Saccharomyces yeast

Mol Cell Biol, 2005 Feb, 25(3), 933 - 44
RAD51-Dependent Break-Induced Replication Differs in Kinetics and Checkpoint Responses from RAD51-Mediated Gene Conversion; Malkova A et al.; Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time . However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms . Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome . Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G(2)/M DNA damage checkpoint . RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process . RAD51-dependent BIR occurs efficiently in G(2)-arrested cells . Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.

Am J Gastroenterol, 2005 Jan, 100(1), 84 - 92
Variants of CARD15 are associated with an aggressive clinical course of Crohn's disease--an IG-IBD study; Annese V et al.; BACKGROUND: Three major variants of the CARD15 gene confer susceptibility to Crohn's disease (CD) . Whether or not these variants correlate with specific clinical features of the disease is under evaluation . AIM: We investigated the possible association of CARD15 variants with specific clinical characteristics, including the occurrence of anti-Saccharomyces cerevisiae antibodies (ASCA) and antineutrophil cytoplasmic antibodies (ANCA), in a large cohort of inflammatory bowel disease (IBD) patients and their unaffected relatives . METHODS: Three hundred and sixteen CD patients (156 with positive family history), 408 ulcerative colitis (UC) patients (206 with positive family history), 588 unaffected relatives, and 205 unrelated healthy controls (HC) were studied . Single nucleotide polymorphisms (SNPs) R702W, G908R, and L1007finsC of the CARD15 gene were investigated and correlated to age at diagnosis, gender, family history, localization, extraintestinal manifestations, previous resective surgery, stenosing/fistulizing pattern, ANCA, and ASCA . RESULTS: Compared to HC, the frequencies of all three variants in CD were significantly increased: 8.7% versus 4.1% for R702W (p < 0.006), 7.3% versus 2.7% for G908R (p < 0.002), 9.3% versus 0.7% for L1007finsC (p < 0.00001) . At least one risk allele was found in 38.2% (p < 0.0001, compared to HC), 13.7% (NS), and 15.1% of CD, UC, and HC, respectively . The L1007finsC risk allele was also significantly increased in unaffected relatives of familial (9.5%; p < 0.00001), and sporadic CD (9%; p < 0.00001), compared to HC (0.7%) . Sixteen healthy relatives, carriers of two risk alleles, were asymptomatic after 5-8 yr of follow-up . CD carriers of at least one variant were younger (p= 0.03), more likely to have ileal localization (p= 0.0001), stenosing pattern (p= 0.01), previous resective surgery (p= 0.0001), and presence of ASCA (p= 0.0001) . No difference in SNPs frequency between familial and sporadic cases of CD was found . CONCLUSION: In our population, both familial and sporadic CD patients carrying at least one major variant of CARD15 had an aggressive clinical course.

Bioinformatics . 2005 Jan 12; {Epub ahead of print}
Detecting clusters of different geometrical shapes in microarray gene expression data; Kim DW et al.; MOTIVATION: Clustering has been used as a popular technique for finding groups of genes that show similar expression patterns under multiple experimental conditions . Many clustering methods have been proposed for clustering gene expression data, including the hierarchical clustering, k-means clustering, and self-organizing map . However, the conventional methods are limited to identify different shapes of clusters because they use a fixed distance norm when calculating the distance between genes . The fixed distance norm imposes a fixed geometrical shape on the clusters regardless of the actual data distribution . Thus, different distance norms are required for handling the different shapes of clusters . RESULTS: We present the Gustafson-Kessel (GK) clustering method for microarray gene expression data . To detect clusters of different shapes in a data set, we use an adaptive distance norm that is calculated by a fuzzy covariance matrix (F) of each cluster in which the eigenstructure of F is used as an indicator of the shape of the cluster . Moreover, the GK method is less prone to falling into local minima than the k-means and self-organizing map because it makes decisions through the use of membership degrees of a gene to clusters . The algorithmic procedure is accomplished by the alternating optimization technique, which iteratively improves a sequence of sets of clusters until no further improvement is possible . To test the performance of the GK method, we applied the GK method and well-known conventional methods to three recently published yeast data sets, and compared the performance of each method using the Saccharomyces Genome Database annotations . The clustering results of the GK method are more significantly relevant to the biological annotations than those of the other methods, demonstrating its effectiveness and potential for clustering gene expression data . AVAILABILITY: The software was developed using Java language, and can be executed on the platforms that JVM (Java Virtual Machine) is running . It is available from the authors upon request . Supplementary data are available at http://dragon.kaist.ac.kr/gk.

Protein J, 2004 Oct, 23(7), 453 - 60
High-activity barley alpha-amylase by directed evolution; Wong DW et al.; Barley alpha-amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae . The gene coding for the wild-type enzyme was subjected to directed evolution . Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate . The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity . After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system . Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B . Two of these mutations . Q44H and R303K, result in amino acids highly conserved in cereal alpha-amylases . R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.

Genome Biol . 2004;5(12):R98 . Epub 2004.
MONKEY: identifying conserved transcription-factor binding sites in multiple alignments using a binding site-specific evolutionary model; Moses AM et al.; We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments . MONKEY employs probabilistic models of factor specificity and binding-site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit . Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

EMBO J, 2004 Dec 8, 23(24), 4847 - 56 Epub 2004 Dec 8.
Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing; Dziembowski A et al.; Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b . While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome . Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex . TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex . Subunits of this complex were not essential for viability . However, they are required for efficient splicing in vitro and in vivo . Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus . The corresponding complex was named pre-mRNA REtention and Splicing (RES) . The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.

Int Rev Cytol, 2004, 241, 53 - 153
Microtubule-associated proteins and their essential roles during mitosis; Maiato H et al.; Microtubules play essential roles during mitosis, including chromosome capture, congression, and segregation . In addition, microtubules are also required for successful cytokinesis . At the heart of these processes is the ability of microtubules to do work, a property that derives from their intrinsic dynamic behavior . However, if microtubule dynamics were not properly regulated, it is certain that microtubules alone could not accomplish any of these tasks . In vivo, the regulation of microtubule dynamics is the responsibility of microtubule-associated proteins . Among these, we can distinguish several classes according to their function: (1) promotion and stabilization of microtubule polymerization, (2) destabilization or severance of microtubules, (3) functioning as linkers between various structures, or (4) motility-related functions . Here we discuss how the various properties of microtubule-associated proteins can be used to assemble an efficient mitotic apparatus capable of ensuring the bona fide transmission of the genetic information in animal cells.

Appl Microbiol Biotechnol . 2004 Nov 5; {Epub ahead of print}
A novel fungal omega3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4; Sakuradani E et al.; A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4 n-6) but also eicosapentaenoic acid (EPA; 20:5 n-3) below a cultural temperature of 20show $132# degrees show $132#C . Here, we describe the isolation and characterization of a gene ( maw3) that encodes a novel omega3-desaturase from M . alpina 1S-4 . Based on the conserved sequence information for M . alpina 1S-4 Delta12-desaturase and Saccharomyces kluyveri omega3-desaturase, the omega3-desaturase gene from M . alpina 1S-4 was cloned . Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M . alpina 1S-4 Delta12-desaturase, whereas it exhibited 36% identity with Sac . kluyveri omega3-desaturase . The cloned cDNA was confirmed to encode the omega3-desaturase by its expression in the yeast Sac . cerevisiae . Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs . The substrate specificity of the M . alpina 1S-4 omega3-desaturase differs from those of the known fungal omega3-desaturases from Sac . kluyveri and Saprolegnia diclina . Plant, cyanobacterial and Sac . kluyveri omega3-desaturases desaturate 18-carbon n-6 PUFAs, Spr . diclina omega3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans omega3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs . The substrate specificity of M . alpina 1S-4 omega3-desaturase is rather similar to that of C . elegans omega3-desaturase, but the M . alpina omega3-desaturase can more effectively convert AA into EPA when expressed in yeast . The M . alpina 1S-4 omega3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.

Cell, 2004 Oct 29, 119(3), 355 - 68
Homologous recombination generates T-loop-sized deletions at human telomeres; Wang RC et al.; The t-loop structure of mammalian telomeres is thought to repress nonhomologous end joining (NHEJ) at natural chromosome ends . Telomere NHEJ occurs upon loss of TRF2, a telomeric protein implicated in t-loop formation . Here we describe a mutant allele of TRF2, TRF2DeltaB, that suppressed NHEJ but induced catastrophic deletions of telomeric DNA . The deletion events were stochastic and occurred rapidly, generating dramatically shortened telomeres that were accompanied by a DNA damage response and induction of senescence . TRF2DeltaB-induced deletions depended on XRCC3, a protein implicated in Holliday junction resolution, and created t-loop-sized telomeric circles . These telomeric circles were also detected in unperturbed cells and suggested that t-loop deletion by homologous recombination (HR) might contribute to telomere attrition . Human ALT cells had abundant telomeric circles, pointing to frequent t-loop HR events that could promote rolling circle replication of telomeres in the absence of telomerase . These findings show that t-loop deletion by HR influences the integrity and dynamics of mammalian telomeres.

Bioinformatics . 2004 Oct 12; {Epub ahead of print}
Understanding protein dispensability through machine-learning analysis of high-throughput data; Chen Y et al.; MOTIVATION: Protein dispensability is fundamental to understanding of gene function and evolution . Recent advances in generating high-throughput data such as genomic sequence data, protein-protein interaction data, gene-expression data, and growth-rate data of mutants allow us to investigate protein dispensability systematically at the genome scale . RESULTS: In our studies, protein dispensability is represented as a fitness score that is measured by the growth rate of gene-deletion mutants . Through analyses of high-throughput data in yeast Saccharomyces cerevisia, we found that a protein's dispensability had significant correlations with its evolutionary rate and duplication rate, as well as its connectivity in protein-protein interaction network and gene-expression correlation network . Neural network and support vector machine were applied to predict protein dispensability through high-throughput data . Our studies shed some lights on global characteristics of protein dispensability and evolution . AVAILABILITY: The original datasets for protein dispensability analysis and prediction, together with related scripts, are available at http://digbio.missouri.edu/~ychen/ProDispen/.

Postepy Hig Med Dosw (Online), 2004 Aug 20, 58, 312 - 20
{RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response}; Lewandowicz AM et al.; RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins . By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction . Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins) . The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies . In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways . They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation . They also take an essential part in inflammation . High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors . The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 95 - 101
Glutamic protease distribution is limited to filamentous fungi; Sims AH et al.; Glutamic proteases are a distinct, and recently re-classified, group of peptidases that are thought to be found only in fungi . We have identified and analysed the distribution of over 20 putative glutamic proteases from all fungal species whose genomes have been sequenced so far . Although absent from the Saccharomycetales class, glutamic proteases appear to be present in all other ascomycetes species examined . A large number of coding regions for glutamic proteases were also found clustered together in the Phanerochaete chrysosporium genome, despite apparently being absent from three other species of Basidiomycota.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1883 - 90
Metschnikowia chrysoperlae sp . nov., Candida picachoensis sp . nov . and Candida pimensis sp . nov., isolated from the green lacewings Chrysoperla comanche and Chrysoperla carnea (Neuroptera: Chrysopidae); Suh SO et al.; Fourteen yeast isolates comprising three taxa were cultured from digestive tracts of adult Chrysoperla species (Neuroptera: Chrysopidae) and their eggs . The yeast taxa were distinguished based on an estimated molecular phylogeny, DNA sequences and traditional taxonomic criteria . The new yeasts are closely related to Metschnikowia pulcherrima but are sufficiently distinguished by sequence comparison of rRNA gene sequences to consider them as novel species . Here, three novel species are described and their relationships with other taxa in the Saccharomycetes are discussed . Metschnikowia chrysoperlae sp . nov . (type strain, NRRL Y-27615T = CBS 9803T) produced needle-shaped ascospores and was the only teleomorph found . Large numbers of chlamydospores similar to those observed in M . pulcherrima were also produced . The other two novel species are asexual yeasts, Candida picachoensis sp . nov . (type strain, NRRL Y-27607T = CBS 9804T) and Candida pimensis sp . nov . (type strain, NRRL Y-27619T = CBS 9805T), sister taxa of M . chrysoperlae and M . pulcherrima . A specialized relationship between yeasts and lacewing hosts may exist, because the yeasts were isolated consistently from lacewings only . Although M . chrysoperlae was isolated from eggs and adult lacewings, suggesting the possibility of vertical transmission, no yeast was isolated from larvae.

Mol Biol Evol, 2005 Jan, 22(1), 174 - 177 Epub 2004 Sep 15.
Adjusting for Selection on Synonymous Sites in Estimates of Evolutionary Distance; Hirsh AE et al.; Evolution at silent sites is often used to estimate the pace of selectively neutral processes or to infer differences in divergence times of genes . However, silent sites are subject to selection in favor of preferred codons, and the strength of such selection varies dramatically across genes . Here, we use the relationship between codon bias and synonymous divergence observed in four species of the genus Saccharomyces to provide a simple correction for selection on silent sites.

Nature, 2004 Sep 2, 431(7004), 99 - 104
Transcriptional regulatory code of a eukaryotic genome; Harbison CT et al.; DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression . Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites . We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species . The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed . We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators.

J Biol Chem, 2004 Oct 29, 279(44), 46182 - 90 Epub 2004 Aug 18.
The Sac3 homologue shd1 is involved in mitotic progression in mammalian cells; Khuda SE et al.; Saccharomyces Sac3 required for actin assembly was shown to be involved in DNA replication . Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression . SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to alpha-tubulin, at M phase . RNA interference suppression of endogenous shd1 caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei . Conversely, increased expression of SHD1 by DNA transfection with shd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells . These results demonstrated that shd1 is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 243 - 8
Stress responses of linear plasmids from Debaryomyces hansenii; Fukuda K et al.; The triplet linear plasmids pDHL1/2/3 from the salt-tolerant yeast Debaryomyces hansenii TK are localized in the cytoplasm and characterized by a unique feature that they require environmental stressors (0.3 M NaCl or solutes such as sorbitol with equivalent osmolarity) for stable replication and maintenance . The degree of osmolarity dependence of pDHLs was greatly affected by growth temperature of the host cells: the stability of pDHLs was maintained in the absence of osmolarity in cells growing at 25 degrees C, and required osmorarity equivalent to 0.3-1.0 M NaCl on shifting to 30-35 degrees C . Although to less extent, similar osmolarity dependence at high temperatures was observed with another system of D . hansenii linear plasmids . Short-term conditioning of cells to heat or high osmolarity resulted in significant improvement in the plasmid stability, suggesting possible involvement of stress proteins and/or high glycerol level in the stabilization process.

J Mol Biol, 2004 Sep 3, 342(1), 19 - 30
Consensus folding of aligned sequences as a new measure for the detection of functional RNAs by comparative genomics; Washietl S et al.; Facing the ever-growing list of newly discovered classes of functional RNAs, it can be expected that further types of functional RNAs are still hidden in recently completed genomes . The computational identification of such RNA genes is, therefore, of major importance . While most known functional RNAs have characteristic secondary structures, their free energies are generally not statistically significant enough to distinguish RNA genes from the genomic background . Additional information is required . Considering the wide availability of new genomic data of closely related species, comparative studies seem to be the most promising approach . Here, we show that prediction of consensus structures of aligned sequences can be a significant measure to detect functional RNAs . We report a new method to test multiple sequence alignments for the existence of an unusually structured and conserved fold . We show for alignments of six types of well-known functional RNA that an energy score consisting of free energy and a covariation term significantly improves sensitivity compared to single sequence predictions . We further test our method on a number of non-coding RNAs from Caenorhabditis elegans/Caenorhabditis briggsae and seven Saccharomyces species . Most RNAs can be detected with high significance . We provide a Perl implementation that can be used readily to score single alignments and discuss how the methods described here can be extended to allow for efficient genome-wide screens.

Inflamm Bowel Dis, 2004 May, 10(3), 270 - 3
Scalloping of duodenal mucosa in Crohn's disease; Culliford A et al.; Scalloping of the duodenal mucosal folds is an endoscopic finding of small bowel mucosal pathology that is generally due to villous atrophy . Though it can be seen in many disease processes, it is most commonly associated with celiac disease . We report three patients with scalloping of duodenal folds and histologic confirmation of villous atrophy due to Crohn's disease . All patients had negative celiac serologies and two had positive markers for Crohn's disease (anti-Saccharomyces cerevisiae antibodies) . Patients had either ileitis or ileocolitis in addition to duodenal abnormalities . These cases illustrate that scalloping can occur in the duodenum in Crohn's disease.

Bioinformatics, 2004 Jul 10, 20(10), 1506 - 11
Bifurcation analysis of the regulatory modules of the mammalian G1/S transition; Swat M et al.; MOTIVATION: Mathematical models of the cell cycle can contribute to an understanding of its basic mechanisms . Modern simulation tools make the analysis of key components and their interactions very effective . This paper focuses on the role of small modules and feedbacks in the gene-protein network governing the G1/S transition in mammalian cells . Mutations in this network may lead to uncontrolled cell proliferation . Bifurcation analysis helps to identify the key components of this extremely complex interaction network . RESULTS: We identify various positive and negative feedback loops in the network controlling the G1/S transition . It is shown that the positive feedback regulation of E2F1 and a double activator-inhibitor module can lead to bistability . Extensions of the core module preserve the essential features such as bistability . The complete model exhibits a transcritical bifurcation in addition to bistability . We relate these bifurcations to the cell cycle checkpoint and the G1/S phase transition point . Thus, core modules can explain major features of the complex G1/S network and have a robust decision taking function.

Genome Biol . 2004;5(6):R43 . Epub 2004 May 28.
TXTGate: profiling gene groups with text-based information; Glenisson P et al.; We implemented a framework called TXTGate that combines literature indices of selected public biological resources in a flexible text-mining system designed towards the analysis of groups of genes . By means of tailored vocabularies, term- as well as gene-centric views are offered on selected textual fields and MEDLINE abstracts used in LocusLink and the Saccharomyces Genome Database . Subclustering and links to external resources allow for in-depth analysis of the resulting term profiles.

Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 9033 - 8 Epub 2004 Jun 02.
Coevolution of gene expression among interacting proteins; Fraser HB et al.; Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions . Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids . Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components . Here, we show that the expression levels of physically interacting proteins coevolve . We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species . We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence . These results demonstrate that gene expression levels can coevolve, adding another dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time . Our results also suggest that expression coevolution can be used for computational prediction of protein-protein interactions.

Methods Mol Biol, 2004, 284, 217 - 27
Assaying phosphoinositide phosphatases; Taylor GS et al.; The roles of phosphoinositide second messengers as signaling molecules in a vast array of cellular processes including cell growth, metabolism, vesicular transport, programmed cell death, and responses to extracellular signals are only beginning to be understood . The recent identification of novel phosphoinositide signaling molecules underscores the need for methodology with which to characterize the enzymes responsible for regulating cellular phosphoinositide levels . One of the ways in which cells control these lipids is through dephosphorylation by phosphoinositide phosphatases, which oppose and regulate the actions of phosphoinositide kinases . We describe herein two rapid and simple assays for characterizing phosphoinositide phosphatases that can be used to provide a basis for understanding the activity and specificity of these enzymes.

Int J Food Microbiol, 2004 Jul 1, 94(1), 55 - 66
Contribution of a selected fungal population to proteolysis on dry-cured ham; Martin A et al.; The proteolytic changes taking place in dry-cured hams lead to increases in free amino acids . Such free amino acids not only contribute to flavour, but also serve as precursors of volatile compounds . Several months of ripening time are required to allow the particular flavour to develop . The fungal population allowed to grow on the surface of some types of dry-cured could play a key role on proteolysis, as it has been shown for dry-cured sausages . The purpose of this work was to study the possible contribution of fungi to proteolysis in dry-cured ham . For this, a strain each of non-toxigenic Penicillium chrysogenum (Pg222) and Debaryomyces hansenii (Dh345), selected for their proteolytic activity on myofibrillar proteins, were inoculated as starter cultures . Changes in the high ionic strength-soluble proteins of an external muscle (adductor) revealed in only 6 months higher proteolysis in the inoculated hams when compared to non-inoculated control hams . Proteolytic strains among the wild fungal population on non-inoculated control hams prevented from obtaining similar differences at the end of processing . However, inoculation with Pg222 and Dh345 led to higher levels for most free amino acids at the external muscle in fully dry-cured hams . In addition, the concentration for some of the more polar free amino acids (i.e . Asp, Glu, Ser and Gln) in inoculated hams was higher at external than at internal (biceps femoris) muscles . These promising results deserve further studies to know the impact of a selected fungal population on the volatile compounds and sensory properties of dry-cured ham.

Trends Cell Biol, 1996 Oct, 6(10), 371 - 5
The subunit-exchange model of histone acetylation; Roth SY et al.; Increased histone acetylation has long been linked to gene activation, but little is known about how acetylation levels are regulated, largely because the histone acetyltrans ferase activities (HATs) responsible for this modification have been cloned only recently . Comparison of the biochemical nature of the Tetrahymena HAT A complex with the genetic and biochemical properties of the Saccharomyces Gcn5p-Ado complex leads us to propose that histone acetylase assemblies may be modular in nature and that this modularity may be an intimate part of the association of these enzymes with chromatin . The 'subunit-exchange' model provides a mechanism for the regulation and targeting of both histone acetylases and deacetylases and has implications for the control of cell growth, proliferation and tumorigenesis.

Bioinformatics, 2004 Sep 22, 20(14), 2242 - 50 Epub 2004 May 06.
Gene co-expression network topology provides a framework for molecular characterization of cellular state; Carter SL et al.; MOTIVATION: Gene expression data have become an instrumental resource in describing the molecular state associated with various cellular phenotypes and responses to environmental perturbations . The utility of expression profiling has been demonstrated in partitioning clinical states, predicting the class of unknown samples and in assigning putative functional roles to previously uncharacterized genes based on profile similarity . However, gene expression profiling has had only limited success in identifying therapeutic targets . This is partly due to the fact that current methods based on fold-change focus only on single genes in isolation, and thus cannot convey causal information . In this paper, we present a technique for analysis of expression data in a graph-theoretic framework that relies on associations between genes . We describe the global organization of these networks and biological correlates of their structure . We go on to present a novel technique for the molecular characterization of disparate cellular states that adds a new dimension to the fold-based methods and conclude with an example application to a human medulloblastoma dataset . RESULTS: We have shown that expression networks generated from large model-organism expression datasets are scale-free and that the average clustering coefficient of these networks is several orders of magnitude higher than would be expected for similarly sized scale-free networks, suggesting an inherent hierarchical modularity similar to that previously identified in other biological networks . Furthermore, we have shown that these properties are robust with respect to the parameters of network construction . We have demonstrated an enrichment of genes having lethal knockout phenotypes in the high-degree (i.e . hub) nodes in networks generated from aggregate condition datasets; using process-focused Saccharomyces cerivisiae datasets we have demonstrated additional high-degree enrichments of condition-specific genes encoding proteins known to be involved in or important for the processes interrogated by the microarrays . These results demonstrate the utility of network analysis applied to expression data in identifying genes that are regulated in a state-specific manner . We concluded by showing that a sample application to a human clinical dataset prominently identified a known therapeutic target . AVAILABILITY: Software implementing the methods for network generation presented in this paper is available for academic use by request from the authors in the form of compiled linux binary executables.

Chem Biol, 2004 Feb, 11(2), 211 - 23
A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors; Becker F et al.; In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors . Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats . For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography . The Y3H system described here may prove generally useful in the discovery of candidate drug targets.

Genetics, 2004 Mar, 166(3), 1177 - 86
The identification of Pcl1-interacting proteins that genetically interact with Cla4 may indicate a link between G1 progression and mitotic exit; Keniry ME et al.; In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes . Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes . However, loss of both proteins is lethal, suggesting some functional overlap . To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4 Delta strain . These mutations define >60 genes . Recently, both Ste20 and Cla4 have been implicated in mitotic exit . Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4 . We further show that the Pho85-coupled G(1) cyclins Pcl1 and Pcl2 contribute to this Pho85 role . We performed a two-hybrid screen with Pcl1 . Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1 . Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1.Pho85 complexes . NCP1, HMS1, and EPA1 also genetically interact with CLA4 . Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins . We propose a model in which the G(1) Pcl1.Pho85 complex regulates mitotic exit machinery.

Mol Cell Proteomics, 2004 Jul, 3(7), 704 - 14 Epub 2004 Apr 07.
Identification of the linker-SH2 domain of STAT as the origin of the SH2 domain using two-dimensional structural alignment; Gao Q et al.; The availability of large volumes of genomic sequences presents an unprecedented proteomic challenge to characterize the structure and function of various protein motifs . Primary structural alignment is often unable to accurately identify a given motif due to sequence divergence; however, with the aid of secondary structural prediction for analysis, it becomes feasible to explore protein motifs on a proteome-wide scale . Here we report the use of secondary structural alignment to characterize the Src homology 2 (SH2) domains of both conventional and divergent sequences and divide them into two groups, Src-type and STAT-type . In addition to the basic "alphabetabetabetaalpha" structure (betaBeta), the Src-type SH2 domain contains an extra beta-strand (betaE or betaE-betaF motif) . Alternatively, the linker domain-conjugated SH2 domain in STAT contains the alphaB' motif . Combining BLAST data from betaBeta core motif sequences with predicted secondary structural alignment, we have screened for SH2 domains in various eukaryotic model systems including Arabidopsis, Dictyostelium, and Saccharomyces . Two novel genes carrying the linker-SH2 domain of STAT were discovered and subsequently cloned from Arabidopsis . These genes, designated as STAT-type linker-SH2 domain factors (STATL), are found in a wide array of vascular and nonvascular plants, suggesting that the linker-SH2 domain evolved prior to the divergence of plants and animals . Using this approach, we expanded the number of putative SH2 domain-bearing genes in Dictyostelium and comparatively studied the secondary structural profiles of both typical and atypical SH2 domains . Our results indicate that the linker-SH2 domain of the transcription factor STAT is one of the most ancient and fully developed functional domains, serving as a template for the continuing evolution of the SH2 domain essential for phosphotyrosine signal transduction.

J Clin Microbiol, 2004 Apr, 42(4), 1832 - 6
Genotyping and antifungal susceptibility profile of Dipodascus capitatus isolates causing disseminated infection in seven hematological patients of a tertiary hospital; Gadea I et al.; Seven cases of disseminated infection due to Dipodascus capitatus are reported . Infections occurred in a hematological unit of a tertiary hospital during a period of 5 years . Five cases were refractory to antifungal therapy . Antifungal susceptibility testing of seven isolates was performed, and strains were typed by PCR fingerprinting with the core sequence of phage M13 and by random amplification of polymorphic DNA with two primers, Ap12h and W-80A . A very short range of MICs of each antifungal agent was observed . The MICs of amphotericin B ranged between 0.50 and 2 microg/ml . Strains were susceptible in vitro to flucytosine and susceptible (dose-dependent) to fluconazole and itraconazole . Voriconazole exhibited an activity in vitro comparable to that of itraconazole . Typing techniques allowed seven additional isolates of D . capitatus neither geographically nor temporally related to be classified into two different genomic patterns . The genomic type of the seven strains from the hematological unit was identical regardless of typing technique utilized . It would indicate that the seven cases of disseminated infection could be related epidemiologically.

Cell, 2004 Apr 2, 117(1), 29 - 45
Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis; Borner GV et al.; Yeast mutants lacking meiotic proteins Zip1, Zip2, Zip3, Mer3, and/or Msh5 (ZMMs) were analyzed for recombination, synaptonemal complex (SC), and meiotic progression . At 33 degrees C, recombination-initiating double-strand breaks (DSBs) and noncrossover products (NCRs) form normally while formation of single-end invasion strand exchange intermediates (SEIs), double Holliday junctions, crossover products (CRs), and SC are coordinately defective . Thus, during wild-type meiosis, recombinational interactions are differentiated into CR and NCR types very early, prior to onset of stable strand exchange and independent of SC . By implication, crossover interference does not require SC formation . We suggest that SC formation may require interference . Subsequently, CR-designated DSBs undergo a tightly coupled, ZMM-promoted transition that yields SEI-containing recombination complexes embedded in patches of SC . zmm mutant phenotypes differ strikingly at 33 degrees C and 23 degrees C, implicating higher temperature as a positive effector of recombination and identifying a checkpoint that monitors local CR-specific events, not SC formation, at late leptotene.

J Exp Clin Cancer Res, 2003 Dec, 22(4), 581 - 9
Free radical scavenging, antitumor and anticarcinogenic activity of gossypin; Babu BH et al.; Antioxidant, antitumor and anticarcinogenic activity of gossypin (3,5,8,3',4'-pentahydroxy-7-O glucosyl flavone) was carried out . The compound needed for 50% inhibition of superoxide, hydroxyl and nitric oxide radicals was 3 microg/ml, 41 microg/ml and 12 microg/ml, respectively . Gossypin also impart 50% inhibition at concentrations 37 microg/ml and 43 microg/ml, respectively for in vitro lipid peroxidation . The compound shows IC50 of 30 microM, 42.5 microM and 45.1 microM concentrations in L 929, HT 29 and K 562 cell lines in 72 hrs MTT assay . The compound shows a zone of inhibition of 8 mm in topo I and topo II inhibition assay in Saccharomyces ceriviseae mutant cultures . It reduces the tumor burden in solid tumor harboring animals (p < 0.001) and effectively inhibits the formation of new blood vessels on tumor mass . 20 mg/kg b.wt of gossypin increase the life span of ascites tumor harboring animals (100%) and (164.7%) respectively by oral and intraperitoneal administration of the drug . Gossypin reduced the incidence of papilloma formation and papilloma/mouse in DMBA/ croton oil induced skin papilloma.

J Biol Chem, 2004 Jun 11, 279(24), 24957 - 64 Epub 2004 Mar 26.
C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats; Jones JC et al.; The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 . In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5 . A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern . We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3'-end formation . Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5 . We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2 . When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD . We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates.

FEMS Yeast Res, 2004 Mar, 4(6), 605 - 7
Metschnikowia kunwiensis comb . nov., the teleomorph of Candida kunwiensis; Brysch-Herzberg M; The teleomorph of Candida kunwiensis Hong, Bae, Herzberg, Titze, Lachance, Metschnikowia kunwiensis, is described . Repeated attempts to obtain ascospore formation succeeded using modified V8 sporulation media and extended incubation times . The asci are ovoid, with only a small protrusion caused by the spore(s) . The species is diplontic, possibly homothallic, with one or two ascospores per ascus . Aside from having atypical ovoid asci, the acicular shape of the spores is characteristic of the genus Metschnikowia . The type strain is CBS 9676(T).

