Curr Opin Chem Biol, 2004 Feb, 8(1), 49 - 53 Protein localization in proteomics; Davis TN; A global analysis of the localization of 4156 yeast proteins has just been accomplished . Smaller scale analyses have been performed in a variety of organisms . These studies typically use green fluorescent protein as a tag for proteins in living cells . Improvements in the yellow and sapphire color variants will increase their utility . Reengineering of the red fluorescent protein has produced faster maturing tetrameric and monomeric variants not prone to aggregation . Techniques for high-throughput tagging of proteins include integration by homologous recombination, integration using mobile elements or recombinational cloning to produce plasmids expressing fusion proteins . Alternatives to localizing tagged proteins are to use antibodies or aptamers to detect the untagged protein. Inflammopharmacology, 2003, 11(2), 127 - 33 Interactions of D-amphetamine with the active site of monoamine oxidase-A; Ramsay RR et al.; Reversible monoamine oxidase A inhibitors (RIMA) are used as antidepressants but little is known about how they interact with the active site of the enzyme . Heterologous expression of human liver MAO-A in yeast provides sufficient protein for molecular studies and direct observation of the changes in the spectrum of the FAD co-factor when inhibitors bind . Using the reversible inhibitor, D-amphetamine, as a model compound, a concentration-dependent change in the spectrum with clean isosbestic points was observed . The decrease in absorbance between 400 and 500 nm gave a dissociation constant for binding similar to the K(i) value . Anaerobic reduction yielded the semiquinone spectrum only and the midpoint potential was the same as the free enzyme . Full reduction was not possible with dithionite as the reductant, suggesting that the semiquinone-reduced couple had a much lower midpoint potential than the free enzyme . In contrast, with substrate, which reduces the enzyme on an equimolar basis, the semiquinone is never seen . In anaerobic stopped-flow experiments, amphetamine inhibits completely the reoxidation of the reduced enzyme in contrast to a substrate such as 2-phenylethylamine (the desmethyl analogue of amphetamine) that accelerates the rate 12-fold . The spectral changes in MAO-A permit the examination of inhibitor interaction with the redox co-factor . Stacking of the inhibitor and flavin rings constitutes part of the interaction but, taking into account other evidence, steric factors may be the clue to the differences between substrate and inhibitor. Mycol Res, 2004 Jan, 108(Pt 1), 93 - 100 Production and processing of Metarhizium anisopliae var . acridum submerged conidia for locust and grasshopper control; Kassa A et al.; Currently, mycopesticide development for locust and grasshopper control depends on aerial conidia or submerged spores of entomopathogenic fungi . In our study, the production of submerged conidia of Metarhizium anisopliae var . acridum (IMI 330189) was investigated in a liquid medium containing 3% biomalt and 1% yeast extract (BH-medium) . The effects of freeze and spray drying techniques on the quality of submerged conidia were determined . The influence of different additives on the viability of fresh submerged conidia and their suitability for oil flowable concentrate formulation development was assessed . In a BH medium maintained at 180 rev min(-1), at 30 degrees C for 72 h, IMI 330189 produced a green pigmented biomass of submerged conidia whereas in Adamek medium it produced a yellowish biomass of submerged spores . The spore concentration was high in both media; however, the size of the spores produced in the BH medium was significantly lower than those produced in Adamek medium (P < 0.001) . Submerged conidia can be effectively dried using either freeze or spray drying techniques . The viability and speed of germination were significantly affected by the drying and pulverizing process (P < 0.001) . The initial viability was significantly higher for spray-dried submerged conidia than for freeze-dried spores . Pulverizing of freeze-dried submerged conidia reduced the speed of germination and the viability by 63-95% . Dried submerged conidia can be stored over 45 wk at low temperatures (< 10 degrees) without suffering a significant loss in viability . Furthermore, we have identified carriers that are suitable for oil flowable concentrate formulation development. J Cell Biochem, 2004 Apr 1, 91(5), 1030 - 42 Characterizing the new transcription regulator protein p60TRP; Heese K et al.; Active cell death ('apoptosis' or 'programmed cell death') is essential in the development and homeostasis of multicellular organisms and abnormal inhibition of apoptosis is an indicator of cancer and autoimmune diseases, whereas excessive cell death might be implicated in neurodegenerative disorders such as Alzheimer's disease (AD) . Using bioinformatics-, Western-blotting-, yeast-two-hybrid-system-, polymerase chain reaction (PCR)-, and fluorescence microscopy-analyses, we demonstrate here that the neuroprotective protein p60TRP (p60-transcription-regulator-protein) is a basic helix-loop-helix (bHLH) domain-containing member of a new protein family that interacts with the Ran-binding-protein-5 (RanBP5) and the protein-phosphatase-2A (PP2A) . The additional findings of its influence on NNT1 and p48ZnF (new-neurotrophin-1, p48-zinc-finger-protein)-signaling and its down-regulation in the brain of AD subjects point to a possible pivotal role of p60TRP in the control of cellular aging and survival . Planta, 2004 Jun, 219(2), 359 - 68 Epub 2004 Mar 19. Regulation and a conserved intron sequence of liguleless3/4 knox class-I homeobox genes in grasses; Bauer P et al.; The nine class-I maize (Zea mays L.) knox genes are putative transcription factors normally expressed in shoot apices, but not in leaves . knotted1 (kn1) seems to function in shoot apical meristem maintenance, and rough sheath1 (rs1)-like genes may act in internode elongation . The function of liguleless3 (lg3)-type genes is still unknown . Here, we characterized lg3 as well as the two most closely related genes liguleless4a (lg4a, formerly knox11) and liguleless4b (lg4b, formerly knox5) . We termed this subclass of knox genes lg3/4 genes . We studied the expression patterns of lg3/4 genes and compared their sequences . We obtained knockout mutants of lg3 by finding Mu transposon insertions into exons . Our results show that lg3 was not essential for plant development, and that lg4a and lg4b were likely to encode the redundant function . In addition, lg4a but not lg4b was ectopically expressed in the Lg4-O mutant, suggesting that this mutant was affected at the lg4a locus . We found that the lg3 gene was unique among knox genes as it was co-induced in the leaves of leaf mutants that ectopically expressed knox genes in the leaves . The leaf phenotype expressed in the dominant Rs1-O mutant was not altered when lg3 function was removed using the knockout . Genomic sequence comparisons of lg3, lg4a and lg4b from maize and the two homologous genes, osh6 and osh71, from rice revealed a 14-bp phylogenetic footprint in intron II . This sequence was conserved in nucleotide composition, position and polarity in the lg3/4 genes of divergent grasses representing six Gramineae subfamilies . In an independent experiment, this same conserved sequence was found in a yeast reverse one-hybrid screen for putative binding sites of the LG3 homeodomain protein . Distribution of this 14-bp sequence was examined within the public rice database . The possible function of this sequence in regulation of lg3/4 genes is discussed . Mol Biol Cell, 2004 Jun, 15(6), 2664 - 73 Epub 2004 Mar 19. Tissue-specific expression and dynamic organization of SR splicing factors in Arabidopsis; Fang Y et al.; The organization of the pre-mRNA splicing machinery has been extensively studied in mammalian and yeast cells and far less is known in living plant cells and different cell types of an intact organism . Here, we report on the expression, organization, and dynamics of pre-mRNA splicing factors (SR33, SR1/atSRp34, and atSRp30) under control of their endogenous promoters in Arabidopsis . Distinct tissue-specific expression patterns were observed, and differences in the distribution of these proteins within nuclei of different cell types were identified . These factors localized in a cell type-dependent speckled pattern as well as being diffusely distributed throughout the nucleoplasm . Electron microscopic analysis has revealed that these speckles correspond to interchromatin granule clusters . Time-lapse microscopy revealed that speckles move within a constrained nuclear space, and their organization is altered during the cell cycle . Fluorescence recovery after photobleaching analysis revealed a rapid exchange rate of splicing factors in nuclear speckles . The dynamic organization of plant speckles is closely related to the transcriptional activity of the cells . The organization and dynamic behavior of speckles in Arabidopsis cell nuclei provides significant insight into understanding the functional compartmentalization of the nucleus and its relationship to chromatin organization within various cell types of a single organism. Ann N Y Acad Sci, 2003 Dec, 1010, 433 - 6 A novel gene, Jpk, induces apoptosis in F9 murine teratocarcinoma cell through ROS generation; Kong KA et al.; A novel gene Jpk (Jopock) has been originally isolated through yeast 1 hybridization technique as a trans-acting factor interacting with the position-specific regulatory element of a murine Hoxa-7 . Northern analysis revealed that the Jpk was expressed at day 7.0 post coitum (p.c.) during early gastrulation . Previously it has been shown that a trace amount of JPK protein led bacterial cells to death . In eukaryotic F9 cells, Jpk also led the cell to death-generating DNA ladder: fewer than 50% of the cells survived after 72-h transfection . Flow cytometric analysis with cells stained with each Annexin V/7-amino-actinomycin D (7-AAD), MitoTracker, and hydroethidine (HE) revealed that Jpk induced apoptotic cell death in a time-dependent manner, reduced mitochondrial membrane potential, and increased ROS (reactive oxygen species) production, respectively . Additionally, Jpk seemed to regulate the Bcl family at the transcriptional level when RT-PCR was performed . Although the precise mechanism is not clear, these results altogether suggest that Jpk is a potent inducer of apoptosis through generation of ROS as well as concomitant reduction of mitochondrial membrane potential. Biochem Biophys Res Commun, 2004 Apr 9, 316(3), 827 - 33 Physical and functional interactions between Daxx and TSG101; Muromoto R et al.; Daxx has been reported to mediate the Fas/JNK-dependent signals in the cytoplasm . However, several evidences have suggested that Daxx is located mainly in the nucleus and functions as a transcriptional regulator . Recently, we identified DMAP1, a TSG101-interacting protein as a Daxx binding partner by yeast two-hybrid screening . TSG101 has been shown to act as transcriptional co-repressor of nuclear hormone receptors . Here we examined whether TSG101also interacts with Daxx directly . The association of Daxx and TSG101 was confirmed using co-expressed tagged proteins . The interaction regions in both proteins were also mapped, and the cellular localization of the interaction was examined . TSG101 formed a complex with Daxx through its coiled-coil domain and co-localized in the nucleus . Furthermore, TSG101 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity . These results provide the novel molecular interactions between Daxx and TSG101, which establish an efficient repressive transcription complex in the nucleus. Mol Cell Neurosci, 2004 Mar, 25(3), 469 - 79 Potentiation of NMDA receptor-mediated excitotoxicity linked with intrinsic apoptotic pathway in YAC transgenic mouse model of Huntington's disease; Zeron MM et al.; Evidence suggests N-methyl-D-aspartate receptor (NMDAR) activation is involved in the degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD) . We tested the hypothesis that enhanced NMDAR-mediated excitotoxicity is mediated by the mitochondrial-associated apoptotic pathway in cultured MSNs from YAC transgenic mice expressing full-length huntingtin (htt) with a polyglutamine (polyQ) expansion of 46 or 72 (YAC46 or YAC72) . NMDAR-mediated Ca(2+) transients and mitochondrial membrane depolarization were significantly increased in YAC compared to wild-type mice MSNs . Inhibitors of the mitochondrial permeability transition (mPT), cyclosporin A and bongkrekic acid, and coenzyme Q10 (an anti-oxidant involved in bioenergetic metabolism) dramatically diminished NMDA-induced cell death and eliminated genotypic differences . In YAC46 MSNs, NMDA stimulated significantly higher activation of caspase-3 and caspase-9 but not caspase-8, and NMDA-induced caspase-3 and -9 activation was markedly attenuated by cyclosporin A . Agents that improve mitochondrial function or inhibit the permeability transition may eliminate increased caspase activation and cell death associated with enhanced NMDAR activity in HD. Curr Med Chem, 2004 Mar, 11(5), 607 - 28 The Catharanthus alkaloids: pharmacognosy and biotechnology; van Der Heijden R et al.; The Catharanthus (or Vinca) alkaloids comprise a group of about 130 terpenoid indole alkaloids . Vinblastine is now marketed for more than 40 years as an anticancer drug and became a true lead compound for drug development . Due to the pharmaceutical importance and the low content in the plant of vinblastine and the related alkaloid vincristine, Catharanthus roseus became one of the best-studied medicinal plants . Consequently it developed as a model system for biotechnological studies on plant secondary metabolism . The aim of this review is to acquaint a broader audience with the recent progress in this research and with its exciting perspectives . The pharmacognostical aspects of the Catharanthus alkaloids cover botanical (including some historical), phytochemical and analytical data . An up-to-date view on the biosynthesis of the alkaloids is given . The pharmacological aspects of these alkaloids and their semi-synthetic derivatives are only discussed briefly . The biotechnological part focuses on alternative production systems for these alkaloids, for example by in vitro culture of C . roseus cells . Subsequently it will be discussed to what extent the alkaloid biosynthetic pathway can be manipulated genetically ("metabolic engineering"), aiming at higher production levels of the alkaloids . Another approach is to produce the alkaloids (or their precursors) in other organisms such as yeast . Despite the availability of only a limited number of biosynthetic genes, the research on C . roseus has already led to a broad scientific spin-off . It is clear that many interesting results can be expected when more genes become available. Cell Death Differ, 2004 Jul, 11(7), 771 - 81 Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32; Sunayama J et al.; Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis . To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria . Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood . In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer . In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32 . Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively . Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis . Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis. Microbiol Immunol, 2004, 48(3), 205 - 10 Human herpesvirus 6 immediate-early 2 protein interacts with heterogeneous ribonucleoprotein K and casein kinase 2; Shimada K et al.; Human herpesvirus 6 (HHV-6) immediate-early (IE) 2 protein (IE2) may play important but incompletely defined roles during infection . We used yeast two-hybrid screening to detect proteins interacting with HHV-6 IE2, and found heterogeneous nuclear ribonucleoprotein K (hnRNP K) and the beta subunit of casein kinase 2 (CK2beta) specifically interacted with HHV-6 IE2 . The interactions were confirmed by GST pull-down assay, coimmunoprecipitation, and colocalization studies . These findings indicate that the HHV-6 IE2 protein interacts with hnRNP K and CK2, and these interactions may affect viral and cellular RNA transcription and translation in viral replication. Nucleic Acids Res, 2004 Mar 18, 32(5), 1774 - 82 Print 2004. Comparative analysis of orthologous eukaryotic mRNAs: potential hidden functional signals; Shabalina SA et al.; Sequencing of multiple, nearly complete eukaryotic genomes creates opportunities for detecting previously unnoticed, subtle functional signals in non-coding regions . A genome-wide comparative analysis of orthologous sets of mammalian and yeast mRNAs revealed distinct patterns of evolutionary conservation at the boundaries of the untranslated regions (UTRs) and the coding region (CDS) . Elevated sequence conservation was detected in approximately 30 nt regions around the start codon . There seems to be a complementary relationship between sequence conservation in the approximately 30 nt regions of the 5'-UTR immediately upstream of the start codon and that in the synonymous positions of the 5'-terminal 30 nt of the CDS: in mammalian mRNAs, the 5'-UTR shows a greater conservation than the CDS, whereas the opposite trend holds for yeast mRNAs . Unexpectedly, a approximately 30 nt region downstream of the stop codon shows a substantially lower level of sequence conservation than the downstream portions of the 3'-UTRs . However, the sequence in this poorly conserved 30 nt portion of the 3'-UTR is non-random in that it has a higher GC content than the rest of the UTR . It is hypothesized that the elevated sequence conservation in the region immediately upstream of the start codon is related to the requirement for initiation factor binding during pre-initiation ribosomal scanning . In contrast, the poorly conserved region downstream of the stop codon could be involved in the post- termination scanning and dissociation of the ribosomes from the mRNA, which requires only the mRNA-ribosome interaction . Additionally, it was found that the choice of the stop codon in mammals, but not in yeasts, and the context in the immediate vicinity of the stop codons in both mammals and yeasts are subject to strong selection . Thus, genome-wide analysis of orthologous gene sets allows detection of previously unrecognized patterns of sequence conservation, which are likely to reflect hidden functional signals, such as ribosomal filters that could regulate translation by modulating the interaction between the mRNA and ribosomes. Genes Dev, 2004 Mar 15, 18(6), 700 - 14 Epub 2004 Mar 18. Natural genetic variation in Arabidopsis identifies BREVIS RADIX, a novel regulator of cell proliferation and elongation in the root; Mouchel CF et al.; Mutant analysis has been tremendously successful in deciphering the genetics of plant development . However, less is known about the molecular basis of morphological variation within species, which is caused by naturally occurring alleles . In this study, we succeeded in isolating a novel regulator of root growth by exploiting natural genetic variation in the model plant Arabidopsis . Quantitative trait locus analysis of a cross between isogenized accessions revealed that a single locus is responsible for approximately 80% of the variance of the observed difference in root length . This gene, named BREVIS RADIX (BRX), controls the extent of cell proliferation and elongation in the growth zone of the root tip . We isolated BRX by positional cloning . BRX is a member of a small group of highly conserved genes, the BRX gene family, which is only found in multicellular plants . Analyses of Arabidopsis single and double mutants suggest that BRX is the only gene of this family with a role in root development . The BRX protein is nuclear localized and activates transcription in a heterologous yeast system, indicating that BRX family proteins represent a novel class of transcription factors . Thus, we have identified a novel regulatory factor controlling quantitative aspects of root growth. Biol Reprod, 2004 Jul, 71(1), 331 - 9 Epub 2004 Mar 17. A dominant-negative isoform of hypoxia-inducible factor-1 alpha specifically expressed in human testis; Depping R et al.; Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption . Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low . We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1alpha in the mouse, termed mHIF-1alphaI.1 . Here, we demonstrate that expression of mHIF-1alphaI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells . Using 5'-rapid amplification of cDNA ends, we identified a novel HIF-1alpha isoform in the human testis, called hHIF-1alphaTe . Like mHIF-1alphaI.1, hHIF-1alphaTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence . Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1alphaTe is exclusively expressed in the testis . As determined by immunofluorescence of ejaculated sperm cells, HIF-1alpha protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa . Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1alphaTe nor hHIF-1alpha associated with mitochondria . In contrast with the ubiquitously expressed HIF-1alpha protein and the mouse testis-specific mHIF-1alphaI.1 isoform, the hHIF-1alphaTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain . As shown by yeast two-hybrid assays, hHIF-1alphaTe still formed heterodimeric complexes with HIF-1beta . However, hHIF-1alphaTe was incapable of forming a DNA-binding HIF-1 complex . Overexpression of exogenous hHIF-1alphaTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1alphaTe isoform is a dominant-negative regulator of normal HIF-1 activity. J Agric Food Chem, 2004 Mar 24, 52(6), 1534 - 8 Considerations on endopolygalacturonase activity and determination of comparison ratios with emphasis on the influence of the degree of substrate esterification; Serrat M et al.; A study on the determination and standardization of endopolygalacturonase (EPG) activity is reported, with emphasis on the influence of the degree of substrate esterification using pure yeast EPG . Differences in the results, depending on how the EPG activity unit was defined, are described and discussed . From a theoretical analysis of the expressions established, a general equation for expressing EPG activity in standard international units was obtained, together with the proportional coefficient for each of the substrates studied . It was observed that for a wide range of enzyme concentrations good linear correlations were obtained . Analysis of the comparison ratio (CR) values calculated revealed that these do not differ significantly, except for low-methoxyl apple pectin, confirming the validity of the general expression obtained for pectins with different degrees of esterification . The anomalous CR value found for low-methoxyl (LM) apple pectin is discussed. EMBO J, 2004 Apr 7, 23(7), 1411 - 21 Epub 2004 Mar 18. Ubiquitin interactions of NZF zinc fingers; Alam SL et al.; Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains . One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes . We now report the solution structure of the NZF domain from Npl4 in complex with Ub . The structure reveals that three key NZF residues ((13)TF(14)/M(25)) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub . Mutations in the (13)TF(14)/M(25) motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub . However, substitution of the (13)TF(14)/M(25) motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold . Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole . Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes. Antonie Van Leeuwenhoek, 2004 Apr, 85(3), 175 - 85 Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers; de Llanos Frutos R et al.; The PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene is applied to the identification of yeasts belonging to the genus Candida . This methodology has previously been used for the identification of some species of this genus, but in the present work this application has been applied to the identification and characterisation of a greater number of species of the genus Candida, with a special survey of species of clinical and biotechnological interest . Among the species of the genus Candida, the high variability observed, both in the length of the amplified region (ranging between 390 and 900 bp) and in their restriction patterns, allows the unequivocal identification to the species level, with the exception of the group of species that comprises C . membranifaciens, C . conglobata, C . atlantica, C . atmosphaerica, and C . oleophila, that required the sequencing of the D1/D2 domain of the 26S rRNA gene or the 5.8S-ITS region for their proper differentiation . The 5.8S-ITS restriction analysis also failed in the differentiation of species within the pairs C.aaseri/C.butyri,C.fructus/C.musae,C.santamariae var . santamariae / C . beechii and C . zeylanoides / C . krissii . In this case, the high sequence similarities obtained for their 26S D1/D2 domain and the 5.8S-ITS region indicate that each pair of species should be considered as a single species . The main purpose of this work is to generate a database for a high number of yeast species, of both biotechnological and clinical interest, and to facilitate their easy, fast, and reliable identification . The present work improves the database available online at the IATA web page with the patterns of 75 species belonging to the genus Candida. Hum Mol Genet, 2004 May 15, 13(10), 1025 - 40 Epub 2004 Mar 17. QRX, a novel homeobox gene, modulates photoreceptor gene expression; Wang QL et al.; A novel paired-like homeobox gene, designated as Qrx, was identified by a yeast one-hybrid screen using the bovine Rhodopsin promoter Ret-1 DNA regulatory element as bait . Qrx is preferentially expressed in both the outer and inner nuclear layers of the retina . Its homeodomain is nearly identical to that of Rx/Rax, a transcription factor that is essential for eye development, but it shares only limited homology elsewhere . Although Qrx and Rx/Rax show similar DNA binding properties in vitro, the two proteins demonstrate distinct target selectivity and functional behavior in promoter activity assays . QRX synergistically increases the transactivating function of the photoreceptor transcription factors Crx and NRL and it physically interacts with CRX . Qrx is present in the bovine and human genomes, but appears to be absent from the mouse genome . Nonetheless, a 5.8 kb upstream region of human QRX is capable of directing expression in presumptive photoreceptor precursor cells in transgenic mice . These results indicate that Qrx may be involved in modulating photoreceptor gene expression . In addition, the finding of rare heterozygous QRX sequence changes in three individuals with retinal degeneration raises the possibility that QRX may be involved in disease pathogenesis. Indian J Pathol Microbiol, 2003 Jul, 46(3), 442 - 3 Sporotrichosis in Amritsar--a case report; Arora U et al.; A case of cutaneous, lymphatic Sporotrichosis, in a farm labourer, is presented . The diagnosis was established by isolating fungus from the lesion . Dimorphic nature of the fungus was established in vitro by demonstrating the mycelial phase at 25-30 degrees C and yeast phase at 37 degrees C . The patient responded well to oral administration of Potassium iodide. Arch Dermatol Res, 2004 Apr, 295(11), 482 - 9 Epub 2004 Mar 13. Involvement of RFX proteins in transcriptional activation from a Ras-responsive enhancer element; Maijgren S et al.; The B10.RRE element has previously been shown to mediate induced transcription in response to an activated Ha- ras gene and epidermal growth factor in keratinocytes but not in fibroblasts . We report the identification of regulatory factor for X box 3 (RFX-3) as a B10.RRE-binding protein, using the yeast one-hybrid assay . We showed that in vitro-translated RFX3, as well as RFX1 and RFX2, was able to bind B10.RRE in a sequence-specific manner . Furthermore, RFX proteins are part of the protein complex in HeLa and HepG2 cells that binds the B10.RRE element . Functional analysis in cell culture demonstrated that a truncated version of RFX1 (*RFX1) lacking the repressor domain was able to activate transcription from B10.RRE . Conversely, a dominant-negative form of RFX1 was able to block Ras-induced transcription . Taken together, these results suggest a novel role for the RFX family of transcription factors as modulators of Ras signalling in epithelial cells . Such an interaction is of potential relevance for cell growth and carcinogenesis in the skin. J Biol Chem, 2004 May 21, 279(21), 22522 - 31 Epub 2004 Mar 15. Role of mammalian vacuolar protein-sorting proteins in endocytic trafficking of a non-ubiquitinated G protein-coupled receptor to lysosomes; Hislop JN et al.; Many signaling receptors require covalent modification by ubiquitin for agonist-induced down-regulation via endocytic trafficking to lysosomes, a process that is mediated by a conserved set of endosome-associating proteins also required for vacuolar protein-sorting (VPS) in yeast . The delta opioid receptor (DOR) is a G protein-coupled receptor that can undergo agonist-induced proteolysis via endocytic trafficking to lysosomes but does not require covalent modification by ubiquitin to do so . This raises the question of whether lysosomal down-regulation of this "ubiquitination-independent" GPCR is mediated by a completely distinct biochemical mechanism or if similar VPS machinery is involved . Agonist-induced proteolysis of DOR was significantly inhibited by dominant negative mutant versions of Vps4/Skd1, an AAA-family ATPase required for a late step in lysosomal sorting of ubiquitinated membrane cargo . Furthermore, overexpression and interfering RNA-mediated knockdown indicated that lysosomal trafficking of opioid receptors is also dependent on Hrs, a VPS protein that mediates an early step in lysosomal sorting of ubiquitinated cargo . However, interfering RNA-mediated knockdown of Tsg101, a VPS protein that is essential for an intermediate step of the conserved lysosomal sorting mechanism, did not detectably affect agonist-induced proteolysis of DOR in the same cells in which (ubiquitination-dependent) lysosomal trafficking of epidermal growth factor receptors was clearly inhibited . These results indicate that opioid receptors, despite their ability to undergo efficient agonist-induced trafficking to lysosomes in the absence of covalent modification by ubiquitin, utilize some (Vps4 and Hrs) but perhaps not all (Tsg101) of the VPS machinery required for lysosomal sorting of ubiquitinated membrane cargo. Asia Pac J Clin Nutr . 2003;12 Suppl:S14. Selenium and iodine interactions with thyroid status; Thomson CD; Background - The adequacy of selenium (Se) status may influence iodine metabolism because of Se's role in the deiodinase enzymes . Se deficiency may exacerbate symptoms of iodine deficiency . There is little research on any detrimental effects of marginal selenium intakes on thyroid status . Objective - This paper reports on two studies investigating (a) the relationship between Se status and thyroid status in a NZ population and (b) the effect of Se supplementation on TSH and the ratio of T(3)/T(4); . Design - Study 1: Plasma Se was determined in 199 Otago residents for which data was available on thyroid volume, plasma TSH, and plasma T(4) . Study 2: TSH, T(4) and T(3) were measured in plasma from two supplementation studies: 57 smokers who received 100 microg Se or a placebo daily as selenomethionine; 172 subjects who received 200 microg daily as high-Se yeast (Precise) or a placebo . Outcomes - Study 1: In contrast to observations in France, preliminary analyses did not show significant associations between plasma Se and measures of thyroid status . Study2: Se supplementation resulted in a trend towards lower T(4) confirming an earlier study of a small but significant fall in T(4) . Conclusions - Lack of association between plasma Se and thyroid status, and non-significant changes in T(4) suggest that Se status in NZ is adequate for optimal activity of the deiodinases Exp Cell Res, 2004 Apr 1, 294(2), 469 - 79 Ligand-regulated association of ErbB-4 to the transcriptional co-activator YAP65 controls transcription at the nuclear level; Omerovic J et al.; It has been proposed that ligand-dependent Regulated Intramembrane Proteolysis (RIP) of ErbB-4 receptors generates 80 kDa Intra-Cellular Domains (E4.ICDs) that relocate to the nuclear compartments where they implement the signaling abilities of the ErbB-4 receptors . The E4.ICD may directly regulate gene transcription or, in an alternative scenario, the tyrosine kinase activity of E4.ICDs may target proteins involved in transcriptional regulation upon its relocation into the nucleus . We have identified the transcriptional coactivator YAP65, here referred as YAP (Yes Associated Protein), as binding partner of ErbB-4 in a two hybrid screening in yeast . Interaction between YAP and ErbB-4 occurs via the WW domain of YAP and the PPPPY at positions 1297-1301 and the PPPAY at positions 1052-1056 of the amino acid sequence of the Cyt-1 isoform of ErbB-4 . Stechiometry of binding is regulated by the ligand-dependent phosphorylation of Tyr 1056 in the PPPAYTPM module that function as "biochemical switch" to decrease the association of YAP to ErbB-4 . In principle, this novel interaction highlights new mechanisms of signaling propagation from the ErbB-4 receptors, offering supporting evidences that the E4.ICDs forms released following ligand-receptor engagement may recruit YAP and relocate to the nucleus to implement or regulate transcription. Plant Physiol, 2004 Mar, 134(3), 927 - 39 Genome-wide identification of Arabidopsis coiled-coil proteins and establishment of the ARABI-COIL database; Rose A et al.; Increasing evidence demonstrates the importance of long coiled-coil proteins for the spatial organization of cellular processes . Although several protein classes with long coiled-coil domains have been studied in animals and yeast, our knowledge about plant long coiled-coil proteins is very limited . The repeat nature of the coiled-coil sequence motif often prevents the simple identification of homologs of animal coiled-coil proteins by generic sequence similarity searches . As a consequence, counterparts of many animal proteins with long coiled-coil domains, like lamins, golgins, or microtubule organization center components, have not been identified yet in plants . Here, all Arabidopsis proteins predicted to contain long stretches of coiled-coil domains were identified by applying the algorithm MultiCoil to a genome-wide screen . A searchable protein database, ARABI-COIL , was established that integrates information on number, size, and position of predicted coiled-coil domains with subcellular localization signals, transmembrane domains, and available functional annotations . ARABI-COIL serves as a tool to sort and browse Arabidopsis long coiled-coil proteins to facilitate the identification and selection of candidate proteins of potential interest for specific research areas . Using the database, candidate proteins were identified for Arabidopsis membrane-bound, nuclear, and organellar long coiled-coil proteins. Mol Biol Cell, 2004 Jun, 15(6), 2537 - 48 Epub 2004 Mar 12. Activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum; Nadanaka S et al.; Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway . The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER . However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus . This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway . We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER . Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER. Genetics, 2004 Jan, 166(1), 151 - 60 sel-7, a positive regulator of lin-12 activity, encodes a novel nuclear protein in Caenorhabditis elegans; Chen J et al.; Suppressor genetics in C . elegans has identified key components of the LIN-12/Notch signaling pathway . Here, we describe a genetic and molecular characterization of the suppressor gene sel-7 . We show that reducing or eliminating sel-7 activity suppresses the effects of constitutive lin-12 activity, enhances the effects of partially reduced lin-12 activity, and causes a synthetic Lin-12(0) phenotype when combined with a null mutation in the sel-12 presenilin gene . These observations suggest that sel-7 is a positive regulator of lin-12 activity . We also show that SEL-7 encodes a novel nuclear protein . Through yeast two-hybrid screening, we identified an apparent interaction partner, K08E3.8, that also interacts with SEL-8, a known component of the nuclear complex that forms upon LIN-12 activation . Our data suggest potential roles for SEL-7 in the assembly or function of this nuclear complex. J Mol Biol, 2004 Mar 26, 337(3), 545 - 60 Nuclear control of cloverleaf structure of human mitochondrial tRNA(Lys); Helm M et al.; The evolutionary loss in eukaryotic cells of mitochondrial (mt) tRNA genes and of tRNA structural information in the surviving genes has led to the appearance of mt-tRNAs with highly unusual structural features . One such mt-tRNA is the human mt-tRNALys, which relies on post-transcriptional base modification to achieve correct three-dimensional structure . It has been shown that the in vitro transcript of human mt-tRNALys adopts a particular, non-cloverleaf structure when devoid of modified bases, while the native, fully modified tRNA shows the expected cloverleaf structure . Furthermore, a methyl group at position A9-N1, introduced chemically in an otherwise unmodified mt-tRNALys transcript, was found to induce a stable cloverleaf conformation, raising the question of how the specific methyltransferase recognizes the unmodified transcript . In order to shed light on this unusual case of tRNA