Yeasts

Anim Biotechnol, 2003 May, 14(1), 51 - 9
Construction of YAC/BAC contig map for the BTA 6q21 region containing a locus for bovine chondrodysplastic dwarfism; Takeda H et al.; To characterize the bovine chromosome 6q21 for bovine chondrodysplastic dwarfism (BCD), we developed 48 new microsatellite markers from yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones using a modified magnetic bead capture method . These new markers were used to construct a high-resolution physical map of the region with a total of 85 loci . The physical map will be a powerful tool for successful positional cloning experiments.

Genes Brain Behav, 2002 Jan, 1(1), 27 - 34
The ultimate chip shot: can microarray technology deliver for neuroscience?
Nisenbaum LK.
The use of cDNA and oligonucleotide microarrays, or 'chips', is emerging as a powerful, new technology in the field of neuroscience for examining gene expression in a high-throughput fashion . The application of microarray technology to the study of brain and behavior has lagged behind other areas of biology such as cancer and yeast genetics due to the challenges presented by the heterogeneous and complex organization of the nervous system . This review provides a brief overview of available microarray technology as well as a description of experimental considerations in planning and implementing a neuroscience-based array study . Successful implementation of microarray technology within the field of neuroscience will provide a molecular approach to studying systems neurobiology, leading to insights into areas ranging from fundamental questions of developmental neurobiology to neurological and psychiatric disorders.

Mem Inst Oswaldo Cruz, 2003 Apr, 98(3), 401 - 5 Epub 2003 Jul 18.
Species distribution and antifungal susceptibility profile of Candida spp . bloodstream isolates from Latin American hospitals; Godoy P et al.; From March 1999 to March 2000, we conducted a prospective multicenter study of candidemia involving five tertiary care hospitals from four countries in Latin America . Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to the National Committee for Clinical Laboratory Standards microbroth assay method . During a 12 month-period we were able to collect a total of 103 bloodstream isolates of Candida spp . C . albicans was the most frequently isolated species accounting for 42% of all isolates . Non-albicans Candida species strains accounted for 58% of all episodes of candidemia and were mostly represented by C . tropicalis (24.2%) and C . parapsilosis (21.3%) . It is noteworthy that we were able to identify two cases of C . lusitaniae from different institutions . In our casuistic, non-albicans Candida species isolates related to candidemic episodes were susceptible to fluconazole . Continuously surveillance programs are needed in order to identify possible changes in the species distribution and antifungal susceptibility patterns of yeasts that may occurs after increasing the use of azoles in Latin American hospitals.

J Biol Chem, 2003 Oct 10, 278(41), 40136 - 43 Epub 2003 Jul 28.
CALEB/NGC interacts with the Golgi-associated protein PIST; Hassel B et al.; CALEB/NGC is a neural member of the epidermal growth factor protein family expressed in axon and synapse-rich areas of the nervous system and shown to be important for neurite formation . It can bind to the extracellular matrix proteins tenascin-R and tenascin-C . Here we show that CALEB/NGC interacts with the Golgi-associated protein PIST . PIST was originally described as an interaction partner of the small GTPase TC10 and was then found to be Golgi-associated by binding to syntaxin-6 and to be important for the transport of frizzled proteins and the cystic fibrosis transmembrane conductance regulator to the plasma membrane . In addition, PIST was demonstrated to be involved in autophagy and linked to processes of neurodegeneration . CALEB/NGC interacts with PIST in the yeast two-hybrid system . This interaction can be confirmed by co-immunoprecipitations and co-localization studies . The juxtamembrane cytoplasmic peptide segment of CALEB/NGC, highly conserved during evolution, mediates the binding to PIST . CALEB/NGC co-localizes with PIST in the Golgi apparatus of transfected COS7 cells and in Golgi-derived vesicles after brefeldin A or nocodazole treatment . Co-localization studies in primary hippocampal cells and analysis of Purkinje cells of colchicine-treated rats, serving as an in vivo model system to block microtubule-dependent transport processes, support the view that PIST is an interaction partner of CALEB/NGC and implicate that this interaction may play a role in the intracellular transport of CALEB/NGC.

J Biol Chem, 2003 Oct 3, 278(40), 38780 - 5 Epub 2003 Jul 28.
The RING finger protein RNF4, a co-regulator of transcription, interacts with the TRPS1 transcription factor; Kaiser FJ et al.; The TRPS1 gene encodes a repressor of GATA-mediated transcription . Mutations in this gene cause the tricho-rhino-phalangeal syndromes, but the affected pathways are unknown . In a yeast two-hybrid screen with the C-terminal part of the murine Trps1 protein as bait, we obtained three yeast clones encoding two overlapping fragments of the 194 amino acids RING finger protein 4 (Rnf4) . The overlap narrows down the Trps1-binding region within Rnf4 to amino acids 6-65 . This region in Rnf4 is also known to interact with several proteins including steroid receptors . By using truncated Trps1 constructs, the Rnf4-binding region in Trps1 could be assigned to amino acids 985-1184 of 1281 . This 200 amino acid region of Trps1 does not contain any predicted protein-protein interacting motif . Complex formation between the human proteins TRPS1 and RNF4 was verified by co-immunoprecipitation from transfected and native mammalian cells . Confocal laser-scanning microscopy revealed that the endogenous proteins are located in distinct structures of the nucleus . Using a luciferase reporter assay, we could demonstrate that the repressional function of TRPS1 is inhibited by RNF4 . This finding suggests that RNF4 is a negative regulator of TRPS1 activity.

Biophys J, 2003 Aug, 85(2), 1215 - 22
Proline can have opposite effects on fast and slow protein folding phases; Osvath S et al.; Proline isomerization is well known to cause additional slow phases during protein refolding . We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10-100 min) and very fast (4 micro s-2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation . Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines . Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle . The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature . Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule . The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales.

Parasitology, 2003 Jul, 127(Pt 1), 69 - 77
Purification and characterization of a saccharide-binding protein from penetration glands of Diplostomum pseudospathaceum--a bifunctional molecule with cysteine protease activity; Mikes L et al.; A beta-1,3-glucan-binding lectin from the penetration glands of Diplostomum pseudospathaceum cercariae was isolated by affinity chromatography using yeast glucan and curdlan as affinity matrices . Further purification to homogeneity was performed by cation-exchange chromatography . The protein migrated as a double band around 24 kDa in gels after SDS-PAGE . The protein is of strongly basic nature--its pI shown by native IEF was around 10 . The mass of the protein determined by MALDI-TOF mass spectrometry was 23.9 kDa . N-terminal sequence as well as some internal sequences showed significant alignments with several cysteine protease sequences found in databases . The protein bound a biotinylated synthetic analogue of the irreversible inhibitor of cysteine proteases, E-64 and, moreover, its proteolytic activity was demonstrated in substrate gels . The enzymatic activity could be inhibited by the cysteine protease inhibitor E-64; therefore, the investigated protein was considered to be a bifunctional molecule possessing both lectin and enzyme activities . Glycanohydrolytic activity was not proved . The detected characters of this molecule lead to a hypothesis on its role in penetration of Diplostomum cercariae into fish hosts--that of binding to the carbohydrates of fish mucus and concurrent cleaving of protein components of the mucus and skin.

Cancer Metastasis Rev, 2003 Dec, 22(4), 451 - 64
The Aurora kinases: role in cell transformation and tumorigenesis; Katayama H et al.; Aurora kinases representing a novel family of serine/threonine kinases have been identified as key regulators of the mitotic cell division process . The three members of this kinase family, identified so far, referred to as Aurora-A, Aurora-B and Aurora-C kinases, are close homologues of the prototypic yeast Ipll and Drosophila aurora kinases, which are known to be involved in the regulation of centrosome function, bipolar spindle assembly and chromosome segregation processes . All three members of the mammalian kinase family have a catalytic domain that is highly conserved with a short C-terminal domain and an N-terminal domain of varying sizes . Following their discovery about five years ago, extensive research has focused on understanding the biological roles of these kinases and elucidation of their pathways, which regulate cell proliferation and maintenance of normal cellular phenotypes . Significant interest in the subject was generated since all three Aurora kinases family members were reported to be overexpressed in many human cancers, and elevated expression has been correlated with chromosomal instability and clinically aggressive disease in some instances . Ectopic overexpression of one member of the family, Aurora-A, was shown to induce oncogenic transformation in cells . Unlike most other putative oncogenes identified, so far, members of this kinase family are expressed and active at the highest level during G2-M phase of the cell cycle . Aurora kinases are localized at the centrosomes of interphase cells, at the poles of the bipolar spindle and in the midbody of the mitotic apparatus . Substrates identified for the Aurora-A and Aurora-B kinases, include a kinesin-like motor protein, spindle apparatus proteins, histone H3 protein, kinetochore protein and the tumor suppressor protein p53 . Identification of Aurora kinases as RasGAP Src homology 3 domain binding protein, also implicates these kinases as potential effectors in the Ras pathway relevant to oncogenesis . Abnormal elevated expression of Aurora kinases detected in human cancer cells could help explain the underlying biological mechanisms responsible for the development of many cellular phenotypes associated with malignant cells . Identification of these mechanisms offers the possibility of designing novel targeted therapies for cancer in the future.

J Biol Inorg Chem, 2003 Sep, 8(7), 777 - 86 Epub 2003 Jul 19.
A further investigation of the cytochrome b5-cytochrome c complex; Banci L et al.; The interaction of reduced rabbit cytochrome b(5) with reduced yeast iso-1 cytochrome c has been studied through the analysis of (1)H-(15)N HSQC spectra, of (15)N longitudinal ( R(1)) and transverse ( R(2)) relaxation rates, and of the solvent exchange rates of protein backbone amides . For the first time, the adduct has been investigated also from the cytochrome c side . The analysis of the NMR data was integrated with docking calculations . The result is that cytochrome b(5) has two negative patches capable of interacting with a single positive surface area of cytochrome c . At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed . At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed . All the species are in fast exchange on the scale of differences in chemical shift . By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b(5).

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jul, 35(7), 661 - 5
Identification of interaction between PAI-2 and IRF-3; Zhang YQ et al.; HeLa cells transfected with plasminogen activator inhibitor-2 ( PAI-2 ) were protected from TNF- alpha-induced apoptosis . The apoptosis protection by PAI-2 is dependent on a 33 amino acids fragment between helix C and D of PAI-2, which may be due to the interaction of PAI-2 with some intracellular proteins . In this study, the yeast two-hybrid system was used to screen a HeLa cells cDNA library constructed during apoptosis with the fragment between helix C and D of PAI-2 as bait . We retrieved a clone that encodes 98 amino acids of C-terminus of interferon regulatory factor-3 (IRF-3) . Co-immunoprecipitation experiments confirmed the interaction between PAI-2 and IRF-3 in vivo . IRF-3 belongs to a family of the IRF transcription factors and has been shown to participate in a large number of biological processes . These data suggest that IRF-3 may be involved in the apoptosis protection and antiviral function of PAI-2.

J Biol Chem, 2003 Sep 26, 278(39), 37471 - 9 Epub 2003 Jul 25.
Repair of UV lesions in silenced chromatin provides in vivo evidence for a compact chromatin structure; Livingstone-Zatchej M et al.; Genes positioned close to telomeres in yeast are silenced by a heterochromatin-like structure containing Sir proteins . To investigate whether silencing also affects DNA repair, we studied removal of UV lesions by photolyase and nucleotide excision repair (NER) in strains containing the URA3 gene inserted 2 kilobases from a telomere . URA3 was transcriptionally active in sir3delta mutants, partially silenced in SIR3 cells, or completely silenced by overexpression of SIR3 or deletion of RPD3 . The active URA3 showed efficient repair by both pathways . Fast repair of the promoter and 3' end by photolyase reflected a non-nucleosomal structure . Partial silencing had no remarkable effect on photolyase but reduced repair by NER, indicating differential accessibility for the two repair reactions . Complete silencing inhibits NER and photolyase in the coding region as well as in the promoter and the 3'-end . Conventional nuclease footprinting analyses revealed subtle changes in the promoter proximal nucleosome under partially silenced conditions but a pronounced reorganization of chromatin extending over the whole gene in silenced chromatin . Thus, both repair systems are sensitive to chromatin changes associated with silencing and provide direct evidence for a compact structure of heterochromatin.

J Biol Chem, 2003 Oct 17, 278(42), 41552 - 6 Epub 2003 Jul 25.
A novel gamma-hydroxybutyrate dehydrogenase: identification and expression of an Arabidopsis cDNA and potential role under oxygen deficiency; Breitkreuz KE et al.; In plants, gamma-aminobutyrate (GABA), a non-protein amino acid, accumulates rapidly in response to a variety of abiotic stresses such as oxygen deficiency . Under normoxia, GABA is catabolized to succinic semialdehyde and then to succinate with the latter reaction being catalyzed by succinic semialdehyde dehydrogenase (SSADH) . Complementation of an SSADH-deficient yeast mutant with an Arabidopsis cDNA library enabled the identification of a novel cDNA (designated as AtGH-BDH for Arabidopsis thaliana gamma-hydroxybutyrate dehydrogenase), which encodes a 289-amino acid polypeptide containing an NADP-binding domain . Constitutive expression of AtGHBDH in the mutant yeast enabled growth on 20 mm GABA and significantly enhanced the cellular concentrations of gamma-hydroxybutyrate, the product of the GHDBH reaction . These data confirm that the cDNA encodes a polypeptide with GHBDH activity . Arabidopsis plants subjected to flooding-induced oxygen deficiency for up to 4 h possessed elevated concentrations of gamma-hydroxybutyrate as well as GABA and alanine . RNA expression analysis revealed that GHBDH transcription was not up-regulated by oxygen deficiency . These findings suggest that GHBDH activity is regulated by the supply of succinic semialdehyde or by redox balance . It is proposed that GHBDH and SSADH activities in plants are regulated in a complementary fashion and that GHBDH and gamma-hydroxybutyrate function in oxidative stress tolerance.

Invest Ophthalmol Vis Sci, 2003 Aug, 44(8), 3424 - 31
Effects of mannose on Acanthamoeba castellanii proliferation and cytolytic ability to corneal epithelial cells; Hurt M et al.; PURPOSE: Acanthamoeba trophozoites express a mannose binding receptor that facilitates adhesion of trophozoites to mannosylated proteins on corneal epithelial cells . This study was undertaken to determine the role that mannose stimulation has in the amoeba's growth, secreted products, and ability to desquamate the corneal epithelium . METHODS: Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose (PYG) and PYG with 100 mM methyl alpha-D-mannopyranoside or galactose . The proliferation of trophozoites and cysts was examined by optical density and direct counts . The molecular weight of the mannose-stimulated protein was examined by SDS/PAGE . The cytolytic protein was purified by fast protein liquid chromatography (FPLC) size exclusion and ionic exchange and then tested for cytopathic effect (CPE) and collagenolytic activity in vitro . Collagenolytic activity was examined by zymography . Proteases and protease inhibitors were used to characterize the nature of the cytolytic protein . RESULTS: Methyl alpha-D-mannopyranoside inhibited the growth of A . castellanii by 50% (P < 0.05) and concomitantly induced a threefold increase in the formation of cysts . SDS-PAGE analysis revealed a mannose-induced protein of approximately 133 kDa (MIP-133) . The MIP-133 protein was found to be highly cytolytic against corneal epithelial cells, but not human intestinal epithelial cells and also degraded collagen in vitro . Serine protease inhibitors abrogated both CPE and collagenolytic activity of the MIP-133 protein (P < 0.001) . CONCLUSIONS: The results suggest that binding of trophozoites to mannosylated proteins on the corneal surface induces A . castellanii to secrete a approximately 133-kDa serine protease that kills both human and hamster corneal epithelium and degrades collagen.

Nat Genet, 2003 Aug, 34(4), 403 - 12
Identification of Stk6/STK15 as a candidate low-penetrance tumor-susceptibility gene in mouse and human; Ewart-Toland A et al.; Linkage analysis and haplotype mapping in interspecific mouse crosses (Mus musculus x Mus spretus) identified the gene encoding Aurora2 (Stk6 in mouse and STK15 in human) as a candidate skin tumor susceptibility gene . The Stk6 allele inherited from the susceptible M . musculus parent was overexpressed in normal cells and preferentially amplified in tumor cells from F(1) hybrid mice . We identified a common genetic variant in STK15 (resulting in the amino acid substitution F31I) that is preferentially amplified and associated with the degree of aneuploidy in human colon tumors . The Ile31 variant transforms rat1 cells more potently than the more common Phe31 variant . The E2 ubiquitin-conjugating enzyme UBE2N was a preferential binding partner of the 'weak' STK15 Phe31 variant form in yeast two-hybrid screens and in human cells . This interaction results in colocalization of UBE2N with STK15 at the centrosomes during mitosis . These results are consistent with an important role for the Ile31 variant of STK15 in human cancer susceptibility.

Plant Cell Physiol, 2003 Jul, 44(7), 735 - 42
An Arabidopsis MADS-box protein, AGL24, is specifically bound to and phosphorylated by meristematic receptor-like kinase (MRLK); Fujita H et al.; Intercellular signaling mediated by receptor-like kinases (RLKs) is important for diverse processes in plant development, although downstream intracellular signaling pathways remain poorly understood . Proteins interacting directly with RLK were screened for by yeast two-hybrid assay with the kinase domain as bait . A MADS-box protein, AGL24 was identified as a candidate substrate of MRLK (Meristematic Receptor-Like Kinase), which was named for its spatial expression in shoot and root apical meristems in ARABIDOPSIS: The AGL24 protein specifically interacted with, and was phosphorylated by, the MRLK kinase domain in in vitro assays . The simultaneous expression of AGL24 and MRLK in shoot apices during floral transition suggested that the interaction occurs in plants . Using plants constitutively expressing a fusion protein of AGL24 and green fluorescent protein, the subcellular localization of AGL24 protein was observed exclusively in the nucleus in apical tissues where MRLK was expressed, while AGL24 was localized in both the cytoplasm and the nucleus in tissues where no MRLK expression was detectable . These results suggest that MRLK signaling promotes translocation of AGL24 from the cytoplasm to the nucleus . We propose that the RLK signaling pathway involves phosphorylation of a MADS-box transcription factor.

Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9578 - 83 Epub 2003 Jul 24.
Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2; Marcora E et al.; NeuroD (ND) is a basic helix-loop-helix transcription factor important for neuronal development and survival . By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt) . Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntington's disease, a neurodegenerative disorder . However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown . Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND . Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2 . To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt . We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.

Clin Chem, 2003 Aug, 49(8), 1347 - 52
Xuezhikang decreases serum lipoprotein(a) and C-reactive protein concentrations in patients with coronary heart disease; Liu L et al.; BACKGROUND: Increased serum lipoprotein(a) {Lp(a)} and high-sensitivity C-reactive protein (hsCRP) concentrations are independent risk factors for coronary heart disease (CHD) . Xuezhikang, an extract of cholestin, effectively lowers fasting cholesterol and triglyceride concentrations . We studied whether xuezhikang lowered Lp(a) and hsCRP concentrations . METHODS: We randomly divided 60 CHD patients into two groups to receive xuezhikang (1200 mg daily) or placebo for 6 weeks . The fasting hsCRP concentration and the postprandial changes of serum lipid concentrations at 2, 4, and 6 h after a high-fat meal (800 calories; 50 g of fat) were measured before and after the 6-week protocol . RESULTS: The two groups had similar baseline fasting lipid and hsCRP concentrations . The postprandial triglyceride and Lp(a) concentrations were significantly increased (P <0.05) . After 6 weeks, the fasting and postprandial lipid concentrations decreased significantly in the xuezhikang group, accompanied by a significant reduction in fasting hsCRP concentration (P <0.001) . The placebo group had no significant change in lipid concentrations, whereas the fasting serum hsCRP concentration was reduced significantly (P <0.05) . The reduction in hsCRP was closely related to the changes in fasting Lp(a) concentration (r = 0.402; P <0.05) and triglyceride area under the curve (r = 0.441; P <0.001) . CONCLUSIONS: Xuezhikang effectively decreased fasting Lp(a) and postprandial triglyceride concentrations, which were associated with reductions of fasting hsCRP concentrations in CHD patients.

Cerebellum, 2003, 2(2), 146 - 53
Iron metabolism in mice with partial frataxin deficiency; Santos MM et al.; Friedreich ataxia (FRDA), the most common autosomal recessive inherited ataxic disorder, is the consequence of deficiency of the mitochondrial protein frataxin, typically caused by homozygous intronic GAA expansions in the corresponding gene . The yeast frataxin homologue (yfh1p) is required for cellular respiration . Yfh1p appears to regulate mitochondrial iron homeostasis and protect from free radical toxicity . Complete loss of frataxin in knockout mice leads to early embryonic lethality, indicating an important role for frataxin during development . Heterozygous littermates with partial frataxin deficiency are apparently healthy and have no obvious phenotype . Here we evaluate iron metabolism and sensitivity to dietary and parenteral iron loading in heterozygote frataxin knockout mice (Fx(+/-)) . Iron concentrations in the liver, heart, pancreas and spleen, and cellular iron distribution patterns were compared between wild type and Fx(+/-) mice . Response to parenteral iron challenge was not different between Fx(+/-) mice and wild type littermates, while sporadic iron deposits were observed in the hearts of dietary iron-loaded Fx(+/-) mice . Finally, we evaluated the effect of partial frataxin deficiency on susceptibility to cardiac damage in the mouse model of hereditary hemochromatosis (HH), the Hfe knockout mice . HH, an iron overload disease, is one of the most frequent genetic diseases in populations of European origin . By breeding Hfe(-/-) with Fx(+/-) mice, we obtained compound mutant mice lacking both Hfe and one frataxin allele . Sparse iron deposits in areas of mild to moderate cardiac fibrosis were found in the majority of these mice . However, they did not develop any neurological symptoms . Our studies indicate an association between frataxin deficiency, iron deposits and cardiac fibrosis, but no obvious association between iron accumulation and neurodegeneration similar to FRDA could be detected in our model . In addition, these results suggest that frataxin mutations may have a modifier role in HH, that predisposes to cardiomyopathy.

Trends Plant Sci, 2003 Jul, 8(7), 317 - 20
Sphingolipids, new players in plant signaling; Worrall D et al.; Sphingolipids are a diverse group of compounds, some of which play important signaling roles in animals and yeast . Results from recent research suggest that not only do plants contain components present in animal sphingolipid signaling pathways but that they might also possess novel plant-specific sphingolipid signaling systems . We suggest that the time is ripe for an in depth investigation of the role of this enigmatic group of compounds in plants.

Brain Res Mol Brain Res, 2003 Jul 23, 115(2), 121 - 9
Interaction of Disabled-1 and the GTPase activating protein Dab2IP in mouse brain; Homayouni R et al.; The Reelin signaling pathway controls neuronal positioning during mammalian brain development by binding to the very low density lipoprotein receptor and apolipoprotein E receptor-2, and signaling through the intracellular adapter protein Disabled-1 (Dab1) . To identify new components in the Reelin signaling pathway, we used a yeast two-hybrid screen to select Dab1-interacting proteins . Here, we report the characterization of a new mouse Dab1-interacting protein that is orthologous to rat Dab2IP, a Ras-GTPase activating protein previously shown to bind to Dab2/DOC . The interaction of Dab1 and Dab2IP was confirmed in biochemical assays and by co-immunoprecipitation from brain lysates . The site of interaction between Dab1 and Dab2IP was narrowed to the Dab1-PTB domain and the NPxY motif in Dab2IP . The deduced amino acid sequence of mouse Dab2IP encompasses 1,208 residues containing several protein interaction motifs as well as a Ras-like GAP-related domain . Northern blot analysis revealed at least two isoforms of Dab2IP mRNA in the brain, both of which exhibited increased expression during development . In situ hybridization analyses indicated that Dab2IP mRNA is diffusely expressed throughout the developing and the adult brain . Using a polyclonal antiserum specific for Dab2IP, we observed protein expression in the soma and processes of neurons in a variety of brain structures, including the developing cerebral cortex . Our findings suggest that Dab2IP may function as a downstream effector in the Reelin signaling pathway that influences Ras signaling during brain development.

J Mol Biol, 2003 Aug 1, 331(1), 167 - 76
Tipin, a novel timeless-interacting protein, is developmentally co-expressed with timeless and disrupts its self-association; Gotter AL; The mouse Timeless gene (mTim) was identified originally on the basis of its similarity to a Drosophila circadian gene, but has no substantiated role in the circadian clock mechanism . The importance of mTim in cellular processes involved in development, however, is undeniable, since targeted mutagenesis of this gene arrests embryonic development . To connect mTim to known pathways controlling cellular processes important for early development, a yeast two-hybrid approach was used to identify embryonic mTIM-interacting proteins . One positive interactor, a previously uncharacterized protein that is here termed TIPIN (TIMELESS interacting protein), was shown to interact with mTIM in vitro and in cultured cells . mTim and Tipin transcripts are co-expressed in similar tissues during embryonic development and in the adult brain . In transiently transfected cultures, mTIM promotes the nuclear localization of TIPIN . Immunoprecipitation experiments suggest that TIPIN is capable of regulating mTIM activity by disrupting the ability of mTIM to form homo-multimeric complexes . Together, these results indicate that mTIM forms a functional complex with TIPIN, and provide a starting point from which to link mTim to biochemical pathways controlling vital cellular functions.

