Yeasts

Mol Biol Cell, 2002 Jul, 13(7), 2289 - 300
Disruption of centrosome structure, chromosome segregation, and cytokinesis by misexpression of human Cdc14A phosphatase; Kaiser BK et al.; In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis . Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B . We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated . hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus . These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events . In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and gamma-tubulin from centrosomes . Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis . Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

J Virol, 2002 Aug, 76(16), 8208 - 17
Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49; Fuchs W et al.; Envelope glycoprotein M (gM) and the complex formed by glycoproteins E (gE) and I (gI) are involved in the secondary envelopment of pseudorabies virus (PrV) particles in the cytoplasm of infected cells . In the absence of the gE-gI complex and gM, envelopment is blocked and capsids surrounded by tegument proteins accumulate in the cytoplasm (A . R . Brack, J . Dijkstra, H . Granzow, B . G . Klupp, and T . C . Mettenleiter, J . Virol . 73:5364-5372, 1999) . Here we demonstrate by yeast two-hybrid analyses that the cytoplasmic domains of gE and gM specifically interact with the C-terminal part of the UL49 gene product of PrV, which represents a major tegument protein and which is homologous to VP22 of herpes simplex virus type 1 . However, deletion of the UL49 gene from PrV had only minor effects on viral replication, and ultrastructural analyses of infected cells confirmed that virus maturation and egress, including secondary envelopment in the cytoplasm, were not detectably affected by the absence of UL49 . Moreover, the UL49 gene product was shown to be dispensable for virion localization of gE and gM, and mutants lacking either gE or gM incorporated the UL49 protein efficiently into virus particles . In contrast, a PrV mutant with deletions of gE-gI and gM failed to incorporate the UL49 protein despite apparently unaltered intracytoplasmic UL49 expression . In summary, we describe specific interactions between herpesvirus envelope and tegument proteins which may play a role in secondary envelopment during herpesvirus virion maturation.

J Virol, 2002 Aug, 76(16), 8019 - 30
RelA-associated inhibitor blocks transcription of human immunodeficiency virus type 1 by inhibiting NF-kappaB and Sp1 actions; Takada N et al.; RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor kappaB (NF-kappaB) newly identified by yeast two-hybrid screen as an interacting protein of the p65 (RelA) subunit . In this study, we attempted to examine the effect of RAI on transcription and replication of human immunodeficiency virus type 1 (HIV-1) . We found that RAI inhibited gene expression from the HIV-1 long terminal repeat (LTR) even at the basal level . Upon in vitro DNA-binding reactions, RAI could directly block the DNA-binding of p65 subunit of NF-kappaB but not that of the p50 subunit or AP1 . We found that RAI could also inhibit the DNA-binding of Sp1 and thus inhibit the basal HIV-1 promoter activity . We further examined the effects of RAI on Sp1 and found that RAI colocalizes with Sp1 in the nucleus and interacts with Sp1 in vitro and in vivo . Moreover, we found that RAI efficiently blocked the HIV-1 replication when cotransfected with a full-length HIV-1 clone . These findings indicate that RAI acts as an efficient inhibitor of HIV-1 gene expression in which both NF-kappaB and Sp1 play major roles.

J Biol Chem, 2002 Sep 27, 277(39), 36296 - 303 Epub 2002 Jul 19.
Differential localization of the vacuolar H+ pump with G subunit isoforms (G1 and G2) in mouse neurons; Murata Y et al.; Vacuolar H(+)-ATPases (V-ATPases), a family of multimeric proton pumps, are involved in a wide variety of physiological processes . We have identified two mouse genes, Atp6g1 and Atp6g2, encoding the G1 and G2 isoforms of the V-ATPase G subunit, respectively . G1 was distributed ubiquitously in the tissues examined, whereas G2 was specifically distributed in central nervous system neurons . G1 was expressed at an early embryonic stage, whereas G2 transcription was significantly induced at 10.5 days postcoitus (embryonic day 10.5, i.e . 2 days before axon outgrowth) . Both G1 and G2 were strongly expressed in cortical and hippocampal neurons, cerebellar granule cells, and Purkinje cells . Immunohistochemistry with isoform-specific antibodies revealed that G2 was localized in cell bodies, dendrites, and axons . In addition, electron microscopy and subcellular fractionation indicated that G2 was localized in synaptic vesicles, whereas G1 was not detectable . G1 and G2 exhibit 62% identity, and both isoforms were immunoprecipitated with the c and A subunits of V-ATPase . G2 could complement the yeast deletion mutant Deltavma10, which lacks the G subunit . The V-ATPases containing the G1 and G2 isoforms, respectively, showed similar K(m)((ATP)) values and maximal velocity . These results indicate that G1 and G2 are bona fide subunits of V-ATPases and that the enzyme with the G2 isoform is involved in synaptic vesicle acidification.

Biochem J, 2002 Nov 15, 368(Pt 1), 213 - 21
Evidence for crucial electrostatic interactions between Bcl-2 homology domains BH3 and BH4 in the anti-apoptotic Nr-13 protein; Lalle P et al.; Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax . The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax . The Bax-interacting domain has been identified and corresponds to alpha-helices 5 and 6 in Nr-13 . Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4) . The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3 . Disruption of these interactions severely affects Nr-13 anti-apoptotic activity . Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.

Biochem J, 2002 Nov 1, 367(Pt 3), 587 - 99
Purification, characterization and catalytic properties of human sterol 8-isomerase; Nes WD et al.; CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a . The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity . The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric . The structural features of zymosterol provided optimal substrate acceptability . Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI . Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products . The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol . Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7) . Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM) <ketoconazole (156 microM)<cholesterol (620 microM) . The results were consistent with stereoelectronic features of isomerization and support the general model for Delta(7)-sterol formation in cholesterol synthesis.

Mol Cells, 2002 Jun 30, 13(3), 493 - 7
Involvement of subcomplexes of 32 and 14 kDa subunits in RPA's DNA binding activity through redox change; Kim A et al.; The eukaryotic replication protein A (RPA) is a heterotrimeric protein complex . It consists of 70, 32, and 14 kDa subunits that are involved in DNA replication, repair, and genetic recombination . RPA is a 4-cysteine type zinc-finger protein . RPA's zinc-finger domain is not essential for DNA binding activity, but it is involved in the regulation of RPA's DNA binding activity through reduction-oxidation (redox) . In this study, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its subcomplexes of 32 and 14 kDa subunits . In contrast, the subunits' complex, RPA70, formed a stable complex with ssDNA, even under non-reducing conditions . The addition of DTT and H202 had no effect on its DNA binding activity . In RPA70, since the addition of the subcomplexes of the 32 and 14 kDa subunits, it restored the modulating ssDNA binding activity to native RPA's DNA binding activity . These results suggest that the subcomplexes of the 32 and 14 kDa subunits may be involved in the modulating RPA's DNA binding activity through redox change . These studies, therefore, show the novel structure and function relationship of a multiprotein complex in that the role of a specific domain (or one subunit) is regulated by the other subunits.

Zoolog Sci, 2002 May, 19(5), 539 - 44
Analyses of mRNA expression patterns of cohesin subunits Rad21 and Rec8 in mice: germ cell-specific expression of rec8 mRNA in both male and female mice; Lee J et al.; A multisubunit protein complex called cohesin is required for the cohesion between sister chromatids in both mitosis and meiosis in yeast . We investigate here the mRNA expression patterns of mouse homologues of the yeast mitotic cohesin rad21 and the meiotic cohesin rec8 in various organs, with special attention to their expression in gonads . Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that, in contrast to the ubiquitous expression of rad21 mRNA in all of the organs examined, rec8 was expressed only in the gonads . We conducted in situ hybridization analysis to identify the cells that express rad21 and rec8 mRNAs in the gonads . In the testis, rad21 mRNA was expressed in somatic cells and spermatogonia but not in spermatocytes, and conversely, rec8 mRNA was expressed in spermatocytes but not in spermatogonia or somatic cells . Spermatids expressed rad21 and rec8 mRNAs simultaneously . In the ovary, rad21 mRNA was detected in all of the ovarian cells including germ cells and somatic cells, whereas rec8 mRNA was detected only in oocytes . Unlike the widespread expression of rad21 gene, therefore, the gene expression of rec8 is strictly confined to spermatocytes and spermatids in male mouse and oocytes in female mouse . The restricted expression pattern of rec8 mRNA implies its essential role in meiosis in both sexes of mammals, as has been reported in yeast . We also discuss the cooperative functions of Rad21 and Rec8 on the basis of the finding that their mRNAs are coexpressed in oocytes and spermatids.

Mol Pharmacol, 2002 Aug, 62(2), 366 - 78
Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors; Makinen J et al.; The constitutive androstane receptor (CAR) regulates mouse and human CYP2B genes through binding to the direct repeat-4 (DR4) motifs present in the phenobarbital-responsive enhancer module (PBREM) . The preference of PBREM elements for nuclear receptors and the extent of cross-talk between CAR and other nuclear receptors are currently unknown . Our transient transfection and DNA binding experiments indicate that binding to DR4 motifs does not correlate with the activation response and that mouse and human PBREM are efficiently 'insulated' from the effects of other nuclear receptors despite their substantial affinity for DR4 motifs . Certain nuclear receptors that do not bind to DR4 motifs, such as peroxisome proliferator-activated receptor-alpha and farnesoid X receptor, can suppress PBREM function via a coactivator-dependent process that may have relevance in vivo . In competition experiments, mouse PBREM is clearly more selective for CAR than human PBREM . Pregnane X, vitamin D, and thyroid hormone receptors can potentially compete with human CAR on human PBREM . In contrast to the selective nature of PBREM, CYP3A enhancers are highly and comparably responsive to CAR, pregnane X receptor, and vitamin D receptor . In addition, the ligand specificities of human and mouse CAR were defined by mammalian cotransfection and yeast two-hybrid techniques . Our results provide new mechanistic explanations to several previously unresolved aspects of CYP2B and CYP3A gene regulation.

Comp Biochem Physiol B Biochem Mol Biol, 2002 Aug, 132(4), 721 - 8
Carotenol fatty acid esters: easy substrates for digestive enzymes?
Breithaupt DE, Bamedi A, Wirt U.
To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied . Digestions were carried out in phosphate buffer at pH 7.4 and 37 degrees C . As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; beta-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; beta-cryptoxanthin esters) as well as retinyl palmitate . These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase . As reference enzyme a yeast lipase from Candida rugosa was applied . A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut . Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed . The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (U/mg solid) . Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility .

Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 910 - 6
Identification of putative mammalian D-lactate dehydrogenase enzymes; Flick MJ et al.; Mammalian L-isomer dehydrogenases represent an expansive and well characterized class of metabolic enzymes . Surprisingly, little is known regarding their evolutionarily distinct counterparts, D-isomer dehydrogenases, since few mammalian D-isomer 2-hydroxy acid enzymes have been isolated . Here we present the identification and initial characterization of putative human and murine D-lactate dehydrogenases (DLD) that can interact with the muscle-specific cysteine-rich protein CRP3/MLP . Sequence analysis reveals that the human and mouse transcripts encode novel proteins that display strong similarities to the yeast D-lactate dehydrogenase proteins DLD1, AIP2, and YEL071W . Expression analysis of the mammalian proteins indicates widespread distribution with transcripts present in striated muscle tissues and a variety of other tissue types . Immunofluorescence subcellular localization of the mouse DLD protein indicates that it resides within mitochondria, a feature shared by many dehydrogenases . The identification of the human and mouse DLD clones provides new insight regarding the activity of D-isomer-specific enzymes in mammalian cells.

Int J Biochem Cell Biol, 2002 Oct, 34(10), 1190 - 206
Possible physiological roles of mitochondrial uncoupling proteins--UCPn; Jezek P; Five mitochondrial uncoupling proteins exist in the human gemone: UCP2, expressed ubiquitously; UCP1, exclusively in brown adipose tissue (BAT); UCP3, predominantly in muscle; UCP4 and BMCP (UCP5), in brain . UCP4 is the ancestral prototype from which the other UCPn diverged . Findings on the level of organism and reconstituted recombinant proteins demonstrated that UCPn exhibit a protonophoric function, documented by overexpression in mice, L6 myotubes, INS1 cells, muscle, and yeast . In a few cases (yeast), this protonophoric function was correlated with elevated fatty acid (FA) levels . Reconstituted UCPn exhibited nucleotide-sensitive FA induced H(+) uniport . Two mechanisms, local buffering or FA cycling were suggested as an explanation . A basic UCPn role with mild uncoupling is to accelerate metabolism and reduce reactive oxygen species . UCP2 (UCP3) roles were inferred from transcriptional up-regulation mediated by FAs via peroxisome proliferator-activated receptors, cytokines, leptin signalling via hypothalamic pathway, and by thyroide and beta2 adrenergic stimulation . The latter indicated a role in catecholamine-induced thermogenesis in skeletal muscle . UCP2 (UCP3) may contribute to body weight regulation, although obesity was not induced in knockout (KO) mice . An obesity reduction in middle-aged humans was associated with the less common allele of -866 G/A polymorphism in the ucp2 gene promoter enhancing the exon 8 insertion: deletion transcript ratio . Up-regulated UCP2 transcription by pyrogenic cytokines (tumour necrosis factor alpha (TNFalpha)) suggested a role in fever . UCP2 could induce type 2 diabetes as developed from obesity due to up-regulated UCP2 transcription by FAs in pancreatic beta-cells . UCPn might be pro-apoptotic as well as anti-apoptotic, depending on transcriptional and biochemical regulation .

J Neurooncol, 2002 May, 57(3), 169 - 77
Expression of Nedd5, a mammalian septin, in human brain tumors; Sakai K et al.; The septins are a family of cytoskeletal GTPases that play an essential role in cytokinesis in yeast and mammalian cells . Nedd5 is a mammalian septin known to associate with actin-based structures such as the contractile ring and stress fibers . In the present study, we examined the expression of Nedd5 in a series of human brain tumor cell lines and surgical specimens by northern and western analyses . The temporal expression of Nedd5 in U373 astrocytoma cells at various timepoints throughout the cell cycle was determined by an analysis of lovastatin- and nocodazole-treated, synchronized cell populations . The intracellular localization of Nedd5 was determined by immunocytochemistry of steady state cultures and nocodazole-treated cultures enriched in M phase cells . The effects of inhibiting Nedd5 expression in human brain tumors was determined by stably transfecting U373 astrocytoma cells with an antisense-Nedd5 cDNA expression vector and by analyzing clones for Nedd5 expression by immunocytochemistry, morphological changes, cell growth and nuclear content . All human brain tumor cell lines and surgical specimens expressed Nedd5 transcript and protein . Synchronized U373 astrocytoma cells showed a relative increase in Nedd5 transcript levels from late Gl to G2M phases; and an increase in Nedd5 protein levels from S to G2M phases . Maximum expression of both transcript and protein levels was observed at the G2M phase . By immunocytochemistry, Nedd5 was concentrated at the cleavage furrow of mitotic cells . Double staining with Nedd5 and F-actin showed co-localization of Nedd5 with actin filaments except during cytokinesis . Antisense-Nedd5 expression led to an accumulation of nuclear content . These data suggest that Nedd5 is involved in the process of cytokinesis in human brain tumours . Nedd5 expression may be cell cycle-dependent with increased levels found at G2M phase . Blocking Nedd5 expression in astrocytoma cells by antisense interferes with the process of cytokinesis during cell division.

J Vector Ecol, 2002 Jun, 27(1), 39 - 43
The effects of diet upon pupal development and cocoon formation by the cat flea (Siphonaptera: Pulicidae); Lawrence W et al.; Cocoon formation by cat flea larvae was directly related to the quantity of eggs or yeast consumed . Larvae consuming either 1-3 eggs or 0.25-1.0 mg of yeast developed as naked pupae or formed incomplete cocoons . Third instar cat flea larvae fed upon naked pupae and pupae within partial cocoons, but complete cocoons protected pupae from cannibalism . First and second instars did not attack pupae . When larvae were provided with a carpet fiber for protection, a greater number of fleas successfully developed to the adult stage.

Curr Mol Med, 2002 Aug, 2(5), 439 - 44
Mutated genes in juvenile and variant late infantile neuronal ceroid lipofuscinoses encode lysosomal proteins; Vesa J et al.; Positional cloning efforts of genes mutated in Batten disease and in the Finnish type of variant late infantile neuronal ceroid lipofuscinosis resulted in the identification of two novel genes, CLN3 and CLN5, and corresponding gene products that proved to be residents of lysosomes . Although the clinical phenotype of these NCL subtypes differs in the age of onset, average life span and EEG findings, the major component of material accumulating in patients' lysosomes is subunit c of mitochondrial ATPase in both these diseases . The CLN3 and CLN5 genes show ubiquitous expression patterns and are targeted to lysosomes in vitro, but the observed synaptosomal localization of the CLN3 protein in neurons would suggest some cell specificity in targeting and function of these proteins . So far, 31 different mutations of the CLN3 gene have been described in Batten patients, with one deletion of 1.02 kb accounting for 75% of disease alleles worldwide . Four CLN5 mutations are known, with one premature stop representing the major founder mutation in the isolated Finnish population . Functional studies of the yeast homolog of CLN3 and increased pH in patients' lysosomes would suggest an involvement of this protein in lysosomal pH homeostasis . Knock-out mouse models for CLN3 have been produced and the histopathology bears a close resemblance to human counterparts with characteristic lysosomal accumulations . Both CLN3 and CLN5 mouse models will provide experimental tools to resolve the pathological cascade in these neurodegenerative diseases.

Yeast, 2002 Aug, 19(11), 973 - 89
PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes; Kanbe T et al.; For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C . albicans, C . dubliniensis, C . tropicalis (genotypes I and II), C . parapsilosis (genotypes I and II), C . krusei, C . kefyr, C . guilliermondii, C . glabrata, C . lusitaniae and Y . lipolytica were designed and their specificities tested in PCR-based identifications . Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp . but also from DNA mixtures containing other DNAs from several fungal species . For a simpler PCR-based identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs . PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp . in the sample DNA . To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes . By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample . Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients . In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests . These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials .

Cancer, 2002 Jul 15, 95(2), 249 - 57
Influence of p53 mutations on prognosis of patients with glioblastoma; Shiraishi S et al.; BACKGROUND: The influence of p53 mutations on the biology of astrocytic tumors is controversial . p53 is thought to be inactivated in the early stage of gliomagenesis; however, what role its inactivation plays in the malignancy of gliomas remains unknown . To understand the significance of p53 inactivation, the authors identified the locus of p53 gene mutation in glioma samples at different stages of progression and studied the correlation between the mutation and clinical behavior . METHODS: Samples from newly diagnosed gliomas, including pure and mixed astrocytomas, were analyzed for p53 mutations using a yeast functional assay . To determine the locus of the gene mutations, DNA sequencing was performed . RESULTS: The incidence of p53 mutations was higher in anaplastic astrocytomas (AA, 48%) than glioblastomas (GBM, 31%) . There was no significant difference in the average ages of GBM patients with and without p53 mutations (54.9 years +/- 2.3 and 53.2 years +/- 4.6, respectively) . In GBM patients, the mutation did not affect progression free survival or overall survival . Astrocytomas and GBM differed in the distribution of p53 mutation loci . CONCLUSIONS: The p53 gene mutation does not markedly affect the survival of GBM patients . The difference in the location of p53 mutations between AA and GBM suggests that in gliomas, the p53 mutation may contribute not only to tumorigenesis (as an early event) but also to progression to malignancy (as a late event) .

Am J Med Genet, 2002 Jul 22, 111(1), 76 - 80
Duplication of (2)(q11.1-q13.2) in a boy with mental retardation and cleft lip and palate: another clefting gene locus on proximal 2q?
Riegel M, Schinzel A.
A 4-year-old boy with left cleft lip and cleft palate, multiple minor anomalies and developmental delay revealed an abnormal chromosome 2 with enlarged proximal long arm, de novo, in his karyotype . Fluorescence in situ hybridization with a chromosome 2 library and band-specific YACs confined the duplicated segment to 2q11.1-q13.2 and indicated a direct tandem duplication due to unbalanced crossover between chromatids .

FEBS Lett, 2002 Jul 17, 523(1-3), 193 - 9
Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta; Engelke M et al.; Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly . However, little is known which domains facilitate the interaction of subunits . Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells . MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants . Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus . These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Neuroscience, 2002, 113(1), 23 - 35
Similar ultrastructural distribution of the 5-HT(2A) serotonin receptor and microtubule-associated protein MAP1A in cortical dendrites of adult rat; Cornea-Hebert V et al.; As visualized by light and electron microscopic immunocytochemistry, the distribution of the neuronal serotonin-2A (5-HT(2A)) receptor is mainly intracellular throughout adult rat brain . This localization is particularly striking in the pyramidal cells of cerebral cortex, the dendrites of which are intensely immunoreactive, but without any labeling of their spines . In view of recent yeast two-hybrid and biochemical results suggesting an association of 5-HT(2A) receptors with the cytoskeletal microtubule-associated protein MAP1A, the respective subcellular distributions of the receptors and of MAP1A were compared by quantitative electron microscopic immunocytochemistry in dendrites of adult rat frontoparietal cortex . Counts of silver-intensified immunogold particles revealed a higher density of 5-HT(2A) receptors in smaller rather than larger dendrites, and an apportionment between pre-defined compartments representing the plasma membrane and the cytoplasm that was proportional to the relative surface area of these compartments . MAP1A immunoreactivity also predominated in smaller versus larger dendrites, but with a slightly lower proportion of labeling in the plasma membrane versus cytoplasmic compartment . The co-localization of 5-HT(2A) receptors and MAP1A protein in the same dendrites could be demonstrated in double immunolabeling experiments . These results confirmed the predominantly somato-dendritic, intracellular localization of 5-HT(2A) receptors in cerebral cortex, showed their higher concentration in distal as opposed to proximal dendrites, and suggested their potential association to the cytoskeleton in cortical neurons in vivo.Such a distribution of 5-HT(2A) receptors reinforces our earlier hypothesis that 5-HT(2A) receptors participate in intraneuronal signaling processes involving the cytoskeleton, and raises the possibility that their activation could be dependent upon that of another co-localized, plasma membrane-bound, 5-HT receptor.

Curr Biol, 2002 Jun 25, 12(12), R415 - 6
Genetic architecture: dissecting the genetic basis of phenotypic variation; Christians JK et al.; Identifying genes that influence phenotypic variation within species has proven to be more difficult than expected . A recent study has achieved exceptional success in yeast, and demonstrates how complex genetic architecture can be.

Curr Biol, 2002 Jun 25, 12(12), 1052 - 8
Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain; Feng Q et al.; The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation . Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity . Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family . Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated . K79 methylation occurs in a variety of organisms ranging from yeast to human . In budding yeast, K79 methylation is mediated by the silencing protein DOT1 . Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms . We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo . Furthermore, we found that K79 methylation level is regulated throughout the cell cycle . Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase.

Sci STKE . 2002 Jul 16;2002(141):PE32.
Multivesicular bodies and multivesicular endosomes: the "ins and outs" of endosomal traffic; Stahl PD et al.; Multivesicular endosomes (MVEs) are complex intracellular organelles that function in endocytosis . A major function of the endocytic pathway is to sort internalized macromolecules and membrane proteins . Appropriately sorted proteins, such as epidermal growth factor (EGF) receptor (EGFR), are incorporated into MVEs before transport to the lysosomal compartment, where degradation occurs . Thus, MVEs operate in the endosome-to-lysosome portion of the pathway . In yeast cells, where MVE formation has been extensively studied, the pathway terminates in the yeast vacuole, which is equivalent to the vertebrate lysosome . MVEs arise by invagination of the limiting membrane of an endosomal vesicle such that many small internal vesicles are formed, hence the term "multivesicular endosome." In part, the internalization and targeting of membrane proteins to the MVE involves ubiquitin, a small protein associated with protein degradation . In reticulocytes and certain antigen-presenting cells, MVEs are routed to the plasma membrane rather than the lysosome, releasing small vesicles called "exosomes" back into the extracellular space.

J Neurosci, 2002 Jul 15, 22(14), 5920 - 30
Neurofilament-M interacts with the D1 dopamine receptor to regulate cell surface expression and desensitization; Kim OJ et al.; We used the yeast two-hybrid assay to identify novel proteins that interact with the D(1) dopamine receptor . The third cytoplasmic loop (residues 217-273) of the rat D(1) receptor was used as bait to identify clones encoding interacting proteins from a rat brain cDNA library . This identified two clones encoding the C terminus of rat neurofilament-M (NF-M) (residues 782-846) . The NF-M clone did not interact with the third cytoplasmic loops of the rat D(2), D(3), or D(4) receptors, but showed weak interaction with that of the D(5) receptor . Coexpression of full-length NF-M with the D(1) receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D(1) receptor-mediated cAMP accumulation . NF-M had no effect on the expression of other dopamine receptor subtypes . Using a D(1) receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D(1) receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol . Interestingly, the D(1) receptors that were expressed at the cell surface in the presence of NF-M were resistant to agonist-induced desensitization . Cellular colocalization of NF-M and the D(1) receptor in the rat brain was examined by epifluorescence microscopy . These experiments showed that approximately 50% of medium-sized striatal neurons expressed both proteins . Colocalization was also observed in pyramidal cells and interneurons within the frontal cortex . Similar immunohistochemical analyses using NF-M-deficient mice showed decrements in D(1) receptor expression compared with control mice . These results suggest that NF-M interacts with the D(1) receptor in vivo and may modify its expression and regulation.

J Biol Chem, 2002 Sep 20, 277(38), 35044 - 9 Epub 2002 Jul 16.
Microfibril-associated glycoprotein-2 interacts with fibrillin-1 and fibrillin-2 suggesting a role for MAGP-2 in elastic fiber assembly; Penner AS et al.; Elastic fibers are composed of the protein elastin and a network of 10-12 nm microfibrils . The microfibrillar proteins include, among others, the fibrillins and microfibril-associated glycoproteins-1 and -2 (MAGP-1 and MAGP-2) . Little is known about how microfibrillar proteins interact to support fiber assembly . We used the C-terminal half of MAGP-2 in a yeast two-hybrid library screen to identify relevant ligands . Six of 13 positive clones encoded known microfibrillar proteins, including fibrillin-1 and -2 . Deletion analysis of partial fibrillin-1 and -2 clones revealed a calcium-binding epidermal growth factor repeat-containing region near the C terminus responsible for binding . This region is distinct from the region of fibrillin-1 reported by others to bind MAGP-1 . The MAGP-2 bait was unable to interact productively with other epidermal growth factor repeats in fibrillin-1, demonstrating specificity of the interaction . Deletion analysis of the MAGP-2 bait demonstrated that binding occurred in a core region containing 48% identity and 7 conserved cysteine residues with MAGP-1 . Immunoprecipitation of MAGP-2 from transfected COS-7 cells resulted in the coprecipitation of fibrillin . These results demonstrate that MAGP-2 specifically interacts with fibrillin-1 and -2 and suggest that MAGP-2 may help regulate microfibrillar assembly . The results also demonstrate the utility of the yeast two-hybrid system to study protein-protein interactions of the extracellular matrix.

J Biol Chem, 2002 Nov 22, 277(47), 45181 - 7 Epub 2002 Jul 16.
A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity; Leavey PJ et al.; Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil . The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O(2)()) . With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity . A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67(phox) . N-terminal sequence analysis of p29 revealed homology to an open reading frame gene described in a myeloid leukemia cell line . A cDNA for p29 identical to the open reading frame protein was amplified from RNA of neutrophils . Significant interaction between p29 and p67(phox) was demonstrated using a yeast two-hybrid system . A recombinant (rh) p29 was expressed in Sf9 cells resulting in a protein with an apparent molecular weight of 34,000 . The rh-p29 showed immunoreactivity with the original rabbit antiserum that detected p47(phox) and p67(phox) . In addition, rh-p29 exhibited PLA(2) activity, which was Ca(2+) independent, optimal at low pH, and preferential for phosphatidylcholine substrates . The recombinant protein protected glutathione synthetase and directly inactivated H(2)O(2) . By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin . Finally, O(2)() production by plasma membrane and recombinant cytosolic oxidase components in the SDS-activated, cell-free NADPH oxidase system were enhanced by rh-p29 . This effect was not inhibited by PLA(2) inhibitors . Thus, p29 is a novel protein that associates with p67 and has peroxiredoxin activity . This protein has a potential role in protecting the NADPH oxidase by inactivating H(2)O(2) or altering signaling pathways affected by H(2)O(2).

J Inorg Biochem, 2002 Jul 25, 91(1), 1 - 8
Fe-only hydrogenases: structure, function and evolution; Nicolet Y et al.; Hydrogenases are enzymes capable of catalyzing the oxidation of molecular hydrogen or its production from protons and electrons according to the reversible reaction: H(2)<==>2H(+)+2e(-) . Most of these enzymes fall into to major classes: NiFe and Fe-only hydrogenases . Extensive spectroscopic, electrochemical and structural studies have shed appreciable light on the catalytic mechanism of hydrogenases . Although evolutionarily unrelated, NiFe and Fe-hydrogenases share a common, unusual feature: an active site low-spin Fe center with CO and CN coordination . We have recently focused our attention on Fe-hydrogenases because from structural studies by us and others, it appears to be a simpler system than the NiFe counterpart . Thus the primary hydrogen binding site has been identified and plausible, electron, proton and hydrogen pathways from and to the buried active site may be proposed from the structural data . The extensive genome sequencing effort currently under way has shown that eukaryotic organisms contain putatively gene coding sequences that display significant homology to Fe-hydrogenases . Here, we summarize the available evidence concerning the mechanism of these enzymes and carry out a structural comparison between Fe-hydrogenases and related proteins of unknown metal content from yeast, plant, worm, insect and mammals.

Traffic, 2002 Aug, 3(8), 513 - 20
Clathrin-protein interactions; Lafer EM; There is a complex network of protein-protein and protein-lipid interactions that underlie clathrin-mediated vesicular traffic in all compartmentalized cells from yeast to man . Major progress has been made in the determination of the three-dimensional structures of many of the components . Recently, there has been an explosion in the identification and characterization of clathrin binding partners . This review integrates the structural and biochemical information that is currently available to present a unified view of how many clathrin binding partners interact with clathrin.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10865 - 9 Epub 2002 Jul 15.
Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants; Peart JR et al.; Homologues of the yeast ubiquitin ligase-associated protein SGT1 are required for disease resistance in plants mediated by nucleotide-binding site/leucine-rich repeat (NBS-LRR) proteins . Here, by silencing SGT1 in Nicotiana benthamiana, we extend these findings and demonstrate that SGT1 has an unexpectedly general role in disease resistance . It is required for resistance responses mediated by NBS-LRR and other R proteins in which pathogen-derived elicitors are recognized either inside or outside the host plant cell . A requirement also exists for SGT1 in nonhost resistance in which all known members of a host species are resistant against every characterized isolate of a pathogen . Our findings show that silencing SGT1 affects diverse types of disease resistance in plants and support the idea that R protein-mediated and nonhost resistance may involve similar mechanisms.

Plant Cell, 2002 Jul, 14(7), 1567 - 77
Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element; Reinders A et al.; Suc represents the major transport form for carbohydrates in plants . Suc is loaded actively against a concentration gradient into sieve elements, which constitute the conduit for assimilate export out of leaves . Three members of the Suc transporter family with different properties were identified: SUT1, a high-affinity Suc proton cotransporter; SUT4, a low-affinity transporter; and SUT2, which in yeast is only weakly active and shows features similar to those of the yeast sugar sensors RGT2 and SNF3 . Immunolocalization demonstrated that all three SUT proteins are localized in the same enucleate sieve element . Thus, the potential of Suc transporters to form homooligomers was tested by the yeast-based split-ubiquitin system . The results show that both SUT1 and SUT2 have the potential to form homooligomers . Moreover, all three Suc transporters have the potential to interact with each other . As controls, a potassium channel and a monosaccharide transporter, expressed in the plasma membrane, did not interact with the SUTs . The in vivo interaction between the functionally different Suc transporters indicates that the membrane proteins are capable of forming oligomeric structures that, like mammalian Glc transporter complexes, might be of functional significance for the regulation of transport.

Plant Cell, 2002 Jul, 14(7), 1509 - 25
Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes; Viehweger K et al.; The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons . Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD . Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters . Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride . In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry . It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.

Gene, 2002 Jun 12, 292(1-2), 81 - 90
Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3-18; Butchbach ME et al.; Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission . The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells . Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening . GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport . In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern . GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates . Mouse and human GTRAP3-18 genes contain three exons separated by two introns . The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy . GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle . Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner . These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.

Bioresour Technol, 2002 Sep, 84(3), 287 - 90
Flocculation properties of pectin in various suspensions; Yokoi H et al.; Pectin had a flocculating activity and its flocculating activities in various suspensions were investigated . Flocculating activity of pectin in a kaolin suspension was markedly stimulated by the addition of Al3+ and Fe3+ to the suspension . Optimum temperature for flocculating activity of pectin in the kaolin suspension was around 30 degrees C and high flocculating activity was obtained when 30 mg/l of pectin and 0.2 mM Fe3+ were added to the suspension . Other inorganic suspensions of activated carbon and acid clay were flocculated by pectin in the presence of Al3+ or Fe3+ . Flocculation of organic suspensions such as cellulose and yeast by pectin occurred when 0.1-0.2 mM Fe3+ was present in the suspensions.