FEMS Yeast Res, 2004 Mar, 4(6), 587 - 96
Speciation in the large-spored Metschnikowia clade and establishment of a new species, Metschnikowia borealis comb . nov; Marinoni G et al.; The reproductive boundaries among species in the large-spored Metschnikowia clade were studied by prototrophic recombinant selection, electrophoretic karyotyping, mitochondrial DNA restriction analysis, and DNA sequence analysis . Inviable ascospores arose from crosses between the two varieties of Metschnikowia continentalis, indicating that they should be recognized as separate species . Prototrophic recombinants were recovered from crosses between auxotrophic mutants of Metschnikowia borealis, M . continentalis, Metschnikowia lochheadii, Metschnikowia sp . UWO(PS)00-154.1, and Candida ipomoeae, showing that some genetic exchange is possible in spite of the sterility of the asci formed in interspecific crosses . Metschnikowia hawaiiensis, although capable of ascus formation when its h(-) mating type is crossed with the h(+) mating type of the other species, did not give rise to recombinants . In the other species, some recombinants acquired the ability to form asci directly from single cells . These often contained the chromosomes of both parents, suggesting formation of allodiploid hybrids . Other recombinants behaved as haploids and were similar to one parent except for having inherited the selectable wild-type allele from the other parent . In most, but not all cases, inheritance of the mitochondrial genome was uniparental and correlated with the inheritance of the nuclear chromosome complement . In some cases, what appeared to be a recombinant mitochondrial genome was observed . Phylogenies derived from the sequences of various DNA regions were not congruent, indicating that hybridization may have taken place in nature as the large-spored species diverged from their common ancestor . Further evidence that C . ipomoeae arose from a natural recombination event was obtained, but a pair of Metschnikowia species that might represent derived forms of the parents could not be identified conclusively . C . ipomoeae and most of its closely related Metschnikowia species contained a group-II intron in the mitochondrial small-subunit ribosomal gene . The intron was absent in M . borealis, M . hawaiiensis, and other species in the genus Metschnikowia.

Appl Environ Microbiol, 2004 Mar, 70(3), 1347 - 55
Molecular detection and identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in spoiled wines; Cocolin L et al.; In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B . anomalus . Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used . No amplification product was obtained when DNA from other Brettanomyces spp . or wine yeasts were used as the templates . The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B . bruxellensis and B . anomalus, and each species could be identified on the basis of the different restriction profiles . After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed . Total agreement between traditional identification and molecular identification was observed . The protocol developed was also used for direct detection of B . bruxellensis and B . anomalus in wines suspected to be spoiled by Brettanomyces spp . Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B . bruxellensis . Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.

BMC Evol Biol . 2004 Jan 28;4(1):2.
A molecular timescale of eukaryote evolution and the rise of complex multicellular life; Hedges SB et al.; BACKGROUND: The pattern and timing of the rise in complex multicellular life during Earth's history has not been established . Great disparity persists between the pattern suggested by the fossil record and that estimated by molecular clocks, especially for plants, animals, fungi, and the deepest branches of the eukaryote tree . Here, we used all available protein sequence data and molecular clock methods to place constraints on the increase in complexity through time . RESULTS: Our phylogenetic analyses revealed that (i) animals are more closely related to fungi than to plants, (ii) red algae are closer to plants than to animals or fungi, (iii) choanoflagellates are closer to animals than to fungi or plants, (iv) diplomonads, euglenozoans, and alveolates each are basal to plants+animals+fungi, and (v) diplomonads are basal to other eukaryotes (including alveolates and euglenozoans) . Divergence times were estimated from global and local clock methods using 20-188 proteins per node, with data treated separately (multigene) and concatenated (supergene) . Different time estimation methods yielded similar results (within 5%): vertebrate-arthropod (964 million years ago, Ma), Cnidaria-Bilateria (1,298 Ma), Porifera-Eumetozoa (1,351 Ma), Pyrenomycetes-Plectomycetes (551 Ma), Candida-Saccharomyces (723 Ma), Hemiascomycetes-filamentous Ascomycota (982 Ma), Basidiomycota-Ascomycota (968 Ma), Mucorales-Basidiomycota (947 Ma), Fungi-Animalia (1,513 Ma), mosses-vascular plants (707 Ma), Chlorophyta-Tracheophyta (968 Ma), Rhodophyta-Chlorophyta+Embryophyta (1,428 Ma), Plantae-Animalia (1,609 Ma), Alveolata-plants+animals+fungi (1,973 Ma), Euglenozoa-plants+animals+fungi (1,961 Ma), and Giardia-plants+animals+fungi (2,309 Ma) . By extrapolation, mitochondria arose approximately 2300-1800 Ma and plastids arose 1600-1500 Ma . Estimates of the maximum number of cell types of common ancestors, combined with divergence times, showed an increase from two cell types at 2500 Ma to approximately 10 types at 1500 Ma and 50 cell types at approximately 1000 Ma . CONCLUSIONS: The results suggest that oxygen levels in the environment, and the ability of eukaryotes to extract energy from oxygen, as well as produce oxygen, were key factors in the rise of complex multicellular life . Mitochondria and organisms with more than 2-3 cell types appeared soon after the initial increase in oxygen levels at 2300 Ma . The addition of plastids at 1500 Ma, allowing eukaryotes to produce oxygen, preceded the major rise in complexity.

Genome Biol . 2004;5(3):R21 . Epub 2004 Feb 13.
MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins; Maurer-Stroh S et al.; We evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences . Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated . We give experimental evidence for in vitro myristoylation of selected predictions . Furthermore, we classify five membrane-attachment factors that occur most frequently in combination with, or even replacing, myristoyl anchors, as some protein family examples show.

Pac Symp Biocomput . 2004;:324-35.
Phylogenetic motif detection by expectation-maximization on evolutionary mixtures; Moses AM et al.; The preferential conservation of transcription factor binding sites implies that non-coding sequence data from related species will prove a powerful asset to motif discovery . We present a unified probabilistic framework for motif discovery that incorporates evolutionary information . We treat aligned DNA sequence as a mixture of evolutionary models, for motif and background, and, following the example of the MEME program, provide an algorithm to estimate the parameters by Expectation-Maximization . We examine a variety of evolutionary models and show that our approach can take advantage of phylogenic information to avoid false positives and discover motifs upstream of groups of characterized target genes . We compare our method to traditional motif finding on only conserved regions . An implementation will be made available at http://rana.lbl.gov.

Med Mycol, 2004 Feb, 42(1), 87 - 92
Co-isolation of Trichosporon inkin and Candida parapsilosis from a scalp white piedra case; Taj-Aldeen SJ et al.; White piedra is a rare fungal infection of the hair shaft characterized by small, firm, irregular white-brown nodules . The infection is caused by basidiomycetous yeasts in the genus Trichosporon . We report a case of a 28-year-old female patient who acquired the infection in Qatar . In this case, the scalp was the only site affected, but infection at that site was extensive . The hair had a Saccharomyces-like yeast odor and appeared to be beaded, with light-brown nodules of varying sizes up to 2 mm long . Trichosporon sp . accompanied by Candida parapsilosis grew out along hair shafts planted in primary isolation media . Molecular identification of the Trichosporon carried out by analyzing the 26S ribosomal gene gave a 100% match with Trichosporon inkin, a major cause of pubic white piedra . The patient was treated with daily applications of ketoconazole shampoo followed by econazole shampoo and cream, and was considered clinically and mycologically cured after 2 months . Novel findings in the present case are the first identification of T . inkin as an agent of scalp white piedra, and the heavy outgrowth of C . parapsilosis from the concretions, although in the latter case it is not clear if the co-occurring yeast was etiologically contributory to the pathogenesis of the white piedra.

Med Mycol, 2004 Feb, 42(1), 27 - 34
Delineation of Clavispora lusitaniae clinical isolates by polymerase chain reaction-single strand conformation polymorphism analysis of the ITSI region: a retrospective study comparing five typing methods; Arabatzis M et al.; Strain delineation of the emerging opportunistic pathogen Clavispora lusitaniae was studied using 12 strains, including two strains of known opposite mating type, CBS 6936 (h+) and CBS 5094 (h-), and 10 strains isolated between 1998 and 2001 from immunocompromized patients . This retrospective study assessed the occurrence of C . lusitaniae subtypes within and among hospitals, and in outpatients who were regularly screened for fungal infections in the course of radio-chemotherapy . Strain typing was accomplished for the first time using single strand conformation polymorphism (SSCP) analysis of amplicons of the ribosomal DNA internal transcribed spacer (ITS) 1 and 2 regions . The results were compared with those produced by three pulsed-field gel electrophoresis (PFGE) methods and PCR fingerprinting with the minisatellite-specific primer M13 . Karyotyping separated 7-9 chromosomes, not 6-8 as previously reported . Pulsotyping of SfiI and NotI digested chromosomes grouped isolates in five and four distinct clusters, respectively . All methods revealed strain heterogeneity, though not as extensive as previously recorded . SSCP analysis of the ITS1 region generated five subtypes, based on a sequencing-confirmed nucleotide polymorphism . The discriminatory power of this method was high . All strains displayed a homogeneous SSCP pattern for the ITS2 region . ITS1 PCR-SSCP appears to allow rapid and reliable delineation of C . lusitaniae strains . Pending examination of a larger sample size and interlaboratory study, this protocol can be recommended for rapid prospective identification of hospital outbreaks.

Am J Hum Genet, 2004 Mar, 74(3), 545 - 51 Epub 2004 Feb 17.
Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I; Kranz C et al.; This study describes the discovery of a new inherited disorder of glycosylation named "CDG-Ik." CDG-Ik (congenital disorder of glycoslyation type Ik) is based on a defect of human mannosyltransferase I (MT-I {MIM 605907}), an enzyme necessary for the elongation of dolichol-linked chitobiose during N-glycan biosynthesis . Mutations in semiconserved regions in the corresponding gene, HMT-1 (yeast homologue, Alg1), in two patients caused drastically reduced enzyme activity, leading to a severe disease with death in early infancy . One patient had a homozygous point mutation (c.773C-->T, S258L), whereas the other patient was compound heterozygous for the mutations c.773C-->T and c.1025A-->C (E342P) . Glycosylation and growth of Alg1-deficient PRY56 yeast cells, showing a temperature-sensitive phenotype, could be restored by the human wild-type allele, whereas only slight restoration was observed after transformation with the patients' alleles.

Fungal Genet Biol, 2004 Mar, 41(3), 285 - 92
Comparative sequence analysis of Sordaria macrospora and Neurospora crassa as a means to improve genome annotation; Nowrousian M et al.; One of the most challenging parts of large scale sequencing projects is the identification of functional elements encoded in a genome . Recently, studies of genomes of up to six different Saccharomyces species have demonstrated that a comparative analysis of genome sequences from closely related species is a powerful approach to identify open reading frames and other functional regions within genomes {Science 301 (2003) 71, Nature 423 (2003) 241} . Here, we present a comparison of selected sequences from Sordaria macrospora to their corresponding Neurospora crassa orthologous regions . Our analysis indicates that due to the high degree of sequence similarity and conservation of overall genomic organization, S . macrospora sequence information can be used to simplify the annotation of the N . crassa genome.

J Biol Chem, 2004 Apr 2, 279(14), 14245 - 55 Epub 2004 Jan 26.
Cell cycle-dependent phosphorylation of the DNA polymerase epsilon subunit, Dpb2, by the Cdc28 cyclin-dependent protein kinase; Kesti T et al.; DNA polymerase epsilon (Polepsilon), one of the three major eukaryotic replicative polymerases, is comprised of the essential catalytic subunit, called Pol2 in budding yeast, and three accessory subunits, only one of which, Dpb2, is essential . Polepsilon is recruited to replication origins during late G(1) phase prior to activation of replication . In this work we show that the budding yeast Dpb2 is phosphorylated in a cell cycle-dependent manner during late G(1) phase . Phosphorylation results in the appearance of a lower mobility species . The appearance of that species in vivo is dependent upon the Cdc28 cyclin-dependent protein kinase (CDK), which can directly phosphorylate Dpb2 in vitro . Either G(1) cyclin (Cln) or B-type cyclin (Clb)-associated CDK is sufficient for phosphorylation . Mapping of phosphorylation sites by mass spectrometry using a novel gel-based proteolysis protocol shows that, of the three consensus CDK phosphorylation sites, at least two, Ser-144 and Ser-616, are phosphorylated in vivo . The Cdc28 CDK phosphorylates only Ser-144 in vitro . Using site-directed mutagenesis, we show that Ser-144 is sufficient for the formation of the lower mobility form of Dpb2 in vivo . In contrast, Ser-616 appears not to be phosphorylated by Cdc28 . Finally, inactivation of all three CDK consensus sites in Dpb2 results in a synthetic phenotype with the pol2-11 mutation, leading to decreased spore viability, slow growth, and increased thermosensitivity . We suggest that phosphorylation of Dpb2 during late G(1) phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Polepsilon

J Mol Evol, 2003 Dec, 57(6), 694 - 701
Mammalian mutation pressure, synonymous codon choice, and mRNA degradation; Duan J et al.; The usage of synonymous codons (SCs) in mammalian genes is highly correlated with local base composition and is therefore thought to be determined by mutation pressure . The usage is nonetheless structured . For instance, mammals share with Saccharomyces and Drosophila most preferences for the C-ending over the G-ending codon (or vice versa) within each fourfold-degenerate SC family and the fact that their SCs are placed along coding regions in ways that minimize the number of T|A and C|G dinucleotides ("|" being the codon boundary) . TA and CG underrepresentations are observed everywhere in the mammalian genome affecting the SC usage, the amino acid composition of proteins, and the primary structure of introns and noncoding DNA . While the rarity of CG is ascribed to the high mutability of this dinucleotide, the rarity of TA in coding regions is considered adaptive because UA dinucleotides are cleaved by endoribonucleases . Here we present in vivo experimental evidence indicating that the number of T|A and/or C|G dinucleotides of a human gene can affect strongly the expression level and degradation of its mRNA . Our results are consistent with indirect evidence produced by other workers and with the detailed work that has been devoted to characterize UA cleavage in vitro and in vivo . We conclude that SC choice can influence strongly mRNA function and gene expression through effects not directly related to the codon-anticodon interaction . These effects should constrain heavily the nucleotide motif composition of the most abundant mRNAs in the transcriptome, in particular, their SC usage, a usage that must be reflected by cellular tRNA concentrations and thus defines for all other genes which SCs are translated fastest and most accurately . Furthermore, the need to avoid such effects genome-wide appears serious enough to have favored the evolution of biases in context-dependent mutation that reduce the occurrence of intrinsically unfavorable motifs, and/or, when possible, to have induced the molecular machinery mediating such effects to rely opportunistically on already existing motif rarities and abundances . This may explain why nucleotide motif preferences are very similar in transcribed and nontranscribed mammalian DNA even though the preferences appear to be adaptive only in transcribed DNA.

Mol Biol Cell, 2004 Apr, 15(4), 1724 - 35 Epub 2004 Jan 23.
S-phase checkpoint genes safeguard high-fidelity sister chromatid cohesion; Warren CD et al.; Cohesion establishment and maintenance are carried out by proteins that modify the activity of Cohesin, an essential complex that holds sister chromatids together . Constituents of the replication fork, such as the DNA polymerase alpha-binding protein Ctf4, contribute to cohesion in ways that are poorly understood . To identify additional cohesion components, we analyzed a ctf4Delta synthetic lethal screen performed on microarrays . We focused on a subset of ctf4Delta-interacting genes with genetic instability of their own . Our analyses revealed that 17 previously studied genes are also necessary for the maintenance of robust association of sisters in metaphase . Among these were subunits of the MRX complex, which forms a molecular structure similar to Cohesin . Further investigation indicated that the MRX complex did not contribute to metaphase cohesion independent of Cohesin, although an additional role may be contributed by XRS2 . In general, results from the screen indicated a sister chromatid cohesion role for a specific subset of genes that function in DNA replication and repair . This subset is particularly enriched for genes that support the S-phase checkpoint . We suggest that these genes promote and protect a chromatin environment conducive to robust cohesion.

J Biol Chem, 2004 Apr 9, 279(15), 15621 - 9 Epub 2004 Jan 21.
Dictyostelium macroautophagy mutants vary in the severity of their developmental defects; Otto GP et al.; Macroautophagy is the major mechanism that eukaryotes use to recycle cellular components during stressful conditions . We have shown previously that the Atg12-Atg5 conjugation system, required for autophagosome formation in yeast, is necessary for Dictyostelium development . A second conjugation reaction, Aut7/Atg8 lipidation with phosphatidylethanolamine, as well as a protein kinase complex and a phosphatidylinositol 3-kinase complex are also required for macroautophagy in yeast . In this study, we characterize mutations in the putative Dictyostelium discoideum orthologues of budding yeast genes that are involved in one of each of these functions, ATG1, ATG6, and ATG8 . All three genes are required for macroautophagy in Dictyostelium . Mutant amoebae display reduced survival during nitrogen starvation and reduced protein degradation during development . Mutations in the three genes produce aberrant development with defects of varying severity . As with other Dictyostelium macroautophagy mutants, development of atg1-1, atg6(-), and atg8(-) is more aberrant in plaques on bacterial lawns than on nitrocellulose filters . The most severe defect is observed in the atg1-1 mutant, which does not aggregate on bacterial lawns and arrests as loose mounds on nitrocellulose filters . The atg6(-) and atg8(-) mutants display almost normal development on nitrocellulose filters, producing multi-tipped aggregates that mature into small fruiting bodies . The distribution of a green fluorescent protein fusion of the autophagosome marker, Atg8, is aberrant in both atg1-1 and atg6(-) mutants.

Bioinformatics, 2004 Jan 22, 20(2), 180 - 5
A comparative phylogenetic approach for dating whole genome duplication events; Chapman BA et al.; MOTIVATION: Whole genome duplications have played a major role in determining the structure of eukaryotic genomes . Current evidence revealing large blocks of duplicated chromatin yields new insights into the evolutionary history of species, but also presents a major challenge for researchers attempting to utilize comparative genomics techniques . Understanding the timing of duplication events relative to divergence among taxa is critical to accurate and comprehensive cross-species comparisons . RESULTS: We describe a large-scale approach to estimate the timing of duplication events in a phylogenetic context . The methodology has been previously utilized for analysis of Arabidopsis and Saccharomyces duplication events . This new implementation provides a more flexible and reusable framework for these analyses . Scripts written in the Python programming language drive a number of freely available bioinformatics programs, creating a no-cost tool for researchers . The usefulness of the approach is demonstrated through genome-scale analysis of Arabidopsis and Oryza (rice) duplications . AVAILABILITY: Software and documentation are freely available from http://plantgenome.agtec.uga.edu/bioinformatics/dating/

Clin Infect Dis, 2004 Feb 1, 38(3), 335 - 41 Epub 2004 Jan 14.
Blastoschizomyces capitatus infection in patients with leukemia: report of 26 cases; Martino R et al.; Twenty-six cases of Blastoschizomyces capitatus infection were diagnosed in 25 patients at 7 tertiary care hematology units in Spain over a 10-year period . Most patients (92%) had acute leukemia and developed infection during a period of severe and prolonged neutropenia . Two patients had esophagitis, and the rest had invasive infection . Fungemia (20 cases) was a common finding, with frequent visceral dissemination . The 30-day mortality associated with this infection was 52%, compared with 57% among patients with systemic infection . In a univariate analysis, the following 3 variables had a positive impact on 30-day survival: removal of the central venous catheter within 5 days after the onset of infection (P=.02), a good performance status (P=.003), and receipt of systemic prophylactic or empirical antifungal therapy before infection onset (P=.006) . Outcome for neutropenic patients with B . capitatus infection is still poor . Rapid removal of the central venous catheter and novel antifungal therapies are recommended for treatment of this rare infection.

BMC Genomics . 2004 Jan 13;5(1):5.
Identification and comparative analysis of components from the signal recognition particle in protozoa and fungi; Rosenblad MA et al.; BACKGROUND: The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane . The SRP of metazoans is well characterized and composed of an RNA molecule and six polypeptides . The particle is organized into the S and Alu domains . The Alu domain has a translational arrest function and consists of the SRP9 and SRP14 proteins bound to the terminal regions of the SRP RNA . So far, our understanding of the SRP and its evolution in lower eukaryotes such as protozoa and yeasts has been limited . However, genome sequences of such organisms have recently become available, and we have now analyzed this information with respect to genes encoding SRP components . RESULTS: A number of SRP RNA and SRP protein genes were identified by an analysis of genomes of protozoa and fungi . The sequences and secondary structures of the Alu portion of the RNA were found to be highly variable . Furthermore, proteins SRP9/14 appeared to be absent in certain species . Comparative analysis of the SRP RNAs from different Saccharomyces species resulted in models which contain features shared between all SRP RNAs, but also a new secondary structure element in SRP RNA helix 5 . Protein SRP21, previously thought to be present only in Saccharomyces, was shown to be a constituent of additional fungal genomes . Furthermore, SRP21 was found to be related to metazoan and plant SRP9, suggesting that the two proteins are functionally related . CONCLUSIONS: Analysis of a number of not previously annotated SRP components show that the SRP Alu domain is subject to a more rapid evolution than the other parts of the molecule . For instance, the RNA portion is highly variable and the protein SRP9 seems to have evolved into the SRP21 protein in fungi . In addition, we identified a secondary structure element in the Saccharomyces RNA that has been inserted close to the Alu region . Together, these results provide important clues as to the structure, function and evolution of SRP.

Curr Biol, 2004 Jan 6, 14(1), R33 - 4
Vesicle transport: a close collaboration of Rabs and effectors; Spang A; COPII vesicles transport proteins destined for secretion from the ER to the Golgi apparatus . A recent study has shown that, in budding yeast, the formation of COPII vesicles requires Yip1p, an effector protein of a Rab GTPase.

Bioinformatics, 2004 Jan 1, 20(1), 5 - 20
Identifying periodically expressed transcripts in microarray time series data; Wichert S et al.; MOTIVATION: Microarray experiments are now routinely used to collect large-scale time series data, for example to monitor gene expression during the cell cycle . Statistical analysis of this data poses many challenges, one being that it is hard to identify correctly the subset of genes with a clear periodic signature . This has lead to a controversial argument with regard to the suitability of both available methods and current microarray data . METHODS: We introduce two simple but efficient statistical methods for signal detection and gene selection in gene expression time series data . First, we suggest the average periodogram as an exploratory device for graphical assessment of the presence of periodic transcripts in the data . Second, we describe an exact statistical test to identify periodically expressed genes that allows one to distinguish periodic from purely random processes . This identification method is based on the so-called g-statistic and uses the false discovery rate approach to multiple testing . RESULTS: Using simulated data it is shown that the suggested method is capable of identifying cell-cycle-activated genes in a gene expression data set even if the number of the cyclic genes is very small and regardless the presence of a dominant non-periodic component in the data . Subsequently, we re-examine 12 large microarray time series data sets (in part controversially discussed) from yeast, human fibroblast, human HeLa and bacterial cells . Based on the statistical analysis it is found that a majority of these data sets contained little or no statistical significant evidence for genes with periodic variation linked to cell cycle regulation . On the other hand, for the remaining data the method extends the catalog of previously known cell-cycle-specific transcripts by identifying additional periodic genes not found by other methods . The problem of distinguishing periodicity due to generic cell cycle activity and to artifacts from synchronization is also discussed . AVAILABILITY: The approach has been implemented in the R package GeneTS available from under the terms of the GNU General Public License.

Adv Space Res, 2003, 31(10), 2181 - 6
Gravitaxis and graviperception in flagellates; Hader DP et al.; There is strong evidence that gravitactic orientation in flagellates and ciliates is mediated by an active physiological gravireceptor rather than by passive alignment of the cells in the water column . In flagellates the threshold for graviorientation was found to be at 0.12 x g on a slow rotating centrifuge during the IML-2 mission on the Shuttle Columbia and a subsequent parabolic rocket flight (TEXUS) . During the IML-2 mission no adaptation to microgravity was observed over the duration of the space flight, while gravitaxis was lost in a terrestrial closed environmental system over the period of almost two years . Sedimenting statoliths are not likely to be involved in graviperception because of the small size of the cells and their rotation around the longitudinal axis during forward locomotion . Instead the whole cytoplasmic content of the cell, being heavier than the surrounding aqueous medium (1.05 g/ml), exerts a pressure on the lower membrane . This force activates stretch-sensitive calcium specific ion channels which can be inhibited by the addition of gadolinium which therefore abolishes gravitaxis . The channels seem to mainly allow calcium ions to pass since gravitaxis is blocked by the addition of the calcium ionophore A23187 and by vanadate which blocks the Ca-ATPase in the cytoplasmic membrane . Recently, a gene for a mechanosensitive channel, originally sequenced for Saccharomyces, was identified in Euglena by PCR . The increase in intracellular free calcium during reorientation can be visualized by the fluorophore Calcium Crimson using laser excitation and image intensification . This result was confirmed during recent parabolic flights . The gated calcium changes the membrane potential across the membrane which may be the trigger for the reorientation of the flagellum . cAMP plays a role as a secondary messenger . Photosynthetic flagellates are suitable candidates for life support systems since they absorb CO2 and produce oxygen . Preliminary experiments are discussed . c2003 COSPAR . Published by Elsevier Ltd . All rights reserved.

Curr Biol, 2003 Dec 16, 13(24), 2190 - 5
EST analysis of the cnidarian Acropora millepora reveals extensive gene loss and rapid sequence divergence in the model invertebrates; Kortschak RD et al.; A significant proportion of mammalian genes are not represented in the genomes of Drosophila, Caenorhabditis or Saccharomyces, and many of these are assumed to have been vertebrate innovations . To test this assumption, we conducted a preliminary EST project on the anthozoan cnidarian, Acropora millepora, a basal metazoan . More than 10% of the Acropora ESTs with strong metazoan matches to the databases had clear human homologs but were not represented in the Drosophila or Caenorhabditis genomes; this category includes a surprising diversity of transcription factors and metabolic proteins that were previously assumed to be restricted to vertebrates . Consistent with higher rates of divergence in the model invertebrates, three-way comparisons show that most Acropora ESTs match human sequences much more strongly than they do any Drosophila or Caenorhabditis sequence . Gene loss has thus been much more extensive in the model invertebrate lineages than previously assumed and, as a consequence, some genes formerly thought to be vertebrate inventions must have been present in the common metazoan ancestor . The complexity of the Acropora genome is paradoxical, given that this organism contains apparently few tissue types and the simplest extant nervous system consisting of a morphologically homogeneous nerve net.

Proteomics, 2003 Dec, 3(12), 2330 - 8
Proteomic analysis of Candida magnoliae strains by two-dimensional gel electrophoresis and mass spectrometry; Lee DY et al.; Candida magnoliae which has been newly isolated from honey comb is an osmotolerant yeast to produce erythritol as a major product . Erythritol is a noncariogenic, low calorie sweetener and safe for diabetics . Strain development by chemical mutation to obtain the improved erythritol yield and productivity relative to the parental strain made it necessary to elucidate the physiological differences between the wild and mutant strains . Proteomic analyses of C . magnoliae wild and mutant strains with two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry were carried out to identify intracellular proteins and to estimate the effects of newly characterized metabolic enzymes on the yeast cell growth and erythritol production . Most of the molecular mass of intracellular proteins were distributed in the range of pI 4-8 and molecular mass of approximately 130 kDa . Six out of nine protein spots expressed at different levels between the wild and mutant strains were analyzed with nanoelectrospray tandem mass spectrometry and identified by comparing amino acid sequences with the National Center for Biotechnology Information and Saccharomyces Genome Databases . Except for Ygr086cp, these proteins were believed to be the metabolic enzymes involved in the citric acid cycle (citrate synthase, succinyl-CoA ligase and fumarase) and the glycolysis pathway (pyruvate decarboxylase and enolase) . Up-regulated enzymes in the citric acid cycle could explain high growth of the C . magnoliae mutant strain owing to the increased NADH and ATP formation . Down-regulated enolase and up-regulated fumarase in the mutant strain seemed to play a role in the improved bioconversion of erythrose-4-phosphate to erythritol compared with the wild strain.

J Gen Appl Microbiol, 2003 Oct, 49(5), 267 - 70
Ribosomal DNA sequencing and reinstatement of the genus Arthroascus von Arx; Naumov GI et al.; Sequence analysis of the D1/D2 domain of 26S rDNA was conducted upon seven Arthroascus strains from different geographic localities . The European and Asian species Arthroascus schoenii was documented from the North-American continent and from the Island of Hawaii . We discuss the heterogeneity of the genus Saccharomycopsis sensu Kurtzman and Robnett 1995 . On the basis of molecular and genetic data the genus Arthroascus von Arx is reinstated.

Genome, 2003 Dec, 46(6), 947 - 52
Unfolding large-scale maps; Jenkins G; This is an account of the development and use of genetic maps, from humble beginnings at the hands of Thomas Hunt Morgan, to the sophistication of genome sequencing . The review charters the emergence of molecular marker maps exploiting DNA polymorphism, the renaissance of cytogenetics through the use of fluorescence in situ hybridisation, and the discovery and isolation of genes by map-based cloning . The historical significance of sequencing of DNA prefaces a section describing the sequencing of genomes, the ascendancy of particular model organisms, and the utility and limitations of comparative genomic and functional genomic approaches to further our understanding of the control of biological processes . Emphasis is given throughout the treatise as to how the structure and biological behaviour of the DNA molecule underpin the technological development and biological applications of maps.

J Cell Sci, 2003 Dec 15, 116(Pt 24), 4883 - 90
Wee1-dependent mechanisms required for coordination of cell growth and cell division; Kellogg DR; Wee1-related kinases function in a highly conserved mechanism that controls the timing of entry into mitosis . Loss of Wee1 function causes fission yeast and budding yeast cells to enter mitosis before sufficient growth has occurred, leading to formation of daughter cells that are smaller than normal . Early work in fission yeast suggested that Wee1 is part of a cell-size checkpoint that prevents entry into mitosis before cells have reached a critical size . Recent experiments in fission yeast and budding yeast have provided new support for this idea . In addition, studies in budding yeast have revealed the existence of highly intricate signaling networks that are required for regulation of Swe1, the budding yeast homolog of Wee1 . Further understanding of these signaling networks may provide important clues to how cell growth and cell division are coordinated.