Chin Med J (Engl), 2003 May, 116(5), 685 - 7
A randomized control trial on interruption of HBV transmission in uterus; Zhu Q et al.; OBJECTIVE: To study the interruptive effect of hepatitis B virus (HBV) specific immunolobulin (HBIG) before delivery in attempt to prevent intrauterine transmission of HBV . METHODS: Nine hundred and eighty HBsAg carrier pregnant women were randomly divided into HBIG group and control group . Each subject in the HBIG group received 200 IU or 400 IU of HBIG intramuscularly at 3, 2 and 1 month before delivery . The subjects in the control group did not receive any specific treatment . All newborn infants received 100 IU of HBIG intramascularly after venous blood samples were taken at birth and 2 weeks after birth, followed by 30 micro g plasma-derived HB vaccine or 5 micro g recombinant yeast-derived hepatitis B vaccine at 1, 2 and 7 months of age . Blood tests were performed for all the lying-in women and their neonates . Blood specimens were tested for HBsAg and HBeAg by enzyme immunoassay . All infants were followed up for 1 year . RESULTS: In the HBIG group, 491 neonates were born to 487 HBV carrier mothers; and in the control group, 496 neonates were born to 493 HBV carrier mothers . The rates of intrauterine transmission in the two groups were 14.3% and 5.7% respectively (chi(2) = 20.280, P < 0.001), and the rates of chronic hepatitis B in the two groups were 2.2% and 7.3% respectively (chi(2) = 13.696, P < 0.001) . The high risk factors of intrauterine HBV infection included HBsAg HBeAg double positive and HBV DNA positive in the peripheral blood of pregnant women . CONCLUSION: HBV infection in the uterus may be interrupted by injecting multiple intramuscular HBIG injections before delivery without causing any side-effects.

Genes Cells, 2003 Aug, 8(8), 677 - 84
Negative regulation of Wnt signalling by HMG2L1, a novel NLK-binding protein; Yamada M et al.; BACKGROUND: Wnt signalling plays a critical role in many developmental processes and tumorigenesis . Wnt/beta-catenin signalling induces the stabilization of cytosolic beta-catenin, which interacts with TCF/LEF-1 transcription factors, thereby inducing expression of Wnt-target genes . Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling . RESULTS: To identify NLK-interacting proteins, we performed yeast two-hybrid screening . We isolated the gene HMG2L1 and showed that injection of Xenopus HMG2L1 (xHMG2L1) mRNA into Xenopus embryos inhibited Wnt/beta-catenin-induced axis duplication and expression of Wnt/beta-catenin target genes . Moreover, xHMG2L1 inhibited beta-catenin-stimulated transcriptional activity in mammalian cells . CONCLUSIONS: Our findings indicate that xHMG2L1 may negatively regulate Wnt/beta-catenin signalling, and that xHMG2L1 may play a role in early Xenopus development together with NLK.

Electrophoresis, 2003 Jul, 24(14), 2359 - 68
Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures; Shang TQ et al.; The field of proteomics requires methods that offer high sensitivity and wide dynamic range . One of the strategies used to improve the dynamic range is sample prefractionation, such as microsolution isoelectric focusing (IEF) . We have modified a commercial solution IEF instrument, the Rotofor, to prefractionate protein mixtures by carrier ampholyte-free solution IEF . The focusing chamber of the Rotofor was divided into several compartments by polyacrylamide membranes with imbedded Immobiline mixtures of specific pH values . When an electric field is applied, each protein migrates to the compartment confined by membranes with pH values flanking its isoelectric point . The approach was demonstrated for the focusing of myoglobin into a predicted compartment, as well as the separation of a complex soluble yeast protein mixture into several distinct fractions . The proteins were dissolved in water or 30% isopropanol . The method is applicable to both gel-based and solution-phase protein identification methods, without the need for further sample preparation.

Mol Psychiatry, 2003 Jul, 8(7), 685 - 94
Disrupted-In-Schizophrenia 1, a candidate gene for schizophrenia, participates in neurite outgrowth; Miyoshi K et al.; Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation that segregated with schizophrenia in a Scottish family . Predicted DISC1 product has no significant homology to other known proteins . Here, we demonstrated the existence of DISC1 protein and identified fasciculation and elongation protein zeta-1 (FEZ1) as an interacting partner of DISC1 by a yeast two-hybrid study . FEZ1 and its nematode homolog are reported to represent a new protein family involved in axonal outgrowth and fasciculation . In cultured hippocampal neurons, DISC1 and FEZ1 colocalized in growth cones . Interactions of these proteins were associated with F-actin . In the course of neuronal differentiation of PC12 cells, upregulation of DISC1/FEZ1 interaction was observed as along with enhanced extension of neurites by overexpression of DISC1 . The present study shows that DISC1 participates in neurite outgrowth through its interaction with FEZ1 . Recent studies have provided reliable evidence that schizophrenia is a neurodevelopmental disorder . As there is a high level of DISC1 expression in developing rat brain, dysfunction of DISC1 may confer susceptibility to psychiatric illnesses through abnormal development of the nervous system.

J Immunol, 2003 Aug 1, 171(3), 1352 - 9
Positive modulation of IL-12 signaling by sphingosine kinase 2 associating with the IL-12 receptor beta 1 cytoplasmic region; Yoshimoto T et al.; IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses . IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3 . In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling . To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rbeta1 cytoplasmic region . Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rbeta1, while the regions including the carboxyl terminus and Box II in the IL-12Rbeta1 cytoplasmic region appear to be involved in the binding to SPHK2 . Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation . Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-gamma production, while that of wild-type SPHK2 enhanced it . In contrast, the expression minimally affected IL-12-induced proliferation . A similar decrease in IL-12-induced IFN-gamma production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system . These results suggest that SPHK2 associates with the IL-12Rbeta1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.

Development, 2003 Sep, 130(17), 3917 - 28
Nil per os encodes a conserved RNA recognition motif protein required for morphogenesis and cytodifferentiation of digestive organs in zebrafish; Mayer AN et al.; Digestive organ development occurs through a sequence of morphologically distinct stages, from overtly featureless endoderm, through organ primordia to, ultimately, adult form . The developmental controls that govern progression from one stage to the next are not well understood . To identify genes required for the formation of vertebrate digestive organs we performed a genetic screen in zebrafish . We isolated the nil per os (npo) mutation, which arrests morphogenesis and cytodifferentiation of the gut and exocrine pancreas in a primordial state . We identified the npo gene by positional cloning . It encodes a conserved protein, with multiple RNA recognition motifs, that is related to the yeast protein Mrd1p . During development npo is expressed in a dynamic fashion, functioning cell autonomously to promote organ cytodifferentiation . Antisense-mediated knockdown of npo results in organ hypoplasia, and overexpression of npo causes an overgrowth of gastrointestinal organs . Thus, npo is a gene essential for a key step in the gut morphogenetic sequence.

Neuron, 2003 Jul 17, 39(2), 227 - 39
Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1; Tang TS et al.; Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt) . The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function . In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A) . We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo . In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt . Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG . Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.

Hum Mutat, 2003 Aug, 22(2), 121 - 8
Mutational analysis of the BRCA1-interacting genes ZNF350/ZBRK1 and BRIP1/BACH1 among BRCA1 and BRCA2-negative probands from breast-ovarian cancer families and among early-onset breast cancer cases and reference individuals; Rutter JL et al.; Two potential breast cancer susceptibility genes, encoding the BRCA1-interacting proteins ZNF350 (or ZBRK1) and BRIP1 (or BACH1), have been identified in yeast two-hybrid screens . We sequenced these genes in probands from 21 families with potentially inherited breast/ovarian cancer, all of which were negative for BRCA1/BRCA2 mutations . Families had at least one case of male breast cancer, two cases of ovarian cancer, or three or more cases of breast and ovarian cancer . In addition, 58 early-onset (before age 35) breast cancer cases and 30 reference individuals were analyzed . Of 17 variants detected in ZBRK1, a missense mutation Val524Ile was identified in the proband of one high-risk family, but no other family members were available for testing . Of 25 variants identified in BRIP1, in addition to four common silent or missense mutations, we identified Gln540Leu, a non-conservative amino acid change, in a single familial proband with inflammatory breast cancer, but this mutation was not present in her three relatives with breast cancer . Haplotype analysis suggests that all ZBRK1 SNPs fall within a single block with two SNPs capturing 92% of the haplotype diversity, while the BRIP1 SNPs fall in two blocks, with five SNPs capturing 89% of the haplotype diversity . Based on sequencing of ZBRK1 and BRIP1 in 21 BRCA1/2-negative probands from inherited breast/ovarian cancer families, it appears unlikely that mutations in these genes account for a significant fraction of inherited breast cancer . Further analysis in unselected cases will be required to know whether the identified variants play a role in genetic predisposition to breast cancer in the general population . Hum Mutat 22:121-128, 2003 . Published 2003 Wiley-Liss, Inc.

J Biol Chem, 2003 Oct 3, 278(40), 38301 - 9 Epub 2003 Jul 18.
Rim, a component of the presynaptic active zone and modulator of exocytosis, binds 14-3-3 through its N terminus; Sun L et al.; Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity . Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain . Both Rim1 and Rim2 contain multiple domains, including an N-terminal zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells . The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain . In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3 . The N-terminal domain of Rim2 also bound 14-3-3 . The binding domains were localized to a short segment just C-terminal to the zinc finger . 14-3-3 proteins bind to specific phosphoserine residues . Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3 . Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding . Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding . Rabphilin3, another Rab3a effector, also bound 14-3-3 . Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds . Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.

Curr Top Med Chem, 2003, 3(12), 1348 - 57
Immunophilin chaperones in steroid receptor signalling; Ratajczak T et al.; The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine/threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling . Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function . Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold . Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors . Association of receptors with different immunophilins leads to differential functional consequences for receptor activity . Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity . Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIase-mediated conformational changes in the ligand-binding domain . A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus.

Mycoses, 2003 Apr, 46(3-4), 145 - 8
A case of subcutaneous phaeohyphomycotic cyst due to Exophiala jeanselmei complicated with systemic lupus erythematosus; Murayama N et al.; We report a case of subcutaneous phaeohyphomycosis by Exophiala jeanselmei that appeared on the extensor surface of the left lower leg of a 34-year-old woman with systemic lupus erythematosus (SLE) . The superficial symptoms were a subcutaneous nodule 2.5 x 2 cm in size discharging a serous exudate from its center . Histopathological examination revealed granulomatous changes including large numbers of neutrophils in the dermis and the subcutaneous tissues . In addition, periodic acid-Schiff-positive fungal elements consisting of many yeast-like cells and chains of cells with hyphae were seen . The statistics on E . jeanselmei infections in Japan indicated that 54 cases (24 in men and 30 in women) had been reported, of which 50 (21 in men and 29 in women) were phaeohyphomycosis, and about half had underlying diseases; and the sites of the lesions were mainly on the extremities.

Curr Opin Neurol, 2003 Aug, 16(4), 465 - 70
Experimental therapeutics in Huntington's disease: are models useful for therapeutic trials?
Bates GP, Hockly E.
PURPOSE OF REVIEW: Research conducted over the past 10 years has uncovered molecular mechanisms that are likely to be important in the early stages of Huntington's disease pathogenesis . This review summarizes the resources and strategies that are in place in order to exploit these new findings and use them to develop novel Huntington's disease therapeutics . The role that disease models will play in this process is discussed . RECENT FINDINGS: A wide variety of models of Huntington's disease have been developed including yeast, Caenorhabditis elegans, Drosophila melanogaster and mouse . These can be developed as screening assays for the identification of chemical compounds that show beneficial effects against a specific phenotype and for the cross validation of potential therapeutics . The first compounds arising through this drug development pipeline have been reported . Similarly, the preclinical screening of compounds in mouse models is being developed in a coordinated manner . SUMMARY: Our understanding of the molecular basis of Huntington's disease is increasing at an exponential rate . Over the next few years an increasing number of potential therapeutic compounds will have been identified . It will only be possible to take a small number of these through to phase III clinical trials . The challenge will be to use the in-vivo models of Huntington's disease to best predict which of these compounds should be pursued in the clinic, to avoid depleting the patient population willing to enter into trials, and demoralizing them by conducting repeated unsuccessful trials.

Science, 2003 Aug 22, 301(5636), 1069 - 74 Epub 2003 Jul 17.
Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing; Schramke V et al.; The expression of short hairpin RNAs in several organisms silences gene expression by targeted mRNA degradation . This RNA interference (RNAi) pathway can also affect the genome, as DNA methylation arises at loci homologous to the target RNA in plants . We demonstrate in fission yeast that expression of a synthetic hairpin RNA is sufficient to silence the homologous locus in trans and causes the assembly of a patch of silent Swi6 chromatin with cohesin . This requires components of the RNAi machinery and Clr4 histone methyltransferase for small interfering RNA generation . A similar process represses several meiotic genes through nearby retrotransposon long terminal repeats (LTRs) . These analyses directly implicate interspersed LTRs in regulating gene expression during cellular differentiation.

Genes Dev, 2003 Aug 1, 17(15), 1882 - 93 Epub 2003 Jul 17.
SKN-1 links C . elegans mesendodermal specification to a conserved oxidative stress response; An JH et al.; During the earliest stages of Caenorhabditis elegans embryogenesis, the transcription factor SKN-1 initiates development of the digestive system and other mesendodermal tissues . Postembryonic SKN-1 functions have not been elucidated . SKN-1 binds to DNA through a unique mechanism, but is distantly related to basic leucine-zipper proteins that orchestrate the major oxidative stress response in vertebrates and yeast . Here we show that despite its distinct mode of target gene recognition, SKN-1 functions similarly to resist oxidative stress in C . elegans . During postembryonic stages, SKN-1 regulates a key Phase II detoxification gene through constitutive and stress-inducible mechanisms in the ASI chemosensory neurons and intestine, respectively . SKN-1 is present in ASI nuclei under normal conditions, and accumulates in intestinal nuclei in response to oxidative stress . skn-1 mutants are sensitive to oxidative stress and have shortened lifespans . SKN-1 represents a connection between developmental specification of the digestive system and one of its most basic functions, resistance to oxidative and xenobiotic stress . This oxidative stress response thus appears to be both widely conserved and ancient, suggesting that the mesendodermal specification role of SKN-1 was predated by its function in these detoxification mechanisms.

J Biol Chem, 2003 Oct 3, 278(40), 38238 - 46 Epub 2003 Jul 17.
Inositol 1,4,5-trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the endoplasmic reticulum-associated degradation pathway, and is inhibited by chelation of intracellular Zn2+; Webster JM et al.; In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome . Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s) . In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p . Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not . These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD . Additional studies showed that the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process . Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.

J Biochem (Tokyo), 2003 Jun, 133(6), 713 - 8
HCC-associated protein HCAP1, a variant of GEMIN4, interacts with zinc-finger proteins; Di Y et al.; The gene HCAP1 (HCC-associated Protein 1), one variant of GEMIN4, has been mapped in a minimum LOH region on chromosome 17p13.3 and encodes a 1047-amino acid protein . Function predictions based on the amino acid sequence of protein HCAP1 revealed it to contain one helix-loop-helix motif and one leucine zipper domain . Using yeast two-hybrid screening, five zinc-finger proteins were identified as HCAP1-interacting proteins . Among them, NDP52 (nuclear dot protein 52) appeared most frequently in positive clones and was the most strongly interacting protein . Then, the interaction between HCAP1 and NDP52 was confirmed by GST pull-down assay and a coimmunoprecipitation experiment . Moreover, an immunofluorescent staining assay indicated that NDP52 colocalizes with HCAP1 in the cytoplasm . By deletion analysis, the leucine zipper domain of HCAP1 and the zinc finger domain of NDP52 were identified as important regions responsible for the interaction.

Clin Dysmorphol, 2003 Apr, 12(2), 123 - 7
Small inherited terminal duplication of 7q with hydrocephalus, cleft palate, joint contractures, and severe hypotonia; Morava E et al.; We report a 14-month-old girl with submucous cleft palate, resolving mild hydrocephalus, severe hypotonia and joint contractures . The finding of extreme hydrocephalus, cleft palate and club feet in a fetus of the mother's previous pregnancy suggested an inherited defect . Chromosome analysis and FISH studies in the proband revealed an abnormal homolog 13 resulting in a duplication of distal chromosome 7q, 7q35-qter, and a very small associated deletion of distal chromosome 13q, 13q34-qter . The mother showed the balanced translocation . Similar clinical signs have been described with larger distal 7q duplications . Our findings suggest that 7q35-qter, and possibly the gene for sonic hedgehog (SHH) on 7q36, is the critical region for the typical facial features and the profound hypotonia observed in the 'trisomy of distal 7q' syndrome.

Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9342 - 7 Epub 2003 Jul 16.
The cell death regulator GRIM-19 is an inhibitor of signal transducer and activator of transcription 3; Zhang J et al.; GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-beta- and retinoic acid-induced cell death, codes for a approximately 16-kDa protein that induces apoptosis in a number of cell lines . Antisense ablation of GRIM-19 caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells . To understand the molecular bases for its cell death regulatory activity, we used a yeast two-hybrid screen and identified that the transcription factor STAT3 (signal transducer and activator of transcription 3) binds to GRIM-19 . GRIM-19 inhibits transcription driven by activation of STAT3, but not STAT1 . It neither inhibits the ligand-induced activation of STAT3 nor blocks its ability to bind to DNA . Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19 binding . Because GRIM-19 does not bind significantly to other STATs, our studies identify a specific inhibitor of STAT3 . Because constitutively active STAT3 up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also demonstrates an antioncogenic effect exerted by biological therapeutics.

J Bacteriol, 2003 Aug, 185(15), 4424 - 31
The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator; Ohta N et al.; Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively . The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC . We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK . The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily . The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases . A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer . We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases . The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO . We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.

Curr Biol, 2003 Jul 15, 13(14), R565 - 7
COP9 signalosome: a provider of DNA building blocks; Nielsen O; In fission yeast, the COP9 signalosome is required to activate ribonucleotide reductase for DNA synthesis . This is mediated via the ubiquitin ligase Pcu4, activation of which leads to degradation of the scaffold protein Spd1, which anchors the small ribonucleotide reductase subunit in the nucleus away from the large subunit in the cytoplasm.

Sci STKE . 2003 Jul 15;2003(191):PE27.
A kelch beta propeller featuring as a G beta structural mimic: reinventing the wheel?
Gettemans J, Meerschaert K, Vandekerckhove J, De Corte V.
New genetic and protein interaction data suggest that G protein alpha subunits may have partners with primary sequences that are quite divergent . How this is achieved may be through the adoption of similar structures, the beta propeller, by both proteins containing WD-40 repeats and kelch domains . Gettemans et al . describe results in yeast that suggest that kelch-domain proteins may serve as previously unrecognized beta subunits in the heterotrimeric G protein complex.

J Biol Chem, 2003 Sep 26, 278(39), 37840 - 8 Epub 2003 Jul 15.
The SIN3 deacetylase complex represses genes encoding mitochondrial proteins: implications for the regulation of energy metabolism; Pile LA et al.; Deacetylation of histones by the SIN3 complex is a major mechanism utilized in eukaryotic organisms to repress transcription . Presumably, developmental and cellular phenotypes resulting from mutations in SIN3 are a consequence of altered transcription of SIN3 target genes . Therefore, to understand the molecular mechanisms underlying SIN3 mutant phenotypes in Drosophila, we used full-genome oligonucleotide microarrays to compare gene expression levels in wild type Drosophila tissue culture cells versus SIN3-deficient cells generated by RNA interference . Of the 13,137 genes tested, 364 were induced and 35 were repressed by loss of SIN3 . The approximately 10-fold difference between the number of induced and repressed genes suggests that SIN3 plays a direct role in regulating these genes . The identified genes are distributed throughout euchromatic regions but are preferentially excluded from heterochromatic regions of Drosophila chromosomes suggesting that the SIN3 complex can only access particular chromatin structures . A number of cell cycle regulators were repressed by loss of SIN3, and functional studies indicate that repression of string, encoding the Drosophila homologue of the yeast CDC25 phosphatase, contributes to the G2 cell cycle delay of SIN3-deficient cells . Unexpectedly, a substantial fraction of genes induced by loss of SIN3 is involved in cytosolic and mitochondrial energy-generating pathways and other genes encode components of the mitochondrial translation machinery . Increased expression of mitochondrial proteins in SIN3-deficient cells is manifested in an increase in mitochondrial mass . Thus, SIN3 may play an important role in regulating mitochondrial respiratory activity.

J Clin Invest, 2003 Jul, 112(2), 189 - 96
Deubiquitination of type 2 iodothyronine deiodinase by von Hippel-Lindau protein-interacting deubiquitinating enzymes regulates thyroid hormone activation; Curcio-Morelli C et al.; The type 2 iodothyronine deiodinase (D2) is an integral membrane ER-resident selenoenzyme that activates the pro-hormone thyroxine (T4) and supplies most of the 3,5,3'-triiodothyronine (T3) that is essential for brain development . D2 is inactivated by selective conjugation to ubiquitin, a process accelerated by T4 catalysis and essential for the maintenance of T3 homeostasis . A yeast two-hybrid screen of a human-brain library with D2 as bait identified von Hippel-Lindau protein-interacting deubiquitinating enzyme-1 (VDU1) . D2 interaction with VDU1 and VDU2, a closely related deubiquitinase, was confirmed in mammalian cells . Both VDU proteins colocalize with D2 in the ER, and their coexpression prolongs D2 half-life and activity by D2 deubiquitination . VDU1, but not VDU2, is markedly increased in brown adipocytes by norepinephrine or cold exposure, further amplifying the increase in D2 activity that results from catecholamine-stimulated de novo synthesis . Thus, deubiquitination regulates the supply of active thyroid hormone to brown adipocytes and other D2-expressing cells.

Mol Cell Biol, 2003 Aug, 23(15), 5331 - 45
RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization; Didier C et al.; RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans . The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C . elegans which shows homology with human paxillin . Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination . RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination . Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination . Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology . Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth . Concomitantly, RNF5 expression results in inhibition of cell motility . Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

Mol Cell Biol, 2003 Aug, 23(15), 5143 - 64
Transcription enhancer factor 1 binds multiple muscle MEF2 and A/T-rich elements during fast-to-slow skeletal muscle fiber type transitions; Karasseva N et al.; In adult mouse skeletal muscle, beta-myosin heavy chain (betaMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression can be induced in fast type II fibers in response to a sustained increase in load-bearing work (mechanical overload {MOV}) . Our previous betaMyHC transgenic and protein-DNA interaction studies have identified an A/T-rich element (betaA/T-rich -269/-258) that is required for slow muscle expression and which potentiates MOV responsiveness of a 293-bp betaMyHC promoter (beta293wt) . Despite the GATA/MEF2-like homology of this element, we found binding of two unknown proteins that were antigenically distinct from GATA and MEF2 isoforms . By using the betaA/T-rich element as bait in a yeast one-hybrid screen of an MOV-plantaris cDNA library, we identified nominal transcription enhancer factor 1 (NTEF-1) as the specific betaA/T-rich binding factor . Electrophoretic mobility shift assay analysis confirmed that NTEF-1 represents the enriched binding activity obtained only when the betaA/T-rich element is reacted with MOV-plantaris nuclear extract . Moreover, we show that TEF proteins bind MEF2 elements located in the control region of a select set of muscle genes . In transient-coexpression assays using mouse C2C12 myotubes, TEF proteins transcriptionally activated a 293-bp betaMyHC promoter devoid of any muscle CAT (MCAT) sites, as well as a minimal thymidine kinase promoter-luciferase reporter gene driven by three tandem copies of the desmin MEF2 or palindromic Mt elements or four tandem betaA/T-rich elements . These novel findings suggest that in addition to exerting a regulatory effect by binding MCAT elements, TEF proteins likely contribute to regulation of skeletal, cardiac, and smooth muscle gene networks by binding select A/T-rich and MEF2 elements under basal and hypertrophic conditions.