maturation, the tRNA modification enzymes contained in protein extracts from either highly purified HeLa cell mitochondria or HeLa cell cytosol were first identified and compared, and then used to analyze the mt-tRNALys . An initial screening for modification activities, using as substrates unmodified in vitro transcripts of tRNA genes with well characterized structures, namely yeast cytosolic tRNAPhe, human cytosolic tRNA3Lys, and human mt-tRNAIle, revealed the presence of nine and 11 modification activities in the mitochondrial and cytosolic protein extracts, respectively, the mitochondrial extract including a tRNA (adenine-9,N1)-methyltransferase activity . The comparison of the level and kinetics of A9-N1 methylation and other secondary modifications in the unmodified, misfolded mt-tRNALys and in a cloverleaf-shaped structural mutant, engineered to adopt the tRNALys cloverleaf structure without post-transcriptional modifications, suggested strongly that the methylation of A9-N1 in tRNALys proceeds via a cloverleaf-shaped intermediate . Therefore, it is proposed that this intermediate is present in the in vitro transcript as part of a dynamic equilibrium, and that the mitochondrial protein extract contains an activity that stabilizes, by secondary modification, such a transient cloverleaf-shaped intermediate . Thus, countering the evolutionary loss of structural information in mt-tRNA genes, the mt-tRNA structure is maintained by a modification enzyme encoded in nuclear DNA. Peptides, 2003 Nov, 24(11), 1705 - 12 Interactions of antifungal plant defensins with fungal membrane components; Thevissen K et al.; Plant defensins are small, basic, cysteine-rich peptides that are generally active against a broad spectrum of fungal and yeast species at micromolar concentrations . Some of these defensins interact with fungal-specific lipid components in the plasmamembrane . Structural differences of these membrane components between fungal and plant cells probably account for the selective activity of plant defensins against fungal pathogens and their nonphytotoxic properties . This review will focus on different classes of complex lipids in fungal membranes and on the selective interaction of plant defensins with these complex lipids. Viral Immunol, 2004, 17(1), 51 - 68 Segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice; Gedvilaite A et al.; Insertion of a short-sized epitope at four different sites of yeast-expressed hamster polyomavirus major capsid protein VP1 has been found to result in the formation of chimeric virus-like particles . Here, we demonstrate that the insertion of 45 or 120 amino acid-long segments from the N-terminus of Puumala hantavirus nucleocapsid protein into sites 1 (amino acids 80-89) and 4 (amino acids 288-295) of VP1 allowed the highly efficient formation of virus-like particles . In contrast, expression level and assembly capacity of fusions to sites 2 (amino acids 222-225) and 3 (amino acids 243-247) were drastically reduced . Immunization of BALB/c mice with chimeric virus-like particles induced a high-titered antibody response against the hantavirus nucleocapsid protein, even in the absence of any adjuvant . The strongest response was observed in mice immunized with virus-like particles harboring 120 amino acids of hantavirus nucleocapsid protein . According to the immunoglobulin subclass distribution of nucleocapsid protein-specific antibodies a mixed Th1/Th2 response was detected . The VP1 carrier itself also induced a mixed Th1/Th2 response, which was found to be reduced in mice immunized with virus-like particles harboring 120 amino acid-long inserts . In conclusion, hamster polyomavirus VP1 represents a promising carrier moiety for future vaccine development. Dermatol Clin, 2004 Jan, 22(1), 33 - 50 Cutaneous fungal infections in the elderly; Loo DS; Because of impaired host defenses and a favorable environment at specific anatomic sites, there is an increased prevalence of seborrheic dermatitis, mucosal and cutaneous candidiasis, tinea pedis, and onychomycosis in the geriatric population compared with other age groups . Both KOH and fungal culture are timely, convenient, and cost-effective methods of diagnosis . Sensitivity of these tests depends on proper technique for specimen collection and experience . KOH 20% with DMSO and DTM are highly recommended . Treatment should be tailored to the diagnosis and the individual patient . This includes the targeted spectrum of coverage (dermatophyte or yeast); topical versus systemic therapy; review of the patient's medication list for potential drug interactions; and likelihood of compliance . Checking baseline laboratories and routine monitoring of complete blood count and liver function tests in healthy patients, without a history of liver disease or active hepatitis, and without potential drug interactions, seems unwarranted for rare adverse events . Successful management requires adequate patient education, correction of underlying predisposing factors, and prophylactic measures against recurrence. J Virol, 2004 Apr, 78(7), 3763 - 76 A peptide from autoantigen La blocks poliovirus and hepatitis C virus cap-independent translation and reveals a single tyrosine critical for La RNA binding and translation stimulation; Izumi RE et al.; La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation . Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein . Here we have identified an 18-amino-acid-long sequence from the N-terminal "La motif" which is required for efficient interaction of La with IRNA and viral 5' untranslated region (5'-UTR) elements . A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro . The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo . The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5'-UTR . Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA . Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP . When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation . However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5'-UTR and stimulate viral IRES-mediated translation significantly . These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation . The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed. J Biol Chem, 2004 May 14, 279(20), 20626 - 35 Epub 2004 Mar 10. Interaction between hex and GATA transcription factors in vascular endothelial cells inhibits flk-1/KDR-mediated vascular endothelial growth factor signaling; Minami T et al.; Recent evidence supports a role for GATA transcription factors as important signal intermediates in differentiated endothelial cells . The goal of this study was to identify proteins that interact with endothelial-derived GATA transcription factors . Using yeast two-hybrid screening, we identified hematopoietically expressed homeobox (Hex) as a GATA-binding partner in endothelial cells . The physical association between Hex and GATA was confirmed with immunoprecipitation in cultured cells . Hex overexpression resulted in decreased flk-1/KDR expression, both at the level of the promoter and the endogenous gene, and attenuated vascular endothelial growth factor-mediated tube formation in primary endothelial cell cultures . In electrophoretic mobility shift assays, Hex inhibited the binding of GATA-2 to the flk-1/KDR 5'-untranslated region GATA motif . Finally, in RNase protection assays, transforming growth factor beta1, which has been previously shown to decrease flk-1 expression by interfering with GATA binding activity, was shown to increase Hex expression in endothelial cells . Taken together, the present study provides evidence for a novel association between Hex and GATA and suggests that transforming growth factor beta-mediated repression of flk-1/KDR and vascular endothelial growth factor signaling involves the inducible formation of inhibitory Hex-GATA complexes. J Mol Med, 2004 Jun, 82(6), 383 - 8 Epub 2004 Mar 10. Sperm membrane protein (hSMP-1) and RanBPM complex in the microtubule-organizing centre; Tang X et al.; hSMP-1 is a human sperm membrane protein expressed during development . It is a testis-specific component produced during male germ cell differentiation . Proteins that interact with hSMP-1 were identified by the application of the yeast two-hybrid system . One of the components, RanBPM, was found to be associated with hSMP-1 under both in vitro and in vivo conditions . In the human testis, RanBPM is produced in spermatogonia and primary spermatocytes, suggesting expression during the early stages of spermatogenesis; whereas in the rat testis, it is located in round and elongated spermatids, similar to hSMP-1, suggesting expression of both components during spermiogenesis . Images obtained by immunofluorescence and confocal scanning microscopy of CHO-K1 cells co-transfected with pEGFP-C1-hSMP-1 and pDsRed1-Nl-RanBPM revealed that RanBPM and hSMP-1 are distributed in discrete loci throughout the cytoplasm . When superimposed, the stained spots appeared as congruent yellow areas, indicative of co-localization and probable complex formation of these two components . This interaction between hSMP-1 and RanBPM may be involved in the process of male germ cell differentiation . In CHO-Kl cells transfected with pEGFP-Cl-hSMP-1, the exogenously expressed hSMP-1 was found to co-localize with alpha-tubulin . Depolymerization of microtubules can be induced in CHO-Kl cells by cold treatment . In cells transfected with the pEGFP-Cl vector, the dispersed tubulins promptly reassembled upon warming . However, in cells transfected with pEGFP-Cl-hSMP-1, reassembly of the dispersed tubulins was blocked even upon rewarming of the cells . These findings suggest that hSMP-1 interacts with tubulins and thereby may modulate microtubule assembly and/or activity . EMBO J, 2004 Mar 24, 23(6), 1348 - 59 Epub 2004 Mar 04. Tandem bromodomains in the chromatin remodeler RSC recognize acetylated histone H3 Lys14; Kasten M et al.; The coordination of chromatin remodeling with chromatin modification is a central topic in gene regulation . The yeast chromatin remodeling complex RSC bears multiple bromodomains, motifs for acetyl-lysine and histone tail interaction . Here, we identify and characterize Rsc4 and show that it bears tandem essential bromodomains . Conditional rsc4 bromodomain mutations were isolated, and were lethal in combination with gcn5Delta, whereas combinations with esa1 grew well . Replacements involving Lys14 of histone H3 (the main target of Gcn5), but not other H3 or H4 lysine residues, also conferred severe growth defects to rsc4 mutant strains . Importantly, wild-type Rsc4 bound an H3 tail peptide acetylated at Lys14, whereas a bromodomain mutant derivative did not . Loss of particular histone deacetylases suppressed rsc4 bromodomain mutations, suggesting that Rsc4 promotes gene activation . Furthermore, rsc4 mutants displayed defects in the activation of genes involved in nicotinic acid biosynthesis, cell wall integrity, and other pathways . Taken together, Rsc4 bears essential tandem bromodomains that rely on H3 Lys14 acetylation to assist RSC complex for gene activation. EMBO J, 2004 Mar 24, 23(6), 1392 - 401 Epub 2004 Mar 04. Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1; Siaud N et al.; Two BRCA2-like sequences are present in the Arabidopsis genome . Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs . In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1 . RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations . We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants . The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks . The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants . Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis . We also propose a role for Rad51 in the dmc1 context. Mol Biol Evol, 2004 Jun, 21(6), 1024 - 31 Epub 2004 Mar 10. Molecular evolution and structure of alpha-actinin; Virel A et al.; The N-terminal actin-binding domain of alpha-actinin is connected to the C-terminal EF-hands by a rod domain . Because of its ability to form dimers, alpha-actinin can cross-link actin filaments in muscle cells as well as in nonmuscle cells . In the prototypic alpha-actinins, the rod domain contains four triple helical bundles, or so-called spectrin repeats . We have found some atypical alpha-actinins in early diverging organisms, such as protozoa and yeast, where the rod domain contains one and two spectrin repeats, respectively . This implies that the four repeats present in modern alpha-actinins arose after two consecutive intragenic duplications from an alpha-actinin with a single repeat . Further, the evolutionary gene tree of alpha-actinins shows that the appearance of four distinct alpha-actinin isoforms may have occurred after the vertebrate-invertebrate split . The topology of the tree lends support to the hypothesis that two rounds (2R) of genome duplication occurred early in the vertebrate radiation . The phylogeny also considers these atypical isoforms as the most basal to alpha-actinins of vertebrates and other eukaryotes . The analysis also positioned alpha-actinin of the fungi Encephalitozoo cuniculi close to the protozoa, supporting the suggestion that microsporidia are early eukaryotes . Because alpha-actinin is considered the basal member of the spectrin family, our studies will improve the understanding of the origin and evolution of this superfamily. Int J Parasitol, 2004 Mar 29, 34(4), 445 - 54 Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions; Uliel S et al.; Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications . Two prevalent types of modifications are 2'-O-methylation and pseudouridylation . The modification is directed by the formation of a canonical small nucleolar RNA-target duplex . Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications . Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host . The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants . However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA . In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites. Annu Rev Plant Physiol Plant Mol Biol, 1998 Jun, 49, 697 - 725 CALMODULIN AND CALMODULIN-BINDING PROTEINS IN PLANTS; Zielinski RE; Calmodulin is a small Ca2+-binding protein that acts to transduce second messenger signals into a wide array of cellular responses . Plant calmodulins share many structural and functional features with their homologs from animals and yeast, but the expression of multiple protein isoforms appears to be a distinctive feature of higher plants . Calmodulin acts by binding to short peptide sequences within target proteins, thereby inducing structural changes, which alters their activities in response to changes in intracellular Ca2+ concentration . The spectrum of plant calmodulin-binding proteins shares some overlap with that found in animals, but a growing number of calmodulin-regulated proteins in plants appear to be unique . Ca2+-binding and enzymatic activation properties of calmodulin are discussed emphasizing the functional linkages between these processes and the diverse pathways that are dependent on Ca2+ signaling. Annu Rev Plant Physiol Plant Mol Biol, 1998 Jun, 49, 669 - 696 MOLECULAR BIOLOGY OF CATION TRANSPORT IN PLANTS; Fox TC et al.; This review summarizes current knowledge about genes whose products function in the transport of various cationic macronutrients (K, Ca) and micronutrients (Cu, Fe, Mn, and Zn) in plants . Such genes have been identified on the basis of function, via complementation of yeast mutants, or on the basis of sequence similarity, via database analysis, degenerate PCR, or low stringency hybridization . Not surprisingly, many of these genes belong to previously described transporter families, including those encoding Shaker-type K+ channels, P-type ATPases, and Nramp proteins . ZIP, a novel cation transporter family first identified in plants, also seems to be ubiquitous; members of this family are found in protozoa, yeast, nematodes, and humans . Emerging information on where in the plant each transporter functions and how each is controlled in response to nutrient availability may allow creation of food crops with enhanced mineral content as well as crops that bioaccumulate or exclude toxic metals. Annu Rev Plant Physiol Plant Mol Biol, 1998 Jun, 49, 127 - 150 PLANT TRANSCRIPTION FACTOR STUDIES; Schwechheimer C et al.; Major advances have been made in understanding the role of transcription factors in gene expression in yeast, Drosophila, and man . Transcription factor modification, synergistic events, protein-protein interactions, and chromatin structure have been successfully integrated into transcription factor studies in these organisms . While many putative transcription factors have been isolated from plants, most of them are only poorly characterized . This review summarizes examples where molecular biological techniques have been successfully employed to study plant transcription factors . The functional analysis of transcription factors is described as well as techniques for studying the interactions of transcription factors with other proteins and with DNA. Annu Rev Plant Physiol Plant Mol Biol, 1998 Jun, 49, 77 - 95 SPLICE SITE SELECTION IN PLANT PRE-mRNA SPLICING; Brown JW et al.; The purpose of this review is to highlight the unique and common features of splice site selection in plants compared with the better understood yeast and vertebrate systems . A key question in plant splicing is the role of AU sequences and how and at what stage they are involved in spliceosome assembly . Clearly, intronic U- or AU-rich and exonic GC- and AG-rich elements can influence splice site selection and splicing efficiency and are likely to bind proteins . It is becoming clear that splicing of a particular intron depends on a fine balance in the "strength" of the multiple intron signals involved in splice site selection . Individual introns contain varying strengths of signals and what is critical to splicing of one intron may be of less importance to the splicing of another . Thus, small changes to signals may severely disrupt splicing or have little or no effect depending on the overall sequence context of a specific intron/exon organization. Annu Rev Plant Physiol Plant Mol Biol, 2000 Jun, 51, 463 - 499 PLANT CELLULAR AND MOLECULAR RESPONSES TO HIGH SALINITY; Hasegawa PM et al.; Plant responses to salinity stress are reviewed with emphasis on molecular mechanisms of signal transduction and on the physiological consequences of altered gene expression that affect biochemical reactions downstream of stress sensing . We make extensive use of comparisons with model organisms, halophytic plants, and yeast, which provide a paradigm for many responses to salinity exhibited by stress-sensitive plants . Among biochemical responses, we emphasize osmolyte biosynthesis and function, water flux control, and membrane transport of ions for maintenance and re-establishment of homeostasis . The advances in understanding the effectiveness of stress responses, and distinctions between pathology and adaptive advantage, are increasingly based on transgenic plant and mutant analyses, in particular the analysis of Arabidopsis mutants defective in elements of stress signal transduction pathways . We summarize evidence for plant stress signaling systems, some of which have components analogous to those that regulate osmotic stress responses of yeast . There is evidence also of signaling cascades that are not known to exist in the unicellular eukaryote, some that presumably function in intercellular coordination or regulation of effector genes in a cell-/tissue-specific context required for tolerance of plants . A complex set of stress-responsive transcription factors is emerging . The imminent availability of genomic DNA sequences and global and cell-specific transcript expression data, combined with determinant identification based on gain- and loss-of-function molecular genetics, will provide the infrastructure for functional physiological dissection of salt tolerance determinants in an organismal context . Furthermore, protein interaction analysis and evaluation of allelism, additivity, and epistasis allow determination of ordered relationships between stress signaling components . Finally, genetic activation and suppression screens will lead inevitably to an understanding of the interrelationships of the multiple signaling systems that control stress-adaptive responses in plants. Oncol Rep, 2004 Apr, 11(4), 923 - 9 Analysis of tumor suppressor p53 status in head and neck squamous cell carcinoma; Smardova J et al.; Head and neck cancer belongs to the most common types of cancer in both males and females with a mortality rate of approximately 50% . More than 90% of head and neck cancers are squamous cell carcinoma (HNSCC) . Carcinogenesis of this disease involves activation of proto-oncogenes and inactivation of tumor suppressor genes . Among them, aberrations of p53 tumor suppressor gene are common events . The aim of this study was to assess the frequency of the tumor suppressor p53 aberrations in Czech population by using a functional test in yeast (FASAY) and by two immunochemical methods . We compared results of the methods and assessed the relationship between the presence of p53 aberration and some clinico-pathological parameters . The following observations were made: i) the accumulated p53 protein was detected in 33 of 50 tested samples (66%) by immunohistochemical analysis and in 27 of 49 tested samples (55.1%) by immunoblotting; ii) the presence of p53 mutation was detected in 36 of 50 tested samples (72%); iii) 6 of 36 p53 mutations detected by FASAY were temperature sensitive (16.7%); iv) 2 independent p53 mutations were found in at least 2 of the 36 positive cases; v) no statistically significant relationship was found between p53 aberration and overall survival. Eur J Biochem, 2004 Mar, 271(5), 1035 - 45 Emerin binding to Btf, a death-promoting transcriptional repressor, is disrupted by a missense mutation that causes Emery-Dreifuss muscular dystrophy; Haraguchi T et al.; Loss of functional emerin, a nuclear membrane protein, causes X-linked recessive Emery-Dreifuss muscular dystrophy . In a yeast two-hybrid screen, we found that emerin interacts with Btf, a death-promoting transcriptional repressor, which is expressed at high levels in skeletal muscle . Biochemical analysis showed that emerin binds Btf with an equilibrium affinity (KD) of 100 nm . Using a collection of 21 clustered alanine-substitution mutations in emerin, the residues required for binding to Btf mapped to two regions of emerin that flank its lamin-binding domain . Two disease-causing mutations in emerin, S54F and Delta95-99, disrupted binding to Btf . The Delta95-99 mutation was relatively uninformative, as this mutation also disrupts emerin binding to lamin A and a different transcription repressor named germ cell-less (GCL) . In striking contrast, emerin mutant S54F, which binds normally to barrier-to-autointegration factor, lamin A and GCL, selectively disrupted emerin binding to Btf . We localized endogenous Btf in HeLa cells by indirect immunoflurorescence using affinity-purified antibodies against Btf . In nonapoptotic HeLa cells Btf was found in dot-like structures throughout the nuclear interior . However, within 3 h after treating cells with Fas antibody to induce apoptosis, the distribution of Btf changed, and Btf concentrated in a distinct zone near the nuclear envelope . These results suggest that Btf localization is regulated by apoptotic signals, and that loss of emerin binding to Btf may be relevant to muscle wasting in Emery-Dreifuss muscular dystrophy. Eur J Biochem, 2004 Mar, 271(5), 972 - 82 NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis; Tanaka T et al.; NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins . Previously, we identified a negative regulator of the NEDD8 conjugation system, NEDD8 ultimate buster-1 (NUB1), that recruits NEDD8 and its conjugates to the proteasome for degradation . Recently, we performed yeast two-hybrid screening with NUB1 as bait and isolated a ubiquitin precursor UbC1 that is composed of nine tandem repeats of a ubiquitin unit through alpha-peptide bonds . Interestingly, NUB1 interacted with UbC1 through its UBA domain . Further study revealed that the UBA domain interacted with alpha-peptide bond-linked polyubiquitin, but not with isopeptide bond-linked polyubiquitin, indicating that the UBA domain of NUB1 is a specific acceptor for the linear ubiquitin precursor . A functional study revealed that an unidentified protein that was immunoprecipitated with NUB1 served as a ubiquitin C-terminal hydrolase for UbC1 . Thus, NUB1 seems to form a protein complex with the unidentified ubiquitin C-terminal hydrolase and recruit UbC1 to this complex . This might allow the ubiquitin C-terminal hydrolase to hydrolyze UbC1, in order to generate ubiquitin monomers . Northern blot analysis showed that the mRNAs of both NUB1 and UbC1 were enriched in the testis . Furthermore, in situ hybridization showed that both mRNAs were strongly expressed in seminiferous tubules of the testis . These results may imply that the UbC1 hydrolysis mediated by NUB1 is involved in cellular functions in the seminiferous tubules such as spermatogenesis. J Mol Evol, 2003, 57 Suppl 1, S277 - 85 Molecular clock and gene function; Saccone C et al.; Molecular