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