FEBS Lett, 2003 Jul 17, 547(1-3), 205 - 11
SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism; Eldstrom J et al.; The functional interaction of the voltage-gated potassium channel hKv1.5 with the PDZ domain containing protein SAP97 has been investigated . In marked contrast with the known dependence of SAP97-induced Kv1 potassium current down-regulation on the channel C-termini, SAP97 increased hKv1.5 current through an indirect interaction with the Kv1.5 N-terminus . Deletion of the Kv1.5 N-terminus eliminated the SAP97-mediated increase in potassium currents whereas deletion of the channel's C-terminal PDZ binding motif had no effect . In contrast with other Kv1-SAP97 interactions, no physical interaction could be detected in vivo or in vitro between the two proteins . The proteins did not co-localize in cardiac myocytes nor did they co-immunoprecipitate from transfected HEK cells . Yeast two-hybrid experiments also failed to detect any interaction between the two proteins, but in one experiment of six, Kv1.5 co-immunoprecipitated very inefficiently with SAP97 from rat ventricular myocytes . Thus, we conclude that the influence of SAP97 on Kv1.5 potassium current levels is dependent upon a novel regulatory mechanism.

Peptides, 2003 Apr, 24(4), 509 - 13
A ubiquitin-like peptide with ribonuclease activity against various polyhomoribonucleotides from the yellow mushroom Cantharellus cibarius; Wang HX et al.; A peptide, with a molecular weight of 9K and an N-terminal sequence identical to that of ubiquitin, was isolated from fresh fruiting bodies of the yellow mushroom Cantharellus cibarius . The peptide manifested ribonucleolytic activity toward poly A, poly C, poly G, and poly U, with the activity toward the first two polyhomoribonucleotides being higher . Maximal activity toward yeast tRNA was observed at a temperature of 70 degrees C and a pH of 7 . The peptide was adsorbed on DEAE-cellulose, CM-Sepharose, and Q-Sepharose . The yield of the peptide was 7mg from 1.8kg mushroom.

Exp Mol Med, 2003 Jun 30, 35(3), 141 - 53
Mouse models for telomere and telomerase biology; Cheong C et al.; Telomeres serve a critical role in maintenance of genomic stability in all eukaryotes, from yeast to human . The maintenance of telomeres is achieved by the telomerase complex, which is largely composed of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) . A variety of mouse models have provided valuable insights into the relationship between the telomerase complex and telomere dysfunction at the organismal level and helped understand their biological significance in human . Recently, in addition to its role in maintenance of the telomeres, novel functions of the telomerase complex have been emerging . In this review, studies of all gene-targeted or transgenic mouse models so far generated for telomerase and telomere biology are comprehensively described, and potential novel functions of telomerase are briefly discussed.

Plant Physiol, 2003 Jul, 132(3), 1623 - 30
PINOID-mediated signaling involves calcium-binding proteins; Benjamins R et al.; The plant hormone auxin is a central regulator of plant development . In Arabidopsis, the PINOID (PID) protein serine/threonine kinase is a key component in the signaling of this phytohormone . To further investigate the biological function of PID, we performed a screen for PID-interacting proteins using the yeast two-hybrid system . Here, we show that PID interacts with two calcium-binding proteins: TOUCH3 (TCH3), a calmodulin-related protein, and PID-BINDING PROTEIN 1 (PBP1), a previously uncharacterized protein containing putative EF-hand calcium-binding motifs . The interaction between PID and the calcium-binding proteins is significant because it is calcium dependent and requires an intact PID protein . Furthermore, the expression of all three genes (PID, TCH3, and PBP1) is up-regulated by auxin . TCH3 and PBP1 are not targets for phosphorylation by PID, suggesting that these proteins act upstream of PID . PBP1 was found to stimulate the autophosphorylation activity of PID, and calcium influx and calmodulin inhibitors where found to enhance the activity of PID in vivo . Our results indicate that TCH3 and PBP1 interact with the PID protein kinase and regulate the activity of this protein in response to changes in calcium levels . This work provides the first molecular evidence for the involvement of calcium in auxin-regulated plant development.

Plant Physiol, 2003 Jul, 132(3), 1475 - 88
Molecular and biochemical characterization of VR-EILs encoding mung bean ETHYLENE INSENSITIVE3-LIKE proteins; Lee JH et al.; ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis . Two full-length cDNA clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by reverse transcriptase-polymerase chain reaction and by screening the cDNA library of mung bean (Vigna radiata) hypocotyls . VR-EIL1 and VR-EIL2 share 70% identity and display varying degrees of sequence conservation (39%-65%) with previously isolated EIN3 homologs from Arabidopsis, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) plants . Gel retardation assay revealed that both VR-EILs were able to interact specifically with optimal binding sequence-1, the recently identified optimal binding sequence for tobacco TEIL, with the binding of VR-EIL2 being more efficient than that of VR-EIL1 . Transient expression analysis using a VR-EIL::smGFP fusion gene in onion (Allium cepa) epidermal cells indicated that the VR-EIL proteins were effectively targeted to the nucleus . The fusion protein of VR-EIL2 with GAL4 DNA-binding domain strongly activated transcription of a reporter gene in yeast cells, and an essential domain for transcription-stimulating activity was localized to the amino-terminal acidic region that consists of 50 amino acid residues . In contrast with what has been previously found in EIN3- and TEIL-overexpressing Arabidopsis plants, transgenic tobacco seedlings expressing the VR-EIL genes under the control of cauliflower mosaic virus 35S promoter did not exhibit a constitutive triple response . Instead, they displayed a markedly enhanced proliferation of root hairs, one of the typical ethylene response phenotypes, and increased sensitivity to exogenous ethylene . In addition, the pathogenesis-related (PR) genes encoding beta-1,3-glucanase, osmotin, and PR1 were constitutively expressed in 35S::VR-EIL lines without added ethylene, and were hyperinduced in response to ethylene treatment . These results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene . The possible physiological role of VR-EILs is discussed in the light of the suggestion that they are active components of the ethylene-signaling pathway and their heterologous expressions constitutively turn on a subset of ethylene responses in tobacco plants.

J Biol Chem, 2003 Sep 19, 278(38), 36603 - 10 Epub 2003 Jul 10.
hMusTRD1alpha1 represses MEF2 activation of the troponin I slow enhancer; Polly P et al.; The novel transcription factor hMusTRD1alpha1 (human muscle TFII-I repeat domain-containing protein 1alpha1; previously named MusTRD1; O'Mahoney, J . V., Guven, K . L., Lin, J., Joya, J . E., Robinson, C . S., Wade, R . P., and Hardeman, E . C . (1998) Mol . Cell . Biol . 18, 6641-6652) was identified in a yeast one-hybrid screen as a protein that binds within an upstream enhancer-containing region of the skeletal muscle-specific gene, TNNI1 (human troponin I slow; hTnIslow) . It has been proposed that hMusTRD1alpha1 may play an important role in fiber-specific muscle gene expression by virtue of its ability to bind to an Inr-like element (nucleotides -977 to -960) within the hTnIslow upstream enhancer-containing region that is necessary for slow fiber-specific expression . In this study we demonstrate that both MEF2C, a known regulator of slow fiber-specific genes, and hMusTRD1alpha1 regulate hTnIslow through the Inr-like element . Co-transfection assays in C2C12 cells and Cos-7 cells demonstrate that hMusTRD1alpha1 represses hTnIslow transcription and prevents MEF2C-mediated activation of hTnIslow transcription . Gel shift analysis shows that hMusTRD1alpha1 can abrogate MEF2C binding to its cognate site in the hTnIslow enhancer . Glutathione S-transferase pull-down assays demonstrate that hMusTRD1alpha1 can interact with both MEF2C and the nuclear receptor co-repressor . The data support the role of hMusTRD1alpha1 as a repressor of slow fiber-specific transcription through mechanisms involving direct interactions with MEF2C and the nuclear receptor co-repressor.

Plant Mol Biol, 2003 May, 52(2), 357 - 69
RNA-binding protein-mediated translational repression of transgene expression in plants; Cerny RE et al.; We have demonstrated that RNA-binding proteins from coliphages and yeast can function as translational repressors in plants . RNA sequences called translational operators were inserted at a cap-proximal position in the 5'-UTR of mRNAs of two reporter genes, gus or aroA:CP4 . Translation of the reporter mRNAs was efficiently repressed when the RNA binding protein that specifically binds to its cognate operator was co-expressed . The efficiency of translational repression by RNA-binding protein positively correlated with the amount of binding protein in transformed plant cells . Detailed studies on coliphage MS2 coat protein-mediated translational repression also suggested that the efficiency of translational repression was position-dependent . A translational operator situated at the cap-proximal position was more efficient in conferring repression than one that was placed cap-distal . Translational repression can be an efficient means for regulation of transgene expression, thereby broadening current approaches for transgene regulation in plants.

Exp Parasitol, 2002 Nov-Dec, 102(3-4), 187 - 90
Entamoeba histolytica: intracellular distribution of the proteasome; Sanchez R et al.; We have studied the intracellular distribution of proteasome subunits, corresponding to the catalytic (20S) core and the regulatory (19S) cap, in the extracellular protozoan parasite Entamoeba histolytica . Contrary to all cell types described to date, notably mammalian and yeast, in which the proteasome is found in the nucleus and actively imported into it, microscopic analysis and subcellular fractionation of E . histolytica trophozoites show that the proteasome is absent from the nucleus of these cells . We speculate that, given the relative abundance of mono- and multinucleated trophozoites in culture, a relationship may exist between this unusual distribution of the proteasome and the frequent lack of synchrony between karyo- and cytokinesis in this primitive eukaryote.

Arch Microbiol, 2003 Sep, 180(3), 204 - 10 Epub 2003 Jul 10.
Characterization of the Penicillium chrysogenum antifungal protein PAF; Kaiserer L et al.; The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF ( Penicillium antifungal protein) . In this study, the antifungal activity of PAF is described . PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected . PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching . Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi . In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.

Sci Aging Knowledge Environ . 2003 Jul 09;2003(27):PE19.
Caloric restriction in trans; Kristal BS et al.; Caloric (or dietary) restriction (CR) is the most potent, robust, and reproducible known means of extending longevity and decreasing morbidity in laboratory mammals . Two of the major questions faced by researchers in this field are the applicability to humans and the biochemical mechanism(s) involved in the actions of CR . Studies in nonhuman primates are beginning to address the former; studies in phylogenetically lower organisms such as yeast and Drosophila are beginning to address the latter . de Cabo and colleagues now provide evidence that some aspects of CR can be reproduced in mammalian tissue culture cells exposed to sera from rats and monkeys subjected to CR . This work presents the initial development of a new model with which to approach mechanistic studies of CR and provides a new form of direct evidence that CR exerts at least some of its effects in trans.

J Biol Chem, 2003 Oct 3, 278(40), 38693 - 8 Epub 2003 Jul 10.
Atrial natriuretic peptide induces natriuretic peptide receptor-cGMP-dependent protein kinase interaction; Airhart N et al.; Circulating natriuretic peptides such as atrial natriuretic peptide (ANP) counterbalance the effects of hypertension and inhibit cardiac hypertrophy by activating cGMP-dependent protein kinase (PKG) . Natriuretic peptide binding to type I receptors (NPRA and NPRB) activates their intrinsic guanylyl cyclase activity, resulting in a rapid increase in cytosolic cGMP that subsequently activates PKG . Phosphorylation of the receptor by an unknown serine/threonine kinase is required before ligand binding can activate the cyclase . While searching for downstream PKG partners using a yeast two-hybrid screen of a human heart cDNA library, we unexpectedly found an upstream association with NPRA . PKG is a serine/threonine kinase capable of phosphorylating NPRA in vitro; however, regulation of NPRA by PKG has not been previously reported . Here we show that PKG is recruited to the plasma membrane following ANP treatment, an effect that can be blocked by pharmacological inhibition of PKG activation . Furthermore, PKG participates in a ligand-dependent gain-of-function loop that significantly increases the intrinsic cyclase activity of the receptor . PKG translocation is ANP-dependent but not nitric oxide-dependent . Our results suggest that anchoring of PKG to NPRA is a key event after ligand binding that determines distal effects . As such, the NPRA-PKG association may represent a novel mechanism for compartmentation of cGMP-mediated signaling and regulation of receptor sensitivity.

Bioinformatics, 2003, 19 Suppl 1, i255 - 63
Using hidden Markov models to analyze gene expression time course data; Schliep A et al.; MOTIVATION: Cellular processes cause changes over time . Observing and measuring those changes over time allows insights into the how and why of regulation . The experimental platform for doing the appropriate large-scale experiments to obtain time-courses of expression levels is provided by microarray technology . However, the proper way of analyzing the resulting time course data is still very much an issue under investigation . The inherent time dependencies in the data suggest that clustering techniques which reflect those dependencies yield improved performance . RESULTS: We propose to use Hidden Markov Models (HMMs) to account for the horizontal dependencies along the time axis in time course data and to cope with the prevalent errors and missing values . The HMMs are used within a model-based clustering framework . We are given a number of clusters, each represented by one Hidden Markov Model from a finite collection encompassing typical qualitative behavior . Then, our method finds in an iterative procedure cluster models and an assignment of data points to these models that maximizes the joint likelihood of clustering and models . Partially supervised learning--adding groups of labeled data to the initial collection of clusters--is supported . A graphical user interface allows querying an expression profile dataset for time course similar to a prototype graphically defined as a sequence of levels and durations . We also propose a heuristic approach to automate determination of the number of clusters . We evaluate the method on published yeast cell cycle and fibroblasts serum response datasets, and compare them, with favorable results, to the autoregressive curves method.

Bioinformatics, 2003, 19 Suppl 1, i108 - 17
Chain functions and scoring functions in genetic networks; Gat-Viks I et al.; One of the grand challenges of system biology is to reconstruct the network of regulatory control among genes and proteins . High throughput data, particularly from expression experiments, may gradually make this possible in the future . Here we address two key ingredients in any such 'reverse engineering' effort: The choice of a biologically relevant, yet restricted, set of potential regulation functions, and the appropriate score to evaluate candidate regulatory relations . We propose a set of regulation functions which we call chain functions, and argue for their ubiquity in biological networks . We analyze their complexity and show that their number is exponentially smaller than all boolean functions of the same dimension . We define two new scores: one evaluating the fitness of a candidate set of regulators of a particular gene, and the other evaluating a candidate function . Both scores use established statistical methods . Finally, we test our methods on experimental gene expression data from the yeast galactose pathway . We show the utility of using chain functions and the improved inference using our scores in comparison to several extant scores . We demonstrate that the combined use of the two scores gives an extra advantage . We expect both chain functions and the new scores to be helpful in future attempts to infer regulatory networks.

J Biomol Struct Dyn, 2003 Aug, 21(1), 43 - 53
Molecular modeling of a Leishmania antigen eIF-4A: identification of a potential epitope implicated in the adjuvant effect; Hamza A et al.; In leishmaniasis, the development of an effective vaccine depends on its capacity to elicit Th1 immune responses . Despite many approaches, recent vaccines do not induce sufficient levels of protection and long-term memory . To overcome this problem, vaccines are used with adjuvants that drive immunity towards Th1 and enhance endogenous production of IL-12, a Th1-promoting cytokine . Although exogenous IL-12 may act as an effective adjuvant, it has an elevated cost and toxic effects . Therefore, it is important to develop cheap and safer adjuvants that trigger endogenous IL-12 . Of particular interest is LmeIF a unique Leishmania protein that provides significant adjuvant effects by stimulating high IL-12 production . This investigation was designed to identify the structural factors responsible for the adjuvant effects of LmeIF by establishing the 3D models of LmeIF and MueIF (mouse) by homology modeling based on the X-ray structure of their homologs in yeast and comparing their stereo-electronic properties . The molecular electrostatic potential was computed around each model and used to localize the active site and the most different amino acids between LmeIF and MueIF . Sequence alignment of LmeIF with eIF-4 from other species showed three residues (Q186, A189, E214) in the active site which were peculiar to the Leishmania protein . Long MD simulation was carried out on LmeIF fragment 129-261 to compare its folding with the native protein . Despite a high degree of sequence similarity with different species, we have identified in LmeIF a set of residues unique to the protozoan parasite Leishmania which may be potentially responsible for its adjuvant property . Using LmeIF model, a plausible surface region for binding with its receptor was also identified.

Nucleic Acids Res, 2003 Jul 15, 31(14), 3993 - 4000
The frequency of gene targeting in Trypanosoma brucei is independent of target site copy number; Wickstead B et al.; We have investigated the effect of target copy number on the efficiency of stable transformation of the protozoan parasite Trypanosoma brucei . Using a single strain of the organism, we targeted integrative vectors to several different genomic sequences, occurring at copy numbers ranging from 1 to approximately 30,000 per diploid genome, and undertook a systematic assessment of both transformation and integration efficiencies . Even over this vast copy number range, frequency of gene targeting was the same for all sites . An independence of targeting frequency and target copy number is characteristic of mammalian homologous recombination and is unlike the situation in budding yeast . It is also not seen in the related parasite Leishmania, a distinction that may be the consequence of the different usage of recombination within the mechanisms of pathogenicity in the two species.

J Biol Chem, 2003 Sep 19, 278(38), 36707 - 17 Epub 2003 Jul 09.
Characterization of periphilin, a widespread, highly insoluble nuclear protein and potential constituent of the keratinocyte cornified envelope; Kazerounian S et al.; While keratinocytes go through the terminal differentiation and move toward the outer layers of epidermis, multiple proteins become sequentially incorporated into the cornified cell envelope . We have identified through yeast two-hybrid screening a novel protein, periphilin, interacting with periplakin, which is known as a precursor of the cornified cell envelope . Periphilin gene at chromosome 12q12 gives rise to multiple alternatively spliced transcripts . A monoclonal antibody detected the keratinocyte-specific periphilin isoform in undifferentiated keratinocytes in speckle-type nuclear granules and at the nuclear membrane, but in differentiated keratinocytes periphilin localized to the cell periphery and at cell-cell junctions, colocalizing there with periplakin . From cultured keratinocytes, periphilin was solubilized only after urea extraction, indicating the highly insoluble character of this protein . The nuclear localization, mediated through the N-terminal sequences of periphilin protein, is a prerequisite for the formation of insoluble complexes . Although the globular N terminus of periphilin was necessary for the interaction with the periplakin tail, the keratinocyte-specific C terminus was responsible for the homodimerization . The C-terminal helical domain, composed of multiple heptad repeats, serves as a substrate for cross-linking by transglutaminases but also was specifically cleaved by caspase-5 in vitro . In conclusion, the localization pattern and insolubility of periphilin indicate that this novel protein is potentially involved in epithelial differentiation and contributes to epidermal integrity and barrier formation.

J Econ Entomol, 2003 Jun, 96(3), 584 - 91
Development of a toxic bait for control of eastern lubber grasshopper (Orthoptera: Acrididae); Barbara KA et al.; This study assessed baits for eastern lubber grasshopper, Romalea guttata (Houttuyn) . When offered a choice among several grain-based baits (rolled oats, wheat bran, oat bran, yeast, corn meal, cornflakes) and vegetable oils (canola, corn, peanut, soybean), eastern lubber grasshopper adults preferred bait consisting of wheat bran carrier with corn oil as an added phagostimulant . Other carriers were accepted but consumed less frequently . Discrimination by eastern lubber grasshoppers among oils was poor . Similarly, addition of flavorings (peppermint, anise, lemon, banana) resulted in few significant effects . The carbaryl, wheat bran, and oil bait developed in this study was effective at causing eastern lubber grasshopper mortality in field-cage studies . Significant mortality occurred even though grasshoppers had to locate dishes of bait in a large cage, and could feed on daylilies, or grass growing through the bottom of the cage, rather than on the bran flakes . Consumption of as little as a single carbaryl-treated bran flake could induce mortality, although individuals varied greatly in their susceptibility . The bait matrix developed in this study was readily consumed when in the presence of some plant species . We expect that wheat bran and corn oil bait would be most effective as protection for less preferred plants (tomato, pepper, eggplant, leek, parsley, fennel, daylily, lily of the Nile, and canna lily) because baits were readily consumed in the presence of these plants . Plants that are readily consumed in the presence of bait (preferred plants) included butter crunch lettuce, carrot, yellow squash, cauliflower, collards, green onion, chive, cucumber, cabbage, cantalope, endive, red leaf lettuce, society garlic, caladium, and amaryllis . Baits are likely to be less effective in the presence of such plants . On average, vegetables in Solanaceae (i.e., tomato, pepper, and eggplant) and Apiaceae (i.e., fennel and parsley) elicited high levels of bait-feeding activity, indicating that these vegetables were not highly preferred . The plants tested from Liliaceae, Cucurbitaceae, Asteraceae, and Brassicaceae elicited an intermediate-to-low level of bait feeding.

Bull Mem Acad R Med Belg, 2003, 158(1-2), 121 - 30; discussion 130-1
{Is the prion abnormality going to involve immunology?}; Bussard A; The behaviour of prion type proteins, be they in mammals (Pr P) or in yeast (URE 3) suggests a non Mendelian heredity based on conformational changes in these proteins . This seems to be an anomaly in regard to molecular Biology (M.B.) which rules information transfer exclusively from DNA to protein with the use of a numeric code . This anomaly may affect M.B . in the future, but also may influence our views in Immunology . For the time being, clonal selection is the only accepted theory to explain the generation of diversity in antibodies; is it full proof?

Cell Cycle, 2003 Jul-Aug, 2(4), 277 - 8
Does a GATA factor make the bed for centromeric nucleosomes?
Chen ES, Yanagida M, Takahashi K.
CENP-A is an evolutionarily conserved, centromere-specific histone H3 variant . It remains a great mystery how CENP-A is correctly incorporated into the centromere, a restricted chromosomal region, despite the presence of an overwhelming amount of histone H3 . We identified a cell cycle-regulated GATA factor, Ams2, as a component of the CENP-A localization pathway in fission yeast . Unexpectedly, this putative transcription factor, which belongs to a protein family containing members that remodel nucleosomes, appeared to bind to and function at the central region of the centromere . Although the centromere has in general been considered transcriptionally inactive, fission yeast's outer centromeric region has recently been shown to encode non-translated snRNAs that are involved in heterochromatin formation . Transcription factors such as Ams2 may directly transcribe some unidentified non-translated centromeric RNAs . Transcription and/or remodeling of the nucleosomes at the centromeres may be important for the precise incorporation of CENP-A in fission yeast.

J Biol Chem, 2003 Oct 3, 278(40), 38847 - 59 Epub 2003 Jul 08.
Down-regulation of RNA helicase II/Gu results in the depletion of 18 and 28 S rRNAs in Xenopus oocyte; Yang H et al.; Genetic manipulations have revealed the functions of RNA helicases in ribosomal RNA (rRNA) biogenesis in yeast . However, no report shows the role of an RNA helicase in rRNA formation in higher eukaryotes . This study reports the functional characterization of the frog homologue of nucleolar RNA helicase II/Gu (xGu or DDX21) . Down-regulation of xGu in Xenopus laevis oocyte using an antisense oligodeoxynucleotide results in the depletion of 18 and 28 S rRNAs . The disappearance of 18 S rRNA is accompanied by an accumulation of 20 S, indicating that xGu is critical in the processing of 20 to 18 S rRNA . The degradation of 28 S rRNA into fragments smaller than 18 S is also associated with a specific decrease in the level of xGu protein . These effects are reversed in the presence of in vitro synthesized wild type xGu mRNA but not its helicase-deficient mutant form . Similar aberrant rRNA processing is observed when antibody against xGu is microinjected . The involvement of xGu in processing of rRNA is consistent with the localization of Gu protein to the granular and dense fibrillar components of PtK2 cell nucleoli by immunoelectron microscopy . Our results show that xGu is involved in the processing of 20 to 18 S rRNA and contributes to the stability of 28 S rRNA in Xenopus oocytes.

Biochim Biophys Acta, 2003 Jul 9, 1628(1), 56 - 63
Cloning and characterization of an eukaryotic initiation factor-2alpha kinase from the silkworm, Bombyx mori; Prasad MD et al.; Eukaryotic initiation factor 2alpha (eIF-2alpha) kinases are involved in the translational regulations that occur in response to various types of environmental stress, and play an important role in the cellular defense system operating under unfavorable conditions . The identification of additional eIF-2alpha kinases and the elucidation of their functions are necessary to understand how different eIF-2alpha kinases can specifically respond to distinct stimuli . Here, we report a novel eIF-2alpha kinase, termed BeK, from the silkworm, Bombyx mori . This gene encodes 579 amino acids and contains all 11 catalytic domains of protein-serine/threonine kinases . Most notably, it contains an "Ile-Gln-Met-Xaa-Xaa-Cys" motif, which is highly conserved from yeast to mammalian eIF-2alpha kinases . BeK does not show any significant homology in the NH(2) terminal regulatory domain, suggesting a distinct regulatory mechanism of this novel eIF-2alpha kinase . BeK is ubiquitously expressed in the various tissues throughout the final larval stage . Importantly, BeK is activated in Drosophila Schneider cells following heat shock and osmotic stress, and activated-BeK has been shown to phosphorylate an eIF-2alpha subunit at the Ser(50) site . However, other forms of stress, such as immune stress, endoplasmic reticulum stress and oxidative stress, cannot significantly elicit BeK activity . Interestingly, the baculovirus gene product, PK2, can inhibit BeK enzymatic activity, suggesting that BeK may be an endogenous target for a viral gene product . Taken together, these data indicate that BeK is a novel eIF-2alpha kinase involved in the stress response in B . mori.