Bioresour Technol, 2002 Sep, 84(3), 259 - 63
Production of laccase by a newly isolated strain of Trametes modesta; Nyanhongo GS et al.; The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by Trametes modesta were evaluated using the one-factor-at-a-time method . The final optimisation was done using a central composite design resulting in a four-fold increase of the laccase activity to 178 nkat ml(-1) . Response-surface analysis showed that 7.34 g l(-1) wheat bran, 0.87 g l(-1) glucose, 2.9 g l(-1) yeast extract, 0.25 g l(-1) ammonium chloride, an initial pH of 6.95 and an incubation temperature of 30.26 degrees C were the optimal conditions for laccase production . Laccase produced by T . modesta was fully active at pH 4 and at 50 degrees C . The laccase was very stable at pH 4.5 and at 40 degrees C but half-lives decreased to 120 and 125 min at higher temperature (60 degrees C) and lower pH (pH 3).

Oncogene, 2002 Jul 25, 21(32), 5024 - 30
Isolation and characterization of a novel gene, hRFI, preferentially expressed in esophageal cancer; Sasaki S et al.; hTID1, a human homologue of Drosophila tumor suppressor, I(2)tid regulates the release of cytochrome c from mitochondria and subsequent alteration of caspase-3 activity on apoptosis induced by exogenous stimuli, such as tumor necrosis factor-alpha and mitomycin C . To search for an interacting molecule with hTid1, we applied two-hybrid yeast screening and isolated a novel gene, which encodes a 46 kDa protein of 373 residues . Within the deduced amino acid sequence, a region showing homology to the Ring Finger domain of X-linked inhibitor of apoptosis protein was identified and the gene was designated as hRFI, standing for human Ring Finger homologous to IAP type . A 2.0 kb hRFI transcript was ubiquitously expressed in all human tissues as well as several cancer cell lines examined . Northern blot analysis showed that in 70% (14 out of 20) of esophageal cancer patients, expression of hRFI in cancerous regions was two or more times higher than in the corresponding normal tissues . HeLa cells transfected with hRFI construct exhibited a tendency to resist TNF-alpha induced apoptosis, suggesting an anti-apoptotic function of the hRFI product . Finally, hRFI protein was shown to be cleaved within the DEDD sequence spanning residues 230-233 by caspase-3 during the apoptotic induction.

Oncogene, 2002 Jul 25, 21(32), 4944 - 56
Activation of mitochondrial voltage-dependent anion channel by apro-apoptotic BH3-only protein Bim; Sugiyama T et al.; Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial membrane permeability . We have previously shown that the voltage-dependent anion channel (VDAC) plays a crucial role in apoptotic changes of the mitochondria and its activity is directly regulated by some Bcl-2 family members, including Bcl-2/Bcl-x(L) and Bax/Bak but not Bid . Here, we showed that in isolated mitochondria, Bim induced loss of membrane potential and cytochrome c release like Bax/Bak, with these changes being inhibited by an anti-VDAC antibody . In addition, microinjection of the anti-VDAC antibody significantly reduced Bim-induced apoptosis . Study using purified proteins indicated that Bim directly interacts with the VDAC . Immunoprecipitation analysis revealed that Bim interacts with the VDAC and the interaction is remarkably enhanced during apoptosis . An experiment using liposomes indicated that Bim enhanced VDAC activity, as did Bax/Bak . Furthermore, Bim (but not tBid) was able to induce apoptotic changes of yeast mitochondria in a VDAC-dependent manner, and also induced the lysis of red blood cells, with this effect being inhibited by the anti-VDAC antibody . These results indicate that Bim has an ability to activate directly the VDAC, which plays an important role in apoptosis of mammalian cells.

Oncogene, 2002 Jul 25, 21(32), 4879 - 84
Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling; Ferrigno O et al.; Members of the TGF-beta family of growth factors signal from the cell surface through serine/threonine kinase receptors . Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family . Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-beta signaling . A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein . The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells . Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains . We demonstrate that YAP65 potentiates the inhibitory activity of Smad7 against TGF-beta-induced, Smad3/4-dependent, gene transactivation . Furthermore, YAP65 augments the association of Smad7 to activated TGF-beta receptor type I (TbetaRI), whereas YAP65(1-301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-beta signaling, reduces these interactions . Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TbetaRI, and enhances the inhibitory activity of Smad7 against TGF-beta signaling.

J Cell Sci, 2002 Aug 1, 115(Pt 15), 3105 - 17
The PtdIns3P phosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1-inducible plasma membrane ruffles; Laporte J et al.; Myotubularin, the phosphatase mutated in X-linked myotubular myopathy, was shown to dephosphorylate phosphatidylinositol 3-monophosphate (PtdIns3P) and was also reported to interact with nuclear transcriptional regulators from the trithorax family . We have characterized a panel of specific antibodies and investigated the subcellular localization of myotubularin . Myotubularin is not detected in the nucleus, and localizes mostly as a dense cytoplasmic network . Overexpression of myotubularin does not detectably affect vesicle trafficking in the mammalian cells investigated, in contrast to previous observations in yeast models . Both mutation of a key aspartate residue of myotubularin and dominant activation of Rac1 GTPase lead to the recruitment of myotubularin to specific plasma membrane domains . Localization to Rac1-induced ruffles is dependent on the presence of a domain highly conserved in the myotubularin family (that we named RID) . We thus propose that myotubularin may dephosphorylate a subpool of PtdIns3P (or another related substrate) at the plasma membrane.

J Biol Chem, 2002 Sep 20, 277(38), 34700 - 7 Epub 2002 Jul 12.
Evidence for a stabilizer element in the untranslated regions of Drosophila glutathione S-transferase D1 mRNA; Akgul B et al.; The neighboring genes gstD1 and gstD21 share 70% sequence identity . gstD1 encodes a 1,1,1-trichloro-2,2-bis-(P-chlorophenyl)ethane dehydrochlorinase; gstD21, a ligandin . Both of their mRNAs are inducible by pentobarbital but otherwise behave very differently . Intact gstD21 mRNA is intrinsically labile, but becomes stabilized when separated from its native untranslated region (UTR) . In contrast, whereas gstD1 mRNA is very stable in its entirety, without its native UTRs it becomes even more labile than that of gstD21 . Decay patterns from four chimeric D1-D21 mRNAs, designed to reveal the individual importance of each molecular region to stability, strongly indicate the presence of destabilizing elements in the coding region of gstD1 mRNA . Thus, the UTRs of this molecule must contain a dominant stabilizer element that overrides the destabilizing influence of the coding region and confers overall stability to the entire molecule . The suspected presence of such a stabilizer element in gstD1 mRNA extends a concept from mRNA metabolism in yeast and cultured mammalian cells to include a multicellular organism, Drosophila melanogaster . The complementary presence of destabilizing and stabilizer elements on the same mRNA reveals a regulatory mechanism by which an abundant mRNA can be further induced by a chemical stimulus, or otherwise be returned to normal levels during recovery.

Annu Rev Biomed Eng, 2002, 4, 349 - 73 Epub 2002 Mar 22.
Advances in proteomic technologies; Yarmush ML et al.; Proteomics is a rapidly emerging set of key technologies that are being used to identify proteins and map their interactions in a cellular context . With the sequencing of the human genome, the scope of proteomics has shifted from protein identification and characterization to include protein structure, function and protein-protein interactions . Technologies used in proteomic research include two-dimensional gel electrophoresis, mass spectrometry, yeast two-hybrids screens, and computational prediction programs . While some of these technologies have been in use for a long time, they are currently being applied to study physiology and cellular processes in high-throughput formats . It is the high-throughput approach that defines and characterizes modern proteomics . In this review, we discuss the current status of these experimental and computational technologies relevant to the three major aspects of proteomics-characterization of proteomes, identification of proteins, and determination of protein function . We also briefly discuss the development of new proteomic technologies that are based on recent advances in analytical and biochemical techniques, engineering, microfabrication, and computational prowess . The integration of these advances with established technologies is invaluable for the drive toward a comprehensive understanding of protein structure and function in the cellular milieu.

J Cell Physiol, 2002 Aug, 192(2), 200 - 8
p115 Rho GTPase activating protein interacts with MEKK1; Christerson LB et al.; Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway . Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization . MEKK1 is activated by molecules that impact cytoskeletal function . MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility . MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins . Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP) . The p115 Rho GAP binds to MEKK1 in vitro and in intact cells . The p115 Rho GAP has selectivity for RhoA over other Rho family members . Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1 . The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity . Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization .

Int J Cancer, 2002 Jul 20, 100(3), 309 - 17
Broadened ligand responsiveness of androgen receptor mutants obtained by random amino acid substitution of H874 and mutation hot spot T877 in prostate cancer; Steketee K et al.; In a subset of endocrine therapy-resistant prostate cancers, amino acid substitutions H874Y, T877A and T877S, which broaden ligand specificity of the ligand binding domain (LBD) of the androgen receptor (AR), have been detected . To increase our knowledge of the role of amino acid substitutions at these specific positions in prostate cancer, codons 874 and 877 were subjected to random mutagenesis . AR mutants were screened in a yeast readout system for responsiveness to 5 alpha-dihydrotestosterone, progesterone and dehydroepiandrosterone . At position 874, only the histidine to tyrosine substitution could broaden AR ligand specificity . At position 877, 4 ligand specificity broadening substitutions were found: T877A, T877S, T877C and T877G . The latter 2 were not found in prostate cancer . The AR mutants were tested in mammalian (Hep3B) cells for responsiveness to 13 different ligands . All mutants displayed their own ligand specificity spectrum . Importantly, AR(H874Y) and AR(T877A) could be activated by cortisol . According to the 3-dimensional structure of the AR LBD, T877 interacts directly with the 17 beta-hydroxyl group of androgens . All amino acid substitutions identified at position 877 had smaller side chains than the threonine in the wild-type receptor, indicating that increased space in the ligand binding pocket is important in broadened ligand specificity . Because H874 does not interact directly with the ligand, its substitution by a tyrosine is expected to change the ligand binding pocket conformation indirectly . For T877C and T877G substitutions, 2-point mutations are required, and for H874Y, T877A and T877S substitutions, only a 1-point mutation is sufficient . This most likely explains that the latter 3 have been found in prostate cancer .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(4), 420 - 424
Isolation and Identification of Membrane Proteins Binding to Transfer RNA; Liu QS et al.; Crude extract of total membrane proteins of yeast was obtained by a method of phase separation in Triton X-114 . The membrane extract was applied to the affinity column on which yeast total tRNAs were covalently coupled to hydrazinyl Sepharose 4B . By eluting with increasing concentration gradient of (NH(4))(2)SO(4), eluted proteins were found between concentrations of (NH(4))(2)SO(4) 0.1 mol/L to 0.3 mol/L . By gel mobility-shift assays, it was also observed that there were more than two mobility-shift bands on the gel electropherogram after incubation of sample of (32)P-labeled tRNA with the eluent proteins and the reaction mixtures were analyzed by non-denaturing polyacrylamide gel electrophoresis . These results confirm that these eluted proteins contain two major tRNA specific binding proteins.

Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 10185 - 90 Epub 2002 Jul 11.
The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis; He JX et al.; Brassinosteroids (BRs) are a class of steroid hormones essential for normal growth and development in plants . BR signaling involves the cell-surface receptor BRI1, the glycogen synthase kinase-3-like kinase BIN2 as a negative regulator, and nuclear proteins BZR1 and BZR2/BES1 as positive regulators . The interactions among these components remain unclear . Here we report that BRs induce dephosphorylation and accumulation of BZR1 protein . Experiments using a proteasome inhibitor, MG132, suggest that phosphorylation of BZR1 increases its degradation by the proteasome machinery . BIN2 directly interacts with BZR1 in yeast two-hybrid assays, phosphorylates BZR1 in vitro, and negatively regulates BZR1 protein accumulation in vivo . These results strongly suggest that BIN2 phosphorylates BZR1 and targets it for degradation and that BR signaling causes BZR1 dephosphorylation and accumulation by inhibiting BIN2 activity.

Eur J Cell Biol, 2002 Jun, 81(6), 335 - 40
Mammalian meiotic telomeres: composition and ultrastructure in telomerase-deficient mice; Franco S et al.; During early meiotic prophase chromosome ends become attached to the nuclear envelope, a process that is essential for faithful homologue pairing and segregation . The factors involved in this attachment are largely unknown . Here we investigated the possible involvement of telomere chromatin by using late generation (G5 and G6) Terc-/- mice . These mice lack telomerase activity and show progressive telomere shortening with increasing mouse generations . We show here that in meiotic chromosome ends of late generation Terc-/- mice telomeric TTAGGG repeats and the TRF1 telomere-binding protein are significantly reduced or below detection level . In spite of this, electron microscopy showed no apparent structural differences at the attachment sites of meiotic chromosomes to the nuclear envelope between wild-type and G6 Terc-/- meiocytes . These results suggest, as already shown in yeast, that most telomere chromatin is dispensable for proper attachment of mammalian meiotic chromosome ends to the nuclear envelope.

Eur J Cell Biol, 2002 Jun, 81(6), 323 - 34
Human Krüppel-like factor5/KLF5: synergy with NF-kappaB/Rel factors and expression in human skin and hair follicles; Sur I et al.; In this report we describe the identification of Kruppel-like factor 5 (KLF5/BTEB2) in a yeast one-hybrid screen using a keratinocyte-specific, NF-kappaB binding site as bait . The KLF5 cDNA encodes a larger protein of 457 aa rather than the earlier reported protein of 209 aa . The full-length KLF5 functions as a transactivator in HepG2 cells, and the stimulation of cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) can modulate its transcriptional activity . Overexpression of KLF5 leads to an increase in the TPA response from VLTRE, a TPA-inducible enhancer element that shows keratinocyte specificity with respect to Rel/NF-kappaB binding . The KLF5-mediated transcriptional increase is not observed in the presence of overexpressed NF-kappaB inhibitor, IkappaBalpha . Cotransfection of KLF5 and the p65 subunit of NF-kappaB, results in a synergistic transactivation of the VLTRE-luciferase reporter . The KLF5 mRNA and the protein is expressed in keratinocytes and throughout the adult human epidermis . Its expression is especially strong in the matrix and the inner root sheath cuticle layer of the hair follicle, sebaceous glands and sweat glands . Considering the TPA-responsiveness and expression pattern, we propose that KLF5 like another member of its family KLF4/GKLF may play an important role in skin morphogenesis and carcinogenesis potentially via its interaction with NF-kappaB factors.

Biochem J, 2002 Jul 1, 365(Pt 1), 147 - 55
The actin-severing activity of cofilin is exerted by the interplay of three distinct sites on cofilin and essential for cell viability; Moriyama K et al.; Cofilin/actin-depolymerizing factor is an essential and conserved modulator of actin dynamics . Cofilin binds to actin in either monomeric or filamentous form, severs and depolymerizes actin filaments, and speeds up their treadmilling . A high turnover rate of F-actin in actin-based motility seems driven largely by cofilin-mediated acceleration of directional subunit release, but little by fragmentation of the filaments . On the other hand, the filament-severing function of cofilin seems relevant for the healthy growth of cells . In this study, we have characterized three mutants of porcine cofilin to elucidate the molecular mechanism that underlies the filament-severing activity of cofilin . The first mutant could neither associate with actin filaments nor sever them, whereas it effectively accelerated their treadmilling and directional subunit release . The second mutant bound to actin filaments, but failed to sever them and to interfere with phalloidin binding to the filament . The third mutant could associate with actin filaments and sever them, although with a very reduced efficacy . Of these mutant proteins, only the last one was able to rescue Deltacof1 yeast cells and to induce thick actin bundles in mammalian cells upon overexpression . Therefore, the actin-severing activity of cofilin is an essential element in its vital function and suggested to be exerted by co-operation of at least three distinct sites of cofilin.

Mol Carcinog, 2002 Jun, 34(2), 64 - 71
A major breakpoint cluster domain in murine radiation-induced acute myeloid leukemia; Finnon R et al.; Cytogenetic and molecular studies have provided evidence of the clustering of chromosome 2 deletion breakpoints in radiation-induced murine acute myeloid leukemia (AML) . Moreover, clustering occurs in at least two fragile domains rich in telomere-like arrays . Here we describe a physical map of the distal breakpoint cluster and confirm the presence of inverted head-to-head telomeric sequence arrays . These potentially recombinogenic sequences were not, however, the direct focus for post-irradiation chromosome breakage in AML . Instead, the two arrays bordered a 2.5-kb sequence with properties expected of a nuclear matrix attachment region (MAR) . The putative MAR co-localized in the fragile domain with genes important to the hemopoietic system (leukocyte tyrosine kinase, zinc finger protein 106, erythrocyte protein band 4.2, and beta(2)-microglobulin (beta2m)); the beta2m subdomain was a particular focus of breakage . On the basis of these and other data, we suggest that AML-associated chromosome 2 fragility in the mouse is a consequence of domain-specific fragility in genomic domains containing numerous genes critical to the hemopoietic system . Copyright Crown

Mol Genet Genomics, 2002 Jun, 267(4), 440 - 6 Epub 2002 May 25.
Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain; Kapetanopoulos A et al.; Lipoprotein(a) {Lp(a)} consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans . To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait . The extracellular matrix protein DANCE {developmental arteries and neural crest epidermal growth factor (EGF)-like} recently implicated in atherogenesis was identified as an interactor . A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells . Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed . Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall . However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.

J Hum Genet, 2002, 47(7), 348 - 54
Homozygous deletion on the chromosomal region 5q12.3 in human lines of small-cell lung cancers; Tamura K et al.; Loss of heterozygosity at the polymorphic loci on the long arm of chromosome 5 is observed in about 80% of human small-cell lung cancer (SCLC) . Absence of inactivating mutations in the APC gene on 5q14 suggests the involvement of another tumor suppressor gene . We found a homozygous deletion of sequence tagged site sequence G73332on 5q12.3 in 2 of 12 human SCLC cell lines, Lu130 and Lu134 . One copy of chromosome 5q was lost in these cell lines, and the remaining allele had a deletion in a more restricted region . A polymerase chain reaction-based analysis of yeast artificial chromosome, bacterial artificial chromosome (BAC), and lambda-phage clones narrowed the region of homozygous deletion to a fragment cloned within one BAC . Sequencing analysis revealed that a DNA fragment of approximately 25 kb was deleted interstitially, probably because of recombination through Alu repetitive sequences in Lu130 and Lu134 cells . This deletion was not detected in normal lymphocyte DNA from 98 unrelated individuals . No candidate genes, however, were detected within this region or in the adjacent 150-kb fragment . The absence of microsatellite instability and the presence of an interstitial deletion as well as gross chromosomal aberration suggest that the genomic integrity of Lu130 and Lu134 cells might possibly be affected by Alu-mediated recombination in addition to chromosomal instability . The identical breakpoints in Lu134 and Lu135 cells as well as the same genotypes at all 33 polymorphic loci examined on various chromosomes strongly suggest that these cell lines share the same genetic materials, at least in part, during the establishment or propagation of cell lines.

Curr Genet, 2002 Jun, 41(3), 150 - 8 Epub 2002 May 28.
Interaction between mating-type proteins from the homothallic fungus Sordaria macrospora; Jacobsen S et al.; Mating-type genes control sexual development in ascomycetes . Little is known about their function in homothallic species, which are self-fertile and do not require a mating partner for sexual reproduction . The function of mating-type genes in the homothallic fungus Sordaria macrospora was assayed using a yeast system in order to find properties typical of eukaryotic transcription factors . We were able to demonstrate that the mating-type proteins SMTA-1 and SMTa-1 have domains capable of activating transcription of yeast reporter genes . Two-hybrid analysis for heterodimerization and homodimerization revealed the ability of SMTA-1 to interact with SMTa-1 and vice versa . These two proteins are encoded by different mating types in the related heterothallic species Neurospora crassa . The interaction between SMTA-1 and SMTa-1 was defined by experiments with truncated versions of SMTA-1 and in vitro by means of protein cross-linking . Moreover, we gained evidence for homodimerization of SMTA-1 . Possible functions of mating-type proteins in the homothallic ascomycete S . macrospora are discussed.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(1), 63 - 68
Ubiquitin Conjugating Enzyme Ubc9 is Involved in Protein Degradation of Redox Factor-1 (Ref-1); Yan MD et al.; Redox factor-1 (Ref-1) is a bifunctional protein playing an important role in both cellular redox regulation and DNA apurinic/apyrimidinic sites' repair . To find Ref-1interacting proteins (Rips), a yeast two-hybrid screening was performed by using Ref-1 redox domain as the 'bait', and five positive clones were obtained . One of them (Rip3) was identified to be the ubiquitin-conjugating enzyme Ubc9 . Simultaneous overexpression of Ubc9 in Hela cells dramatically inhibited the enhancement of AP-1 reporter gene by Ref-1 . Western blot indicated that the protein level of Ref-1 dropped down as the result of simultaneous overexpression of Ubc9 . These results suggest that Ubc9 is involved in the protein degradation of Ref-1, resulting in the downregulation of Ref-1 physiological function.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(1), 4 - 8
Interaction between Jak3 and Nucleosome Assembly Protein 1; Ji HB et al.; Tyrosine kinase Jak3 plays a critical role in the interleukin 2 IL-2 signaling because it not only participates the Jak-Stat pathway, but also interacts with unidentified signal transducers and regulates expression of some oncogenes such as c-fos and c-myc . Abundant evidence demonstrated that phosphorylated tyrosine was necessary for the interaction between two proteins . Therefore, in order to clarify the role of Jak3 in IL-2 signal transduction, the tyrosine-phosphorylation-involved yeast two-hybrid system was constructed and the N-terminal region JH3-JH7 of Jak3 was used as a bait to screen a peripheral blood cDNA library . About 50 double-positive colonies were obtained . Sequence analysis indicated that one of them was from nucleosome assembly protein 1 gene (Nap1), and encoded a protein of 392 amino acid residues . Two-hybrid system results demonstrated that interaction between Jak3 and Nap1 depended on the level of tyrosine phosphorylation . Furthermore, immunoprecipitation and Western blot experiments confirmed that Jak3 really interacted with Nap1 in murine pro-B lymphocyte BAF/BO3beta cells.

Nature, 2002 Jul 11, 418(6894), 195 - 9
Reciprocal regulation of CD4/CD8 expression by SWI/SNF-like BAF complexes; Chi TH et al.; Thymic development produces two sub-lineages of T cells expressing either CD4 or CD8 co-receptors that assist antibody production and mediate cell killing, respectively . The mechanisms for mutually exclusive co-receptor expression remain poorly defined . We find that mutations in the high mobility group (HMG) domain of BAF57--a DNA-binding subunit of the mammalian SWI/SNF-like chromatin-remodelling BAF complexes--or in the BAF complex ATPase subunit Brg, impair both CD4 silencing and CD8 activation . Brg is haploinsufficient for CD8 activation, but not for CD4 silencing, whereas BAF57 mutations preferentially impair CD4 silencing, pointing to target- and subunit-specific mechanisms of chromatin remodelling . BAF complexes directly bind the CD4 silencer, but the BAF57 HMG domain is dispensable for tethering BAF complexes to the CD4 silencer or other chromatin loci in vivo, or for remodelling reconstituted templates in vitro, suggesting that chromatin remodelling in vivo requires HMG-dependent DNA bending . These results indicate that BAF complexes contribute to lineage bifurcation by reciprocally regulating lineage-specific genes, reminiscent of the role of the yeast SWI/SNF complex in mediating mating-type switching.

J Biol Chem, 2002 Sep 27, 277(39), 35896 - 905 Epub 2002 Jul 10.
Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508; DeCarvalho AC et al.; The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients . Deletion of a phenylalanine at amino acid position 508 (DeltaF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems . Using a STE6/CFTRDeltaF508 chimera system in yeast, we isolated two novel DeltaF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively . Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRDeltaF508 defect . Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDeltaF508-mediated chloride current, representing 29% of wild type channel activity . The G550E mutation increased the sensitivity of CFTRDeltaF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDeltaF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine . The data show that the DeltaF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.

EMBO J, 2002 Jul 15, 21(14), 3681 - 93
Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii; Novoselov SV et al.; Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants . Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs . Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA . Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals . These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems . We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals.

Can J Microbiol, 2002 May, 48(5), 437 - 42
Utilization and cell-surface binding of hemin by Histoplasma capsulatum; Foster LA; Histoplasma capsulatum, a dimorphic fungus capable of causing severe respiratory illness in immunocompromised individuals, resides in macrophages during mammalian infection . Previous studies suggest that siderophore-mediated iron transport may be important for the acquisition of iron from transferrin while the organism resides in macrophages . However, iron is also present as hemin in the intracellular environment of the macrophage and may serve as a major source of iron during infection . Thus the ability of H . capsulatum to use hemin and heme-containing compounds was examined . Histoplasma capsulatum G217B was iron-starved by adding the iron chelator deferoxamine mesylate to the culture . The addition of 10 microM hemin in the presence of deferoxamine mesylate restored growth to the levels seen in the absence of the chelator . Histoplasma capsulatum was also cultivated in an iron-limited, chemically defined medium without the addition of chelators and it was determined that the organism could also use hemoglobin as a sole source of iron . The method of iron internalization from heme was examined by measuring hemin binding to the yeast-cell surface . The ability of H . capsulatum to bind hemin was related to the nutritional status of the cells . Cells grown under iron-limited conditions bound more heme to the cell surface than did cells grown in medium without chelator . Pretreatment of iron-starved cells with proteinase K eliminated the ability of the organism to bind hemin . Additionally, the pre-incubation of iron-starved H . capsulatum with hemin eliminated the ability of these cells to remove hemin from the solution, although pre-incubation of cells with the iron-free form of hemin, protoporphyrin IX, only modestly affected the ability of the organism to bind hemin . These results suggest that H . capsulatum uses hemin as a sole source of iron and that one mechanism of iron acquisition involves a cell-surface receptor for hemin.

Sci Total Environ, 2002 Jul 3, 293(1-3), 69 - 83
Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays; Legler J et al.; Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment . Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen . The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen . Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast . Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay . Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment) . The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested . Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity . Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2 . The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity . As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats . Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

J Biol Chem, 2002 Sep 20, 277(38), 34870 - 8 Epub 2002 Jul 09.
Interaction between UV-damaged DNA binding activity proteins and the c-Abl tyrosine kinase; Cong F et al.; The c-Abl tyrosine kinase is activated by some forms of DNA damage, including ionizing radiation, but not UV radiation . The functions of this activation in the damage response pathways remain obscure . To identify potential targets of c-Abl kinase, we utilized the yeast two-hybrid system to screen a murine cDNA library . One c-Abl binding protein of particular interest was the large subunit (DDB1) of the heterodimeric complex with UV-damaged DNA binding activity (UV-DDB) . This complex binds with high specificity to DNA damaged by UV, is absent in a subset of xeroderma pigmentosum group E cells, and is required for global genomic repair of UV-induced damage . The association of c-Abl with DDB1 required the kinase domain of c-Abl and preserved the interaction between DDB1 and the small subunit (DDB2) of the UV-DDB complex . Significantly, overexpression of c-Abl increased tyrosine phosphorylation of DDB2 and suppressed UV-DDB activity . Conversely, a dominant negative, kinase-deficient allele of c-Abl decreased tyrosine phosphorylation of DDB2 and dramatically stimulated UV-DDB activity . These results suggest that one role of c-Abl may be to negatively regulate UV-DDB activity by phosphorylation of DDB2.

Gastroenterology, 2002 Jul, 123(1), 314 - 24
Distribution of the novel eNOS-interacting protein NOSIP in the liver, pancreas, and gastrointestinal tract of the rat; Konig P et al.; BACKGROUND & AIMS: Recently, a yeast 2-hybrid screen served to identify a new endothelial nitric oxide synthase (eNOS)-interacting protein (NOSIP), which causes redistribution of eNOS from the plasma membrane to intracellular compartments and reduces eNOS activity . Its in situ distribution is unknown and is reported here in comparison with that of eNOS and neuronal NOS for the rat gastrointestinal tract . METHODS: Immunofluorescence was performed on acetone-fixed cryosections by using a polyclonal antiserum raised against a NOSIP-glutathione S-transferase fusion protein; specificity was verified by Western blotting . RESULTS: Cytoplasmic NOSIP immunoreactivity was observed in endothelial cells of some locations, e.g., the hepatic central vein, but it was mainly observed in the striated esophageal muscle; vascular, gastric, and intestinal smooth muscle; and in interstitial cells of Cajal . Nuclear NOSIP immunoreactivity was more widespread, including some myenteric neurons and several epithelial cell types of esophagus, stomach, pancreas, liver, and gut . This cellular distribution matched with that of its potential binding partner eNOS, as determined by immunohistochemistry and reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, and eNOS, but not neuronal NOS, could be coimmunoprecipitated with NOSIP from small intestine . CONCLUSIONS: NOSIP coimmunoprecipitates and is widely codistributed with eNOS in nonvascular cells in the gastrointestinal tract, suggesting an involvement of eNOS/NOSIP in the regulation of gastrointestinal secretion and motility.

Nat Cell Biol, 2002 Aug, 4(8), 547 - 55
Beta-arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization; Bhattacharya M et al.; beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton . We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs) . Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins . In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, beta-arrestin Ral-GDS protein complexes dissociate and Ral-GDS translocates with beta-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement . The subsequent re-association of beta-arrestin Ral-GDS complexes is associated with the inactivation of Ral signalling . Thus, beta-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.

J Biol Chem, 2002 Sep 6, 277(36), 32722 - 9 Epub 2002 Jun 24.
Mammalian suppressor of Sec4 modulates the inhibitory effect of Rab15 during early endocytosis; Strick DJ et al.; Rab15 is a novel endocytic Rab that counters the stimulatory effect of Rab5-GTP on early endocytic trafficking . Rab15 may interfere with Rab5 function directly by sequestering Rab5 effectors or indirectly through novel sets of effector interactions . To distinguish between these possibilities, we examined the effector binding properties of Rab15 . Rab15 does not interact directly with the Rab5 effectors rabex-5 and rabaptin-5 in a yeast two-hybrid binding assay . Rather mammalian suppressor of Sec4 (Mss4) was identified as a binding partner for Rab15 . Mss4 preferentially binds GDP-bound (T22N) and nucleotide-free (N121I) Rab15, consistent with the proposed role of Mss4 as a chaperone that stabilizes target Rabs in their nucleotide-free form . Mutational analysis of Rab15 indicates that lysine at position 48 (K48Q) is important for the binding of Rab15-GDP to Mss4 . Moreover, the mutation K48Q counters the inhibitory phenotype of wild type Rab15 on receptor-mediated endocytosis in HeLa cells and homotypic endosome fusion in vitro without altering the relative amount of cell surface-associated transferrin receptor . Together, these data indicate a novel role for Mss4 as an effector for Rab15 in early endocytic trafficking.

J Biol Chem, 2002 Sep 27, 277(39), 36399 - 407 Epub 2002 Jun 24.
The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals; Brooks AJ et al.; Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication . We have shown previously that the 37- amino acid interdomain spacer sequence (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS) . In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor . We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus . Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.

J Biol Chem, 2002 Sep 20, 277(38), 35671 - 81 Epub 2002 Jul 08.
Decorin binds to a narrow region of the epidermal growth factor (EGF) receptor, partially overlapping but distinct from the EGF-binding epitope; Santra M et al.; Decorin, a small leucine-rich proteoglycan, is a key regulator of tumor growth by acting as an antagonist of the epidermal growth factor receptor (EGFR) tyrosine kinase . To search for cell surface receptors interacting with decorin, we generated a decorin/alkaline phosphatase chimeric protein and used it to screen a cDNA library by expression cloning . We identified two strongly reactive clones that encoded either the full-length EGFR or its ectodomain . A physiologically relevant interaction between decorin and EGFR was confirmed in the yeast two-hybrid system and further validated by experiments using EGF/EGFR interaction and transient cell transfection assays . Using a panel of deletion mutants, decorin binding was mapped to a narrow region of the EGFR within its ligand-binding L2 domain . Moreover, the central leucine-rich repeat 6 of decorin was required for interaction with the EGFR . Site-directed mutagenesis of the EGFR L2 domain showed that a cluster of residues, His(394)-Ile(402), was essential for both decorin and EGF binding . In contrast, K465, previously shown to be cross-linked to epidermal growth factor (EGF), was required for EGF but not for decorin binding . Thus, decorin binds to a discrete region of the EGFR, partially overlapping with but distinct from the EGF-binding domain . These findings could lead to the generation of protein mimetics capable of suppressing EGFR function.

Biochemistry, 2002 Jul 16, 41(28), 8785 - 95
The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis; Kavanagh KL et al.; Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins . It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways . It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway . No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase . The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively . Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319 . Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families . An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid . The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding . A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate . Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase . Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

Genes Dev, 2002 Jul 1, 16(13), 1610 - 5
The Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo patterning; Hamann T et al.; Developmental responses to the plant hormone auxin are thought to be mediated by interacting pairs from two protein families: short-lived inhibitory IAA proteins and ARF transcription factors binding to auxin-response elements . monopteros mutants lacking activating ARF5 and the auxin-insensitive mutant bodenlos fail to initiate the root meristem during early embryogenesis . Here we show that the bodenlos phenotype results from an amino-acid exchange in the conserved degradation domain of IAA12 . BODENLOS and MONOPTEROS interact in the yeast two-hybrid assay and the two genes are coexpressed in early embryogenesis, suggesting that BODENLOS inhibits MONOPTEROS action in root meristem initiation.