Curr Biol, 2003 Nov 11, 13(22), 1979 - 84
Spindle checkpoint component Mad2 contributes to biorientation of homologous chromosomes; Shonn MA et al.; Cell cycle checkpoints sense defects in chromosome metabolism, halt the cell cycle, and activate pathways that repair the defects . The spindle checkpoint arrests the cell cycle in response to defects in the interaction between microtubules and kinetochores (the proteinaceous complex assembled on centromeric DNA), but no repair function has been demonstrated for this checkpoint . We show that the roles of two spindle checkpoint components, Mad2 and Mad3, differ in meiosis I . In the absence of Mad2, meiosis I nondisjunction occurs at a high frequency and can be corrected by delaying the onset of anaphase . The absence of Mad3 does not induce nondisjunction, even though mad3Delta cells cannot arrest the cell cycle in response to kinetochores that lack either microtubules or tension on the linkage between chromosomes and microtubules . The two proteins have different roles in chromosome alignment . Compared to wild type and mad3Delta cells, mad2Delta mutants are slower to attach homologous chromosomes to opposite poles of the spindle . This observation suggests that Mad2 plays a role in reorienting chromosomes that are incorrectly attached to the spindle as well as delaying the cell cycle, whereas Mad3 is needed for the cell cycle delay, but not for chromosome reorientation.

Curr Biol, 2003 Nov 11, 13(22), 1954 - 62
The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over; Rockmill B et al.; BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases . Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis . The sgs1 null mutant sporulates poorly and displays reduced spore viability . RESULTS: We have identified novel functions for Sgs1 in meiosis . Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation . In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis . Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion . The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase . A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation . CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions . The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis . Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange . We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase.

Curr Biol, 2003 Nov 11, 13(22), 1930 - 40
A model for ATP hydrolysis-dependent binding of cohesin to DNA; Weitzer S et al.; BACKGROUND: Cohesion between sister chromatids is promoted by the chromosomal cohesin complex that forms a proteinaceous ring, large enough in principle to embrace two sister strands . The mechanism by which cohesin binds to DNA, and how sister chromatid cohesion is established, is unknown . RESULTS: Biochemical studies of cohesin have largely been limited to protein isolated from soluble cellular fractions . Here, we characterize cohesin purified from budding yeast chromatin, suggesting that chromosomal cohesin is sufficiently described by its known distinctive ring structure . We present evidence that the two Smc subunits of cohesin by themselves form a ring, closed at interacting ATPase head domains . A motif in the Smc1 subunit implicated in ATP hydrolysis is essential for loading cohesin onto DNA . In addition to functional ATPase heads, an intact cohesin ring structure is indispensable for DNA binding, suggesting that ATP hydrolysis may be coupled to DNA transport into the cohesin ring . DNA is released in anaphase when separase cleaves cohesin's Scc1 subunit . We show that a cleavage fragment of Scc1 disrupts the interaction between the two Smc heads, thereby opening the ring . CONCLUSIONS: We present a model for cohesin binding to chromatin by ATP hydrolysis-dependent transport of DNA into the cohesin ring . After DNA replication, two DNA strands may be trapped to promote sister chromatid cohesion . In anaphase, Scc1 cleavage opens the ring to release sister chromatids.

J Biol Chem, 2004 Feb 27, 279(9), 8452 - 9 Epub 2003 Nov 10.
Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes; Kanazawa T et al.; Autophagy, a major bulk proteolytic pathway, contributes to intracellular protein turnover, together with protein synthesis . Both are subject to dynamic control by amino acids and insulin . The mechanisms of signaling and cross-talk of their physiological anabolic effects remain elusive . Recent studies established that amino acids and insulin induce p70 S6 kinase (p70(S6k)) phosphorylation by mTOR, involved in translational control of protein synthesis . Here, the signaling mechanisms of amino acids and insulin in macroautophagy in relation to mTOR were investigated . In isolated rat hepatocytes, both regulatory amino acids (RegAA) and insulin coordinately activated p70(S6k) phosphorylation, which was completely blocked by rapamycin, an mTOR inhibitor . However, rapamycin blocked proteolytic suppression by insulin, but did not block inhibition by RegAA . These contrasting results suggest that insulin controls autophagy through the mTOR pathway, but amino acids do not . Furthermore, micropermeabilization with Saccharomyces aureus alpha-toxin completely deprived hepatocytes of proteolytic responsiveness to RegAA and insulin, but still maintained p70(S6k) phosphorylation by RegAA . In contrast, Leu(8)-MAP, a non-transportable leucine analogue, did not mimic the effect of leucine on p70(S6k) phosphorylation, but maintained the activity on proteolysis . Finally, BCH, a System L-specific amino acid, did not affect proteolytic suppression or mTOR activation by leucine . All the results indicate that mTOR is not common to the signaling mechanisms of amino acids and insulin in autophagy, and that the amino acid signaling starts extracellularly with their "receptor(s)," probably other than transporters, and is mediated through a novel route distinct from the mTOR pathway employed by insulin.

Chem Commun (Camb), 2003 Oct 21, (20), 2636 - 7
Stereoinversion of beta- and gamma-substituted alpha-amino acids using a chemo-enzymatic oxidation-reduction procedure; Enright A et al.; Both D- and L-beta- and gamma-substituted alpha-amino acids can be interconverted to their respective L- and D- diastereoisomers by treatment with an enantioselective amino acid oxidase and a chemical reducing agent.

J Cell Sci, 2003 Nov 15, 116(Pt 22), 4501 - 12
Cytoplasmic dynein in fungi: insights from nuclear migration; Yamamoto A et al.; Cytoplasmic dynein is a microtubule motor that mediates various biological processes, including nuclear migration and organelle transport, by moving on microtubules while associated with various cellular structures . The association of dynein with cellular structures and the activation of its motility are crucial steps in dynein-dependent processes . However, the mechanisms involved remain largely unknown . In fungi, dynein is required for nuclear migration . In budding yeast, nuclear migration is driven by the interaction of astral microtubules with the cell cortex; the interaction is mediated by dynein that is probably associated with the cortex . Recent studies suggest that budding yeast dynein is first recruited to microtubules, then delivered to the cortex by microtubules and finally activated by association with the cortex . Nuclear migration in many other fungi is probably driven by a similar mechanism . Recruitment of dynein to microtubules and its subsequent activation upon association with cellular structures are perhaps common to many dynein-dependent eukaryotic processes, including organelle transport.

Nature, 2003 Oct 23, 425(6960), 798 - 804
Genome-scale approaches to resolving incongruence in molecular phylogenies; Rokas A et al.; One of the most pervasive challenges in molecular phylogenetics is the incongruence between phylogenies obtained using different data sets, such as individual genes . To systematically investigate the degree of incongruence, and potential methods for resolving it, we screened the genome sequences of eight yeast species and selected 106 widely distributed orthologous genes for phylogenetic analyses, singly and by concatenation . Our results suggest that data sets consisting of single or a small number of concatenated genes have a significant probability of supporting conflicting topologies . By contrast, analyses of the entire data set of concatenated genes yielded a single, fully resolved species tree with maximum support . Comparable results were obtained with a concatenation of a minimum of 20 genes; substantially more genes than commonly used but a small fraction of any genome . These results have important implications for resolving branches of the tree of life.

Carbohydr Res, 2003 Oct 10, 338(21), 2221 - 5
Preparation and reactivity of a novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose); Yoshinaga K et al.; A novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose, GAF) was enzymatically prepared from 1,5-anhydro-D-fructose (1,5-AF) and cyclomaltoheptaose (beta-cyclodextrin) . Cyclodextrin glucanotransferase transferred various sizes of maltooligosaccharide to 1,5-AF . Glucoamylase digested the maltooligosyl chain of the products to a glucosyl residue giving a final product, GAF . An NMR analysis of GAF elucidated that the glucose residue was linked to C-3 of the 1,5-AF residue with an ether linkage . Reactivity on the aminocarbonyl reaction of GAF with bovine serum albumin was lower than that of 1,5-AF, but was higher than that of glucose.

Dev Cell, 2003 Oct, 5(4), 528 - 30
Ac'septin' a signal: kinase regulation by septins; Moffat J et al.; Budding yeast monitor shape and the assembly of cytoskeletal structures and convey this information to regulators of cell division, but the molecular mechanisms responsible for monitoring and interpreting spatial information about the cytoskeleton remain poorly understood . A paper in the September issue of Molecular Cell shows that direct binding of components of the septin cytoskeleton may relieve autoinhibition of a conserved checkpoint kinase, creating a simple molecular device for sensing septin cytoskeleton organization.

Clin Exp Allergy, 2003 Oct, 33(10), 1429 - 38
Sensitization to fungi: epidemiology, comparative skin tests, and IgE reactivity of fungal extracts; Mari A et al.; BACKGROUND: Several fungal species are known to cause severe respiratory and cutaneous allergic diseases . Extracts from several allergenic fungi are used for in vivo and in vitro tests, as standard preparations are still not available . OBJECTIVE: The aims are to define the pattern of in vivo and in vitro IgE reactivity to fungal species in an allergic population with respiratory symptoms; to determine the influence of different extract preparations on diagnostic results; and to evaluate whether there exists a relationship between the diagnostic pattern of reactivity and the pattern of specific IgE reactivity in immunoblots . METHODS: Skin prick tests were applied to a cohort of 4962 respiratory subjects, aged 3-80 years . Fungal extracts from Alternaria, Aspergillus, Candida, Cladosporium, Penicillium, Saccharomyces, and Trichophyton were used, along with extracts from pollens, mites, and animal dander . Demographical and diagnostic data were recorded . IgE detection was carried out with the same allergenic extracts plus Malassezia . Comparative skin tests and IgE detection were carried out using extracts from three commercial suppliers . IgE immunoblots were carried out with the same panel of commercial fungal extracts and were compared with in-house extracts . Data analysis was carried out by grouping the population on the basis of their reactivity to a single, to two or to more than two, mould species . RESULTS: Nineteen percent of the allergic population reacted to at least one fungal extract by means of the skin test . Alternaria and Candida accounted for the largest number of positive tests, and along with Trichophyton they were the main sensitizers in the subset of patients with an isolated sensitization . The prevalence of skin test reactivity increased for these three fungi in the subsets with two associated reactivities and, furthermore, in the subset showing reactivity to more than two mould species . In the latter group, a steady increase of the skin test reactivity was recorded for all the other fungal sources, suggesting a clustered reactivity . Comparative skin and IgE testing with different groups of subjects with a simple pattern of skin reactivity resulted in sensitivity differences between in vivo and in vitro tests, whereas discrepant results were recorded in the subsets of patients with multiple fungi sensitization . Although hampered by the limited reliability of fungal extracts, IgE immunoblots revealed differing patterns of reactivity when sera from the three subsets were used . This suggests a link between the diagnostic reactivity pattern and the IgE sensitization to extracts' components . Age and gender distribution differed among the Alternaria-, Candida-, and Trichophyton-sensitized subjects, but not in the subset with more than two fungi sensitizations . CONCLUSIONS: The preliminary assessment of a new classification of the mould-sensitized population has been reached . The limiting quality of fungal extracts requires future studies using an allergenic molecule-based approach . The diagnostic process and the definition of the reactivity pattern would thus be easy, and it could lead to a novel specific immunotherapy approach.

J Clin Gastroenterol, 2003 Oct, 37(4), 315 - 29
Autoantibodies in the diagnosis and management of liver disease; Czaja AJ et al.; Autoantibodies are nonpathogenic manifestations of immune reactivity, and they may occur in acute and chronic liver diseases . Autoantibodies may be consequences rather than causes of the liver injury, and they should be regarded as diagnostic clues rather than etiologic markers . Conventional autoantibodies used in the categorization of autoimmune liver disease are antinuclear antibodies, smooth muscle antibodies, antibodies to liver/kidney microsome type 1, antimitochondrial antibodies, and atypical perinuclear anti-neutrophil cytoplasmic antibodies . Ancillary autoantibodies that enhance diagnostic specificity, have prognostic connotation, or direct treatment are antibodies to endomysium, tissue transglutaminase, histones, doubled-stranded DNA, and actin . Autoantibodies that have an emerging diagnostic and prognostic significance are antibodies to soluble liver antigen/liver pancreas, asialoglycoprotein receptor, liver cytosol type 1, and nuclear pore complex antigens . Autoantibodies of uncertain clinical value that remain under investigation are antibodies to chromatin, lactoferrin, and Saccharomyces cervisiae . Continued recognition and characterization of autoantibodies should improve diagnostic precision, provide prognostic indices, and elucidate target autoantigens . These advances may in turn clarify pathogenic mechanisms, facilitate the development of animal models, and generate novel site-specific therapies.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1665 - 70
Metschnikowia vanudenii sp . nov . and Metschnikowia lachancei sp . nov., from flowers and associated insects in North America; Gimenez-Jurado G et al.; Two new species of the ascosporic yeast genus Metschnikowia were isolated from nectaries and associated muscoid flies of flowers from the common milkweed (Asclepias syriaca) in North America, and are described as Metschnikowia vanudenii {type strain=PYCC 4650(T)=CBS 9134(T)=NRRL Y-27243(T)=UWO(PS) 86A4.1(T)} and Metschnikowia lachancei {type strain=PYCC 4605(T)=CBS 9131(T)=NRRL Y-27242(T)=UWO(PS) 7ASB2.3(T)} . As with the previously described Metschnikowia gruessii, M . vanudenii has vegetative cells with an 'aeroplane' or cross-like configuration, produces ovoid chlamydospores and forms ellipsoidopedunculate asci with two acicular ascospores . Metschnikowia lachancei is distinguished from other Metschnikowia species by formation of club-shaped asci with 1-2 thick clavate ascospores . The phylogenetic positions of the proposed new species within Metschnikowia were determined from sequence analysis of the D1/D2 domain of 26S rDNA . The new species show low nuclear DNA relatedness with neighbouring taxa.

J Biol Chem, 2003 Oct 31, 278(44), 43178 - 87 Epub 2003 Aug 22.
Human mitochondrial C1-tetrahydrofolate synthase: gene structure, tissue distribution of the mRNA, and immunolocalization in Chinese hamster ovary calls; Prasannan P et al.; C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase . The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces . Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase . The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon . The gene encodes a protein of 978 amino acids, including an N-terminal mitochondrial targeting sequence . The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme . Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain . Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue . The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8 . Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal . This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity . Both transcripts initiate at the same 5'-site, 107 nucleotides up-stream of the ATG start codon . The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells . Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein . Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase.

Mol Cell Biol, 2003 Sep, 23(17), 6327 - 37
Specific inhibition of Elm1 kinase activity reveals functions required for early G1 events; Sreenivasan A et al.; In budding yeast, the Elm1 kinase is required for coordination of cell growth and cell division at G(2)/M . Elm1 is also required for efficient cytokinesis and for regulation of Swe1, the budding yeast homolog of the Wee1 kinase . To further characterize Elm1 function, we engineered an ELM1 allele that can be rapidly and selectively inhibited in vivo . We found that inhibition of Elm1 kinase activity during G(2) results in a phenotype similar to the phenotype caused by deletion of the ELM1 gene, as expected . However, inhibition of Elm1 kinase activity earlier in the cell cycle results in a prolonged G(1) delay . The G(1) requirement for Elm1 kinase activity occurs before bud emergence, polarization of the septins, and synthesis of G(1) cyclins . Inhibition of Elm1 kinase activity during early G(1) also causes defects in the organization of septins, and inhibition of Elm1 kinase activity in a strain lacking the redundant G(1) cyclins CLN1 and CLN2 is lethal . These results demonstrate that the Elm1 kinase plays an important role in G(1) events required for bud emergence and septin organization.

Mol Cell Biol, 2003 Aug, 23(16), 5939 - 46
Replication checkpoint kinase Cds1 regulates recombinational repair protein Rad60; Boddy MN et al.; Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks . How Cds1 accomplishes this task is unknown . We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast . Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization . A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest . Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair . These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1 . We propose that control of Rad60 regulates recombination events at stalled forks.

Int J Food Microbiol, 2003 Aug 1, 84(3), 327 - 38
Effect of Penicillium chrysogenum and Debaryomyces hansenii on the volatile compounds during controlled ripening of pork loins; Martin A et al.; During ripening of meat products such as dry-cured ham, the moulds and yeasts that proliferate on the surface may contribute to flavour development . However, their contribution to volatile components of dry-cured meat products is not known . One strain each of Penicillium chrysogenum and Debaryomyces hansenii, selected from dry-cured ham by their proteolytic activity, were tested to determine their effect on the volatile compounds during ripening . Sterile pork loins were inoculated and ripened for 106 days . Volatile compounds collected with a Solid Phase Micro-Extraction (SPME) fibre were analysed by GC/MS . Inoculation of pork loins with P . chrysogenum lead to a decrease in compounds attributed to lipid oxidation and to an increase of compounds derived from free amino acids . Inoculation with D . hansenii seemed to favour the formation of complex alcohols.

Genome Biol . 2003;4(6):R40 . Epub 2003 May 30.
A method to assess compositional bias in biological sequences and its application to prion-like glutamine/asparagine-rich domains in eukaryotic proteomes; Harrison PM et al.; We have derived a novel method to assess compositional biases in biological sequences, which is based on finding the lowest-probability subsequences for a given residue-type set . As a case study, the distribution of prion-like glutamine/asparagine-rich ((Q+N)-rich) domains (which are linked to amyloidogenesis) was assessed for budding and fission yeasts and four other eukaryotes . We find more than 170 prion-like (Q+N)-rich regions in budding yeast, and, strikingly, many fewer in fission yeast . Also, some residues, such as tryptophan or isoleucine, are unlikely to form biased regions in any eukaryotic proteome.

Folia Microbiol (Praha), 2003, 48(2), 177 - 82
Colocalization of cortical microtubules and F-actin in Dipodascus magnusii using confocal laser scanning microscopy; Hasek J et al.; Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin . A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei . In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex . In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites . Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network . The actin system of D . magnusii consisted of patches and filaments . Patches were found to accumulate at the tips of growing cells . Bands of fine actin filaments were usually observed before F-actin rings were established . A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells . Cells with developing septa showed medial F-actin discs associated at both sides with microtubules . Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D . magnusii.

J Biol Chem, 2003 Aug 15, 278(33), 31184 - 91 Epub 2003 Jun 03.
Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase: an enzyme that preferentially regulates metabolism of very long chain ceramides; Mao C et al.; Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids . In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity . maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively . Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin . maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry . In vitro biochemical characterization determined that maCER1 hydrolyzed D-erythro-ceramide exclusively but not D-erythro-dihydroceramide or D-ribo-phytoceramide . Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+ . maCER1 was also inhibited by sphingosine, one of its products . Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells . Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of D-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P . Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids.

Nat Rev Mol Cell Biol, 2003 Jun, 4(6), 468 - 78
A fuzzy mitochondrial fusion apparatus comes into focus; Mozdy AD et al.; Membrane fusion is fundamental to eukaryotic life . Unlike the predominant intracellular fusion machineries that fuse compartments bounded by a single membrane, the mitochondrial fusion machinery must sequentially fuse the outer and inner mitochondrial membranes . These coordinated fusion events rely on a transmembrane GTPase that is known as fuzzy onions or Fzo . Recent studies have revealed that Fzo has an evolutionarily conserved role in mitochondrial fusion, and they take the first strides in determining the molecular nature of such a role.

Carbohydr Res, 2003 May 23, 338(11), 1175 - 82
A new mannoheptaose containing alpha and beta-(1-->2) linkages isolated from the mannan of Torulaspora delbrueckii: ELISA inhibition studies; Okawa Y et al.; Torulaspora delbrueckii starin IFO 0955 was examined with respect to its structural and serological properties of the cell wall mannan (Td-0955-M) . Td-0955-M revealed significant reactivities with sera from a commercially available factor serum kit (Candida Check) in ELISA . Td-0955-M was investigated for its chemical structure by acetolysis under conventional and mild conditions . NMR and GC techniques were used as analytical techniques . The mannooligosaccharide fractions eluted from a Bio-Gel P-2 column were found to consist of Man(alpha1-2)Man, M2, Man(alpha1-2)Man(alpha1-2)Man and Man(beta1-2)Man(alpha1-2)Man, M3, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M5, and a new mannoheptaose, which possesses the structure, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M7 . The results of the inhibition ELISA showed that the M7 oligosaccharide significantly inhibited the reactivities in the Td-0955-M-factor serum systems.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 528 - 35 Epub 2003 May 06.
The constitutive AHSB4 promoter--a novel component of the Arxula adeninivorans-based expression platform; Wartmann T et al.; An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized . The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes . The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones . The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions . The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment . Plasmids incorporating respective expression cassettes were used to transform the host strain A . adeninivorans LS3, which forms budding cells at 30 degrees C, and strain 135, which forms mycelia under these conditions . Transformants of both types were found to harbor a single copy of the heterologous DNA . Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4 . In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l(-1) culture medium were achieved . This productivity could be increased to 50 mg l(-1 )in strains harboring two copies of the expression cassette . The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions.

FEMS Yeast Res, 2003 Apr, 3(2), 211 - 6
Physiological behaviour of Hanseniaspora guilliermondii in aerobic glucose-limited continuous cultures; Albergaria H et al.; The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures . By both cultivation techniques this yeast was found to be Crabtree-positive . Up to a dilution rate of 0.25 h(-1), glucose was completely metabolised into biomass, glycerol and carbon dioxide . Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids . Biomass yield during the purely oxidative growth was 0.49 g g(-1) and decreased to 0.26 g g(-1) for D=0.34 h(-1) . A maximal specific ethanol production rate of 1.36 mmol g(-1) h(-1) and a maximal ethanol yield of 0.05 g g(-1) were achieved at D=0.34 h(-1).

FEMS Yeast Res, 2002 Jan, 1(4), 279 - 89
Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera; Cadez N et al.; Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions . Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri . Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes . Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species . Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.

FEMS Yeast Res, 2003 May, 3(3), 301 - 11
Genome comparisons in the genus Dipodascus de Lagerheim; Smith MT et al.; Phenotypic characteristics of physiology and morphology of 71 strains belonging to the genus Dipodascus de Lagerheim were examined . The GC contents of genomic DNAs of 46 strains were calculated from the thermal denaturation curves using the spectrophotometric method . The first derivatives of the melting curves revealed that the DNAs of these strains are heterogeneous; four categories could be recognized . However, DNA similarity values calculated by using DNA-DNA reassociation kinetics showed that each category could be subdivided further . Two categories were separated into four subgroups each; the other two yielded five subgroups each . Strains belonging to the same subgroup exhibited high levels of DNA similarity ranging from 82 to 100% . The 18 subgroups represented 13 currently accepted Dipodascus species and five anamorphic Geotrichum species, four representing novel taxa . A phenotypic key to distinguish the taxa of Dipodascus, Galactomyces and Geotrichum is presented.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 410 - 7
Application of waste activated bleaching earth containing rapeseed oil on riboflavin production in the culture of Ashbya gossypii; Ming H et al.; Waste activated bleaching earth (ABE) that contained 40% rapeseed oil and was discharged by an oil refinery plant, was used for riboflavin production in a culture of Ashbya gossypii . When 125 g/L waste ABE that contained 50 g/L rapeseed oil was added into the culture, the riboflavin concentration was 1.12 g/L, which was almost 1.6-fold as high as that of pure rapeseed oil . However, in waste ABE concentration higher than 125 g/L, the produced riboflavin concentration decreased, which was due to the difficulty in mixing due to the presence of a high amount of solid material in the culture . The surface of the waste ABE was smooth without a hitch, because of being covered with rapeseed oil . However, after the culture, the surface of the waste ABE seemed like that of new one, and the oil content was nearly zero grams per liter . The waste ABE, oily clay, and its black color gradually fade and yellow little by little, and finally the waste ABE changed to yellow powder . Of the riboflavin produced during the culture, 70% was adsorbed in the oil free waste ABE . With diluted alkali solution, extraction only two times yielded 90% recovery of riboflavin adsorbed in the waste ABE . The waste ABE containing waste vegetable oil was suitable for raw material for production of the value-added useful bioproducts, which might be a good model for reuse of the waste resource.

Anal Chem, 2003 Mar 15, 75(6), 1300 - 6
Fast-response proteomics by accelerated in-gel digestion of proteins; Havlis J et al.; Kinetics of in-gel digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards . The effect of the temperature, enzyme concentration, digestion time, and surface area of gel pieces on the yield of digestion products was characterized . Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage . The accelerated digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.

Appl Microbiol Biotechnol, 2003 Apr, 61(2), 157 - 62 Epub 2003 Jan 14.
Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production; Aguilar Uscanga MG et al.; The influence of the oxygen supply on the growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0-300 l h(-1 ) x (0-0.5 vvm) . This study shows that growth of this yeast is stimulated by moderate aeration . The optimal oxygen supply for cellular synthesis was an oxygen transfer rate (OTR) of 43 mg O(2) l(-1) x h(-1) . In this case, there was an air flow rate of 60 l h(-1) (0.1 vvm) . Above this value, the maximum biomass concentration decreased . Ethanol and acetic acid production was also dependent on the level of aeration: the higher the oxygen supply, the greater the acetic acid production and the lower the ethanol production . At the highest aeration rates, we observed a strong inhibition of the ethanol yield . Over 180 l h(-1) x (0.3 vvm, OTR =105 mg O(2) l(-1) x h(-1)), glucose consumption was inhibited and a high concentration of acetic acid (6.0 g x l(-1)) was produced . The ratio of "ethanol + acetic acid" produced per mole of consumed glucose using carbon balance calculations was analyzed . It was shown that this ratio remained constant in all cases . This makes it possible to establish a stoichiometric equation between oxygen supply and metabolite production.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Mar, 35(3), 215 - 8
Monomeric B27 Lys destripeptide insulin: semisynthesis, characterization and biological activity; Ding JG et al.; In this paper, we report the semisynthesis of B27 Lys destripeptide insulin (B27 Lys DTrI), i.e . destetrapeptide insulin with an additional Lys residue at the C-terminus of B-chain . B27 Lys DTrI is also monomeric as shown by gel filtration . Its in vivo biological activity is 80% in comparison with that of native insulin . The addition of a Lys residue at the C-terminus of B-chain makes it possible to obtain monomeric B27 Lys DTrI from a precursor expressed in Saccharomyces cerevesiae by tryptic hydrolysis instead of the less efficient tryptic transpeptidation.

Fungal Genet Biol, 2003 Mar, 38(2), 220 - 7
Mutation in a calpain-like protease affects the posttranslational mannosylation of phosphatases in Aspergillus nidulans; Nozawa SR et al.; In this communication, we show that the palB7 mutation drastically reduced the mannose and N-acetylgalactosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans at pH 5.0, compared to a control strain . By using mRNA differential display reverse transcription and polymerase chain reaction, we isolated two cDNAs from the control pabaA1 strain that were not detected in the palB7 mutant strain that encode a mannosyl transferase and a NADH-ubiquinone oxidoreductase . Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the palB gene, which encodes for a nuclear calpain-like protease that may have specific functions in the processing of transcription factor(s) similar to its homolog, RIM13, in Saccharomyces cereviseae.

J Mol Evol, 2003 Mar, 56(3), 341 - 50
Macronuclear molecules encoding actins in spirotrichs; Croft KE et al.; The nucleotide sequences of 16 newly reported and 8 previously reported actin-encoding macronuclear DNA molecules in spirotrichs have been compared . As described for the eight previously reported molecules, the first 50 bases (noncoding) inside the telomere at both 5' strands in additional actin molecules are purine-rich . This anomalous base composition might serve as a signal to identify macronuclear molecules in micronuclear DNA during development . The 50-base segment upstream of the ATG in the 5' leaders of the actin molecules contains extensive, conserved sequence motifs that are possibly promoter elements . The 3' noncoding trailers contain virtually no conserved sequence motifs . With one exception, the 3' trailers contain a second stop codon (TGA) 36 bases on average downstream of the primary stop codon . Excluding Moneuplotes crassus, amino acid identities in actin I range from 78 to 100%, with variations distributed nonrandomly along the sequence . Phylogenetic trees based on the actin nucleotide sequences of 22 spirotrichs define the evolutionary relationships of their actin-encoding molecules . The actin phylogeny, while well supported by posterior probabilities, does not always coincide with the phylogeny defined in rDNA analyses or classical taxonomic classifications.

Cell, 2003 Feb 21, 112(4), 407 - 21
Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling; Cleveland DW et al.; The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division . Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined . The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement . Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).

Cell, 2003 Feb 21, 112(4), 403 - 6
Considering nuclear compartmentalization in the light of nuclear dynamics; Chubb JR et al.; Many proteins are concentrated in compartments within the nucleus . Chromatin is also compartmentalized at different nuclear sites . However, nuclear proteins have now been shown to be highly mobile . This review considers the formation and function of nuclear compartments in a situation in which proteins are rapidly moving through the nuclear volume.

Genome Biol . 2002;3(12):PREPRINT0012 . Epub 2002 Nov 21.
Osprey: a network visualization system; Breitkreutz BJ et al.; We have developed a software platform called Osprey for visualization and manipulation of complex interaction networks . Osprey builds data-rich graphical representations that are color-coded for gene function and experimental interaction data . Mouse-over functions allow rapid elaboration and organization of network diagrams in a spoke model format . User-defined large-scale data sets can be readily combined with Osprey for comparison of different methods.

Biochim Biophys Acta, 2002 Dec 30, 1585(2-3), 146 - 52
Serine palmitoyltransferase: role in apoptotic de novo ceramide synthesis and other stress responses; Perry DK; Serine palmitoyltransferase is the first and rate-limiting enzyme of sphingolipid synthesis . As such, it is a central control point in the synthesis of bioactivate sphingolipids, and it plays an important role in mediating cellular stress responses . In this review, its role in mediating these responses is discussed within the context of de novo ceramide synthesis . Furthermore, a discussion is provided of its regulation as discerned from both yeast and mammalian studies.