Neural Netw, 2003 Jun-Jul, 16(5-6), 633 - 40
Adaptive double self-organizing maps for clustering gene expression profiles; Ressom H et al.; This paper introduces a new model of self-organizing map (SOM) known as adaptive double self-organizing map (ADSOM) . ADSOM has a flexible topology and performs data partitioning and cluster visualization simultaneously without requiring a priori knowledge about the number of clusters . It combines features of the popular SOM with two-dimensional position vectors, which serve as a visualization tool to detect the number of clusters present in the data . ADSOM updates its free parameters and allows convergence of its position vectors to a fairly consistent number of clusters provided its initial number of nodes is greater than the expected number of clusters . A novel index is introduced based on hierarchical clustering of the final locations of position vectors . The index allows automated detection of the number of clusters, thereby reducing human error that could be incurred from counting clusters visually . To test ADSOM's consistency in data partitioning, we examine the number of common profiles found in the clusters that were obtained by varying the initial number of nodes . This provides a confidence measure for the clusters formed by ADSOM and illustrates the effect of different initial number of nodes on data partitioning . The reliance of ADSOM in identifying number of clusters is demonstrated by applying it to publicly available yeast gene expression data.

Biochem Biophys Res Commun, 2003 Jul 18, 307(1), 86 - 91
The protein of a new gene, Tctex4, interacts with protein kinase CK2beta subunit and is highly expressed in mouse testis; Bai X et al.; Casein kinase 2 (CK2) is a ubiquitous, multifunctional eukaryotic serine/threonine kinase that phosphorylates an array of proteins . CK2 is a heterotetramer composed of two catalytic (alpha,alpha(')) and two regulatory (beta) subunits . CK2 plays an essential role in regulatory pathways in cell transformation and proliferation . But the role and function of the individual subunits of CK2, which are not in the holoenzyme, are not yet clear . Northern blot analysis reveals the highest CK2beta activity in mouse testicles and brain . By employing a yeast two-hybrid screen to identify the proteins that interact with CK2beta, we have isolated a cDNA clone encoding a 14-kDa protein with homology to dynein light chains and have designated it as Tctex4 . CK2beta interacts specifically with Tctex4 both in a yeast two-hybrid system and in an in vitro interaction assay . Northern blot and in situ hybridization showed that Tctex4 is a novel gene that is expressed in mouse testis.

Reprod Toxicol, 2003 Jul-Aug, 17(4), 365 - 75
Mitochondrial benzodiazepine receptors regulate oxygen homeostasis in the early mouse embryo; O'Hara MF et al.; The peripheral benzodiazepine receptor (Bzrp) has been implicated in the control of several processes, including mitochondrial biogenesis and embryo development . The present study examined the impact that specific Bzrp ligands have on oxygen homeostasis in the early mouse embryo . Day 9 embryos at the 16-18 somite pair stage were exposed to standard (21% oxygen) and suboptimal (5% oxygen) oxygen tensions in whole embryo culture . Analysis of gene expression used relative PCR to monitor changes in nuclear respiratory factor-1 (Nrf1), mitochondrial 16S ribosomal RNA (16S rRNA), and genes for several glycolytic enzymes . Ocular development was highly sensitive to periods of hypoxia through a mechanism blocked with the potent Bzrp ligand PK11195 . Hypoxia led to a decline of Nrf1 and 16S rRNA levels also through a mechanism blocked with PK11195 . Similar activity was observed for FGIN-1-27 whereas Ro5-4864 had contradictory effects . Morpholino-based gene knockdown of Nrf1 (anti-NRF1) produced a sequence-specific decrease in 16S rRNA insensitive to PK11195 . These functional relationships suggest that Bzrp-dependent signals regulate the Nrf1 --> Tfam1 --> mtDNA --> 16S rRNA pathway in response to oxygen levels . The activity of PK11195 most likely has a pharmacodynamic basis with regards to specific embryonic precursor target cell populations, transducing a mitochondrial signal to an Nrf1 response analogous to retrograde regulation in yeast for mitochondria-to-nucleus signaling.

Ann N Y Acad Sci, 2003 Jun, 991, 101 - 6
Parkin and endoplasmic reticulum stress; Takahashi R et al.; Autosomal-recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkin gene . Parkin protein is characterized by a ubiquitin-like domain at its NH(2) terminus and by two RING finger motifs and one IBR (in between RING finger) motif at its COOH-terminus (RING-IBR-RING) . We showed that the parkin protein is an E3 ubiquitin ligase, which binds to ubiquitin-conjugating enzymes (E2s) through its RING-IBR-RING motif . The pathogenesis of AR-JP, therefore, was hypothesized to be accumulation of unidentified neurotoxic protein (a substrate of parkin) . On the basis of this hypothesis, the substrate of parkin was sought using a yeast two-hybrid system . A putative G protein-coupled transmembrane polypeptide, named Pael (parkin-associated endothelin receptor-like) receptor, was identified as a parkin binding protein . When overexpressed in cells, this receptor tends to become unfolded, insoluble, and ubiquitinated . The insoluble Pael receptor leads to endoplasmic reticulum (ER) stress-induced cell death . Parkin specifically ubiquitinates this receptor in the presence of ER-resident E2s and promotes the degradation of unfolded Pael receptor, resulting in suppression of the cell death induced by the accumulation of unfolded Pael receptor in the ER . Moreover, the insoluble form of Pael receptor accumulates in the brain of AR-JP patients . This protein is highly expressed in the dopaminergic neurons in the substantia nigra, which is specifically affected in Parkinson's disease; although it is also widely expressed in oligodendroglias in the fiber tract . In conclusion, we showed that the accumulation of unfolded Pael receptor (a substrate of parkin) may cause selective death of dopaminergic neurons in AR-JP.

Br J Haematol, 2003 Jul, 122(2), 333 - 40
The Rh complex exports ammonium from human red blood cells; Hemker MB et al.; The Rh blood group system represents a major immunodominant protein complex on red blood cells (RBC) . Recently, the Rh homologues RhAG and RhCG were shown to promote ammonium ion transport in yeast . In this study, we showed that also in RBC the human Rh complex functions as an exporter of ammonium ions . We measured ammonium import during the incubation of RBC in a solution containing a radiolabelled analogue of NH4Cl (14C-methyl-NH3Cl) . Rhnull cells of the regulator type (expressing no Rh complex proteins) accumulated significantly higher levels (P = 0.05) of radiolabelled methyl-ammonium ions than normal RBC, at room temperature . Rhnull cells of the amorph type (expressing limited amounts of Rh complex proteins) accumulated an intermediate amount of methyl-ammonium ions . To show that decreased ammonium export contributes to its accumulation, the release of intracellular methyl-ammonium from the cells was measured over time . In 30 s, normal RBC released 87% of the intracellular methyl-ammonium ions, whereas Rhnull cells of the regulator type released only 46% . We conclude that the Rh complex is involved in the export of ammonium from RBC.

Immunol Rev, 2003 Aug, 194, 29 - 38
The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response; Iwakoshi NN et al.; X-box binding protein-1 (XBP-1) is a transcription factor essential for plasma cell differentiation . XBP-1 transcripts are found at high levels in plasma cells from rheumatoid synovium and myeloma cell lines . Lymphoid chimeras deficient in XBP-1 have a profound defect in plasma cell differentiation, with few plasma cells in their periphery and severely reduced serum immunoglobulin levels . When introduced into B-lineage cells, XBP-1 initiates plasma cell differentiation . XBP-1 is also the mammalian homologue of the yeast transcription factor Hac1p, an important component of the unfolded protein response (UPR) . The UPR allows cells to tolerate conditions of endoplasmic reticulum (ER) stress caused by misfolded proteins . Studies examining the relationship between plasma cell differentiation, XBP-1, and the UPR demonstrate that this novel signaling system is vital for plasma cell differentiation . Signals that induce plasma cell differentiation and the UPR cooperate via XBP-1 to induce terminal B-cell differentiation . Additionally, XBP-1 plays an important role in the regulation of interleukin-6 production, a cytokine essential for plasma cell survival.

Biochemistry, 2003 Jul 15, 42(27), 8298 - 306
Reverse protonation is the key to general acid-base catalysis in enolase; Sims PA et al.; The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs . Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H(2)O) at different pHs . E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a (1)H/(2)H exchange reaction at different pDs . Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 A resolution, respectively . Analysis of the pH profile of k(cat)/K(Mg) for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1 . These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acid/base catalytic groups in the active site . The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH(2), Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH(3)(+), Glu211-COO(-)) . These two forms of the enzyme coexist in a constant, pH-independent ratio . The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein . Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction.

Yeast, 2003 Jul 15, 20(9), 771 - 80
The effect of under- and overexpressed CoEXG1-encoded exoglucanase secreted by Candida oleophila on the biocontrol of Penicillium digitatum; Yehuda H et al.; The yeast, Candida oleophila, is acknowledged for its biocontrol activity against postharvest moulds . However, the mechanism of this activity is not fully understood . One of the conjectured modes of action is associated with extracellular lytic enzymes, such as beta-exoglucanase . The relationship of beta-exoglucanase in the biocontrol activity of C . oleophila was investigated by generating C . oleophila CoEXG1-knockouts and double-CoEXG1 transformants . The knockout transformants secreted 0-13% of the exoglucanase activity detected in the medium of the untransformed yeast (depending on the medium), indicating that CoEXG1 is the main gene responsible for the production of the secreted exoglucanase . Correspondingly, the double-CoEXG1 transformants secreted approximately twice as much 1,3-beta-exoglucanase as the untransformed C . oleophila . The biocontrol activity of the CoEXG1-knockout and the double-CoEXG1 transformants against Penicillium digitatum did not differ from that of the untransformed C . oleophila on kumquats . These results imply that the 1,3-beta-exoglucanase encoded by the gene CoEXG1 is not involved in the biocontrol activity of C . oleophila against P . digitatum under these experimental terms . However, these findings do not rule out the possibilities, that the participation of CoEXG1 in biocontrol is dependent on the activity of other gene products, or that its effect may be manifested under altered environmental conditions .

Sci Aging Knowledge Environ . 2003 Feb 12;2003(6):RE1.
Subfield history: use of model organisms in the search for human aging genes; Warner HR; The National Institute on Aging (NIA) started a program in 1993 to identify genes involved in the regulation of longevity in a variety of species, including yeast, nematodes, fruit flies, and mice . The initial success of this program has attracted the interest of many investigators working with these organisms . Of primary interest are single-gene mutants that have identified genes and processes involved in longevity regulation across species . These processes include the insulin-like signaling pathway, stress resistance, and most recently, chromosome and nuclear architecture . Mutations in genes that regulate these processes indirectly are also being identified in this program . The ultimate goal of this program is to extend these results to humans to identify the major biological risk factors for age-related decline of function in human physiological systems.

Carcinogenesis, 2003 Sep, 24(9), 1541 - 8 Epub 2003 Jul 04.
Chemopreventive n-3 fatty acids activate RXRalpha in colonocytes; Fan YY et al.; The underlying mechanisms by which n-3 polyunsaturated fatty acids (PUFA) exert a chemopreventive effect in the colon have not been elucidated . Retinoid X receptors (RXR) are a family of nuclear receptors implicated in cancer chemoprevention . Since docosahexaenoic acid (DHA), an n-3 PUFA enriched in fish oil, reduces colonocyte proliferation and enhances apoptosis relative to n-6 PUFA-treated cells, we determined whether DHA can serve as a specific ligand for RXRalpha activation relative to n-6 PUFA in colonocytes . In a mammalian one-hybrid assay, immortalized young adult mouse colonic (YAMC) cells were co-transfected with a yeast galactose upstream activating sequence (UAS)4-tk-Luciferase (Luc) reporter plasmid, plus either GAL4 DNA-binding domain fused to RXRalpha, retinoic acid receptor alpha or GAL4 alone, followed by an n-3, n-6 or n-9 fatty acid incubation . Luc activity levels were dose-dependently elevated only in n-3 PUFA (DHA)-treated RXRalpha . Since RXR homodimers and RXR/peroxisome proliferator-activated receptor (PPAR) heterodimers bind consensus direct repeat (DR1) motifs, YAMC and NCM460 (a normal human colonic cell line), were respectively, co-transfected with RXRalpha and DR1-Luc, followed by different PUFA treatment . Luc activity levels were increased (P < 0.05) only in DHA groups . The DHA-dependent induction of DR-1-Luc was reduced to basal levels upon RXRalpha antagonist-treatment, with no effect on PPARgamma antagonist-treatment . A role for select RXR isoforms in colonocyte biology was also determined by examining nuclear receptor mRNA levels in rat colon following dietary lipid and carcinogen exposure over time . RXRalpha, RXRbeta and RXRgamma were detected in rat colonic mucosa, and the levels of RXRalpha and RXRgamma were elevated in fish oil (n-3 PUFA) versus corn oil (n-6 PUFA) fed animals after 16 weeks . These data indicate that, RXRalpha, an obligatory component of various nuclear receptors, preferentially binds n-3 PUFA in colonocytes, and that the nuclear receptor targets for PUFA in the colon are modulated by dietary lipid exposure.

Chem Pharm Bull (Tokyo), 2003 Jul, 51(7), 794 - 7
Phenyl ethers from cultured lichen mycobionts of Graphis scripta var . serpentina and G . rikuzensis; Takenaka Y et al.; Spore-derived mycobionts of the lichen Graphis scripta var . serpentina and G . rikuzensis were cultivated on a malt-yeast extract medium supplemented with 10% sucrose and their metabolites were investigated . 3,3'-Dihydroxy-5,5'-dimethyldiphenyl ether was isolated from the cultures of the mycobionts of G . scripta var . serpentina, while a new phenyl ether, rikuzenol, along with two known diphenyl ethers, violaceol-I and violaceol-II, were isolated from those of G . rikuzensis . The structure of the new compound was determined by spectroscopic methods . Violaceol-I was chemically synthesized and interconversion between violaceol-I and violaceol-II was proven.

J Clin Microbiol, 2003 Jul, 41(7), 3423 - 6
Isolation of Candida dubliniensis in an aboriginal community in Ontario, Canada; Montour L et al.; This study reports the first isolation of Candida dubliniensis from North American Indians . Of 39 healthy human hosts sampled, two had C . dubliniensis . Genotypic analysis identified polymorphisms in these strains and differences from two reference strains . Our results suggest that yeast populations from indigenous communities in North America may be unique.

J Clin Microbiol, 2003 Jul, 41(7), 3022 - 7
Sequence diversity of the intergenic spacer region of the rRNA gene of Malassezia globosa colonizing the skin of patients with atopic dermatitis and healthy individuals; Sugita T et al.; The lipophilic yeast Malassezia globosa is one of the major constituents of the mycoflora of the skin of patients with atopic dermatitis (AD) . We compared the genotypes of M . globosa colonizing the skin surface of 32 AD patients and 20 healthy individuals for polymorphism of the intergenic spacer (IGS) 1 region of the rRNA gene . Sequence analysis demonstrated that M . globosa was divided into four major groups, which corresponded to the sources of the samples, on the phylogenetic tree . Of the four groups, two were from AD patients and one was from healthy subjects . The remaining group included samples from both AD patients and healthy subjects . In addition, the IGS 1 region of M . globosa contained short sequence repeats: (CT)(n), and (GT)(n) . The number of sequence repeats also differed between the IGS 1 of M . globosa from AD patients and that from healthy subjects . These findings suggest that a specific genotype of M . globosa may play a significant role in AD, although M . globosa commonly colonizes both AD patients and healthy subjects.

Genome Res, 2003 Jul, 13(7), 1638 - 45
Divergence in the spatial pattern of gene expression between human duplicate genes; Makova KD et al.; Microarray gene expression data provide a wealth of information for elucidating the mode and tempo of molecular evolution . In the present study,we analyze the spatial expression pattern of human duplicate gene pairs by using oligonucleotide microarray data,and study the relationship between coding sequence divergence and expression divergence . First,we find a strong positive correlation between the proportion of duplicate gene pairs with divergent expression (as presence or absence of expression in a tissue) and both synonymous (K(S)) and nonsynonymous divergence (K(A)) . The divergence of gene expression between human duplicate genes is rapid, probably faster than that between yeast duplicates in terms of generations . Second,we compute the correlation coefficient (R) between the expression levels of duplicate genes in different tissues and find a significant negative correlation between R and K(S) . There is also a negative correlation between R and K(A), when K(A) <or= 0.2 . These results indicate that protein sequence divergence and divergence of spatial expression pattern are initially coupled . Finally,we compare the functions of those duplicate genes that show rapid divergence in spatial expression pattern with the functions of those duplicate genes that show no or little divergence in spatial expression.

Appl Environ Microbiol, 2003 Jul, 69(7), 3758 - 66
Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics; Gomes NC et al.; The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data . Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years . A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing . A rhizosphere effect was observed for fungal populations at all stages of plant development . In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE . Similar types of fingerprints were observed in two consecutive growth periods . No major differences were detected in the fungal patterns of the two cultivars . Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands . The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced . The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands . Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.

Appl Environ Microbiol, 2003 Jul, 69(7), 3710 - 8
Purification and characterization of a novel erythrose reductase from Candida magnoliae; Lee JK et al.; Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol . Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics . In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis . The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric . Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases . C . magnoliae erythrose reductase catalyzes the reduction of various aldehydes . Among aldoses, erythrose was the preferred substrate (K(m) = 7.9 mM; k(cat)/K(m) = 0.73 mM(-1) s(-1)) . This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k(cat)/K(m) = 450 mM(-1) s(-1)) than with NADPH (k(cat)/K(m) = 5.5 mM(-1) s(-1)), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily . Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism . The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu(2+) and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase . These data indicate that the C . magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.

Genes Cells, 2003 Jul, 8(7), 631 - 44
Cloning and characterization of mCRIP2, a mouse LIM-only protein that interacts with PDZ domain IV of PTP-BL; van Ham M et al.; BACKGROUND: In the mouse submembranous protein tyrosine phosphatase PTP-BL five PDZ domains are present in between the N-terminal FERM domain, which directs the protein to the cell cortex, and the C-terminal catalytic phosphatase domain . To understand more on the physical role of PTP-BL in this microenvironment, we started to search for PTP-BL PDZ domain-interacting proteins . RESULTS: Yeast two-hybrid screening for PTP-BL targets resulted in the identification of a novel mouse LIM-only protein termed CRIP2 that is highly homologous to rat ESP1 and human CRP2 sequences . Mouse CRIP2 has a predicted molecular weight of 23 kD and consists of two LIM domains spaced by 68 amino acids . The fourth PDZ domain of PTP-BL is responsible for the binding of CRIP2 protein . Both PTP-BL and CRIP2 mRNAs display a wide, overlapping tissue distribution . Western blot analysis revealed a more restricted expression pattern for CRIP2 with high expression in lung, heart and brain . CRIP2 protein is localized at cell cortical, actin-rich structures, which is concurrent with the subcellular localization of PTP-BL . CONCLUSIONS: The observed characteristics of the LIM domain-containing adaptor protein CRIP2 are consistent with a potential role of PTP-BL in the dynamics of the cortical actin cytoskeleton.

Genes Cells, 2003 Jul, 8(7), 573 - 86
High dosage Rhp51 suppression of the MMS sensitivity of DNA structure checkpoint mutants reveals a relationship between Crb2 and Rhp51; Smeets MF et al.; BACKGROUND: In eukaryotic cells DNA structure checkpoints organize the cellular responses of DNA repair and transient cell cycle arrest and thereby ensure genomic stability . To investigate the exact role of crb2+ in the DNA damage checkpoint response, a genetic screen was carried out in order to identify suppressors of the conditional MMS sensitivity of a crb2-1 mutant . Here we report the isolation of rhp51+ as a multicopy suppressor . RESULTS: We show that suppression is not specific for the checkpoint mutant while it is specific for the MMS treatment . Rescue by rhp51+ over-expression is not a consequence of increased recombination repair or checkpoint compensation and epistasis analysis confirms that crb2+ and rhp51+ function in different pathways . A tight linkage between the two pathways is nevertheless suggested by the complementary expression or modification of Crb2 and Rhp51 proteins . Crb2 protein stability is down-regulated when Rhp51 is over-expressed and up-regulated in the absence of Rhp51 . The up-regulation of Crb2 is independent of the activation of DNA structure checkpoints . Conversely Rhp51 is more readily activated and differentially modified in the absence of Crb2 or other checkpoint proteins . CONCLUSIONS: We conclude that fission yeast Crb2 and Rhp51 function in two parallel, tightly connected and coordinately regulated pathways.

Cell Mol Biol (Noisy-le-grand), 2003 Feb, 49(1), 13 - 21
Claudin-8 interacts with multi-PDZ domain protein 1 (MUPP1) and reduces paracellular conductance in epithelial cells; Jeansonne B et al.; The claudin family is a set of integral membrane proteins found at cell-cell interactions in tight junctions . To identify proteins that interact with claudin-8, we used the yeast two-hybrid system to search for binding partners . Using the C-terminal 37 amino acids of claudin-8 as bait, we screened a human kidney cDNA library and identified multi-PDZ domain protein 1 (MUPP1) as a claudin-8 binding protein . MUPP1 contains 13 PDZ domains and binds to claudin-8 though its PDZ9 domain . When MDCK cells were transfected with epitope-tagged claudin-8 or MUPP1, both molecules were concentrated at cell-cell junctions . The interaction of claudin-8 and MUPP1 in vivo was confirmed by co-immunolocalization and co-immunoprecipitation in MDCK cells . Expression of claudin-8-myc increased transepithelial electrical resistance (TER) and reduced paracellular flux using FITC-dextran as a tracer . Over-expression of FLAG-MUPP1 in MDCK cells also reduced the epithelial paracelhular conductance . Our results indicate that claudin-8 and MUPP1 interact in tight junctions of epithelial cells and are involved in the tight junction barrier function.

J Bacteriol, 2003 Jul, 185(14), 4110 - 8
Structure of a coenzyme A pyrophosphatase from Deinococcus radiodurans: a member of the Nudix family; Kang LW et al.; Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA) . Nudix enzymes with the same specificity have been found in yeast, humans, and mice . The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily . The structure of the complex of the enzyme with Mg(2+), its activating cation, reveals the position of the catalytic site . A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding . Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.

J Biol Chem, 2003 Sep 26, 278(39), 37545 - 52 Epub 2003 Jul 01.
The adenosine A2A receptor interacts with the actin-binding protein alpha-actinin; Burgueno J et al.; Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling . Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR) . Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue . A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface . In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization . A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist . Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor . These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.

J Biol Chem, 2003 Sep 19, 278(38), 36513 - 21 Epub 2003 Jul 01.
Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2; Arias DA et al.; CCR5 is a G protein-coupled receptor for RANTES, MIP-1alpha, MIP-1beta, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection . To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis . Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells . This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4 . The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity . Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT . The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased . The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar . These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling . CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not . The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases.

Hum Mol Genet, 2003 Jul 15, 12(14), 1713 - 23
Myotubularin-related 2 protein phosphatase and neurofilament light chain protein, both mutated in CMT neuropathies, interact in peripheral nerve; Previtali SC et al.; Charcot-Marie-Tooth disease type 4B1, CMT4B1, is a severe, autosomal-recessive, demyelinating peripheral neuropathy, due to mutations in the Myotubularin-related 2 gene, MTMR2 . MTMR2 is widely expressed and encodes a phosphatase whose substrates include phosphoinositides . However, this does not explain how MTMR2 mutants specifically produce demyelination in the peripheral nerve . Therefore, we analysed the cellular and subcellular distribution of Mtmr2 in nerve . Mtmr2 was detected in all cytoplasmic compartments of myelin-forming Schwann cells, as well as in the cytoplasm of non-myelin-forming Schwann cells and both sensory and motorneurons . In contrast, Mtmr2 was detected in the nucleus of Schwann cells and motorneurons, but not in the nucleus of sensory neurons . As Mtmr2 is diffusely present also within the nerve, a specific function could derive instead from nerve-specific interacting proteins . Therefore, we performed two yeast two-hybrid screenings, using either fetal brain or peripheral nerve cDNA libraries . The neurofilament light chain protein, NF-L, was identified repeatedly in both screenings, and found to interact with MTMR2 in both Schwann cells and neurons . Interestingly, NF-L, encoding NF-L, is mutated in CMT2E . These data may provide a basis for the nerve-specific pathogenesis of CMT4B1, and further support for the notion that hereditary demyelinating and axonal neuropathies may represent different clinical manifestations of a common pathological mechanism.

Am J Physiol Renal Physiol, 2003 Oct, 285(4), F784 - 91 Epub 2003 Jul 01.
Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells; Pribanic S et al.; An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells . Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1 . Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the NH2-terminal part of NaPi-IIa . We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys . Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders . As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments . No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets) . In agreement, no change in the amount of MAP17 mRNA was observed . Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1 . In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the NH2 terminus of NaPi-IIa within the PDZK1/NaPi-IIa/MAP17 complex, and 3) acts as an apical anchoring site for PDZK1.