Cancer Epidemiol Biomarkers Prev, 2002 Jul, 11(7), 630 - 9
Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the Nutritional Prevention of Cancer Trial; Duffield-Lillico AJ et al.; The Nutritional Prevention of Cancer Trial was a randomized, clinical trial designed to evaluate the efficacy of selenium as selenized yeast (200 microg daily) in preventing the recurrence of nonmelanoma skin cancer among 1312 residents of the Eastern United States . Original secondary analyses through December 31, 1993 showed striking inverse associations between treatment and the incidence of total {hazard ratio (HR) = 0.61, 95% confidence interval (CI) = 0.46-0.82}, lung, prostate, and colorectal cancer and total cancer mortality . This report presents results through February 1, 1996, the end of blinded treatment . Effect modification by baseline characteristics is also evaluated . The effects of treatment overall and within subgroups of baseline age, gender, smoking status, and plasma selenium were examined using incidence rate ratios and Cox proportional hazards models . Selenium supplementation reduced total (HR = 0.75, 95% CI = 0.58-0.97) and prostate (HR = 0.48, 95% CI = 0.28-0.80) cancer incidence but was not significantly associated with lung (HR = 0.74, 95% CI = 0.44-1.24) and colorectal (HR = 0.46, 95% CI = 0.21-1.02) cancer incidence . The effects of treatment on other site-specific cancers are also described . The protective effect of selenium was confined to males (HR = 0.67, 95% CI = 0.50-0.89) and was most pronounced in former smokers . Participants with baseline plasma selenium concentrations in the lowest two tertiles (<121.6 ng/ml) experienced reductions in total cancer incidence, whereas those in the highest tertile showed an elevated incidence (HR = 1.20, 95% CI = 0.77-1.86) . The Nutritional Prevention of Cancer trial continues to show a protective effect of selenium on cancer incidence, although not all site-specific cancers exhibited a reduction in incidence . This treatment effect was restricted to males and to those with lower baseline plasma selenium concentrations.

Acta Pharmacol Sin, 2002 Jul, 23(7), 667 - 72
Structure-activity relationship of natural flavonoids in hydroxyl radical-scavenging effects; Chen JW et al.; AIM: To study the relationship between the structure and hydroxyl radical (*OH)-scavenging activity of twelve natural flavonoids . METHODS: The hydroxyl radical-generating chemiluminescence system with ascorbate-CuSO4-yeast-H2O2 was used to determine the hydroxyl radical-scavenging activity of twelve natural flavonoids . RESULTS: Guercetin, heliosin, hyperoside, kaempferol, baicalin, corylifolin, lysionotin, matteucinol, corylifolinin, and genistein could effectively scavenge . OH and inhibit the chemiluminescence of the system . The IC50 values (95 % confidence limits) of the flavonoids were 12.1 (9.9-14.5) g/L, 15.8(14.0-19.2) g/L, 19.5 (16.8-27.4) g/L, 20.1 (13.6-29.0) g/L, 34.6 (28.4-43.4) g/L, 66.8 (63.2-74.4) g/L, 187 (147-235) g/L, 211 (165-284) g/L, 262 (190-346) g/L, and 708 (498-994) g/L, respectively; whereas nobilelin and corylifolin-Ac could not scavenge *OH . CONCLUSION: (1) Phenolic hydroxyls in flavonoids were the main active groups capable of scavenging *OH; (2) Hydroxyl groups in ring B and A were important *OH-scavenging active groups; (3) The ortho-dihydroxyl groups in ring A and/or B could greatly enhance the *OH-scavenging activity of the rings; (4) Comparing the IC50 values of guercetin, heliosin, hyperoside, baicalin, lysionotin, and matteucinol, it was suggested that the hydroxyl groups on 3',4' position of ring B possessed high *OH-scavenging activity and the scavenging activity of hydroxyl groups in ring B was higher than that of hydroxyl groups in ring A . The hydroxyl group or glucoside on 3 position of ring C of the above mentioned 6 flavonoids was also related to the . OH-scavenging ability . (5) The structural types of flavonoids themselves could influence their *OH-scavenging activity.

Mol Microbiol, 2002 Jul, 45(1), 89 - 97
Histone deacetylases in Trypanosoma brucei: two are essential and another is required for normal cell cycle progression; Ingram AK et al.; Reversible protein acetylation is established as a modification of major regulatory significance . In particular, histone acetylation regulates access to genetic information in eukaryotes . For example, class I and class II histone deacetylases are regulatory components of corepressor complexes involved in cell cycle progression and differentiation . Here, we have investigated the function of such enzymes in Trypanosoma brucei, mono-flagellated parasitic protozoa that branched very early from the eukaryotic lineage . Four T . brucei genes encoding histone deacetylase orthologues have been identified, cloned and characterized . The predicted deacetylases, DAC1-4 are approximately 43, 61, 75 and 64 kDa respectively . They share significant similarity with mammalian and yeast class I (DAC1 and DAC2) and class II (DAC3 and DAC4) histone deacetylases, and all except DAC2 have the critical residues predicted to be required for deacetylase activity . In gene targeting experiments, DAC1 and DAC3 appear to be essential whereas DAC2 and DAC4 are not required for viability . Of the two mutant cell types, the dac4 mutant displays a delay in the G2/M phase of the cell cycle . Our results provide genetic validation of DAC1 and DAC3 as potential chemotherapy targets and demonstrate that T . brucei expresses at least three probable histone deacetylases with distinct function.

Biochem Biophys Res Commun, 2002 Jul 19, 295(3), 663 - 7
C-propeptide region of human pro alpha 1 type 1 collagen interacts with thioredoxin; Matsumoto K et al.; Thioredoxin (TRX) is one of major components of thiol reducing systems . To investigate the molecular mechanism of TRX function in the lung tissue, we screened a human lung epithelial cell cDNA library for TRX-binding protein by yeast two-hybrid systems . We isolated a plasmid containing C-propeptide region of human pro alpha 1 type 1 collagen (CP-pro alpha 1(1)) . CP-pro alpha 1(1) stably binds to wild type TRX but not to mutant TRX, in which redox-active cysteine residues are substituted . Failure of the interaction of mutant TRX with CP-pro alpha 1(1) was confirmed in yeast two-hybrid systems . The CP-pro alpha 1(1)/TRX interaction was increased by dithiothreitol treatment, but was markedly inhibited by hydrogen peroxide or diamide treatment . These data showed that the reducing status of TRX active site cysteine residues is important for the TRX-CP-pro alpha 1(1) interaction, indicating that collagen biosynthesis is under the regulation of TRX-dependent redox control . (c) 2002 Elsevier Science (USA).

J Parasitol, 2002 Jun, 88(3), 541 - 7
Host selenium deficiency increases the severity of chronic inflammatory myopathy in Trypanosoma cruzi-inoculated mice; Gomez RM et al.; Weanling C3H/HeN mice were fed either a torula yeast-based diet deficient in selenium (Se) or the same diet supplemented with 0.2 ppm Se as sodium selenite . After 4 wk of feeding, the mice were inoculated intraperitoneally with the CA-I strain (clone K98) of Trypanosoma cruzi (TC) . Before inoculation, mean serum Se levels were 430 versus 61 ng/ml in adequate and deficient mice, respectively . During the ascending phase of parasitemia, the Se-deficient mice exhibited significantly higher levels of parasites at 22-34 days postinfection (PI) . However, no difference was found in the subsequent descending phase . As judged by visual examination at 2-mo-PI, some Se-deficient infected mice presented clinical signs of motor dysfunction . At 3-mo-PI, the end of the observation period, this chronic disease developed into a hind limb flaccid paralysis affecting 5 of 8 infected deficient mice . No signs of paralysis were seen in noninfected mice fed either diet or in infected mice fed the Se-adequate diet . At the histological level, both Se-adequate and Se-deficient infected mice showed mild myocarditis and moderate to severe myositis, with increasing intensity from 1- to 3-mo-PI in both groups . However, the severity of myositis was always more intense in the Se-deficient mice so that prominent areas of skeletal muscle replaced by fibrotic tissue were frequently observed . Thus, it can be concluded that Se deficiency in the murine host increases the severity of TC-induced myositis.

Cell Calcium, 2002 Jun, 31(6), 253 - 64
Current understanding of mammalian TRP homologues; Vennekens R et al.; Calcium influx into the cell from the extracellular medium is crucial for important processes including muscle contraction, secretion and gene expression . This calcium influx is mainly mediated through calcium influx channels, which on the basis of their activation mechanism can be subdivided in voltage-gated calcium channels, which have already been thoroughly characterized and non-voltage-gated calcium permeable channels . This latter group includes ion channels activated by binding of extra and intracellular messengers, mechanical stress or depletion of intracellular calcium stores . Currently little molecular data is available concerning this class of calcium influx channels . However, recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels can function as calcium influx channels both in excitable and non-excitable tissues . On the basis of structural information the TRP family is subdivided in three main subfamilies: the TRPC (canonical) group, the TRPV (vanilloid) group and the TRPM (melastatin) group . The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of tissues and species, including mammals, insects and yeast . This review summarizes the currently available information concerning members of the TRP family expressed in mammalian tissues.

J Neurosci, 2002 Jul 1, 22(13), 5393 - 402
Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes; Fuhrmann JC et al.; The clustering of glycine receptors and major subtypes of GABA(A) receptors at inhibitory synapses is mediated by the tubulin-binding protein gephyrin . In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using gephyrin as bait . Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2 . Dlc-1 protein bound efficiently to gephyrin in in vitro binding assays and colocalized with gephyrin during coexpression in HEK293 cells . The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of gephyrin . In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and gephyrin immunostaining . Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus . Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of gephyrin.

J Neurosci, 2002 Jul 1, 22(13), 5253 - 8
Association of the kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95), synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs large/zona occludens-1 proteins; Mok H et al.; Mutation in KIF1B, a kinesin superfamily motor protein, causes a peripheral neuropathy known as Charcot-Marie-Tooth disease type 2A (CMT2A) . Little is known, however, about how a defective KIF1B gene leads to CMT2A . Here we report that KIF1Balpha, one of the two splice variants of KIF1B, directly interacts through its C-terminal postsynaptic density-95 (PSD-95)/discs large/zona occludens (PDZ) domain-binding motif with PDZ proteins including PSD-95/synapse-associated protein-90 (SAP90), SAP97, and synaptic scaffolding molecule (S-SCAM)-90 (SAP90) . KIF1Balpha selectively interacts with PSD-95, SAP97, and S-SCAM in yeast two-hybrid, pull-down, and in vivo coimmunoprecipitation experiments . KIF1Balpha, SAP97, and S-SCAM are widely distributed to both dendrites and axons of cultured neurons and are enriched in the small membrane fraction of the brain . In the flotation assay, KIF1Balpha cofractionates and coimmunoprecipitates with PSD-95, SAP97, and S-SCAM . These results suggest that the PSD-95 family proteins and S-SCAM have a novel function as KIF1Balpha receptors, linking KIF1Balpha to its specific cargos, and are involved in peripheral neuropathies.

J Biol Chem, 2002 Sep 13, 277(37), 34287 - 94 Epub 2002 Jul 03.
An endoplasmic reticulum stress-specific caspase cascade in apoptosis . Cytochrome c-independent activation of caspase-9 by caspase-12; Morishima N et al.; Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B . A., and Yuan, J . (2000) Nature 403, 98-103), yet the functional consequences of caspase-12 activation have been unclear . We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts . The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor . Although cytochrome c released from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W . J., Liu, X., Lutschg, A., and Wang, X . (1997) Cell 90, 405-413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S . M., Ahmad, M., Alnemri, E . S., and Wang, X . (1997) Cell 91, 479-489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress . These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c . To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation . The binding protein protects procaspase-12 from processing in vitro . Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3 . These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner.

Cancer Res, 2002 Jul 1, 62(13), 3716 - 21
Novel mutation of topoisomerase I in rendering cells resistant to camptothecin; Chang JY et al.; To identify mechanisms of camptothecin (CPT) resistance and the relationship between CPT-resistant cells and other anticancer agents, a CPT-resistant cell line (CPT30) and its partial revertant cell line (CPT30R) were established from a human nasopharyngeal carcinoma cell line (HONE-1) . CPT30 and CPT30R cells displayed a 14- and 3.5-fold resistance to CPT compared with HONE-1 cells, respectively . The resistant and partial revertant cell lines showed cross-resistance to topotecan and increased sensitivity to cisplatin, carboplatin, and 1,3-bis(chloroethyl)-1-nitrosurea . The topoisomerase (Top) I catalytic activity of CPT30 and CPT30R cells was 30% and 200%, respectively, compared with that of HONE-1 cells . The expression of Top I protein and mRNA levels in CPT30 cells was 40% and 30% less than that in HONE-1 cells, respectively, whereas in CPT30R cells, the levels of Top I protein and mRNA were 50% and 20% higher, respectively, than that in HONE-1 cells . Both the resistant and revertant cell line whole-cell lysates demonstrated different levels of sensitivity to CPT in in vitro assays in comparison with that of HONE-1 cells . Furthermore, CPT exhibited 15- and 7-fold better binding affinity in stabilizing protein-linked DNA breaks in HONE-1 cells than in CPT30 and CPT30R cells, respectively . Direct DNA sequencing of the reverse transcription-PCR product and genomic DNA revealed a point mutation resulting in E418K mutation in the Top I of both CPT30 and CPT30R cells . Wild-type Top I RNA and genomic DNA were also detected in these two cell lines . A yeast system was used to examine whether this mutation could be responsible for CPT resistance . Our results showed that a single amino acid change (E418K) resulted in CPT resistance . Therefore, quantitative and qualitative changes in Top I were responsible for CPT resistance in CPT30 cells . CPT resistance in CPT30R cells was caused by mutation of Top I.

Biochem J, 2002 Oct 15, 367(Pt 2), 347 - 57
Role of endocytosis in the internalization of spermidine-C(2)-BODIPY, a highly fluorescent probe of polyamine transport; Soulet D et al.; The mechanism of transmembrane polyamine internalization in mammalian cells remains unknown . A novel fluorescent spermidine conjugate {Spd-C(2)-BODIPY; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-N'-(S -{spermidine-(N(4)-ethyl)}thioacetyl)ethylenediamine} was synthesized from N(4)-(mercaptoethyl)spermidine by a simple, one-step coupling procedure . In Chinese-hamster ovary (CHO) cells, Spd-C(2)-BODIPY accumulation was inhibited by exogenous putrescine, spermidine and spermine, was subject to feedback transport inhibition and was up-regulated by prior polyamine depletion achieved with a biosynthetic inhibitor . Probe internalization was decreased by about 85% in a polyamine-transport-deficient CHO mutant cell line . Using confocal laser scanning fluorescence microscopy, internalized Spd-C(2)-BODIPY was concentrated in vesicle-like structures similar to the recycling endosomes observed with fluorescent transferrin, which partly co-localized with the polyamine probe . In yeast, Spd-C(2)-BODIPY uptake was stringently dependent on receptor-mediated endocytosis, as determined with a mutant defective in early- endosome formation . On the other hand, Spd-C(2)-BODIPY did not mimic the substrate behaviour of natural polyamines in yeast, as shown by the lack of correlation of its uptake characteristics with the phenotypes of mutants defective in either polyamine transport or biosynthesis . These data suggest that endocytosis might be an integral part of the mechanism of polyamine transport in mammalian cells, and that the mammalian and yeast transport systems use qualitatively different transport mechanisms . However, the current data do not rule out the possibility that sequestration of the probe into vesicular structures might be secondary to its prior uptake via a "classical" plasma membrane carrier . Spd-C(2)-BODIPY, a highly sensitive probe of polyamine transport with biochemical parameters qualitatively similar to those of natural polyamines in mammalian cells, should be very useful for dissecting the pathway responsible for polyamine internalization.

J Mol Biol, 2002 Jul 12, 320(3), 663 - 75
A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs; Cardone L et al.; A-kinase anchor proteins (AKAPs) assemble multi-enzyme signaling complexes in proximity to substrate/effector proteins, thus directing and amplifying membrane-generated signals . S-AKAP84 and AKAP121 are alternative splicing products with identical NH(2) termini . These AKAPs bind and target protein kinase A (PKA) to the outer mitochondrial membrane . Tubulin was identified as a binding partner of S-AKAP84 in a yeast two-hybrid screen . Immunoprecipitation and co-sedimentation experiments in rat testis extracts confirmed the interaction between microtubules and S-AKAP84 . In situ immunostaining of testicular germ cells (GC2) shows that AKAP121 concentrates on mitochondria in interphase and on mitotic spindles during M phase . Purified tubulin binds directly to S-AKAP84 but not to a deletion mutant lacking the mitochondrial targeting domain (MT) at residues 1-30 . The MT is predicted to form a highly hydrophobic alpha-helical wheel that might also mediate interaction with tubulin . Disruption of the wheel by site-directed mutagenesis abolished tubulin binding and reduced mitochondrial attachment of an MT-GFP fusion protein . Some MT mutants retain tubulin binding but do not localize to mitochondria . Thus, the tubulin-binding motif lies within the mitochondrial attachment motif . Our findings indicate that S-AKAP84/AKAP121 use overlapping targeting motifs to localize signaling enzymes to mitochondrial and cytoskeletal compartments . (c) 2002 Elsevier Science Ltd.

Oncogene, 2002 Jul 11, 21(30), 4673 - 9
hBUB1 defects in leukemia and lymphoma cells; Ru HY et al.; Tumorigenesis is a multi-step process involving a series of changes of cellular genes . Most solid tumors and hematopoietic malignancies often show abnormal chromosome numbers, the aneuploidy . The chromosomal aneuploidy keeps cells in the state of chromosomal instability (CIN) that will increase the mutation rate of cells affected and thus push them deeper into the process of tumorigenesis . The yeast genetic studies showed that normal distribution of chromosome during mitosis is under the surveillance of a set of genes, the spindle assembly checkpoint genes, that include the BUB and MAD gene groups and MPS . In some colorectal cancers with CIN it was found to have hBUB1 gene mutated and the mutated gene functions dominantly . We have examined a series of breast cancer cell lines with or without CIN for the hBUB1 gene mutation and found none . However, we detected various degrees of deletion in the coding sequences of the hBUB1 gene in cells from T lymphoblastic leukemia cell lines, Molt3 and Molt4, and cells from some acute lymphoblastic leukemia and Hodgkin's lymphoma patients . So far the lesions of deletion are in the kinetochore localization domain of the hBUB1 gene that may explain why the deletion lesions in the BUB1 gene cause aneuploidy in lymphoma and lymphoma cells . The deletions are heterozygous in nature . Like the mutated hBUB1 gene in colorectal cancer, the mutant hBUB1 cDNA from lymphoblastic leukemia cells behaves dominantly.

J Vasc Surg, 2002 Jul, 36(1), 1 - 12
Prolonged administration of doxycycline in patients with small asymptomatic abdominal aortic aneurysms: report of a prospective (Phase II) multicenter study; Baxter BT et al.; BACKGROUND: The primary purpose of this study was to evaluate compliance, side effects, and safety associated with prolonged administration of doxycycline in patients with small asymptomatic abdominal aortic aneurysms (AAAs) . A secondary goal was to determine how treatment with doxycycline influences circulating levels of matrix metalloproteinase-9 (MMP-9) in this patient population . METHODS: Thirty-six patients with AAAs (30 men and 6 women; mean age, 69 +/- 1 years) were enrolled into a 6-month phase II study to evaluate treatment with doxycycline (100 mg orally twice a day) . Aneurysm size was measured before and after treatment, and compliance and side effects were monitored . Plasma levels of doxycycline were measured midway through the study, and plasma MMP-9 concentrations were measured at baseline, 3 months, and 6 months . RESULTS: Thirty-three of the 36 patients (92%) completed 6 months of doxycycline treatment . Significant treatment-related side effects occurred in five patients (13.9%), including three with cutaneous photosensitivity reactions (8.3%), one with tooth discoloration (2.8%), and one with yeast infection (2.8%) . A high rate of compliance with treatment was seen, despite minor but frequent side effects, including nonspecific gastrointestinal symptoms (25%), easily managed episodes of photosensitivity (22.2%), and reversible tooth discoloration (5.5%) . The mean plasma doxycycline level after 3 months was 4.62 +/- 0.68 ug/mL (median, 3.64 microg/mL; range, 1.31 to 14.39 microg/mL; n = 23 patients) . No significant change was seen in AAA diameter (42.7 +/- 1.3 mm at 6 months versus 41.0 +/- 0.9 mm at baseline), and the overall rate of AAA expansion was 0.63% +/- 0.25% per month . The mean plasma MMP-9 level (n = 19 patients) was elevated at baseline (118.9 +/- 37.9 ng/mL; upper limit of normal, 85 ng/mL) but subsequently decreased to 83.8 +/- 32.9 ng/mL at 3 months (not significant versus baseline) and to 66.4 +/- 24.2 ng/mL at 6 months (P =.022 versus baseline) . Only 21% of patients had an elevated level of plasma MMP-9 after 6 months of treatment compared with 47% at baseline (P <.05) . CONCLUSION: Prolonged administration of doxycycline is safe and well tolerated by patients with small asymptomatic AAAs and is associated with a gradual reduction in plasma MMP-9 levels . Further studies are needed to evaluate the long-term effects of doxycycline on the rate and extent of aneurysm growth and the potential use of plasma MMP-9 levels as a biomarker of aneurysm disease progression.

Mol Cell Proteomics, 2002 Feb, 1(2), 104 - 16
Describing biological protein interactions in terms of protein states and state transitions: the LiveDIP database; Duan XJ et al.; Biological protein-protein interactions differ from the more general class of physical interactions; in a biological interaction, both proteins must be in their proper states (e.g . covalently modified state, conformational state, cellular location state, etc.) . Also in every biological interaction, one or both interacting molecules undergo a transition to a new state . This regulation of protein states through protein-protein interactions underlies many dynamic biological processes inside cells . Therefore, understanding biological interactions requires information on protein states . Toward this goal, DIP (the Database of Interacting Proteins) has been expanded to LiveDIP, which describes protein interactions by protein states and state transitions . This additional level of characterization permits a more complete picture of the protein-protein interaction networks and is crucial to an integrated understanding of genome-scale biology . The search tools provided by LiveDIP, Pathfinder, and Batch Search allow users to assemble biological pathways from all the protein-protein interactions collated from the scientific literature in LiveDIP . Tools have also been developed to integrate the protein-protein interaction networks of LiveDIP with large scale genomic data such as microarray data . An example of these tools applied to analyzing the pheromone response pathway in yeast suggests that the pathway functions in the context of a complex protein-protein interaction network . Seven of the eleven proteins involved in signal transduction are under negative or positive regulation of up to five other proteins through biological protein-protein interactions . During pheromone response, the mRNA expression levels of these signaling proteins exhibit different time course profiles . There is no simple correlation between changes in transcription levels and the signal intensity . This points to the importance of proteomic studies to understand how cells modulate and integrate signals . Integrating large scale, yeast two-hybrid data with mRNA expression data suggests biological interactions that may participate in pheromone response . These examples illustrate how LiveDIP provides data and tools for biological pathway discovery and pathway analysis.

J Biol Chem, 2002 Sep 20, 277(38), 35386 - 92 Epub 2002 Jul 02.
Role of ubiquilin associated with protein-disulfide isomerase in the endoplasmic reticulum in stress-induced apoptotic cell death; Ko HS et al.; Up-regulation of several stress proteins such as heat-shock proteins and glucose-regulated proteins participate in tolerance against environmental stress . Previously, we found that protein-disulfide isomerase (PDI) is specifically up-regulated in response to hypoxia/brain ischemia in astrocytes . In addition, the overexpression of this gene into neurons protects against apoptotic cell death induced by hypoxia/brain ischemia . To address the detailed function of PDI, we screened for proteins that interact with PDI using the yeast two-hybrid system . We report here that PDI interacts with ubiquilin, which has a ubiquitin-like domain and a ubiquitin-associated domain . Interestingly, ubiquilin is also up-regulated in response to hypoxia in glial cells with a time course similar to that of PDI induction . In hypoxia-treated glial cells, the endogenous ubiquilin and PDI were almost completely co-localized, suggesting that ubiquilin is an endoplasmic reticulum-associated protein . Overexpression of this gene in neuronal cells resulted in significant inhibition of the DNA fragmentation triggered by hypoxia, but not that induced by nitric oxide or staurosporine . Moreover, ubiquilin has the ability to attenuate CHOP induction by hypoxia . These observations suggested that ubiquilin together with PDI have critical functions as regulatory proteins for CHOP-mediated cell death, and therefore up-regulation of these proteins may result in acquisition of tolerance against ischemic stress in glial cells.

Gene, 2002 May 29, 291(1-2), 135 - 47
Structure, upstream promoter region, and functional domains of a mouse and human Mix paired-like homeobox gene; Sahr K et al.; Mix/Bix proteins represent a vertebrate subgroup of paired-like homeodomain proteins which are known to function around the time of gastrulation . Here we report the structures of the genomic and upstream promoter regions of a mouse and human Mix-like gene . Both genes map to syntenic regions of chromosome 1 and contain two coding exons, with the paired-type homeodomain split between the exons within helix 3 . Differentiating mouse embryonic stem cells transcribe a messenger RNA of approximately 2.6 kb . The first exon encodes the translation initiation codon and a 5' untranslated region of approximately 90 bp . Sequence analysis of the 960 bp upstream of the transcription start site of the mouse Mix gene revealed the presence of a putative initiator region and TATA box as well as potential Smad, FoxH1/FAST, T-box, COUP-TF, C/EBP, GATA, HNF3 binding sites and retinoic acid response elements . A number of these sites are conserved in the human Mix promoter . We find that most paired-related homeodomain proteins, including mouse and human Mix, contain a proline-rich region within their amino termini which may interact with other proteins . Mouse and human Mix proteins contain highly conserved carboxy-terminal polar/acidic regions with the potential to form an amphipathic helix and the ability to activate transcription in yeast . Mouse Mix expressed in COS cells or in vitro binds a DNA consensus sequence identified previously for paired class homeodomain proteins . These studies suggest that a number of features of paired-like protein structure and function are conserved across diverse species and provide a useful framework for studying the function and regulation of the mouse Mix gene.

J Mol Biol, 2002 Jul 19, 320(4), 741 - 50
Kinetic model of DNA replication in eukaryotic organisms; Herrick J et al.; We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition . The model describes well a large amount of different data within a simple theoretical framework . This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis . In particular, we have determined the frequency of origin activation in time and space during the cell cycle . Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast . (c) 2002 Elsevier Science Ltd.

Am J Hum Genet, 2002 Aug, 71(2), 375 - 88 Epub 2002 Jul 01.
Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17; Kehrer-Sawatzki H et al.; A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development . The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres . Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level . During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17 . Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs) . By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements . Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation . No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences . Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13) . The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.

J Biol Chem, 2002 Sep 13, 277(37), 34480 - 8 Epub 2002 Jul 01.
Identification of cytoplasmic domains within the epithelial Na+ channel reactive at the plasma membrane; Hendron E et al.; The activity of membrane proteins is controlled, in part, by protein-protein interactions localized to the plasma membrane . In the current study, domains within the epithelial Na(+) channel (ENaC) reactive at the plasma membrane were identified using a novel yeast one-hybrid screen . The cytosolic N terminus of alphaENaC and the cytosolic C termini of alpha-, beta-, and gammaENaC contained domains reactive at the plasma membrane . Fluorescent micrographs of epithelial cells overexpressing fusion proteins of enhanced green fluorescent protein and mENaC cytosolic domains were consistent with those in yeast . A novel membrane reactive domain within the cytosolic C terminus of gamma-mENaC was localized to the 17 amino acids between residues Thr(584)-Pro(600) . Two overlapping internalization signals within the C terminus of gamma-mENaC, a WW-binding domain (PY motif) and a tyrosine-based endocytic signal, were additive with respect to decreasing complementation and expression levels of hybrid proteins . Decreases in expression levels of hybrid proteins containing the PY and endocytic motif were reversed with latrunculin A, an inhibitor of endosomal movement . Decreases in complementation and expression levels of hybrid proteins mediated by the combined PY and overlapping endocytic motif proceeded in the absence of established ubiquitination sites within ENaC . In addition, the endocytic motif was active in the absence of the PY motif, demonstrating that these two domains, while possibly interacting, also have discrete functions . The novel domains within the cytosolic N terminus of alphaENaC and the C termini of alpha-, beta-, and gammaENaC identified here are likely to be involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane . We hypothesize that these newly identified domains play a role in modulating ENaC activity.

EMBO J, 2002 Jul 1, 21(13), 3516 - 25
PA200, a nuclear proteasome activator involved in DNA repair; Ustrell V et al.; We have identified a novel 200 kDa nuclear protein that activates the proteasome . The protein, which we call PA200, has been purified to homogeneity from bovine testis and has been shown to activate proteasomal hydrolysis of peptides, but not proteins . Following gamma-irradiation of HeLa cells the uniform nuclear distribution of PA200 changes to a strikingly punctate pattern, a behavior characteristic of many DNA repair proteins . Homologs of PA200 are present in worms, plants and yeast . Others have shown that mutation of yeast PA200 results in hypersensitivity to bleomycin, and exposure of yeast to DNA damaging agents induces the PA200 message . Taken together, these findings implicate PA200 in DNA repair, possibly by recruiting proteasomes to double strand breaks.

EMBO J, 2002 Jul 1, 21(13), 3464 - 75
The TRAP100 component of the TRAP/Mediator complex is essential in broad transcriptional events and development; Ito M et al.; The multisubunit TRAP/Mediator complex is a mammalian counterpart of the yeast Mediator that shows diverse coactivation functions . Genetic ablation of the murine TRAP100 component of this complex has revealed that it is not essential for cell viability per se . However, null mutant mice die at an early developmental stage with severe malformations, and cultured TRAP100-deficient cells exhibit attenuated functions of a wide variety of transcriptional activators on ectopic reporters . The TRAP100-deficient TRAP/Mediator complex also lacks TRAP95 and TRAP150 beta/SUR2, which together with TRAP100 may form a submodule, and contains a reduced amount of SRB10/CDK8 . Nevertheless, the residual complex shows unaltered binding both to RNA polymerase II and, with the exception of the oncoprotein E1A, to various activators . These findings suggest that TRAP/Mediator is broadly involved in transcription and that a TRAP100-containing submodule plays a secondary role, beyond primary activator interactions and RNA polymerase recruitment by the TRAP complex, in magnifying effects of activators on the general transcriptional machinery.

EMBO J, 2002 Jul 1, 21(13), 3296 - 306
Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth; Samaj J et al.; Mitogen-activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals . We have analyzed the function of the stress-activated alfalfa MAP kinase SIMK in root hairs . In epidermal cells, SIMK is predominantly nuclear . During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F-actin meshworks . Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus . Conversely, upon actin stabilization with jasplakinolide, SIMK co-localized with thick actin cables in the cytoplasm . Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root-derived cell culture . Loss of tip-focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs . UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs . In contrast, overexpression of gain-of-function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126 . These data indicate that SIMK plays a crucial role in root hair tip growth.

Genome Biol . 2002;3(6):RESEARCH0030 . Epub 2002 May 30.
Phylogenetic analysis of the human basic helix-loop-helix proteins; Ledent V et al.; BACKGROUND: The basic helix-loop-helix (bHLH) proteins are a large and complex multigene family of transcription factors with important roles in animal development, including that of fruitflies, nematodes and vertebrates . The identification of orthologous relationships among the bHLH genes from these widely divergent taxa allows reconstruction of the putative complement of bHLH genes present in the genome of their last common ancestor . RESULTS: We identified 39 different bHLH genes in the worm Caenorhabditis elegans, 58 in the fly Drosophila melanogaster and 125 in human (Homo sapiens) . We defined 44 orthologous families that include most of these bHLH genes . Of these, 43 include both human and fly and/or worm genes, indicating that genes from these families were already present in the last common ancestor of worm, fly and human . Only two families contain both yeast and animal genes, and no family contains both plant and animal bHLH genes . We suggest that the diversification of bHLH genes is directly linked to the acquisition of multicellularity, and that important diversification of the bHLH repertoire occurred independently in animals and plants . CONCLUSIONS: As the last common ancestor of worm, fly and human is also that of all bilaterian animals, our analysis indicates that this ancient ancestor must have possessed at least 43 different types of bHLH, highlighting its genomic complexity.

Biochem J, 2002 Oct 1, 367(Pt 1), 179 - 86
A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion; Pan DA et al.; We have identified single genes encoding homologues of the alpha, beta and gamma subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster . Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast . Expression of double stranded (ds) RNAs targeted at any of the putative alpha, beta or gamma subunits ablated this activity, and abolished expression of the alpha subunit . The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells . Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the alpha subunit . We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia . By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK alpha subunit was mainly detected in the nucleus . Our results show that the AMPK system is highly conserved between insects and mammals . Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs.