Nucleic Acids Res, 2003 Jan 1, 31(1), 374 - 8
TRANSFAC: transcriptional regulation, from patterns to profiles; Matys V et al.; The TRANSFAC database on eukaryotic transcriptional regulation, comprising data on transcription factors, their target genes and regulatory binding sites, has been extended and further developed, both in number of entries and in the scope and structure of the collected data . Structured fields for expression patterns have been introduced for transcription factors from human and mouse, using the CYTOMER database on anatomical structures and developmental stages . The functionality of Match, a tool for matrix-based search of transcription factor binding sites, has been enhanced . For instance, the program now comes along with a number of tissue-(or state-)specific profiles and new profiles can be created and modified with Match Profiler . The GENE table was extended and gained in importance, containing amongst others links to LocusLink, RefSeq and OMIM now . Further, (direct) links between factor and target gene on one hand and between gene and encoded factor on the other hand were introduced . The TRANSFAC public release is available at For yeast an additional release including the latest data was made available separately as TRANSFAC Saccharomyces Module (TSM) at For CYTOMER free download versions are available at http://www.biobase.de:8080/index.html.

J Struct Biol, 2002 Oct-Dec, 140(1-3), 17 - 30
Initiation of DNA replication in multicellular eukaryotes; Gerbi SA et al.; Three questions central to understanding the initiation of DNA replication in eukaryotes are: (1) Does DNA synthesis begin at a defined place? (2) What determines replication initiation sites? (3) What regulates an origin to fire only once per cell cycle? A key player in this is the origin recognition complex (ORC), required for assembly of the pre-replication complex (pre-RC), that is converted later to the initiation complex (IC) . In both yeast ARS1 and DNA puff II/9A of the metazoan fly Sciara, there is a defined start site of replication adjacent to an ORC-binding site . Although ORC has some inherent preference for certain DNA sequences, other factors may also modulate its binding to DNA . The preferred site where DNA synthesis starts at Sciara II/9A and the boundaries of the initiation zone change during development, when DNA puff amplification occurs . The position of the initiation zone may be influenced by the transcriptional machinery and/or chromatin structure . With regard to the third question, rereplication of the whole genome in yeast occurs when components of the pre-RC are stabilized by mutation . In contrast, a locus-specific amplification factor probably exists to account for site-specific DNA amplification in flies.

Transpl Infect Dis, 2002, 4 Suppl 3, 32 - 7
Diagnosis of fungal infection: new technologies for the mycology laboratory; Alexander BD; The dramatic increase in nosocomial invasive mycoses over the past two decades has led to increased interest in the area of antifungal development . Unfortunately, the infusion of new diagnostic technology into the clinical mycology laboratory has lagged behind . Although newer, automated, continuous-monitoring blood culture systems are as sensitive as the older, manual "gold standard" system, the recovery of fungi from blood, as well as other clinical specimens, remains an insensitive marker for invasive fungal infection . Antigen assays for the rapid diagnosis of invasive fungal infections are in development, and galactomannan and glucan are two such promising antigens . Glucan may be present in the blood of patients with infection secondary to a wide variety of fungal pathogens, including Candida, Aspergillus, Fusarium, Saccharomyces, Trichosporon and Acremonium species . Early data suggest galactomannan may be present in the blood in detectable levels very early in the course of invasive aspergillosis . The galactomannan assay currently undergoing evaluations may actually be positive prior to the clinical suspicion for infection and may be useful in monitoring therapeutic response as well; however, the etiology of false-positive results following cytotoxic chemotherapy still has to be elucidated . PCR assays are also being developed in the research laboratory, however, the PCR assays will require a significant amount of adaptation and validation before they are ready for clinical care . Well-planned studies to evaluate the performance characteristics as well as appropriate clinical and cost-effective application of these new tests are needed.

Curr Opin Cell Biol, 2002 Dec, 14(6), 756 - 62
Genome-wide histone modifications: gaining specificity by preventing promiscuity; van Leeuwen F et al.; More than 20 residues within the four core histone proteins of the nucleosome are potential sites of post-translational modifications, such as methylation, acetylation, ubiquitination and phosphorylation . It has been hypothesized that specific patterns of these modifications on the nucleosome facilitate recruitment of non-histone proteins to chromatin . When such modifications are restricted to particular regions of the genome, they seem to play an important role in creating specific chromatin domains . However, more recent results suggest that some histone modifications, particularly those that exist on a genome-wide scale, act to reduce nonspecific binding by chromatin proteins involved in silencing . This decrease of promiscuous binding ensures that the silent chromatin proteins are not titrated away from their normal locations on chromosomes . We suggest that preventing such promiscuous binding of chromatin proteins is an important part of generating specificity to create chromatin domains and overall chromosome organization.

Carbohydr Res, 2002 Oct 11, 337(19), 1757 - 62
Electrophoretically mediated microscale reaction of glycosidases: kinetic analysis of some glycosidases at the nanoliter scale; Kanie Y et al.; Capillary electrophoresis (CE) is one of the extremely important analytical techniques known for its high sensitivity and resolution . We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of some native glycosidases . Under optimized conditions, the enzymatic reactions of alpha-glucosidase, beta-galactosidase and beta-N-acetylglucosaminidase were carried out, and the Michaelis constants were obtained . The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme and substrate required for a reaction.

Biochem J, 2003 Jan 15, 369(Pt 2), 263 - 73
Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii; Schlupen C et al.; Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism . Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity . Determination of the enzyme's specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein . The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial . A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine . Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments . When 500 microM 99% {1-(13)C}threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity . More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase . Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux.

Curr Genet, 2002 Sep, 41(6), 401 - 6 Epub 2002 Aug 17.
Old yellow enzyme confers resistance of Hansenula polymorpha towards allyl alcohol; Komduur JA et al.; In the methylotrophic yeast, Hansenula polymorpha, peroxisomes are formed during growth on methanol as sole carbon and energy source and contain the key enzymes for its metabolism, one of the major enzymes being alcohol oxidase (AO) . Upon a shift of these cells to glucose-containing medium, peroxisomes become redundant for growth and are rapidly degraded via a highly selective process designated macropexophagy . H . polymorpha pdd mutants are disturbed in macropexophagy and hence retain high levels of peroxisomal AO activity upon induction of this process . To enable efficient isolation of PDD genes via functional complementation, we make use of the fact that AO can convert allyl alcohol into the highly toxic compound acrolein . When allyl alcohol is added to cells under conditions that induce macropexophagy, pdd mutants die, whereas complemented pdd mutants and wild-type cells survive . Besides isolating bona fide PDD genes, we occasionally obtained pdd transformants that retained high levels of AO activity although their allyl alcohol sensitive phenotype was suppressed . These invariably contained extra copies of a gene cluster encoding homologues of Saccharomyces carlsbergensis old yellow enzyme . Our data suggest that the proteins encoded by these genes detoxify acrolein by converting it into less harmful components.

J Org Chem, 2002 Sep 20, 67(19), 6816 - 9
Dynamic kinetic resolution of 2-oxocycloalkanecarbonitriles: chemoenzymatic syntheses of optically active yclic beta- and gamma-amino alcohols; Dehli JR et al.; A series of fungi and yeasts have been tested for the stereoselective bioreduction of 2-oxocycloalkanecarbonitriles, 1 . The yeast Saccharomyces montanus CBS 6772 yielded the corresponding cis-hydroxy nitriles, 2, in >90% ee and de and in high chemical yields . Through simple and efficient procedures, they were transformed into optically active 2-amino and 2-aminomethyl cycloalkanols.

J Virol, 2002 Oct, 76(19), 9806 - 18
Identification of a G(2) arrest domain in the E1 wedge E4 protein of human papillomavirus type 16; Davy CE et al.; Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma . Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle . The 16E1 wedge E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification . Using an inducible mammalian expression system, we have shown that 16E1 wedge E4 arrests HeLa cervical epithelial cells in G(2) . 16E1 wedge E4 also caused a G(2) arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm . However, whereas 16E1 wedge E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S . pombe cells that lack keratins, 16E1 wedge E4 had a punctate distribution . Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1 wedge E4 to be important for arrest . This region, which we have termed the "arrest domain," contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site . A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1 wedge E4-mediated G(2) arrest . Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint . G(2) arrest was also mediated by the E1 wedge E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions . The E1 wedge E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G(2) arrest . We conclude that, like other papillomavirus proteins, 16E1 wedge E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.

Am J Gastroenterol, 2002 Aug, 97(8), 2005 - 10
Inflammatory bowel disease in children 5 years of age and younger; Mamula P et al.; OBJECTIVES: Clinicians are becoming increasingly aware that inflammatory bowel disease (IBD) can affect all age groups, although it has not been well described in infants and young children . Our aim was to evaluate early onset IBD in patients 5 yr of age and younger . METHODS: Medical records of patients diagnosed with early onset IBD at The Children's Hospital of Philadelphia between 1977 and 2000 were reviewed . Patients were divided into three categories: those with Crohn's disease (CD), those with ulcerative colitis (UC), and those with indeterminant colitis (IC) . RESULTS: A total of 82 patients fulfilled the criteria . In 12 patients (15%), the IBD diagnosis was changed during the course of illness . At the end of the follow-up period, linear growth failure was present in 10 of 35 (29%) children with CD, one of 30 (3%) with UC, and three of 17 (18%) with IC . Failure to thrive was a frequent presenting symptom in children with CD (44%) and IC (39%), whereas in all four patients with UC and failure to thrive the diagnosis was subsequently changed to CD or IC . A high proportion of patients with CD had large bowel (89%), and perianal (34%) disease . None of the tested patients were positive for anti-Saccharomyces cerevisiae antibody (ASCA), and 10 tested positive for perinuclear antineutrophil cytoplasmic antibody (three of five patients with CD, five of seven with UC, and two of three with IC) . CONCLUSIONS: Failure to thrive, at the time of presentation, is indicative of a final diagnosis of CD or IC, not UC . Linear growth failure is a common finding in patients with early onset CD . A high proportion of patients with CD have failure to thrive, colonic, and perianal disease . The IBD serology panel is of limited clinical relevance in providing definitive diagnostic information in this pediatric population.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 509 - 16 Epub 2002 Jun 25.
A physiological and enzymatic study of Debaryomyces hansenii growth on xylose- and oxygen-limited chemostats; Nobre A et al.; The effect of changing growth rate and oxygen transfer rate (OTR) on Debaryomyces hansenii physiology was studied using xylose-limited and oxygen-limited chemostat cultures, respectively, and complemented with enzymatic assays . Under xylose-limited chemostat (oxygen-excess), neither ethanol nor xylitol was produced over the entire range of dilution rate ( D) . The maximal volumetric biomass productivity was 2.5 g x l(-1) x h(-1) at D =0.25 h(-1) and cell yield was constant at all values of D . The respiratory rates and xylose consumption rate increased linearly with growth rate but, above 0.17 h(-1), oxygen consumption rate had a steeper increase compared to carbon dioxide production rate . Enzymatic analysis of xylose metabolism suggests that internal fluxes are redirected as a function of growth rate . For values of D up to 0.17 h(-1), the xylose reductase (XR) titre is lower than the xylitol dehydrogenase (XDH) titre, whereas above 0.17 h(-1) XR activity is about twice that of XDH and the NADPH-producing enzymes sharply increase their titres indicating an internal metabolic flux shift to meet higher NADPH metabolic requirements . Moreover, the enzymes around the pyruvate node also exhibited different patterns if D was above or below 0.17 h(-1) . Under oxygen-limited chemostat (xylose-excess) the metabolism changed drastically and, due to oxidative phosphorylation limitation, cell yield decreased to 0.16 g g(-1) for an OTR of 1.4 mmol l(-1) h(-1) and xylitol became the major extracellular product along with minor amounts of glycerol . The enzymatic analysis revealed that isocitrate dehydrogenase is not regulated by oxygen, whereas XR, XDH and the NADPH-producing enzymes changed their levels according to oxygen availability.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1370 - 3
Casein digestion by Debaryomyces hansenii isolated from cheese; Kumura H et al.; To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein . Growth and casein degradation were recognized at the cheese-ripening temperature . Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on beta-casein . The cytosol contained more than 90% of the total proteolytic activity which was responsible for the degradation of both alpha(s)- and beta-casein . These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.

Poult Sci, 2002 Jul, 81(7), 966 - 75
Effects of feeding blends of grains naturally contaminated with Fusarium mycotoxins on production and metabolism in broilers; Swamy HV et al.; Three hundred sixty, 1-d-old male broiler chicks were fed diets containing grains naturally contaminated with Fusarium mycotoxins for 56 d . The four diets included control (0.14 mg/kg deoxynivalenol, 18 mg/ kg fusaric acid, < 0.1 mg/kg zearalenone), low level of contaminated grains (4.7 mg/kg deoxynivalenol, 20.6 mg/kg fusaric acid, 0.2 mg/kg zearalenone), and high level of contaminated grains without (8.2 mg/kg deoxynivalenol, 20.3 mg/kg fusaric acid, 0.56 mg/kg zearalenone) and with (9.7 mg/kg deoxynivalenol, 21.6 mg/kg fusaric acid, 0.8 mg/kg zearalenone) 0.2% esterified-glucomannan polymer derived from Saccharomyces cerevisiae1026 (E-GM) . Body weight gain and feed consumption responded in a significant quadratic fashion to the inclusion of contaminated grains during the finisher period . Efficiency of feed utilization, however, was not affected by diets . The feeding of contaminated grains in the finisher period also caused significant linear increases in blood erythrocyte count and serum uric acid concentration and a significant linear decline in the serum lipase activity . Dietary inclusion of contaminated grains resulted in a significant quadratic effect on serum albumin and y-glutamyltransferase activity . Blood hemoglobin and biliary IgA concentrations, however, responded in significant linear and quadratic fashions . Supplementation of E-GM counteracted most of the blood parameter alterations caused by the Fusarium mycotoxin-contaminated grains and reduced breast muscle redness . It was concluded that broiler chickens may be susceptible to Fusarium mycotoxicoses when naturally contaminated grains are fed containing a combination of mycotoxins.

Dev Cell, 2002 Jul, 3(1), 4 - 6
Top-SUMO wrestles centromeric cohesion; Pinsky BA et al.; Sister chromatid cohesion at the centromere is distinct from cohesion at the chromosome arms . In the June issue of Molecular Cell, Bachant et al . have shown that centromeric cohesion in budding yeast is specifically regulated by SUMO-1 modification of Topoisomerase II.

Biochem Biophys Res Commun, 2002 Jun 28, 294(5), 923 - 5
Neuritogenesis: polarization of constitutive exocytosis by effectors of Rho-family GTPases?
Teng FY, Tang BL.
The sprouting of neurites from a neuron represents a highly specialized form of cellular morphogenesis that must involve coordinated changes in two major cellular processes at two membrane locations: reorganization of the cytoskeleton and redirection of membrane traffic from the trans-Golgi network to the plasma membrane of the growth tip . How exactly are these two processes linked and how is spatial and temporal coordination achieved at the first instance of neurite sprouting? Recent advances may have already revealed some, if not most of the pieces in the puzzle . We discuss below, with some extrapolations, of what has recently come to light, and what more is needed to construct a coherent picture.

J Ind Microbiol Biotechnol, 2002 Feb, 28(2), 81 - 7
Purification and characterization of a Galactomyces reessii hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid; Dhar A et al.; Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid . Highest levels of hydratase activity were obtained during growth on isovaleric acid . The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200 . During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates . The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1) . The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification . The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1) . The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer . The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS . Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.

Lik Sprava, 2002, (2), 106 - 11
{Use of enterol in the treatment of diseases complicated by diarrhea syndrome}; Svintsitskii AS et al.; The article analyses data on clinical efficacy of the drug enterol in patients with chronic pancreatitis and chronic atropic gastritis . The use of enterol in a complex therapy has been shown to favour quick dispelling of the pain and dyspeptic syndromes, normalization of stools, elimination of intestinal dysbacteriosis or alleviation of its gravity.

Curr Opin Cell Biol, 2002 Jun, 14(3), 351 - 6
Mechanisms of chromosome-end protection; Cervantes RB et al.; Telomeres must protect chromosome ends from being recognized and processed as double-strand breaks . Identification of the factors involved in end protection, and the mechanisms by which they "cap" chromosome termini, is crucial in understanding how the cell distinguishes between a double-strand break and a normal telomere end . Recent work has characterized the similarities and potential differences between the pathways utilized by multiple organisms in maintaining telomere ends . One unifying concept that has clearly emerged is that chromosome-end protection is necessary in maintaining genetic stability and preventing oncogenesis.

Fungal Genet Biol, 2002 Jun, 36(1), 22 - 34
A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii; Montanini B et al.; An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii . The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1) . Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus(OUT)/C terminus(IN) orientation . As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K(m) for ammonium of 2 microM . The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T . borchii mycelia . Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia.

Int J Food Microbiol, 2002 Jun 5, 76(1-2), 117 - 26
Purification and characterisation of a glutaminase from Debaryomyces spp.; Dura MA et al.; A glutaminase was purified from the cell-free extract of Debaryomyces spp . CECT 11815 by protamine sulphate treatment and several chromatographic procedures including anion exchange chromatography and gel filtration . The purified enzyme consisted of two subunits, with molecular masses of 65 and 50 kDa, respectively . Activity was optimal at 40 degrees C and pH 8.5, and the Km value for L-glutamine was 4.5 mM . The glutaminase exhibited activity against L-gamma-Glu-methyl ester, L-gamma-Glu-hydrazide, and L-albiziin, while L-asparagine, CBZ-L-Gln, CBZ-L-Gln-Gly, glutathione, L-gamma-Glu-pNA and L-gamma-Glu-AMC were not hydrolysed . The enzyme was not affected by PMSF, DTT and EDTA . However, the enzyme was inhibited by sulfhydryl group reagents, DON, L-albizziin, L-asparagine and high concentrations of L-glutamine and ammonium, while L-aspartate did not affect the activity . Phosphate and acetate did not produce any significant effect on the glutaminase activity, but it was slightly stimulated by lactate and borate.

Appl Biochem Biotechnol, 2002 Apr, 101(1), 15 - 29
Effect of starting xylose concentration on the microaerobic metabolism of Debaryomyces hansenii: the use of carbon material balances; Converti A et al.; Xylitol production by Debaryomyces hansenii NRRL Y-7426 was performed on synthetic medium varying the initial xylose concentration between 50 and 300 g/L . The experimental results of these tests were used to investigate the effect of substrate level on xylose consumption by this yeast . Satisfactory values of product yield on substrate (0.74-0.83 g/g) as well as volumetric productivity (0.481-0.694 g/L x h) were obtained over a wide range of xylose levels (90-200 g/L), while a worsening of kinetic parameters took place at higher concentration, likely due to a substrate inhibition phenomenon . The metabolic behavior of D . hansenii was studied, under these conditions, through a carbon material balance to estimate the fractions of xylose consumed by the cell for different activities (xylitol production, biomass growth, and respiration) during the lag, exponential, and stationary phases.

Bioresour Technol, 2002 May, 82(3), 219 - 24
Influence of type and concentration of flavinogenic factors on production of riboflavin by Eremothecium ashbyii NRRL 1363; Kalingan AE et al.; A study was conducted to determine the effect of various low cost organic wastes as flavinogenic factors and the various concentrations at which they induced flavinogenecity resulting in higher yields of riboflavin . A high-yielding riboflavin strain; Eremothecium ashbyii NRRL 1363 was chosen to determine the flavinogenicity . Carbon source at 50 g l(-1) (dextrose equivalents) of molasses and nitrogen source at 50 g l(-1) (weight/volume) of peanut seed cake were found to be optimal levels to yield higher riboflavin . Among the organic wastes, (beef extract, hog casings, blood meal, fish meal) hog casings in association with fish meal supported the highest yield of riboflavin . Among the different recovery processes studied, a vacuum drying process was the most efficient allowing maximum yield, followed by drying at 90 degrees C and freeze-drying . It is apparent from this study that inexpensive or waste organic materials could induce E . ashbyii to synthesize and secrete riboflavin at higher levels in the medium and this could be purified using a vacuum drying process . This bioconversion process allows us to recycle the biomaterials and produce a value-added product of economic importance.

Comp Biochem Physiol B Biochem Mol Biol, 2002 Mar, 131(3), 465 - 74
Dolichol phosphate mannose synthase is differentially expressed in male and female worms of Schistosoma mansoni; Tempone AJ et al.; The cDNA encoding the Schistosoma mansoni dolichol phosphate mannose synthase was completely sequenced, displaying the highest homology with Cricetulus griseus and Saccharomyces pombe genes . The Schistosome enzyme had a K(m) of 0.127 microM, a value that is within the range of those reported for several other species . Thin-layer chromatography of the radiolabelled schistosome lipid intermediate showed it was identical to dolichol-phosphate (C80-C105) . Expression of dolichol phosphate mannose synthase of S . mansoni (SmDPMS) was analysed by Northern blot and quantified by semi-quantitative RT-PCR with cDNA from mature and immature male and female worms . Northern blot analysis revealed a single 1-kb band . Both approaches confirmed a higher level of expression in mature female worms, as compared to immature and male worms.

Oncogene, 2002 Feb 28, 21(10), 1611 - 5
Mutational analysis of the transcriptional activation domains of v-Myb; Wang DM et al.; A minimal transcription activation domain of the v-Myb oncoprotein was initially mapped to a central cluster of charged residues using GAL4-Myb fusion proteins . This region has been proposed to interact directly with the CBP co-activator in animal cells . Regions flanking this central domain of v-Myb are required for transcriptional activation by the native, unfused protein in both mammalian cells and in budding yeast . To identify the critical residues for transcriptional activation, we have now subjected the minimal activation domain and flanking regions including the heptad leucine repeat to random PCR-mediated mutagenesis . We found that the entire region examined can endure extensive substitutions without affecting transcriptional activation by v-Myb in budding yeast . The few mutations that did affect transcriptional activation altered acidic residues within the minimal activation domain or the heptad leucine repeat region, rather than leucine residues . Remarkably, there was a strong concordance between transcriptional activation in animal cells and in budding yeast, even though budding yeast have no known homologue of CBP or related co-activators . In contrast, there was not a strong correlation between transcriptional activation and oncogenic transformation.

Lett Appl Microbiol, 2002, 34(2), 95 - 9
(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin; Santos A et al.; AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin . METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action . Periodate oxidation and 1H-NMR was used to determine the receptor nature . Affinity chromatography on pustulan-Sepharose column was used to purify D . hansenii killer toxin, probably a 23-kDa protein . The killer toxin character was cureless . CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans . This is a low molecular weight protein, probably encoded by chromosomal genes . SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin . This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.

Antonie Van Leeuwenhoek, 2001 Dec, 80(3-4), 263 - 73
Molecular differentiation of sibling species in the Galactomyces geotrichum complex; Naumova ES et al.; PCR-analysis, multilocus enzyme electrophoresis and molecular karyotyping were used to characterize 52 strains belonging to the genus Galactomyces . The resultant data revealed that a PCR method employing the universal primer N21 and microsatellite primer (CAC)5 is appropriate for the distinction of four Ga . geotrichum sibling species, Ga . citri-aurantii and Ga . reessii . Better separation was achieved with the UP primer N21; each species displayed a specific pattern with very low intraspecific variation . We propose to use the primer N21 for the differentiation of the six taxa composing the genus Galactomyces . Multilocus enzyme electrophoresis revealed genetic homogeneity of each sibling species within the Ga . geotrichum complex . On the other hand, the four sibling species, having from 41 to 59% of nDNA homology and similar phenotypic characteristics, are clearly distinguished based on their electrophoretic profiles using two enzymes: mannose-6-phosphate isomerase (MPI) and phosphoglucomutase (PGM) . Despite the same number of chromosomal bands, different karyotype patterns were found in Ga . geotrichum sensu stricto and its two sibling species A and B . Within each sibling species, chromosome length polymorphism was observed, in particular for small bands, allowing discrimination to the strain level.

Mol Genet Genomics, 2002 Jan, 266(5), 787 - 95 Epub 2001 Oct 16.
The sterol C-14 reductase encoded by the Neurospora crassa erg-3 gene: essential charged and polar residues identified by site-specific mutagenesis; Prakash A et al.; Sterol C-14 reductase catalyses the reduction of the Delta(14,15) bond in intermediates in the sterol biosynthesis pathway using NADPH as a cofactor . We have undertaken a systematic site-directed mutational analysis of all the conserved charged and potentially proton-donating residues of the sterol C-14 reductase from Neurospora crassa . The effect of each mutation was determined using an in vivo assay based on the complementation of the corresponding N . crassa mutant ( erg-3) . The non-complementing mutations were also tested in the erg24 mutant of Saccharomyces cervisiae . The results are discussed with reference to the predicted topology of the enzyme and to its proposed catalytic mechanism, which involves addition of a proton from an appropriately positioned charged or polar residue to the substrate double bond, followed by addition of hydride ion from NADPH.

Mol Genet Genomics, 2001 Dec, 266(4), 664 - 71 Epub 2001 Oct 11.
Analysis of beta3-endonexin mutants for their ability to interact with cyclin A; Ohtoshi A et al.; We have recently identified beta(3)-endonexin as a molecule that interacts with cyclin A-associated kinase . In this study, beta(3)-endonexin mutants were constructed by PCR-based site-directed mutagenesis, and characterized . Beta(3)-endonexin has a cyclin binding motif, RxL, in its N-terminal region, and two SP sequences which resemble a known target site for cyclin-dependent kinases (Cdks) . The R5A/L7A mutant of beta(3)-endonexin, in which the RxL motif has been changed to AxA, is unable to bind to cyclin A, as revealed by two-hybrid experiments and in vitro pull-down assays . A GST-beta(3)-endonexin fusion, but not the corresponding R5A/L7A mutant, inhibits phosphorylation of Rb protein by cyclin A/Cdk2 in vitro . A cyclin A/Cdk2 kinase complex produced in, and purified from, insect cells phosphorylated GST-beta(3)-endonexin in vitro . The S33A or S46A mutant is partially phosphorylated by cyclin A/Cdk2, whereas no phosphorylation of the S33A/S46A double mutant is detectable . This demonstrates that these two serine residues, each of which is followed by a proline residue, are target sites for phosphorylation by cyclin A-associated kinase . The R5A/L7A mutant form of beta(3)-endonexin, which is defective for binding to cyclin A, is also not phosphorylated by cyclin A/Cdk2, confirming that the phosphorylation requires binding to cyclin A in the kinase complex . The neutralizing effect of beta(3)-endonexin on the toxicity associated with the expression of full-length human cyclin A in budding yeast is correlated with its ability to bind to cyclin A . Taken together, these data suggest that beta(3)-endonexin is phosphorylated by cyclinA/Cdk2 in vitro and that cyclin A-associated kinase activity is inhibited by the binding of beta(3)-endonexin to the kinase complex.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 2000 Apr, 20(4), 261 - 3
{Study on regulatory effect of composite taixian tablet on immune function of red blood cell in patients with oral lichen planus}; Wu Y et al.; OBJECTIVE: To explore the regulatory effect of Composite Taixian tablet (CTXT) on red blood cell (RBC) immune function in patients with oral lichen planus (OLP) for the sake of providing the basis of clinical medication . METHODS: Sixty patients with OLP were assigned randomly into three groups and were treated by CTXT, Tripterygium hypoglaucum tablet and polyactin-A tablet respectively . And the changes of RBC-C3b receptor and immune circulating rosette complex on the surface of erythrocytes (RBC-ICR) were measured by saccharomycetic assay . RESULTS: Effect of CTXT was superior to that of Tripterygium hypoglaucum and polyactin-A tablets . CONCLUSION: CTXT is a relatively effective remedy with less side effect, it is worthy to be studied further.

Dig Liver Dis, 2001 Nov, 33(8), 680 - 5
Intestinal permeability in Crohn's disease patients and their first degree relatives; Secondulfo M et al.; BACKGROUND: Family studies suggested that an altered intestinal permeability plays a role in the genesis of Crohn's disease . AIM: Aim of the present study was to investigate a possible genetic alteration of the mucosal barrier in Crohn's disease . SUBJECTS: 16 Crohn's disease patients and 26 of their cohabiting first degree relatives were studied . METHODS: To investigate intestinal permeability, Cellobiose/Mannitol test was administered to both groups . RESULTS: In the two groups, we found that the median intestinal permeability values were higher and statistically different from those obtained in 32 healthy control subjects as well as in five healthy control families . Six (37.5%) Crohn's disease patients and three (11.5%) of their first degree relatives showed increased individual intestinal permeability values . Intestinal permeability alteration in Crohn's disease patients was unrelated to sex, age, disease activity, localisation, duration, treatment schedule, as well as to serum anti-Saccharomyces cervisiae antibody positivity in a pilot study conducted in 7 Crohn's disease patients; anti-Saccharomyces cervisiae antibody values were negative in all 10 first degree relatives investigated . CONCLUSIONS: These findings demonstrate the increase in IP in 37% of the patients and in 11% of their relatives . More extensive investigation of the correlation between ASCA alterations and IP will be needed in both patients with Crohn's disease and their relatives.

Trends Biotechnol, 2001 Oct, 19(10 Suppl), S23 - 7
Exploring the protein interactome using comprehensive two-hybrid projects; Ito T et al.; Large-scale two-hybrid projects were used in an approach to examine protein-protein interactions . Despite the various limitations of this approach, these projects revealed a wealth of novel interactions, and the protein interactome may be much larger than expected.

Antonie Van Leeuwenhoek, 2001 Oct, 80(1), 85 - 92
Molecular characterisation of Hanseniaspora species; Esteve-Zarzoso B et al.; The sequences of the internal transcribed spacers (ITS regions) and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 27 strains representative of the six species belonging to the genus Hanseniaspora, were examined . From the analysis of the 5.8S rRNA gene and the ITS regions, the genus Hanseniaspora is monophyletic and can be divided into two subgroups . This subdivision was supported by electrophoretic chromosome patterns . Hanseniaspora guilliermondii, H . uvarum and H . valbyensis show 6-7 bands (8 to 9 chromosomes), while the second group comprises the species H . occidentalis, H . osmophila and H . vineae which have only 5 chromosomes.

