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