Biochem J, 2002 Oct 1, 367(Pt 1), 57 - 65
A novel Rho GTPase-activating-protein interacts with Gem, a member of the Ras superfamily of GTPases; Aresta S et al.; Gem is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli . With the aim of isolating the cellular partners of Gem that mediate its biological activity we performed a yeast two-hybrid screen and identified a novel protein of 970 amino acids, Gmip, that interacts with Gem through its N-terminal half, and presents a cysteine-rich domain followed by a Rho GTPase-activating protein (RhoGAP) domain in its C-terminal half . The RhoGAP domain of Gmip stimulates in vitro the GTPase activity of RhoA, but is inactive towards other Rho family proteins such as Rac1 and Cdc42; it is also specific for RhoA in vivo . The same is true for the full-length protein, which is furthermore able to down-regulate RhoA-dependent stress fibres in Ref-52 rat fibroblasts . These findings suggest that the signalling pathways controlled by two proteins of the Ras superfamily, RhoA and Gem, are linked via the action of the RhoGAP protein Gmip (Gem-interacting protein).

Virology, 2002 Jun 20, 298(1), 106 - 15
Subcellular localisation, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins; Cowan GH et al.; Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied . The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector . Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata . In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures . Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3 . No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay . TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner . The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata . However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2 . (c) 2002 Elsevier Science (USA).

Mol Cell Neurosci, 2002 Jun, 20(2), 298 - 306
USP7, a ubiquitin-specific protease, interacts with ataxin-1, the SCA1 gene product; Hong S et al.; Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration . SCA1 has been known to associate with elongated polyglutamine tract in ataxin-1, the SCA1 gene product . Using the yeast two-hybrid system, we have found that USP7, a ubiquitin-specific protease, binds to ataxin-1 . Further experiments with deletion mutants indicated that the C-terminal region of ataxin-1 was essential for the interaction . Liquid beta-galactosidase assay and coimmunoprecipitation experiments revealed that the strength of the interaction between USP7 and ataxin-1 is influenced by the length of the polyglutamine tract in the ataxin-1; weaker interaction was observed in mutant ataxin-1 with longer polyglutamine tract and USP7 was not recruited to the mutant ataxin-1 aggregates in the Purkinje cells of SCA1 transgenic mice . Our results suggest that altered function of the ubiquitin system can be involved in the pathogenesis of spinocerebellar ataxia type 1 . (c) 2002 Elsevier Science (USA).

J Am Vet Med Assoc, 2002 Jun 15, 220(12), 1807 - 12
Comparison of pulse administration versus once daily administration of itraconazole for the treatment of Malassezia pachydermatis dermatitis and otitis in dogs; Pinchbeck LR et al.; OBJECTIVE To compare clinical efficacy of pulse administration with itraconazole versus once daily administration for the treatment of cutaneous and otic M pachydermatis infection in dogs . DESIGN: Randomized controlled trial . ANIMALS: 20 dogs . PROCEDURE: Dogs were treated with itraconazole orally (n = 10/group), using a pulse administration regimen (5 mg/kg {2.3 mg/lb}, PO, q 24 h for 2 consecutive days per week for 3 weeks) or once daily administration (5 mg/kg, PO, q 24 h for 21 days) . No other treatment was permitted . On days 0 and 21, clinical severity of cutaneous and otic disease was assessed, and samples were collected for cytologic examination and yeast culture . Cytology (sum of the mean number of yeast organisms per oil immersion field for affected sites) and culture (mean of the score for extent of yeast growth for samples from affected sites) scores were calculated . RESULTS: For dogs in both treatment groups, clinical severity of cutaneous and otic disease was significantly decreased by day 21, but decrease in severity was not significantly different between groups . Similarly, skin cytology, skin culture, and ear culture scores were significantly decreased on day 21, compared with day 0, for both groups, but decreases were not significantly different between groups except that dogs in the pulse administration group had a significantly greater decrease in ear culture scores than did dogs in the daily administration group . However, when cytology scores only for ear samples were analyzed, day 21 score was not significantly decreased, compared with day 0 score, for either group . CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that both pulse administration and once daily administration of itraconazole were efficacious in the treatment of M pachydermatis cutaneous infection in dogs . However, adjunctive treatment may be needed in dogs with M pachydermatis otitis.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1093 - 6
Short-step syntheses of all stereoisomers of preussin and their bioactivities; Okue M et al.; All the eight stereoisomers of (+)-preussin (1b), an antifungal agent inhibiting the growth of fission yeast and human cancer cells, were synthesized in two steps by non-stereoselective reactions and chromatographic separation, starting from L- and D-N-protected-phenylalaninal (2) . Their bioassay revealed all of the stereoisomers to be almost equally bioactive.

Nat Genet, 2002 Aug, 31(4), 385 - 90 Epub 2002 Jul 01.
Meiotic arrest and aneuploidy in MLH3-deficient mice; Lipkin SM et al.; MutL homolog 3 (Mlh3) is a member of a family of proteins conserved during evolution and having dual roles in DNA mismatch repair and meiosis . The pathway in eukaryotes consists of the DNA-binding components, which are the homologs of the bacterial MutS protein (MSH 2 6), and the MutL homologs, which bind to the MutS homologs and are essential for the repair process . Three of the six homologs of MutS that function in these processes, Msh2, Msh3 and Msh6, are involved in the mismatch repair of mutations, frameshifts and replication errors, and two others, Msh4 and Msh5, have specific roles in meiosis . Of the four MutL homologs, Mlh1, Mlh3, Pms1 and Pms2, three are involved in mismatch repair and at least two, Pms2 and Mlh1, are essential for meiotic progression in both yeast and mice . To assess the role of Mlh3 in mammalian meiosis, we have generated and characterized Mlh3(-/-) mice . Here we show that Mlh3(-/-) mice are viable but sterile . Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid pachynema stage of prophase I . Mlh3(-/-) spermatocytes reach metaphase before succumbing to apoptosis, but oocytes fail to complete meiosis I after fertilization . Our results show that Mlh3 has an essential and distinct role in mammalian meiosis.

Plant Mol Biol, 2002 Jul, 49(5), 459 - 71
Regulation and characterization of four CBF transcription factors from Brassica napus; Gao MJ et al.; Four orthologues of the Arabidopsis CBF/Dreb transcriptional activator genes were isolated from the winter Brassica napus, cv . Jet neuf . All four BNCBF clones encode a putative DRE/CRT (LTRE)-binding protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain . Deduced amino acid sequences suggested that BNCBFs 5, 7and 16 are very similar to the Arabidopsis CBFI whereas BNCBF17 is different in that it contains two extra regions of 16 and 21 amino acids in the acidic domain . Transcripts hybridizing specifically to BNCBF17 and to one or more of the other BNCBFs accumulated in leaves within 30 min of cold exposure of the Brassica seedlings and preceded transcript accumulation of the cold-inducible BN28 gene, a Brassica orthologue of the cor6.6 or KIN gene from Arabidopsis . Cold-induced accumulation of BNCBF17 mRNA was rapid but was short-lived compared to transcripts hybridizing to BNCBF5/7/16 . Transcripts hybridizing to one or more of BNCBF5/7/16 accumulated at low levels after the plants were subjected to prolonged exposure to salt stress . BNCBF17 was not responsive to salt stress . BNCBF transcript accumulation was similar in both spring and winter Brassica but the persistence of the transcripts in the cold were generally shorter in the spring than in the winter type . BNCBF5 and 17 proteins bind in vitro to the LTRE domains of the cold-inducible BN115 (cor15a orthologue) or BN28 promoters . Differential binding preferences, however, to LTREs between BNI 15 and BN28 were observed . Mutation of the core CCGAC sequence of the LTRE indicated that BNCBF17 had a lower sequence binding specificity than BNCBF5 . Furthermore, experiments indicated that the LTREs were able to drive BNCBF5 and 17 trans-activation of the Lac-Z reporter gene in yeast . We conclude that the BNCBFs reported here could function as trans-acting factors in low-temperature responses in Brassica, controlling the expression of cold-induced genes through an ABA-independent pathway.

J Clin Microbiol, 2002 Jul, 40(7), 2483 - 9
Seminested PCR for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification; Ahmad S et al.; The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis . The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity . In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens . The universal outer primers amplified the 3' end of 5.8S ribosomal DNA (rDNA) and the 5' end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350- to 410-bp fragments from the four commonly encountered Candida species, viz., C . albicans, C . tropicalis, C . glabrata, and C . parapsilosis . The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR . The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml . Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system . Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n = 12), suspected (n = 16), and superficially colonized (n = 10) patients and healthy subjects (n = 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients . In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity . In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture . In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C . albicans and either C . tropicalis or C . glabrata . In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens . Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.

J Biochem Biophys Methods, 2002 May 31, 51(3), 243 - 9
Monitoring and control of pullulan production using vision sensor; Ronen M et al.; The production of the polysaccharide pullulan by the yeast-like fungi, Aureobasidium pullulans, is accompained by cellular morphogenetic changes . High productivity and yield of the process have been found to correlate with high concentration of yeast-like cells in the culture . The morphogenetic changes of A . pullulans cells depend on the culture conditions, e.g., dissolved oxygen, shear rate and medium composition . In order to improve the productivity of the process, a novel control law was formulated . A feeding strategy dependent on the culture cellular composition was designed and aimed to keep the yeast-like cell concentration high . The culture morphogenetic composition during the process was monitored by a recently developed vision sensor . Feeding was actuated when the yeast-like cell concentration decreased below a threshold . The proposed control strategy improved pullulan production by increasing both productivity and yield of the cells by 67% and 80%, correspondingly . The results point to the advantage and the potential of using the monitoring and control system and algorithm to increase productivity and yield in cellular bioprocesses.

Ann Agric Environ Med, 2002, 9(1), 59 - 63
Indoor fungi and their ciliostatic metabolites; Pieckova E et al.; According to epidemiological studies, it is possible that some secondary metabolites of indoor airborne fungi could be responsible for health troubles which occupants suffer from . In our previous experiments, a model with tracheal rings of 1-day-old chicks in vitro was shown to be a very suitable method to study the ciliostatic chloroform-extractable endo- and/or exometabolites of filamentous fungi . In this study we isolated the filamentous fungi from walls of "mouldy" dwellings and schools (cultivation on dichloran 18% glycerol agar at 25 and 37 degrees C for 10 d) in Slovakia . We studied the ciliostatic effect of the chloroform-extractable endo- and exometabolites of 96 representative isolates (stationary cultivation on the liquid medium with 2% of yeast extract and 10% of sucrose at 25 degrees C for 10 days) on the cilia movement in tracheal organ cultures of 1-day-old chickens in vitro after 24, 48 and 72 hrs (incubation in the minimal essential medium according to Eagle with Earl s salts and 20 microg of extract of metabolites dissolved in dimethylsulfoxide per 1 mL) . Strains of Penicillium Link: Fr . sp., Aspergillus versicolor (Vuill.) Tiraboschi, A . flavus Link, Cladosporium sphaerospermum Penzig and C . cladosporioides (Fres.) de Vries were isolated most frequently . Two A . flavus isolates were able to produce aflatoxins B1, B2, G1, G2 in vitro after cultivation on the liquid medium with 20% sucrose and 2% yeast extract . This is the first isolation of aflatoxigenic A . flavus strains from dwellings in Slovakia . All frequently isolated strains produced secondary metabolites with the strongest ciliostatic activity -- their exo- and endometabolites stopped tracheal ciliary movement in chicks till 24 h . There are some toxic fungal metabolites in the indoor air not only with the ability to destroy ciliary movement in the upper airways in vitro but, probably, during long-lasting exposure to cause general intoxication of macroorganism via lung tissue.

J Biol Chem, 2002 Sep 6, 277(36), 32781 - 90 Epub 2002 Jun 26.
Role of Dok1 in cell signaling mediated by RET tyrosine kinase; Murakami H et al.; Using a yeast two-hybrid screen, we identified Dok1 as a docking protein for RET tyrosine kinase . Dok1 bound more strongly to RET with a multiple endocrine neoplasia (MEN) 2B mutation than RET with a MEN2A mutation and was highly phosphorylated in the cells expressing the former mutant protein . Analysis by site-directed mutagenesis revealed that tyrosine 361 in mouse Dok1 represents a binding site for the Nck adaptor protein and tyrosines 295, 314, 361, 376, 397, and 408 for the Ras-GTPase-activating protein . We replaced tyrosine 361 or these six tyrosines with phenylalanine (designated Y361F or 6F) in Dok1 and introduced the mutant Dok1 genes into the cells expressing the wild-type RET or RET-MEN2B protein . Overexpression of Dok1 or Dok1-Y361F, but not Dok1-6F, suppressed the Ras/Erk activation induced by glial cell line-derived neurotrophic factor or RET-MEN2B, implying that this inhibitory effect requires the Ras-GTPase-activating protein binding to Dok1 . In contrast, overexpression of Dok1, but not Dok1-Y361F or Dok1-6F, enhanced the c-Jun amino-terminal kinase (JNK) and c-Jun activation . This suggested that the association of Nck to tyrosine 361 in Dok1 is necessary for the JNK and c-Jun activation by glial cell line-derived neurotrophic factor or RET-MEN2B . Because a high level of the JNK phosphorylation was observed in the cells expressing RET-MEN2B, its strong activation via Nck binding to Dok1 may be responsible for aggressive properties of medullary thyroid carcinoma developed in MEN 2B.

Mol Cell, 2002 Jun, 9(6), 1191 - 200
The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly; Ahmad K et al.; Two very similar H3 histones-differing at only four amino acid positions-are produced in Drosophila cells . Here we describe a mechanism of chromatin regulation whereby the variant H3.3 is deposited at particular loci, including active rDNA arrays . While the major H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition . In contrast to replication-coupled (RC) deposition, RI deposition does not require the N-terminal tail . H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast . RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification . Inheritance of newly deposited nucleosomes may then mark sites as active loci.

Mol Cell, 2002 Jun, 9(6), 1183 - 90
Efficient supercoiling of DNA by a single condensin complex as revealed by electron spectroscopic imaging; Bazett-Jones DP et al.; Condensin, a five-subunit protein complex essential for mitotic chromosome condensation from yeast to humans, introduces positive supercoils into DNA in an ATP-dependent manner in vitro . We report here the direct visualization of this supercoiling reaction by electron spectroscopic imaging . In the presence of ATP, a single condensin complex is capable of introducing two or more compensatory supercoils into the protein-free region of a closed circular DNA . Within the condensin-bound region, approximately 190 bp of DNA is organized into a compact structure with two distinct domains, indicative of the formation of two oriented gyres . The current results suggest that the action of condensin is more dynamic and more efficient than that postulated before, providing fundamental insight into the energy-dependent mechanism of higher order chromatin folding.

Syst Appl Microbiol, 2002 Apr, 25(1), 162 - 72
Characterization of Alternaria and Penicillium species from similar substrata based on growth at different temperature, pH and water activity; Andersen B et al.; Fifty-eight Alternaria isolates representing 10 species or species-groups and 66 Penicillium isolates representing 18 species were examined for their growth response to the combined effects of water activity, temperature and pH in an extended Central Composite Design . Growth responses were recorded as colony diameter after one and two weeks of growth and analysed using different multivariate statistical analyses . The isolates, when analysed by Principal Component Analysis, clustered according to their genus and to some degree to species or species groups and not according to substratum as excepted . Soft Independent Modelling of Class Analogy and Response Surface Analysis showed that growth responses or growth profiles may be used as classification tool . Partial Least Squares Regression showed that a combination of two different media based on Dichloran Rose bengal Yeast Extract Sucrose agar incubated at two different temperatures were enough to get genus segregation and to some extent species segregation . The results also showed that water activity, temperature and pH interact strongly in their effect on growth rates and that the squared products (optima) of water activity, temperature and pH for each isolate were important for modelling the data sufficiently.

J Appl Genet, 2002, 43(1), 1 - 17
Cucumber: a model angiosperm for mitochondrial transformation?
Havey MJ, Lilly JW, Bohanec B, Bartoszewski G, Malepszy S.
Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus . The chloroplast genomes of higher plants tend to be of similar sizes and structure . In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species . The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes . Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs . The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes . Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants . A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome . Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant . Firstly, its mitochondria show paternal transmission . Secondly, microspores possess relatively few, huge mitochondria . Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes . The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome . These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.

Plant Cell, 2002 Jun, 14(6), 1377 - 89
In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis; Fan W et al.; The Arabidopsis NPR1 protein is a key regulator of salicylic acid (SA)-mediated gene expression in systemic acquired resistance . Based on yeast two-hybrid analysis, NPR1 has been suggested to interact with members of the TGA family of transcription factors, including TGA2 (AHBP-1b) . However, genetic evidence demonstrating that the NPR1-TGA interaction occurs in planta is still lacking, and the role of this interaction in SA-mediated gene activation has yet to be determined . In this study, we expressed a truncated form of TGA2 in Arabidopsis and found that the resulting transgenic lines displayed phenotypes similar to those of npr1 mutants . This dominant-negative effect of the TGA2 mutant shows that TGA2 and NPR1 interact in planta . We also present biochemical evidence indicating that this interaction is specific and enhanced by SA treatment . Moreover, using a chimera reporter system, we found that a chimeric TGA2GAL4 transcription factor activated a UAS(GAL)::GUS reporter gene in response to SA and that this activation was abolished in the npr1 mutant . NPR1 is required for the DNA binding activity of the transcription factor . These genetic data clearly demonstrate that TGA2 is a SA-responsive and NPR1-dependent transcription activator.

J Biol Chem, 2002 Sep 20, 277(38), 35323 - 32 Epub 2002 Jun 25.
Phorbol ester-regulated oligomerization of diacylglycerol kinase delta linked to its phosphorylation and translocation; Imai S et al.; Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid . In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKdelta containing a sterile alpha-motif (SAM) domain . We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures . Moreover, gel filtration and co-immunoprecipitation analyses demonstrated that DGKdelta formed homo-oligomeric structures in intact cells and that the SAM domain was critically involved in the oligomerization . Interestingly, phorbol ester stimulation induced dissociation of the oligomeric structures with concomitant phosphorylation of DGKdelta . Furthermore, we found that DGKdelta was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation . In this case, DGKdelta mutants lacking the ability of self-association were localized at the plasma membranes even in the absence of phorbol ester . A protein kinase C inhibitor, staurosporine, blocked all of the effects of phorbol ester, i.e . oligomer dissociation, phosphorylation, and translocation . We confirmed that tumor-promoting phorbol esters did not directly bind to DGKdelta . The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKdelta and that DGKdelta is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.

Biochim Biophys Acta, 2002 Jul 19, 1576(3), 306 - 15
cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester; Mogayzel PJ Jr et al.; The cystic fibrosis transmembrane conductance regulator gene (CFTR) is regulated in a tissue-specific and developmental fashion . Although it has been known for some time that phorbol esters decrease CFTR expression in cell lines that have high CFTR mRNA levels, the cis-acting elements that control this down-regulation remain ill-defined . The role of cis-acting elements within the CFTR minimal promoter in modulating responses to phorbol 12-myristate 13-acetate (PMA) and forskolin was assessed using luciferase reporter gene (luc)-containing plasmids transfected into Calu-3 and HT-29 cells . PMA treatment had no effect on luciferase activity in Calu-3 cells transiently transfected with plasmids containing luc driven by up to 2.3 kb of CFTR 5'-flanking DNA . PMA increased luciferase activity in transfected HT-29 cells . A more extensive region of DNA was evaluated using a yeast artificial chromosome (YAC) containing luc driven by approximately 335 of CFTR 5'-flanking DNA (y5'luc) stably introduced into HT-29 cells . Clonal cell lines containing y5'luc were created and assessed for luciferase activity at baseline and in response to forskolin and PMA . There was a wide range of baseline luciferase activities among the clones (42-1038 units/microg protein) that was not entirely due to the number of luc copies present within the cells . Treatment with both PMA and forskolin led to increased luciferase activity in six randomly selected clonal cell lines . As expected, endogenous CFTR expression increased in response to forskolin and decreased in response to PMA . These studies demonstrate that luc-containing YAC vectors can be used to study CFTR expression in human cells . In addition, these data suggest that important regulatory elements responsible for decreased CFTR expression in response to PMA are not located upstream of CFTR in the approximately 335 kb 5'-flanking sequence included in this YAC construct.

Eur J Biochem, 2002 Jul, 269(13), 3237 - 45
Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle; Lezzi M et al.; The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP . We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization . We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other . Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner . This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay . Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically . Ligand binding was greatly enhanced by the presence of a USP ligand binding domain . Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur . This capability may be a regulated aspect of ecdysteroid action during insect development.

J AOAC Int, 2002 May-Jun, 85(3), 736 - 43
Determination of cadmium, aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry; Vinas P et al.; Procedures were developed for determining cadmium, aluminium, and copper in beer and the products used in its manufacture by electrothermal atomic absorption spectrometry . Beer samples were injected into the furnace and solid samples were introduced as suspensions after preparation in a medium containing hydrogen peroxide, nitric acid, and ammonium dihydrogen phosphate for cadmium atomization . Calibration was performed with aqueous standards, and characteristic masses and detection limits were, respectively, 1 and 0.3 pg for cadmium, 18 and 5.4 pg for aluminium, and 5.6 and 6.8 pg for copper . Different samples of beer, wort, brewer's yeast, malt, raw grain, and hops were analyzed by the proposed procedures . Cadmium was found in low concentrations (0.001-0.08 microg/g and 0-1.3 ng/mL); copper (3-13 microg/g and 25-137 ng/mL) and aluminium (0.6-9 microg/g and 0.1-2 microg/mL) were found at higher levels . The reliability of the procedure was confirmed by comparing the results obtained with others based on microwave oven sample digestion, and by analyzing several certified reference materials.

Curr Top Microbiol Immunol, 2002, 268, 175 - 84
The intracellular localization of the proteasome; Gordon C; In summary, localization of proteasomes does appear to be important in the regulation of proteolysis . In yeast, a discrete localization is observed at the nuclear periphery for cells undergoing mitotic growth . This localization is clearly important as degradation by the ubiquitin/proteasome pathway is impaired in mutants that mislocalize proteasomes . In mammalian cells, proteasomes are present throughout the cell . However, the proteasome does appear to be enriched at the MTOC upon aggresome formation . The inhibition of the ubiquitin/proteasome pathway in aggresome-containing cells could provide an explanation for the pathogenicity of a number of neurodegenerative diseases.

Lakartidningen, 2002 May 16, 99(20), 2293 - 4
{Antifungal agents can change the balance between resistant and susceptible Candida species}; Petrini B et al.; The use of fluconazole against yeast infections eradicates susceptible but promotes naturally resistant candida strains as C glabrata and C krusei . C albicans dominated in blood cultures taken at the Karolinska Hospital during the years 1989-2001 . There was no trend towards an increase of Candida non-albicans . Restrictive use of fluconazole may explain why the balance between different Candida species with varying susceptibility to fluconazole has not shifted.

Oncogene, 2002 May 9, 21(20), 3112 - 20
Chromatin remodeling factor encoded by ini1 induces G1 arrest and apoptosis in ini1-deficient cells; Ae K et al.; Ini1/hsnf5 gene encodes INI1 protein, a chromatin remodeling factor associated with the SWI/SNF complex . In yeast, this complex modifies chromatin condensation to coactivate various transcriptional factors . However, in human, little is known about the SWI/SNF complex and INI1 . To elucidate cellular functions of ini1, we constructed a recombinant adenovirus (AdexHA-INI1) capable of overexpressing INI1 in ini1-deficient cells . AdexHA-INI1 produced intranuclear INI1 in three ini1-deficient cell lines, changed their morphology, and decreased the proportion of viable cells . Flow cytometry and a BrdU incorporation assay showed that after the infection, growth of these cells was partially arrested at G1 . In two of the three ini1-deficient cell lines, apoptosis was found to occur after the infection, as detected by the presence of cleaved poly (ADP-ribose) polymerase . To determine functional domains of INI1, we constructed plasmids expressing INI1 and its deletion mutants, which were used for a colony formation assay . Repeats 1 and 2 of INI1 were found to be required to suppress the growth of the three ini1-deficient cell lines . The results support the hypothesis that ini1 is a tumor suppressor gene and suggest a novel link between human SWI/SNF chromatin remodeling complex and apoptosis.

Oncogene, 2002 May 2, 21(19), 3003 - 10
Interaction and colocalization of PGP9.5 with JAB1 and p27(Kip1); Caballero OL et al.; PGP9.5 (UCH-L1) is a member of the ubiquitin C-terminal hydrolase (UCH) family of proteins that is expressed in neuronal tissues . Our previous studies have shown that PGP9.5 was highly expressed in primary lung cancers and lung cancer cell lines . Additionally, the frequency of PGP9.5 over expression increases with tumor stage, indicating that PGP9.5 may play a role in lung cancer tumorigenesis . We used the yeast two-hybrid system to identify proteins that interact with PGP9.5 . We show that PGP9.5 interacts with at least three proteins, one of which is JAB1, a Jun activation domain binding protein that can bind to p27(Kip1) and is involved in the cytoplasmic transportation of p27(Kip1) for its degradation . We also show that PGP9.5 is associated with JAB1 in vitro and in vivo; and that both proteins can be a part of a heteromeric complex containing p27(Kip1) in the nucleus in lung cancer cells . Furthermore, under serum-restimulation, nuclear translocation of both PGP9.5 and JAB1 coincides with a reduced level of p27(Kip1) in the nucleus . In contrast, when cells are contact inhibited, both PGP9.5 and JAB1 became more perinuclear and cytoplasmic in localization while p27(Kip1) was present only in the nucleus . Therefore, PGP9.5 may contribute to p27(Kip1) degradation via its interaction and nuclear translocation with JAB1.

J Cell Sci, 2002 Jul 15, 115(Pt 14), 2893 - 905
Dynamics of the vacuolar H(+)-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells; Clarke M et al.; The vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit enzyme that plays important roles in eukaryotic cells . In Dictyostelium, it is found primarily in membranes of the contractile vacuole complex, where it energizes fluid accumulation by this osmoregulatory organelle and also in membranes of endolysosomes, where it serves to acidify the endosomal lumen . In the present study, a fusion was created between vatM, the gene encoding the 100 kDa transmembrane subunit of the V-ATPase, and the gene encoding Green Fluorescent Protein (GFP) . When expressed in Dictyostelium cells, this fusion protein, VatM-GFP, was correctly targeted to contractile vacuole and endolysosomal membranes and was competent to direct assembly of the V-ATPase enzyme complex . Protease treatment of isolated endosomes indicated that the GFP moiety, located on the C-terminus of VatM, was exposed to the cytoplasmic side of the endosomal membrane rather than to the lumenal side . VatM-GFP labeling of the contractile vacuole complex revealed clearly the dynamics of this pleiomorphic vesiculotubular organelle . VatM-GFP labeling of endosomes allowed direct visualization of the trafficking of vacuolar proton pumps in this pathway, which appeared to be entirely independent from the contractile vacuole membrane system . In cells whose endosomes were pre-labeled with TRITC-dextran and then fed yeast particles, VatM-GFP was delivered to newly formed yeast phagosomes with the same time course as TRITC-dextran, consistent with transfer via a direct fusion of endosomes with phagosomes . Several minutes were required before the intensity of the VatM-GFP labeling of new phagosomes reached the level observed in older phagosomes, suggesting that this fusion process was progressive and continuous . VatM-GFP was retrieved from the phagosome membrane prior to exocytosis of the indigestible remnants of the yeast particle . These data suggest that vacuolar proton pumps are recycled by fusion of advanced with newly formed endosomes.

Mol Biol Evol, 2002 Jul, 19(7), 1041 - 52
Identification and phylogenetic analysis of Drosophila melanogaster myosins; Tzolovsky G et al.; Myosins constitute a superfamily of motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments . Phylogenetic analysis currently places myosins into 17 classes based on class-specific features of their conserved motor domain . Traditionally, the myosins have been divided into two classes depending on whether they form monomers or dimers . The conventional myosin of muscle and nonmuscle cells forms class II myosins . They are complex molecules of four light chains bound to two heavy chains that form bipolar filaments via interactions between their coiled-coil tails (type II) . Class I myosins are smaller monomeric myosins referred to as unconventional myosins . Now, at least 15 other classes of unconventional myosins are known . How many myosins are needed to ensure the proper development and function of eukaryotic organisms? Thus far, three types of myosins were found in budding yeast, six in the nematode Caenorhabditis elegans, and at least 12 in human . Here, we report on the identification and classification of Drosophila melanogaster myosins . Analysis of the Drosophila genome sequence identified 13 myosin genes . Phylogenetic analysis based on the sequence comparison of the myosin motor domains, as well as the presence of the class-specific domains, suggests that Drosophila myosins can be divided into nine major classes . Myosins belonging to previously described classes I, II, III, V, VI, and VII are present . Molecular and phylogenetic analysis indicates that the fruitfly genome contains at least five new myosins . Three of them fall into previously described myosin classes I, VII, and XV . Another myosin is a homolog of the mouse and human PDZ-containing myosins, forming the recently defined class XVIII myosins . PDZ domains are named after the postsynaptic density, disc-large, ZO-1 proteins in which they were first described . The fifth myosin shows a unique domain composition and a low homology to any of the existing classes . We propose that this is classified when similar myosins are identified in other species.

J Biol Chem, 2002 Sep 20, 277(38), 35314 - 22 Epub 2002 Jun 24.
Interaction of p58(PITSLRE), a G2/M-specific protein kinase, with cyclin D3; Zhang S et al.; The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression . Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase . To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE) . In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3 . This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle . The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution . Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3 . Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1 . These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.

Lett Appl Microbiol, 2002, 35(1), 74 - 7
Conidium production by insect pathogenic fungi on commercially available agars; Kamp AM et al.; AIMS: Conidium production by three species of insect pathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, was assessed on various depths and types of commercially available agars . METHODS: Conidium production was assessed after 14 d of growth on commercially available media as well as at three different agar depths . RESULTS: Metarhizium anisopliae and B . bassiana isolates showed greatest conidium production on potato dextrose agar (PDA) at a depth of 2 mm, whereas V . lecanii showed greatest conidium production on yeast extract-peptone-dextrose agar (YPDA) regardless of agar depth . Optimum conidium production for M . anisopliae and B . bassiana was not only dependent upon the isolate used but also on the medium type and agar depth . SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia are the infective structures for insect pathogenic fungi and this study suggests a rationale basis for consistent conidium production for laboratory and commercial practices.

Gene Ther, 2002 Jul, 9(13), 844 - 9
The potential of 5-fluorocytosine/cytosine deaminase enzyme prodrug gene therapy in an intrahepatic colon cancer model; Nyati MK et al.; Colorectal cancer can metastasize to the liver, but remain liver confined for years . A critical step in developing treatments for intrahepatic cancer involves assessment in an orthotopic intrahepatic model . The purpose of this study was to develop a noninvasive intrahepatic tumor model to study the efficacy of 5-flucytosine/yeast cytosine deaminase (5FC/yCD)-based gene therapy for liver tumors . Luciferase expressing human colorectal carcinoma (HT-29luc) cells were generated by retroviral infection and implanted in the left liver lobe of nude mice . The bioluminescence was measured every week for a period of 1 month, then animals were killed and tumors were measured by calipers . After we found a correlation between photon counts and tumor size, animals were implanted with tumors composed of either 0%, 10%, or 100% yCD/HT-29luc cells, and treated with 5FC . Tumor bioluminescence was measured during treatment and tumor histology examined at the time of death . We found that 5FC caused significant regression of yCD expressing tumors . Furthermore, visible tumors at the time of death, which emitted little bioluminescence, contained little or no viable tumor . We then developed an adenoviral vector for yCD . Intraperitoneal administration of adenovirus containing yCD led to the production of yCD enzyme within intrahepatic tumors . These results suggest that (1) intrahepatic cancer responds to 5FC when cells express yCD; (2) the luciferin-luciferase system permits non-invasive real time imaging of viable intrahepatic cancer; and (3) this system can be used to carry out gene therapy experiments using yCD adenovirus.

Mol Med, 2002 Feb, 8(2), 75 - 87
A novel natural product compound enhances cAMP-regulated chloride conductance of cells expressing CFTR{delta}F508; deCarvalho AV et al.; BACKGROUND: Cystic fibrosis (CF) results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel localized at the plasma membrane of diverse epithelia . The most common mutation leading to CF, Delta F508, occurs in the first nucleotide-binding domain (NBD1) of CFTR . The Delta F508 mutation disrupts protein processing, leading to a decreased level of mutant channels at the plasma membrane and reduced transepithelial chloride permeability . Partial correction of the Delta F508 molecular defect in vitro is achieved by incubation of cells with several classes of chemical chaperones, indicating that further investigation of novel small molecules is warranted as a means for producing new therapies for CF . MATERIALS AND METHODS: The yeast two-hybrid assay was used to study the effect of CF-causing mutations on the ability of NBD1 to self-associate and form dimers . A yeast strain demonstrating defective growth as a result of impaired NBD1 dimerization due to Delta F508 was used as a drug discovery bioassay for the identification of plant natural product compounds restoring mutant NBD1 interaction . Active compounds were purified and the chemical structures determined . The purified compounds were tested in epithelial cells expressing CFTR Delta F508 and the resulting effect on transepithelial chloride permeability was assessed using short-circuit chloride current measurements . RESULTS: Wild-type NBD1 of CFTR forms homodimers in a yeast two-hybrid assay . CF-causing mutations within NBD1 that result in defective processing of CFTR (Delta F508, Delta I507, and S549R) disrupted NBD1 interaction in yeast . In contrast, a CF-causing mutation that does not impair CFTR processing (G551D) had no effect on NBD1 dimerization . Using the yeast-based assay, we identified a novel limonoid compound (TS3) that corrected the Delta F508 NBD1 dimerization defect in yeast and also increased the chloride permeability of Fisher Rat Thyroid (FRT) cells stably expressing CFTR Delta F508 . CONCLUSION: The establishment of a phenotype for the Delta F508 mutation in the yeast two-hybrid system yielded a simple assay for the identification of small molecules that interact with the mutant NBD1 and restore dimerization . The natural product compound identified using the system (TS3) was found to increase chloride conductance in epithelial cells to an extent comparable to genistein, a known CFTR activator . The yeast system will thus be useful for further identification of compounds with potential for CF drug therapy.