Nucleic Acids Res, 2002 Jan 1, 30(1), 145 - 8
euGenes: a eukaryote genome information system; Gilbert DG; euGenes is a genome information system and database that provides a common summary of eukaryote genes and genomes, at Seven popular genomes are included: human, mouse, fruitfly, Caenorhabditis elegans worm, Saccharomyces yeast, Arabidopsis mustard weed and zebrafish, with more planned . This information, automatically extracted and updated from several source databases, offers features not readily available through other genome databases to bioscientists looking for gene relationships across organisms . The database describes 150 000 known, predicted and orphan genes, using consistent gene names along with their homologies and associations with a standard vocabulary of molecular functions, cell locations and biological processes . Usable whole-genome maps including features, chromosome locations and molecular data integration are available, as are options to retrieve sequences from these genomes . Search and retrieval methods for these data are easy to use and efficient, allowing one to ask combined questions of sequence features, protein functions and other gene attributes, and fetch results in reports, computable tabular outputs or bulk database forms . These summarized data are useful for integration in other projects, such as gene expression databases . euGenes provides an extensible, flexible genome information system for many organisms.

Microbiology, 2001 Dec, 147(Pt 12), 3377 - 86
Transcriptional regulation of 3,4-dihydroxy-2-butanone 4-phosphate synthase; Schlosser T et al.; The filamentous hemiascomycete Ashbya gossypii is a strong riboflavin overproducer . A striking but as yet uninvestigated phenomenon is the fact that the overproduction of this vitamin starts when growth rate declines, which means that most of the riboflavin is produced in the stationary phase, the so-called production phase . The specific activity of 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase, the first enzyme in the biosynthetic pathway for riboflavin, was determined during cultivation and an increase during the production phase was found . Furthermore, an increase of RIB3 mRNA, encoding DHBP synthase, was observed by competitive RT-PCR in the production phase . The mRNAs of two housekeeping genes, ACT1 (encoding actin) and TEF (encoding translation elongation factor-1 alpha), served as standards in the RT-PCR . Reporter studies with a RIB3 promoter-lacZ fusion showed an increase of beta-galactosidase specific activity in the production phase . This investigation verified that the increase of RIB3 mRNA in the production phase is caused by an induction of promoter activity . These data suggest that the time course of riboflavin overproduction of A . gossypii is correlated with a transcriptional regulation of the DHBP synthase.

J Mol Evol, 2002 Jan, 54(1), 42 - 53
Maximum likelihood methods reveal conservation of function among closely related kinesin families; Lawrence CJ et al.; We have reconstructed the evolution of the anciently derived kinesin superfamily using various alignment and tree-building methods . In addition to classifying previously described kinesins from protists, fungi, and animals, we analyzed a variety of kinesin sequences from the plant kingdom including 12 from Zea mays and 29 from Arabidopsis thaliana . Also included in our data set were four sequences from the anciently diverged amitochondriate protist Giardia lamblia . The overall topology of the best tree we found is more likely than previously reported topologies and allows us to make the following new observations: (1) kinesins involved in chromosome movement including MCAK, chromokinesin, and CENP-E may be descended from a single ancestor; (2) kinesins that form complex oligomers are limited to a monophyletic group of families; (3) kinesins that crosslink antiparallel microtubules at the spindle midzone including BIMC, MKLP, and CENP-E are closely related; (4) Drosophila NOD and human KID group with other characterized chromokinesins; and (5) Saccharomyces SMY1 groups with kinesin-I sequences, forming a family of kinesins capable of class V myosin interactions . In addition, we found that one monophyletic clade composed exclusively of sequences with a C-terminal motor domain contains all known minus end-directed kinesins.

Curr Gastroenterol Rep, 2001 Dec, 3(6), 491 - 9
Diagnostic methodologies: serology, endoscopy, and radiology; Dassopoulos T; The two major inflammatory bowel diseases, Crohn's disease (CD) and ulcerative colitis (UC), represent clinicopathologic entities that traditionally have been diagnosed on the basis of a combination of clinical, radiologic, endoscopic, and histologic features . Serum perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA) have recently been added to our diagnostic armamentarium . Several studies have demonstrated that UC-associated pANCAs recognize nuclear antigens . Additional studies have demonstrated that the pANCA human monoclonal antibody (mAb) Fab 5-3 reacts with histone H1 and with bacterial and mycobacterial antigens . Several reports have suggested that, in CD, pANCA and ASCA are correlated with colonic and small bowel disease respectively . One study found that higher ASCA levels were correlated with more aggressive CD . Serology may prove to be useful in predicting the evolution of indeterminate colitis . Magnetic resonance imaging (MRI) and leukocyte scintigraphy hold promise in identifying inflammatory CD . MRI enteroclysis is useful in identifying both luminal small bowel disease and extraluminal complications . A recent study of surveillance colonoscopy in extensive Crohn's colitis showed a high risk of dysplasia and cancer.

Appl Biochem Biotechnol, 2001 Aug, 95(2), 83 - 92
Simultaneous purification and reversible immobilization of D-amino acid oxidase from Trigonopsis variabilis on a hydrophobic support; Dsouza SF et al.; Purification and reversible immobilization of D-amino acid oxidase from Trigonopsis variabilis could be simultaneously accomplished by hydrophobic interaction on Phenyl Sepharose CL-4B in the presence of 50 mM pyrophosphate buffer (pH 8.5) . The presence of a high salt concentration of 2 M, which is generally required for the hydrophobic interactions, was not essential for the hydrophobic immobilization . The enzyme in free as well as immobilized form was optimally active between pH 7.0 and 9.0 . The immobilized preparation could be reused in a batch process for the conversion of D-amino acids to alpha-keto acids . When the activity of the preparation dropped below practical limits, the gel could be regenerated by water wash and recharged with fresh crude extract from yeast.

Mol Cell Biol, 2001 Dec, 21(23), 7995 - 8006
Highly frequent frameshift DNA synthesis by human DNA polymerase mu; Zhang Y et al.; DNA polymerase mu (Polmu) is a newly identified member of the polymerase X family . The biological function of Polmu is not known, although it has been speculated that human Polmu may be a somatic hypermutation polymerase . To help understand the in vivo function of human Polmu, we have performed in vitro biochemical analyses of the purified polymerase . Unlike any other DNA polymerases studied thus far, human Polmu catalyzed frameshift DNA synthesis with an unprecedentedly high frequency . In the sequence contexts examined, -1 deletion occurred as the predominant DNA synthesis mechanism opposite the single-nucleotide repeat sequences AA, GG, TT, and CC in the template . Thus, the fidelity of DNA synthesis by human Polmu was largely dictated by the sequence context . Human Polmu was able to efficiently extend mismatched bases mainly by a frameshift synthesis mechanism . With the primer ends, containing up to four mismatches, examined, human Polmu effectively realigned the primer to achieve annealing with a microhomology region in the template several nucleotides downstream . As a result, human Polmu promoted microhomology search and microhomology pairing between the primer and the template strands of DNA . These results show that human Polmu is much more prone to cause frameshift mutations than base substitutions . The biochemical properties of human Polmu suggest a function in nonhomologous end joining and V(D)J recombination through its microhomology searching and pairing activities but do not support a function in somatic hypermutation.

Annu Rev Cell Dev Biol, 2001, 17, 351 - 86
Animal cell cytokinesis; Glotzer M; Cytokinesis creates two daughter cells endowed with a complete set of chromosomes and cytoplasmic organelles . This conceptually simple event is mediated by a complex and dynamic interplay between the microtubules of the mitotic spindle, the actomyosin cytoskeleton, and membrane fusion events . For many decades the study of cytokinesis was driven by morphological studies on specimens amenable to physical manipulation . The studies led to great insights into the cellular structures that orchestrate cell division, but the underlying molecular machinery was largely unknown . Molecular and genetic approaches have now allowed the initial steps in the development of a molecular understanding of this fundamental event in the life of a cell . This review provides an overview of the literature on cytokinesis with a particular emphasis on the molecular pathways involved in the division of animal cells.

Curr Biol, 2001 Oct 16, 11(20), R824 - 7
Retinoblastoma protein: combating algal bloom; Cross FR et al.; The discovery of a homolog of the retinoblastoma protein (Rb) in a single-celled eukaryote--the alga Chlamydomonas--promises new and surprising insights into Rb's function in cell-cycle regulation.

Aquat Toxicol, 2001 Nov 12, 55(3-4), 149 - 56
Toxicity of domoic acid in the marine mussel Mytilus edulis; Dizer H et al.; The neurotoxic, immunotoxic and genotoxic effects of domoic acid (DA) on the blue mussel Mytilus edulis were investigated by biomarkers, acethylcholinesterase (ChE) activity in gills, DNA fragmentation in digestive glands, vitality and phagocytosis activity of haemocytes in haemolymph of mussels . After intra muscular injection of DA at the concentrations ranging from 1-500 ng/g body weight (bw), no neurotoxic effect was detected within incubation times of 48 h and 7 d . The vitality of haemocytes remained in all mussels at the level of control samples within 48 h, and increased significantly after 7 d (P<0.05) . At DA concentrations ranging from 1 to 100 ng/g bw haemocytes suggested a great phagocytosis activity, but no alteration in their number by both incubation times . By increasing DA concentration of 500 ng/g bw, the number of haemocytes doubled in 48 h without any change in phagocytosis activity . Primary DNA lesions in digestive glands of all injected mussels were determined in acute phase of poisoning within 48 h, and rapidly repaired after 7 d of incubation.

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 79 - 89
Variability of the lipolytic activity in Yarrowia lipolytica and its dependence on environmental conditions; Guerzoni ME et al.; This work was aimed to the evaluation of the variability of lipolytic activity in Yarrowia lipolytica strains, as well as to asses for a selected strain, the response to the changes of physico-chemical variables (such as pH, NaCl and lipid content), in order to obtain predictive models describing their effects on the lipolysis pattern . The strains tested, having different environmental origin, showed different patterns of the free fatty acids (FFA) released . The clustering of the free fatty acids profiles evidenced that the unweighted average distance within the strains of the same species did not exceeded 30% . However, the lipolytic activity of some strains generated FFA profiles that differentiated from the majority of the strains considered . Also, when a single strain was inoculated in model systems in which pH, NaCl and milk fat were modulated according to a Central Composite Design (CCD), chemico-physical characteristics of the system led to marked variations in the lipolytic activity with consequent changes in individual fatty acids released . In most cases, when the same Y . lipolytica strain was used, under the experimental conditions adopted, the modulation of the lactic acid, NaCl and lipid content did not generate differences in the fatty acid release exceeding 20-21% . However, some combinations of factors remarkably affected lipase expression or activity, and generated differences in the fatty acid released higher than those observed among different strains of the same species.

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 69 - 77
Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese; Suzzi G et al.; This work studied the qualitative and quantitative proteolytic and lipolytic activities of Yarrowia lipolytica strains isolated from two cheese types . Randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to compare the cheese strains of Y . lipolytica with strains isolated from other food products and with the type strain of the species in order to investigate the genetic diversity and occurrence of specific environmental groups . Diversity of proteolytic and especially lipolytic activity within Y . lipolytica strains isolated from dairy products was observed . In particular, the degree of specificity for saturated or unsaturated fatty acids as well as for even- or odd-numbered carbon free fatty acids (FFAs) varied among the strains . The RAPD-PCR profiles showed low genetic relatedness between many of the food isolates and the type strain of the species . Such genetic variability needs to be further evaluated . Most of the Y . lipolytica strains appeared to be specific to the particular environment from which they were isolated . However, phenotypic characteristics having technological importance in dairy products and, particularly, lipolytic activities did not correspond to the genetic differences observed by RAPD-PCR analysis.

Yeast, 2001 Sep 30, 18(13), 1227 - 38
Cloning and sequencing the genomic encoding region of copper-zinc superoxide dismutase enzyme from several marine strains of the genus Debaryomyces (Lodder & Kreger-van Rij); Hernandez-Saavedra NY et al.; Copper-zinc superoxide dismutase (SODC) is a cytosolic enzyme which catalyses the dismutation of the superoxide radical . Due to its physiological importance, the encoding genes have been cloned from several species of higher eukaryotes . However, genes from moulds and yeast have not been studied extensively . In this paper, the encoding region of this gene (sod1) has been cloned from several strains of marine yeast belonging to the genus Debaryomyces (dvv sod1, dvy sod1 and dh sod1-61) through genomic DNA-PCR amplification . Fragments of 480-486 nucleotides were obtained, which contain information for products of 153-156 amino acids with calculated molecular masses of 15.8-16.6 kDa . The deduced amino acid sequence shows that D . vanrijiae enzymes present three additional amino acids not closely related to the active site conformation . In addition, in D . vanrijiae var . vanrijiae (strain 020), one histidine residue is apparently replaced by a proline; the incidence and function of other aromatic or heterocyclic amino acids is discussed . Homology and phylogenetic trees were constructed from amino-acid sequence multi-alignment analyses; the interrelationships among fungi are discussed . The sod-1 sequences reported in this paper were deposited in the public data library of the NCBI under Accession Nos AF301019, AF327449 and AF327448 .

Mol Reprod Dev, 2001 Sep, 60(1), 128 - 36
Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin; Lessman CA et al.; Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominantly in megadalton (MDa) fractions and that fails to readily assemble in vitro . In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used . The results revealed the presence of alpha-, beta-, and gamma-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes . These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as gamma-tubulin ring complexes . This seems true since both size (estimates, i.e . approximately 2MDa) and protein components are similar . Furthermore, both alpha- and gamma-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction . These putative Rana gamma-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species . Rana appears to belong to a select group in which gamma-tubulin complexes contain significant alpha- and beta-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components . The heterogeneity in both size and protein components of Rana oocyte gamma-tubulin ring complexes may reflect different states of tubulin complex assembly . The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of gamma-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo . While the gamma-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble gamma-tubulin complexes in vertebrate ovarian oocytes .

Enzyme Microb Technol, 1985 Jul, 7, 339 - 45
Determining a carbohydrate profile for Hansenula polymorpha; Petersen GR; The determination of the levels of carbohydrates in the yeast Hansenula polymorpha required the development of new analytical procedures . Existing fractionation and analytical methods were adapted to deal with the problems involved with the lysis of whole cells . Using these new procedures, the complete carbohydrates profiles of H . polymorpha and selected mutant strains were determined and shown to correlate favourably with previously published results.

Int J Food Microbiol, 2001 Sep 1, 68(3), 199 - 206
Hydrolysis of pork muscle sarcoplasmic proteins by Debaryomyces hansenii; Santos NN et al.; Strains of Debaryomyces hansenii originally isolated from sausages were screened for proteinase and aminopeptidase activity towards synthetic substrates . On the basis of these results, D . hansenii CT12487 was selected for further assays . The activities of the whole cells (WC), cell-free extracts (CFE) and a combination of both from the selected strain on pork muscle sarcoplasmic protein extracts were determined by protein, peptide and free amino acid analyses . There was a pronounced hydrolysis of protein bands of 110 kDa and 27-64 kDa regardless the incorporation of WC, CFE or a combination of both . The proteolytic activity also resulted in the generation of polar and non-polar peptides showing noticeable differences depending on the addition of WC or CFE . Whole cells generated greater amounts of free amino acids than the cell-free extracts.

Invest Ophthalmol Vis Sci, 2001 Sep, 42(10), 2324 - 31
Characterization of myocilin-myocilin interactions; Fautsch MP et al.; PURPOSE: To determine whether myocilin (MYOC; also referred to as TIGR) is present as a complex in human aqueous humor, whether part of the complex formation may be due to MYOC-MYOC interactions and to characterize the sites of interaction . METHODS: Human aqueous humor was analyzed by using a gel filtration column for the identification of MYOC complexes . MYOC-MYOC interactions were studied with a yeast two-hybrid system . Expression of full-length and truncated MYOC proteins in AH109 yeast was analyzed for growth and color on minimal medium . Site-directed mutagenesis was used to selectively mutate eight leucine residues within the leucine zipper motif . In vitro transcription and translation was used to verify yeast two-hybrid analysis . RESULTS: MYOC was found to be present in human aqueous humor as a complex ranging from 120 to 180 kDa . Expression of full-length MYOC in yeast as well as in vitro binding studies revealed that MYOC can interact with itself . MYOC-MYOC interactions occurred mainly within amino acids 117-166, a region containing a leucine zipper domain . Glycine substitution for selective leucine residues confirmed that MYOC-MYOC interactions occurred mainly within the leucine zipper domain . CONCLUSIONS: MYOC is present in human aqueous humor, not as a monomer but as a complex . Part of this complex may form due to MYOC-MYOC interactions that take place mainly within the leucine zipper domain.

Genome Res, 2001 Aug, 11(8), 1425 - 33
Creating the gene ontology resource: design and implementation; Gene Ontology Consortium; The exponential growth in the volume of accessible biological information has generated a confusion of voices surrounding the annotation of molecular information about genes and their products . The Gene Ontology (GO) project seeks to provide a set of structured vocabularies for specific biological domains that can be used to describe gene products in any organism . This work includes building three extensive ontologies to describe molecular function, biological process, and cellular component, and providing a community database resource that supports the use of these ontologies . The GO Consortium was initiated by scientists associated with three model organism databases: SGD, the Saccharomyces Genome database; FlyBase, the Drosophila genome database; and MGD/GXD, the Mouse Genome Informatics databases . Additional model organism database groups are joining the project . Each of these model organism information systems is annotating genes and gene products using GO vocabulary terms and incorporating these annotations into their respective model organism databases . Each database contributes its annotation files to a shared GO data resource accessible to the public at The GO site can be used by the community both to recover the GO vocabularies and to access the annotated gene product data sets from the model organism databases . The GO Consortium supports the development of the GO database resource and provides tools enabling curators and researchers to query and manipulate the vocabularies . We believe that the shared development of this molecular annotation resource will contribute to the unification of biological information.

Appl Environ Microbiol, 2001 Aug, 67(8), 3463 - 8
Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid; Carreira A et al.; Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism . During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium . Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased . With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1) . p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde . The catabolic pathway of tyrosine in Y . lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid . Brown pigments are produced when homogentisic acid accumulates in the medium . This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins . Mn(2+) accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn(2+) . Alkaline conditions also accelerated pigment formation . The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.

In Silico Biol, 1999, 1(3), 163 - 73
Threading analysis of prospero-type homeodomains; Banerjee-Basu S et al.; The homeodomain is a common structural motif found in many transcription factors involved in cell fate determination during development . We have used threading analysis techniques to predict whether the atypical homeodomain of prospero (pros) family members could form the three-helical homeodomain structural motif, even though these proteins are not statistically similar to canonical homeodomains as assessed by BLAST searches . Amino acid sequences of these divergent homeodomain proteins were threaded through the X-ray coordinates of the Drosophila engrailed homeodomain protein {23} . The analysis confirms that the prospero class of homeodomain proteins is indeed capable of forming the homeodomain structure despite its low degree of sequence identity to the canonical homeodomain . Energy calculations indicate that the homeodomain structure is stabilized primarily by hydrophobic interactions between residues at the helical interfaces . Although the atypical prospero-type homeodomain shows very little sequence similarity when compared to other homeodomain proteins, the critical amino acids responsible for maintaining the three-dimensional structure are highly conserved . A number of other homeodomain proteins, such as PHO2p from Saccharomyces and Pax6 from human, were also included in the threading analysis and were shown to be able to form the engrailed structure, indicating that there are no rigid overall sequence requirements for the formation of the homeodomain structural motif . Based on the threading experiments and the subsequent structural alignment, a new amino acid signature that unambiguously identifies the prospero-type proteins was deduced.

Mikrobiologiia, 2001 May-Jun, 70(3), 365 - 9
{Williopsis saturnus and Williopsis beijerinckii-different taxons from polymerase chain reaction data with nonspecific primers}; Naumova ES et al.; Fifteen strains of the yeast Williopsis sensu stricto were analyzed by means of UP-PCR . With the N21 universal primer, this approach showed that the strains could be clearly divided into two groups corresponding to the species W . saturnus (Klocker) Zender and W . beijerinckii (van der Walt) Naumov et Vustin . The results obtained are in good agreement with data of genetic and isoenzyme analyses and provide no support for the conspecificity of W . saturnus and W . beijerinckii commonly accepted in modern determination manuals.

J Cell Biol, 2001 Jun 25, 153(7), 1391 - 402
Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export; Lindsay ME et al.; Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP . This complex translocates through the nuclear pores and dissociates in the cytosol . The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action has not been established . We show that the human orthologue of Yrb2p, Ran-binding protein 3 (RanBP3), acts as a cofactor for Crm1-mediated export in a permeabilized cell assay . RanBP3 binds directly to Crm1, and the complex possesses an enhanced affinity for both Ran:GTP and cargo . RanBP3 shuttles between the nucleus and the cytoplasm by a Crm1-dependent mechanism, and the Crm1--RanBP3-NES-Ran:GTP quarternary complex can associate with nucleoporins . We infer that this complex translocates through the nuclear pore to the cytoplasm where it is disassembled by RanBP1 and Ran GTPase--activating protein.

J Biol Chem, 2001 Aug 24, 276(34), 31515 - 20 Epub 2001 Jun 15.
Water and ion permeation of aquaporin-1 in planar lipid bilayers . Major differences in structural determinants and stoichiometry; Saparov SM et al.; The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled . Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers . Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane . Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels . Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect . The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance . Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags . Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)) . Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions . Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers . The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.

J Neurosci, 2001 Jun 1, 21(11), 3764 - 70
Bipartite interaction between neurofibromatosis type I protein (neurofibromin) and syndecan transmembrane heparan sulfate proteoglycans; Hsueh YP et al.; The neurofibromatosis type 1 (NF1) gene encodes a large tumor suppressor protein (neurofibromin) . Although it is known to possess Ras GTPase-activating protein (GAP) activity, the cellular role of neurofibromin remains unclear . Here we used yeast two-hybrid screening to identify neurofibromin-interacting proteins . Syndecan-2, a transmembrane heparan sulfate proteoglycan (HSPG), was isolated as a binding partner for two distinct regions of the neurofibromin protein . We subsequently found that neurofibromin can bind all four mammalian syndecans . NF1 interaction requires the transmembrane domain and a membrane-proximal region of the cytoplasmic tail of syndecan, but not the C terminus of syndecan known to bind to CASK, a membrane-associated guanylate kinase (MAGUK) . Neurofibromin, syndecans, and CASK have overlapping subcellular distributions in axons and synapses of neurons, as shown by biochemical fractionation and immunostaining . Moreover, neurofibromin exists in a complex with syndecan and CASK in vivo, as evidenced by their coimmunoprecipitation from rat brain . Our findings suggest that interaction with different members of the syndecan family may be a mechanism for localizing neurofibromin to specialized domains of the plasma membrane.

Mol Biochem Parasitol, 2001 Apr 25, 114(1), 41 - 52
Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors; Lima AP et al.; Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases . In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51 . Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation . We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms . We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T . cruzi . Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases . We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T . cruzi adaptation to vertebrate hosts.

J Biol Chem, 2001 Jun 29, 276(26), 24082 - 7 Epub 2001 May 07.
A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I; Kashani-Poor N et al.; We have analyzed a series of eleven mutations in the 49-kDa protein of mitochondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica to identify functionally important domains in this central subunit . The mutations were selected based on sequence homology with the large subunit of {NiFe} hydrogenases . None of the mutations affected assembly of complex I, all decreased or abolished ubiquinone reductase activity . Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors . Unexpectedly, seven mutations affected the properties of iron-sulfur cluster N2, a prosthetic group not located in the 49-kDa subunit . In three of these mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy . The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a straightforward explanation for the observed, unforeseen effects on this iron-sulfur cluster . We propose that the fold around the hydrogen reactive site of {NiFe} hydrogenase is conserved in the 49-kDa subunit of complex I and has become part of the inhibitor and ubiquinone binding region . We discuss that the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.

Nat Rev Genet, 2001 May, 2(5), 360 - 9
Meiotic recombination hot spots and cold spots; Petes TD; Meiotic recombination events are distributed unevenly throughout eukaryotic genomes . This inhomogeneity leads to distortions of genetic maps that can hinder the ability of geneticists to identify genes by map-based techniques . Various lines of evidence, particularly from studies of yeast, indicate that the distribution of recombination events might reflect, at least in part, global features of chromosome structure, such as the distribution of modified nucleosomes.

Life Sci, 2001 Mar 23, 68(18), 2131 - 9
cDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6; Iwano H et al.; A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5'-RACE from a cDNA library prepared from the bovine liver . The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus . The encoded protein has a potential asparagine-linked glycosylation site (Asn291) . The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity . BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme . The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, confirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase . Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis . These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3814 - 9 Epub 2001 Mar 20.
The human brm protein is cleaved during apoptosis: the role of cathepsin G; Biggs JR et al.; The human brm (hbrm) protein (homologue of the Drosophila melanogaster brahma and Saccharomyces cervisiae SNF-2 proteins) is part of a polypeptide complex believed to regulate chromatin conformation . We have shown that the hbrm protein is cleaved in NB4 leukemic cells after induction of apoptosis by UV-irradiation, DNA damaging agents, or staurosporine . Because hbrm is found only in the nucleus, we have investigated the nature of the proteases that may regulate the degradation of this protein during apoptosis . In an in vitro assay, the hbrm protein could not be cleaved by caspase-3, -7, or -6, the "effector" caspases generally believed to carry out the cleavage of nuclear protein substrates . In contrast, we find that cathepsin G, a granule enzyme found in NB4 cells, cleaves hbrm in a pattern similar to that observed in vivo during apoptosis . In addition, a peptide inhibitor of cathepsin G blocks hbrm cleavage during apoptosis but does not block activation of caspases or cleavage of the nuclear protein polyADP ribose polymerase (PARP) . Although localized in granules and in the Golgi complex in untreated cells, cathepsin G becomes diffusely distributed during apoptosis . Cleavage by cathepsin G removes a 20-kDa fragment containing a bromodomain from the carboxyl terminus of hbrm . This cleavage disrupts the association between hbrm and the nuclear matrix; the 160-kDa hbrm cleavage fragment is less tightly associated with the nuclear matrix than full-length hbrm.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 237 - 47
Phylogenetic structure of the Sporopachydermia cereana species complex; Lachance MA et al.; A large number of isolates previously referred to as members of the 'Sporopachydermia cereana species complex' were examined by various DNA characterization methods, leading to the conclusion that the complex is in fact made up of 10 species, one of which contains three varieties . The sequences of the internal transcribed spacer (ITS) region and the D1/D2 divergent domains of the large subunit rDNA were determined for representatives of each taxon and specific primers based on differences in the ITS were designed for rapid identification of five of the taxa . Whereas the data provide additional elements for the calibration of the ITS as a criterion for species delineation, the emerging pattern is that the ITS region does not function as well as the D1/D2 domains as an evolutionary clock . Some taxa appear to be specific for the geographical regions where they were isolated, and the distribution of many taxa is mutually exclusive.

Photochem Photobiol, 2001 Jan, 73(1), 1 - 5
Bound transcription factor suppresses photoproduct formation in the NF-kappa B promoter; Ghosh R et al.; The relationship between purified transcription factor p50 binding and ultraviolet light-induced DNA damage formation in the NF-kappa B promoter element was investigated . The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam-Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V . Two methods were employed for cleaving (6-4) photoproducts . Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers . In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites . Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction . These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters.

Br Poult Sci, 2000 Dec, 41(5), 640 - 50
Influence of esterified-glucomannan on performance and organ morphology, serum biochemistry and haematology in broilers exposed to individual and combined mycotoxicosis (aflatoxin, ochratoxin and T-2 toxin); Raju MV et al.; 1 . A study was conducted to evaluate the individual and combined effects of aflatoxin B1 (AF), ochratoxin A (OA) and T-2 toxin (T-2) on performance, organ morphology serum biochemistry and haematology of broiler chickens and the efficacy of esterified-glucomannan (E-GM), a cell wall derivative of Saccharomyces cerevisiae1026 in their counteraction . 2 . Two dietary inclusion rates of AF (0 and 0.3 mg/kg), OA (0 and 2 mg/kg), T-2 (0 and 3 mg/kg) and E-GM (0 and 1 g/kg) were tested in a 2 x 2 x 2 x 2 factorial manner on a total of 960 broiler chickens from 1 to 35 d of age in an open sided deep litter pen house . 3 . Body weight and food intake were depressed by all the mycotoxins, OA being the most toxic during early life . 4 . Weights of kidney and adrenals were increased by AF and OA . Liver weight was increased by AF (17.8%), while OA increased gizzard weight (14.6%) and reduced bone ash content (8.1%) . T-2 toxin showed no effect on these variables . 5 . Serum cholesterol content was decreased and activity of serum gamma glutamyl transferase (GGT) was increased by AF and OA while serum protein content was decreased by AF . These effects were more pronounced at 21 d than at 35 d of age . Inconsistent responses were seen in the other variables: blood urea nitrogen (BUN) content, activities of serum alanine amino transferase and aspertate amino transferase . Blood haemoglobin content was depressed by AF and T-2, whereas blood coagulation time was prolonged by OA . 6 . Significant interactions were observed between any 2 toxins for their additive effects on body weight, food intake, bone ash content and serum GGT activity at 21 d . Conversely, antagonistic interactions were observed among any 2 of the toxins for their effects on variables such as serum protein and serum cholesterol content . Simultaneous feeding of all 3 mycotoxins did not show increased toxicity above that seen with any 2 . 7 . Esterified-glucomannan increased body weight (2.26%) and food intake (1.6%), decreased weights of liver (32.5%) and adrenals (18.9%) and activity of serum GGT (8.7%), and increased serum protein (14.7%), cholesterol (21.9%), BUN (20.8%) and blood haemoglobin (3.1%) content, indicating its possible beneficial effect on mycotoxicosis in broiler chickens.

IUBMB Life, 2000 Aug, 50(2), 151 - 5
Phosphate-uptake systems in Yarrowia lipolytica cells grown under alkaline conditions; Zvyagilskaya R et al.; In this study we used a newly isolated Yarrowia lipolytica strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying phosphate transport systems in cells grown under alkaline conditions . Phosphate uptake by Y . lipolytica yeast cells grown at pH 9.5-10 was shown to be mediated by several kinetically discrete Na+-dependent systems . One of these, a low-affinity transporter, operates at high Pi concentrations and is, to our knowledge, here kinetically characterized for the first time . The other two high-affinity systems are derepressible, come into play under conditions of Pi-starvation, and appear to be controlled by the availability of extracellular Pi . They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor the bacterial kingdoms.

Plant Cell, 2001 Jan, 13(1), 139 - 51
TRH1 encodes a potassium transporter required for tip growth in Arabidopsis root hairs; Rigas S et al.; Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth . Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization . Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA-tagging method . Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth . A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family . The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport . In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake . Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations . These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation.

