusual SMS deletion might suggest the presence of a JS gene in close proximity to the SMS locus .

Exp Hematol, 2000 Dec, 28(12), 1432 - 40
The involvement of human-nuc gene in polyploidization of K562 cell line; Cavalloni G et al.; During megakaryocyte differentiation, the immature megakaryocyte increases its ploidy to a 2(x) DNA content by a process called endomitosis . This leads to the formation of a giant cell, the mature megakaryocyte, which gives rise to platelets . We investigated the role of human-nuc (h-nuc), a gene involved in septum formation in karyokynesis in yeast, during megakaryocytic polyploidization . Nocodazole and 12-O-tetradecanoylphorbol-13-acetate (TPA) were used to induce megakaryocytic differentiation in K562 cell line . The ploidy distribution and CD41 expression of treated K562 cells were evaluated by flow cytometry . Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we analyzed the h-nuc mRNA expression on treated K562 cells.Mature megakaryocyte-like polyploid cells were detected at day 5-7 of treatment with nocodazole . TPA also had a similar effect on K562 cells, but it was much weaker than that of nocodazole . The analysis of ploidy of nocodazole-treated K562 cells showed that nocodazole preferentially induced polyploidization of K562 cell line with a pronounced increase of the cells 8N at day 7 of culture . Expression of CD41, a differentiation-related phenotype, was significantly induced by TPA after 7 days of treatment, showing that functional maturation was mainly induced by TPA . In contrast, there was no significant increase in CD41 expression in nocodazole-treated K562 cells, suggesting that polyploidization and functional maturation are separately regulated during megakaryocytopoiesis . RT-PCR analysis indicated that h-nuc mRNA increased after 72 hours in the presence of nocodazole, preceding the induction of polyploidization . Our data indicate that h-nuc might play a role in polyploidization during megakaryocytic differentiation via inhibition of septum formation.

Brain Res Mol Brain Res, 2000 Dec 28, 85(1-2), 68 - 76
Characterization of mouse Ire1 alpha: cloning, mRNA localization in the brain and functional analysis in a neural cell line; Miyoshi K et al.; In yeast, an endoplasmic reticulum (ER)-associated protein, Ire1p, is believed to initiate the unfolded protein response (UPR), that is responsible for protein folding in the ER under stressed conditions . Two mammalian homologs of Ire1p have been identified, Ire1 alpha and Ire1 beta . We have previously reported that familial Alzheimer's disease linked presenilin-1 variants downregulate the signaling pathway of the UPR by affecting the phosphorylation of Ire1 alpha . In the present study, we cloned the mouse homolog of Ire1 alpha for generating genetically modified mice . Ire1 alpha was ubiquitously expressed in all mouse tissues examined, and was expressed preferentially in neuronal cells in mouse brain . This led us to investigate the effects of the downregulation of the UPR on the survival of neuronal cells under conditions of ER stress . Morphological and biochemical studies using a dominant-negative form of mouse Ire1 alpha have revealed that cell death caused by ER stress can be attributed to apoptosis, and that the downregulation of the UPR enhances the apoptotic process in the mouse neuroblastoma cell line, Neuro2a . Our results indicate that genetically modified mice such as transgenic mice with a dominant-negative form of Ire1 alpha might provide further understanding of the pathogenic mechanisms of Alzheimer's disease and other neurodegenerative disorders.

Brain Res Mol Brain Res, 2000 Dec 28, 85(1-2), 1 - 12
Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation; Okazaki N et al.; We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening . Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3) . One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport {39} . The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport {46} . Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions . However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction . Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum . In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized . These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.

J Neurochem, 2001 Jan, 76(1), 155 - 65
Two rat brain staufen isoforms differentially bind RNA; Monshausen M et al.; In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2) . Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs . Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE . A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the DROSOPHILA: RNA-binding protein Staufen . Mammalian Staufen appears to be expressed in most tissues and brain areas . Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs . Both isoforms contain four double-stranded RNA-binding domains (dsRBD) . In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD . Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6 . Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex . Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules . Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

Semin Cell Dev Biol, 2000 Dec, 11(6), 457 - 66
Phytochrome-interacting factors; Quail PH; The phytochrome family of sensory photoreceptors transduces environmental light signals to responsive nuclear genes by poorly defined pathways . The recent application of yeast two-hybrid library screens to the identification of components that physically interact with members of the phytochrome family has dramatically altered previous views of the likely intracellular signaling pathways . The evidence indicates that one pathway involves light-triggered translocation of the photoreceptor molecule from cytoplasm to nucleus where it binds specifically in its biologically active form to a promoter-bound basic helix-loop-helix protein . The phytochrome molecules are proposed to function as integral, light-switchable components of transcriptional regulator complexes targeting environmental light signals directly and instantly to specific gene promoters .

Mol Endocrinol, 2001 Jan, 15(1), 69 - 79
The DEAD box protein DP103 is a regulator of steroidogenic factor-1; Ou Q et al.; The nuclear receptor steroidogenic factor-1 (SF-1) is essential for development of the gonads, adrenal gland, and the ventromedial hypothalamic nucleus . It also regulates the expression of pivotal steroidogenic enzymes and other important proteins in the reproductive system . We sought to elucidate the mechanisms that govern the transcriptional activity of SF-1 . We demonstrate here that a previously uncharacterized domain, located C-terminal to the DNA binding domain of SF-1, exhibits transcriptional repression function . Point mutations in this domain markedly potentiate the transcriptional activity of native SF-1 . Using an SF-1 region that spans this proximal repression domain as bait in a yeast two-hybrid system, we cloned an SF-1 interacting protein that is homologous to human DP103, a member of the DEAD box family of putative RNA helicases . DP103 directly interacts with the proximal repression domain of SF-1, and mutations in this domain abrogate its interaction with DP103 . DP103 is expressed predominantly in the testis and is also expressed at a lower level in other steroidogenic and nonsteroidogenic tissues . Functionally, DP103 exhibits a native transcriptional repression function that localizes to the C-terminal region of the protein and represses the activity of wild-type, but not mutant, SF-1 . Together, the physical and functional interaction of DP103 with a previously unrecognized repression domain within SF-1 represents a novel mechanism for regulation of SF-1 activity.

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