Genes Dev, 2002 Jun 15, 16(12), 1498 - 508
Characterization of HCP-6, a C . elegans protein required to prevent chromosome twisting and merotelic attachment; Stear JH et al.; Previous studies of mitosis show that capture of single kinetochores by microtubules from both centrosomes (merotelic orientation) is a major cause of aneuploidy . We have characterized hcp-6, a temperature-sensitive chromosome segregation mutant in C . elegans that exhibits chromosomes attached to both poles via a single sister kinetochore . We demonstrate that the primary defect in this mutant is a failure to fully condense chromosomes during prophase . Although centromere formation and sister centromere resolution remain unaffected in hcp-6, the chromosomes lack the rigidity of wild-type chromosomes and twist around the long axis of the chromosome . As such, they are unable to establish a proper orientation at prometaphase, allowing individual kinetochores to be captured by microtubules from both poles . We therefore propose that chromosome rigidity plays an essential role in maintaining chromosome orientation to prevent merotelic capture.

J Biol Chem, 2002 Sep 6, 277(36), 32640 - 9 Epub 2002 Jun 21.
Chromatin structural analyses of the mouse Igkappa gene locus reveal new hypersensitive sites specifying a transcriptional silencer and enhancer; Liu ZM et al.; To identify new regulatory elements within the mouse Igkappa locus, we have mapped DNase I hypersensitive sites (HSs) in the chromatin of B cell lines arrested at different stages of differentiation . We have focused on two regions encompassing 50 kilobases suspected to contain new regulatory elements based on our previous high level expression results with yeast artificial chromosome-based mouse Igkappa transgenes . This approach has revealed a cluster of HSs within the 18-kilobase intervening sequence, which we cloned and sequenced in its entirety, between the Vkappa gene closest to the Jkappa region . These HSs exhibit pro/pre-B cell-specific transcriptional silencing of a Vkappa gene promoter in transient transfection assays . We also identified a plasmacytoma cell-specific HS in the far downstream region of the locus, which in analogous transient transfection assays proved to be a powerful transcriptional enhancer . Deletional analyses reveal that for each element multiple DNA segments cooperate to achieve either silencing or enhancement . The enhancer sequence is conserved in the human Igkappa gene locus, including NF-kappaB and E-box sites that are important for the activity . In summary, our results pinpoint the locations of presumptive regulatory elements for future knockout studies to define their functional roles in the native locus.

J Biol Chem, 2002 Aug 30, 277(35), 31887 - 92 Epub 2002 Jun 21.
Identification of a tankyrase-binding motif shared by IRAP, TAB182, and human TRF1 but not mouse TRF1 . NuMA contains this RXXPDG motif and is a novel tankyrase partner; Sbodio JI et al.; Tankyrase-1 and -2 are closely related poly(ADP-ribose) polymerases that use an ankyrin-repeat domain to bind diverse proteins, including TRF (telomere-repeat binding factor)-1, IRAP (insulin-responsive aminopeptidase), and TAB182 (182-kDa tankyrase-binding protein) . TRF1 binding allows tankyrase to regulate telomere dynamics in human cells, whereas IRAP binding presumably allows tankyrase to regulate the targeting of IRAP . The mechanism by which tankyrase binds to diverse proteins has not been investigated . Herein we describe a novel RXXPDG motif shared by IRAP, TAB182, and human TRF1 that mediates their binding to tankyrases . Interestingly, mouse TRF1 lacks this motif and thus does not bind either tankyrase-1 or -2 . Using the ankyrin domain of tankyrase as a bait in a yeast two-hybrid screen, we also found the RXXPDG motif in six candidate tankyrase partners, including the nuclear/mitotic apparatus protein (NuMA) . We verified NuMA as an RXXPDG-mediated partner of tankyrase and suggest that this interaction contributes to the known colocalization of tankyrase and NuMA at mitotic spindle poles.

J Biol Chem, 2002 Sep 6, 277(36), 32947 - 53 Epub 2002 Jun 21.
Cloning and characterization of a protein kinase A anchoring protein (AKAP)-related protein that interacts with and regulates sphingosine kinase 1 activity; Lacana E et al.; Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has novel dual actions . S1P is the ligand for a family of G protein-coupled receptors known as S1PRs that mediate various physiological functions . Growth factors rapidly activate sphingosine kinase type 1 (SPHK1) resulting in phosphorylation of sphingosine to form S1P, which plays important roles in cell growth regulation and protection from apoptosis . However, little is known of the mechanism(s) by which SPHK activity is regulated . Using a yeast two-hybrid screening approach, we cloned a 3-kb cDNA encoding a SPHK1-interacting protein (SKIP) . BLAST analysis revealed that SKIP corresponded to the C-terminal region of a larger ( approximately 7 kb) cDNA that encoded a protein with a high degree of similarity to a family of protein kinase A anchor proteins (AKAP) . In confirmation of the yeast two-hybrid assay, glutathione S-transferase (GST)-SPHK1 specifically pulled down SKIP, whereas GST did not . Moreover, immunoprecipitation of in vitro translated SPHK1 and SKIP revealed that SKIP and SPHK1 are tightly associated . Furthermore, SKIP overexpression in NIH 3T3 fibroblasts reduced SPHK1 activity and interfered with its biological functions . The apoptotic-sparing effect of SPHK1 against serum deprivation was reduced when co-transfected with SKIP . In addition, SPHK1-enhanced cell proliferation was also abolished by SKIP, with a corresponding decrease in activation of ERK . Taken together, these results indicate that SKIP is a novel protein likely to play a regulatory role in the modulation of SPHK1 activity.

J Mol Biol, 2002 Jun 21, 319(5), 1243 - 55
Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions; Mueller TD et al.; UBA domains are a commonly occurring sequence motif of approximately 45 amino acid residues that are found in diverse proteins involved in the ubiquitin/proteasome pathway, DNA excision-repair, and cell signaling via protein kinases . The human homologue of yeast Rad23A (HHR23A) is one example of a nucleotide excision-repair protein that contains both an internal and a C-terminal UBA domain . The solution structure of HHR23A UBA(2) showed that the domain forms a compact three-helix bundle . We report the structure of the internal UBA(1) domain of HHR23A . Comparison of the structures of UBA(1) and UBA(2) reveals that both form very similar folds and have a conserved large hydrophobic surface patch . The structural similarity between UBA(1) and UBA(2), in spite of their low level of sequence conservation, leads us to conclude that the structural variability of UBA domains in general is likely to be rather small . On the basis of the structural similarities as well as analysis of sequence conservation, we predict that this hydrophobic surface patch is a common protein-interacting surface present in diverse UBA domains . Furthermore, accumulating evidence that ubiquitin binds to UBA domains leads us to the prediction that the hydrophobic surface patch of UBA domains interacts with the hydrophobic surface on the five-stranded beta-sheet of ubiquitin . Detailed comparison of the structures of the two UBA domains, combined with previous mutagenesis studies, indicates that the binding site of HIV-1 Vpr on UBA(2) does not completely overlap the ubiquitin binding site . (c) 2002 Elsevier Science Ltd.

Genomics, 2002 Jul, 80(1), 54 - 61
The zinc-finger transcription factor INSM1 is expressed during embryo development and interacts with the Cbl-associated protein; Xie J et al.; Here we describe the isolation and characterization of the mouse homolog of the human zinc-finger transcription factor INSM1 (IA-1) and identify an interacting protein . A 2.9-kb cDNA with an open reading frame of 1563 nucleotides, corresponding to a translated protein of 521 amino acids, was isolated from a mouse beta TC-1 cDNA library . Mouse INSM1 was found to be 86% identical to human INSM1 and both proteins contain proline-rich regions and multiple zinc-finger DNA-binding motifs . Sequencing of mouse Insm1 genomic DNA revealed that it is an intronless gene . Chromosomal mapping localized Insm1 to chromosome 2 . Northern blot analysis showed that mouse Insm1 expression begins at 10.5 days in the embryo, decreases after 13.5 days, and is barely detected at 18.5 days . In mouse brain, Insm1 is strongly expressed for 2 weeks after birth but shows little or no expression thereafter . Transfection of cells with GFP-tagged INSM1 revealed that INSM1 is expressed exclusively in the nucleus . We identified proteins that interacted with INSM1 by the yeast two-hybrid system and the binding of one of them, Cbl-associated protein (CAP), to INSM1 was confirmed by in vitro pull-down experiments, nuclear colocalization, and co-immunoprecipitation assays . Further studies showed that both INSM1 and CAP proteins were present in the nucleus of insulinoma cells and that endogenous INSM1 protein was co-precipitated with antibody to CAP . These findings raise the possibility that during embryo development CAP may enter the nucleus through its own nuclear localization signal or by binding to INSM1.

Bioorg Khim, 2002 May-Jun, 28(3), 277 - 83
{Novel synthesis of zymosterol}; Baranovskii AV et al.; A modified scheme for the synthesis of zymosterol, one of the biosynthetically important yeast sterols, starting from from 3-benzoyloxyergosta-8(14),22-dien-15-one has been suggested.

J Cell Sci, 2002 Jul 1, 115(Pt 13), 2619 - 22
Molecular evolution of the actin family; Goodson HV et al.; Members of the actin family have well-characterized cytoskeletal functions, but actin and actin-related proteins (ARPs) have also been implicated in nuclear activities . Previous analyses of the actin family have identified four conserved subfamilies, but many actin-related proteins (ARPs) do not fall into these groups . A new systematic phylogenetic analysis reveals that at least eight ARP subfamilies are conserved from humans to yeast, indicating that these ARPs are part of the core set of eukaryotic proteins . Members of at least three subfamilies appear to be involved in chromatin remodeling, suggesting that ARPs play ancient, fundamental roles in this nuclear process.

J Immunol, 2002 Jul 1, 169(1), 248 - 53
Transcriptional regulation of GATA-3 by an intronic regulatory region and fetal liver zinc finger protein 1; Hwang ES et al.; GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage . The transcriptional regulation of GATA-3, however, remains elusive . In this study, we report the identification of a regulatory region located within the first intron of the murine GATA-3 gene . The intronic regulatory region contains both a positive and a negative cis-acting element but, as a whole, serves as a potent T cell-specific enhancer and is essential for the promoter activity in vitro . By using yeast one-hybrid screening, we discovered that fetal liver zinc finger protein 1 (Fliz1) could bind specifically to the negative cis-acting element, the sequence of which is conserved between the mouse and human GATA-3 genes . More importantly, overexpression of Fliz1 repressed the expression of GATA-3 in vivo and in vitro . Our data suggest that the expression of GATA-3 might be partly regulated by the intronic regulatory region and Fliz1 in a developmental stage-specific fashion.

J Biol Chem, 2002 Sep 6, 277(36), 32939 - 46 Epub 2002 Jun 20.
The roadblock light chain binds a novel region of the cytoplasmic Dynein intermediate chain; Susalka SJ et al.; Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells . It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock . The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined . There are two roadblock genes in mammals, ROBL-1 and ROBL-2 . We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein . This was determined with three complementary approaches . A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo . This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay . The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region . The binding domain is downstream of each of the two alternative splice sites in the intermediate chains . This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform . In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.

J Biol Chem, 2002 Sep 6, 277(36), 33319 - 24 Epub 2002 Jun 20.
Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein; Hayashi S et al.; Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms . However, the molecular mechanism of the intracellular actions of SPP remains unclear . Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening . RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family . The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies . RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins . Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate . These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell.

Mol Biochem Parasitol, 2002 Jun, 122(1), 81 - 9
Two families of RNA binding proteins from Trypanosoma brucei associate in a direct protein-protein interaction; Pitula JS et al.; We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei, termed p34 and p37 . The predicted primary structures of the two proteins are highly homologous with one major difference, an 18 amino acid insertion in the N-terminal region of p37 . These two proteins are localized to the nucleus based on immunofluorescence microscopy . Recently, we have shown that p34 and p37 interact with T . brucei 5S rRNA . In order to gain further insight into their function, we have utilized protein affinity chromatography and immune capture approaches to identify T . brucei proteins which associate with p34 and p37 . We demonstrate here an interaction of both p34 and p37 with the NOPP44/46 proteins, identified in T . brucei as a family of tyrosine-phosphorylated RNA binding proteins primarily localized to the nucleolus . This interaction was mapped to the RNA-binding region of p34/p37 and an acidic region of NOPP44/46 by protein affinity chromatography using recombinant deletion constructs of p34 and p37 and yeast two-hybrid analysis . These data may suggest a role for p34 and p37 and NOPP44/46 in the import and/or assembly pathway of T . brucei 5S rRNA in ribosome biogenesis.

Int J Parasitol, 2002 Jul, 32(8), 961 - 7
Characterisation of hexokinase in Toxoplasma gondii tachyzoites; Saito T et al.; We have cloned the hexokinase {E.C . 2.7.1.1} gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82 . Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T . gondii . The sequence of T . gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms . The specific activity of the homogeneously purified recombinant enzyme was 4.04 micromol/mg protein/min at 37 degrees C under optimal conditions . The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose . Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate . K(m) values for glucose and adenosine triphosphate were 8.0+/-0.8 microM and 1.05+/-0.25mM, respectively . No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T . gondii hexokinase activity . Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T . gondii hexokinase activity . Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T . gondii tachyzoites . This result suggests that T . gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.

Bioinformatics, 2002 Jun, 18(6), 880 - 3
Global analysis of tandem aromatic octapeptide repeats: the significance of the aromatic-glycine motif; Gazit E; MOTIVATION: Tandem peptide repeats play a key role in self-assembly and aggregation processes . A notable example is the occurrence of tandem peptide repeats in prionic proteins and their role in the aggregation process that leads to the formation of the prion . One of the structural characteristics that is evident from the comparison of mammalian and yeast prion proteins is the presence of aromatic residues in their tandem repeats . These residues are accompanied by glycine residues before and/or after the aromatic amino acid . Such aromatic-glycine conjugates are also present in the tandem repeats of the large family of the bacterial ice nucleation proteins . To study the significance of such aromatic-glycine occurrences, a global analysis of all the aromatic octapeptide repeats in the Swiss-Prot and TrEMBL databases was conducted . The search pattern was formulated to compare the number of conjugates of each of the 20 natural amino acids before or after the different aromatic residues . RESULTS: The presence of aromatic-glycine conjugates appears to be significantly higher than aromatic conjugates to any other amino acid . Furthermore, all the six various combination of glycine occurrences before or after the three aromatic residues are present . No such pattern was observed for any other amino acid . The significance of the findings is being discussed in the context of the physicochemical properties of aromatic-glycine conjugates and its possible role in the facilitation of aggregates formation.

Bioinformatics, 2002 Jun, 18(6), 813 - 8
Visualizing metabolic activity on a genome-wide scale; Luyf AC et al.; MOTIVATION: To enhance the exploration of gene expression data in a metabolic context, one requires an application that allows the integration of this data and which represents this data in a (genome-wide) metabolic map . The layout of this metabolic map must be highly flexible to enable discoveries of biological phenomena . Moreover, it must allow the simultaneous representation of additional information about genes and enzymes . Since the layout and properties of existing maps did not fulfill our requirements, we developed a new way of representing gene expression data in metabolic charts . RESULTS: ViMAc generates user-specified (genome-wide) metabolic maps to explore gene expression data . To enhance the interpretation of these maps information such as sub-cellular localization is included . ViMAc can be used to analyse human or yeast expression data obtained with DNA microarrays or SAGE . We introduce our metabolic map method and demonstrate how it can be applied to explore DNA microarray data for yeast . Availability: ViMAc is freely available for academic institutions on request from the authors.

Dev Comp Immunol, 2002 Sep, 26(7), 603 - 14
Thioester function is conserved in SpC3, the sea urchin homologue of the complement component C3; Smith LC; The amino acid sequence of the thioester site in the alpha chain of SpC3, the sea urchin homologue of C3, is conserved . This implies a conserved function of covalent bond formation with amine or hydroxyl groups on target molecules . When coelomic fluid (CF) was incubated with 14C-methylamine, a classic assay for thioester binding function, the alpha chain became labeled . When CF was treated to induce autolysis, peptide bond cleavage occurred at the thioester site . Autolysis could be blocked or reduced by pre-treating CF with either methylamine or yeast, both of which are known to bind to thioester sites C3 proteins from other organisms . The data suggest that SpC3 can bind to target cell surfaces, constituting indirect evidence that it can covalently bind to pathogen surfaces and function as an opsonin in vivo . This activity may be an important aspect of host defense in the sea urchin.

Trends Cell Biol, 2002 Jun, 12(6), 267 - 73
XMAP215: a key component of the dynamic microtubule cytoskeleton; Kinoshita K et al.; Microtubules are essential for various cellular processes including cell division and intracellular organization . Their function depends on their ability to rearrange their distribution at different times and places . Microtubules are dynamic polymers and their behaviour is described as dynamic instability . Rearrangement of the microtubule cytoskeleton is made possible by proteins that modulate the parameters of dynamic instability . Studies using Xenopus egg extracts led to identification of a microtubule-associated protein called XMAP215 as a major regulator of physiological microtubule dynamics . XMAP215 belongs to an evolutionarily conserved protein family present in organisms ranging from yeast to mammals . Together with members of the Kin I family of kinesins, XMAP215 and its orthologues form an essential circuit for generating dynamic microtubules in vivo.

Vet Dermatol, 2002 Jun, 13(3), 151 - 6
A semiquantitative cytological evaluation of normal and pathological samples from the external ear canal of dogs and cats; Ginel PJ et al.; Numbers of desquamated epithelial cells, yeast cells and bacterial organisms were counted in samples collected from the external ear canal of 37 normal dogs and 16 normal cats, and from 24 dogs and 22 cats with otitis externa . The aims of the study were to establish quantitative reference ranges and to correlate these data with the clinical status of the dogs and cats . Numbers of yeast cells and bacterial organisms were significantly increased in dogs (P = 0.05; P = 0.0001) and cats (P = 0.0001; P = 0.0001) with otitis externa, and in most cases high counts were correlated with clinical signs . Mean Malassezia counts per high-power dry field of > or = 5 in the dog and > or = 12 in the cat were considered abnormal . Mean bacterial counts per high-power dry field of > or = 25 in the dog and > or = 15 in the cat were considered abnormal . When used to differentiate normal from inflamed external ear canals, these figures provided a low sensitivity but a specificity of > or = 95%.

Biochem Biophys Res Commun, 2002 Jun 28, 294(5), 1087 - 92
Brain-derived neurotrophic factor promotes interaction of the Nck2 adaptor protein with the TrkB tyrosine kinase receptor; Suzuki S et al.; Brain-derived neurotrophic factor (BDNF) binds to and activates the TrkB tyrosine kinase receptor to regulate cell differentiation, survival, and neural plasticity in the nervous system . However, the identities of the downstream signaling proteins involved in this process remain unclear . Using a yeast two-hybrid screen with the intracellular domain (ICD-TrkB) of the TrkB BDNF receptor, we identified the Nck2 adaptor protein as a novel interaction partner of the active form of TrkB . Additionally, we identified three tyrosines in ICD-TrkB (Y694, Y695, and Y771) that are crucial for this interaction . Similar results were obtained for Nck1, an Nck2 homolog . We also found that TrkB could be co-precipitated with GST-Nck2 recombinant protein or anti-Nck antibody in BDNF-activated cortical neurons . These results suggest that BDNF stimulation promotes interaction of Ncks with TrkB in cortical neurons.

Biochem Biophys Res Commun, 2002 Jun 28, 294(5), 1071 - 8
YlALK1 encoding the cytochrome P450ALK1 in Yarrowia lipolytica is transcriptionally induced by n-alkane through two distinct cis-elements on its promoter; Sumita T et al.; The YlALK1 gene, which encodes cytochrome P450ALK1, plays a primary role in the assimilation of n-decane by yeast Yarrowia lipolytica and is inducible by n-decane at the transcriptional level . Deletion analysis of the YlALK1 promoter revealed that a 95-bp region on the YlALK1 promoter (from the position -400 to -304 upstream of the ATG codon) is essential for the induction by n-decane and we named this region ARR1 (alkane-responsive region) . ARR1 was found to be made up of two different elements, ARE1 (alkane-responsive element 1; from -394 to -371) and ARE2 (from -325 to -305) . By electrophoretic mobility shift assay, we found that the respective elements gave specific shift bands with the extracts from Y . lipolytica cells grown on n-alkane, but not much evidently from the cells grown on glycerol or glucose . This suggests that proteins that specifically bind to these elements are present and their binding or synthesis is dependent on n-alkane.

J Am Coll Nutr, 2002 Jun, 21(3), 223 - 32
Selenocompounds in plants and animals and their biological significance; Whanger PD; There are several selenocompounds in tissues of plants and animals . Selenate is the major inorganic selenocompound found in both animal and plant tissues . Selenocysteine is the predominant selenoamino acid in tissues when inorganic selenium is given to animals . Selenomethionine is the major selenocompound found initially in animals given this selenoamino acid, but is converted with time afterwards to selenocysteine . Selenomethionine is the major selenocompound in cereal grains, grassland legumes and soybeans . Selenomethionine can also be the major selenocompound in selenium enriched yeast, but the amount can vary markedly depending upon the growth conditions . Se-methylselenocysteine is the major selenocompound in selenium enriched plants such as garlic, onions, broccoli florets and sprouts, and wild leeks.

J Ind Microbiol Biotechnol, 2002 Mar, 28(3), 180 - 5
Microarray technology GEM microarrays and drug discovery; Reynolds MA; Incyte Genomics' GEM Gene Expression Microarray is a proven genomics tool used by a large number of pharmaceutical companies to speed up the drug discovery and development process . The development and integration of this technology, together with Incyte's sequence databases and clone resources, have resulted in GEM microarrays that span approximately 60,000 human genes as well as approximately 60,000 plant, rat, mouse, yeast, and bacterial genes . The technology underlying the use of these arrays and their application to the drug discovery process is highlighted.

Genetics, 2002 Jun, 161(2), 825 - 34
Comparative mapping of the barley Ppd-H1 photoperiod response gene region, which lies close to a junction between two rice linkage segments; Dunford RP et al.; Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions . We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments . RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs . Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley . The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome . Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region . The implications of the results for comparative trait mapping in junction regions are discussed.

Genetics, 2002 Jun, 161(2), 721 - 31
Ectopic expression of the Drosophila Cdk1 inhibitory kinases, Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development; Price DM et al.; Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2 . Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1) . We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development . Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype . The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1 . To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers . Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development . We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis.

Genetics, 2002 Jun, 161(2), 661 - 72
Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster; Sun J et al.; A transgenic system ("FLP-out") based on yeast FLP recombinase allowed induced overexpression of MnSOD enzyme in adult Drosophila melanogaster . With FLP-out a brief heat pulse (HP) of young, adult flies triggered the rearrangement and subsequent expression of a MnSOD transgene throughout the adult life span . Control (no HP) and overexpressing (HP) flies had identical genetic backgrounds . The amount of MnSOD enzyme overexpression achieved varied among six independent transgenic lines, with increases up to 75% . Life span was increased in proportion to the increase in enzyme . Mean life span was increased by an average of 16%, with some lines showing 30-33% increases . Maximum life span was increased by an average of 15%, with one line showing as much as 37% increase . Simultaneous overexpression of catalase with MnSOD had no added benefit, consistent with previous observations that catalase is present in excess in the adult fly with regard to life span . Cu/ZnSOD overexpression also increases mean and maximum life span . For both MnSOD and Cu/ZnSOD lines, increased life span was not associated with decreased metabolic activity, as measured by O2 consumption.

J Cell Biol, 2002 Jun 24, 157(7), 1105 - 12 Epub 2002 Jun 17.
Functional binding interaction identified between the axonal CAM L1 and members of the ERM family; Dickson TC et al.; A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function . The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor . Binding of bacterial fusion proteins confirmed this interaction . To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins . Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed . Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development . Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.

Antimicrob Agents Chemother, 2002 Jul, 46(7), 2145 - 54
Molecular characterization of recombinant Pneumocystis carinii topoisomerase I: differential interactions with human topoisomerase I poisons and pentamidine; van Dross RT et al.; The Pneumocystis carinii topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized . The P . carinii topoisomerase I protein contains 763 amino acids and has a molecular mass of ca . 90 kDa . The expressed enzyme relaxes supercoiled DNA to completion and has no Mg2+ requirement . Cleavage assays reveal that both the human and P . carinii enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48 . As with the human enzyme, no cleavage is stimulated in the presence of 4',6'-diamidino-2-phenylindole (DAPI) or berenil . A yeast cytotoxicity assay shows that P . carinii topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin . In contrast to the human enzyme, P . carinii topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons . The differences in the sensitivities of P . carinii and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development . Additionally, we have characterized the interaction of pentamidine with P . carinii topoisomerase I . We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.

Biochim Biophys Acta, 2002 May 23, 1582(1-3), 45 - 51
Roles for lipid phosphate phosphatases in regulation of cellular signaling; Sciorra VA et al.; Lipid phosphate phosphatases (LPPs) are a family of integral membrane glycoproteins that catalyze the dephosphorylation of a number of bioactive lipid mediators including lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and phosphatidic acid (PA) . These mediators exert complex effects on cell function through both actions at cell surface receptors and on intracellular targets . The LPP-catalyzed dephosphorylation of these substrates can both terminate their signaling actions and itself generate further molecules with biological activity . Recent advances have revealed that a family of structurally related genes is responsible for LPP activities in species from yeast to mammals . These genes exhibit distinct but overlapping expression patterns and their products appear to be heterogeneous with respect to their posttranslational modification and subcellular localizations . Here we review the structure and catalytic properties of the LPPs and consider recent developments in understanding their cellular biology and functions.

Chromosoma, 2002 Feb, 110(8), 519 - 31
Identification of a multicopy chromatin boundary element at the borders of silenced chromosomal domains; Cuvier O et al.; The insulating properties required to delimit higher-order chromosomal domains have been shown to be shared by a variety of chromatin boundary elements (BEs) . Boundary elements have been described in several species, from yeast to human, and we have previously reported the existence of a class of chromatin BEs in Drosophila melanogaster whose insulating activity requires the DNA-binding protein BEAF (boundary element-associated factor) . Here we focus on the characterization of a moderately repeated 1.2 kb DNA sequence that encompasses boundary element 28 (BE28) . We show that it directionally blocks enhancer/promoter communication in transgenic flies . This sequence contains a BEAF-binding sequence juxtaposed to an AT-rich sequence that harbors a strong nuclease-hypersensitive site . Using a combination of DNA-protein and protein blotting techniques, we found that this region is recognized by the A+T-binding D1 non-histone chromosomal protein of D . melanogaster, and we provide evidence that D1 and BEAF physically interact . In addition, the multicopy BE28 element maps to pericentric regions of the D . melanogaster 2L, 2R and X chromosome arms to which D1 has been shown to localize . In yeast, BEs that mark the periphery of silenced chromosomal domains have recently been shown to block the spreading of heterochromatin assembly . We propose that the BE28 repeat clusters could fulfill a similar function, acting as a local boundary between hetero- and euchromatin in a process involving interactions between the BEAF and D1 proteins.

Mol Biol (Mosk), 2002 May-Jun, 36(3), 397 - 407
{Synaptonemal complex proteins: specific proteins of meiotic chromosomes}; Penkina MV et al.; The review considers proteins of the synaptonemal complex (SC), a specific structure formed between homologous chromosomes in maturing germline cells during meiotic prophase I . The structure and functions are described for proteins that form ultrastructural SC elements in mammals, in yeast, and in higher plants . The roles of cohesions and of the SC proteins in meiotic sister-chromatid cohesion are considered . Though still scarce, data are summarized on the SC self-assembly and dissociation and on the molecular composition of SC-associated recombination nodules, which provide a compartment for meiotic recombination enzymes . The accumulating data on the SC molecular components and on their structure, properties, and interactions improve the understanding of the SC function.

FEBS Lett, 2002 Jun 19, 521(1-3), 36 - 8
The LIM domain protein Lmo2 binds to AF6, a translocation partner of the MLL oncogene; Begay-Muller V et al.; The LIM only protein Lmo2 plays an important role in hematopoiesis and leukemogenesis . Lmo2 acts as a bridging molecule between components of hematopoietic gene regulatory protein complexes . We used the yeast two-hybrid system to identify novel Lmo2 interacting proteins and found that the AF6 protein binds to Lmo2 . AF6 is a recurrent fusion partner of MLL, the human homolog of Drosophila trithorax chromatin remodeling protein that is involved in childhood leukemia and mixed lineage leukemia . Our data support the notion that recurrent fusion partners of chimeric MLL proteins recruit hematopoietic gene regulatory complexes.

FEBS Lett, 2002 Jun 19, 521(1-3), 170 - 4
Regulation of the Werner helicase through a direct interaction with a subunit of protein kinase A; Nguyen DT et al.; Werner syndrome is a hereditary disease characterized by cancer predisposition, genetic instability, and the premature appearance of features associated with normal aging . At the molecular level this syndrome has been related to mutations in the Werner helicase, a member of the RecQ family of DNA helicases which are required to maintain genomic stability in cells . Here we show by a yeast two-hybrid screen that the Werner helicase can directly interact with the regulatory subunit (RIbeta) of cAMP protein kinase A (PKA) . We confirm that this interaction occurs in vivo . Interestingly, serum withdrawal causes a redistribution of the Werner helicase within the nucleus of mammalian cells . Raising intracellular cAMP levels or increased expression of the regulatory but not the catalytic subunit of PKA inhibits this nuclear redistribution stimulated by serum deprivation . These results suggest that similar to lower organisms, gene products linked to genomic instability and aging may be directly regulated by growth factor-sensitive, PKA-dependent pathways.

Curr Opin Cell Biol, 2002 Jun, 14(3), 372 - 6
Nuclear organisation and gene expression; Baxter J et al.; The development of increasingly sophisticated tools to track chromosomes and proteins in living cells offers the possibility of visualising gene regulation in the nucleus with minimal distortion . This, in conjunction with powerful genetic approaches available in yeast, is beginning to allow functional definition of nuclear "compartments".

Curr Opin Cell Biol, 2002 Jun, 14(3), 279 - 85
Centromeres and variant histones: what, where, when and why?
Smith MM.
The CENP-A histone H3-like variants are centromere-specific histones found in all eukaryotes examined to date, from budding yeast to man . New experiments using antibodies, green fluorescent protein fusions, and epitope tags show that CENP-A replaces the major histone H3 subunits in a specialized histone octamer and that it does so with histones H4, and probably H2A and H2B . One of the classic hallmarks of chromatin molecular biology is that nucleosomes are deposited on DNA during replication in S phase . However, dramatic new results in mammalian and Drosophila cells show that CENP-A deposition is uncoupled from the replication of centromere DNA . Furthermore, genetic and phenotypic knockout experiments over the past year have demonstrated that the deposition of CENP-A at newly duplicated sister centromeres is an early step in the biogenesis of new centromeres and is required for the recruitment of other proteins to the centromere and kinetochore . In organisms with complex regional or holocentric centromeres, centromere identity was thought to be defined by the epigenetic state of centromere chromatin . Now, new experiments solidify this model and show that the epigenetic state can be spread in cis experimentally, creating a neocentromere, in a mechanism reminiscent of chromatin transcriptional silencing . Finally, a new report provides a glimpse into the potential regulation of CENP-A through specific post-translational phosphorylation, suggesting a broad level of control through histone tail modifications.

Curr Opin Cell Biol, 2002 Jun, 14(3), 269 - 78
Chromatin as a eukaryotic template of genetic information; Cavalli G; In eukaryotes, chromatin is essential for heredity . Chromatin architecture is sometimes "epistatic" over the DNA and imparts a different heritable state to the same DNA sequence or the same functional state to unrelated DNA sequences . This has been documented recently in a wide variety of studies focused on regulation of the yeast mating type, the function of Polycomb and trithorax group proteins, the specification of eukaryotic centromeres and neocentromeres, and genomic imprinting.