Trends Cell Biol, 2001 Jan, 11(1), 22 - 29
Dynamics of peroxisome assembly and function; Titorenko VI et al.; Recent studies in human cells and in the yeast Yarrowia lipolytica have shown that peroxisomes consist of numerous structurally distinct subcompartments that differ in their import competency for various proteins and are related through a time-ordered conversion of one subcompartment to another . Our studies have implicated the fusion of small peroxisomal precursors as an early event in the multistep assembly of peroxisomes operating in Y . lipolytica . Newly discovered unexpected roles for peroxisomes in specific developmental programs have expanded the remarkable plasticity of peroxisomal functions . Here, we highlight recent discoveries on the highly dynamic nature of peroxisome assembly and function and suggest questions for future research in these areas.

Biochimie, 2000 Dec, 82(12), 1123 - 7
Human homeodomain-interacting protein kinase-2 (HIPK2) is a member of the DYRK family of protein kinases and maps to chromosome 7q32-q34; Hofmann TG et al.; Here we identified the human serine/threonine kinase HIPK2 as a novel member of the DYRK kinase subfamily . Alignment of several DYRK family proteins including the kinases minibrain, MJAK, PKY, the Dictyostelium kinase YakA and Saccharomyces YAK1 allowed the identification of several evolutionary conserved DYRK consensus motifs within the kinase domain . A lysine residue conserved between all DYRK kinase family members was found to be essential for the kinase function of HIPK2 . Human HIPK2 was mapped to chromosome 7q32-q34 and murine HIPK2 to chromosome 6B, the homologue to human chromosome 7.

Can J Microbiol, 2000 Nov, 46(11), 1046 - 50
Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch and chemostat cultures; Aguilar Uscanga MG et al.; The nutritional requirements of Brettanomyces bruxellensis have been investigated . Batch culture and chemostat pulse techniques were used to identify growth-limiting nutrients . The study included determination of the essential components of the culture medium and quantification of the effects of the components . Among the components tested, ammonium sulfate and yeast extract had a significant effect on glucose consumption, growth, and ethanol production . However, if the ammonium sulfate concentration is above 2 g/L, an inhibitory effect on B . bruxellensis growth is observed . The yeast extract appears to be the most important and significant component for growth . The maximum amount of synthesized biomass is proportional to the concentration of yeast extract added to the culture broth (in the tested range) . Magnesium and phosphate ions are probably not essential for B . bruxellensis . These ions appear to be supplied in sufficient amounts by the yeast extract in the culture medium . Brettanomyces bruxellensis appears to have very low nutritional requirements for growth.

Mycoses, 2000 Sep, 43(7-8), 269 - 72
The incidence of opportunistic fungi in patients suspected of tuberculosis; Chadeganipour M et al.; The incidence of opportunistic fungi in bronchoalveolar lavage specimens from patients suspected of tuberculosis in Isfahan, Iran, was determined . From 200 patients 36 yeasts (18%) and seven filamentous fungi (3.5%) were isolated . Out of 44 patients who had fungal infections, 12 cases were affected with definite tuberculosis.

J Neurochem, 2000 Nov, 75(5), 2221 - 4
Rat alpha-synuclein interacts with Tat binding protein 1, a component of the 26S proteasomal complex; Ghee M et al.; The alpha-synuclein gene, which encodes a brain presynaptic nerve terminal protein of unknown function, is linked to familial early-onset Parkinson's disease (PD) . The finding that alpha-synuclein forms the major fibrillary component of Lewy bodies in brains of PD patients suggests that the two point mutations in alpha-synuclein (Ala(53)Thr, Ala(30)Pro) may promote the aggregation of alpha-synuclein into filaments . To address the role of alpha-synuclein in neurodegenerative diseases, we performed a yeast two-hybrid screen of a rat adult brain cDNA library using rat alpha-synuclein 2 (alphaSYN2) . Here we report that alphaSYN2 interacts specifically with Tat binding protein 1, a subunit of the 700-kDa proteasome activator (PA700), the regulatory complex of the 26S proteasome and of the modulator complex, which enhances PA700 activation of the proteasome.

Microbiol Immunol, 2000, 44(8), 711 - 3
First isolation of Stephanoascus ciferrii from a cat; Kano R et al.; The present study deals with the first isolation of Stephanoascus ciferrii from a cat . A 2-year-old female Persian cat weighing 2.25 kg was referred to an animal hospital with a chief complaint of otitis externa of the left ear . Microscopic examination of specimen from the left ear disclosed yeast cells . The colony of the clinical isolate was cream-colored, rough, raised and wrinkled . The microscopic examination of the clinical isolate revealed abundant branched and septated mycelia with small ramified chains of oval blastoconidia, variable in size, and arranged alongside the hyphae . Amplification of the isolate DNA with LSU rDNA primers yielded a fragment of about 570 bp, whose nucleotide sequence of the isolate showed 100% similarity to that of Stephanoascus ciferrii in the GenBank database . Therefore, the isolate was identified as Stephanoascus ciferrii, confirming the result of mycological examination by molecular analysis.

Clin Infect Dis, 2000 Sep, 31(3), 822 - 4
The use of adjuvant interferon-gamma therapy for hepatosplenic Blastoschizomyces capitatus infection in a patient with leukemia; DeMaio J et al.; Hepatosplenic fungal infections are a devastating complication of neutropenia . Despite aggressive antifungal therapy, clinical response may be poor . We describe a case of hepatosplenic Blastoschizomyces capitatus infection that responded to adjuvant interferon-gamma therapy.

Genes Cells, 2000 Jul, 5(7), 525 - 41
Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint; Kondoh H et al.; BACKGROUND: In normal somatic cell cycle, growth and cell cycle are properly coupled . Although CDK (cyclin-dependent kinase) activity is known to be essential for cell cycle control, the mechanism to ensure the coupling has been little understood . RESULTS: We here show that fission yeast Mis3, a novel evolutionarily highly conserved protein with the RNA-interacting KH motif, is essential for ribosome RNA processing, and implicated in initiating the cell growth . Growth arrest of mis3-224, a temperature sensitive mutant at the restrictive temperature, coincides with the early G2 block in the complete medium or the G1/S block in the release from nitrogen starvation, reflecting coupling of cell growth and division . Genetic interactions indicated that Mis3 shares functions with cell cycle regulators and RNA processing proteins, and is under the control of Dsk1 kinase and PP1 phosphatase . Mis3 is needed for the formation of 18S ribosome RNA, and may hence direct the level of proteins required for the coupling . One such candidate is Mik1 kinase . mis3-224 is sensitive to hydroxyurea, and the level of Mik1 protein increases during replication checkpoint in a manner dependent upon the presence of Mis3 and Cds1 . CONCLUSIONS: Mis3 is essential for ribosome biogenesis, supports S phase checkpoint, and is needed for the coupling between growth and cell cycle . Whether Mis3 interacts solely with ribosomal precursor RNA remains to be determined.

Indian J Exp Biol, 2000 Mar, 38(3), 278 - 9
Hypoglycemic activity of bio-tea in mice; Shenoy C; Administration of bio-tea (1.71 ml/kg) to normal albino mice caused hypoglycemia after 30 min which reached to maximum after 2 hr with a significant decrease in blood sugar level (BSL) and became normal beyond 8 hr . In alloxan-induced diabetic albino mice, repeated treatments of bio-tea for 3 days (five doses) brought about a significant fall in mean BSL . Continuous decrease in BSL was observed after 4 hr of administration of last dose of bio-tea . Hypoglycemic effect was persistent in alloxan-induced diabetic mice . Effect on glucose tolerance test showed a significant fall in BSL of bio-tea treated animals after 1 hr of glucose treatment indicating hypoglycemic effect of bio-tea.

Appl Microbiol Biotechnol, 2000 May, 53(5), 525 - 9
Biosynthesis of citric acid by Yarrowia lipolytica repeat-batch culture on ethanol; Arzumanov TE et al.; After analysis of batch culture and identification of the ways for prolongation of citric acid active synthesis by yeast, repeat-batch (RB) cultivation was suggested . Yarrowia lipolytica strain RB cultivation was studied and optimal conditions for cultivation selected . It was shown that when applying RB cultivation, better results were obtained than for batch cultivation . The activity of the culture remained stable after cultivation for more than 700 h . Comparative analysis of enzyme activities confirmed the regularity of the effect described, as the activity of practically of all the enzymes participating in ethanol oxidation and citric acid biosynthesis remained stable over time during RB cultivation . Advantages of RB cultivation for the production of citric acid by yeast are discussed.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 543 - 59
Coproduction of ethanol and glycerol; Gong CS et al.; Ethanol and glycerol are both metabolic products of yeasts . There are occasions when coproduction of both is considered desirable in industrial operations . In this article, we describe the potential of integrating the two processes . A LORRE Y8 yeast culture isolated from molasses is capable of efficient glycerol production from glucose, and a yeast Culture 1400 is an excellent producer of ethanol . By controlling the process conditions, the ratio of ethanol and glycerol production can be varied.

J Biol Chem, 2000 Sep 15, 275(37), 29042 - 52
Human Cdc7-related kinase complex . In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 bY Cdks; Masai H et al.; huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human . ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D . , Seghezzi, W., Lees, E., Arai, K., and Masai, H . (1999) Mol . Cell . Biol . 19, 5083-5095) . We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system . To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified . GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit . It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents . Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro . MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent . MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase . huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro . Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue . In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

Mol Cell Biochem, 2000 Mar, 206(1-2), 169 - 75
Differential regulation of testosterone vs . 5alpha-dihydrotestosterone by selective androgen response elements; Hsiao PW et al.; There are two major physiological androgens, testosterone (T), and 5alpha-dihydrotestosterone (DHT), which induce different responses in mammals . These androgens regulate the target gene transcription via binding to and activating the same androgen receptor (AR) . The molecular mechanisms that differ between these two very close androgens through the same AR protein to target the distinct genomic responses remain unknown . Using yeast genetic selection, we identified two kinds of androgen response elements (ARE), which could respond differentially to T vs . DHT . These two AREs also show different T- vs . DHT-induced AR transactivation in mammalian Chinese hamster ovary (CHO) cells in terms of copy number and comparisons with the classic mouse mammary tumor virus ARE . Together, our results suggest that the selective ARE sequence may play an important role in the differential T- vs . DHT-induced AR transactivation.

J Biol Chem, 2000 Aug 4, 275(31), 23577 - 82
Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica; Ahlers PM et al.; Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain . Complex I from bovine mitochondria consists of more than forty different polypeptides . Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding . Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping . To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica . Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone . When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region . This indicates that Glu(89) is not a ligand of N-2 . The results are discussedin the light of structural similarities to the homologous {NiFe} hydrogenases.

J Mol Biol, 2000 May 19, 298(5), 749 - 64
Geometry of site alignment during int family recombination: antiparallel synapsis by the Flp recombinase; Grainge I et al.; The Flp site-specific recombinase functions in the copy number amplification of the yeast 2 microm plasmid . The recombination reaction is catalyzed by four monomers of Flp bound to two separate, but identical, recombination sites (FRT sites) and occurs in two sequential pairs of strand exchanges . The relative orientation of the two recombination sites during synapsis was examined . Topoisomerase relaxation and nick ligation were used to detect topological nodes introduced by the synapse prior to the chemical steps of recombination . A single negative supercoil was found to be trapped by Flp in substrates with inverted FRT sites whereas no trapped supercoils were observed with direct repeats . The topology of products resulting from Flp-mediated recombination adjacent to a well characterised synapse, that of Tn3 resolvase/res, was analyzed . The deletion and inversion reactions yielded the four noded catenane and the three noded knot, respectively, as the simplest and the most abundant products . The linking number change introduced by the Flp-mediated inversion reaction was determined to be +/-2 . The most parsimonious explanation of these results is that Flp aligns its recombination sites with antiparallel geometry . The majority of synapses appear to occur without entrapment of additional random plectonemic DNA supercoils between the sites and no additional crossings are introduced as a result of the chemical steps of recombination .

C R Acad Sci III, 2000 Mar, 323(3), 257 - 66
Time-co-ordinated control of glycogen synthase, protein phosphatase 2A and protein kinase CK2 during culture growth in Yarrowia lipolytica in relation to glycogen metabolism; Queiroz-Claret C et al.; In the growth course of the lipolytic yeast Yarrowia lipolytica, the activities of protein phosphatase 2A (PP2A) and glycogen synthase (GS) rise during the exponential phase and concomitantly glycogen storage occurs in the cells . There is also an increase in the independence ratio (RI) indicating a shift from an inactive phosphorylated GS form to an active dephosphorylated GS form . During the early stationary phase, an increase in protein kinase CK2 (CK2) activity, a reversion of RI variation and a glycogen content decrease are observed . GS activity proved to be a good indicator of early culture growth phase . Experiments carried out with enzymes purified from Y . lipolytica show strong RI variations upon the action of CK2 and PP2Ac, and 32P incorporation into GS protein through phosphorylation by CK2 . GS activity would be controlled by the sequential action of PP2A and CK2.

J Bacteriol, 2000 May, 182(10), 2802 - 10
Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica; Pignede G et al.; We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica . It was found to encode a 334-amino-acid precursor protein . The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 3.1.1.3) . The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6 . Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme . Thus, the pro region does not inhibit lipase secretion and activity . However, it does play an essential role in the production of a stable enzyme . Processing was found to be correct in LIP2(A) (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting . No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene . Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y . lipolytica.

Proc Natl Acad Sci U S A, 2000 Apr 25, 97(9), 4603 - 8
Functional interaction between the Werner Syndrome protein and DNA polymerase delta; Kamath-Loeb AS et al.; Werner Syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability . The WS gene encodes a 1,432-amino acid polypeptide (WRN) with a central domain homologous to the RecQ family of DNA helicases . Purified WRN unwinds DNA with 3'-->5' polarity, and also possesses 3'-->5' exonuclease activity . Elucidation of the physiologic function(s) of WRN may be aided by the identification of WRN-interacting proteins . We show here that WRN functionally interacts with DNA polymerase delta (pol delta), a eukaryotic polymerase required for DNA replication and DNA repair . WRN increases the rate of nucleotide incorporation by pol delta in the absence of proliferating cell nuclear antigen (PCNA) but does not stimulate the activity of eukaryotic DNA polymerases alpha or epsilon, or a variety of other DNA polymerases . Moreover, we show that functional interaction with WRN is mediated through the third subunit of pol delta: i.e., Pol32p of Saccharomyces cerevisae, corresponding to the recently identified p66 subunit of human pol delta . Absence of the third subunit abrogates stimulation by WRN, and stimulation is restored by reconstituting the three-subunit enzyme . Our findings suggest that WRN may facilitate pol delta-mediated DNA replication and/or DNA repair and that disruption of WRN-pol delta interaction in WS cells may contribute to the previously observed S-phase defects and/or the unusual sensitivity to a limited number of DNA damaging agents.

Analyst, 1999 Jun, 124(6), 907 - 10
Nephelometric determination of micro amounts of nucleic acids with protamine sulfate; Li Z et al.; Nucleic acids can form large particle complexes with protamine sulfate by electrostatic forces, which results in strong light scattering . Based on this, a nephelometric method is described for sensitive and convenient determination of nucleic acids with protamine sulfate by using a common spectrofluorimeter . Maximum light scattering is produced in the range of pH 2.2-4.4 with the same excitation and emission wavelengths at 365 nm . Under optimal conditions, the calibration curves are linear in the range 0.05-60.0 micrograms cm-3 for nucleic acids . The corresponding detection limits are 12.5 ng cm-3 for calf thymus DNA, 9.0 ng cm-3 for fish sperm DNA, and 18.0 ng cm-3 for yeast RNA, respectively . Six synthetic samples are determined with satisfactory results . The relative standard deviation of five replicate measurements is 3.2% for 2.0 micrograms cm-3 calf thymus DNA.

Analyst, 1999 Jun, 124(6), 901 - 6
Interaction of a novel red-region fluorescent probe, Nile blue, with DNA and its application to nucleic acids assay; Chen QY et al.; A novel fluorimetric method was developed for the rapid determination of DNA and RNA based on their quenching effect on the cationic red-region fluorescent dye Nile Blue (NB) . In the investigation of the interaction of NB with DNA by steady-state polarization measurements, thermal denaturing study, determination of absorption and fluorescence characteristics, salt effect study and electrophoresis experiments, the results supported the suggestion that NB served as an intercalator to the stack base pairs of nucleic acids . Further evidence showed that the quenching could be ascribed to the static quenching mode . A binding constant of about 10(6) M-1 and a binding site size of about three base pairs were obtained by spectral methods . Under optimum conditions, the calibration curves for the determination of calf thymus DNA (CT DNA) and yeast RNA were linear over the ranges 3.0 ng mL-1-2.0 micrograms mL-1 and 27 ng mL-1-10 micrograms mL-1, respectively . The detection limits were 3.0 ng mL-1 for CT DNA and 27 ng mL-1 for RNA . The relative standard deviation (n = 6) was within 2.1% in the middle of the linear range . Interferences from some interesting co-existing substances in the determination of DNA were also examined.

Appl Environ Microbiol, 2000 Mar, 66(3), 1233 - 6
Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica; Wache Y et al.; We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H . Wang et al., J . Bacteriol . 181:5140-5148, 1999) . Here, these mutants were studied for lactone production . Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Deltapox3, which produced 220 mg of gamma-decalactone per liter after 24 h . The Deltapox2 Deltapox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation . The Deltapox2 Deltapox3 Deltapox5 triple-disrupted strain produced and consumed lactone very slowly . On the contrary, the Deltapox2 Deltapox3 Deltapox4 Deltapox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into gamma-decalactone, demonstrating that Aox4p is essential for the biotransformation.

Biochem J, 2000 Feb 15, 346 Pt 1, 177 - 84
Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p; Szilard RK et al.; Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes . The Yarrowia lipolytica pex5-1 mutant fails to import a subset of peroxisomal matrix proteins, including those with a type 1 peroxisomal targeting signal (PTS1) . Pex5p family members interact with a PTS1 through their characteristic tetratricopeptide repeat (TPR) domain . We used binding assays in vitro to investigate the nature of the association of Y . lipolytica Pex5p (YlPex5p) with the PTS1 signal . A purified recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1 . Wild-type YlPex5p translated in vitro recognized functional PTS1s specifically . This activity is abrogated by the substitution of an aspartic residue for a conserved glycine residue in the TPR domain (G455D) of YlPex5p encoded by the pex5-1 allele . Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary but not sufficient for both interaction with a PTS1 and functional complementation of a strain lacking YlPex5p.

Nucleic Acids Res, 2000 Feb 15, 28(4), 952 - 9
Involvement of multiple subunit-subunit contacts in the assembly of RNA polymerase II; Kimura M et al.; RNA polymerase II from the fission yeast Schizo-saccharomyces pombe consists of 12 species of subunits, Rpb1-Rpb12 . We expressed these subunits, except Rpb4, simultaneously in cultured insect cells with baculovirus expression vectors . For the isolation of subunit complexes formed in the virus-infected cells, a glutathione S -transferase (GST) sequence was fused to the rpb3 cDNA to produce GST-Rpb3 fusion protein and a decahistidine-tag sequence was inserted into the rpb1 cDNA to produce Rpb1H protein . After successive affinity chromatography on glutathione and Ni(2+)columns, complexes consisting of the seven subunits, Rpb1H, Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, were identified . Omission of the GST-Rpb3 expression resulted in reduced assembly of the Rpb11 into the complex . Direct interaction between Rpb3 and the other six subunits was detected by pairwise coexpression experiments . Coexpression of various combinations of a few subunits revealed that Rpb11 enhances Rpb3-Rpb8 interaction and consequently Rpb8 enhances Rpb1-Rpb3 interaction to some extent . We propose a mechanism in which the assembly of RNA poly-merase II is stabilized through multiple subunit-subunit contacts.

J Cell Biol, 2000 Jan 10, 148(1), 29 - 44
Fusion of small peroxisomal vesicles in vitro reconstructs an early step in the in vivo multistep peroxisome assembly pathway of Yarrowia lipolytica; Titorenko VI et al.; We have identified and purified six subforms of peroxisomes, designated P1 to P6, from the yeast, Yarrowia lipolytica . An analysis of trafficking of peroxisomal proteins in vivo suggests the existence of a multistep peroxisome assembly pathway in Y . lipolytica . This pathway operates by conversion of peroxisomal subforms in the direction P1, P2-->P3-->P4-->P5-->P6 and involves the import of various peroxisomal proteins into distinct vesicular intermediates . We have also reconstituted in vitro the fusion of the earliest intermediates in the pathway, small peroxisomal vesicles P1 and P2 . Their fusion leads to the formation of a larger and more dense peroxisomal vesicle, P3 . Fusion of P1 and P2 in vitro requires cytosol and ATP hydrolysis and is inhibited by antibodies to two membrane-associated ATPases of the AAA family, Pex1p and Pex6p . We provide evidence that the fusion in vitro of P1 and P2 peroxisomes reconstructs an actual early step in the peroxisome assembly pathway operating in vivo in Y . lipolytica.

Rev Argent Microbiol, 1999 Oct-Dec, 31(4), 205 - 18
{Prion biology: update}; Weber EL; The word "prion" was created in 1982 to name the etiological agent of the transmissible spongiform encephalopathies (TSE), a group of degenerative diseases affecting central nervous system of man and animals, including bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) . Prions present two isoforms: PrPC, cellular or normal, which exists in all vertebrates and is sensitive to detergents and proteases, and PrPSc, disease associated, partially resistant . The molecular weight of both PrPC and PrPSc is 30-35 kD; after treatment with detergents and proteases PrPSc originates PrP27-30 (27-30 kD) . PrPC is also denominated PrPsens, and PrPSc is PrPres . PrPSc and PrP27-30 cause disease . PrPC presents polymorphisms specifically associated with some TSE . The "prion hypothesis" says that PrPSc transmits its characteristic resistance to PrPC through conformational changes, and accumulation of the protein, without involvement of nucleic acids, causes disease . Most of the hypothesis has been demonstrated with transgenic mice, computer models and recombinant proteins, but the existence of strains of the TSE agents has not been explained . The description of similar mechanisms of propagation of protein conformational properties in Saccharomyces cereviseae has extended the meaning of the prion definition . Although the transmission of conformational changes between PrPC and PrPSc was experimentally shown, the pathogenesis of the TSE remains unknown . The relationship between BSE and vCJD is mentioned.

Mol Biol Evol, 1999 Dec, 16(12), 1799 - 808
Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit; Liu YJ et al.; In an effort to establish a suitable alternative to the widely used 18S rRNA system for molecular systematics of fungi, we examined the nuclear gene RPB2, encoding the second largest subunit of RNA polymerase II . Because RPB2 is a single-copy gene of large size with a modest rate of evolutionary change, it provides good phylogenetic resolution of Ascomycota . While the RPB2 and 18S rDNA phylogenies were highly congruent, the RPB2 phylogeny did result in much higher bootstrap support for all the deeper branches within the orders and for several branches between orders of the Ascomycota . There are several strongly supported phylogenetic conclusions . The Ascomycota is composed of three major lineages: Archiascomycetes, Saccharomycetales, and Euascomycetes . Within the Euascomycetes, plectomycetes, and pyrenomycetes are monophyletic groups, and the Pleosporales and Dothideales are distinct sister groups within the Loculoascomycetes . We confirm the placement of Neolecta within the Archiascomycetes, suggesting that fruiting body formation and forcible discharge of ascospores were characters gained early in the evolution of the Ascomycota . These findings show that a slowly evolving protein-coding gene such as RPB2 is useful for diagnosing phylogenetic relationships among fungi.

Clin Microbiol Rev, 1999 Jul, 12(3), 454 - 500
Developments in fungal taxonomy; Guarro J et al.; Fungal infections, especially those caused by opportunistic species, have become substantially more common in recent decades . Numerous species cause human infections, and several new human pathogens are discovered yearly . This situation has created an increasing interest in fungal taxonomy and has led to the development of new methods and approaches to fungal biosystematics which have promoted important practical advances in identification procedures . However, the significance of some data provided by the new approaches is still unclear, and results drawn from such studies may even increase nomenclatural confusion . Analyses of rRNA and rDNA sequences constitute an important complement of the morphological criteria needed to allow clinical fungi to be more easily identified and placed on a single phylogenetic tree . Most of the pathogenic fungi so far described belong to the kingdom Fungi; two belong to the kingdom Chromista . Within the Fungi, they are distributed in three phyla and in 15 orders (Pneumocystidales, Saccharomycetales, Dothideales, Sordariales, Onygenales, Eurotiales, Hypocreales, Ophiostomatales, Microascales, Tremellales, Poriales, Stereales, Agaricales, Schizophyllales, and Ustilaginales).

Nucleic Acids Res, 1999 Jul 15, 27(14), 2905 - 11
Plasmid linking number change induced by topoisomerase I-mediated DNA damage; Duann P et al.; The state of cellular chromatin in response to DNA damage has been examined by monitoring the change in the linking number of circular episomes . COS cells transfected with an SV40-based vector were treated with camptothecin (CPT), a eukaryotic DNA topoisomerase I (TOP1) poison which induces TOP1-mediated DNA damage . Within minutes, a large increase in the linking number (over 10 linking number) of a small fraction (5-15%) of the episomal DNA was observed . A similar CPT-induced increase in plasmid DNA linking number was observed in Saccharomyces cerevisae expressing human DNA TOP1 . In this case, the majority of the plasmid DNA can undergo rapid relaxation . The large increase in the plasmid linking number suggests major chromatin structural reorganization in response to TOP I-mediated DNA damage.

Med Mycol, 1999 Apr, 37(2), 101 - 4
Rapid identification of Debaryomyces hansenii/Candida famata by polymerase chain reaction; Nishikawa A et al.; Primers designed in this study were used in a polymerase chain reaction test to amplify a species-specific fragment of approximately 340 bp of the large subunit ribosomal DNA of Debaryomyces hansenii/Candida famata . None of the other medically relevant yeasts including C . guilliermondii, and also the related species, D . nepalensis and C . saitoana, were amplified by this primer pair.

FEBS Lett, 1999 May 14, 451(1), 1 - 4
Selective peroxisome degradation in Yarrowia lipolytica after a shift of cells from acetate/oleate/ethylamine into glucose/ammonium sulfate-containing media; Gunkel K et al.; We have shown that peroxisomes of the yeast Yarrowia lipolytica are subject to specific degradation after exposure of acetate/oleate-grown cells to glucose excess conditions . Electron microscopic analysis has revealed that the peroxisomes were degraded by uptake in the vacuole . Our results suggest that peroxisomes are taken up by macroautophagic processes, because sequestration of individual peroxisomes, which occurs typically at the beginning of microautophagy, was never observed . The observation that a peroxisomal amine oxidase activity is specifically induced by ethylamine was used for the development of a plate assay screening procedure to isolate peroxisome degradation-defective mutants.

J Immunol, 1999 May 15, 162(10), 6148 - 54
Regulation of the macrophage vacuolar ATPase and phagosome-lysosome fusion by Histoplasma capsulatum; Strasser JE et al.; Histoplasma capsulatum (Hc) maintains a phagosomal pH of about 6.5 . This strategy allows Hc to obtain iron from transferrin, and minimize the activity of macrophage (Mo) lysosomal hydrolases . To determine the mechanism of pH regulation, we evaluated the function of the vacuolar ATPase (V-ATPase) in RAW264.7 Mo infected with Hc yeast or the nonpathogenic yeast Saccharomyces cerevisae (Sc) . Incubation of Hc-infected Mo with bafilomycin, an inhibitor of the V-ATPase, did not affect the intracellular growth of Hc, nor did it affect the intraphagosomal pH . In contrast, upon addition of bafilomycin, phagosomes containing Sc rapidly changed their pH from 5 to 7 . Hc-containing phagosomes had 5-fold less V-ATPase than Sc-containing phagosomes as quantified by immunoelectron microscopy . Furthermore, Hc-containing phagosomes inhibited phagolysosomal fusion as quantified by the presence of acid phosphatase, accumulation of LAMP2, and fusion with rhodamine B-isothiocyanate-labeled dextran-loaded lysosomes . Finally, in Hc-containing phagosomes, uptake of ferritin was equivalent to phagosomes containing Sc, indicating that Hc-containing phagosomes have full access to the early "bulk flow" endocytic pathway . Thus, Hc yeasts inhibit phagolysosomal fusion, inhibit accumulation of the V-ATPase in the phagosome, and actively acidify the phagosomal pH to 6.5 as part of their strategy to survive in Mo phagosomes.

Res Microbiol, 1999 Mar, 150(2), 95 - 103
Yarrowia lipolytica cell wall architecture: interaction of Ywp1, a mycelial protein, with other wall components and the effect of its depletion; Ramon AM et al.; Linkages of Ywp1 to other components of the Yarrowia lipolytica mycelial cell wall were studied by extraction with beta-mercaptoethanol and zymolyase (a beta-glucanase complex) and by the use of rabbit polyclonal antibody preparation raised against Ywp1 . Ywp1 complexed with an N-glycosylated cell wall protein(s) to form supramolecular complexes through disulphide bridges (extractable with beta-mercaptoethanol) or bonded to beta-1,3-glucan (extractable with zymolyase) . The lack of a specific morphological phenotype when YWP1 was knocked out by gene disruption might indicate that other proteins present in the cell wall of Y . lipolytica compensated for its loss . In this mutant, the electrophoretic pattern of proteins, detected with polyclonal antibodies against the entire cell wall, was different from that obtained with the parental strain, but sensitivity to calcofluor white, zymolyase and chitinase did not change . Quantitative analysis of fluorescence emitted by cells in the presence of fluorescent wheat germ agglutinin (FITC-WGA) indicated that chitin was organized in the cell wall of the mutant cells in a form different from that in the parental strain.

Science, 1999 Apr 9, 284(5412), 328 - 30
The Pex16p homolog SSE1 and storage organelle formation in Arabidopsis seeds; Lin Y et al.; Mature Arabidopsis seeds are enriched in storage proteins and lipids, but lack starch . In the shrunken seed 1 (sse1) mutant, however, starch is favored over proteins and lipids as the major storage compound . SSE1 has 26 percent identity with Pex16p in Yarrowia lipolytica and complements pex16 mutants defective in the formation of peroxisomes and the transportation of plasma membrane- and cell wall-associated proteins . In Arabidopsis maturing seeds, SSE1 is required for protein and oil body biogenesis, both of which are endoplasmic reticulum-dependent . Starch accumulation in sse1 suggests that starch formation is a default storage deposition pathway.