phylogenies based on the molecular clock require the comparison of orthologous genes . Orthologous and paralogous genes usually have very different evolutionary fates . In general, orthologs keep the same functions in species, whereas, particularly over a long time span, paralogs diverge functionally and may become pseudogenes or get lost . In eukaryotic genomes, because of the degree of redundancy of genetic information, homologous genes are grouped in gene families, the evolution of which may differ greatly between the various organisms . This implies that each gene in a species does not always have an ortholog in another species and thus, due to multiple duplication events following a speciation, many orthologous clades of paralogs are generated . We are often dealing with a one-to-many or many-to-many relationship between genes . In this paper, we analyze the evolution of two gene families, the p53 gene family and the porin gene family . The evolution of the p53 family shows a one-to-many gene relationship going from invertebrates to vertebrates . In invertebrates only a single gene has been found, while in vertebrates three members of the family, namely p53, p63, and p73, are present . The evolution of porin (VDAC) genes (VDAC1, VDAC2, and VDAC3) is an example of a many-to-many gene relationship going from yeast to mammals . However, the porin gene redundancy found in invertebrates and possibly in some fishes may indicate a tendency to duplicate the genetic material, rather than a real need for function innovation. Curr Genet, 2004 May, 45(5), 289 - 301 Epub 2004 Mar 09. Sequence heterology and gene conversion at his-3 of Neurospora crassa; Yeadon PJ et al.; Although sequence heterology clearly reduces crossing over in yeast, conflicting studies suggest that mismatches may increase or decrease gene conversion . To investigate this issue in an additional species, we measured the effect of local sequence heterology on conversion in his-3 of Neurospora crassa . Mismatches close to the cog recombination initiator or within his-3 reduce conversion to 70% and 30% of the homologous level, respectively, while heterologous insertions between his-3 and cog increase conversion by 20% . We suggest that, in both Neurospora and yeast, mismatches reduce the efficiency of the establishment and resolution stages of recombination, but substantial heterology may increase the progress of already established events by preventing repair synthesis from switching between templates . These data provide additional support that recombination at his-3 (and perhaps at yeast hotspots) proceeds by a synthesis-dependent strand-annealing mechanism, during which synthesis can switch templates, with the process being more tolerant of sequence mismatch in Neurospora. Microbiol Mol Biol Rev, 2004 Mar, 68(1), 109 - 31, table of contents Eukaryotic MCM proteins: beyond replication initiation; Forsburg SL; The minichromosome maintenance (or MCM) protein family is composed of six related proteins that are conserved in all eukaryotes . They were first identified by genetic screens in yeast and subsequently analyzed in other experimental systems using molecular and biochemical methods . Early data led to the identification of MCMs as central players in the initiation of DNA replication . More recent studies have shown that MCM proteins also function in replication elongation, probably as a DNA helicase . This is consistent with structural analysis showing that the proteins interact together in a heterohexameric ring . However, MCMs are strikingly abundant and far exceed the stoichiometry of replication origins; they are widely distributed on unreplicated chromatin . Analysis of mcm mutant phenotypes and interactions with other factors have now implicated the MCM proteins in other chromosome transactions including damage response, transcription, and chromatin structure . These experiments indicate that the MCMs are central players in many aspects of genome stability. Cancer Epidemiol Biomarkers Prev, 2004 Mar, 13(3), 391 - 7 DNA stability and serum selenium levels in a high-risk group for prostate cancer; Karunasinghe N et al.; The essential micronutrient, selenium, is at low levels in the New Zealand diet . Selenium is a component of a number of proteins involved in the maintenance of genomic stability, and recommended daily allowances (RDA) are set on saturation levels for glutathione peroxidase (GPx), a key enzyme in surveillance against oxidative stress . It has been assumed but not proven that this level will be adequate for other key selenoenzymes . The "Negative Biopsy Trial" identifies a group of New Zealand individuals at high risk of prostate cancer, whose serum selenium levels will be monitored and who will be supplemented with a yeast-based tablet, with or without selenium, over an extended time . Access to patients on this trial provides the opportunity to ask the more generic question as to whether selenium levels in this population are adequate to maintain genomic stability . The single cell gel electrophoresis (comet) assay was used to study DNA damage in blood leukocytes harvested from these volunteers . Average serum selenium levels before randomization was 97.8 +/- 16.6 ng/ml, low by international standards . For the half of the population below this mean value, lower serum selenium levels showed a statistically significant inverse relationship (P = 0.02) with overall accumulated DNA damage . Although other interpretations cannot be excluded, the data suggest that the selenium intake in half of this population is marginal for adequate repair of DNA damage, increasing susceptibility to cancer and other degenerative diseases . It also raises the question as to whether glutathione peroxidase saturation levels are appropriate indicators of the optimal selenium levels for a given population. Nat Struct Mol Biol, 2004 Apr, 11(4), 330 - 7 Epub 2004 Mar 07. The structural basis for the interaction between nonsense-mediated mRNA decay factors UPF2 and UPF3; Kadlec J et al.; Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism by which eukaryotic cells detect and degrade transcripts containing premature termination codons . Three 'up-frameshift' proteins, UPF1, UPF2 and UPF3, are essential for this process in organisms ranging from yeast to human . We present a crystal structure at a resolution of 1.95 A of the complex between the interacting domains of human UPF2 and UPF3b, which are, respectively, a MIF4G (middle portion of eIF4G) domain and an RNP domain (ribonucleoprotein-type RNA-binding domain) . The protein-protein interface is mediated by highly conserved charged residues in UPF2 and UPF3b and involves the beta-sheet surface of the UPF3b RNP domain, which is generally used by these domains to bind nucleic acids . We show that the UPF3b RNP does not bind RNA, whereas the UPF2 construct and the complex do . Our results advance understanding of the molecular mechanisms underlying the NMD quality control process. Cell Cycle, 2004 Apr, 3(4), 513 - 8 Epub 2004 Apr 01. The first green lineage cdc25 dual-specificity phosphatase; Khadaroo B et al.; The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G(2)/M transition in metazoans and yeast . However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G(2)/M transition . In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri . It encodes a protein which is able to rescue the yeast S . pombe cdc25-22 conditional mutant . Furthermore, microinjection of GST-tagged O . tauri Cdc25 specifically activates prophase-arrested starfish oocytes . In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O . tauri Cdc25 . We propose that there has been coevolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages. Cell Cycle, 2004 Apr, 3(4), 449 - 51 Epub 2004 Apr 01. Akt-induced cellular senescence: implication for human disease; Minamino T et al.; Reduction-of-function mutations in components of the insulin/insulin-like growth factor-1/Akt pathway have been shown to extend the lifespan in organisms ranging from yeast to mice . It has also been reported that activation of Akt induces proliferation and survival of mammalian cells, thereby promoting tumorigenesis . We have recently shown that Akt activity increases with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells . Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, leading to endothelial dysfunction . This novel role of Akt in regulating the cellular lifespan may contribute to various human diseases including atherosclerosis and diabetes mellitus. Cytogenet Genome Res, 2003, 103(1-2), 3 - 7 The application of region-specific probes for the resolution of duplication 8p: a case report and a review of the literature; Pabst B et al.; The structural rearrangement in the short arm of a chromosome 8 in a clinically affected patient has been reinvestigated by FISH using whole chromosome painting and region specific YAC probes . An inverted duplication of the segment p22-->p11.2 and a deletion of the subtelomeric region were demonstrated . By this approach, a more detailed resolution of the duplication/deletion 8p was possible . With the application of molecular cytogenetic methods the existence of different duplication segments within the clinical entity of duplication/deficiency 8p can be shown. J Immunol, 2004 Mar 15, 172(6), 3644 - 51 A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies {correction of interspecie} cross-reactivity; Barral P et al.; Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America . Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized . Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure . Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced . The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues . Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity) . Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation . Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens . Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach . These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome. Virology, 2004 Mar 1, 320(1), 99 - 106 The Epstein-Barr virus BFRF1 and BFLF2 proteins interact and coexpression alters their cellular localization; Lake CM et al.; The BFRF1 protein of Epstein-Barr virus (EBV) is a recently identified membrane protein that is the homolog of the alphaherpesvirus UL34 gene product . We report here that a yeast two-hybrid screen identified the BFLF2 gene product, a homolog of alphaherpesvirus UL31, as a protein that interacts with BFRF1 . Expression of BFLF2 in mammalian cells revealed a protein of approximately 28 kDa that associated with BFRF1 in a noncovalently linked complex . When expressed alone, the BFRF1 protein was found in the cytoplasm and perinuclear region . BFLF2 was found diffusely in the nucleus in the absence of BFRF1, but coexpression of BFRF1 and BFLF2 resulted in colocalization of the two proteins at the nuclear rim . These data recapitulate the behavior of the alphaherpesvirus homologs of BFRF1 and BFLF2 and suggest that functional as well as structural and positional homology may be conserved. Biochem Biophys Res Commun, 2004 Mar 26, 316(1), 182 - 8 NF-kappaB binds to a polymorphic repressor element in the MMP-3 promoter; Borghaei RC et al.; A 5T/6T polymorphic site in the matrix metalloproteinase-3 (MMP-3) promoter has been identified as a repressor element involved in inhibiting induction of MMP-3 transcription by interleukin 1; and the 6T allele has been associated with decreased expression of MMP-3 as compared to the 5T allele . Zinc-binding protein-89 (ZBP-89) was cloned from a yeast one-hybrid assay via its ability to interact with this site, but when the protein was over-expressed, it resulted in activation of the MMP-3 promoter rather than repression . Here we show that in nuclear extracts isolated from human gingival fibroblasts stimulated with IL-1, this site is bound by p50 and p65 components of NF-kappaB in addition to ZBP-89, and that recombinant p50 binds preferentially to the 6T binding site . These results are consistent with a role for NF-kappaB in limiting the cytokine induced expression of MMP-3. Neuron, 2004 Mar 4, 41(5), 687 - 99 Androgen receptor YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration; Sopher BL et al.; X-linked spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration . SBMA is caused by polyglutamine repeat expansions in the androgen receptor (AR) . To determine the basis of AR polyglutamine neurotoxicity, we introduced human AR yeast artificial chromosomes carrying either 20 or 100 CAGs into mouse embryonic stem cells . The AR100 transgenic mice developed a late-onset, gradually progressive neuromuscular phenotype accompanied by motor neuron degeneration, indicating striking recapitulation of the human disease . We then tested the hypothesis that polyglutamine-expanded AR interferes with CREB binding protein (CBP)-mediated transcription of vascular endothelial growth factor (VEGF) and observed altered CBP-AR binding and VEGF reduction in AR100 mice . We found that mutant AR-induced death of motor neuron-like cells could be rescued by VEGF . Our results suggest that SBMA motor neuronopathy involves altered expression of VEGF, consistent with a role for VEGF as a neurotrophic/survival factor in motor neuron disease. Cell Death Differ, 2004 Jun, 11(6), 645 - 54 Regulation of DNaseY activity by actinin-alpha4 during apoptosis; Liu QY et al.; DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated . We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity . Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26 . However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation . Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways . Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis. FASEB J, 2004 May, 18(7), 890 - 2 Epub 2004 Mar 04. Human LZIP binds to CCR1 and differentially affects the chemotactic activities of CCR1-dependent chemokines; Ko J et al.; Signaling molecules that bind to chemokine receptors should play key roles in regulation of cell migration induced by chemokines . To characterize the CCR1-mediated cellular signal transduction mechanism, we used the yeast two-hybrid system to identify a cellular ligand for CCR1 . LZIP, which has been known as a transcription factor in various cell types, was identified as a CCR1 binding protein . Although the ability of LZIP to bind DNA is possibly what allows it to function as a transcription factor, its detailed function and participation in chemotaxis have not been established . We found that LZIP binds to CCR1 based on results of a mammalian two-hybrid assay and immunoprecipitation experiments . The 21-260 residues of LZIP were essential for interaction with CCR1 . Results from a chemotaxis assay using LZIP transfected cells showed that LZIP enhanced Lkn-1-induced chemotaxis, whereas the chemotactic activities induced by other CC chemokines that bind to CCR1, including MIP-1alpha, RANTES, or HCC-4, were not affected by LZIP overexpression . These data indicate that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn-1-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1. Circ Res, 2004 Mar 19, 94(5), E46 - 54 Epub 2004 Mar 04. Evidence that IGF binding protein-5 functions as a ligand-independent transcriptional regulator in vascular smooth muscle cells; Xu Q et al.; Insulin-like growth factor binding protein (IGFBP)-5 is a conserved protein synthesized and secreted by vascular smooth muscle cells (VSMCs) . IGFBP-5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival . IGFBP-5 also stimulates VSMC migration through an IGF-independent mechanism, but the molecular basis underlying this ligand-independent action is unknown . In this study, we show that endogenous IGFBP-5 or transiently expressed IGFBP-5-EGFP, but not IGFBP-4-EGFP, is localized in the nuclei of VSMCs . Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we demonstrated that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical . Intriguingly, inhibition of protein secretion abolishes IGFBP-5 nuclear localization, suggesting the nuclear IGFBP-5 is derived from the secreted protein . When added exogenously, (125)I- or Cy3-labeled IGFBP-5 is capable of cellular entry and nuclear translocation . To identify potential transcriptional factor(s) that interact with IGFBP-5, a human aorta cDNA library was screened by a yeast two-hybrid screening strategy . Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP-5, no known transcription factors were found . Further motif analysis revealed that the IGFBP-5 N-domain contains a putative transactivation domain . When fused to GAL-4 DNA dinging domain and tested, the IGFBP-5 N-domain has strong transactivation activity . Mutation of the IGF binding domain or treatment of cells with IGF-I has little effect on transactivation activity . These results suggest that IGFBP-5 is localized in VSMC nucleus and possesses transcription-regulatory activity that is IGF independent. J Mol Biol, 2004 Mar 12, 337(1), 157 - 65 An altered-specificity ubiquitin-conjugating enzyme/ubiquitin-protein ligase pair; Winkler GS et al.; The human CCR4-NOT complex is a global regulator of RNA polymerase II transcription . Recently, we showed that the RING domain CNOT4 subunit contains intrinsic ubiquitin-protein ligase (E3) activity . Here we show that binding of the CNOT4 RING finger to the ubiquitin-conjugating enzyme (E2) UbcH5B is highly selective . To understand the basis for this interaction, we identified several basic residues of UbcH5B important for binding to CNOT4 by mutational analysis . Subsequently, we tested pairs of UbcH5B and CNOT4 mutants for restoration of interaction . Concomitant charge-alteration of E49 of CNOT4 and K63 of UbcH5B restored binding and re-created a functional enzyme pair, indicative of an electrostatic interaction between these residues . The corresponding amino acids in the yeast orthologues can also be used to create a similarly designed E2-E3 enzyme pair . These are the first examples of altered-specificity E2-E3 enzyme pairs and give further insight into how E2-E3 specificity is obtained. J Mol Biol, 2004 Mar 12, 337(1), 93 - 104 Cofilin (ADF) affects lateral contacts in F-actin; Bobkov AA et al.; The effect of yeast cofilin on lateral contacts between protomers of yeast and skeletal muscle actin filaments was examined in solution . These contacts are presumably stabilized by the interactions of loop 262-274 of one protomer with two other protomers on the opposite strand in F-actin . Cofilin inhibited several-fold the rate of interstrand disulfide cross-linking between Cys265 and Cys374 in yeast S265C mutant F-actin, but enhanced excimer formation between pyrene probes attached to these cysteine residues . The possibility that these effects are due to a translocation of the C terminus of actin by cofilin was ruled out by measurements of fluorescence resonance energy transfer (FRET) from tryptophan residues and ATP to acceptor probes at Cys374 . Such measurements did not reveal cofilin-induced changes in FRET efficiency, suggesting that changes in Cys265-Cys374 cross-linking and excimer formation stem from the perturbation of loop 262-274 by cofilin . Changes in lateral interactions in F-actin were indicated also by the cofilin-induced partial release of rhodamine phalloidin . Disulfide cross-linking of S265C yeast F-actin inhibited strongly and reversibly the release of rhodamine phalloidin by cofilin . Overall, this study provides solution evidence for the weakening of lateral interactions in F-actin by cofilin. Microb Pathog, 2004 Apr, 36(4), 177 - 88 The in vitro interaction of Sporothrix schenckii with human endothelial cells is modulated by cytokines and involves endothelial surface molecules; Figueiredo CC et al.; Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis that can evolve to systemic complications in immunocompromised patients . Interactions with endothelium are thought to be essential for systemic infections . In the present work, we studied the interaction between S . schenckii and human umbilical vein endothelial cells (HUVECs) . S . schenckii interacts with HUVECs in a time-dependent manner . Morphological analysis showed that yeasts locate to interendothelial junctions . Ultrastructural studies showed that internalized yeasts were found inside endocytic vacuoles as early as 2 h, without causing any detectable damage to HUVECs after 24 h of infection . The viability of infected HUVECs was confirmed by the MTT assay . When HUVECs were treated with different concentrations of Interleukin-1beta or transforming growth factor-beta, a significant dose-dependent increase in cell-associated yeasts was observed . The preliminary analysis of the endothelial surface ligands for S . schenckii cells revealed two major molecules, with Mr of approximately 90 and 135 kDa . The interaction of endothelial cell surface molecules with S . schenckii yeast cells was modulated by divalent cations . This is the first demonstration that S . schenckii is able to adhere and invade endothelial cells without significantly affect cellular integrity . Our results suggest the contribution of cytokine-modulated calcium-dependent molecules to this process. Semin Cell Dev Biol, 1997 Feb, 8(1), 71 - 8 Regulation of the export of RNA from the nucleus; Cole CN et al.; Transport of macromolecules across the nuclear envelope is an essential activity in eukaryotic cells . RNA molecules within cells are found complexed with proteins and the bound proteins likely contain signals for RNA export . RNAs microinjected into Xenopus oocyte nuclei are readily exported, and their export can be competed by self RNA but not by RNAs of other classes . This indicates that the rate-limiting step in RNA export is the interaction of RNAs with class-specific proteins, at least when substrate RNAs are present at saturating levels . Export of host mRNAs is inhibited following infection by some animal viruses, while the export of viral RNAs occurs . The HIV-1 RNA-binding protein, Rev, mediates the export of intron-containing viral RNAs that would normally be retained in nuclei . This requires a nuclear export signal (NES) within Rev and an element within the RNA to which Rev binds . In yeast, heat shock causes accumulation of poly(A)(+)RNA within nuclei but heat-shock mRNAs are transcribed and exported efficiently . This requires elements within heat shock mRNA that probably interact with a cellular protein to facilitate RNA export . In these cases, the proteins that recognize critical sequences in the RNAs probably direct the RNAs to an RNA export pathway not generally used for mRNA export . This would circumvent the general retention of most poly(A)(+)mRNAs following heat shock in yeast and the need for complete splicing of viral mRNAs that travel through the normal mRNA export pathway. Biotechnol Lett, 2004 Jan, 26(2), 103 - 8 Factors enhancing lycopene production by a new Mycobacterium aurum mutant; Kerr S et al.; A mutant strain of Mycobacterium aurum was isolated that produced mainly lycopene (>80%) with a total carotenoid content of 1.2 mg g(-1) dry biomass when grown on yeast extract and glucose . Lycopene content of the cells could be significantly increased, up to 7.4 mg g(-1) biomass, by growing the cells at suboptimal initial culture pH (pH 6-6.4) or by using high salt concentration (85 mM NaCl) in the culture medium, although a 25-40% decrease in biomass production occurred in both cases . Highestproductivity (4 mg lycopene l(-1) d(-1)) was obtained by cultivating the cells at pH 6. J Anim Sci, 2003, 81 Suppl 3, 48 - 57 Proteomics methods for probing molecular mechanisms in signal transduction; Sheffield LG et al.; mRNA splicing and various posttranslational modifications to proteins result in a larger number of proteins than genes . Assessing the dynamic nature of this proteome is the challenge of modern proteomics . Recent advances in high throughput methods greatly facilitate the analysis of proteins involved in signal transduction, their production, posttranslational modifications and interactions . Highly reproducible two dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods, coupled with matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS) allow rapid separation and identification of proteins . These methods, alone or in conjunction with other techniques such as immunoprecipitation, allow identification of various critical posttranslational modifications, such as phosphorylation . High throughput identification of important protein-protein interactions is accomplished by yeast two hybrid approaches . In vitro and in vivo pulldown assays, coupled with MALDI-TOF-MS, provide an important alternative to two hybrid approaches . Emerging advances in production of protein-based arrays promise to further increase throughput of proteomics-based approaches to signal transduction. Nucleic Acids Res, 2004 Mar 03, 32(4), 1527 - 38 Print 2004. Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase; Calmels C et al.; HIV-1 integrase (IN) catalyzes the integration of the proviral DNA into the cellular genome . The catalytic triad D64, D116 and E152 of HIV-1 IN is involved in the reaction mechanism and the DNA binding . Since the integration and substrate binding processes are not yet exactly known, we studied the role of amino acids localized in the catalytic site . We focused our interest on the V151E152S153 region . We generated random mutations inside this domain and selected mutated active INs by using the IN-induced yeast lethality assay . In vitro analysis of the selected enzymes showed that the IN nuclease activities (specific 3'-processing and non-sequence-specific endonuclease), the integration and disintegration reactions and the binding of the various DNA substrates were affected differently . Our results support the hypothesis that the three reactions may involve different DNA binding sites, enzyme conformations or mechanisms . We also show that the V151E152S153 region involvement in the integration reaction is more important than for the 3'-processing activity and can be involved in the recognition of DNA . The IN mutants may lead to the development of new tools for studying the integration reaction, and could serve as the basis for the discovery of integration-specific inhibitors. Nucleic Acids Res, 2004 Mar 03, 32(4), 1492 - 501 Print 2004. RING1 inhibits transactivation of RBP-J by Notch through interaction with LIM protein KyoT2; Qin H et al.; The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC) . In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors . KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation . Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells . The interaction between KyoT2 and RING1 was detected both in vitro and in vivo . By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively . These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells . Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC . Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2 . Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. J Biochem (Tokyo), 2004 Jan, 135(1), 117 - 28 The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1; Shibata H et al.; ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca(2+)-dependent manner . The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix . The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2 . This region includes four-tandem PxY repeats . Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2 . Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca(2+) with FLAG-tagged Alix or FLAG-tagged Alix Delta EBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged Alix Delta ABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct . FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca(2+)-dependent fashion, whereas FLAG-tagged ALG-2(E47A/E114A), a Ca(2+)-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca(2+) . Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCT Delta ABS is observed diffusely in the cytoplasm . These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca(2+)-dependent interaction with Alix. J Biol Chem, 2004 May 7, 279(19), 19755 - 63 Epub 2004 Mar 03. Salicylihalamide A inhibits the V0 sector of the V-ATPase through a mechanism distinct from bafilomycin A1; Xie XS et al.; The newly identified specific V-ATPase inhibitor, salicylihalamide A, is distinct from any previously identified V-ATPase inhibitors in that it inhibits only mammalian V-ATPases, but not those from yeast or other fungi (Boyd, M . R., Farina, C., Belfiore, P., Gagliardi, S., Kim, J . W., Hayakawa, Y., Beutler, J . A., McKee, T . C., Bowman, B . J., and Bowman, E . J . (2001) J . Pharmacol . Exp . Ther . 297, 114-120) . In addition, salicylihalamide A does not compete with concanamycin or bafilomycin for binding to V-ATPase, indicating that it has a different binding site from those classic V-ATPase inhibitors (Huss, M., Ingenhorst, G., Konig, S., Gassel, M., Drose, S., Zeeck, A., Altendorf, K., and Wieczorek, H . (2002) J . Biol . Chem . 277, 40544-40548) . By using purified bovine brain V-pump and its dissociated V(1) and V(0) sectors, we identified the recognition and binding site for salicylihalamide to be within the V(0) domain . Salicylihalamide does not inhibit the ATP hydrolysis activity of the dissociated V(1)-ATPase but inhibits the ATPase activity of the holoenzyme by inhibiting the V(0) domain . Salicylihalamide causes a dramatic redistribution of cytosolic V(1) from soluble to membrane-associated form, a change not observed in cells treated with either bafilomycin or NH(4)Cl . By synthesizing and characterizing a series of salicylihalamide derivatives, we investigated the structural determinants of salicylihalamide inhibition in terms of potency and reversibility, and used this information to suggest a possible binding mechanism. J Biol Chem, 2004 May 7, 279(19), 19764 - 74 Epub 2004 Mar 03. Transcriptional regulation of mouse mu opioid receptor gene by PU.1; Hwang CK et al.; We previously reported that the 34-bp cis-acting element of the mouse micro opioid receptor (MOR) gene represses transcription of the MOR gene from the distal promoter . Using a yeast one-hybrid screen to identify potential transcription factors of the MOR promoter, we have identified PU.1 as one of the candidate genes . PU.1 is a member of the ets family of transcription factors, expressed predominantly in hematopoietic cells and microglia of brain . PU.1 plays an essential role in the development of both lymphoid and myeloid lineages . Opioids exert neuromodulatory as well as immunomodulatory effects, which are transduced by MOR . Moreover, MOR-deficient mice exhibit increased proliferation of hematopoietic cells, suggesting a possible link between the opioid system and hematopoietic development . The PU.1 protein binds to the 34-bp element of the MOR gene in a sequence-specific manner confirmed by electrophoretic mobility shift assay and supershift assays . We have also determined endogenous PU.1 interactions with the 34-bp element of MOR promoter by chromatin immunoprecipitation assays . In co-transfection studies PU.1 represses MOR promoter reporter constructs through its PU.1 binding site . When the PU.1 gene is disrupted as in PU.1 knock-out mice and using small interfering RNA-based strategy in RAW264.7 cells, the transcription of the endogenous target MOR gene is increased significantly . This increase is probably mediated through modification of the chromatin structure, as suggested by the reversal of the PU.1-mediated repression of MOR promoter activity after trichostatin A treatment in neuroblastoma NMB cells . Our results suggest that PU.1 may be an important regulator of the MOR gene, particularly in brain and immune cells. Fungal Genet Biol, 2004 Apr, 41(4), 428 - 42 Aspergillus nidulans as a model system to characterize the DNA damage response in eukaryotes; Goldman GH et al.; Interest in DNA repair in Aspergillus nidulans had mainly grown out of studies of three different biological processes, namely mitotic recombination, inducible responses to detrimental environmental changes, and genetic control of the cell cycle . Ron Morris started the investigation of the genetic control of the cell cycle by screening hundreds of cell cycle temperature sensitive Aspergillus mutants . The sequencing and innovative analysis of these genes revealed not only several components of the cell cycle machinery that are directly involved in checkpoint response, but also components required for DNA replication and DNA damage response machinery . Here, we will provide an overview about currently known aspects of the DNA damage response in A . nidulans . Emphasis is put on analyzed mutants that are available and review epistatic relationships and other interactions among them . Furthermore, a comprehensive list of A . nidulans genes involved in different processes of the DNA damage response, as identified by homology of genome sequences with well-characterized human and yeast DNA repair genes, is shown. Oecologia . 2004 Mar 3; {Epub ahead of print} Lipid content and carbon assimilation in Collembola: implications for the use of compound-specific carbon isotope analysis in animal dietary studies; Chamberlain PM et al.; In an effort to understand the relationships between both the lipid content and delta(13)C values of Collembola and their diet, isotopically labelled (C(3) and C(4)) bakers' yeasts were cultured and fed to two Collembolan species, Folsomia candida and Proisotoma minuta . The fatty acid composition of Collembola generally reflected that of the diet with the addition of the polyunsaturated components 18:2(n-6), 20:4(n-6) and 20:5(n-3), which appeared to be biosynthesised by the Collembola . Whilst ergosterol was the only sterol detected in the yeast diets, only cholesterol was detected in Collembola, and although the delta(13)C values of diet and consumer sterols differed by >2 per thousand, the delta(13)C values indicated that cholesterol was derived entirely from dietary sterol . The bulk delta(13)C values of Collembola were similar to those of the diets, but fatty acid delta(13)C values did not necessarily reflect those of the dietary fatty acids, indicating significant de novo biosynthesis of fatty acids within Collembola . Switching the Collembola from C(3) to C(4) yeast enabled the determination of the rates of incorporation of dietary carbon into Collembolan lipids, and showed that half-lives of the incorporation of dietary carbon varied between 1.5 and 5.8 days at 20 degrees C . Cholesterol exhibited the slowest rate of incorporation in both species, while bulk carbon in F . candida possessed an intermediate rate . These results demonstrate that an understanding of the sources of isotopic fractionation and the role of biochemistry in regulating the delta(13)C values of individual compounds is important in the application of compound-specific isotopic analysis to the study of animal trophic activities. J Agric Food Chem, 2004 Mar 10, 52(5), 1410 - 4 Evaluation of estrogenic activity in diets for experimental animals using in vitro assay; Kato H et al.; We used a modified yeast-based human estrogen receptor alpha (ER alpha) bioassay to determine the estrogenic activity in 22 kinds of diets for experimental animals . The estrogenic activity of each diet was reevaluated by comparison with a calibration curve of 17 beta-estradiol . Almost all of the diets had estrogenic activity . The diets for rabbits and guinea pigs had the highest estrogenic activity compared to any other diets, including those for rats and mice . Estrogenic activity was found in dried skim milk, fishmeal, soybean meal, and alfalfa meal . In the NIH-07 diet opened for the ingredients, estrogenic activity was nearly all derived from the alfalfa meal . Multiple assays were performed to evaluate potential seasonal variations in the estrogenic potency in the raw materials of the rat and mouse diets . We found that the estrogenic activity in these raw materials changed throughout the year. J Agric Food Chem, 2004 Mar 10, 52(5), 1385 - 9 Antioxidant effects of chromium supplementation with type 2 diabetes mellitus and euglycemic subjects; Cheng HH et al.; To determine the effects of chromium (Cr) supplementations on oxidative stress of type 2 diabetes and euglycemic (EU) subjects, adult having HbA(1C) values of <6.0% (EU), 6.8-8.5% (mildly hyperglycemic, MH), and >8.5% (severely hyperglycemic, SH) were supplemented for 6 months with 1000 microg/day of Cr (as Cr yeast) or with a placebo . In the beginning, the levels of the plasma Cr in the MH and SH groups were 25-30% lower than those of the EU subjects . The values of thiobarbituric acid reactive substances (TBARS) and total antioxidative status (TAS) of the MH and SH groups were significantly higher than those of the EU ones . Following supplementations, the levels of plasma TBARS in the Cr groups of MH and SH groups were significantly decreased (the inverse was found in the EU) and showed no significant changes in the placebo group . The levels of plasma TAS in the Cr groups of EU and MH were significantly decreased (the inverse was found in the SH) and showed no significant changes in the placebo group . No significant difference was found in the antioxidant enzyme (superoxide dismutase, glutathione peroxidase, catalase) activities during supplementations . These data suggest that Cr supplementation was an effective treatment strategy to minimize increased oxidative stress in type 2 diabetes mellitus patients whose HbA(1C) level was >8.5%, and the Cr in EU groups might act as a prooxidant. Cell Mol Biol (Noisy-le-grand), 2003, 49 Online Pub, OL467 - 71 Human reticulon 1-A and 1-B interact with a medium chain of the AP-2 adaptor complex; Iwahashi J et al.; Human reticulon family gene 1 (RTN1) is expressed predominantly in neuroendocrine tissues, and produces three proteins termed RTN1-A, RTN1-B, and RTN1-C . Yeast two-hybrid screening indicated that RTN1-A and RTN1-B interacted with A