Ageing Res Rev, 2002 Jun, 1(3), 443 - 63
Replicative enzymes and ageing: importance of DNA polymerase alpha function to the events of cellular ageing; Srivastava VK et al.; A hallmark of cellular ageing is the failure of senescing cells to initiate DNA synthesis and transition from G1 into S phase of the cell cycle . This transition is normally dependent on or concomitant with expression of a set of genes specifying cellular proteins, some of which directly participate in DNA replication . Deregulation of this gene expression may play a pivotal role in the ageing process . The number of known enzymes and co-factors required to maintain integrity of the genome during eukaryotic DNA replication has increased significantly in the past few years, and includes proteins essential for DNA replication and repair, as well as for cell cycle regulation . In eukaryotic cells, ranging from yeast to man, a replicative enzyme essential for initiation of transcription is DNA polymerase alpha (pol alpha), the activity of which is coordinately regulated with the initiation of DNA synthesis . DNA pol alpha, by means of its primase subunit, has the unique ability to initiate de novo DNA synthesis, and as a consequence, is required for the initiation of continuous (leading-strand) DNA synthesis at an origin of replication, as well as for initiation of discontinuous (lagging-strand) DNA synthesis . The dual role of the pol alpha-primase complex makes it a potential interactant with the regulatory mechanisms controlling entry into S phase . The purpose of this review is to address the regulation and/or modulation of DNA pol alpha during ageing that may play a key role in the cascade of events which ultimately leads to the failure of old cells to enter or complete S phase of the cell cycle.

Ageing Res Rev, 2002 Jun, 1(3), 313 - 26
Regulation of lifespan by histone deacetylase; Chang KT et al.; Aging is a universal biological phenomenon in eukaryotes, but why and how we age still remain mysterious . It would be of great biological interest and practical importance if we could uncover the molecular mechanism of aging, and find a way to delay the aging process while maintaining physical and mental strengths of youth . Histone deacetylases (HDACs) such as SIR2 and RPD3 are known to be involved in the extension of lifespan in yeast and Caenorhabditis elegans . An inhibitor of HDACs, phenylbutyrate, also can significantly increase the lifespan of Drosophila, without diminution of locomotor vigor, resistance to stress, or reproductive ability . Treatment for a limited period, either early or late in adult life, is also effective . Alteration in the pattern of gene expression, including induction or repression of numerous genes involved in longevity by changing the level and the pattern of histone acetylation may be an important factor in determining the longevity of animals.

J Neurochem, 2002 Apr, 81(1), 36 - 45
Identification and characterization of a novel Nogo-interacting mitochondrial protein (NIMP); Hu WH et al.; Nogo is a potent inhibitor of regeneration following spinal cord injury . To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two-hybrid approach to try and identify proteins that interact with Nogo . We identified a novel mitochondrial protein designated Nogo-interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library . This interaction was confirmed by co-immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed) . In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain . The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria . NIMP is highly conserved and ubiquitously expressed in mitochondria-enriched tissues . Within the CNS, NIMP-like immunoreactivity is present in neurons and astrocytes . These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo . The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo-induced inhibition of neurite growth.

Eur J Cell Biol, 2002 May, 81(5), 273 - 80
The SNAREs vti1a and vti1b have distinct localization and SNARE complex partners; Kreykenbohm V et al.; Two mammalian proteins, vtila and vtilb, are homologous to the yeast Q-SNARE Vtilp which is part of several SNARE complexes in different transport steps . In vitro experiments suggest distinct functions for vtila and vtilb . Here we compared the subcellular localization of endogenous vtila and vtilb by immunofluorescence and immuno-electron microscopy . Both proteins had a distinct but overlapping localization . vtila was found predominantly on the Golgi and the TGN, vtilb mostly on tubules and vesicles in the TGN area and on endosomes . vti1a coimmunoprecipitated with VAMP-4, syntaxin 6, and syntaxin 16 . These four SNAREs could assemble into a SNARE complex of conserved structure because one SNARE motif of each subgroup is present . vtila-beta, VAMP-4, syntaxin 6, and syntaxin 16 are coenriched with small synaptic vesicles and with clathrin-coated vesicles isolated from rat brain synaptosomes . Therefore, this SNARE complex may have a role in synaptic vesicle biogenesis or recycling.

Res Microbiol, 2002 May, 153(4), 249 - 52
Influence of abiotic factors on the bacteriocinogenic activity of Actinobacillus actinomycetemcomitans; Lima FL et al.; We evaluated the influence of abiotic factors on antagonistic activity of Actinobacillus actinomycetemcomitans strains isolated from periodontal pockets and two reference strains (ATCC 29523 and FDC Y4) . Antagonistic assays were performed by the overlayer method on tryptic soy agar (TSA), brain heart infusion agar, thioglycollate agar and brucella agar, added with yeast extract and supplemented (S) or not with L-cystine and sodium bicarbonate . Iso-, auto-, and heteroantagonism against a wide range of indicator strains were assayed . The influence of incubation atmosphere (anaerobic chamber, anaerobic and candle jars) and pH (5.0 to 11.0) was also evaluated . Autoantagonism was not observed . TSA-S was shown to be the most adequate medium for antagonistic activity expression . The widest spectrum of heteroantagonistic activity was also observed on TSA-S . The incubation atmosphere affected only the isoantagonistic activity expression . Only at pH 8.0 did A . actinomycetemcomitans express bacteriocinogenic activity . The lack of standardized methodology to detect antagonistic activity can lead to discrepant results and can make data difficult to be compared . This study provides information on abiotic factors that influence bacteriocinogenic activity expression and suggests adequate culture conditions for testing A . actinomycetemcomitans bacteriocin production, contributing to the establishment of a reproducible and reliable methodology.

Int J Prosthodont, 2001 Sep-Oct, 14(5), 457 - 60
Adherence of Candida species to newly polymerized and water-stored denture base polymers; Waltimo T et al.; PURPOSE: The aim of this in vitro study was to examine the adherence of yeasts to newly polymerized and water-stored denture base polymers using four Candida strains with different cell surface hydrophobicities . MATERIALS AND METHODS: Thirty-two autopolymerized denture base polymer specimens were stored in distilled water at 37 degrees C for 7 days and 32 were newly polymerized . Sixteen specimens of each group were pretreated with unstimulated mixed saliva (saliva group), and 16 were pretreated with phosphate-buffered saline ({PBS} PBS group) at 37 degrees C for 1 hour . Hydrophobicity of the newly polymerized and water-stored specimen surfaces was determined by contact angle measurements . C guilliermondii, C albicans, C glabrata, and C tropicalis were grown on tryptic soy agar . They were pretreated either with saliva or PBS and suspended in PBS . Four parallel test specimens were incubated without agitation in each yeast suspension at 37 degrees C for 1 hour, washed, and allowed to dry in air . Adherent cells were fixed, gram stained, and counted from 10 high-power light-microscopy fields of each specimen . RESULTS: Enhanced adherence of yeasts was observed in water-stored test specimens in comparison to newly polymerized test specimens . Salivary pellicle reduced the hydrophobicity of test specimens and the adherence of yeasts . No differences in contact angles of distilled water between newly polymerized and water-stored polymers were observed . CONCLUSION: Yeasts seem to adhere less to newly polymerized than water-stored denture base polymer . This may be due to the release of residual monomer from the newly polymerized material.

J Neurochem, 2002 May, 81(3), 557 - 64
The RNA-binding protein Staufen from rat brain interacts with protein phosphatase-1; Monshausen M et al.; In mammalian neurones, homologues of the Drosophila RNA-binding protein Staufen are part of ribonucleoprotein complexes that move bidirectionally along dendritic microtubules and appear to regulate mRNA translocation and translation . In this study, putative components of Staufen granules were identified in a yeast two-hybrid screen of a rat brain cDNA library with a rat Staufen bait . Protein phosphatase-1 was found as an interacting partner . Binding appears to be mediated by a five amino acid residue sequence motif (R-K-V-T-F) in Staufen that is conserved in a number of proteins interacting with the phosphatase . A two amino acid residue mutation within this motif (R-K-V-G-A) disrupted the interaction . A cytoplasmic interaction of both proteins was shown by coimmunoprecipitation of rat Staufen and protein phosphatase-1 from the cytoplasm of transfected cells and rat brain homogenates . In mammalian brain, the phosphatase represents the first described endogenous interaction partner of Staufen . In primary hippocampal neurones, both proteins partially colocalize in somata and neuronal processes . Staufen does not modulate the in vitro protein phosphatase activity . These findings show that protein phosphatase-1 is a native component of Staufen particles . Cellular functions of Staufen may be regulated via phosphorylation or Staufen may recruite the phosphatase into specific ribonucleoprotein complexes.

J Neurochem, 2002 Jun, 81(5), 1130 - 40
The metabotropic glutamate receptor mGluR7b binds to the catalytic gamma-subunit of protein phosphatase 1; Enz R; Correct targeting of enzymes represents an important biological mechanism to control post-translational modifications of neurotransmitter receptors . The metabotropic glutamate receptor type 7 (mGluR7) exists in two splice variants (mGluR7a and mGluR7b), defined by different C-termini that are phosphorylated by protein kinase C (PKC) . Recently, the search for mGluR7a binding partners yielded several proteins that interacted with its C-terminus . Here, a yeast two-hybrid screen using the mGluR7b C-terminus identified both variants of the catalytic gamma-subunit of protein phosphatase 1 (PP1gamma1 and PP1gamma2) as binding partners . The minimal interacting region of PP1gamma1/2 contained the core domain and was homologous to a region of PP1alpha that is needed for functional expression . Although this core domain is highly conserved within the protein phosphatase family, PP1alpha1 and PP1beta did not interact with mGluR7b . Binding between PP1gamma1 and mGluR7b might be regulated by alternative splicing, as the variant-specific distal part of the mGluR7b C-terminus mediated the interaction . Within this domain, amino acids involved in the binding to PP1gamma1 were mapped and biochemical assays using recombinant and native proteins verified the proposed interaction . Finally, the expression pattern of PP1gamma1, PP1gamma2 and mGluR7b was analysed in various CNS regions . In summary, these results suggest a regulation of mGluR7b by PP1gamma.

Infect Immun, 2002 Jul, 70(7), 3759 - 67
Cellular and molecular regulation of vaccination with heat shock protein 60 from Histoplasma capsulatum; Deepe GS Jr et al.; Vaccination with heat shock protein 60 (Hsp60) from Histoplasma capsulatum induces a protective immune response in mice . We explored the cellular and molecular requirements for the efficacy of recombinant Hsp60 in mice . Depletion of CD4(+), but not CD8(+), cells during the inductive phase of vaccination abolished protection, as assessed by survival and by the fungal burden in lungs and spleens . In the expressive phase, the elimination of CD4(+) or CD8(+) cells after immunization did not significantly alter fungal recovery or survival from a lethal challenge . Depletion of both subpopulations after Hsp60 vaccination resulted in a failure to control a lethal infection and a higher fungal burden in lungs and spleens . Cytokine release by spleen cells from mice vaccinated with Hsp60 produced substantially more gamma interferon and interleukin-10 and -12 than that of cells from mice immunized with either H . capsulatum recombinant Hsp70 or bovine serum albumin . The generation of gamma interferon, but not of interleukin-10, was dependent on T cells, in particular CD4(+) cells . Treatment of Hsp60-immunized mice with monoclonal antibody to gamma interferon or interleukin-10 or -12 in the inductive phase of vaccination was accompanied by increased recovery of yeast cells from lungs and spleens and 100% mortality . Likewise, the neutralization of gamma interferon or interleukin-12 abolished the protective effect of Hsp60 in the expressive phase . These results delineate the complexity of the regulatory elements necessary for vaccination against this fungus.

EMBO J, 2002 Jun 17, 21(12), 3171 - 81
Drf1, a novel regulatory subunit for human Cdc7 kinase; Montagnoli A et al.; Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs . Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins . Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity . This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells . Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4 . Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase . Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits . Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome.

EMBO J, 2002 Jun 17, 21(12), 2843 - 53
Structural basis for the interaction between NTF2 and nucleoporin FxFG repeats; Bayliss R et al.; Interactions with nucleoporins containing FxFG-repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2) . We describe here the 1.9 A resolution crystal structure of yeast NTF2-N77Y bound to a FxFG-nucleoporin core, which provides a basis for understanding this interaction and its role in nuclear trafficking . The two identical FxFG binding sites on the dimeric molecule are formed by residues from each chain of NTF2 . Engineered mutants at the interaction interface reduce the binding of NTF2 to nuclear pores and cause reduced growth rates and Ran mislocalization when substituted for the wild-type protein in yeast . Comparison with the crystal structure of FG-nucleoporin cores bound to importin-beta and TAP/p15 identified a number of common features of their binding sites . The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.

J Endocrinol, 2002 Jun, 173(3), 493 - 506
Implication of alpha4 phosphoprotein and the rapamycin-sensitive mammalian target-of-rapamycin pathway in prolactin receptor signalling; Boudreau RT et al.; A prolactin (PRL)-responsive 3'-end cDNA encoding rat alpha4 phosphoprotein was previously isolated from a rat lymphoma cDNA library . Rat alpha4 is a homologue of yeast Tap42 and is a component of the mammalian target-of-rapamycin (mTOR) signalling pathway that stimulates translation initiation and G1 progression in response to nutrients and growth factors . In the present study, the full-length rat alpha4 cDNA was obtained by 5'-RACE and the 1023 bp open reading frame predicted a 340 amino acid protein of 39.1 kDa . The alpha4 mRNA was expressed in quiescent PRL-dependent Nb2 lymphoma cells deprived of PRL for up to 72 h but expression was downregulated within 4 h of PRL treatment . In contrast, PRL-independent Nb2-Sp cells showed constitutive expression of alpha4 that was not affected by PRL . Western analysis of Nb2 cell lysates or of V5-tagged-alpha4 expressed in COS-1 cells detected a single immunoreactive band of approximately 45 kDa . Enzymatic deglycosylation of affinity-purified 45 kDa alpha4 yielded the predicted 39 kDa protein . Phosphorylation of Nb2 alpha4 was induced by PRL or 2-O-tetradecanoyl-phorbol-13-acetate (TPA) and further enhanced by a combination of PRL and TPA . The Nb2 alpha4 associated with the catalytic subunit of protein phosphatase 2A and localized predominantly in Nb2 nuclear fractions with trace amounts in the cytosol . The immunosuppressant drug rapamycin inhibited proliferation of Nb2 cells in response to PRL or interleukin-2, but had no effect on Nb2-Sp cells . Furthermore, transient overexpression of alpha4 in COS-1 cells inhibited PRL stimulation of the immediate-early gene interferon regulatory factor-1 promoter activity . Therefore, PRL downregulation of alpha4 expression and/or PRL-inducible phosphorylation of alpha4 may be necessary for PRL receptor (PRLr) signalling to the interferon regulatory factor-1 promoter in the Nb2 cells and, furthermore, implicates cross-talk between the mTOR and PRLr signalling cascades during Nb2 cell mitogenesis.

Food Chem Toxicol, 2002 Jul, 40(7), 1023 - 32
Comparison of acute toxicities of indolo{3,2-b}carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-sensitive rats; Pohjanvirta R et al.; 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons are environmental toxicants that act via the AH receptor (AHR) . In vitro studies have demonstrated that some indole derivatives present in cruciferous vegetables also bind to the AHR . One of the highest AHR binding affinities is exhibited by indolo{3,2-b}carbazole (ICZ) . Since exposure to these dietary indoles is quantitatively far larger than that to halogenated aromatic compounds, their potential toxic risks have raised concern . In the present study, we compared the effects of ICZ with those of a single dose of 20 microg/kg TCDD in the most TCDD-sensitive rat strain (Long-Evans {Turku AB}) (L-E) . Whereas TCDD elicited the expected toxicity syndrome, ICZ, either as a single subcutaneous dose (63.5, 127 or 508 microg/kg) or with repeated sc dosing (508 microg/kg for 5 days) failed to reproduce any toxic impacts of TCDD . Furthermore, a simultaneous ICZ treatment (63.5 or 127 microg/kg for 10 days) did not interfere with TCDD (20 microg/kg; single exposure) action . A moderate hepatic induction of CYP1A1 could be triggered by repeated intragastric administration of ICZ (127 microg/kg for 4 days, the last treatment 2.5 h prior to termination) . In control experiments in a reconstituted yeast system, ICZ potently and dose-dependently activated L-E rat AHR function demonstrating that it represents a bona fide high-affinity ligand for the rat receptor in vivo . Thus, the present study does not support the view that dietary exposure to ICZ would present a hazard of AHR-mediated adverse health effects to humans.

J Struct Biol, 2002 Jan-Feb, 137(1-2), 119 - 27
Molecular dissection of the interaction of desmin with the C-terminal region of nebulin; Bang ML et al.; In vertebrate skeletal muscle, ultrastructural studies have suggested that the Z-line and extracellular intermediate filaments are linked, although a structural basis for this has remained elusive . We searched for potential novel ligands of the Z-line portion of nebulin by a yeast two-hybrid (Y2H) approach . This identified that the nebulin modules M160 to M170 interact with desmin . In desmin, deletion series experiments assigned a 19-kDa central coiled-coil domain as the nebulin-binding site . The specific interactions of nebulin and desmin were confirmed in vitro by GST pull-down experiments . In situ, the nebulin modules M176 to M181 colocalize with desmin in a Z-line-associated, striated pattern as shown by immunofluorescence studies . Our data are consistent with a model that desmin attaches directly to the Z-line through its interaction with the nebulin repeats M163-M170 . This interaction may link myofibrillar Z-discs to the intermediate filament system, thereby forming a lateral linkage system which contributes to maintain adjacent Z-lines in register . (c) 2002 Elsevier Science (USA).

J Biol Chem, 2002 Aug 23, 277(34), 31115 - 23 Epub 2002 Jun 12.
Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen; Kedar PS et al.; Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery . As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM . Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo . Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins . Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA . The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.

J Biol Chem, 2002 Aug 23, 277(34), 30488 - 94 Epub 2002 Jun 12.
trans-Lesion synthesis past bulky benzo{a}pyrene diol epoxide N2-dG and N6-dA lesions catalyzed by DNA bypass polymerases; Rechkoblit O et al.; The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro . The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (a metabolite of the environmental carcinogen benzo{a}pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long . In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts . In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed . Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts . In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure . Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion . None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.

Gene, 2002 May 15, 290(1-2), 63 - 71
A gene trap Dissociation insertion line, associated with a RING-H2 finger gene, shows tissue specific and developmental regulated expression of the gene in Arabidopsis; Lechner E et al.; Real interesting new gene (RING) finger proteins act as E3 ubiquitin-protein ligases and play critical roles in targeting the destruction of proteins of diverse functions in all eukaryotes, ranging from yeast to mammals . Arabidopsis genome contains a large number of genes encoding RING finger proteins . In this report we describe the identification of more than 40 RING-H2 finger proteins that are of small size, not more than 200 amino acids, and contain no other recognizable protein-protein interaction domain(s) . We characterize RHA2b, one of these small RING-H2 finger genes . A gene trap line, SGT6304, was identified to contain a Dissociation (Ds) insertion in RHA2b gene . No RHA2b transcript was detected in the homozygous SGT6304 plants . Despite the elimination of RHA2b function, homozygous SGT6304 plants lacked detectable growth or development defects, suggesting functional redundancy of RHA2b with other RING finger genes . Expression of RHA2b was specifically active in vascular tissue and in upper pistil of inflorescence as well as in root tip and shoot apical meristem region . Potential functions of ubiquitin-proteolysis pathway in vascular formation and in fertilization are discussed.

Int J Parasitol, 2002 Jun, 32(6), 749 - 58
HcSTK, a Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, Haemonchus contortus; Nikolaou S et al.; A putative serine/threonine protein kinase (HcSTK) from the parasitic nematode Haemonchus contortus was characterised at the mRNA and amino acid levels . HcSTK displays a high level of identity (85-93% in the catalytic domain) with proteins of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily, which represent signal transduction molecules involved in establishing and maintaining polarity in proliferating and differentiating cells . The transcript of hcstk is expressed in different developmental stages (second-, third-, fourth-stage larvae and adults) and various organs (muscle, intestine and reproductive) of H . contortus . In addition, there are several isoforms which appear to relate to a single gene . The expression profile of hcstk is similar to that of Caenorhabditis elegans PAR-1, and the level of sequence identity among members of the PAR-1/MARK STK subfamily, representing a range of species of vertebrates (e.g . humans and rodents), invertebrates (e.g . insects and C . elegans) and yeast, suggests that HcSTK may be involved in a conserved signal transduction pathway.

Neuromuscul Disord, 2002 May, 12(4), 343 - 9
Muscular dystrophy into the new millennium; Emery AE; Since the identification of the gene for Duchenne muscular dystrophy and its protein product some 15 years ago, the basic defects in all the commoner forms of dystrophy have now been identified . It is thus possible, on the basis of this information, to make a precise diagnosis in an affected individual and to offer accurate genetic counselling and prenatal diagnosis . Now newer technologies are being applied to the investigation of these disorders . These include studies of single nucleotide polymorphisms, microarray analysis and expression profiling, the yeast two-hybrid assay, and proteomics . A great deal of new information is emerging in this way which will hopefully help us to understand the causes of inter-familial and intra-familial variation and particularly pathogenesis, a detailed understanding of which could be the first step in finding effective treatments.

J Biochem Biophys Methods, 2002 Apr 18, 51(2), 165 - 77
Fluorescence polarization discriminates green fluorescent protein from interfering autofluorescence in a microplate assay for genotoxicity; Knight AW et al.; An unconventional use for the polarization optics, associated with a variety of commercially available fluorescence microplate readers, is reported . This novel application has allowed the discrimination of green fluorescent protein (GFP) fluorescence in genetically modified yeast cells from interfering autofluorescent species . The method exploits the unusually high fluorescence anisotropy of GFP compared to smaller fluorophores and autofluorescent species . The principle was successfully applied to resolve the induced GFP signal from that of autofluorescent test compounds, in an assay for genotoxic species . The use of fluorescence polarization enabled both proflavin and methapyrilene to be identified as genotoxic agents in the yeast assay . This would not have been possible using conventional fluorescence alone since these compounds were found to be intensely autofluorescent at the same wavelength as GFP and thus effectively mask the GFP signal.

Cell, 2002 May 31, 109(5), 563 - 73
Cid13 is a cytoplasmic poly(A) polymerase that regulates ribonucleotide reductase mRNA; Saitoh S et al.; Fission yeast Cid13 and budding yeast Trf4/5 are members of a newly identified nucleotidyltransferase family conserved from yeast to man . Trf4/5 are thought to be essential DNA polymerases . We report that Cid13 is a poly(A) polymerase . Unlike conventional poly(A) polymerases, which act in the nucleus and indiscriminately polyadenylate all mRNA, Cid13 is a cytoplasmic enzyme that specifically targets suc22 mRNA that encodes a subunit of ribonucleotide reductase (RNR) . cid13 mutants have reduced dNTP pools and are sensitive to hydroxyurea, an RNR inhibitor . We propose that Cid13 defines a cytoplasmic form of poly(A) polymerase important for DNA replication and genome maintenance.

Curr Biol, 2002 Jun 4, 12(11), 951 - 6
A direct interaction between IP(3) receptors and myosin II regulates IP(3) signaling in C . elegans; Walker DS et al.; Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signalling machinery as an important mechanism for the regulation of calcium signalling . However, little is known about the functions of such mechanisms in animals . A key component of the calcium signalling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP(3)), mediated by the IP(3) receptor (IP(3)R) . We show that C . elegans IP(3)Rs, encoded by the gene itr-1, interact directly with myosin II . The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro . We defined the interaction sites on both the IP(3)R and MYO-1 . To test the effect of disrupting the interaction in vivo we overexpressed interacting fragments of both proteins in C . elegans . This decreased the animal's ability to upregulate pharyngeal pumping in response to food . This is a known IP(3)-mediated process {15} . Other IP(3)-mediated processes, e.g., defecation, were unaffected . Thus it appears that interactions between IP(3)Rs and myosin are required for maintaining the specificity of IP(3) signalling in C . elegans and probably more generally.

Curr Biol, 2002 Jun 4, 12(11), 925 - 9
The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes; Bickel SE et al.; Accurate chromosome partitioning during cell division requires that cohesion hold sister chromatids together until kinetochores correctly attach to spindle microtubules . In 1932, Darlington noted that sister-chromatid cohesion distal to the site of exchange also could play a vital role in maintaining the association of chiasmate homologs during meiosis . Cohesion linking a recombinant chromatid with a sister of each homologous pair would resist spindle forces that separate kinetochores of homologous chromosomes (see Figure 1) . Although centromeric cohesion must be retained to ensure proper segregation during meiosis II, dissolution of arm cohesion would be required for anaphase I to occur . This hypothesis is supported by recent evidence in yeast and C . elegans that separase activity is essential for the segregation of recombinant homologs during meiosis I . We present evidence that Drosophila oocytes require sister-chromatid cohesion to maintain a physical attachment between recombinant chromosomes . Using FISH to monitor cohesion directly, we confirm that oocytes lacking ORD activity exhibit cohesion defects, consistent with previous genetic results . We also show that ord(null) oocytes that have undergone recombination are unable to arrest at metaphase I, indicating that chiasmata are unstable in the absence of cohesion . Our results support the model that arm cohesion provides a conserved mechanism that ensures physical attachment between recombinant homologs until anaphase I.

Curr Biol, 2002 Jun 4, 12(11), 900 - 5
Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis; Kallio MJ et al.; How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear . In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion . In C . elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis . To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects . After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes . Injected cells exited mitosis with no evidence of anaphase or cytokinesis . Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules . Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs . Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7986 - 91
Obligatory heterotetramerization of three previously uncharacterized Kv channel alpha-subunits identified in the human genome; Ottschytsch N et al.; Voltage-gated K(+) channels control excitability in neuronal and various other tissues . We identified three unique alpha-subunits of voltage-gated K(+)-channels in the human genome . Analysis of the full-length sequences indicated that one represents a previously uncharacterized member of the Kv6 subfamily, Kv6.3, whereas the others are the first members of two unique subfamilies, Kv10.1 and Kv11.1 . Although they have all of the hallmarks of voltage-gated K(+) channel subunits, they did not produce K(+) currents when expressed in mammalian cells . Confocal microscopy showed that Kv6.3, Kv10.1, and Kv11.1 alone did not reach the plasma membrane, but were retained in the endoplasmic reticulum . Yeast two-hybrid experiments failed to show homotetrameric interactions, but showed interactions with Kv2.1, Kv3.1, and Kv5.1 . Co-expression of each of the previously uncharacterized subunits with Kv2.1 resulted in plasma membrane localization with currents that differed from typical Kv2.1 currents . This heteromerization was confirmed by co-immunoprecipitation . The Kv2 subfamily consists of only two members and uses interaction with "silent subunits" to diversify its function . Including the subunits described here, the "silent subunits" represent one-third of all Kv subunits, suggesting that obligatory heterotetramer formation is more widespread than previously thought.

Am J Physiol Renal Physiol, 2002 Jul, 283(1), F142 - 9
Regulation of the voltage-gated K+ channel KCNA10 by KCNA4B, a novel beta-subunit; Tian S et al.; Voltage-gated K+ (Kv) channels are heteromultimeric complexes consisting of pore-forming alpha-subunits and accessory beta-subunits . Several beta-subunits have been identified and shown to interact with specific alpha-subunits to modify their levels of expression or some of their kinetic properties . The aim of the present study was to isolate accessory proteins for KCNA10, a novel Kv channel alpha-subunit functionally related to Kv and cyclic nucleotide-gated cation channels . Because one distinguishing feature of KCNA10 is a putative cyclic nucleotide-binding domain located at the COOH terminus, the entire COOH-terminal region was used to probe a human cardiac cDNA library using the yeast two-hybrid system . Interacting clones were then rescreened in a functional assay by coinjection with KCNA10 in Xenopus oocytes . One of these clones (KCNA4B), when injected alone in oocytes, produced no detectable current . However, when coinjected with KCNA10, it increased KCNA10 current expression by nearly threefold . In addition, the current became more sensitive to activation by cAMP . KCNA4B can be coimmunoprecipitated with the COOH terminus of KCNA10 and full-length KCNA10 . It encodes a soluble protein (141 aa) with no amino acid homology to known beta-subunits but with limited structural similarity to the NAD(P)H-dependent oxidoreductase superfamily . KCNA4B is located on chromosome 13 and spans approximately16 kb, and its coding region is made up of five exons . In conclusion, KCNA4B represents the first member of a new class of accessory proteins that modify the properties of Kv channels.

J Dermatolog Treat, 2002 Jun, 13(2), 51 - 60
A randomised, single-blind, single-centre clinical trial to evaluate comparative clinical efficacy of shampoos containing ciclopirox olamine (1.5%) and salicylic acid (3%), or ketoconazole (2%, Nizoral) for the treatment of dandruff/seborrhoeic dermatitis; Squire RA et al.; BACKGROUND: The association between seborrhoeic dermatitis and dandruff and the yeast Malassezia furfur is well recognized . Symptoms include scalp itchiness and scaling . Due to its antimycotic activity, ciclopirox olamine is established as an effective treatment for these scalp conditions . Salicylic acid has keratolytic properties and aids in the removal of scales . OBJECTIVE: To compare the therapeutic efficacy of a shampoo containing 1.5% ciclopirox olamine and 3% salicylic acid (CPO/SA) with Nizoral (2.0% ketoconazole shampoo) in a study involving 154 subjects with dandruff - 70 of whom also had seborrhoeic dermatitis of the scalp . Nizoral is currently a registered treatment for dandruff and seborrhoeic dermatitis . METHODS: The shampoos were used three times week for 4 weeks, with 2-week washout and follow-up periods . Clinical and self-assessments were made throughout treatment and after follow-up (day 43) . Within and between-treatment assessments of signs and symptoms were analysed . RESULTS: In the two groups, seborrhoeic dermatitis and dandruff improved significantly throughout treatment, with lower clinical and self-assessment scores at both the end of treatment (day 29) and follow-up (day 43) . Only the subjects treated with CPO/SA shampoo showed a significant reduction in the itching of seborrhoeic dermatitis at these times . CONCLUSION: The study demonstrated that both CPO/SA and Nizoral were safe and effective in the treatment of dandruff and seborrhoeic dermatitis.

J Invest Dermatol, 2002 Jun, 118(6), 1044 - 51
Positive atopy patch test reaction to Malassezia furfur in atopic dermatitis correlates with a T helper 2-like peripheral blood mononuclear cells response; Johansson C et al.; The yeast Malassezia furfur belongs to the normal cutaneous flora, but is also a triggering allergen that can contribute to atopic dermatitis . To illuminate the effect of circulating allergen-specific T cells in atopic dermatitis, the peripheral mononuclear cell response was correlated with the in vivo skin prick test and atopy patch test reactivity to M . furfur . None of 16 healthy controls showed any positive in vivo reaction . The 40 atopic dermatitis patients, of whom 18 had serum IgE reactivity to M . furfur, were subdivided according to their in vivo reaction to M . furfur extract into three groups: skin prick test positive/atopy patch test positive (n = 12), skin prick test positive/atopy patch test negative (n = 12), and skin prick test negative/atopy patch test negative (n = 16) . The skin prick test positive/atopy patch test positive and the skin prick test positive/atopy patch test negative groups had a significantly higher peripheral mononuclear cell stimulation index than the healthy controls . Interestingly, the stimulation index values in the skin prick test positive/atopy patch test positive group were significantly higher than in the skin prick test positive/atopy patch test negative group . In the M . furfur skin prick test positive atopic dermatitis patients (n = 24) a correlation was found between stimulation index and the M . furfur atopy patch test reactions, but not between stimulation index and M . furfur-specific serum IgE levels . Skin prick test positive and/or atopy patch test positive reactions to the recombinant M . furfur allergens rMal f 1, rMal f 5, and rMal f 6 were observed in 7, 14, and 16 of the 40 atopic dermatitis patients, respectively . Further, there was a correlation between production of the T helper 2-related cytokines interleukins 4, 5, and 13 and stimulation index to M . furfur extract, but not between the T helper 1-related interferon-gamma and stimulation index to M . furfur extract . Our data strongly suggest a relationship between circulating specific T cells with a T helper 2-like cytokine profile and positive atopy patch test reactions.

J Agric Food Chem, 2002 Jun 19, 50(13), 3867 - 73
Assessment of selenium bioavailability from high-selenium spirulina subfractions in selenium-deficient rats; Cases J et al.; It was previously found that the bioavailability of Se from Se-rich spirulina (SeSp) was lower than that from selenite or selenomethionine when fed to Se-deficient rats . The present study examined the bioavailability of Se from SeSp subfractions: a pellet (P) issuing from the centrifugation of a suspension of broken SeSp and a retentate (R) resulting from ultrafiltration of the supernatant through a 30 kDa exclusion membrane . Animals were fed a torula yeast based diet with no Se (deficients) or supplemented with 75 microg of Se/kg of diet as sodium selenite (controls) for 42 days . Se-deficient rats were then repleted for 56 days with Se (75 microg/kg of diet) supplied as sodium selenite, SeSp, P, or R . During this period, controls continued to receive sodium selenite . Speciation of Se in subfractions showed that the majority was present in the form of high molecular weight compounds; free selenomethionine was only a minor constituent . Gross absorption of Se from sodium selenite, P, and R was not different and was higher than from SeSp . Only retentate allowed full replenishment of Se concentration in liver and kidney (as did sodium selenite) and glutathione peroxidase (GSHPx) activity in liver, kidney, plasma, and erythrocytes . The bioavailabilities of Se in retentate, as assessed by slope ratio analysis using selenite as a reference Se, were 89 and 112% in the tissue Se content and 106-133% in the GSHPx activities . SeSp and P exhibited a gross bioavailability of <100% . These results indicate that Se in retentate is highly bioavailable and represents an interesting source of Se for food supplementation.