J Struct Funct Genomics, 2003, 3(1-4), 27 - 34
Detection of gene duplications and block duplications in eukaryotic genomes; Li WH et al.; Several eukaryotic genomes have been completely sequenced and this provides an opportunity to investigate the extent and characteristics (e.g., single gene duplication, block duplication, etc.) of gene duplication in a genome . Detecting duplicate genes in a genome, however, is not a simple problem because of several complications such as domain shuffling, the existence of isoforms derived from alternative splicing, and annotational errors in the databases . We describe a method for overcoming these difficulties and the extents of gene duplication in the genomes of Drosophila melanogaster, Caenorhabditis elegans, and yeast inferred from this method . We also describe a method for detecting block duplications in a genome . Application of this method showed that block duplication is a common phenomenon in both yeast and nematode . The patterns of block duplication in the two species are, however, markedly different . Yeast shows much more extensive block duplication than nematode, with some chromosomes having more than 40% of the duplications derived from block duplications . Moreover, in yeast the majority of block duplications occurred between chromosomes, while in nematode most block duplications occurred within chromosomes.

Med Sci (Paris), 2003 Mar, 19(3), 299 - 307
{Triggering cell mitosis in higher eukaryotes}; Doree M; Dramatic changes of cell organisation occur at onset of mitosis . Genetic analysis of fission yeast and physiological studies of vertebrate and invertebrate oocytes showed that activation of cyclin B-cdc2 kinase triggers mitosis . Nevertheless, upstream mechanisms responsible for this activation remain largely unknown in somatic cells of higher eukaryotes . This review discusses possible pathways and mechanisms involved in triggering onset of mitosis in such cells, including inhibitory checkpoint mechanisms that detect defects in structural organisation of the cell.

Nucleic Acids Res Suppl, 2001, (1), 273 - 4
Structure of the DNA-binding domain of human telomeric protein, TRF1 and its interaction with telomeric DNA; Nishikawa T et al.; TRF1, a key player in regulation of the telomere length, is a double-stranded telomeric DNA binding factor in vertebrate . The DNA-binding domain of TRF1 shows a sequence similarity to each of the tandem repeat in the DNA-binding domain of the c-Myb protein . Here, the solution structure of the DNA-binding domain of human TRF1 has been determined by NMR . It consists of three helices, and its topological arrangement is very close to that of each c-Myb repeat and also to that of each subdomain of the DNA-binding domain in yeast telomeric protein, Rap1p . The interaction with DNA has been investigated by chemical shift perturbations . The result suggests that TRF1 recognizes the sequence centered on AGGGTTA mainly with its N-terminal arm, N-terminal portion of second helix and both ends of third helix.

Nucleic Acids Res Suppl, 2001, (1), 179 - 80
Control of tRNA function by antisense PNA derivatives; Ninomiya K et al.; The interaction of antisense peptide nucleic acid (PNA) with yeast tRNA(Phe) was investigated . A 6-mer PNA complementary to the 3'-terminal sequence including the 73ACCA end hybridized to the tRNA . While the PNA with a single mismatch did not . PNA is a promising candidate for controlling tRNA functions by the sequence-specific hybridization.

Mech Dev, 2003 Jun, 120(6), 681 - 9
Trl-GAGA directly interacts with lola like and both are part of the repressive complex of Polycomb group of genes; Mishra K et al.; Epigenetic inheritance to maintain the expression state of the genome is essential during development . In Drosophila, the cis regulatory elements, called the Polycomb Response Elements (PREs) function to mark the epigenetic cellular memory of the corresponding genomic region with the help of PcG and trxG proteins . While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins . The observations that some proteins may function both as PcG and trxG member and that both these group of proteins act upon common cis elements indicate at least a partial functional overlap among these proteins . Trl-GAGA was initially identified as a trxG member but later was shown to be essential for PcG function on several PREs . In order to understand how Trl-GAGA functions in PcG context, we have looked for the interactors of this protein . We identified lola like, aka batman, as a strong interactor of GAGA factor in a yeast two-hybrid screen . lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene . These observations suggest a possible mechanism of how Trl-GAGA plays a role in maintaining the repressed state of target genes involving lolal, which may function as a mediator to recruit PcG complexes.

Biochemistry, 2003 Jul 8, 42(26), 8085 - 93
Influence of cytosolic AGS3 on receptor--G protein coupling; Ma H et al.; Activator of G protein signaling 3 (AGS3) activates the Gbetagamma mating pathway in yeast in a manner that is independent of heptahelical receptors . It competes with Gbetagamma subunits to bind GDP-bound Gi/o(alpha) subunits via four repeated G protein regulatory (GPR) domains in the carboxyl-terminal half of the molecule . However, little is known about the functional role of AGS3 in cellular signaling . Here the effect of AGS3 on receptor-G protein coupling was examined in an Sf9 cell membrane-based reconstitution system . A GST-AGS3-GPR fusion protein containing the four individual AGS3-GPR domains inhibits receptor coupling to Galpha subunits as effectively as native AGS3 and more effectively than GST fusion proteins containing the individual AGS3-GPR domains . While none of the GPR domains distinguished among the three G(i)alpha subunits, both individual and full-length GPR domains interacted more weakly with G(o)alpha than with G(i)alpha . Cytosolic AGS3, but not membrane-associated AGS3, can interact with G(i)alpha subunits and disrupt their receptor coupling . Immunoblotting studies reveal that cytosolic AGS3 can remove G(i)alpha subunits from the membrane and sequester G(i)alpha subunits in the cytosol . These findings suggest that AGS3 may downregulate heterotrimeric G protein signaling by interfering with receptor coupling.

IEEE Trans Inf Technol Biomed, 2003 Jun, 7(2), 93 - 100
Database of repetitive elements in complete genomes and data mining using transcription factor binding sites; Horng JT et al.; Approximately 43% of the human genome is occupied by repetitive elements . Even more, around 51% of the rice genome is occupied by repetitive elements . The analysis presented here indicates that repetitive elements in complete genomes may have been very important in the evolutionary genomics . In this study, a database, called the Repeat Sequence Database, is first designed and implemented to store complete and comprehensive repetitive sequences . See for more information . The database contains direct, inverted and palindromic repetitive sequences, and each repetitive sequence has a variable length ranging from seven to many hundred nucleotides . The repetitive sequences in the database are explored using a mathematical algorithm to mine rules on how combinations of individual binding sites are distributed among repetitive sequences in the database . Combinations of transcription factor binding sites in the repetitive sequences are obtained and then data mining techniques are applied to mine association rules from these combinations . The discovered associations are further pruned to remove insignificant associations and obtain a set of associations . The mined association rules facilitate efforts to identify gene classes regulated by similar mechanisms and accurately predict regulatory elements . Experiments are performed on several genomes including C . elegans, human chromosome 22, and yeast.

Nat Biotechnol, 2003 Aug, 21(8), 885 - 90 Epub 2003 Jun 29.
Nanoparticles for the delivery of genes and drugs to human hepatocytes; Yamada T et al.; Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans . Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model . In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues . When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B . The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes . These advantages should facilitate targeted delivery of genes and drugs to the human liver.

Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1154 - 64 Epub 2003 Jun 27.
Structures of NAD(+)- and NADH-bound 1-l-myo-inositol 1-phosphate synthase; Jin X et al.; 1-l-myo-Inositol 1-phosphate synthase catalyzes the conversion of d-glucose 6-phosphate to 1-l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds . It involves an oxidation, an intramolecular aldol cyclization and a reduction . Here, the structure of the enzyme in its NAD(+)-bound, NADH-bound and apo forms is presented . These structures confirm that a significant portion of the active site is disordered in the absence of a small molecule, as none of the NAD(+)-bound forms of the enzyme have ordered active sites . On the other hand, the NADH-bound form contains two small molecules in the active site: a phosphate and glycerol . The entire active site is ordered in the presence of these two molecules, completely encapsulating them within the interior cavity . Significant changes in the structure of the active site are also seen, including repositioning of the nicotinamide ring and a motion of a loop region to accommodate the bound phosphate . These changes call into question the mechanism previously proposed for the enzyme . A comparison of the yeast and mycobacterial enzymes shows a surprisingly large change in the relative orientation of the catalytic and Rossmann-fold domains in the two enzymes.

J Neurosci, 2003 Jun 15, 23(12), 4975 - 83
Identification of a novel, membrane-associated neuronal kinase, cyclin-dependent kinase 5/p35-regulated kinase; Kesavapany S et al.; Here we characterize a novel neuronal kinase, cyclin-dependent kinase 5 (cdk5)/p35-regulated kinase (cprk) . Cprk is a member of a previously undescribed family of kinases that are predicted to contain two N-terminal membrane-spanning domains and a long C terminus, which harbors a dual-specificity serine/threonine/tyrosine kinase domain . Cprk was isolated in a yeast two-hybrid screen using the neuronal cdk5 activator p35 as "bait." Cprk interacts with p35 in the yeast-two hybrid system, binds to p35 in glutathione S-transferase fusion pull-down assays, and colocalizes with p35 in cultured neurons and transfected cells . In these cells, cprk is present with p35 in the Golgi apparatus . Cprk is expressed in a number of tissues but is enriched in brain and muscle and within the brain is found in a wide range of neuronal populations . Cprk displays catalytic activity in in vitro kinase assays and is itself phosphorylated by cdk5/p35 . Cdk5/p35 inhibits cprk activity . Cdk5/p35 may therefore regulate cprk function in the brain.

Mol Cell Biol, 2003 Jul, 23(14), 4859 - 69
MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent manner; Michels AA et al.; Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II . P-TEFb is essential for transcriptional elongation in human cells . A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome . In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA . We now report on a novel protein termed MAQ1 (for menage a quatre) that is also present in this complex . Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed . Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb . Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor . According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2 . Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.

Mol Cell Biol, 2003 Jul, 23(14), 4805 - 13
Rapid deadenylation triggered by a nonsense codon precedes decay of the RNA body in a mammalian cytoplasmic nonsense-mediated decay pathway; Chen CY et al.; Nonsense-mediated mRNA decay (NMD) is an RNA surveillance pathway that detects and destroys aberrant mRNAs containing nonsense or premature termination codons (PTCs) in a translation-dependent manner in eukaryotes . In yeast, the NMD pathway bypasses the deadenylation step and directly targets PTC-containing messages for decapping, followed by 5'-to-3' exonuclease digestion of the RNA body . In mammals, most PTC-containing mRNAs are subject to active nucleus-associated NMD . Here, using two distinct transcription-pulsing approaches to monitor mRNA deadenylation and decay kinetics, we demonstrate the existence of an active cytoplasmic NMD pathway in mammalian cells . In this pathway, a nonsense codon triggers accelerated deadenylation that precedes decay of the PTC-containing mRNA body . Transcript is stabilized when accelerated deadenylation is impeded by blocking translation initiation; by ectopically expressing two RNA-binding proteins, UNR and NSAP1; or by ectopically expressing a UPF1 dominant-negative mutant . These results are consistent with the notion that the nonsense codon can function in the cytoplasm by promoting rapid removal of the poly(A) tail as a necessary first step in the decay process.

J Biol Chem, 2003 Sep 19, 278(38), 36621 - 7 Epub 2003 Jun 28.
Binding surface mapping of intra- and interdomain interactions among hHR23B, ubiquitin, and polyubiquitin binding site 2 of S5a; Ryu KS et al.; hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein . However, hHR23B and RAD23 also have many important functions related to general proteolysis . hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains . The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin . On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome . We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation . Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops . This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B . The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated . The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B . The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.

J Biol Chem, 2003 Sep 12, 278(37), 35093 - 101 Epub 2003 Jun 27.
Tomosyn interacts with the t-SNAREs syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation; Widberg CH et al.; The Sec1p-like/Munc18 (SM) protein Munc18a binds to the neuronal t-SNARE Syntaxin1A and inhibits SNARE complex assembly . Tomosyn, a cytosolic Syntaxin1A-binding protein, is thought to regulate the interaction between Syntaxin1A and Munc18a, thus acting as a positive regulator of SNARE assembly . In the present study we have investigated the interaction between b-Tomosyn and the adipocyte SNARE complex involving Syntaxin4/SNAP23/VAMP-2 and the SM protein Munc18c, in vitro, and the potential involvement of Tomosyn in regulating the translocation of GLUT4 containing vesicles, in vivo . Tomosyn formed a high affinity ternary complex with Syntaxin4 and SNAP23 that was competitively inhibited by VAMP-2 . Using a yeast two-hybrid assay we demonstrate that the VAMP-2-like domain in Tomosyn facilitates the interaction with Syntaxin4 . Overexpression of Tomosyn in 3T3-L1 adipocytes inhibited the translocation of green fluorescent protein-GLUT4 to the plasma membrane . The SM protein Munc18c was shown to interact with the Syntaxin4 monomer, Syntaxin4 containing SNARE complexes, and the Syntaxin4/Tomosyn complex . These data suggest that Tomosyn and Munc18c operate at a similar stage of the Syntaxin4 SNARE assembly cycle, which likely primes Syntaxin4 for entry into the ternary SNARE complex.

Virology, 2003 Jun 20, 311(1), 60 - 71
The protein kinase pUL97 of human cytomegalovirus interacts with and phosphorylates the DNA polymerase processivity factor pUL44; Marschall M et al.; The protein kinase pUL97 of human cytomegalovirus plays important but incompletely defined roles in viral replication . Concerning the early phase of infection, it is postulated that pUL97 possesses regulatory functions in gene expression and/or DNA synthesis . Here we report that pUL97 interacts with an essential component of the replication complex, the DNA polymerase processivity factor pUL44 . Interaction was determined by yeast two-hybrid and coimmunoprecipitation analyses and was mapped to the pUL97 region 366-459 . In vitro kinase assays demonstrated that pUL44, coimmunoprecipitated either from transfected or from infected cells, is phosphorylated by pUL97 (but not by a catalytically inactive pUL97-mutant) . In infected fibroblasts, immunofluorescence analysis revealed that pUL97 and pUL44 accumulate in the nucleus and are both incorporated into viral replication centers . The treatment with inhibitors of DNA synthesis or pUL97 kinase activity largely prevented colocalization . Thus, pUL97 may be indirectly involved in viral genome replication by modifying the replication cofactor pUL44.

Curr Opin Struct Biol, 2003 Jun, 13(3), 383 - 8
Genome-wide studies of protein-protein interaction; Janin J et al.; Recent large-scale studies of protein complexes in yeast have demonstrated that the wide majority of proteins exist in the cell as parts of multicomponent assemblies, mostly novel and of unknown function . The structural and functional analysis of these complexes should be a priority for structural biologists in coming years . In silico methods such as docking simulations, which may contribute to this analysis, are being tested in the CAPRI community-wide experiment, which assesses blind predictions of the structure of protein-protein complexes.

J Virol, 2003 Jul, 77(14), 8019 - 30
The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus manipulates the activity of glycogen synthase kinase-3beta; Fujimuro M et al.; The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in all KSHV-associated malignancies . LANA is essential for replication and maintenance of the viral episomes during latent infection . However, LANA also has a transcriptional regulatory role and can affect gene expression both positively and negatively . A previously performed yeast two-hybrid screen identified glycogen synthase kinase 3 (GSK-3) as a LANA-interacting protein . Interaction with both GSK-3alpha and GSK-3beta was confirmed in transfected cells with coprecipitation assays . GSK-3beta also interacted with the herpesvirus saimiri homolog ORF73 . GSK-3beta is an intermediate in the Wnt signaling pathway and a negative regulator of beta-catenin . In transfected cells, LANA was shown to overcome GSK-3beta-mediated degradation of beta-catenin . Examination of primary effusion lymphoma (PEL) cells found increased levels of beta-catenin relative to KSHV-negative B cells, and this translated into increased activity of a beta-catenin-responsive reporter containing Tcf/Lef binding sites . In tetradecanoyl phorbol acetate-treated PEL cells, loss of LANA expression correlated temporally with loss of detectable beta-catenin . LANA was found to alter the intracellular distribution of GSK-3beta so that nuclear GSK-3beta was more readily detectable in the presence of LANA . Mapping experiments with coimmunoprecipitation assays revealed that both N-terminal and C-terminal LANA sequences were required for efficient GSK-3beta interaction . LANA mutants that were defective for GSK-3beta interaction were unable to mediate GSK-3beta relocalization or activate a beta-catenin-responsive Tcf-luciferase reporter . This study identified manipulation of GSK-3beta activity as a mechanism by which LANA may modify transcriptional activity and contribute to the phenotype of primary effusion lymphoma.

J Biol Chem, 2003 Sep 26, 278(39), 38040 - 50 Epub 2003 Jun 26.
A novel double-stranded RNA-binding protein, disco interacting protein 1 (DIP1), contributes to cell fate decisions during Drosophila development; DeSousa D et al.; We report the identification of the Disco Interacting Protein 1 (DIP1) gene isolated in a yeast interaction trap screen using the zinc finger protein disconnected (disco) as a bait . DIP1 encodes a protein containing two double-stranded RNA binding domains (dsRBD) . Consistent with the presence of dsRBD, DIP1 binds dsRNA or structured RNAs in Northwestern assays . DIP1 is found in nuclear subdomains resembling speckles known to accumulate transcription and splicing factors . In early embryos, nuclear localization of DIP1 protein coincides with the onset of zygotic gene expression . Later in development DIP1 expression is decreased in dividing cells in different tissues . Overexpression of DIP1 in the eye-antennal imaginal disc, early in embryonic and larval development, causes the formation of supernumerary structures in the head capsule . A role for DIP1 in epigenetic mechanisms that lead to the establishment and/or maintenance of cell fate specification is discussed.

J Biol Chem, 2003 Sep 12, 278(37), 35299 - 310 Epub 2003 Jun 26.
Depletion of GIM5 causes cellular fragility, a decreased glycosome number, and reduced levels of ether-linked phospholipids in trypanosomes; Voncken F et al.; Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins . The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial . PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei . The compartmentation of glycosomal enzymes is essential in trypanosomes . Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death . We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B . The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect . Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers . Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced . Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway . We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.

FEBS Lett, 2003 Jul 3, 546(1), 73 - 80
NDR family of AGC kinases--essential regulators of the cell cycle and morphogenesis; Tamaskovic R et al.; The nuclear Dbf2-related (NDR) family of protein serine/threonine kinases comprises mammalian NDR and large tumor suppressor (LATS) kinases, their orthologs from Drosophila melanogaster and Caenorhabditis elegans, and a number of related kinases from yeast and plants . The members of this family were independently implicated in various aspects of the control of cell division and morphogenesis . They are crucial regulators of the actin and tubulin cytoskeletal organization during polarized growth and cytokinesis in yeast . Furthermore, they are key players in control of proliferation and morphology of many cell types in D . melanogaster and C . elegans . In mammalians, the LATS kinase is a tumor suppressor, negatively regulating the cyclin-dependent kinase CDK1, cell proliferation rate, and modulating cell survival.

FEBS Lett, 2003 Jul 3, 546(1), 65 - 72
The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks; Bockaert J et al.; All cell types express a great variety of G protein-coupled receptors (GPCRs) that are coupled to only a limited set of G proteins . This disposition favors cross-talk between transduction pathways . However, GPCRs are organized into functional units . They promote specificity and thus avoid unsuitable cross-talk . New methodologies (mostly yeast two-hybrid screens and proteomics) have been used to discover more than 50 GPCR-associated proteins that are involved in building these units . In addition, these protein networks participate in the trafficking, targeting, signaling, fine-tuning and allosteric regulation of GPCRs . To date, proteins that interact with the GPCR C-terminus are the most abundant and are the focus of this review.

Arch Oral Biol, 2003 Aug, 48(8), 597 - 604
Intracellular localisation of SNARE proteins in rat parotid acinar cells: SNARE complexes on the apical plasma membrane; Imai A et al.; Intracellular localisation of soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) is an important factor in clarifying whether SNAREs regulate exocytosis in salivary glands . We investigated intracellular localisation of syntaxins 2, 3 and 4 and SNAP-23, which are thought to be target membrane (t)-SNAREs, in rat parotid gland by Western blotting and immunocytochemistry . Syntaxins 2 and 3 were localised in the apical plasma membrane (APM), and syntaxin 4 was localised in the plasma membrane . SNAP-23 was localised in the APM and intracellular membrane (ICM) . In a yeast two-hybrid assay, syntaxins 2, 3 and 4 interacted with SNAP-23 and VAMP-3 . Using immunoprecipitation methods, syntaxins 3 and 4 were seen to interact with VAMP-8 and SNAP-23 at the APM, respectively . SNAP-23 interacted with syntaxin 3, syntaxin 4, VAMP-2, VAMP-3 and VAMP-8 . Many SNARE complexes were detected under non-stimulated/basic conditions in the parotid APM . Some of these complexes may have a role in exocytosis from parotid acinar cells.

Mol Microbiol, 2003 Jul, 49(2), 319 - 29
SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development; Dessens JT et al.; An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall . For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion . After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components . Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP) . The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus . It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains . Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds . Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays . Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts . These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes.

Protein Eng, 2003 May, 16(5), 365 - 72
Complex chimeras to map ligand binding sites of GPCRs; Gearing KL et al.; Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices . In this study, receptor-ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors . Regions of P2Y(1), P2Y(2) and BLT(1) predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor . Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay . Binding of ligand to a P2Y(2)/BLT(1) chimera confirmed that the ligand binding determinants of BLT(1) are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.

Plant Cell Physiol, 2003 Jun, 44(6), 619 - 29
A Link between circadian-controlled bHLH factors and the APRR1/TOC1 quintet in Arabidopsis thaliana; Yamashino T et al.; APRR1 (ARABIDPSIS PSUEDO-RESPONSE REGULATOR 1) (or TOC1, TIMING OF CAB EXPRESSION 1) is believed to be a crucial component of biological clocks of Arabidopsis thaliana . Nevertheless, its molecular function remains to be fully elucidated . Based on the results of yeast two-hybrid and in vitro binding assays, we previously showed that APRR1/TOC1 interacts with certain bHLH factors (i.e . PIF3 and PIL1, which are PHYTOCHROME INTERACTING FACTOR 3 and its homolog (PIF3-LIKE 1), respectively) . To critically examine the relevance of PIL1 with reference to the function of APRR1/TOC1, T-DNA insertion mutants were isolated for PIL1 . No phenotype was observed for such homozygous pil1 mutants, in terms of circadian-associated events in plants . We then examined more extensively a certain set of bHLH factors, which are considerably similar to PIL1 in their structural designs . The results of extensive analyses of such bHLH factors (namely, HFR1, PIL2, PIF4, PIL5 and PIL6) in wild-type and APRR1-overexressing (APRR1-ox) transgenic lines provided us with several new insights into a link between APRR1/TOC1 and these bHLH factors . In yeast two-hybrid assays, APRR1/TOC1 showed the ability to interact with these proteins (except for HFR1), as well as PIL1 and PIF3 . Among them, it was found that the expressions of PIF4 and PIL6 were regulated in a circadian-dependent manner, exhibiting free-running robust rhythms . The expressions of PIF4 and PIL6 were regulated also by light in a manner that their transcripts were rapidly accumulated upon exposure of etiolated seedlings to light . The light-induced expressions of PIF4 and PIL6 were severely impaired in APRR1-ox transgenic lines . Taken together, here we propose the novel view that these bHLH factors (PIF4 and PIL6) might play roles, in concert with APRR1/TOC1, in the integration of light-signals to control both circadian and photomorphogenic processes.

Biol Reprod, 2003 Nov, 69(5), 1572 - 9 Epub 2003 Jun 25.
Binding and inactivation of the germ cell-specific protein phosphatase PP1gamma2 by sds22 during epididymal sperm maturation; Mishra S et al.; Testis- and sperm-specific protein phosphatase, PP1gamma2, is a key enzyme regulating sperm function . Its activity decreases during sperm maturation in the epididymis . Inhibition of PP1gamma2 leads to motility initiation and stimulation . Our laboratory is focused on identifying mechanisms responsible for the decline in PP1gamma2 activity during sperm motility initiation in the epididymis . Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1gamma2 in motile caudal spermatozoa (Huang Z, et al . Biol Reprod 2002; 67:1936-1942) . The objectives of this study were to determine: 1) stoichiometry of PP1gamma2-sds22 binding and 2) whether PP1gamma2 in immotile caput epididymal spermatozoa is bound to sds22 . The enzyme from caudal and caput sperm extracts was purified by column chromatography . Immunoreactive PP1gamma2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column . However, PP1gamma2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active . The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column . Caudal sperm sds22 and PP1gamma2 coeluted in the gradient fraction . In contrast, caput sperm sds22 and PP1gamma2 were separated in the flow-through and gradient fractions, respectively . Further purification through a Superose 6 column showed that PP1gamma2-sds22 complex from caudal sperm was 88 kDa in size . Caput sperm sds22 and PP1gamma2 eluted at 60 kDa and 39 kDa, respectively . SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP1gamma2 (39 kDa), suggesting a 1:1 complex between these two proteins . PP1gamma2 bound to sds22 in this complex was inactive . Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17) . This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP1gamma2 . Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP1gamma2 during epididymal sperm maturation.