Biotechnol Bioeng, 1998 Aug 5, 59(3), 379 - 85
Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy; Chang CC et al.; A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated . A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity . This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein . The volumetric enzyme productivity (1, 960 units/L . h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L . h) .

Antonie Van Leeuwenhoek, 1998 Nov, 74(4), 283 - 91
Diversity and affinities among species and strains of Lipomyces; Gouliamova DE et al.; Phylogenetic relationships of the yeast genus Lipomyces were studied using sequences from fragments of 5.8S rRNA gene and from internal transcribed spacer region ITS2 of 13 strains (7 type strains included) representing five species and subtaxa, and originating from different geographical locations (Japan, Trinidad, Nigeria, North America, Western Europe, Russia, South Africa, Mauritius) . Parsimony and distance analyses were performed . Tree topology from the parsimony and distance analyses of the sequence confirmed the results of nDNA reassociation . Results segregate the 13 isolates of Lipomyces into five major clades.

Antonie Van Leeuwenhoek, 1998 Nov, 74(4), 229 - 35
Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum: genomic characterization; Smith MT et al.; The G + C contents of 25 strains of Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum were found to be heterogeneous on basis of derivative graphs of the melting profiles . Strains showing similar derivative graphs of the melting curve exhibited high levels of DNA homology (80-100%); strains showing dissimilar derivative graphs exhibited low levels of DNA homology (5 to 45%) . Being considered separate taxa on basis of these parameters, D . capitatus, D . spicifer and G . clavatum could be identified by a combination of the key characteristics growth on xylose, cellobiose, salicin and arbutin.

Biochim Biophys Acta, 1999 Feb 4, 1449(1), 1 - 24
Signal perception and transduction: the role of protein kinases; Schenk PW et al.; Cells can react to environmental changes by transduction of extracellular signals, to produce intracellular responses . Membrane-impermeable signal molecules are recognized by receptors, which are localized on the plasma membrane of the cell . Binding of a ligand can result in the stimulation of an intrinsic enzymatic activity of its receptor or the modulation of a transducing protein . The modulation of one or more intracellular transducing proteins can finally lead to the activation or inhibition of a so-called 'effector protein' . In many instances, this also results in altered gene expression . Phosphorylation by protein kinases is one of the most common and important regulatory mechanisms in signal transmission . This review discusses the non-channel transmembrane receptors and their downstream signaling, with special focus on the role of protein kinases.

J AOAC Int, 1999 Jan-Feb, 82(1), 112 - 8
Determination of neutral lactase activity in industrial enzyme preparations by a colorimetric enzymatic method: collaborative study; Engelen AJ et al.; Thirteen laboratories participated in a collaborative study to validate a colorimetric assay for determining neutral lactase activity in industrial enzyme preparations . Each laboratory received 5 duplicate samples with activity levels of 2000 and 5000 neutral lactase units provided by 4 commercial suppliers . Two laboratories did not return results . Method performance was calculated according to AOAC guidelines . From the 11 remaining laboratories, 3 were excluded from statistical analysis because of invalid data determined during initial review by Youden pair, value versus laboratory . Repeatability relative standard deviation (RSDr) values ranged from 3.20 to 8.62%, and reproducibility relative standard deviation (RSDR) values ranged from 8.77 to 16.35% . With outliers excluded, RSDr values ranged from 2.94 to 5.01%, and RSDR values ranged from 7.50 to 13.84% . The colorimetric enzymatic method for determining neutral lactase activity in industrial enzyme preparations has been adopted first action by AOAC INTERNATIONAL.

Arch Biochem Biophys, 1999 Feb 15, 362(2), 339 - 45
Purification and properties of mycobacterial GDP-mannose pyrophosphorylase; Ning B et al.; The enzyme that catalyzes the formation of GDP-d-mannose from GTP and alpha-d-mannose-1-P was purified about 2300-fold to near homogeneity from the soluble fraction of Mycobacterium smegmatis . At the final stage of purification, a major protein band of 37 kDa was observed and this band was specifically labeled, and in a concentration-dependent manner, by the photoaffinity probe 8-N3-GDP{32P}-d-mannose . The purified enzyme was stable for several months when kept in the frozen state . The 37-kDa band was subjected to protein sequencing and one peptide sequence of 25 amino acids showed over 80% identity to GDP-mannose pyrophosphorylases of pig liver and Saccharomyces cerevesiae . In contrast to some other bacterial GDP-mannose pyrophosphorylases, the mycobacterial enzyme was not multifunctional and did not have phosphomannose isomerase or phosphoglucose isomerase activity . Also, in contrast to the pig liver enzyme which uses mannose-1-P or glucose-1-P plus GTP to synthesize either GDP-mannose or GDP-glucose, the mycobacterial enzyme was specific for mannose-1-P as the sugar phosphate substrate . The enzyme was also relatively specific for GTP as the nucleoside triphosphate substrate . ITP was about 18% as effective as GTP, but ATP, CTP, and UTP were inactive . The activity of the enzyme was inhibited by GDP-glucose and glucose-1-P, although neither was a substrate for this enzyme . The pH optimum for the enzyme was 8.0, and Mg2+ was the best cation with optimum activity at about 5 mM . This enzyme is important for producing the activated form of mannose for formation of cell wall lipoarabinomannan and various mannose-containing glycolipids and polysaccharides .

Mycoses, 1998 Nov, 41(9-10), 425 - 6
Infection of an intravenous port system with Metschnikowia pulcherrima Pitt et Miller; Mohl W et al.; A patient with short bowel syndrome as a consequence of multiple intestinal resections for Crohn's disease, had a port system implanted to improve her nutritional status . One year later she presented with fever, weakness and nighttime sweating . Metschnikowia pulcherrima Pitt et Miller was grown in blood cultures from the port system . After antifungal chemotherapy using fluconazole and removal of the implant, the patient's condition improved markedly and her fever and sweating disappeared . We conclude that Metschnikowia pulcherrima can turn into a human pathogen in patients with indwelling catheters for parenteral nutrition . Chemotherapy with fluconazole and, whenever possible, removal of the implant, appear to be adequate treatment.

Yeast, 1998 Nov, 14(15), 1387 - 97
Cloning and characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica; Iida T et al.; A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B . The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family . The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B . The nucleotide sequence of one of the positive clones was determined . An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified . It was of 523 amino acid residues, 60.2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed . The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol . A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source . Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation . These results suggest that the YlALK1 gene product is the major P450A1k to metabolize short-chain n-alkanes such as decane and dodecane in Y . lipolytica.

FEMS Microbiol Lett, 1998 Oct 15, 167(2), 209 - 14
Organelle purification and selective permeabilisation of the plasma membrane: two different approaches to study vacuoles of the filamentous fungus Ashbya gossypii; Forster C et al.; Two different approaches to prepare and characterise vacuoles from the filamentous fungus Ashbya gossypii are described, i.e . the isolation of vacuoles from hyphal cells and the controlled permeabilisation of the plasma membrane . By mechanical lysis of protoplasts and separation of the organelles on a stepped density gradient, we obtained a vacuolar fraction virtually free of contamination by other organelles, unlysed protoplasts and cell debris . The integrity of the isolated organelles was characterised by vital-staining, the presence of alpha-mannosidase, and retained accumulation of basic amino acids . In a second approach, the cell membrane of the fungus was selectively permeabilised by use of the saponin digitonin leaving the vacuoles in their physiological surrounding, i.e . protected by the rigid cell wall . The permeabilisation was monitored by the latency of predominantly cytosolic amino acids and the ATP status of the cells . Functional intactness of the vacuoles within the permeabilised hyphae was demonstrated by maintenance of the pH gradient across the vacuolar membrane as detected by accumulation of the fluorescent dye, Acridine orange . These two methods are well-suited tools for the in situ assay of intracellular compartmentation of metabolites, for vacuolar transmembrane fluxes in Ashbya gossypii, as well as for the direct access to vacuolar membranes and enzymes of this fungus.

J Am Soc Nephrol, 1998 Sep, 9(9), 1574 - 80
Function and expression of a novel rat salt-tolerant protein: evidence of a role in cellular sodium metabolism; Tsuji E et al.; Higher dietary salt intake in humans is associated with higher BP, but the BP response to NaCl, so-called salt sensitivity, is heterogeneous among individuals . It has been postulated that modifications in cellular cation metabolism may be related to salt sensitivity in mammalian hypertension . The authors have isolated a novel rat complementary DNA, called salt-tolerant protein (STP), that can functionally complement Saccharomyces cervisiae HAL1, which improves salt tolerance by modulating the cation transport system . On high-salt (8% NaCl) diets, both Dahl salt-sensitive and salt-resistant rats displayed an elevated BP and increased STP mRNA expression . Immunohistochemistry using an anti-rat STP antibody demonstrated the presence of STP immunoreactivity in the proximal tubules . In cells that transiently expressed STP, the intracellular {Na+}/{K+} ratio was higher than that in control cells . STP contains predicted coiled-coil and Src homology 3 domains, and shows a partially high degree of nucleotide identity to human thyroid-hormone receptor interacting protein . These results suggest that STP may play an important role in salt sensitivity through cellular sodium metabolism by mediating signal transduction and a hormone-dependent transcription mechanism.

Nippon Rinsho, 1998 May, 56(5), 1097 - 101
{Mammalian proteins that associate with telomeres}; Uchiumi F et al.; Telomeres are the DNA-protein complexes found at the ends of linear chromosomes . The structure is believed to be important for chromosome stability and cell integrity, and thereby for cell senescence and immortality . Telomeric DNA consists of tandemly repeated sequences which are observed from human to yeast . For example, human and Saccharomyces telomeres have T2AG3 and TG1-3 repeats, respectively . Recently, various protein factors including replication factor C (RFC) have been found as telomere repeat sequence binding-proteins . Characterization of these proteins and their interactions with telomeres may provide a new insight into not only telomere functions but also aging, immortality and apoptosis of the cells.

Plant Cell, 1998 Jan, 10(1), 105 - 17
Molecular characterization of a carbon transporter in plastids from heterotrophic tissues: the glucose 6-phosphate/phosphate antiporter; Kammerer B et al.; Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy . Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example . We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT) . The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers . Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only approximately 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families . Thus, the GPTs represent a third group of plastidic phosphate antiporters . All three classes of phosphate translocator genes show differential patterns of expression . Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues . Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates . Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates.

Curr Opin Genet Dev, 1998 Feb, 8(1), 112 - 26
Genetic analysis of protein tyrosine phosphatases; Van Vactor D et al.; Genetic analysis has enhanced our understanding of the biological roles of many protein tyrosine kinases (PTKs) . More recently, studies utilizing both spontaneous mutants and mutants induced by homologous recombination techniques have begun to yield key insights into the role of specific protein tyrosine phosphatases (PTPs) and to suggest how PTKs and PTPs interact . Specific PTPs in Saccharomyces cerevesiae and Schizomyces pombe regulate MAP kinase pathways . Several Drosophila receptor PTPs control axonal targeting pathways, whereas the non-receptor PTP Corkscrew (Csw), plays an essential positive signaling role in multiple developmental pathways directed by receptor PTKs . The vertebrate homolog of Csw, SHP-2, also is required for growth factor signaling and normal development . Finally, very recent studies of other mammalian PTPs suggest that they have critical roles in processes as diverse as hematopoiesis and liver and pituitary development.

J Eukaryot Microbiol . 1997 Nov-Dec;44(6):80S.
Ultrastructural features of spindle microtubule organization during the nuclear division of Encephalitozoon hellem; Sacchi L et al.; Ultrastructural studies were carried out to describe the nuclear division cycle of a strain of Encephalitozoon hellem isolated from an Italian AIDS patient . The nuclear division occurs during the proliferative vegetative phase and it is characterized by the intranuclear mitosis and by the lack of centrioles . The spindle termini are electron dense spindle plaques (ESPs), resembling to the spindle pole bodies (SPBs) of Saccharomycetes . The ESPs are bifacial organella forming microtubules on both nucleoplasic and cytoplasmic faces . In the outer layer of the spindle plaque are present vesicular elements lined by a double membrane of unknown function . The peculiar morphological features of E . hellem ESPs indicate that both intranuclear spindle and cytoplasmic microtubules are involved in the nuclear division.

Gene, 1997 Dec 5, 203(1), 59 - 63
A homologue of the 19 kDa signal recognition particle protein locus in Drosophila melanogaster; Lai C et al.; A homologue of 19 kDa signal recognition particle locus (SRP19) was cloned and molecularly characterized in Drosophila melanogaster . It is located in the 65F region of the left arm on the third chromosome, approx . 500 bp 5' of the quemao locus . The SRP19 transcript was determined from cDNA clones, Northern blot analysis, and the 5' rapid amplification of cDNA end method . SRP19 was expressed in all the developmental stages of Drosophila . The predicted amino acid sequence (163 aa) shows that SRP19 of Drosophila shares 44%, 29%, 17% and 19% identity with the homologues from human, rice and two yeast species (Saccharomyces and Yarrowia), respectively . The most conserved amino acid residues across these species are located at those sites required for in vitro association with the 7S RNA component of the SRP.

Endocrinology, 1997 Dec, 138(12), 5184 - 8
Spot 14 protein-protein interactions: evidence for both homo- and heterodimer formation in vivo; Cunningham BA et al.; Spot 14 (S14) is a nuclear protein that is abundant only in lipogenic tissues (liver, adipose, lactating mammary), where its expression is rapidly regulated by hormones and dietary constituents . We recently showed that S14 acts at the transcriptional level in the transduction of signals for increased expression of genes encoding lipogenic enzymes . To better understand the mechanism of the regulation of gene transcription by S14, we employed a yeast two-hybrid system to identify hepatic proteins that physically interact with S14 . We found that S14 has a strong propensity for homodimerization, as is the case for many transcription factors . Relevance of this finding to mammalian cells was established by transient cotransfection of S14 constructs bearing two different epitope tags . Glutathione-S-transferase-S14 and hemagglutinin-S14 fusions copurified from the transfected cells by glutathione-affinity chromatography, indicating their association in vivo . Analysis of S14 deletion mutants in the yeast system showed that an evolutionarily conserved hydrophobic heptad repeat (zipper) near the carboxyl terminus was necessary for homodimerization . In parallel studies, we observed a 36-kDa protein that specifically coimmunoprecipitated with S14 from extracts of radiolabeled rat hepatocytes . We propose that S14 is an acidic transcriptional activator that acts as a homodimer to modulate gene expression as a component of a tripartite complex with a 36-kDa hepatic protein.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11472 - 7
Characterization of the CHD family of proteins; Woodage T et al.; The murine gene CHD1 (MmCHD1) was previously isolated in a search for proteins that bound a DNA promoter element . The presence of chromo (chromatin organization modifier) domains and an SNF2-related helicase/ATPase domain led to speculation that this gene regulated chromatin structure or gene transcription . This study describes the cloning and characterization of three novel human genes related to MmCHD1 . Examination of sequence databases produced several more related genes, most of which were not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals . The major region of sequence variation is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1 . Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil . This finding suggested that enhanced transcriptional arrest at RNA polymerase II pause sites due to 6-azauracil-induced nucleotide pool depletion was reduced in the deletion strain and that ScCHD1 inhibited transcription . This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template.

Microbiology, 1997 Sep, 143 ( Pt 9), 3045 - 54
pH-regulated expression of the acid and alkaline extracellular proteases of Yarrowia lipolytica; Glover DJ et al.; The pH-regulated expression of the acid (AXP) and alkaline (AEP) extracellular proteases of the yeast Yarrowia lipolytica 148 was analysed . Expression in batch and continuous cultures was determined at the mRNA level by Northern blotting, and at the enzyme level by enzyme assays and Western blotting . Culture pH regulated AEP and AXP expression predominantly at the level of mRNA content . Highest levels of AEP mRNA were detected at pH 6.5 whereas highest levels of AXP mRNA were detected at pH 5.5 . At pH values either side of these maxima AEP and AXP expression were progressively down-regulated . For both enzymes, the variation in mRNA levels with culture pH occurred progressively rather than by discrete steps . AXP expression did not occur above pH 7.0 . Some degree of AEP expression occurred at all pH values tested in two unrelated strains of Y . lipolytica.

J Biol Chem, 1997 Sep 26, 272(39), 24594 - 8
Yarrowia lipolytica TSR1 gene product . A novel endoplasmic reticulum membrane component involved in the signal recognition particle-dependent translocation pathway; Mamoun CB et al.; The tsr1-1 mutation has been initially identified as an extragenic suppressor of the scr2.II-13 mutation that alters the 7SL RNA component of the signal recognition particle (SRP) and results in severe defects in protein translocation and SRP stability . We showed previously that the TSR1 gene was essential and that the tsr1-1 mutation allowed complete recovery of scr2.II-13-associated secretory defects . We show here that the tsr1-1 mutation also restores SRP stability in an scr2.II-13 context . The TSR1 gene product (Tsr1p) is stably associated with rapidly sedimenting material and cofractionates with the lumenal protein Kar2p of the endoplasmic reticulum; it behaves in protease protection assays as a transmembrane component . Coimmunoprecipitation experiments revealed a physical interaction with Kar2p and with ribosomal components associated to the 5.8S rRNA as well as with SRP components like Sec65p and 7SL RNA . We propose that Tsr1p is an important component of the endoplasmic reticulum membrane, interacting both with the SRP-ribosome complex in the cytosol and with Kar2p in the lumen of the endoplasmic reticulum.

Comp Biochem Physiol B Biochem Mol Biol, 1997 Aug, 117(4), 599 - 604
Comparative studies of the indoleamine dioxygenase-like myoglobin from the abalone Sulculus diversicolor; Suzuki T et al.; The abalone Sulculus diversicolor contains abundant myoglobin in its buccal mass . The myoglobin is homodimeric and the molecular mass of the constituent polypeptide chain is 41,000 Da . The amino acid sequence and gene structure are highly homologous with those of a vertebrate tryptophan-degrading enzyme, indoleamine dioxygenase (IDO) . Thus Sulculus myoglobin evolved from an IDO gene, and represents a typical case of functional convergence . The oxygen equilibrium properties of Sulculus myoglobin were examined and compared with those of myoglobins from other sources . It binds oxygen reversibly, and the P50 was determined to be 3.8 mmHg at 20 degrees C and pH 7.4, showing that the oxygen affinity of Sulculus myoglobin is significantly lower than those of usual 16 kDa myoglobins . It also displays no cooperativity (nmax: 1.02-1.06) and no alkaline Bohr effect between pH 7.0 and 7.9 . The cDNA-derived amino acid sequences of vertebrate IDOs, molluscan IDO-like myoglobins and a homolog in the yeast Saccharomyces were aligned, and several amino acid residues were proposed as candidates for key residues to control the function of IDO or myoglobin.

J Mol Evol, 1997 Jul, 45(1), 50 - 9
Analysis of donor splice sites in different eukaryotic organisms; Rogozin IB et al.; We present here a new algorithm for functional site analysis . It is based on four main assumptions: each variation of nucleotide composition makes a different contribution to the overall binding free energy of interaction between a functional site and another molecule; nonfunctioning site-like regions (pseudosites) are absent or rare in genomes; there may be errors in the sample of sites; and nucleotides of different site positions are considered to be mutually dependent . In this algorithm, the site set is divided into subsets, each described by a certain consensus . Donor splice sites of the human protein-coding genes were analyzed . Comparing the results with other methods of donor splice site prediction has demonstrated a more accurate prediction of consensus sequences AG/GU(A,G), G/GUnAG, /GU(A,G)AG, /GU(A,G)nGU, and G/GUA than is achieved by weight matrix and consensus (A,C)AG/GU(A,G)AGU with mismatches . The probability of the first type error, E1, for the obtained consensus set was about 0.05, and the probability of the second type error, E2, was 0.15 . The analysis demonstrated that accuracy of the functional site prediction could be improved if one takes into account correlations between the site positions . The accuracy of prediction by using human consensus sequences was tested on sequences from different organisms . Some differences in consensus sequences for the plant Arabidopsis sp., the invertebrate Caenorhabditis sp., and the fungus Aspergillus sp . were revealed . For the yeast Saccharomyces sp . only one conservative consensus, /GUA(U,A,C)G(U,A,C), was revealed (E1 = 0.03, E2 = 0.03) . Yeast is a very interesting model to use for analysis of molecular mechanisms of splicing.

Oncogene, 1997 Jun 19, 14(24), 2943 - 50
ayk1, a novel mammalian gene related to Drosophila aurora centrosome separation kinase, is specifically expressed during meiosis; Yanai A et al.; A novel murine gene, designated ayk1, which encodes a putative serine/threonine kinase has been cloned and characterized . The predicted catalytic domain of the protein is highly similar to that of Drosophila aurora (62.9% identity), and to that of Saccharomyces Ipl1 (49.4% identity) . All three proteins also have very basic calculated isoelectric points (higher than 10) . aurora has been recently shown to be crucial for centrosome separation and chromosome segregation, while Ipl1 is essential for yeast viability and accurate chromosome segregation . The results of Northern analysis and in situ RNA localization support a similar role for ayk1 . The gene is specifically expressed in meiotically active cells, and during spermatogenesis, ayk1 transcripts accumulate just before the first meiotic division . Much lower levels are found in mitotically active cells . We propose that Ayk1, aurora and Ipl1 belong to a distinct new subfamily of kinases . These results suggest that the pathways controlling chromosome segregation are evolutionary conserved, and that similar control mechanisms operate in mitosis and meiosis.

J Cell Biol, 1997 Jun 16, 137(6), 1265 - 78
Enlarged peroxisomes are present in oleic acid-grown Yarrowia lipolytica overexpressing the PEX16 gene encoding an intraperoxisomal peripheral membrane peroxin; Eitzen GA et al.; Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly . The mutant strain pex16-1 lacks morphologically recognizable peroxisomes . Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes . The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D) . Pex16p has no known homologues . Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane . Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes . Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid-containing medium . Overexpression of the PEX16 gene in oleic acid- grown Y . lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

J Ind Microbiol Biotechnol, 1997 Jun, 18(6), 379 - 83
Derepressed utilization of L-malic acid and succinic acid by mutants of Pachysolen tannophilus; Harrod CJ et al.; Utilization of the tricarboxylic acid (TCA) cycle intermediates, L-malic acid and succinic acid, by the yeast Pachysolen tannophilus is repressed in the presence of glucose . Strains of P . tannophilus containing mutations in two hexokinases and a glucokinase were characterized for growth on glucose plus L-malic acid or succinic acid . Increased specific utilization rates of malic acid and succinic acid in the presence of glucose were observed in mutants containing a lesion in hexokinase A, an enzyme associated with catabolite repression . Such derepressed mutants may have application in winemaking in which utilization of a major grape acid, L-malic acid, is often desirable for acidity reduction.

J Membr Biol, 1997 May 15, 157(2), 105 - 15
Protein transport from the cytoplasm into the vacuole; Klionsky DJ; The fungal vacuole is integrally involved in various cellular processes that include protein and organellar degradation and recycling . The ability to sequester numerous hydrolases within the cell makes the hydrolytic capacity of the vacuole critical under certain environmental conditions . Accordingly, cellular constituents destined for degradation are delivered to the vacuole through the secretory pathway, by endocytosis and from the cytoplasm . Different mechanisms have evolved to accommodate these multiple transport pathways . Protein transport from the cytoplasm into the vacuole in particular relies on the dynamic nature of the vacuole membrane . This review describes recent research on this topic from yeast systems and points out the direction of future studies aimed at understanding this complex organelle.

Antonie Van Leeuwenhoek, 1997 May, 71(4), 325 - 8
Lipomyces mesembrius sp . nov., a member of the L . starkeyi species-complex; van der Walt JP et al.; Lipomyces starkeyi is known to be associated with three strains-clusters showing high mutual nDNA reassociation within each cluster, but which reassociate ambiguously with the type of L . starkeyi . Representative strains of L . starkeyi and Cluster alpha were examined for possible genetic exchange by the prototrophic selection technique . Since no genetic recombination was detected, the strains are presumed to be genetically isolated . Cluster alpha is consequently assigned to the rank of species as Lipomyces mesembrius . A description of the new species is given . Lipomyces kononenkoae ssp . spencermartinsiae has been raised to the rank of species as L . spencermartinsiae.

Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 114 - 7
Enolases are highly conserved fungal allergens; Breitenbach M et al.; BACKGROUND: Lack of knowledge of the identity of fungal allergens still is a major obstacle for improvement of diagnosis and therapy of allergies to moulds . We have therefore further analyzed the allergens of the two moulds, Alternaria alternata and Cladosporium herbarum and found that enolases (EC 4.2.1.11) are major allergens, at least of the two fungal species just mentioned . METHODS: The enolases of Alternaria and Cladosporium were cloned from cDNA libraries constructed from vegetative cells of the two moulds by immunological screening with sera from selected patients allergic to the moulds . The two enolases were expressed as recombinant nonfusion proteins and used for determination of the incidence of allergy to enolase among a cohort of patients . RESULTS: Sequencing of the two enolases showed very close relationships with other known fungal enolase sequences . Competition experiments using immunoblots of the recombinant nonfusion proteins showed nearly complete identity of the epitopes on both enolases . Serum from a patient reactive to Cladosporium enolase reacted equally well with the enolases of Alternaria, Saccharomyces and Candida . About 50% each of the sera from patients reactive to Cladosporium and Alternaria were strongly reactive to the recombinant enolases . CONCLUSIONS: Enolases are therefore considered to be highly conserved major fungal allergens.

J Biochem (Tokyo), 1997 Apr, 121(4), 690 - 5
Amino acid sequence and chemical modification of a novel alpha-neurotoxin (Oh-5) from king cobra (Ophiophagus hannah) venom; Lin SR et al.; A novel alpha-neurotoxin, Oh-5, was isolated from king cobra (Ophiophagus hannah) venom and purified by successive SP-Sephadex C-25 column chromatography and reversed-phase HPLC . The complete sequence of Oh-5 was determined by Edman degradation of peptide fragments generated by endopeptidases, i.e., trypsin, Saccharomyces aureus V8 protease and lysyl endopeptidase . This novel toxin comprises 72 amino acid residues with 10 cysteines . The sequence shows 89% sequence homology with Oh-4, and 60% with Toxins a and b from the same venom . The tyrosine, tryptophan, lysine and arginine residues in Oh-5 were modified with tetranitromethane (TNM), 2-nitrophenylsulfenyl (NPS) chloride, trinitrobenzene sulfonate (TNBS), and p-hydroxyphenylglyoxal (HPG), respectively . Modification of Tyr-4 or Trp-27 did not affect the lethal toxicity at all, while the Tyr-4 and 23 nitrated derivative retained about 50% of the lethality of native toxin . Selective trinitrophenylation of Lys-51 or 69 resulted in a decrease in lethality by 29%, and 50% lethality was retained after modification of Lys-2, 51, and 69 . A drastic decrease in lethality to 26% was observed when both Arg-35 and 37 were modified . The neurotoxicity was further decreased when Arg-9 was additionally modified . These results suggest that the aromatic residues, Tyr-4 and Trp-27, are not crucial for the neurotoxicity, whereas the cationic residues are involved in multipoint contact between the toxin molecule and the nicotinic acetylcholine receptor (nAChR) . The residues Tyr-23 and Arg-35 and 37 in the central loop of Oh-5 seem to contribute greatly to the neurotoxicity.

Nucleic Acids Res, 1997 Mar 1, 25(5), 965 - 73
Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes; Ladomery M et al.; In investigating the composition of stored (maternal) mRNP particles in Xenopus oocytes, attention has focussed primarily on the phosphoproteins pp60/56, which are Y-box proteins involved in a general packaging of mRNA . We now identify a third, abundant, integral component of stored mRNP particles, Xp54, which belongs to the family of DEAD-box RNA helicases . Xp54 was first detected by its ability to photocrosslink ATP . Subsequent sequence analysis identifies Xp54 as a member of a helicase subfamily which includes: human p54, encoded at a chromosomal breakpoint in the B-cell lymphoma cell line, RC-K8; Drosophila ME31B, encoded by a maternally-expressed gene, and Saccharomyces pombe Ste13, cloned by complementation of the sterility mutant ste13 . Expression studies reveal that the gene encoding Xp54 is transcribed maximally at early oogenesis: no transcripts are detected in adult tissues, other than ovary . Using a monospecific antibody raised against native Xp54, its presence in mRNP particles is confirmed by immunoblotting fractions bound to oligo(dT)-cellulose and separated by rate sedimentation and buoyant density . On isolating Xp54 from mRNP particles, it is shown to possess an ATP-dependent RNA helicase activity . Possible functions of Xp54 are discussed in relation to the assembly and utilization of mRNP particles.

Mol Gen Genet, 1997 Feb 27, 253(6), 703 - 10
Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphisms; Reyna-Lopez GE et al.; A modification of the amplified fragment length polymorphism technique was developed for the determination of DNA methylation in dimorphic fungi representative of three of the major fungal taxa: Mucor rouxii, a zygomycete; Yarrowia lipolytica, an ascomycete; and Ustilago maydis, a basidiomycete . DNA obtained from the yeast or mycelial stages of the fungi was digested with a mixture of EcoRI, and one of the isoschizomers MspI and HpaII, whose ability to cleave at the sequence CpCpGpG is affected by the methylation state . The resulting fragments were ligated to primers and subjected to a double round of amplification by the polymerase chain reaction, radiolabeled in the second round, and separated by polyacrylamide gel electrophoresis . Comparison of patterns revealed differences indicative of fragments whose methylation state did or did not change during the dimorphic transition . These results indicate the usefulness of the method for the study of DNA methylation, demonstrate the universality of DNA methylation in fungi, and confirm that differential DNA methylation occurs during fungal morphogenesis.