J Mol Neurosci, 2002 Jun, 18(3), 229 - 38
Mutant and wild-type alpha-synuclein interact with mitochondrial cytochrome C oxidase; Elkon H et al.; Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson's disease (PD) . Furthermore, rare mutations of alpha-synuclein cause autosomal-dominant PD . However, it is unknown how alpha-synuclein is involved in the pathogenesis of nigral degeneration in PD . In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) a-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique . We found that both normal and mutant alpha-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX) . Wild-type and mutant alpha-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate . Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant alpha-synuclein, coimmunoprecipitated with COX . We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death . In conclusion, we found specific protein-protein interactions of alpha-synuclein, a major LB protein, to COX, a key enzyme of the mithochondrial respiratory system . This interaction suggests that alpha-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.

Planta Med, 2002 May, 68(5), 449 - 51
Estrogenic activity of the phytoestrogens naringenin, 6-(1,1-dimethylallyl)naringenin and 8-prenylnaringenin; Zierau O et al.; Chemically synthesized naringenin derivatives, identical to natural occurring compounds, were tested for their estrogenic activity using two independent estrogen screening assays . Using a yeast based estrogen receptor assay, strong estrogenic activities were demonstrated for 6-(1,1-dimethylallyl)naringenin and 8-prenylnaringenin, while the parent compound naringenin did not show recognizable estrogenic activity . In MVLN cells, a bioluminescent MCF-7-derived cell line, the estrogenic activity of 8-prenylnaringenin and 6-(1,1-dimethylallyl)naringenin was detected at concentrations of 10(-6) M and 5 x 10(-6) M respectively . Naringenin demonstrated estrogenic activity but only at a concentration of 10(-5) M . These estrogenic effects are mediated by the ER, as the antiestrogen 4-hydroxytamoxifen inhibited these activities . In summary, this study provides the further confirmation that 8-prenylnaringenin demonstrates high estrogenic activity, and demonstrated for the first time for 6-(1,1-dimethylallyl)naringenin a reasonable high estrogenic activity, while naringenin exhibit low or no estrogenic activity.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(6), 633 - 636
A Recombinant Monomeric Human Insulin Mutant with Resistance to Trypsin Design, Preparation and Characterization; Zhang H et al.; By using a gapped duplex DNA method, the four amino acids on the B chain of insulin, i.e . B22Arg, B28Pro, B29Lys and B30Thr, were mutagenized simultaneously into B22Asp, B28Lys, B29Pro and B30Lys, respectively . The recombinant B22Asp B28Lys B29Pro B30Lys human insulin was obtained simply by the treatment with trypsin of the precursor that was expressed in yeast . This human insulin analog had only 6% of receptor binding activity as that of the porcine insulin, but retained 50% of in vivo biological activity, compared with the latter . The self-association ability of the mutant measured through FPLC gel filtration chromatography showed that it was in monomeric state . As a monomeric insulin analog with resistance to the trypsin digestion, this compound may be promising in practical application.

J Cell Biol, 2002 Jun 10, 157(6), 1029 - 39 Epub 2002 Jun 10.
The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics; Facanha AL et al.; Here we describe the phenotypic characterization of the cta4+ gene, encoding a novel member of the P4 family of P-type ATPases of fission yeast . The cta4Delta mutant is temperature sensitive and cold sensitive lethal and displays several morphological defects in cell polarity and cytokinesis . Microtubules are generally destabilized in cells lacking Cta4p . The microtubule length is decreased, and the number of microtubules per cell is increased . This is concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell . These defects are likely due to a general imbalance in cation homeostasis . Immunofluorescence microscopy and membrane fractionation experiments revealed that green fluorescent protein-tagged Cta4 localizes to the ER . Fluorescence resonance energy transfer experiments in living cells using the yellow cameleon indicator for Ca2+ indicated that Cta4p regulates the cellular Ca2+ concentration . Thus, our results reveal a link between cation homeostasis and the control of cell shape, microtubule dynamics, and cytokinesis, and appoint Ca2+ as a key ion in controlling these processes.

Structure (Camb), 2002 Jun, 10(6), 751 - 61
Crystal structure of the C-terminal WD40 repeat domain of the human Groucho/TLE1 transcriptional corepressor; Pickles LM et al.; Groucho (Gro)/TLE proteins are transcriptional corepressors that lack inherent DNA binding but interact with DNA-bound transcription factors and histones, and recruit histone deacetylases . Groucho-mediated repression is essential in embryonic development and involved in regulation of Wnt signaling in adult tissue . We have determined the 1.6 A crystal structure of a C-terminal fragment of human Groucho/TLE1, comprising part of the Ser/Pro-rich region and a seven-bladed beta propeller WD40 repeat domain, implicated in protein-protein interactions . The structure confirms the relationship to the yeast Tup1 corepressor, but reveals important structural differences specific to the metazoan system . Analysis of missense mutations in the C . elegans Groucho homolog UNC-37 identifies sites of interaction with repression effectors, and suggests an induced fit binding site for eh1 domains of Engrailed-type transcription factors.

DNA Res, 2002 Apr 30, 9(2), 31 - 4
Overexpression of the AtmybL2 gene represses trichome development in Arabidopsis; Sawa S; Leaf trichome formation is known to be regulated by the TTG, GL1, GL2, and GL3 genes in Arabidopsis . GL1 and GL3 encode proteins with Myb and bHLH domains, respectively . Overexpression of the AtmybL2 gene, which encodes a single Myb-like DNA-binding domain, repressed trichome development in transgenic Arabidopsis plants . The amount of GL2 transcription was clearly reduced in the transgenic plants . Consistent with this, overexpression of AtmybL2 decreased beta-glucuronidase (GUS) activity in transgenic plants carrying a GUS-reporter gene regulated by the GL2 promoter . These findings, together with the results from our yeast two-hybrid analysis, suggest that GL3 gene function and overexpression of AtmybL2 act synergistically to inhibit trichome formation by negatively regulating GL2 expression.

Hong Kong Med J, 2002 Jun, 8(3), 212 - 4
Malassezia furfur fungaemia in a ventilator-dependent patient without known risk factors; Chu CM et al.; Malassezia furfur is the lipophilic yeast which causes tinea versicolor and is an uncommon cause of fungaemia . It usually occurs in the context of hyperalimentation with lipid emulsion, immunosuppression, or the presence of a central venous catheter . We report a case of a ventilator-dependent patient who developed Malassezia furfur fungaemia in the absence of these known risk factors . A likely risk factor in this patient was receipt of multiple courses of broad-spectrum antibiotics . This case highlights the importance of recognising Malassezia furfur as a cause of fungaemia, as well as the need for special culture techniques to aid identification.

Methods, 2002 Jan, 26(1), 84 - 92
The use of altered specificity mutants to probe specific protein-protein interactions involved in the activation of GATA-1 target genes; Crispino JD et al.; With the increasing popularity of the yeast two-hybrid screen, a large number of protein-protein interactions have been identified . In many cases, single proteins have been found to associate with a large number of cofactors . For example, the hematopoietic transcription factor GATA-1 interacts with a multitude of other nuclear proteins, including Friend of GATA-1 (FOG-1), EKLF, CBP/p300, and Lmo2 . Furthermore, p300, besides associating with GATA-1, interacts with at least seven other hematopoietic transcription factors . Despite the numerous pairwise and higher-order interactions identified, assessment of their contribution to transcriptional control has been lacking . Here we describe a strategy that can be applied to assess the functional relevance of any protein-protein association . This approach involves the creation of altered specificity mutants though the use of a combination of two yeast two-hybrid screens . Once altered specificity factors are obtained, a researcher can then proceed to functional assays that address the role of a specific protein-protein interaction . (c) 2002 Elsevier Science (USA).

Methods, 2002 Mar, 26(3), 254 - 9
Immunoprecipitation techniques for the analysis of transcription factor complexes; Klenova E et al.; Interactions among transcription factors can be detected and analyzed by a variety of in vitro and in vivo approaches . In many studies, the existence of putative interactions among transcription factor partners is initially established from yeast two-hybrid screening and in vitro protein association analysis . The ability to detect candidate interacting proteins in coimmunoprecipitates from cell lysates provides an important criterion for establishing the authenticity of such protein interactions in vivo . This article describes methodology developed for detecting interactions between the helix-loop-helix protein, Id3, and the paired homeodomain protein, Pax5, and interactions involving the zinc finger transcription factor, CTCF . The importance of empirically establishing optimum conditions for cell lysis, selection of appropriate antibodies, conditions for immunoprecipitation, and detection of interacting partners are discussed.

J Mol Biol, 2002 Apr 19, 318(1), 45 - 58
A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase; de Soultrait VR et al.; The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome . In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy . To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides . Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme . I33 inhibited both in vitro IN activities, i.e . 3' end processing and strand transfer . Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33 . Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate . Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition . The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties . Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells . Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN .

Biochem Biophys Res Commun, 2002 May 24, 293(5), 1426 - 30
Identification and genetic analysis of human and mouse activated Cdc42 interacting protein-4 isoforms; Wang L et al.; By yeast two-hybrid screening with the Src kinase Lyn as bait, we identified a novel gene product with features of a scaffolding protein . Reported as Felic ( es-related, with homology to Ezrin, Lyn interactor with Cdc42), it is related to the CIP4 (Cdc42 Interacting Protein-4) gene . Southern blotting for CIP4/Felic of genomic DNA shows a single band, suggesting no gene duplication . Felic differs from CIP4 because of a 29 nucleotide sequence derived from the end of intron 13 . Consequently, there is an out-of-frame translation that destroys an SH3 domain . Analysis of various tissues shows that the original CIP4 is the predominant transcript . Therefore, we propose to call that, CIP4a and Felic, CIP4b . During screening of the colorectal CaCo2 cell line, clones corresponding to a third CIP4-related transcript (CIP4c) were identified . CIP4c encodes a premature stop codon, resulting in the loss of the SH3 domain . A fourth, relatively abundant transcript (CIP4h) was isolated from heart, lung, and trachea tissue . CIP4h retains the SH3 domain . CIP4 levels are modified by all-trans-retinoic acid . The presence of alternative splice transcripts, with or without SH3 domains, suggests that CIP4 regulates cytoskeletal organization through structural-functional differences in a tissue-specific manner.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 617 - 21
Up-regulation of PDCD4 in senescent human diploid fibroblasts; Kang MJ et al.; Programmed cell death 4 (PDCD4) has a common MI domain sharing with death associated protein 5 (DAP5) and a component of eukaryotic translation initiation factor (eIF4G) complex and it might also work as a tumor suppressor . We could find that the message and product of Pdcd4 gene were up-regulated in senescent human diploid fibroblasts . In yeast two hybrid analysis, the C-terminal region of PDCD4 interacted with ribosomal protein S13 (RPS13), ribosomal protein L5 (RPL5), and TI-227H . In in vitro binding assay, RPS13, a component of 40S ribosome was stably bound to PDCD4 . We also found that PDCD4 was localized to polysome fractions . We could pull out eIF4G with GST-PDCD4, but eIF4E did not interact with PDCD4 . From these results, we could assume that PDCD4 might regulate the eIF4G-dependent translation through direct interactions with eIF4G and RPS13 in senescent fibroblasts.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 274 - 83
Genetic analysis of four novel peroxisome proliferator activated receptor-gamma splice variants in monkey macrophages; Zhou J et al.; Peroxisome proliferator activated receptor-gamma (PPAR-gamma) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis . The existence of three splice variants, PPAR-gamma 1, PPAR-gamma 2, and PPAR-gamma 3 has been established . Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-gamma exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5(') terminal region to generate four novel PPAR-gamma subtypes, PPAR-gamma 4, -gamma 5, -gamma 6, and -gamma 7 . PPAR-gamma 4 and gamma 5 were detected only in macrophages whereas gamma 6 and gamma 7 were expressed both in macrophages and adipose tissues . None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys . We found sequences identical to exons C and D in the human genome database . These and all PPAR-gamma exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3 . We cloned and expressed PPAR-gamma 1, PPAR-gamma 4, and PPAR-gamma 5 proteins in yeast using the expression vector pPICZB . As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa . We also investigated the effect of a high-fat diet on the level of macrophage PPAR-gamma expression in monkeys . RT-PCR showed a significant increase in total PPAR-gamma and ABCA1 mRNA levels in macrophages of fat-fed monkeys (n=7) compared to those maintained on a normal diet (n=2) . However, none of the novel isoforms seemed to be induced by fat-feeding . We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-gamma 4 and -gamma 5 in CHO cells . In these cells, expression of PPAR-gamma 5 but not -gamma 4 repressed the expression of ABCA1 . Neither isoform modulated the expression of lipoprotein lipase . Our results suggest that individual PPAR-gamma isoforms may be responsible for unique tissue-specific biological effects and that PPAR-gamma 4 and -gamma 5 may modulate macrophage function and atherogenesis.

Biochem Biophys Res Commun, 2002 May 3, 293(2), 816 - 26
Identification and characterization of a novel murine multigene family containing a PHD-finger-like motif; Trappe R et al.; The genes Phf5a and Phf5b-ps are the first two members of a novel murine multigene family that is highly conserved during evolution and belongs to the superfamily of PHD-finger genes . The Phf5 gene family contains an active locus on mouse chromosome 15, region E and several processed pseudogenes on different chromosomes . The active locus, Phf5a, is expressed ubiquitously in pre- and postnatal murine tissues and encodes a protein of 110 amino acids . The protein is localized in the nucleus in a non-homogenous pattern as the nucleolar subcompartment is almost free of Phf5a . The molecular and biological functions of Phf5a are unknown up-to-date, but the systematic deletion of its yeast homolog is lethal, pointing out that the protein is required for cell viability . Interpretation of our data and review of the literature suggest both basic and essential cellular functions of the Phf5a protein, possibly acting as a chromatin-associated protein .

Biochem Biophys Res Commun, 2002 May 3, 293(2), 759 - 65
The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/utrophin complex; Buechler C et al.; Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues . ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1 . Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains . Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex . ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains . Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin .

Biochem Biophys Res Commun, 2002 May 3, 293(2), 698 - 704
Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis; Iwahashi J et al.; Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER) . At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown . Each RTN gene produces 1-3 proteins by different promoters and alternative splicing . In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C . mRNA of nRTN-C is expressed in germ cells and embryos . However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch . By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated . These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis .

Biochem Biophys Res Commun, 2002 May 17, 293(4), 1191 - 6
Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction; Dai F et al.; Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K) . SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling . However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established . In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library . The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation . Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody . The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9 . (c) 2002 Elsevier Science (USA).

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 851 - 9
Leisingera methylohalidivorans gen . nov., sp . nov., a marine methylotroph that grows on methyl bromide; Schaefer JK et al.; A marine methylotroph, designated strain MB2T, was isolated for its ability to grow on methyl bromide as a sole carbon and energy source . Methyl chloride and methyl iodide also supported growth, as did methionine and glycine betaine . A limited amount of growth was observed with dimethyl sulfide . Growth was also noted with unidentified components of the complex media marine broth 2216, yeast extract and Casamino acids . No growth was observed on methylated amines, methanol, formate, acetate, glucose or a variety of other substrates . Growth on methyl bromide and methyl iodide resulted in their oxidation to CO2 with stoichiometric release of bromide and iodide, respectively . Strain MB2T exhibited growth optima at NaCl and Mg2+ concentrations similar to that of seawater . Phylogenetic analysis of the 16S rDNA sequence placed this strain in the alpha-Proteobacteria in proximity to the genera Ruegeria and Roseobacter . It is proposed that strain MB2T (= ATCC BAA-92T = DSM 14336T) be designated Leisingera methylohalidivorans gen . nov., sp . nov..

EMBO Rep, 2002 Jun, 3(6), 521 - 6
Peptidyl-prolyl isomerases: a new twist to transcription; Shaw PE; Peptidyl-prolyl isomerases (PPIs) catalyse the cis-trans isomerisation of peptide bonds N-terminal to proline residues in polypeptide chains . They have roles in the folding of newly synthesised proteins and in the function of the immune system . In addition, members of the parvulin-like family of PPIs have been implicated in cell cycle control . Their activity is directed by the prior phosphorylation of target proteins in both yeast and mammalian cells . More recent data have illustrated that they may also influence other nuclear events . This review examines PPI activity in the context of eukaryotic transcriptional regulation . The findings are consistent with a two-step model of conformational control, in which the outcome depends on the transcription factor involved.

Diagn Microbiol Infect Dis, 2002 May, 43(1), 85 - 6
Hypertonic sabouraud broth as a simple and powerful test for Candida dubliniensis screening; Alves SH et al.; We developed a new screening test for C . dubliniensis based on its inability to grow on Sabouraud dextrose broth with 6.5% NaCl . A total of 266 clinical yeast isolates and 3 reference strains were tested, including 250 C . albicans and 19 C . dubliniensis strains . All C . albicans isolates tested exhibited significant growth on hypertonic Sabouraud broth up to 96 h, while, all C . dubliniensis isolates did not exhibit any visually detectable growth during the same period.

Biotechnol Prog, 2002 May-Jun, 18(3), 597 - 603
Evaluation of process-induced dimensional changes in the membrane structure of biological cells using impedance measurement; Angersbach A et al.; The impact of high intensity electric field pulses, high hydrostatic pressure, and freezing-thawing on local structural changes of the membrane was determined for potato, sugar beet tissue, and yeast suspensions . On the basis of the electrophysical model of cell systems in biological tissues and suspensions, a method was derived for determining the extent of local damage of cell membranes . The method was characterized by an accurate and rapid on-line determination of frequency-dependent electrical conductivity properties from which information on microscopic events on cellular level may be deduced . Evaluation was based on the measurement of the relative change in the sample's impedance at characteristically low (f(l)) and high (f(h)) frequencies within the beta-dispersion range . For plant and animal cells the characteristic frequencies were f(l) approximately 5 kHz and f(h) > 5 MHz and for yeast cells in the range f(l) approximately 50 kHz and f(h) > 25 MHz . The observed phenomena were complex . The identification of the underlying mechanisms required consideration of the time-dependent nature of the processing effects and stress reactions of the biological systems, which ranged from seconds to several hours . A very low but significantly detectable membrane damage (0.004% of the total area) was found after high hydrostatic pressure treatment of potato tissue at 200 MPa . The membrane rupture in plant tissue cells was higher after freezing and subsequent thawing (0.9% of total area for potato cells and 0.05-0.07% for sugar beet cells determined immediately after thawing), which increased substantially during the next 2 h.

J Mol Biol, 2002 May 24, 319(1), 209 - 27
Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA; Herrmann T et al.; Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra . CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers . The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra . The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle . CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e . network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle . Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments . Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation . CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates . The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins . The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis . The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination.

J Mol Biol, 2002 May 31, 319(2), 395 - 406
DNA-repair by photolyase reveals dynamic properties of nucleosome positioning in vivo; Suter B et al.; Nucleosomes exert a repressive influence on the biological functions of DNA by restricting the access of proteins to DNA . To investigate how intrinsic properties of nucleosomes modulate DNA-accessibility in vivo, we studied DNA repair by photolyase in the yeast URA3 gene . Formation of DNA lesions (cyclobutane pyrimidine dimers, CPDs) and photolyase activity are controlled precisely by light . Preceding work revealed that photolyase repairs nucleosome-free DNA rapidly, while repair of nucleosomes is inhibited severely . The high-resolution data presented here show slow repair in the center of nucleosomes and a gradual increase towards the periphery . This pattern was observed in all nucleosomes and demonstrates that dynamic properties facilitate DNA accessibility . Since the URA3 nucleosomes can occupy alternate positions, the repair data are most consistent with nucleosome mobility that moves CPDs in linker DNA where they are repaired rapidly . A partial and transient unfolding or disruption of nucleosomes, however, may not be excluded . In addition, repair heterogeneity was found between closely spaced sites, indicating that structural properties of nucleosomes contribute to damage processing . Moreover, nucleosome-specific modulation of photolyase was found on the transcribed and non-transcribed strand . This is in contrast to homogeneous repair of the transcribed strand by nucleotide excision repair, and reveals fundamental differences in how both repair systems interact with nucleosomes and transcription .

Biochem Biophys Res Commun, 2002 May 10, 293(3), 1047 - 52
Protein Ser/Thr phosphatases PPEF interact with calmodulin; Kutuzov MA et al.; Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin . Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases . We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains . We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis . We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro . The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro . These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM . (c) 2002 Elsevier Science (USA).

Biochem Biophys Res Commun, 2002 Jun 7, 294(2), 272 - 9
Interaction of the C-terminal region of the rat serotonin transporter with MacMARCKS modulates 5-HT uptake regulation by protein kinase C; Jess U et al.; The serotonin transporter (SERT) mediates the re-uptake of released serotonin into presynaptic nerve terminals . Its activity is regulated by different mechanisms including protein kinase C (PKC) triggered internalization . Here, we used yeast 2-hybrid screening and cotransfection into 293 cells to identify a homologue of the myristoylated alanine-rich C kinase substrate (MARCKS), MacMARCKS, as a C-terminally interacting protein of SERT . Upon cotransfection with SERT, MacMARCKS caused a reduction in the maximal rate of {(3)H}serotonin uptake and reduced its down-regulation elicited by activation of PKC . Our data are consistent with MARCKS proteins regulating the plasma membrane dynamics of neurotransmitter transporters . (c) 2002 Elsevier Science (USA).

Contemp Top Lab Anim Sci, 2002 May, 41(3), 33 - 7
A technique for creating localized subcutaneous Blastomyces granulomas in rats; Bassett CL et al.; Our purpose was to develop a simple, reliable method for creating subcutaneous Blastomyces dermatitidis nodules in rats and to describe the histologic appearance of these lesions . We used B . dermatitidis isolated from a dog with blastomycosis to prepare a Blastomyces yeast suspension . Four rats were used to test initial dose concentrations of 10(5), 10(6), 10(9), and 10(10) yeast organisms . The dose was administered subcutaneously over the distal tibia in a volume of 0.1 ml . We then inoculated 35 additional rats with 10(9) or 10(10) yeast organisms . Rats were euthanized 7, 10, 14, 21, or 28 days after inoculation, and the histologic appearance of the nodules was described . A full post-mortem examination sought evidence of systemic spread of Blastomyces organisms . We successfully induced subcutaneous Blastomyces abscesses in 34 of 37 rats injected with 10(9) or 10(10) organisms . Nodules first appeared 3 to 7 days after injection and reached 2 to 15 mm in diameter by 7 to 28 days after inoculation . Histologically the lesions were characterized by a necrotic center surrounded by a layer of viable yeast and granulomatous inflammation . Live yeast organisms were recovered from all lesions . No adverse effects or systemic spread of Blastomyces organisms were observed . We conclude that subcutaneous Blastomyces abscesses can be induced safely and reliably in rats after injection of 10(9) and 10(10) organisms . Histologically, the experimentally induced lesions share both similarities to and differences with lesions of naturally occurring blastomycosis.

Int J Food Microbiol, 2002 Jun 25, 76(3), 215 - 21
Nystatin and osmotica as chemical enhancers of the phenotypic adaptation to freeze-thaw stress in Geotrichum candidum ATCC 204307; Dubernet S et al.; Geotrichum candidum is a yeast-like fungus used as ripening starter in cheese making . The present study focused on chemical stress pretreatments affecting survival of G . candidum ATCC 204307 to freeze-thaw stress . Cryotolerance of G . candidum cells was induced by pretreatment with NaCl, CaCl2, or MgCl2, indicating heterologous phenotypic adaptation to freeze-thaw stress (- 20 to 25 degrees C) by osmotic stress . Furthermore, the nystatin, an antifungal compound, was shown to be a cryotolerance inducer.

J Biol Chem, 2002 Aug 23, 277(34), 30928 - 34 Epub 2002 Jun 05.
Interaction of SAP97 with minus-end-directed actin motor myosin VI . Implications for AMPA receptor trafficking; Wu H et al.; SAP97 is a modular protein composed of three PDZ domains, an SH3 domain, and a guanylate kinase-like domain . It has been implicated functionally in the assembly and structural stability of synaptic junctions as well as in the trafficking, recruitment, and localization of specific ion channels and neurotransmitter receptors . The N terminus of SAP97 (S97N) has been shown to play a key role in the selection of binding partners and the localization of SAP97 at adhesion sites, as well as the clustering of ion channels in heterologous cells . Using the S97N domain as bait in a yeast two-hybrid screen, we identified the minus-end-directed actin-based motor, myosin VI, as an S97N binding partner . Moreover, in light membrane fractions prepared from rat brain, we found that myosin VI and SAP97 form a trimeric complex with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR1 . These data suggest that SAP97 may serve as a molecular link between GluR1 and the actin-dependent motor protein myosin VI during the dynamic translocation of AMPA receptors to and from the postsynaptic plasma membrane.

Bioelectrochemistry, 2002 Jul, 57(1), 83 - 7
Calculation of dielectric spectra of suspensions of rod-shaped cells using boundary element method; Sekine K et al.; The boundary element method (BEM) has been applied to the calculation of dielectric spectra of suspensions of rod-shaped cells using two kinds of models: model-R consisting of a cylinder and two hemispheres and model-PU of prolate spheroid shape . Both models have an insulating shell phase of a uniform thickness . The calculations were compared with those using a conventional spheroidal model with a confocal shell (model-PC) and previous observations on rod-shaped yeast cells . The differences among the three models were not considerable and all the models succeeded in interpreting the observed data on yeast cells.

Mol Cell, 2002 May, 9(5), 1113 - 23
Aberrant nuclear trafficking of La protein leads to disordered processing of associated precursor tRNAs; Intine RV et al.; Eukaryotic precursor tRNAs undergo extensive processing prior to nuclear export . The first of multiple factors to interact with pre-tRNAs and other nascent transcripts is the La protein . Using suppressor and wild-type tRNAs, we demonstrate that the normal distribution of cellular end-processed and spliced tRNA species is disordered by La proteins that lack a conserved nuclear retention element . Fission yeast or human La mutants that lack this element enter nuclei and stabilize nascent pre-tRNA but are aberrantly exported and fail to support normal tRNA processing . Instead, anomalous 5' and 3' end-containing, spliced tRNAs accumulate, complexed with the mutant La protein . Thus, appropriate nuclear trafficking by La affects the normal order of pre-tRNA processing.

Genome Biol . 2002;3(5):reviews3007 . Epub 2002 Apr 26.
The ADF/cofilin family: actin-remodeling proteins; Maciver SK et al.; The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined . Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin . The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end . The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity . Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins . Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene . Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.

Biochem J, 2002 Jun 15, 364(Pt 3), 669 - 77
Human tastin, a proline-rich cytoplasmic protein, associates with the microtubular cytoskeleton; Nadano D et al.; Tastin was originally identified as an accessory protein for trophinin, a cell adhesion molecule that potentially mediates the initial attachment of the human embryo to the uterine epithelium . However, no information regarding tastin's function is available to date . The present study is aimed at understanding the role of tastin in mammalian cells . Hence, we examined the intracellular localization of tastin in human cell lines transfected with an expression vector encoding influenza virus haemagglutinin (HA)-tagged tastin . Ectopically expressed HA-tastin was seen as a pattern resembling the fibres that overlap the microtubular cytoskeleton . When HA-tastin-expressing cells were cultured with nocodazole to disrupt microtubule (MT) polymerization, tastin was dispersed to the entire cytoplasm and an MT sedimentation assay showed tastin in the supernatant; however, tastin was sedimented with polymeric MTs in cell lysates not treated with nocodazole . Sedimentation assays using HA-tastin mutants deleted at the N- or C-terminus revealed MT-binding activity associated with the N-terminal basic region of tastin . A yeast two-hybrid screen for tastin-interacting proteins identified Tctex-1, one of the light chains of cytoplasmic dynein, as a tastin-binding protein . Immunoprecipitation and Western-blot analysis confirmed binding of HA-tagged tastin and FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys epitope)-tagged Tctex-1 in human cells . Furthermore, in vitro assays have demonstrated the binding between a fusion protein, glutathione S-transferase-Tctex-1, and in vitro translated (35)S-labelled tastin . As Tctex-1 is a component of a MT-based molecular motor, these results suggest that tastin plays an important role in mammalian cells by associating with the microtubular cytoskeleton.

Fish Shellfish Immunol, 2002 Apr, 12(4), 335 - 51
Ubiquitin genes in rainbow trout (Oncorhynchus mykiss); Okubo K et al.; Ubiquitin is a small protein involved in intracellular proteolysis . It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man . A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla . In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.

J Biol Chem, 2002 Aug 23, 277(34), 30454 - 62 Epub 2002 Jun 04.
ILPIP, a novel anti-apoptotic protein that enhances XIAP-mediated activation of JNK1 and protection against apoptosis; Sanna MG et al.; We have previously described a new aspect of the Inhibitor of Apoptosis (IAP) family of proteins anti-apoptotic activity that involves the TAK1/JNK1 signal transduction pathway (1,2) . Our findings suggest the existence of a novel mechanism that regulates the anti-apoptotic activity of IAPs that is separate from caspase inhibition but instead involves TAK1-mediated activation of JNK1 . In a search for proteins involved in the XIAP/TAK1/JNK1 signaling pathway we isolated by yeast two-hybrid screening a novel X chromosome-linked IAP (XIAP)-interacting protein that we called ILPIP (hILP-Interacting Protein) . Whereas ILPIP moderately activates JNK family members when expressed alone, it strongly enhances XIAP-mediated activation of JNK1, JNK2, and JNK3 . The expression of a catalytically inactive mutant of TAK1 blocked XIAP/ILPIP synergistic activation of JNK1 thereby implicating TAK1 in this signaling pathway . ILPIP moderately protects against interleukin-1beta converting enzyme- or Fas-induced apoptosis and significantly potentiates the anti-apoptotic activity of XIAP . In vivo co-precipitation experiments show that both ILPIP and XIAP interact with TAK1 and tumor necrosis factor receptor-associated factor 6 . Finally, expression of ILPIP did not affect the ability of XIAP to inhibit caspase activation, further supporting the idea that XIAP protection against apoptosis is achieved by two separate mechanisms: one requiring JNK1 activation and a second involving caspase inhibition.

Plant J, 2002 Jun, 30(5), 555 - 66
Zinc-dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria; Lister R et al.; Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins . Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin-sensitive manner without processing . Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import . Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS-depleted mitochondria, but did not stimulate import of a non-carrier control precursor protein, the alternative oxidase . Anion-exchange separation of IMS components before addition to IMS-depleted mitochondria revealed a correlation between several 9-10 kDa proteins and stimulation of carrier import . MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc-finger carrier import components Tim9 and Tim10 . Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N-ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein . This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.

Plant J, 2002 Jun, 30(5), 529 - 39
Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stress response; Yokoi S et al.; The Arabidopsis thaliana vacuolar Na+/H+ antiporter AtNHX1 is a salt tolerance determinant . Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na+/H+ exchangers led to the identification of five additional AtNHX genes (AtNHX2-6) . The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots . A lower-abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots . AtNHX4 and AtNHX6 mRNAs were detected only by RT-PCR . AtNHX1, 2 or 5 suppress, with differential efficacy, the Na+/Li+-sensitive phenotype of a yeast mutant that is deficient in the endosomal/vacuolar Na+/H+ antiporter ScNHX1 . Ion accumulation data indicate that these AtNHX proteins function to facilitate Na+ ion compartmentalization and maintain intracellular K+ status . Seedling steady-state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi-osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment . Hyper-osmotic up-regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis . However, steady-state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress . AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2-1, mutant indicating that the osmotic responsiveness of these genes is ABA-dependent . An as yet undefined stress signal pathway that is ABA- and SOS-independent apparently controls transcriptional up-regulation of AtNHX5 expression by hyper-saline shock . Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells . Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na+.

Traffic, 2002 Jul, 3(7), 472 - 82
Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24; Munafo DB et al.; Rab GTPases comprises a large family of proteins, with more than 50 gene products localized in distinct subcellular compartments . Rab24 is a member of this family whose function is not presently known . In order to elucidate the role of this protein we have generated a GFP-tagged Rab24 and studied the distribution of this chimera by fluorescence microscopy . GFP-Rab24 showed a perinuclear reticular localization that often encircled the nucleus . This reticular pattern partially overlapped with ER markers, cis-Golgi, and the ER-Golgi intermediate compartment . Surprisingly, when GFP-Rab24-transfected cells were starved to induce autophagy the distribution of the protein changed dramatically . GFP-Rab24 localized in large dots, cup-shaped structures and ring-shaped vesicles . Some of these vesicles were labeled with monodansylcadaverine, a specific autophagosome marker . In the presence of vinblastine, an agent that induces the formation of very large autophagic vesicles, GFP-Rab24 accumulated in the large vacuoles that were also labeled by monodansylcadaverine . Furthermore, Rab24 colocalized with LC3, a mammalian homolog of the yeast protein Apg8/Aut7, an essential gene for autophagy . This is the first report indicating that Rab24 localizes on autophagosomes, suggesting that this Rab protein is involved in the autophagic pathway.