J Infect Chemother, 2003 Jun, 9(2), 122 - 5
Isolation of Legionella anisa from multiple sites of a hospital water system: the eradication of Legionellacontamination; Yamamoto N et al.; For the prevention of nosocomial cases of legionellosis in the Ryukyu University Hospital neonatal wards, we examined nine shower units and a sink tap water unit for the presence of Legionella, over a 6-year period . We isolated Legionella-like organisms (LLO) from showerheads by culturing sediments from the water samples on buffered charcoal yeast extracts (BCYE) . We used DNA-DNA hybridization to determine that the organisms were L . anisa . A fingerprinting technique called random amplified polymorphism DNA (RAPD) also showed that all the organisms were identical at the genome level . Replacement of the shower heads harboring colonies of L . anisa prevented further contamination . Nosocomial cases of legionellosis have not been reported from the wards during the period of this survey . This is the first description of the isolation of L . anisa from multiple sites within a hospital, and RAPD analysis suggests that these may be the spread of a single clone.

Mol Microbiol, 2003 Jul, 49(1), 131 - 41
Glycerol dehydrogenase, encoded by gldB is essential for osmotolerance in Aspergillus nidulans; de Vries RP et al.; We have characterized the Aspergillus nidulans gldB gene encoding a NADP+-dependent glycerol dehydrogenase . A basal expression level was observed for gldB, which increased significantly under conditions of hyper-osmotic shock (1 M NaCl) . Growth of strains in which gldB was disrupted was severely reduced on plates containing 1% glucose and 1 M NaCl, but these strains were able to grow on plates containing 1 M NaCl and 1% glycerol, arabitol, mannitol or erythritol . Uptake of these polyols compensated for the inability of the gldB disruptants to produce glycerol . Presence of 1% glucose in these plates prevented growth restoration by all the polyols tested with the exemption of glycerol, indicating that uptake of mannitol, arabitol and erythritol is subject to glucose repression, whereas uptake of glycerol is significantly less or not repressed . No intracellular glycerol dehydrogenase activity could be detected in the gldB disruption strains . Intracellular glycerol levels in these strains were strongly decreased compared to wild type, whereas intracellular mannitol, erythritol and arabitol levels were increased . Conidia of the gldB disruption strain did not accumulate glycerol upon germination in glucose media with or without 1 M NaCl and germ tube emergence was significantly delayed in this strain in the presence of 1 M NaCl in comparison to the wild type . These data indicate that gldB is essential for osmotolerance in A . nidulans and that the pathways for glycerol biosynthesis under osmotic stress differ between yeast and filamentous fungi.

Mol Microbiol, 2003 Jul, 49(1), 117 - 30
A binuclear zinc transcription factor binds the host isoflavonoid-responsive element in a fungal cytochrome p450 gene responsible for detoxification; Khan R et al.; The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes pisatin demethylase, a cytochrome P450 . Pisatin is a fungistatic isoflavonoid produced by garden pea (Pisum sativum), a host for this fungus . Pisatin demethylase detoxifies pisatin and functions as a virulence factor for this fungus . Pisatin induces PDA1 expression both in cultured mycelia as well as during pathogenesis on pea . The regulatory element within PDA1 that provides pisatin-responsive expression was identified using a combination of in vivo functional analysis and in vitro binding analysis . The 40 bp pisatin-responsive element is located 635 bp upstream of the PDA1 transcription start site . This element was sufficient to provide strong pisatin-induced expression to a minimal promoter in vivo and was required for pisatin regulation of the PDA1 promoter . A gene encoding a DNA-binding protein specific to this 40 bp element was isolated from a N . haematococca cDNA library using the yeast one-hybrid screen . The cloned gene possesses sequence motifs found in the binuclear zinc (Cys 6-Zn 2) family of transcription factors unique to fungi . The results suggest that it is a regulator of this fungal cytochrome P450 gene and may provide pisatin-responsive regulation.

Exp Dermatol, 2003 Jun, 12(3), 268 - 77
Increased epidermal functioning wild-type p53 expression in vitiligo; Schallreuter KU et al.; Despite the lack of protective melanin and increased oxidative stress due to mM concentrations of epidermal H2O2 in vitiligo, there is no significantly increased risk for chronic actinic damage and non-melanoma skin cancer . Therefore the question arises, which protective mechanisms could be involved in the skin of these patients preventing the initiation of these cancers . Recently an overexpression of p53 has been shown in vitiligo . Unfortunately there was no further characterization of this elevated p53 . Employing a functional colour yeast assay, the study presented herein demonstrates for the first time the overexpression of a functioning wild-type p53 protein in both depigmented and 'normal' pigmented epidermis of patients with vitiligo compared with healthy controls . Surprisingly long-term narrowband UVB (311 nm) treatment does not alter this expression . Moreover, MDM-2, PCNA and p21 protein expression remain unchanged compared with healthy controls . This increased epidermal p53 in vitiligo coincides with decreased thioredoxin reductase (TR) protein levels in both depigmented and pigmented skin whereas mRNA expression is unaffected . Because TR is one transcriptional target of p53, these results support a wild-type functionality, which was further supported by the specific p53 FASAY yeast test . To our knowledge this is the first example of persistent elevated functioning wild-type p53 in humans . Based on our results we hypothesize that the low incidence for actinic damage, basal cell and squamous cell carcinoma as documented in vitiligo could well reside in a protective function of up-regulated wild-type p53.

J Biol Chem, 2003 Sep 5, 278(36), 33708 - 13 Epub 2003 Jun 23.
Calmodulin is a phospholipase C-beta interacting protein; McCullar JS et al.; Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways . Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function . A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM) . Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3 . Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site . Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates . CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity . There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.

J Biol Chem, 2003 Sep 5, 278(36), 34211 - 8 Epub 2003 Jun 23.
IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia; Baker SA et al.; Intraflagellar transport (IFT) is an evolutionarily conserved mechanism thought to be required for the assembly and maintenance of all eukaryotic cilia and flagella . Although IFT proteins are present in cells with sensory cilia, the organization of IFT protein complexes in those cells has not been analyzed . To determine whether the IFT complex is conserved in the sensory cilia of photo-receptors, we investigated protein interactions among four mammalian IFT proteins: IFT88/Polaris, IFT57/Hippi, IFT52/NGD5, and IFT20 . We demonstrate that IFT proteins extracted from bovine photoreceptor outer segments, a modified sensory cilium, co-fractionate at approximately 17 S, similar to IFT proteins extracted from mouse testis . Using antibodies to IFT88 and IFT57, we demonstrate that all four IFT proteins co-immunoprecipitate from lysates of mouse testis, kidney, and retina . We also extended our analysis to interactions outside of the IFT complex and demonstrate an ATP-regulated co-immunoprecipitation of heterotrimeric kinesin II with the IFT complex . The internal architecture of the IFT complex was investigated using the yeast two-hybrid system . IFT20 exhibited a strong interaction with IFT57/Hippi and the kinesin II subunit, KIF3B . Our data indicate that all four mammalian IFT proteins are part of a highly conserved complex in multiple ciliated cell types . Furthermore, IFT20 appears to bridge kinesin II with the IFT complex.

J Biol Chem, 2003 Sep 5, 278(36), 34725 - 32 Epub 2003 Jun 23.
Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes; Gao Z et al.; The plant hormone ethylene is perceived by a five-member family of receptors related to the bacterial histidine kinases . The Raf-like kinase CTR1 functions downstream of the ethylene receptors as a negative regulator of ethylene signal transduction . CTR1 is shown here to be associated with membranes of the endoplasmic reticulum in Arabidopsis as a result of its interactions with ethylene receptors . Membrane association of CTR1 is reduced by mutations that eliminate ethylene receptors and by a mutation in CTR1 that reduces its ability to bind to the ethylene receptor ETR1 . Direct evidence that CTR1 is part of an ethylene receptor signaling complex was obtained by co-purification of the ethylene receptor ETR1 with a tagged version of CTR1 from an Arabidopsis membrane extract . The histidine kinase activity of ETR1 is not required for its association with CTR1, based on co-purification of tagged ETR1 mutants and CTR1 after expression in a transgenic yeast system . These data demonstrate that CTR1 is part of an ethylene receptor signaling complex in Arabidopsis and support a model in which localization of CTR1 to the endoplasmic reticulum is necessary for its function . Additional data that demonstrate a post-transcriptional effect of ethylene upon the expression of CTR1 suggest that production of ethylene receptor signaling complexes may be coordinately regulated.

Biochem Biophys Res Commun, 2003 Jul 11, 306(4), 1106 - 11
A novel TBP-interacting zinc finger protein functions in early development of Xenopus laevis; Kim M et al.; A zinc finger protein that interacts with Xenopus TATA-binding protein was previously isolated by a yeast two-hybrid screen and found to serve as a transcriptional repressor . The gene was designated the negatively regulating zinc finger protein gene (NZFP) . Herein, NZFP was found to be expressed maternally . After gastrulation, the level of NZFP mRNA decreased significantly throughout the neurula stage . However, mRNA levels increased at stage 35 and then began to decrease at stage 48 . Eventually, no NZFP mRNA was observed in adult tissues except in the ovary . NZFP mRNA was detected in the animal hemisphere during gastrulation and observed in the neural ectoderm at the neurula stage . At the tailbud stage, NZFP was highly expressed in the head tissues such as brain, eyes, otic vesicles, lateral line placodes, and branchial arches, but weakly in somites . Depletion of NZFP in the embryos using RNA interference caused premature death at the gastrula stage or induced secondary partial axis after gastrulation . These results strongly suggest that NZFP is an essential transcription factor involved in the cell movement during gastrulation and the formation of the dorsal axis during early development in Xenopus.

Cell Stress Chaperones, 2003 Spring, 8(1), 93 - 104
Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans; David CL et al.; In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol . Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases . To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake . Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span . However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90 . Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system . Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA . Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background . It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions . Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant . These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.

J Biol Chem, 2003 Sep 5, 278(36), 34641 - 53 Epub 2003 Jun 19.
GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2; Zhao C et al.; Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk . To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait . This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases . The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro . The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42 . GC-GAP is predominantly expressed in the brain with low levels detected in other tissues . Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa . Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA . Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells . In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro . We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities . Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.

Infect Immun, 2003 Jul, 71(7), 4026 - 33
Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection; Morris-Jones R et al.; Melanin has been implicated in the pathogenesis of several important human fungal pathogens . Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells . S . schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically . However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins . Melanin particles extracted from S . schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles . Tissue from hamster testicles infected with S . schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive . Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles . These findings indicate that S . schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo . Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.

Infect Immun, 2003 Jul, 71(7), 4003 - 10
Host-adapted Borrelia burgdorferi in mice expresses OspA during inflammation; Crowley H et al.; Antibody responses to outer surface protein A (OspA) of Borrelia burgdorferi may occur during periods of arthritis late in the clinical course of untreated Lyme disease . These antibody responses are paradoxical, given the conclusive evidence demonstrating that B . burgdorferi transmitted to the mammalian host expresses little or no OspA . The parallel occurrence of OspA antibodies and arthritic episodes suggests that OspA expression is upregulated during infection with B . burgdorferi . We hypothesized that this was due to the inflammatory environment caused by the immune response to the spirochete . To test our hypothesis, we adapted an in vivo model that mimics the host-pathogen interaction . Dialysis chambers containing B . burgdorferi were implanted into the peritoneal cavities of mice in the presence or absence of zymosan, a yeast cell wall extract that induces inflammation . Spirochetes were harvested 2 days later, and OspA expression was assessed at the protein and transcription level by Western blotting and real-time reverse transcription-PCR, respectively . Flow cytometry was also utilized to evaluate OspA protein expression on individual spirochetes . B . burgdorferi maintained in an inflammatory in vivo environment show an increased OspA expression relative to B . burgdorferi kept under normal in vivo conditions . Furthermore, host-adapted B . burgdorferi with a low OspA phenotype upregulates OspA expression when transferred to an inflammatory in vivo environment . The results obtained by these techniques uniformly identify inflammation as a mediator of in vivo OspA expression in host-adapted B . burgdorferi, providing insights into the behavior of live spirochetes in the mammalian host.

Infect Immun, 2003 Jul, 71(7), 3787 - 93
Overexpression of interleukin-4 in lungs of mice impairs elimination of Histoplasma capsulatum; Gildea LA et al.; Protection against the pathogenic fungus Histoplasma capsulatum requires Th1 cytokines . Since interleukin-4 (IL-4) can inhibit both Th1 cytokine production and activity, we examined the effects of overproduction of IL-4 in the lung on the course of pulmonary histoplasmosis . IL-4 lung transgenic mice manifested a higher fungal burden in their lungs, but not spleens, compared to wild-type infected controls . Despite the higher burden, the transgenic animals were ultimately capable of controlling infection . The adverse effects of IL-4 on H . capsulatum elimination were not observed during the early phase of infection (days 1 to 3) but were maximal at day 7 postinfection, prior to the induction of cell-mediated immunity . Analysis of total body and lung cytokine levels revealed that gamma interferon and tumor necrosis factor alpha production were not inhibited in the presence of excess IL-4 . Our results with transgenic mice were supported by additional in vivo studies in which allergen induction of pulmonary IL-4 was associated with delayed clearance of H . capsulatum yeast and increased fungal burden . These findings demonstrate that excess production of endogenous IL-4 modulates protective immunity to H . capsulatum by delaying clearance of the organism but does not prevent the generation of a Th1 response that ultimately controls infection.

Am J Pathol, 2003 Jul, 163(1), 243 - 51
ASK1 associates with troponin T and induces troponin T phosphorylation and contractile dysfunction in cardiomyocytes; He X et al.; There is increasing support for the idea that excessive production of proinflammatory mediators such as tumor necrosis factor (TNF) and reactive oxygen species (ROS) contribute to the pathogenesis of cardiac dysfunction . However, the mechanisms by which cytokine/ROS production mediates cardiac dysfunction have not been established . Given that apoptosis signal-regulating kinase 1 (ASK1) is highly expressed in cardiac muscle and that ASK1 is an important mediator in the signaling pathways induced by tumor necrosis factor, interleukin-1, and ROS, we used the yeast two-hybrid system with ASK1 as bait to identify ASK1 substrates from a human heart cDNA library . The cDNA encoding the cardiac troponin T (cTnT) was isolated . ASK1 specifically interacted with cTnT, but not cTnI, in vitro and in vivo via the C-terminal ASK1 domain . ASK1 specifically phosphorylated cTnT in vitro and in vivo . Mutations in cTnT (T194/S198) at an ASK1-phosphorylation consensus sequence significantly reduced phosphorylation by ASK1 . ROS-induced ASK1 activation, cTnT phosphorylation, and contractile dysfunction in cardiomyocytes showed similar kinetics . Moreover, overexpression of constitutively active ASK1 induces cTnT phosphorylation and inhibits shortening and calcium transient in adult cardiomyocytes . We conclude that ASK1 plays an important role in regulation of cardiac contractile function by phosphorylating cTnT and may participate in cytokine/ROS-induced pathogenesis of cardiomyopathy and heart failure.

Biol Chem, 2003 May, 384(5), 811 - 5
Fabin, a novel calcyon-like and glucanase-like protein with mitogenic, antifungal and translation-inhibitory activities from broad beans; Ng TB et al.; A protein with an N-terminal sequence displaying similarities to N-terminal sequences of human calcyon and barley endo-1,4-glucanase, and to C-terminal sequences of human translation initiation factor 4 gamma and yeast superkiller viralicidic activity, was isolated from the broad bean Vicia faba . The protein, termed fabin, has a molecular mass of 34 kDa in SDS-polyacrylamide gel electrophoresis . Antifungal activity of the protein was observed against several fungal species including Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola . Fabin inhibits HIV-1 reverse transcriptase with an IC50 of 34 microM and translation in a rabbit reticulocyte lysate with an IC50 of 2.4 microM . At a concentration of about 1.5 microM fabin is able to elicit a 9-fold increase in the mitogenic response of murine splenocytes.

Biol Chem, 2003 May, 384(5), 689 - 95
The circadian clock of the unicellular eukaryotic model organism Chlamydomonas reinhardtii; Mittag M et al.; The green unicellular alga Chlamydomonas reinhardtii, also called 'green yeast', emerged in the past years as a model organism for specific scientific questions such as chloroplast biogenesis and function, the composition of the flagella including its basal apparatus, or the mechanism of the circadian clock . Sequencing of its chloroplast and mitochondrial genomes have already been completed and a first draft of its nuclear genome has also been released recently . In C . reinhardtii several circadian rhythms are physiologically well characterized, and one of them has even been shown to operate in outer space . Circadian expression patterns of nuclear and plastid genes have been studied . The mode of regulation of these genes occurs at the transcriptional level, although there is also evidence for posttranscriptional control . A clock-controlled, phylogenetically conserved RNA-binding protein was characterized in this alga, which interacts with several mRNAs that all contain a common cis-acting motif . Its function within the circadian system is currently under investigation . This review summarizes the current state of the knowledge about the circadian system in C . reinhardtii and points out its potential for future studies.

Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8589 - 94 Epub 2003 Jun 18.
A SNARE complex containing SGR3/AtVAM3 and ZIG/VTI11 in gravity-sensing cells is important for Arabidopsis shoot gravitropism; Yano D et al.; Plants can sense the direction of gravity and change the growth orientation of their organs . The molecular mechanisms of gravity sensing and signal transduction during gravitropism are not well known . We have isolated several shoot gravitropism (sgr) mutants of Arabidopsis . The sgr3-1 mutant exhibits a reduced gravitropic response in the inflorescence stems . In the inflorescence stems of Arabidopsis, gravity is sensed in endodermal cells that contain sedimentable amyloplasts . In sgr3-1, some amyloplasts in the endodermis failed to sediment in the direction of gravity . SGR3 encodes a syntaxin, AtVAM3, which had previously been cloned as a homologue of yeast Vam3p . AtVAM3 is localized to the prevacuolar compartment and vacuole and is suggested to function in vesicle transport to the vacuole . We have also cloned another soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), ZIG/AtVTI11, a mutation that causes abnormal gravitropism . This mutant displayed an abnormal distribution of amyloplasts in the endodermal cells similar to that in sgr3-1 . Endodermis-specific expression of SGR3 and ZIG by using the SCR promoter could complement the abnormal shoot gravitropism of each mutant . Protein-protein interaction between AtVAM3 and AtVTI11 in the endodermal cells was detected immunologically . The sgr3-1 mutation appeared to reduce the affinity of AtVAM3 for AtVTI11 or SYP5 . These results suggest that vesicle transport to the prevacuolar compartment/vacuole in the endodermal cells, mediated by a specific SNARE complex containing AtVAM3 and AtVTI11, plays an important role in shoot gravitropism.

Genes Dev, 2003 Jul 1, 17(13), 1630 - 45 Epub 2003 Jun 18.
A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein; Ng JM et al.; Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex . HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown . Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development . Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability . Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation . Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair . These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry.

Vet Microbiol, 2003 Jul 17, 94(3), 219 - 24
Diagnosis of histoplasmosis by detection of the internal transcribed spacer region of fungal rRNA gene from a paraffin-embedded skin sample from a dog in Japan; Ueda Y et al.; The lesions of histoplasmosis in dogs in Japan differ from those in dogs in North America . Affected dogs in Japan have had multiple granulomatous or ulcerated foci in skin or gingiva and have not had pulmonary or gastrointestinal lesions . The present report introduces a polymerase chain reaction (PCR) diagnosis of canine histoplasmosis and the characteristic of disease in Japan . The surgically removed skin ulcerate samples from a 5-years-old female Shiba-inu native to Japan without traveling out of the country were evaluated . Tissue samples had many yeast-like organisms in the macrophages . DNA was extracted from paraffin-embedded tissue samples . A nested PCR technique was applied . The detected sequence of the internal transcribed spacer of ribosomal RNA gene had 99.7% in homology with Ajellomyces capsulatus (the teleomorph of Histoplasma capsulatum) . Clinical manifestations, historical background of equine epizootic lymphangitis in Japan, and a human autochthonous case of histoplasmosis farciminosi indicated that this dog might have been infected with H . capsulatum var . farciminosum as a heteroecism.

Hum Mol Genet, 2003 Jul 1, 12(13), 1591 - 608
DISC1 (Disrupted-In-Schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation; Morris JA et al.; Disrupted-In-Schizophrenia 1 (DISC1) is a novel gene associated with schizophrenia by multiple genetic studies . In order to determine how mutations in DISC1 might cause susceptibility to schizophrenia, we undertook a comprehensive study of the cellular biology of DISC1 in its full-length and disease-associated mutant forms . DISC1 interacts by yeast two-hybrid, mammalian two-hybrid, and co-immunoprecipitation assays with multiple proteins of the centrosome and cytoskeletal system, including MIPT3, MAP1A and NUDEL; proteins which localize receptors to membranes, including alpha-actinin2 and beta4-spectrin; and proteins which transduce signals from membrane receptors, including ATF4 and ATF5 . Truncated mutant DISC1 fails to interact with ATF4, ATF5 or NUDEL . Deletion mapping demonstrated that DISC1 has distinct interaction domains: MAP1A interacts via its LC2 domain with the N-terminus of DISC1, whereas MIPT3 and NUDEL bind via their C-terminal domains to the central coiled-coil domain of DISC1, and ATF4/5 bind via their C-terminal domains to the C-terminus of DISC1 . In its full-length form, DISC1 protein localizes to predominantly perinuclear punctate structures which extend into neurites in some cells; mutant truncated DISC1, by contrast, is seen in a diffuse pattern throughout the cytoplasm and abundantly in neurites . Both forms co-localize with the centrosomal complex, although truncated less abundantly than full-length DISC1 . Although both full-length and mutant DISC1 are found in microtubule fractions, neither form of DISC1 appears to bind directly to microtubules, but rather do so in a MIPT3-dependent fashion that is stabilized by taxol . Based on these data, we propose that DISC1 is a multifunctional protein whose truncation contributes to schizophrenia susceptibility by disrupting intracellular transport, neurite architecture and/or neuronal migration, all of which have been hypothesized to be pathogenic in the schizophrenic brain.

Hum Mol Genet, 2003 Jul 1, 12(13), 1555 - 67
Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease; Slow EJ et al.; An expanded CAG repeat is the underlying genetic defect in Huntington disease, a disorder characterized by motor, psychiatric and cognitive deficits and striatal atrophy associated with neuronal loss . An accurate animal model of this disease is crucial for elucidation of the underlying natural history of the illness and also for testing experimental therapeutics . We established a new yeast artificial chromosome (YAC) mouse model of HD with the entire human HD gene containing 128 CAG repeats (YAC128) which develops motor abnormalities and age-dependent brain atrophy including cortical and striatal atrophy associated with striatal neuronal loss . YAC128 mice exhibit initial hyperactivity, followed by the onset of a motor deficit and finally hypokinesis . The motor deficit in the YAC128 mice is highly correlated with striatal neuronal loss, providing a structural correlate for the behavioral changes . The natural history of HD-related changes in the YAC128 mice has been defined, demonstrating the presence of huntingtin inclusions after the onset of behavior and neuropathological changes . The HD-related phenotypes of the YAC128 mice show phenotypic uniformity with low inter-animal variability present, which together with the age-dependent striatal neurodegeneration make it an ideal mouse model for the assessment of neuroprotective and other therapeutic interventions.

Oncol Res, 2003, 13(11), 491 - 502
Mode of cell death induced in Chinese hamster cells by sequence-selective DNA minor groove binding nitrogen mustards: comparison with untargeted mustards and with Hoechst 33342; Whiteside G et al.; The clinical antitumor efficacy of nitrogen mustards such as chlorambucil may relate to their ability to cause programmed cell death (apoptosis), probably through their DNA cross-linking properties . In contrast, bisbenzimidazoles such as Hoechst 33342 interact noncovalently with the minor groove of DNA, and appear to cause apoptosis in a fundamentally different way, which may involve the inhibition of topoisomerase (topo) I enzymes . A series of DNA minor groove binding nitrogen mustards with selective DNA affinity and in vivo antitumor activity in animal models was studied . Although two examples of such compounds proved to inhibit topo I enzymes in vitro, they were equally toxic towards topo I-proficient and- deficient strains of yeast, suggesting that topo I inhibition was not involved in cell killing . Flow cytometric analysis of Chinese hamster cells highlighted the differences in the propensity to cause apoptosis by chlorambucil compared with Hoechst 33342, revealing two distinct apoptotic populations in cells treated with the latter drug . Unexpectedly, the bisbenzimidazole mustards showed a novel peak of apoptotic activity, distinct from that shown by either parent drug . Exploring these different mechanisms of apoptosis may provide new directions for the development of antitumor drugs.