Eur J Biochem, 1997 Feb 15, 244(1), 220 - 5
Regulation and properties of a fungal lipase showing interfacial inactivation by gas bubbles, or droplets of lipid or fatty acid; Stahmann KP et al.; Ashbya gossypii can grow on triacyglycerol as carbon source . A degradation rate of 0.05 g x g-1 mycelial dry mass x h-1 was detected for soybean oil . Although this rate was within the sensitivity range of lipase assays no activity was detectable . On the other hand, extracellular lipase activity could be visualized by clearance halos round the growing mycelium when trioleoylglycerol was emulsified as the sole carbon source in agar plates . Variation of the culture conditions revealed that reduced shaking speed and decreased fat content in the medium led to detectable amounts of lipase in the supernatant of flask cultures . A maximal activity of 800 U x l-1 was obtained after 32 h of cultivation in flasks containing 1% yeast extract and incubated at 60 rpm . Because of its pI of 9.0, the enzyme could be purified in a single step by preparative isoelectric focusing . It appeared as a homogeneous protein in analytical isoelectric focusing and SDS/PAGE (M 35,000) . The lipase was inactivated within minutes in stirred gas/water, trioleoylglycerol/water or oleic acid/water mixtures . These effects suggested an interface inactivation . This idea was supported by a stability modulation observed with the surfactant Pluronic F-68 . Inactivation by oleic acid led to an aggregation of the lipase shown by gel filtration . Growth experiments performed under lipase-stabilizing conditions revealed a negative influence of glucose, glycerol or oleic acid on detectable lipase activity, probably due to a regulation of lipase formation . Inactivation and regulation thus explained the lack of detectable lipase activity in cultures of A . gossypii growing on triacylglycerol.

Lipids, 1997 Jan, 32(1), 73 - 8
Synthesis of a novel vitamin E derivative, 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol, by alpha-glucosidase-catalyzed transglycosylation; Murase H et al.; A novel derivative of vitamin E, vitamin E glucoside, was synthesized from 2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol and maltose in a solution containing DMSO by transglycosylation with alpha-glucosidase from Saccharomyces species . The glycosylated product was identified as 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG) by mass spectrometry and nuclear magnetic resonance spectroscopy . The optimal pH of transglycosylation was 5.5, and the yield of TMG increased as the concentration of maltose increased . TMG has high solubility in water (> 1 x 10(3) mg/mL) . The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of TMG was found to be nearly the same as those of alpha-tocopherol, Trolox (2-carboxy-2,5,7,8-tetramethylchroman-6-ol), and ascorbic acid.

Arch Biochem Biophys, 1996 Dec 1, 336(1), 49 - 58
Structure and antigenicity of the mannans of Candida famata and Candida saitoana: comparative study with the mannan of Candida guilliermondii; Shibata N et al.; The chemical structure of the mannans of antigenic factor 9-expressing yeast, Candida famata and Candida saitoana, was analyzed by acetolysis and NMR . The structural study of the oligosaccharides and mannans using one- and two-dimensional NMR indicated that the mannan of C . saitoana contains a third type of beta-1,2-linked mannose unit . On the other hand, the mannan of C . famata does not contain any beta-1,2-linked mannose units . The mannan of C . saitoana gave two groups of beta-1,2 linkage-containing oligosaccharides by acetolysis . One contains one beta-1,2-linked mannose unit and the other contains two consecutive beta-1,2-linked mannose units at the nonreducing terminal . The inhibition of the reactivity of factor 9 serum on an enzyme-linked immunosorbent assay (ELISA) with these oligosaccharides indicated that the inhibition activity of the former oligosaccharide is 1/20 of that of the latter ones . The ELISA of the mannans of Candida guilliermondii, C . saitoana, and Saccharomyces kluyveri, all of which contain the third type of beta-1,2-linked mannose unit, indicated that Man(beta)1--> 2Man(beta)1-->2Man(alpha)1-->3Man(alpha)1-->works as the antigenic factor 9 but Man(beta)1-->2Man(alpha)1-->3Man(alpha)1--> weakly behaves as both antigenic factors 6 and 9 . The epitope structures of the side chain oligosaccharides agreed well with that proposed from the 2D-HOHAHA spectra of the mannans . This result demonstrates the usefulness of the H-1 - H-2-correlated cross-peak pattern, which was reported in a preceding paper (Shibata, N., Akagi, R., Hosoya, T., Kawahara, K., Suzuki, A., Ikuta, K., Kobayashi, H., Hisamichi, K., Okawa, Y., and Suzuki, S . (1996) J . Biol . Chem . 271, 9259-9266) for the determination of the epitope structure of Candida mannans without any chemical fragmentation.

Mutat Res, 1996 Nov, 366(2), 129 - 35
The involvement of telomeric sequences in chromosomal aberrations; Bouffler SD et al.; Three functional elements are required for the stable transmission of eukaryotic chromosomes: replication origins, centromeres and telomeres . In the yeast Saccharomyces cerivisiae the DNA sequences defining each of these elements are known . The simplest and most widely conserved of these sequences is that of the telomere . As the name implies, the telomere is the end of a linear eukaryotic chromosome . Two of the main functions of the telomere are to prevent DNA loss as a consequence of replication and to prevent interactions with other chromosomal ends . Thus, telomeres play a major role in maintaining chromosome stability and consequently they have been considered as likely to be involved in some aspects of chromosomal aberration formation . The involvement of telomeric DNA sequences in stabilizing normal and broken chromosome ends, in "hot spots' for aberration formation and in delayed chromosomal instability will be reviewed here drawing on material presented at the Workshop and the published literature.

Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1686 - 9
Cloning of stress tolerance gene in Torulaspora delbrueckii No . 3110; Nakata K et al.; A cryptic plasmid (pSRY1) was found in Torulaspora delbrueckii No . 3110 . A plasmid vector (pSRY21) was constructed by ligating it to pPR1-RIM-C ORF with a cycloheximide resistance gene (RIM-C) . The transformation efficiency of the strain No . 3110 with pSRY21 by the protoplast-PEG method was increased by 3000-fold by treatment of the protoplasts with polylysine {poly(epsilon-L-lysine)} . Previously we have reported a mutant No . 3110, T1, which can neither assimilate nor accumulate trehalose . The mutant was salt- and temperature-sensitive . We cloned a DNA fragment from No . 3110, which, when introduced, complemented these mutant characters of T1 . These results suggested an important role of metabolism of trehalose for the stress tolerance.

Microbiology, 1996 Oct, 142 ( Pt 10), 2913 - 21
The extracellular acid protease gene of Yarrowia lipolytica: sequence and pH-regulated transcription; Young TW et al.; The gene encoding an acid extracellular protease (AXP) from Yarrowia lipolytica (Candida olea) 148 was cloned and the complete nucleotide sequence was determined . The amino acid sequence deduced from the nucleotide sequence reveals that the mature AXP consists of 353 amino acids with an M, of 37427 . The gene also encodes a putative 17 amino acid hydrophobic prepeptide and a 27 amino acid propeptide containing no potential N-glycosylation sites . The mature extracellular enzyme is produced by cleavage between Phe and Ala . AXP is a member of the aspartyl family of proteases . AXP shows homology to proteases of several fungal genera and to human progastricin . The coding sequence is preceded by a potential regulatory region of 1982 bp . Transcription of both AXP and alkaline extracellular protease genes of Y . lipolytica 148 is regulated by the pH of culture.

Biotechnol Appl Biochem, 1996 Oct, 24 ( Pt 2), 171 - 6
Comparative studies of recombinant human albumin and human serum albumin derived by blood fractionation; Dodsworth N et al.; Recombinant human albumin produced in the yeast Saccharomyces cerevisial and human serum albumin derived from blood fractionation were compared by a series of analytical techniques . These demonstrated that the two proteins were equivalent structurally . However, differences observed in some of the assays indicated that the recombinant product had lower levels of structural heterogeneity than the blood-derived protein.

J Biol Chem, 1996 Aug 23, 271(34), 20300 - 6
The Yarrowia lipolytica gene PAY5 encodes a peroxisomal integral membrane protein homologous to the mammalian peroxisome assembly factor PAF-1; Eitzen GA et al.; Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes . One mutant strain, pay5-1, lacks normal peroxisomes and instead contains irregular vesicular structures surrounded by multiple unit membranes . The pay5-1 mutant is not totally deficient in peroxisomal matrix protein targeting, as a subset of matrix proteins continues to localize to a subcellular fraction enriched for peroxisomes . The functionally complementing gene PAY5 encodes a protein, Pay5p, of 380 amino acids (41,720 Da) . Pay5p is a peroxisomal integral membrane protein homologous to mammalian PAF-1 proteins, which are essential for peroxisome assembly and whose mutation in humans results in Zellweger syndrome . Pay5p is targeted to mammalian peroxisomes, demonstrating the evolutionary conservation of the targeting mechanism for peroxisomal membrane proteins . Our results suggest that in pay5 mutants, normal peroxisome assembly is blocked, which leads to the accumulation of the membranous vesicular structures observed.

J Mol Evol, 1996 Jul, 43(1), 46 - 57
The evolution of the RNase P- and RNase MRP-associated RNAs: phylogenetic analysis and nucleotide substitution rate; Sbisa E et al.; We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs . The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs . For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences . RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock . Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny . However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals . The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains . The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments.

Transfus Med, 1996 Jun, 6(2), 125 - 31
Antibodies to human major histocompatibility complex products inhibit Fc gamma receptors types I and II; Neppert J et al.; We have analysed the effect of polyclonal and monoclonal antibodies of distinct IgG isotypes directed against products of the human major histocompatibility complex (MHC) on the function of Fc gamma receptors types I (Fc gamma RI) and II (Fc gamma RII) . Human anti-D sensitized red blood cells (RBC), selectively binding to Fc gamma RI, and bovine or murine IgG1 (bIgG1 or mIgG1) sensitized RBC, selectively binding to Fc gamma RII, were employed as targets for the effector cell in order to distinguish the inhibition of both types . Using these targets it could be shown that human antibodies to products of the human MHC or murine monoclonal antibodies with the same specificity and mIgG2a isotype inhibited both the Fc gamma RI-and the Fc gamma RII-mediated function . In contrast, murine monoclonal antibodies with the same specificity but of the mIgG1 isotype only inhibited the Fc gamma RII-mediated function . In a control experiment, human and murine antibodies to products of the MHC did not impair the Fc gamma R-independent phagocytosis of Saccharomyces by monocytes and neutrophils . The present study suggests that this mechanism involves at least two different Fc gamma receptors.

J Biochem (Tokyo), 1996 May, 119(5), 926 - 33
Sterol 14-demethylase P450 (P45014DM*) is one of the most ancient and conserved P450 species; Aoyama Y et al.; To determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51) . The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae) . All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available . The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7 alpha-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8 . These lines of evidence show that fungal and mammalian P45014DMs are really orthologous . This is the first example of orthologous P450s occurring in distinct kingdoms . P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution . The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes . In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells . These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.

Genomics, 1996 May 1, 33(3), 389 - 408
Assembly of high-resolution restriction maps based on multiple complete digests of a redundant set of overlapping clones; Gillett W et al.; An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented . Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme . Synchronization between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible . Basic concepts that underlie multiple-complete-digest mapping--including the match/merge approach to map incorporation, extension vs assimilation, ambiguity, and clone-end compatibility--are presented . An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects.

J Biol Chem, 1996 Apr 19, 271(16), 9259 - 66
Existence of novel branched side chains containing beta-1,2 and alpha-1,6 linkages corresponding to antigenic factor 9 in the mannan of Candida guilliermondii; Shibata N et al.; Isolation of beta-linkage-containing side chain oligosaccharides from the mannan of Candida guilliermondii IFO 10279 strain has been conducted by acetolysis under mild conditions . A structural study of these oligosaccharides by one- and two-dimensional NMR and methylation analyses indicated the presence of extended oligosaccharide side chains with two consecutive beta-1,2-linked mannose units at the nonreducing terminal of alpha-linked oligosaccharides . The linkage sequence present in this mannan, Man beta 1-->2Man alpha 1-->3Man alpha-->, has also been found in the mannan of Saccharomyces kluyveri but not in the mannan of Candida species . Furthermore, these oligosaccharides are branched at position 6 of the 3-O-substituted mannose units as follows . (Carbohydrate sequence in text) Structure 1 and (Carbohydrate sequence in text) Structure 2 The H-1 signals of the mannose units substituted by a 3,6-di-O-substituted unit showed a significant upfield shift (delta delta = 0.04-0.08 ppm) due to a steric effect . The inhibition of an enzyme-linked immunosorbent assay between the mannan of C . guilliermondii and factor 9 serum with oligosaccharides obtained from several mannans indicated that only the oligosaccharides with the above structure were active, suggesting that these correspond to the epitope of antigenic factor 9.

Yeast, 1996 Mar 15, 12(3), 237 - 40
Saccharomycodes ludwigii has seven chromosomes; Yamazaki T et al.; Tetrad distributions for 108 different gene pairs in 1346 asci of 113 diploids heterozygous for various combinations of 24 genes in Saccharomycodes ludwigii were investigated . Tetratype tetrads occurred only rarely and the 24 genes tested were classified into seven linkage groups . Electrophoretic karyotypes of three independent strains of S'codes ludwigii showed seven bands of chromosome-sized DNA having molecular sizes of 0 center dot 8 to 2 center dot 3 Mb with strain-specific polymorphic chromosomal DNAs as determined based on their migration distances.

Z Ernahrungswiss, 1996 Mar, 35(1), 39 - 44
Studies on date waste dietary fibers as hypolipidemic agent in rats; Jwanny EW et al.; Date waste dietary fibers were examined as a hypolipidemic agent . White albino rats were fed on three experimental diets: I) high carbohydrate diet free of fiber; II) and III) diets consisted of diet I substituted with 100 g/kg of date waste dietary fibers cultured with Endomycopsis fibuligera at zero time and after 60 h of culturing respectively for 8 weeks . The total lipids, total cholesterol, triglycerides and phospholipids in the liver of rats given diets II and III were significantly decreased over those rats fed the control diet throughout the feeding period (8 weeks) . The highest decrease in content of all these parameters was produced by diet III . Comparing diets II and III with the control diet I, total serum lipids and low density lipoprotein cholesterol (LDL-cholesterol) were decreased by 32-48%, while serum triglycerides and total cholesterol levels were lowered in the groups fed diets II and III by 23-35% respectively . Concerning high density lipoprotein cholesterol (HDL-cholesterol), the decrease was only 2-6% in rats fed diets II and III . The highest decrease level was shown in the phospholipids content (51-56%) during all of the experimental period (8 weeks).

Plant Foods Hum Nutr, 1996 Feb, 49(2), 113 - 7
Influence of sundrying on the chemical composition, aflatoxin content and fungal counts of two pepper varieties--Capsicum annum and Capsicum frutescens; Adegoke GO et al.; Samples of sundried, matured red pepper, Capsicum annum with a moisture content (MC) of 12.7-26.8 percent had on dry weight basis, vitamin C, 5.0-6.4 mg/100 g; crude protein, 0.8-1.2 percent; total soluble solids, 3.3-4.1 percent, and fungal counts of log 4.4-4.5/g . Ordinary matured red C . annum had MC, 75.7-78.2 percent vitamin C, 36.1-38.5 mg/100 g; crude protein, 2.4-2.8 percent; total soluble solids, 9.3-9.9 percent and fungal count of log 3.32-3.39/g . Sundried matured red C . frutescens had corresponding values of 9.4-18.7 percent; 5.8-6.3 mg/100 g; 0.8-1.1 percent; 0.9-2.6 percent and log 3.2-3.4/g . No aflatoxins were detected in sundried, matured red C . frutescens, but aflatoxin B1 values obtained from C . annum varied from non-detectable to 2.2 micrograms/kg . Dominant fungi isolated from C . annum and C . frutescens were Rhizopus oryzaze, Aspergillus niger, A . flavus, Geotrichum candidum and Saccharomyces spp.

Chromosome Res, 1996 Feb, 4(2), 133 - 40
The synaptonemal complex--the chaperone of crossing over; Hasenkampf CA; During meiosis homologous chromosomes pair and exchange homologous chromosome segments . The synaptonemal complex (SC) forms between paired chromosomes . The role of the SC in the process of reciprocal exchange of flanking markers is a matter of debate . I propose a dual pathway for reciprocal exchange of flanking markers (REFM) . In the first, SC-independent, path, two 'half-nodules' and an independent REFM protein combine to form a functional recombination nodule (RN) . The RN binds to paired chromosomes and accomplishes reciprocal exchange of flanking markers . In the other, SC-dependent, pathway 'half-nodules' occur at pairing initiation sites . 'Half-nodules' move along the SC as it forms . Assisted by an SC-bound REFM protein, 'half-nodules' combine to form functional RNs . I propose that different organisms rely to different extents on the two pathways, and hence rely to different extents on the SC.

Biochimie, 1996, 78(6), 466 - 73
Function of a pseudoknot in the suppression of an alternative splicing event in a group I intron; Jaeger L et al.; Like most mitochondrial group I introns with a free-standing open reading frame (ORF) located downstream of their catalytic core, the Sd.cob, 1 intron in the gene coding for the cytochrome b of Saccharomyces douglasii mitochondria possesses a putative proximal 3' splice site . However, incubation of Sd.cob, 1 preRNA transcripts under optimal in vitro splicing conditions essentially results in splicing at the authentic, distal 3' splice junction . The mechanism by which the proximal splicing event is suppressed in vitro involves formation of a tertiary interaction which is only found in the Sd.cob, 1 intron . Core nucleotides located in loop L5 block proximal splicing by forming Watson-Crick base pairs with the nucleotide sequence of the proximal 3' splice site . This tertiary base pairing, also important for the folding of the intron into an active conformation, may be regarded as equivalent to the L9/P5, GNRA-loop/helix interaction found in more than one-third of known group I introns.

Curr Genet, 1995 Dec, 29(1), 81 - 7
Electrophoretic karyotype of Dipodascus (Endomyces) magnusii: two main intraspecific chromosomal polymorphisms associated with the difference in total genome size; Filipp D et al.; This study describes the karyotype of strain 270 of the yeast-like fungus Endomyces magnusii . It consists of 13 chromosomal DNA molecules, the size of which range between 1.2 and 5.7Mb producing a genome size of approximately 38Mb . By comparing the karyotype of six strains of E . magnusii, we revealed two main chromosome length polymorphisms (CLPs) associated with a pronounced difference in the total genome size (roughly 50%) . Karyotype heterogeneity between two main CLPs was demonstrated by Southern analysis with three heterologous probes . The same species affiliation of six E . magnusii strains was confirmed by morphological and cytological studies, protein fingerprint comparisons, as well as restriction analysis of mitochondrial DNA and genomic Southern analysis.

Curr Genet, 1995 Nov, 28(6), 534 - 45
DNA polymerase delta of Physarum polycephalum; Achhammer G et al.; DNA polymerase delta from the phylogenetically ancient slime mold Physarum polycephalum has been 380-fold enriched from amoebae . It was found to have the properties typical for this type of DNA polymerase from higher eukaryotes with regard to effectors, template-primer acceptance, co-purification with 3'-5'-exonuclease activity, as well as the effect of endogenous proliferating cell nuclear antigen (PCNA) from amoebae on the stimulation and processivity of DNA synthesis . An identified cDNA fragment shows 65.5% identical amino acides with DNA polymerase delta from Saccharomyces pombe . The molecular mass of the polymerase is 125 kDa while that of PCNA is 35 kDa . During size-exclusion chromatography, the highly purified polymerase eluted in the position of 125 kDa, suggesting that no other proteins were tightly complexed with the enzyme . The DNA polymerases from the (mononucleate) amoebae and from the (multinucleate) plasmodia of P . polycephalum have very similar properties in contrast to their differences in phenotype and their mode of nuclear division . The polymerase shows a higher degree of similarity than DNA polymerase alpha, and especially the beta-like DNA polymerase, with the corresponding polymerases of higher eukaryotes . According to antibody staining, DNA polymerase delta is readily fragmented by proteases, even in the presence of inhibitor cocktails . Including freshly prepared cell lysates, proteolytic fragments are reproducible, the most abundant being 50 kDa in size . The DNA polymerase is recognized by the antisera against two peptides which have been derived by PCR-screening of plasmodial cDNA . One of the proteolytic splitting sites is located within an eight amino-acid stretch between the two antigenic sequences.

Development, 1995 Oct, 121(10), 3477 - 86
The pelota locus encodes a protein required for meiotic cell division: an analysis of G2/M arrest in Drosophila spermatogenesis; Eberhart CG et al.; During Drosophila spermatogenesis, germ cells undergo four rounds of mitosis, an extended premeiotic G2 phase and two meiotic divisions . In males homozygous for mutations in pelota, the germline mitotic divisions are normal, but the cell cycle arrests prior to the first meiotic division; pelota males are therefore sterile . Chromosomes begin to condense in these mutants, but other meiotic processes, including nuclear envelope breakdown and spindle formation, do not occur . The arrest phenotype closely resembles that of mutations in the Drosophila cdc25 homolog twine . Although meiosis is blocked in pelota and twine homozygotes, spermatid differentiation continues . pelota is also required for patterning in the eye and mitotic divisions in the ovary . We have cloned the pelota locus and show it encodes a 44 x 10(3) M(r) protein with yeast, plant, worm and human homologs.

Zhonghua Yi Xue Za Zhi, 1995 Sep, 75(9), 552 - 6, 575-6
{Clinical significance of fungi-bearing status of hospitalized patients}; Zheng Y et al.; The samples for fungi culture were collected from 11 body parts of 1109 patients who had been admitted to hospitals in Wuhan for more than 3 days . The body parts included the cavities and foramina (inner canthus, nasal vestibule, external auditory canal), skin (finger raphe, cubital fossa, axillary fossa, nipple), and mucosa (pharynx, vagina, coronary sulcus and anal canal) . 201 healthy subjects were also examined and served as controls . It was found that the total fungus-bearing rate in hospitalized patients was 88.73% (984/1109), while the rate in healthy subject was 76.62% (154/201) (X2 = 21.61) . Among the hospitalized patients, the fungus-bearing rate in patients with leukemia, tumors, connective tissue diseases or severe infectious diseases was significantly higher than other patients (X2 = 4.30-9.87) . In the hospitalized patients, the body parts with highest fungus-bearing rates were cavities and foramina, 80.70% (895/1109) for patients and 71.64% (144/201) for healthy subjects respectively . The skin had lowest fungus-bearing rate . 20.02% (222/1109) and 13.43% (26/201), and the mucosa had a rate of 50.77% (563/1109) for patients and 32.34% (65/201) respectively . The fungi in the hospitalized patients included 1884 strains (mean = 1.92 strains per case) in which the Candida accounted for 29.78% (561/1884) and Aspergilla 24.31% (458/1884) and those identified in healthy subjects covered 234 strains (mean = 1.52 strains per person) in which Penicillia accounted for 45.30% (106/234) and Saccharomycetes 26.07% (61/234) . The patients who had been hospitalized for 3-10 days and over 20 days had higher fungus-bearing rate as compared with those who had been hospitalized for 10 to 20 days (X2 = 5.633-97.09) . The patients before the administration of antibiotics and adrenocortical steroid and those who had been given these medicines for over 20 days had a higher fungus-bearing rate than those who had been on these medicines for 1-20 days.

Mikrobiologiia, 1995 Sep-Oct, 64(5), 705 - 13
{Determining the concentration of eukaryotic cells by their integral fluorescence}; Poglazova MN; A proportional relationship was established between the total luminescence intensity of eukaryotic cells trapped on membrane filters and stained with fluorescent dyes and the number of cells present on the specimen area corresponding to the size of the cytofluorometric unit probe . An integral fluorescent method was developed that allows determination of the concentration of somatic cells in milk samples with the help of a calibration curve constructed on the basis of counting yeast cells . The method allows determination of the cell concentration in a 1 x 10(5)-5 x 10(6) cells/ml range . The results obtained using the integral fluorescent method are compared with data of direct visual count and with readings of the automatic FOSSOMATIK device.

Biosci Biotechnol Biochem, 1995 Sep, 59(9), 1732 - 6
Synthesis of 2-deoxy-glucooligosaccharides through condensation of 2-deoxy-D-glucose by glucoamylase and alpha-glucosidase; Nakano H et al.; Glucoamylases from Aspergillus niger and Rhizopus niveus catalyzed condensation of 2-deoxy-D-glucose (dGlc) to yield deoxy-glucooligosaccharides with polymerization degrees of 2-5 . The enzymes also gave a small amount of products from 3-deoxy-D-glucose, but no products from 6-deoxy-D-glucose . A . niger alpha-glucosidase also catalyzed condensation of dGlc, while Torula and Saccharomyces alpha-glucosidases had low activity . alpha-1,4-, 1,6-, and 1,3-linked deoxy-glucobioses were isolated and identified as the products of A . niger glucoamylase and A . niger alpha-glucosidase . In the reaction of the glucoamylase, 1,4- and 1,3-linked saccharides decreased with an increase of 1,6-linked one . A . niger alpha-glucosidase produced alpha-1,6-linked disaccharide predominantly during the whole course of the reaction.

Ann Thorac Surg, 1995 Sep, 60(3), 538 - 43
Surgical and long-term antifungal therapy for fungal prosthetic valve endocarditis; Muehrcke DD et al.; BACKGROUND . Fungal prosthetic valve endocarditis is an uncommon but serious disease . We have developed a strategy of treatment that includes perioperative amphotericin B, radical debridement of infected tissue, reconstruction using biologic tissue when possible, and prolonged oral suppressive antifungal therapy . METHODS . We retrospectively reviewed the charts of 12 patients reoperated on for fungal prosthetic valve endocarditis involving the aortic valve (10 patients: six porcine valves, two mechanical valves, two homografts) and the mitral valve (2 patients, both porcine valves) . Prosthetic valve endocarditis developed in 7 within 12 months after the first valve procedure . The organisms included Candida species (9 patients), Scopulariopsis brevicaulis (1), Saccharomyces cervisiae (1), and histoplasmosis (1) . RESULTS . At operation, all patients had prosthetic vegetations, 8 had abscesses, and 4 had sinus tracts . Seven received aortic homografts, 4 received porcine valves (two mitral), and 1 received a mechanical prosthesis . Two patients died in the hospital after prolonged illnesses (83% hospital survival) . Four patients had recurrence an average of 25 months later and 3 underwent further surgical intervention . One patient had recurrence and died 17 months postoperatively . One other late death occurred 96 months after operation, and there was no evidence of recurrence . Eight patients (67%) are alive and well 51.5 +/- 61.0 months (range, 1 to 189 months) after the first redo procedure for fungal prosthetic valve endocarditis . CONCLUSIONS . We conclude that preoperative treatment with amphotericin B, radical resection of all infected tissue, cardiac reconstruction using biologic tissue when possible, and life-long oral antifungal therapy is effective for fungal prosthetic valve endocarditis.

Development, 1995 Sep, 121(9), 3035 - 43
Expression of the cell cycle control gene, cdc25, is constitutive in the segmental founder cells but is cell-cycle-regulated in the micromeres of leech embryos; Bissen ST; The identifiable cells of leech embryos exhibit characteristic differences in the timing of cell division . To elucidate the mechanisms underlying these cell-specific differences in cell cycle timing, the leech cdc25 gene was isolated because Cdc25 phosphatase regulates the asynchronous cell divisions of postblastoderm Drosophila embryos . Examination of the distribution of cdc25 RNA and the zygotic expression of cdc25 in identified cells of leech embryos revealed lineage-dependent mechanisms of regulation . The early blastomeres, macromeres and teloblasts have steady levels of maternal cdc25 RNA throughout their cell cycles . The levels of cdc25 RNA remain constant throughout the cell cycles of the segmental founder cells, but the majority of these transcripts are zygotically produced . Cdc25 RNA levels fluctuate during the cell cycles of the micromeres . The levels peak during early G2, due to a burst of zygotic transcription, and then decline as the cell cycles progress . These data suggest that cells of different lineages employ different strategies of cell cycle control.

Plant Mol Biol, 1995 Sep, 28(6), 1011 - 25
Characterization of a family of genes encoding a fruit-specific wound-stimulated protein of bell pepper (Capsicum annuum): identification of a new family of transposable elements; Pozueta-Romero J et al.; Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome . Both genes have a single intron and numerous unusual long inverted repeat sequences . The introns share 87% homology and Sn-2 contains one 450 bp additional sequence with structural features of a transposable element, which is highly repetitive in the bell pepper genome . Surprisingly, analysis in data banks showed that genes encoding the potato starch phosphorylase (EC 2.4.1.1) and patatin contain a similar element, named Alien, in their 5'-upstream region . Alien elements are characterized by a conserved 28 bp terminal inverted repeat (TIR), small size, high AT content, potential to form stable DNA secondary structures and they have probably been inserted in TA target sites . Interestingly, the TIR of the Alien elements shares high homology with sequences existing in the TIR of extrachromosomal linear pSKL DNA plasmid of Saccharomyces kluyveri . Northern blot analyses detected Sn-1 transcripts principally in the red fruit whereas no Sn-2 transcripts were detected in neither of the samples monitored . Western blot analyses detected a 16.8 kDa Sn protein principally in the ripe red fruit and wounded areas of green unripe fruit . A comparison of the deduced amino acid sequence of Sn-1 with protein sequences in data banks revealed a significant homology with proteins likely involved in the plant's disease resistance response . Analyses at the subcellular level showed that Sn-1 is localized in the membrane of vacuoles.

Antonie Van Leeuwenhoek, 1995 Jul, 68(1), 75 - 87
Species delimitation in the genus Lipomyces by nuclear genome comparison; Smith MT et al.; Species delimitation in Lipomyces was attempted by nuclear genome comparison in conjunction with the re-evaluation of 48 physiological characters of 65 strains . High intraspecific (> 75%) and low interspecific (< 28%) similarity values established that L . japonicus, L . lipofer and L . tetrasporus are genetically isolated, and also distinct from L . kononenkoae and L . starkeyi . Ambiguous similarity values were obtained with L . kononenkoae and L . starkeyi . Strains previously assigned to L . kononenkoae constitute two related clusters . While similarity values within each cluster range from 76-99%, representatives of the two clusters reassociate for only 47% . Since these clusters are differentiated by their ecologically relevant maximum growth temperature, L . kononenkoae is subdivided . Strains previously assigned to L . starkeyi resolve into four closely related clusters . While similarity values within each cluster range from 78-100%, representatives of the four clusters reassociate for only 59-69% . Since these four clusters are poorly differentiated, the subdivision of L . starkeyi does not appear possible without recourse to other criteria . Four unassigned strains constitute a further two clusters . Reassociation within these clusters is of the order of 91-100%, while reassociation between them occurs only at 59% . Reassociation of representatives of these clusters with those of the L . kononenkoae and L . starkeyi complexes is around 40% and 31%, respectively . These two clusters consequently appear to be intermediate between L . kononenkoae and L . starkeyi, and will, as such, have to be considered in any delimitation of these two species . A key to the taxa of Lipomyces and related genera of the Lipomycetaceae is given.

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