Genes Cells, 2002 May, 7(5), 475 - 85
ERBIN associates with p0071, an armadillo protein, at cell-cell junctions of epithelial cells; Izawa I et al.; BACKGROUND: ERBIN, an ErbB2 receptor-interacting protein, belongs to a recently described family of proteins termed the LAP {leucine-rich repeats and PSD-95/dLg-A/ZO-1 (PDZ) domains} family which has essential roles in establishment of cell polarity . RESULTS: To identify new ERBIN-binding proteins, we screened a yeast two-hybrid library, using the carboxyl-terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin-4) as an ERBIN-interacting protein . p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell-cell border . The ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence . Endogenous ERBIN was co-immunoprecipitated with p0071 . In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localized largely with beta-catenin and partly with desmoplakin along the lateral plasma membrane domain . At these cell-cell contact regions, ERBIN co-localizes with p0071 . Over-expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, resulted in a marked accumulation of ERBIN at the cell-cell contacts of MDCK and HeLa cells . CONCLUSION: These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia . In addition, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.

Anim Genet, 2002 Apr, 33(2), 91 - 6
Precise mapping of breakpoints in conserved synteny between human chromosome 1 and pig chromosomes 4, 6 and 9; Sun HS et al.; Previous comparative mapping suggested that at least five pig chromosomes (Sscr4, 6, 9, 10 and 14) share homology with human chromosome 1 (Hsap1) . A significant quantitative trait loci (QTL) for fat deposition has been identified on Sscr4 that appears to be near the junction region between Sscr4 and Sscr9 relative to Hsap1 . It is of interest to define the boundaries of conserved synteny between pig chromosomes and Hsap1 to use human map information to identify putative comparative positional candidates for this QTL . Eleven genes, including Janus kinase 1 (JAK1), Prostaglandin E receptor3 (PTGER3), urate oxidase (UOX), coagulation factor 3 (F3), vascular cell adhesion molecule 1 (VCAM1), ribosomal protein L5 (RPL5), POU domain, class 2, transcription factor 1 (POU2F1), coagulation factor 5 (F5), Prostaglandin endoperoxide synthase-2 (PTGS2), myosin binding protein H (MYBPH) and Antithrombin III (SERPINC1), were selected to refine the boundaries of the blocks of conserved synteny between Hsap1 and pig chromosomes . Pig sequence tagged sites (STSs) were developed and used to physically map these 11 genes using a somatic cell hybrid panel . Eight loci have been mapped by using fluorescent in situ hybridization (FISH) to improve map resolution . Heterologous FISH was used to refine the location of VCAM1 on human chromosomes . In addition, human yeast artificial chromosomes (YACs) were mapped by heterologous FISH on pig metaphases to refine the boundaries of the regions of homology between Sscr4 and Sscr9 on Hsap1 . Results from this study suggest the precise break in conserved synteny on Hsap1 corresponding to the Sscr4/6 and Sscr4/9 transitions are most likely on the Hsap1p22 and Hsap1q24-25 regions, respectively . Further, our data predict that Hsap1q21-24 is a candidate region for the backfat QTL localized to Sscr4.

Biometals, 2002 Jun, 15(2), 167 - 74
The extremely high Al resistance of Penicillium janthineleum F-13 is not caused by internal or external sequestration of Al; Zhang D et al.; Penicillium janthinellum F-13 has been isolated in previous work as a fungus tolerating the presence of high concentrations of Al (as high as 100 mM AlCl3) . Here its growth rate and yield in three acidic (pH 3.0) media of different composition with varying concentrations of Al are reported . The presence of Al did not affect these parameters . except that the growth yield was somewhat lower in GM (a glucose/peptone/yeast extract-containing medium) with the highest concentration tested (100 mM AlCl3) . The amount of Al found in the mycelium was so low that it cannot lead to a significant decrease in the medium for the higher Al concentrations applied . Although citric acid was excreted at growth on GM, and the presence of Al even promoted this, the concentration of this was far too low to diminish (by chelation) the high Al concentrations in the medium to a non-toxic level, i.e . the level (of approx . 1 mM) that is tolerated by low-resistance fungi . At growth on SLBM (a peptone/yeast extract/soil extract-containing medium), a rise in pH occurred . The same was found for SM (a glucose/mineral salts-containing medium), although in this case the picture was more complicated because the initial rise in pH was followed by a lowering due to the excretion of oxalic acid . Although both phenomena can diminish Al toxicity (by decreasing the external concentration of monomeric Al, regarded to be the toxic species), again the decrease is far too low to attain a non-toxic level when high Al concentrations are applied . Therefore, although in principal the metabolic phenomena observed for P . janthinellum F-13 at growth on different media can diminish Al toxicity, the tolerance of this organism for high external Al concentrations must be caused by another mechanism.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(4), 579 - 600
The emission soot of biomass fuels combustion as a source of endocrine disrupters; Wu WZ et al.; Emissions of biomass fuel combustion in residential stove from Chinese countryside were analyzed to determine the endocrine disrupters by using recombinant yeast bioassay . The results showed that there were significant steroid modulating activities found in combustion soot of five kinds of biomass fuels, which were wood, crop residue, grass, bush and rice straw . The steroid activities in the different sub-fractions from chromatographic separation were also determined, and the results indicated that polar fraction extracted by methanol and aromatic fraction extracted by benzene had relatively high steroid activities, and aliphatic fraction almost had no activity . The GC/MS results showed that polycyclic aromatic compounds and their derivatives, substituted phenolic compounds and their derivatives, aromatic carbonyl compounds, and higher molecular weight alcohols and ketones may be the main steroid disrupters in these soots.

AIDS, 2002 Jun 14, 16(9), 1237 - 44
Adherence of different Candida dubliniensis isolates in the presence of fluconazole; Borg-von Zepelin M et al.; BACKGROUND: The recently described yeast species Candida dubliniensis is closely related to C . albicans and has been recovered predominantly from the oral cavities of HIV-infected individuals and AIDS patients who are often receiving fluconazole as prophylactic or therapeutic treatment for oropharyngeal candidiasis . Like C . albicans, C . dubliniensis secretes aspartic proteinases which in C . albicans have been shown to be involved in adherence . OBJECTIVE: To explain the increasing prevalence of C . dubliniensis in AIDS patients and to investigate the virulence factors of this yeast . METHODS: An in vitro assay was developed to compare the adherence to epithelial cells of C . dubliniensis from HIV-patients with that of C . albicans . RESULTS: All C . albicans isolates adhered better than the 22 C . dubliniensis isolates . In the presence of fluconazole, the C . dubliniensis isolates tested showed increased adherence as compared with controls without fluconazole . In contrast, all C . albicans isolates decreased in adherence to epithelial cells in the presence of fluconazole independently of their in vitro susceptibility to this drug . Proteinase antigens are present on the surface of C . dubliniensis cells adherent to epithelial target cells . In the presence of fluconazole this proteinase antigen was more strongly expressed . CONCLUSION: Increased adherence of C . dubliniensis strains in the presence of fluconazole could explain its high recovery rate from HIV-positive patients in recent years . The induction of proteinase secretion in the presence of fluconazole found for most of the C . dubliniensis isolates could be one of the factors involved in adherence.

J Clin Invest, 2002 Jun, 109(11), 1445 - 52
Thyrostimulin, a heterodimer of two new human glycoprotein hormone subunits, activates the thyroid-stimulating hormone receptor; Nakabayashi K et al.; Human thyrotropin (TSH), luteotropin (LH), follitropin (FSH), and chorionic gonadotropin are members of the heterodimeric glycoprotein hormone family . The common alpha subunit forms noncovalent heterodimers with different beta subunits . Two novel human glycoprotein hormonelike genes, alpha2 (A2) and beta5 (B5), recently have been identified . Using a yeast two-hybrid assay, the two subunits were found as potential heterodimerization partners . Immunological analyses confirmed the heterodimerization of A2 and B5 in transfected cells and their colocalization in the anterior pituitary . Recombinant A2/B5 heterodimeric glycoproteins, purified using cation exchange and size fractionation chromatography, activated human TSH receptors, but not LH and FSH receptors, and showed high affinity to TSH receptors in a radioligand receptor assay . The heterodimer also stimulated cAMP production and thymidine incorporation by cultured thyroid cells and increased serum thyroxine levels in TSH-suppressed rats in vivo . This new heterodimeric glycoprotein hormone was named as thyrostimulin based on its thyroid-stimulating activity . The expression of thyrostimulin in the anterior pituitary known to express TSH receptors suggested a paracrine mechanism . The present discovery of a new ligand based on genomic approaches could facilitate the understanding of the physiological roles of extra-thyroid TSH receptor systems and the structural-functional basis of receptor signaling by related glycoprotein hormones.

J Cell Sci, 2002 Jun 15, 115(Pt 12), 2461 - 4
From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner?
Doree M, Hunt T.
The idea that Cdc2 and cyclins play a key role in the control of the G2/M transition of the cell cycle came largely from genetic analysis of fission yeast and physiological studies of clam, frog, sea urchin and starfish eggs and oocytes . However, it took a long time to realise that Cdc2 and cyclins form a stoichiometric complex and that a cyclin subunit is necessary for the Cdc2 subunit to gain its protein kinase activity . Cyclins were first recognized as proteins whose abundance oscillates during the early cell cycles of marine invertebrate eggs and their connection with MPF (maturation-promoting factor), the entity defined in frog and starfish oocytes whose activity controls entry into M phase, was far from clear at first . Indeed, it was a long time before MPF was shown to be a protein kinase, and direct proof that MPF is a heterodimer comprising one molecule of cyclin and one molecule of Cdc2 was finally obtained only when the Cdc2-associated component of purified starfish MPF was sequenced and found to be cyclin B . When this fundamental discovery was confirmed in vertebrates and mammalian members of the Cdc2 family were also shown to bind cyclins, Cdc2 became Cdk1, the first cyclin-dependent protein kinase.

Annu Rev Biochem, 2002, 71, 847 - 85 Epub 2001 Nov 09.
Catalytic proficiency: the unusual case of OMP decarboxylase; Miller BG et al.; Enzymes are called upon to differ greatly in the difficulty of the tasks that they perform . The catalytic proficiency of an enzyme can be evaluated by comparing the second-order rate constant (kcat/Km) with the rate of the spontaneous reaction in neutral solution in the absence of a catalyst . The proficiencies of enzymes, measured in this way, are matched by their affinity constants for the altered substrate in the transition state . These values vary from approximately approximately 10(9) M(-1) for carbonic anhydrase to approximately 10(23) M(-1) for yeast orotidine 5'-phosphate decarboxylase (ODCase) . ODCase turns its substrate over with a half-time of 18 ms, in a reaction that proceeds in its absence with a half-time of 78 million years in neutral solution . ODCase differs from other decarboxylases in that its catalytic activity does not depend on the presence of metals or other cofactors, or on the formation of a covalent bond to the substrate . Several mechanisms of transition state stabilization are considered in terms of ODCase crystal structures observed in the presence and absence of bound analogs of the substrate, transition state, and product . Very large connectivity effects are indicated by the results of experiments testing how transition state stabilization is affected by the truncation of binding determinants of the substrate and the active site.

Annu Rev Biochem, 2002, 71, 755 - 81 Epub 2001 Nov 09.
Nuclear actin and actin-related proteins in chromatin remodeling; Olave IA et al.; The existence and function of actin in the nucleus has been hotly debated for forty years . Recently, beta-actin was found to be a component of mammalian SWI/SNF-like BAF chromatin remodeling complexes and still more recently other SWI/SNF-related chromatin remodeling complexes in yeast, flies, and man . Although the function of actin in these chromatin remodeling complexes is only starting to be explored, the fact that actin is one of the most regulated proteins in the cell suggests that control of nuclear actin may be a critical regulatory point in the control of chromatin remodeling . Actin rapidly shuttles between the nucleus and the cytoplasm offering additional sites and modes of regulation . In addition, actin-related proteins (Arps) are also components of these chromatin remodeling complexes and have been implicated in transcriptional control in yeast . The observation that the BAF chromatin remodeling complex in which actin was originally identified, is also a human tumor suppressor complex necessary for the actions of the retinoblastoma protein indicates that the study of nuclear actin is likely to contribute to understanding cell growth control.

Trends Genet, 2002 Jun, 18(6), 295 - 301
A critical analysis of the role of growth hormone and IGF-1 in aging and lifespan; Carter CS et al.; Studies in Caenorhabditis elegans demonstrate that disruption of the daf-2 signaling pathways extends lifespan . Similarities among the daf-2 pathway, insulin-like signaling in flies and yeast, and the mammalian insulin-like growth factor 1 (IGF-1) signaling cascade raise the possibility that modifications to IGF-1 signaling could also extend lifespan in mammals . In fact, growth hormone (GH)/IGF-1-deficient dwarf mice do live significantly longer than their wild-type counterparts . However, multiple endocrine deficiencies and developmental anomalies inherent in these models confound this interpretation . Here, we critique the current mammalian models of GH/IGF-1 deficiency and discuss the actions of GH/IGF-1 on biological aging and lifespan.

Nat Biotechnol, 2002 Jun, 20(6), 613 - 8
Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality, and vine life; Mehta RA et al.; Polyamines, ubiquitous organic aliphatic cations, have been implicated in a myriad of physiological and developmental processes in many organisms, but their in vivo functions remain to be determined . We expressed a yeast S-adenosylmethionine decarboxylase gene (ySAMdc; Spe2) fused with a ripening-inducible E8 promoter to specifically increase levels of the polyamines spermidine and spermine in tomato fruit during ripening . Independent transgenic plants and their segregating lines were evaluated after cultivation in the greenhouse and in the field for five successive generations . The enhanced expression of the ySAMdc gene resulted in increased conversion of putrescine into higher polyamines and thus to ripening-specific accumulation of spermidine and spermine . This led to an increase in lycopene, prolonged vine life, and enhanced fruit juice quality . Lycopene levels in cultivated tomatoes are generally low, and increasing them in the fruit enhances its nutrient value . Furthermore, the rates of ethylene production in the transgenic tomato fruit were consistently higher than those in the nontransgenic control fruit . These data show that polyamine and ethylene biosynthesis pathways can act simultaneously in ripening tomato fruit . Taken together, these results provide the first direct evidence for a physiological role of polyamines and demonstrate an approach to improving nutritional quality, juice quality, and vine life of tomato fruit.

J Nutr, 2002 Jun, 132(6), 1122 - 8
Selenium deficiency in Fisher-344 rats decreases plasma and tissue homocysteine concentrations and alters plasma homocysteine and cysteine redox status; Uthus EO et al.; The purpose of the present study was to determine the effect of graded amounts of dietary selenium on plasma and tissue parameters of methionine metabolism including homocysteine . Male weanling Fisher-344 rats (n = 7-8/group) were fed a selenium-deficient, torula yeast-based diet, supplemented with 0 (selenium deficient), 0.02, 0.05 or 0.1 microg (adequate) selenium (as selenite)/g diet . After 61 d, plasma total homocysteine and cysteine were decreased (P < 0.0001) and glutathione increased (P < 0.0001) by selenium deficiency . The concentrations of homocysteine in kidney and heart were decreased (P = 0.02) by selenium deficiency . The activities of liver betaine homocysteine methyltransferase, methionine synthase, S-adenosylmethionine synthase, cystathionine synthase and cystathionase were determined; selenium deficiency affected only betaine homocysteine methyltransferase, which was decreased (P < 0.0001) . The ratios of plasma free reduced homocysteine (or cysteine) to free oxidized homocysteine (or cysteine) or to total homocysteine (or cysteine) were increased by selenium deficiency, suggesting that selenium status affects the normally tightly controlled redox status of these thiols . Most differences due to dietary selenium were between rats fed 0 or 0.02 microg selenium/g diet and those fed 0.05 or 0.1 microg selenium/g diet . The metabolic consequences of a marked decrease in plasma homocysteine and smaller but significant decreases in tissue homocysteine are not known.

J Biol Chem, 2002 Aug 9, 277(32), 28934 - 41 Epub 2002 May 31.
Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3; Kato A et al.; Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets . We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells . Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm . Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1 . Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm) . Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits . Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling . As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.

J Biol Chem, 2002 Aug 9, 277(32), 28663 - 8 Epub 2002 May 31.
Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent; Monti P et al.; Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA . We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target . We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1 . When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1 . The Me-lex-induced mutation frequency increased in the following order: wild type < deltamag1< deltaapn1apn2 = deltaapn1apn2mag1 . A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced . Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected . The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2) . The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants . This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined . The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.

J Biol Chem, 2002 Aug 23, 277(34), 31005 - 13 Epub 2002 May 31.
Characterization of regions in hsMAD1 needed for binding hsMAD2 . A polymorphic change in an hsMAD1 leucine zipper affects MAD1-MAD2 interaction and spindle checkpoint function; Iwanaga Y et al.; In eukaryotes, the mitotic spindle assembly checkpoint provides a monitor for the fidelity of chromosomal segregation . In this context, the mitotic arrest deficiency protein 2 (MAD2) censors chromosomal mis-segregation by monitoring microtubule attachment/tension, a role that requires its attachment to kinetochores . Studies in yeast have shown that binding of MAD1 to MAD2 is important for the checkpoint function of the latter . The interactions between human MAD1 (hsMAD1) and human MAD2 (hsMAD2) have, however, remained poorly characterized . Here we report that two leucine zipper domains (amino acids 501-522 and 557-571) in hsMAD1 are required for its contact with hsMAD2 . Interestingly, in several cancer cell lines, we noted the frequent presence of a coding single nucleotide Arg to His polymorphism at codon 558 located within the second leucine zipper of hsMAD1 . We found that hsMAD1H558 is less proficient than hsMAD1R558 in binding hsMAD2 and in enforcing mitotic arrest . We also document a first example of loss-of-heterozygosity for a spindle checkpoint gene (at the hsMAD1 558 locus) in a human breast cancer . Based on our findings, it is possible that hsMAD1H558 could be an at-risk polymorphism that contributes to attenuated spindle checkpoint function in human cells.

J Biol Chem, 2002 Jul 19, 277(29), 26351 - 5 Epub 2002 May 31.
Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family; Hewitson KS et al.; Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues . Hydroxylation of specific prolyl residues by 2-oxoglutarate (2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-alpha . Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-alpha abrogates interaction with p300, preventing transcriptional activation . Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-alpha . Since FIH contains certain motifs present in iron- and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase . Assays using recombinant FIH and HIF-alpha fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen . Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.

Microbiol Mol Biol Rev, 2002 Jun, 66(2), 155 - 78
Cytokinesis in eukaryotes; Guertin DA et al.; Cytokinesis is the final event of the cell division cycle, and its completion results in irreversible partition of a mother cell into two daughter cells . Cytokinesis was one of the first cell cycle events observed by simple cell biological techniques; however, molecular characterization of cytokinesis has been slowed by its particular resistance to in vitro biochemical approaches . In recent years, the use of genetic model organisms has greatly advanced our molecular understanding of cytokinesis . While the outcome of cytokinesis is conserved in all dividing organisms, the mechanism of division varies across the major eukaryotic kingdoms . Yeasts and animals, for instance, use a contractile ring that ingresses to the cell middle in order to divide, while plant cells build new cell wall outward to the cortex . As would be expected, there is considerable conservation of molecules involved in cytokinesis between yeast and animal cells, while at first glance, plant cells seem quite different . However, in recent years, it has become clear that some aspects of division are conserved between plant, yeast, and animal cells . In this review we discuss the major recent advances in defining cytokinesis, focusing on deciding where to divide, building the division apparatus, and dividing . In addition, we discuss the complex problem of coordinating the division cycle with the nuclear cycle, which has recently become an area of intense research . In conclusion, we discuss how certain cells have utilized cytokinesis to direct development.

J Biol Chem, 2002 Aug 16, 277(33), 30031 - 9 Epub 2002 May 30.
Activation function-1 domain of androgen receptor contributes to the interaction between subnuclear splicing factor compartment and nuclear receptor compartment . Identification of the p102 U5 small nuclear ribonucleoprotein particle-binding protein as a coactivator for the receptor; Zhao Y et al.; In the androgen receptor (AR), most of its transactivation activity is mediated via the activation function-1 (AF-1) . By employing yeast two-hybrid assay, we isolated a cDNA sequence encoding a protein binding to AR-AF-1 . This protein, named ANT-1 (AR N-terminal domain transactivating protein-1), enhanced the ligand-independent autonomous AF-1 transactivation function of AR or glucocorticoid receptor but did not enhance that of estrogen receptor alpha . In contrast, the ANT-1 did not enhance any ligand-dependent AF-2 activities . Furthermore, the ligand-independent interaction between AR-AF-1 and ANT-1 was confirmed in vivo and in vitro . The ANT-1 sequence was identical to that of a protein that binds to U5 small nuclear ribonucleoprotein particle, a human homologue of yeast splicing factor Prp6p, involved in spliceosome . ANT-1 was compartmentalized into 20-40 coarse splicing factor compartment speckles against the background of the diffuse reticular distribution . AR colocalized with ANT-1 only in the diffusely distributed area, whereas the ANT-1 speckles were spatially distinct from but surrounded by the AR compartments . The active gene transcription has been shown to couple simultaneously with pre-mRNA processing at the periphery of the splicing factor compartment . The molecular interaction between two spatially distinct subnuclear compartments mediated by ANT-1 may therefore recruit AR into the transcription-splicing-coupling machinery.

J Biol Chem, 2002 Aug 16, 277(33), 29856 - 64 Epub 2002 May 30.
The Arabidopsis AtSTE24 is a CAAX protease with broad substrate specificity; Bracha K et al.; Following prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting . First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety . An Arabidopsis homologue of the yeast CAAX protease STE24 (AFC1) was cloned and expressed in rce1 Delta ste24 Delta mutant yeast to demonstrate functional complementation . The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box . Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24 . In addition, AtSTE24 promoted plasma membrane association of the GFP-Rac fusion protein, which terminates with a CLLM CAAX box . Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data . Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established . Transient expression of GFPAtSTE24 in leaf epidermal cells of Nicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation.

J Biol Chem, 2002 Aug 9, 277(32), 28624 - 30 Epub 2002 May 30.
Identification of protein arginine methyltransferase 2 as a coactivator for estrogen receptor alpha; Qi C et al.; In an attempt to isolate cofactors capable of influencing estrogen receptor alpha (ERalpha) transcriptional activity, we used yeast two-hybrid screening and identified protein arginine methyltransferase 2 (PRMT2) as a new ERalpha-binding protein . PRMT2 interacted directly with three ERalpha regions including AF-1, DNA binding domain, and hormone binding domain in a ligand-independent fashion . The ERalpha-interacting region on PRMT2 has been mapped to a region encompassing amino acids 133-275 . PRMT2 also binds to ERbeta, PR, TRbeta, RARalpha, PPARgamma, and RXRalpha in a ligand-independent manner . PRMT2 enhanced both ERalpha AF-1 and AF-2 transcriptional activity, and the potential methyltransferase activity of PRMT2 appeared pivotal for its coactivator function . In addition, PRMT2 enhanced PR, PPARgamma, and RARalpha-mediated transactivation . Although PRMT2 was found to interact with two other coactivators, the steroid receptor coactivator-1 (SRC-1) and the peroxisome proliferator-activated receptor-interacting protein (PRIP), no synergistic enhancement of ERalpha transcriptional activity was observed when PRMT2 was coexpressed with either PRIP or SRC-1 . In this respect PRMT2 differs from coactivators PRMT1 and CARM1 (coactivator-associated arginine methyltransferase) . These results suggest that PRMT2 is a novel ERalpha coactivator.

Mol Cell Endocrinol, 2002 Mar 28, 189(1-2), 201 - 11
Bombyx mori orphan receptor, BmHR78: cDNA cloning, testis abundant expression and putative dimerization partner for Bombyx ultraspiracle; Hirai M et al.; We have identified a novel member of the nuclear receptor superfamily from the silkworm Bombyx mori, and named it as BmHR78, the B . mori hormone receptor . The DNA binding domain of BmHR78 shows high similarities to those of Tenebrio molitor hormone receptor 78, Drosophila hormone receptor 78, and mammalian testicular receptor 2, whereas the ligand binding domain is not well conserved . Northern blot analysis showed that BmHR78 gene was most abundantly expressed in the testis . From the fourth to fifth instar, BmHR78 gene was constantly expressed in the testis . In the anterior silk gland, the level of BmHR78 gene expression was developmentally changed . From day 10.0 to 11.0 in the fifth instar, another BmHR78 transcript with the smaller size appeared . Ultraspiracle (USP) isoform also appeared at the same stages in this tissue . BmHR78 forms not only a homodimer, but also a heterodimer with USP in a yeast two hybrid assay . The direct interaction between BmHR78 and USP was confirmed by pull down assay . Deletion mutant analysis showed that BmHR78 interacts with USP via the ninth heptad repeat in helix ten of the E region . This repeat is well conserved in RXR and its heterodimer partners, and shown to be an interface for their dimerization . In insect, only the ecdysone receptor and hormone receptor 38 are known thus far to dimerize with USP . Thus, BmHR78 is a third dimerization partner for USP and may modulate the molecular action of USP, including the ecdysone signal cascades.

J Clin Microbiol, 2002 Jun, 40(6), 2199 - 206
Genetic structure of Candida glabrata populations in AIDS and non-AIDS patients; de Meeus T et al.; The genotypes of 63 strains (11 reference strains and 52 strains from hospitalized patients) of the haploid yeast Candida glabrata were determined from 33 putative gene enzymatic loci . This enabled the characterization of 26 different multilocus genotypes . Genetic differentiation was found between distant hospitals (located in Montpellier and Paris, France) but not for other parameters (anatomic origins or human immunodeficiency virus-positive {HIV+} and HIV- patients) . Strong nonrandom association between loci could be seen . Such statistical linkages were confirmed upon comparing the patterns of 14 RAPD {random(ly) amplified polymorphic DNA} primers from 20 of these strains to results obtained from multilocus enzyme electrophoresis analysis . This finding suggests a mainly clonal mode of reproduction of C . glabrata . The consequences of the clonality displayed by C . glabrata populations on the epidemiology of this yeast are also discussed.

J Biol Chem, 2002 Aug 16, 277(33), 30236 - 43 Epub 2002 May 29.
Structural characterization of the closed conformation of mouse guanylate kinase; Sekulic N et al.; Guanylate kinase (GMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryl transfer from ATP to GMP to yield ADP and GDP . In addition to phosphorylating GMP, antiviral prodrugs such as acyclovir, ganciclovir, and carbovir and anticancer prodrugs such as the thiopurines are dependent on GMPK for their activation . Hence, structural information on mammalian GMPK could play a role in the design of improved antiviral and antineoplastic agents . Here we present the structure of the mouse enzyme in an abortive complex with the nucleotides ADP and GMP, refined at 2.1 A resolution with a final crystallographic R factor of 0.19 (R(free) = 0.23) . Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions . Previous studies on the yeast enzyme have shown that these parts move as rigid bodies upon substrate binding . It has been proposed that consecutive binding of substrates leads to "closing" of the active site bringing the NMP-binding and LID regions closer to each other and to the CORE region . Our structure, which is the first of any guanylate kinase with both substrates bound, supports this hypothesis . It also reveals the binding site of ATP and implicates arginines 44, 137, and 148 (in addition to the invariant P-loop lysine) as candidates for catalyzing the chemical step of the phosphoryl transfer.

J Biol Chem, 2002 Jul 12, 277(28), 24847 - 50 Epub 2002 May 29.
A vitamin D receptor-Ser/Thr phosphatase-p70 S6 kinase complex and modulation of its enzymatic activities by the ligand; Bettoun DJ et al.; We provide evidence of a cross-talk between nuclear receptor and Ser/Thr protein phosphatases and show that vitamin D receptor (VDR) interacts with the catalytic subunit of protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner . PP1c specifically interacts with VDR but not retinoic acid receptor alpha and retinoid X receptor alpha in yeast . Although VDR-PP1c and VDR-PP2Ac interaction is ligand-independent in vivo, 1alpha,25-dihydroxy-vitamin D(3) induces VDR-associated phosphatase activity . Further, VDR modulation of PP1c/PP2Ac activity results in a rapid and specific dephosphorylation and inactivation of their substrate, p70 S6 kinase (p70(S6k)) . Finally, we demonstrate that the endogenous VDR, PP1c or PP2Ac, and p70(S6k) are present in a ternary complex in vivo, and the interaction of p70(S6k) with the VDR-PP complex is modulated by the phosphorylation state of the kinase . Since p70(S6k) is essential for G(1)-S transition, our results provide a molecular basis of 1alpha,25-dihydroxyvitamin D(3)-induced G(1) block in colon cancer cells.

Genomics, 2002 Jun, 79(6), 809 - 17
The thymocyte-specific MAR binding protein, SATB1, interacts in vitro with a novel variant of DNA-directed RNA polymerase II, subunit 11; Durrin LK et al.; A yeast two-hybrid screen of a Jurkat (T cell) derived cDNA library, using SATB1 (a matrix attachment region binding protein) as the bait, yielded four independent isolates of a novel variant of the DNA directed RNA polymerase II, subunit 11 (RPB11) . Absence of lysine-17 from the amino terminus of this variant cannot be explained by alternative mRNA splicing . Instead, allele-specific PCR, combined with GenBank database searches, suggests that a recent gene duplication event has resulted in distinct loci encoding three variant forms of RPB11 . Differential splicing of mRNA transcripts accounts for unique carboxy termini among the RPB11 proteins . The exclusive association of SATB1 with one form of RPB11 is influenced primarily by the N-terminal amino acid disparity, as deletion of the entire C terminus does not alter interaction affinity . Association of RPB11 with SATB1 maps between amino acids 58 and 222 of SATB1, a region that includes a PDZ-like dimerization motif . (c)2002 Elsevier Science (USA).

Genomics, 2002 Jun, 79(6), 799 - 808
Identification and characterization of cDNAs encoding four novel proteins that interact with translin associated factor-X; Bray JD et al.; Translin-associated factor X (TRAX) is the predominantly cytoplasmic binding partner of TB-RBP/translin in mouse testis . Four mouse testis cDNAs encoding specific TRAX-interacting proteins were isolated from a yeast two-hybrid library screen . One novel cDNA designated Tsnaxip1 (TRAX-interacting protein-1) encodes 709 amino acids . We isolated a cDNA encoding the 427 carboxy-terminal amino acids of MEA-2, a Golgi-associated, maleenhanced autoantigen; a cDNA encoding 429 amino acids with 73% homology to centrosomal Akap9; and a cDNA encoding 346 amino acids with 75% homology to SUN1, a predicted human protein that contains a SUN domain (which is present in some perinuclear proteins) . Interactions were verified using in vitro synthesized fusion proteins . All four genes were expressed in the testis and enriched in germ cells . Confocal microscopy studies using green fluorescent protein fusion proteins determined that these TRAX-interacting proteins colocalize with TRAX . The data suggest that TRAX may have a function associated with perinuclear organelles during spermatogenesis . (c)2002 Elsevier Science (USA).

Ukr Biokhim Zh, 2001 Sep-Oct, 73(5), 128 - 34
{Is it reality that the endonuclease that cleaves pre-mRNA on polyadenylation has not been discovered?}; Zarudnaia MI et al.; Specific cleavage of transcript by a complex of multisubunit proteins is the first stage of polyadenylation of eukaryotic pre-mRNAs . The main participant of this reaction--endonuclease--is not discovered yet . However it is known that proteins CPSF-30 (mammalian) and Yth 1p (yeast) are homologues of the drosofila protein clipper (CLP), which displays endoribonucleolytic activity . In the N-terminal region all three proteins contain five copies of CCCH zinc finger motif that are associated with nucleolytic activity in the case of CLP . Literature data on the three above-mentioned proteins has been analysed . The results of these works do not contradict the hypothesis that exactly CPSF-30 and its homologues are the actual nucleases that cleave pre-mRNA in the process of polyadenylation.

Plant Cell, 2002 May, 14(5), 1173 - 83
Extensive interallelic polymorphisms drive meiotic recombination into a crossover pathway; Dooner HK; Recombinants isolated from most meiotic intragenic recombination experiments in maize, but not in yeast, are borne principally on crossover chromosomes . This excess of crossovers is not explained readily by the canonical double-strand break repair model of recombination, proposed to account for a large body of yeast data, which predicts that crossovers (COs) and noncrossovers (NCOs) should be recovered equally . An attempt has been made here to identify general rules governing the recovery of the CO and NCO classes of intragenic recombinants in maize . Recombination was analyzed in bz heterozygotes between a variety of mutations derived from the same or different progenitor alleles . The mutations include point mutations, transposon insertions, and transposon excision footprints . Consequently, the differences between the bz heteroalleles ranged from just two nucleotides to many nucleotides, indels, and insertions . In this article, allelic pairs differing at only two positions are referred to as dimorphic to distinguish them from polymorphic pairs, which differ at multiple positions . The present study has revealed the following effects at these bz heteroalleles: (1) recombination between polymorphic heteroalleles produces mostly CO chromosomes; (2) recombination between dimorphic heteroalleles produces both CO and NCO chromosomes, in ratios apparently dependent on the nature of the heteroalleles; and (3) in dimorphic heterozygotes, the two NCO classes are recovered in approximately equal numbers when the two mutations are point mutations but not when one or both mutations are insertions . These observations are discussed in light of a recent version of the double-strand break repair model of recombination that postulates separate pathways for the formation of CO and NCO products.

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