Yi Chuan Xue Bao, 2003 Jan, 30(1), 76 - 80
{Genetic analysis of isozyme loci of intraspecific hybrid in Auricularia auricula}; Bian YB et al.; Monokaryon strains H2 and J3 were respectively developed from the cultivated strains He-1 and Ju-1 of Auricularia auricula through protoplasted monokaryon technique . Dicaryon strain H2J3 was bred through crossbreeding of H2 and J3 as parents . Fifty-two F1 monokaryon strains were obtained from the fruitbodies of dicaryon H2J3 of A . auricula by the single-spore isolation and culture . All the tested strains, parent monokaryon strains (H2,J3) and fifty-two F1 monokaryon strains, were cultured in liquid Complete Yeast Medium(CYM) for twenty days, and the mycelia of tested strains were respectively analysed by polyacrylamide gel electrophoresis . Bian Y B et al . have reported the results of isozyme zymogram polymorphisms of cultured strains including He-1 and Ju-1, and the specific expression of esterase genes on the different stages of mycelial development in A . auricula . The same named methods of isozyme loci and alleles suggested by Gottlieb (1973) were used in this study . The relative fitness of allele segregation and theoretical expected ratio was examined by the contingency chi 2 test . The linkage relationship was assessed according to the method suggested by Rudin and Ekberg (1978) . The isozyme analysis of tested strains showed that the isozymes of esterase (EST), malate dehydrogenase(MDH) and formate dehydrogenase(FDH) of A . auricula were respectively controlled by 7, 5 and 4 polymorphic loci . The isozyme bands which were controlled by EST-3, EST-4, MDH-3 and MDH-4 loci were stably expressed in the parental strains and most tested F1 monokaryon strains . Genetic analysis was impossible for these four loci . The relative fitness between allele observed value and theoretical expected value was further examined by means of contingency chi 2 at 1% level, the result indicated that significant difference existed from the chi 2 value of MDH-5 and FDH-1 in 7 polymorphic loci of EST, MDH and FDH, and denied the hypothesis in which the isozyme bands controlled by MDH-5 and FDH-1 loci were presumed to be allozyme bands . The allele observed values (100:SA) of EST-7, MDH-1, MDH-5, FDH-1, FDH-2 and FDH-4 were significant deviation of theoretical expected ratio (1:1), which showed that these variations were not due to the allele expressions . The significant difference was not be showed at the 5% level from the chi 2 values of EST-1, EST-2, EST-5, EST-6 and MDH-2, this result indicated the allele segregation was corresponding to the theoretical expected ratio at these 5 loci, these 5 loci were allozyme loci which was corresponding to Mendel's law of segregation . A total of 10 possible pair combinations from the 5 allozyme loci were tested . The numbers of monokaryon strains with parental type zymogram or recombinant type zymogram were counted up, and the chi 2I, chi 2II and chi 2L were calculated for examining the linkage relationships of these allozyme loci, the chi 2L value attained to significant difference at 1% level between EST-5 and EST-6 loci, it indicated that the linkage relationship existed between the two loci, non-linkage relationships existed between the other pairing loci of 5 allozyme loci.

J Cell Physiol, 2003 Aug, 196(2), 312 - 8
Pro-apoptotic ASY/Nogo-B protein associates with ASYIP; Qi B et al.; We have previously shown that ectopic expression of the ASY/Nogo-B gene induced apoptosis in various cancer cell lines . Nogo-A, a splice variant of the ASY, has been reported to have an inhibitory effect on neuronal regeneration in the central nervous system . To investigate the mechanism of ASY-induced apoptosis or inhibition of neuronal regeneration, we cloned a cDNA for the ASY-interacting protein from the human cDNA library using the yeast two-hybrid method, and obtained a cDNA we designated as ASYIP . The ASYIP protein contains two hydrophobic regions and a double lysine endoplasmic reticulum (ER) retrieval motif at its C-terminus, which was shown to be identical to RTN3, a reticulon family protein of unknown function . We showed that ASY and ASYIP proteins formed a complex also in human cells . Mutational analysis indicated that both of the hydrophobic regions of the ASYIP protein were required for the association . By immunofluorescence analysis, the ASYIP protein was shown to be co-localized with ASY in the ER . Characterization of the ASYIP gene may be very useful in clarifying the mechanism of ASY-induced apoptosis or Nogo-involved inhibition of neuronal regeneration in the central nervous system .

J Gen Virol, 2003 Jul, 84(Pt 7), 1931 - 9
Interaction of replicase components between Cucumber mosaic virus and Peanut stunt virus; Suzuki M et al.; Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) each have genomes consisting of three single-stranded RNA molecules: RNA 1, 2 and 3 . RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome . Although RNA 3 is exchangeable between CMV and PSV, exchange of RNA 1 and 2 between the two viruses has been unsuccessful . In this study, reassortants containing PSV RNA 1 and CMV RNA 2 together with RNA 3 of CMV or PSV were shown to be able to replicate their genomic RNA, but not to transcribe subgenomic RNA 4 in tobacco protoplasts . Conversely, the reassortant consisting of CMV RNA 1 and PSV RNA 2 together with RNA 3 of CMV or PSV could not replicate . Subsequently, a yeast two-hybrid system was used to analyse the in vivo interaction between the 1a and 2a proteins . The C-terminal half of PSV-1a protein interacted with the N-terminal region of 2a protein of both PSV and CMV, but the C-terminal half of CMV-1a and the N-terminal region of PSV-2a did not interact . These results suggest that RNA replication in the interspecific reassortant between CMV and PSV requires compatibility between the C-terminal half of the 1a protein and the N-terminal region of the 2a protein, and this compatibility is insufficient for transcription of subgenomic RNA 4.

J Gen Virol, 2003 Jul, 84(Pt 7), 1789 - 97
Flock house virus replicates and expresses green fluorescent protein in mosquitoes; Dasgupta R et al.; Flock house virus (FHV) is a non-enveloped, positive-sense RNA virus of insect origin that belongs to the family Nodaviridae . FHV has been shown to overcome the kingdom barrier and to replicate in plants, insects, yeast and mammalian cells . Although of insect origin, FHV has not previously been shown to replicate in mosquitoes . We have tested FHV replication in vitro in C6/36 cells (derived from neonatal Aedes albopictus) and in vivo in four different genera of mosquitoes, Aedes, Culex, Anopheles and Armigeres . FHV replicated to high titres in C6/36 cells that had been subcloned to support maximum growth of FHV . When adult mosquitoes were orally fed or injected with the virus, FHV antigen was detected in various tissues and infectious virus was recovered . Vectors developed from an infectious cDNA clone of a defective-interfering RNA, derived from FHV genomic RNA2, expressed green fluorescent protein in Drosophila cells and adult mosquitoes . This demonstrates the potential of FHV-based vectors for expression of foreign genes in mosquitoes and possibly other insects.

Cancer Res, 2003 Jun 15, 63(12), 3289 - 95
The G2-phase decatenation checkpoint is defective in Werner syndrome cells; Franchitto A et al.; It has been proposed that cells monitor chromatid catenation status after DNA replication and inhibit progression into mitosis until chromatids are correctly decatenated by topoisomerase II (TopoII) . Studies in yeast have suggested that TopoII may interact with RecQ helicases during this process . Using ICRF187, a TopoII catalytic inhibitor that prevents chromatid decatenation without producing DNA strand breaks, we demonstrated that cells deficient of WRN, a human RecQ helicase, displayed a defect in decatenation checkpoint activation, which was corrected by ectopic expression of wild-type WRN . We also provide evidence that BRCA1 is phosphorylated in an ATR-dependent manner in response to decatenation checkpoint activation and that this phosphorylation is not detectable in Werner syndrome cells . Furthermore, ICRF187 treatment resulted in coimmunoprecipitation of WRN and TopoII . Finally, we demonstrated that override of the decatenation checkpoint resulted in enhanced chromosomal damage and apoptosis only in the absence of WRN, but not in normal cells . Our findings suggest that WRN plays a role in the activation of G(2) decatenation checkpoint and that the abortive function of this pathway itself does not appear to be sufficient to cause genomic instability but rather predisposes to genomic instability and apoptotic cell death in the absence of other "caretaker" genes, such as WRN.

Cancer Res, 2003 Jun 15, 63(12), 3049 - 53
Both DNA topoisomerase II-binding protein 1 and BRCA1 regulate the G2-M cell cycle checkpoint; Yamane K et al.; Cell cycle checkpoints play a central role in genomic stability . The human DNA topoisomerase II-binding protein 1 (TopBP1) protein contains eight BRCA1 COOH terminus motifs and shares similarities with Cut5, a yeast checkpoint Rad protein . TopBP1 also shares many features with BRCA1 . We report that, when expression of TopBP1 protein is inhibited in BRCA1 mutant cells, mimicking a TopBP1, BRCA1 double-negative condition, the G(2)-M checkpoint is strongly abrogated and apoptosis is increased after ionizing radiation . However, a BRCA1-negative or a TopBP1-negative background resulted in only partial abrogation of the G(2)-M checkpoint . The BRCA1 mutant and TopBP1-reduced condition specifically destroys regulation of the Chk1 kinase but not the Chk2 kinase, suggesting involvement in the ataxia telangiectasia-related pathway . These results indicate that both TopBP1 and BRCA1 specifically regulate the G(2)-M checkpoint, partially compensating each function.

Endocrinology, 2003 Jul, 144(7), 3148 - 58
Ini, a small nuclear protein that enhances the response of the connexin43 gene to estrogen; Oltra E et al.; This article describes the structural and functional characterization of Ini (AF495522), a novel highly conserved zinc-finger protein that had been identified by screening an estrogen-induced rat myometrial expression library . Ini localizes to the nucleus of HeLa cells and binds to the proximal connexin43 (cx43) promoter, as demonstrated by EMSA . In addition, transient transfection experiments performed with estrogen receptor alpha (ERalpha) cDNA show that overexpression of Ini enhances, in a dose-dependent fashion, the up-regulation of the cx43 gene by estrogen . On binding to the cx43 promoter, Ini stimulates the transcriptional activating function (AF)-1, but not the AF-2, of the ERalpha . This makes Ini one of the few known coactivators specific for AF-1 . Because estrogen up-regulates Ini mRNA in the myometrium, it is likely that Ini's physiological role in this tissue is to modulate the response of the cx43 gene to estrogen . Transfection studies with an Ini antisense construct seem to indicate that Ini plays an additional role in the cellular response to estrogen affecting both AF-1 and AF-2 activities of the ERalpha . This broader effect may be associated with cell cycle progression that in yeast has been shown to require Ini.

Int J Food Microbiol, 2003 Aug 15, 85(1-2), 83 - 5
Isolation of moulds capable of producing mycotoxins from blue mouldy Tulum cheeses produced in Turkey; Erdogan A et al.; A total of 16 moulds was isolated and identified from 12 blue mouldy Tulum cheeses collected from retailers in Erzurum, Turkey; 12 were Penicillium roqueforti and 4 were Geotrichum candidum . The P . roqueforti isolates were grown in yeast sucrose broth at 5, 12 and 25 degrees C for 10 days, then extracted with chloroform and acetone and the extracts were examined for the presence of patulin, penicillic acid, roquefortine and PR toxin using thin layer chromatography . All of the P . roqueforti strains had toxin-producing ability at 5, 12 and 25 degrees C, eight produced only at 5 and 12 degrees C and six could not produce toxin at 5 degrees C.

Cancer Genet Cytogenet, 2003 Jul 1, 144(1), 23 - 30
Tetrasomy 6 and 6q14 deletion are associated with better survival in hepatocellular carcinomas . a fluorescence in situ hybridization study of 77 cases; Huang SF et al.; We previously described frequent 6q14 deletion and polysomy 6 in 25 hepatocellular carcinomas (HCC) by fluorescence in situ hybridization (FISH) . A more favorable prognosis was noted in patients with 6q14 deletions . To confirm this clinical association, a two-colored FISH study was carried out on 77 HCC using a combination of a yeast artificial chromosome probe (813_E_12) of the 6q14 region and a chromosome 6 centromeric probe (D6Z1) for simultaneous evaluation of the copy number change and chromosome arm deletion . The 77 HCC were divided into four groups according to the copy number of the centromeric signal: 26 with HCC (33.7%) were disomy for chromosome 6, 9 with HCC (11.7%) were trisomy, 29 with HCC (37.6%) were tetrasomy, and 13 with HCC (17%) were classified as hypersomy with presence of one major clone (>7%) of pentasomy or hexasomy of chromosome 6 . Allelic loss at 6q14 was found in 40 with HCC (52%) . The distribution of sex, age, stage, tumor size, tumor grade, and viral markers in each group showed no significant differences . An association with cirrhosis, however, was significantly lower in the hypersomy group (P = 0.001) . The tetrasomy group had the best survival . An interaction between 6q14 deletion and numerical change of chromosome 6 on patients' survival were also noted . For patients with 6q14 deletion, both disomy and tetrasomy groups had significantly better survival rates than trisomy and hypersomy groups . In contrast, no differences in survival rates could be observed among these four groups for patients without the 6q14 deletion . The association with more favorable prognosis shown in this study indicates that tetrasomy 6 and 6q14 deletion may play an important role in the tumorigenesis of hepatocellular carcinoma and is worthy of further investigation.

Biochemistry, 2003 Jun 24, 42(24), 7618 - 25
Troponin I binds polycystin-L and inhibits its calcium-induced channel activation; Li Q et al.; Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel . We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor . In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively) . Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells . Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other . GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins . The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I . Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I.

Biochemistry, 2003 Jun 24, 42(24), 7604 - 10
Rupture of the hydrogen bond linking two Omega-loops induces the molten globule state at neutral pH in cytochrome c; Sinibaldi F et al.; His26Tyr and His33Tyr mutants were obtained from the Cys102Thr variant of yeast iso-1-cytochrome c . Spectroscopic studies show that a mutation at position 26 at pH 7.0 enhances flexibility of the peptide, alters the heme pocket region and the axial coordination to heme-iron, and reduces protein stability . The His26Tyr mutant shows properties typical of the molten globule . Further, formation of an axially misligated minor low spin species occurs with partial displacement of Met80, the axial ligand of the heme-iron in the native protein . The pK(a) determined for the alkaline transition of this mutant is 7.48 (+/- 0.05), approximately 0.5 lower than that of the wild-type protein . Hence, the alkaline conformer is populated at pH 7.0, and the sixth ligand of the misligated species is proposed to be a lysine . Furthermore, a reduction in catalytic activity indicates that the functional properties are altered . The results suggest that the structural and functional changes observed in the His26Tyr mutant are because the mutation frees the two Omega-loops that, in the native protein, are linked by the hydrogen bond between His26 and Glu44 . Hence, one may infer that the His26-Glu44 hydrogen bond is essential for the rigidity and stability of the native protein . In its absence, the heightened flexibility of the peptide fold results in conversion of the macromolecule to a molten globule state, even at neutral pH . Ligand exchange at the sixth coordination position of the heme-iron(III) observed as the minor species (i.e., the alkaline conformer) is therefore induced by a long-range effect . This result is of interest since mutations reported to date, which stabilize the alkaline conformer, all occur in the loop including Met80 . By contrast, only very minor spectroscopic (and, thus, structural) changes are observed for the His33Tyr mutant . This suggests that His33 does not form intramolecular bonds considered important for the protein structure and stability, and is consistent with the high variability of residues at position 33 in cytochromes c.

Biochemistry, 2003 Jun 24, 42(24), 7270 - 82
ST7 is a novel low-density lipoprotein receptor-related protein (LRP) with a cytoplasmic tail that interacts with proteins related to signal transduction pathways; Battle MA et al.; In 1997, McCormick and co-workers identified a novel putative tumor suppressor gene, designated ST7, encoding a unique protein with transmembrane receptor characteristics {Qing et al . (1999) Oncogene 18, 335-342} . Using degenerate primers corresponding to the highly conserved region of the ligand-binding domains of members of the low-density lipoprotein receptor (LDLR) superfamily, Ishii et al . {Genomics (1998) 51, 132-135} discovered a low-density lipoprotein receptor-related protein (LRP) that closely resembles ST7 . Later, another LRP closely resembling ST7 and LRP3 was found (murine LRP9) {Sugiyama et al . (2000) Biochemistry 39, 15817-15825} . These results strongly suggested that ST7 was also a novel member of the low-density lipoprotein receptor superfamily . Proteins of this superfamily have been shown to function in endocytosis and/or signal transduction . To evaluate the relationship of ST7 to the LDLR superfamily proteins and to determine whether ST7 may function in endocytosis and/or signal transduction, we used proteomic tools to analyze the functional motifs present in the protein . Our results indicate that ST7 is a member of a subfamily of the LDLR superfamily and that its cytoplasmic domain contains several motifs implicated in endocytosis and signal transduction . Use of the yeast two-hybrid system to identify proteins that associate with ST7's cytoplasmic domain revealed that this domain interacts with three proteins involved in signal transduction and/or endocytosis, viz., receptor for activated protein C kinase 1 (RACK1), muscle integrin binding protein (MIBP), and SMAD anchor for receptor activation (SARA), suggesting that ST7, like other proteins in the LDLR superfamily, functions in these two pathways . Clearly, ST7 is an LRP, and therefore, it should now be referred to as LRP12.

Mol Biol Cell, 2003 Jun, 14(6), 2399 - 409 Epub 2003 Feb 06.
Distinct developmental function of two Caenorhabditis elegans homologs of the cohesin subunit Scc1/Rad21; Mito Y et al.; Cohesin, which mediates sister chromatid cohesion, is composed of four subunits, named Scc1/Rad21, Scc3, Smc1, and Smc3 in yeast . Caenorhabditis elegans has a single homolog for each of Scc3, Smc1, and Smc3, but as many as four for Scc1/Rad21 (COH-1, SCC-1/COH-2, COH-3, and REC-8) . Except for REC-8 required for meiosis, function of these C . elegans proteins remains largely unknown . Herein, we examined their possible involvement in mitosis and development . Embryos depleted of the homolog of either Scc3, or Smc1, or Smc3 by RNA interference revealed a defect in mitotic chromosome segregation but not in chromosome condensation and cytokinesis . Depletion of SCC-1/COH-2 caused similar phenotypes . SCC-1/COH-2 was present in cells destined to divide . It localized to chromosomes in a cell cycle-dependent manner . Worms depleted of COH-1 arrested at either the late embryonic or the larval stage, with no indication of mitotic dysfunction . COH-1 associated chromosomes throughout the cell cycle in all somatic cells undergoing late embryogenesis or larval development . Thus, SCC-1/COH-2 and the homologs of Scc3, Smc1, and Smc3 facilitate mitotic chromosome segregation during the development, presumably by forming a cohesin complex, whereas COH-1 seems to play a role important for development but unrelated to mitosis.

Mol Biol Cell, 2003 Jun, 14(6), 2314 - 26 Epub 2003 Mar 20.
Identifying phase-specific genes in the fungal pathogen Histoplasma capsulatum using a genomic shotgun microarray; Hwang L et al.; A fundamental feature of the fungal pathogen Histoplasma capsulatum is its ability to shift from a mycelial phase in the soil to a yeast phase in its human host . Each form plays a critical role in infection and disease, but little is understood about how these two morphologic phases are established and maintained . To identify phase-regulated genes of H . capsulatum, we carried out expression analyses by using a genomic shotgun microarray representing approximately one-third of the genome, and identified 500 clones that were differentially expressed . Genes induced in the mycelial phase included several involved in conidiation, cell polarity, and melanin production in other organisms . Genes induced in the yeast phase included several involved in sulfur metabolism, extending previous observations that sulfur metabolism influences morphology in H . capsulatum . Other genes with increased expression in the yeast phase were implicated in nutrient acquisition and cell cycle regulation . Unexpectedly, differential regulation of the site of transcript initiation was also observed in the two phases . These findings identify genes that may determine some of the major characteristics of the mycelial and yeast phases.

J Biol Chem, 2003 Sep 5, 278(36), 33848 - 56 Epub 2003 Jun 14.
Fidelity of primate cell repair of a double-strand break within a (CTG).(CAG) tract . Effect of slipped DNA structures; Marcadier JL et al.; At least 15 human diseases are caused by the instability of gene-specific (CTG).(CAG) repeats . The precise mechanism of instability remains unknown, though bacterial and yeast models have suggested a role for aberrant repair of double-strand breaks (DSBs) . Using an established primate DSB repair system, we have investigated the fidelity of repair of a DSB within a (CTG).(CAG) repeat tract . DSB repair substrates were generated from plasmids that are stably replicated in their circular form, permitting us to highlight the effects of DSB repair on repeat stability and minimize the contribution of replication . DSBs were introduced into repeat-containing plasmids using a unique BsmI site, such that the entire repeat tract comprised one free end of the linearized plasmid . Substrates containing 17, 47, and 79 repeats, in either their linear duplex form or containing slipped structures (out-of-register interstrand mispairings at repeat sequences), were transiently transfected into primate cells . Linearized plasmids with repeats were repaired with mildly reduced efficiency, while the presence of slipped structures considerably reduced repair efficiency . The repaired products were characterized for alterations within the repeat tract and flanking sequence . DSB repair induced predominantly repeat deletions . Notably, a polarized/directional deletion effect was observed, in that the repetitive end of the DSB was preferentially removed . This phenomenon was dramatically enhanced when slipped structures were present within the repeat tract, providing the first evidence for error-prone processing of slipped-strand structures . These results suggest the existence of primate nuclease activities that are specific for (CTG).(CAG) repeats and the structures they form.

J Biol Chem, 2003 Aug 22, 278(34), 31479 - 85 Epub 2003 Jun 13.
Stability of the Rel homology domain is critical for generation of NF-kappa B p50 subunit; Lin L et al.; The NF-kappa B transcription factor p50 and the Rel protein-specific transcription inhibitor p105 are both encoded by the nfkb1 gene . The p50 protein is incorporated within the N-terminal portion of p105 and is a unique product of proteasomal processing . Because proteasome-mediated proteolysis generally results in complete degradation of the substrate, how p50 survives the proteasomal processing remains unknown . Survival of proteasomal processing has also been observed recently for the yeast transcription factors SPT23 and MGA2, but the mechanism is also unclear . Here we show evidence that stability of the Rel homology domain (RHD) within the N-terminal portion of the NF-kappa B 1 protein is required for p50 generation . We demonstrated that proteolysis initiated at an internal location of the NF-kappa B 1 protein, which normally generates p50, degrades the N-terminal portion of the NF-kappa B 1 protein when the RHD is destabilized . Our findings highlight the critical role of the unique structure of the RHD for the survival of p50 during proteosomal processing.

J Appl Microbiol, 2003, 95(1), 13 - 22
Molecular detection of Candida krusei contamination in fruit juice using the citrate synthase gene cs1 and a potential role for this gene in the adaptive response to acetic acid; Casey GD et al.; AIMS: To develop a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect viable Candida krusei contaminations and examine the potential role of the citrate synthase (cs1) gene in adaptation to acetic acid . METHODS AND RESULTS: Fruit juice artificially contaminated with C . krusei cells was heat treated to inactivate the yeast cells, after which the improved ability of the RT-PCR over the PCR assay, through the amplification of the cs1 gene, to differentiate viable contaminations was shown . The sensitivity of the detection assay was 6 x 104 CFU ml-1 . RT-PCR and densitometric analysis of the cs1 gene throughout the process of adaptation to acetic acid highlighted a potential role for the gene in the yeast's adaptive response . CONCLUSIONS: The RT-PCR assay through the targeting of the cs1 gene proved to be a specific, sensitive and direct method for the identification of a C . krusei contamination in a food environment . The cs1 gene was shown to play a potential role in the adaptation of the culture to the weak-acid preservative acetic acid . SIGNIFICANCE AND IMPORTANCE OF THE STUDY: The development of a direct, sensitive and specific identification assay for C . krusei from a food environment and understanding the mechanism employed in adapting to a preservative challenge, represent important tools to the food industry in attempting to limit spoilage by this important food spoilage yeast.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 847 - 51
Thermococcus gammatolerans sp . nov., a hyperthermophilic archaeon from a deep-sea hydrothermal vent that resists ionizing radiation; Jolivet E et al.; Enrichments for anaerobic organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected at the Guaymas Basin (27 degrees 01' N, 111 degrees 24' W) . Positive enrichments were submitted to gamma-irradiation at a dose of 30 kGy . One of the resistant strains, designated strain EJ3(T), formed regular motile cocci . The new strain grew between 55 and 95 degrees C, with an optimum growth temperature of 88 degrees C . The optimal pH for growth was 6.0, and the optimum NaCl concentration for growth was around 20 g l(-1) . Strain EJ3(T) was an obligately anaerobic heterotroph that utilized yeast extract, tryptone and peptone . Elemental sulfur or cystine was required for growth and reduced to hydrogen sulfide . The G + C content of the genomic DNA was 51.3 mol% . As determined by 16S rRNA gene sequence analysis, the organism was most closely related to Thermococcus celer, Thermococcus guaymasensis, Thermococcus hydrothermalis, Thermococcus profundus and Thermococcus gorgonarius . However, no significant homology was observed between them by DNA-DNA hybridization . The novel organism also possessed phenotypic traits that differ from those of its closest phylogenetic relatives . Therefore, it is proposed that this isolate, which constitutes the most radioresistant hyperthermophilic archaeon known to date, should be described as the type strain of a novel species, Thermococcus gammatolerans sp . nov . The type strain is EJ3(T) (= DSM 15229(T) = JCM 11827(T)).

ScientificWorldJournal, 2002 May 29, 2, 1444 - 8
OptiPrep density gradient solutions for nonmammalian organelles; Graham JM; Any density gradient for the isolation of nonmammalian organelles should ideally only expose the sedimenting biological particles to an increasing concentration of the gradient solute . Thus they will experience only an increasing density and viscosity, other parameters such as osmolality, pH, ionic strength and the concentration of important additives (such as EDTA and DTT) should remain as close to constant as possible . This Protocol Article describes the strategies for the dilution of OptiPrep in order to prepare such solutions for organelles and membranes from nonmammalian sources such as yeast.

Plant Physiol, 2003 Jun, 132(2), 666 - 80
The Arabidopsis CDPK-SnRK superfamily of protein kinases; Hrabak EM et al.; The CDPK-SnRK superfamily consists of seven types of serine-threonine protein kinases: calcium-dependent protein kinase (CDPKs), CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PEP carboxylase kinase-related kinases (PEPRKs), calmodulin-dependent protein kinases (CaMKs), calcium and calmodulin-dependent protein kinases (CCaMKs), and SnRKs . Within this superfamily, individual isoforms and subfamilies contain distinct regulatory domains, subcellular targeting information, and substrate specificities . Our analysis of the Arabidopsis genome identified 34 CDPKs, eight CRKs, two PPCKs, two PEPRKs, and 38 SnRKs . No definitive examples were found for a CCaMK similar to those previously identified in lily (Lilium longiflorum) and tobacco (Nicotiana tabacum) or for a CaMK similar to those in animals or yeast . CDPKs are present in plants and a specific subgroup of protists, but CRKs, PPCKs, PEPRKs, and two of the SnRK subgroups have been found only in plants . CDPKs and at least one SnRK have been implicated in decoding calcium signals in Arabidopsis . Analysis of intron placements supports the hypothesis that CDPKs, CRKs, PPCKs and PEPRKs have a common evolutionary origin; however there are no conserved intron positions between these kinases and the SnRK subgroup . CDPKs and SnRKs are found on all five Arabidopsis chromosomes . The presence of closely related kinases in regions of the genome known to have arisen by genome duplication indicates that these kinases probably arose by divergence from common ancestors . The PlantsP database provides a resource of continuously updated information on protein kinases from Arabidopsis and other plants.

J Virol, 2003 Jul, 77(13), 7236 - 43
A nuclear kinesin-like protein interacts with and stimulates the activity of the leucine-rich nuclear export signal of the human immunodeficiency virus type 1 rev protein; Venkatesh LK et al.; The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE) . In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof . REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells . Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells . REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP . Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

J Biol Chem, 2003 Aug 22, 278(34), 31843 - 7 Epub 2003 Jun 12.
Autosomal recessive hypercholesterolemia protein interacts with and regulates the cell surface level of Alzheimer's amyloid beta precursor protein; Noviello C et al.; The familial Alzheimer's disease gene product amyloid beta protein precursor (A beta PP) is sequentially processed by beta- and gamma-secretases to generate the A beta peptide . Although much is known about the biochemical pathway leading to A beta formation, because extracellular aggregates of A beta peptides are considered the cause of Alzheimer's disease, the biological role of A beta PP processing is only recently being investigated . Cleavage of A beta PP by gamma-secretase releases, together with A beta, a COOH-terminal A beta PP intracellular domain, termed AID . Hoping to gain clues about proteins that regulates A beta PP processing and function, we used the yeast two-hybrid system to identify proteins that interact with the AID region of A beta PP . One of the interactors isolated is the autosomal recessive hypercholesterolemia (ARH) adapter protein . This molecular interaction is confirmed in vitro and in vivo by fluorescence resonance energy transfer and in cell lysates . Moreover, we show that reduction of ARH expression by RNA interference results in increased levels of cell membrane A beta PP . These data assert a physiological role for ARH in A beta PP internalization, transport, and/or processing.

Vaccine, 2003 Jul 4, 21(23), 3179 - 85
Immunogenicity of 10 and 20 microgram hepatitis B vaccine in a two-dose schedule; ul-Haq N et al.; Healthy subjects (15-50 years old) were randomised to receive, intramuscularly, 10 or 20 micrograms of a yeast-derived recombinant hepatitis B vaccine (Heberbiovac HB) on a two-dose (0-1) schedule . Anti-hepatitis B surface antigen antibodies (HBsAb) were determined at 2, 3 and 6 months . Twenty micrograms immunisation yielded 96.3% seroprotection (HBsAb >10 IU/l) at t=3, which persisted to 97.2% at t=6 . The 10 microgram group resulted in 87.4% seroprotection at t=3 and 81% at t=6, but reached >95% among subjects <21 years old, both at t=3 and 6 . It is concluded that the vaccine used is highly immunogenic and the two-dose 20 microgram immunisation can be used in all age groups and 10 micrograms in subjects up to 20 years . These schedules can result in significantly better compliance and cost-effectiveness of the vaccination programs.

FEBS Lett, 2003 Jun 19, 545(2-3), 151 - 4
The PEF family proteins sorcin and grancalcin interact in vivo and in vitro; Hansen C et al.; The penta-EF hand (PEF) family of calcium binding proteins includes grancalcin, peflin, sorcin, calpain large and small subunits as well as ALG-2 . Systematic testing of the heterodimerization abilities of the PEF proteins using the yeast two-hybrid and glutathione S-transferase pull-down assays revealed the new finding that grancalcin interacts strongly with sorcin . In addition, sorcin and grancalcin can be co-immunoprecipitated from lysates of human umbilical vein endothelial cells . Our results indicate that heterodimerization, in addition to differential interactions with target proteins, might be a way to regulate and fine tune processes mediated by calcium binding proteins of the penta-EF hand type.

Biochem Biophys Res Commun, 2003 Jun 27, 306(2), 394 - 401
AKAP7gamma is a nuclear RI-binding AKAP; Brown RL et al.; Spatial regulation of protein kinase A (PKA) is accomplished by its sequestration via A-kinase anchor proteins (AKAPs) . PKA activity is critical for mammalian oocyte development, suggesting that PKA must be appropriately positioned in these large cells . A screen for AKAPs in oocytes identified AKAP7gamma, an AKAP originally found in pancreas . Yeast two-hybrid analysis and co-immunoprecipitation studies showed that AKAP7gamma bound the type I PKA regulatory subunit (RI) and that the RI-binding domain overlapped the previously identified type II PKA regulatory subunit (RII) binding domain . Overexpressed AKAP7gamma localized to the nuclei of HEK 293 cells via a nuclear localization signal . In addition, endogenous AKAP7gamma protein was found in both the nucleus and cytoplasm of oocytes . This work identifies AKAP7gamma as the first nuclear AKAP to bind RI and suggests that AKAP7gamma may be responsible for positioning PKA via RI and/or RII to regulate PKA-mediated gene transcription in both somatic cells and oocytes.

Curr Genet, 2003 Sep, 43(6), 439 - 46 Epub 2003 Jun 11.
Evaluation of Aspergillus niger as host for virus-like particle production, using the hepatitis B surface antigen as a model; Pluddemann A et al.; The filamentous fungus Aspergillus niger was transformed with the hepatitis B virus S gene encoding the major viral envelope protein under control of the constitutive A . nidulans glyceraldehyde-3-phosphate dehydrogenase ( gpdA) promoter . Approximately seven copies of the expression cassette were integrated on the genome, resulting in high-level transcription of the S gene . Production of the 24-kDa S protein and a 48-kDa S protein dimer in the membrane-associated protein fraction of the recombinant A . niger strain was shown through Western analysis . Electron microscopy of partially purified recombinant S protein revealed the formation of spherical pseudoviral particles with a diameter of 22 nm . The production level of hepatitis B pseudoviral particles was estimated to be 0.4 mg/l culture, which compares favourably with the reported levels initially obtained in yeast, indicating the potential of the Aspergillus expression system as an alternative, cost-effective vaccine production system.

Mol Biol Cell, 2003 May, 14(5), 1978 - 92 Epub 2003 Jan 26.
Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus; Fontao L et al.; The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers . Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs) . We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays . The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch . In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin . Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus . Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present . The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP . On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.

Mol Biol Cell, 2003 May, 14(5), 1923 - 40
Direct interaction with nup153 mediates binding of Tpr to the periphery of the nuclear pore complex; Hase ME et al.; Tpr is a 267-kDa protein forming coiled coil-dominated homodimers that locate at the nucleoplasmic side of the nuclear pore complex (NPC) . The proteins that tether Tpr to this location are unknown . Moreover, the question whether Tpr itself might act as a scaffold onto which other NPC components need to be assembled has not been answered to date . To assess Tpr's role as an architectural element of the NPC, we have studied the sequential disassembly and reassembly of NPCs in mitotic cells, paralleled by studies of cells depleted of Tpr as a result of posttranscriptional tpr gene silencing by RNA interference (RNAi) . NPC assembly and recruitment of several nucleoporins, including Nup50, Nup93, Nup96, Nup98, Nup107, and Nup153, in anaphase/early telophase is shown to precede NPC association of Tpr in late telophase . In accordance, cellular depletion of Tpr by RNAi does not forestall binding of these nucleoporins to the NPC . In a search for proteins that moor Tpr to the NPC, we have combined the RNAi approach with affinity-chromatography and yeast two-hybrid interaction studies, leading to the identification of nucleoporin Nup153 as the binding partner for Tpr . The specificity of this interaction is demonstrated by its sensitivity to Tpr amino acid substitution mutations that abolish Tpr's ability to adhere to the NPC and affect the direct binding of Tpr to Nup153 . Accordingly, cellular depletion of Nup153 by RNAi is shown to result in mislocalization of Tpr to the nuclear interior . Nup153 deficiency also causes mislocalization of Nup50 but has no direct effect on NPC localization of the other nucleoporins studied in this investigation . In summary, these results render Tpr a protein only peripherally attached to the NPC that does not act as an essential scaffold for other nucleoporins.

Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7666 - 71 Epub 2003 Jun 11.
Origin and evolutionary process of the CNS elucidated by comparative genomics analysis of planarian ESTs; Mineta K et al.; Among the bilateral animals, a centralized nervous system is found in both the deuterostome and protostome . To address the question of whether the CNS was derived from a common ancestor of deuterostomes and protostomes, it is essential to know kinds of genes existed in the CNS of the putative common ancestor and to trace the evolutionary divergence of genes expressed in the CNS . To answer these questions, we took a comparative approach using different species, particularly focusing on one of the lower bilateral animals, the planarian (Platyhelminthes, Tricladida), which is known to possess a CNS . We determined the nucleotide sequence of ESTs from the head portion of planarians, obtaining 3,101 nonredundant EST clones . As a result of homology searches, we found that 116 clones had significant similarity to known genes related to the nervous system . Here, we compared these 116 planarian EST clones with all ORFs of the complete genome sequences of the human, fruit fly, and nematode, and showed that >95% of these 116 nervous system-related genes, including genes involved in brain or neural morphogenesis, were commonly shared among these organisms, thus providing evidence at the molecular level for the existence of a common ancestral CNS . Interestingly, we found that approximately 30% of planarian nervous system-related genes had homologous sequences in Arabidopsis and yeast, which do not possess a nervous system . This implies that the origin of nervous system-related genes greatly predated the emergence of the nervous system, and that these genes might have been recruited toward the nervous system.

J Biochem (Tokyo), 2003 May, 133(5), 659 - 64
Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle; Keira Y et al.; Calpain 3/p94, the skeletal muscle-specific isoform of the calpain large subunit family, is a protein product of the gene responsible for limb-girdle muscular dystrophy type 2A (LGMD2A) . Through yeast two-hybrid experiments, calpain 3 has been shown to bind to titin in myofibrils {Sorimachi et al . (1995) J . Biol . Chem . 270, 31158-31162} . However, because of extensive autolysis activity, calpain 3 localization in skeletal muscle has been undefined . In this study, we generated a polyclonal antibody against an N-terminal 98-amino-acid calpain 3 fragment, which is not homologous to the corresponding regions of other conventional calpains . This antibody stained myofibrils with a unique repeated doublet-pattern . Confocal microscopic observation with marker antibodies confirmed that calpain 3 is localized in the N2 region of myofibrils . Furthermore, using this antibody, we examined the localization of calpain 3 in LGMD2A muscles.

J Biochem (Tokyo), 2003 May, 133(5), 641 - 9
Molecular cloning, expression, and characterization of a novel class of synaptotagmin (Syt XIV) conserved from Drosophila to humans; Fukuda M; Synaptotagmins (Syts) represent a large family of putative membrane trafficking proteins found in various species from different phyla . In this study, I identified a novel class of Syt (named Syt XIV) conserved from Drosophila to humans and its highly related molecule, Strep14 (Syt XIV-related protein) . Although both Syt XIV and Strep14 belong to the C-terminal-type (C-type) tandem C2 protein family, only Syt XIV has a single transmembrane domain at the N-terminus and a putative fatty-acylation site just downstream of the transmembrane domain . Biochemical analyses have indicated that Syt XIV is a Ca(2+)-independent Syt (e.g., Syts VIII, XII, and XIII) and that, like other Syt family proteins, it is capable of forming a Ca(2+)-independent oligomer . Unlike other Syt isoforms, however, expression of Syt XIV and Strep14 mRNA is highly restricted to mouse heart and testis and absent in the brain, where most other Syts are abundantly expressed, suggesting that Syt XIV and Strep14 may be involved in membrane trafficking in specific tissues outside the brain . I also identified all of the C-type tandem C2 proteins in humans, the mouse, the fruit fly, a nematode, a plant, and a yeast and discuss the molecular evolution of the C-type tandem C2 protein families, including the Syt family, the Syt-like protein (Slp) family, and the Doc2 family.

J Biochem (Tokyo), 2003 May, 133(5), 615 - 23
A novel rat orthologue and homologue for the Drosophila crooked neck gene in neural stem cells and their immediate descendants; Amada N et al.; The crooked neck (crn) gene of Drosophila melanogaster encodes a scaffold protein carrying multiple tetratricopeptide repeat (TPR) motifs, and its mutation results in a reduction in the number of neuroblasts and lethality during larval stages . Here, we isolated two structurally related genes from a rat embryonic brain cDNA library . One gene is the rat orthologue of crn, which encodes 690 amino acids including 16 copies of TPR . The other gene, ATH55, encodes an 855 amino acid protein including 21 TPR motifs, which presumably represents a rat crn homologue and an orthologue of human XAB2 . Both genes are highly expressed in embryonic brain but their expressions decrease during development . ATH55-like immunoreactivity is present in the ventricular zone and newly formed cortical plate, while CRN-like immunoreactivity is more abundant in a younger ventricular zone . In agreement, both proteins were found to be enriched in cultured neural stem cells and to decrease in response to cell differentiation signals . As indicated for the yeast CRN-like protein, ATH55 and CRN immunoreactivities were both recovered in the nuclear fraction and detected in the splicing complex carrying pre-mRNA . These findings suggest that both TPR-motif-containing proteins are involved in RNA processing of mammalian neural stem cells and their immediate descendants.

Trends Genet, 2003 Jun, 19(6), 330 - 8
Biased gene conversion: implications for genome and sex evolution; Marais G; Classical genetic studies show that gene conversion can favour some alleles over others . Molecular experiments suggest that gene conversion could favour GC over AT basepairs, leading to the concept of biased gene conversion towards GC (BGC(GC)) . The expected consequence of such a process is the GC-enrichment of DNA sequences under gene conversion . Recent genomic work suggests that BGC(GC) affects the base composition of yeast, invertebrate and mammalian genomes . Hypotheses for the mechanisms and evolutionary origin of such a strange phenomenon have been proposed . Most BGC(GC) events probably occur during meiosis, which has implications for our understanding of the evolution of sex and recombination.

Gene, 2003 May 22, 310, 1 - 15
The 'ORC cycle': a novel pathway for regulating eukaryotic DNA replication; DePamphilis ML; The function of the 'origin recognition complex' (ORC) in eukaryotic cells is to select genomic sites where pre-replication complexes (pre-RCs) can be assembled . Subsequent activation of these pre-RCs results in bi-directional DNA replication that originates at or close to the ORC DNA binding sites . Recent results have revealed that one or more of the six ORC subunits is modified during the G1 to S-phase transition in such a way that ORC activity is inhibited until mitosis is complete and a nuclear membrane is assembled . In yeast, Cdk1/Clb phosphorylates ORC . In frog eggs, pre-RC assembly destabilizes ORC/chromatin sites, and ORC is eventually hyperphosphorylated and released . In mammals, the affinity of Orc1 for chromatin is selectively reduced during S-phase and restored during early G1-phase . Unbound Orc1 is ubiquitinated during S-phase and in some cases degraded . Thus, most, perhaps all, eukaryotes exhibit some manifestation of an 'ORC cycle' that restricts the ability of ORC to initiate pre-RC assembly to the early G1-phase of the cell cycle, making the 'ORC cycle' the premier step in determining when replication begins.

Atherosclerosis, 2003 Jun, 168(2), 375 - 80
Effect of xuezhikang, a cholestin extract, on reflecting postprandial triglyceridemia after a high-fat meal in patients with coronary heart disease; Zhao SP et al.; The effect of xuezhikang on postprandial triglyceride (TG) level was investigated in patients with coronary heart disease (CHD) after a high-fat meal (800 cal; 50 g fat) . Fifty CHD patients were randomly divided into two groups to accept xuezhikang (xuezhikang group) 1200 mg/day (600 mg twice daily) or not (control group) on the base of routine therapy which included aspirin, metoprolol and fosinopril and nitrates during the whole 6 weeks following-up . Xuezhikang significantly reduced fasting serum total cholesterol (TC) (-20%), low-density lipoprotein cholesterol (LDL-C, -34%), TG (-32%) and apoB (-27%) levels, and raised fasting high-density lipoprotein cholesterol (HDL-C, 18%) and apoA-I (13%) levels (P<0.001) . The postprandial serum TG levels at 2, 4 and 6 h decreased 32, 38 and 43%, respectively, in xuezhikang group (P<0.001) . The TG area under the curve over the fasting TG level (TG-AUC) significantly decreased in CHD patients accepted xuezhikang with normal (less than 1.7 mmol/l) and elevated (1.74 to 2.92 mmol/l) fasting TG levels by 45 and 50%, respectively (P<0.001) . Routine therapy had no significant effect on the fasting and postprandial lipid and apolipoprotein levels . The change of TG-AUC was significantly related to the changes of fasting TG, TC, LDL-C, and HDL-C levels after the treatment, which were related to the changes of fasting apoA-I and apoB levels significantly (P<0.001) . Xuezhikang was shown to be beneficial in the treatment of reflecting postprandial triglyceridemia in CHD patients with normal and mildly elevated fasting TG levels.

Leuk Res, 2003 Aug, 27(8), 687 - 94
Tumor necrosis factor receptor-associated factor (TRAF) 4 is a new binding partner for the p70S6 serine/threonine kinase; Fleckenstein DS et al.; p70S6K is an intracellular serine/threonine kinase that mediates cell cycle progression and gene transcription . Immunofluorescent staining shows in factor-dependent hematopoietic M-07e cells that p70S6K is localized both in the cytosol and, after cytokine stimulation, also in the nucleus . We hypothesized that the p70S6K might interact with a transcription factor in the nucleus or with other proteins in the cytosol besides the S6 protein . By screening a yeast two-hybrid HeLa cDNA library with full-length p70S6K cDNA as bait, we identified tumor necrosis factor receptor-associated factor (TRAF) 4 as a new binding partner for this kinase . TRAF4 is a member of the TRAF family of putative signal-transducing proteins . Members of this family are capable of negatively regulating apoptotic pathways by inducing the expression of genes that promote cell survival . Immunoprecipitation experiments showed that stimulation of receptors of the tumor necrosis factor (TNF) family induced the formation of TRAF4/p70S6K complexes . Transfection studies showed that TRAF4 functions in p70S6K activation: TNF induced phosphorylation of S6, the main intracellular substrate of the kinase, in cells stably expressing TRAF4, but not in TRAF4-negative cells . In addition to its role in p70S6K activation, we postulate an anti-apoptotic role for TRAF4, because the agonistic anti-Fas antibody CH-11 induced apoptosis in untransfected HEK-293 cells, but not in TRAF4-expressing HEK-293 cells . In conclusion: (i) TNF-receptor activation leads to activation of the p70S6K; (ii) TRAF4 is a mediator in this TNF-induced signaling pathway; and (iii) TRAF4 inhibits Fas-induced apoptosis.

Genome Biol . 2003;4(6):218 . Epub 2003 May 28.
What makes a mitochondrion?
Heazlewood JL, Millar AH, Day DA, Whelan J.
Experimental analyses of the proteins found in the mitochondria of yeast, humans and Arabidopsis have confirmed some expectations but given some surprises and some insights into the evolutionary origins of mitochondrial proteins.

Genes Chromosomes Cancer, 2003 Aug, 37(4), 421 - 6
Identification of candidate tumor-suppressor genes in 6q27 by combined deletion mapping and electronic expression profiling in lymphoid neoplasms; Steinemann D et al.; Deletions in the long arm of chromosome 6 (6q) are among the most frequent chromosome aberrations in lymphoid neoplasms . Recently, the region of minimal deletion (RMD1) in 6q27 was narrowed down to 5-9 Mb . In the present study, we aimed to define the distal border of the commonly lost region in 6q27 more precisely and to identify and investigate tumor-suppressor genes (TSGs) from this region . Twenty-nine cases, in which our previous fluorescence in situ hybridization (FISH) screening that used a set of 36 YAC probes revealed loss in 6q25-27, were further investigated by means of FISH . In all cases, deletions of 6q27 extended from yeast artificial chromosome (YAC) 977e10 spanning the proximal border of RMD1 to the most telomeric YAC 933f7 within the recently established YAC-contig of this region . An interstitial homozygous deletion, flanked by the telomeric probe TelVysion6q and YAC 971g12, was detected, which substantially narrows down the RMD1 . To identify candidate TSGs down-regulated in malignant lymphomas from this region of homozygous loss, we performed electronic profiling of expressed sequences mapped to this region . This analysis suggested the gene PDCD2 originally thought to be involved in programmed cell death to be probably down-regulated in malignant B-cell lymphomas compared to normal B lymphocytes . Nevertheless, mutation analyses failed to identify mutations in the coding region of PDCD2 in nine lymphomas with FISH-proved 6q27 deletions . Furthermore, epigenetic studies in these nine and an additional 48 lymphomas did not show altered methylation of the PDCD2 locus in these tumors . Possibly haploinsufficiency is effectual in accelerating tumor progression .

Genome Res, 2003 Jun, 13(6A), 1133 - 45
Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila; Cohen S et al.; One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA) . This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens . eccDNA is heterogeneous in size and composed of chromosomal sequences . In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila . We found eccDNA throughout the fly's life cycle . These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb . The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate . Multimers of centromeric heterochromatin sequences are included in eccDNA as well . Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation . The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells . This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time . Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

Mol Cell Neurosci, 2003 May, 23(1), 28 - 38
Mutant huntingtin increases nuclear corepressor function and enhances ligand-dependent nuclear hormone receptor activation; Yohrling GJ et al.; There is increasing evidence that transcriptional dysregulation is important in Huntington's disease pathogenesis . The transcriptional protein, nuclear corepressor (NCoR), acts to repress transcription of nuclear hormone receptors, such as the thyroid hormone receptor (TR) and retinoic acid receptor, in the absence of their appropriate ligand . NCoR has been shown to bind to the mutated huntingtin protein in a yeast two-hybrid screen . This aberrant interaction may have profound effects on both the function of the NCoR protein and on its control of nuclear hormone receptor-mediated transcription . To test this hypothesis, reporter gene assays were performed in inducible PC12 cell lines expressing exon 1 of the human huntingtin protein (Htt) with either a 25 or 103 polyglutamine (Q) repeat . Expression of mutant 103Q protein appears to enhance the ability of NCoR to repress TR-mediated transcription in the absence of ligand . Western analyses indicated that the expression of the mutant 103Q Htt protein did not change the endogenous NCoR levels in the HD103Q PC12 cells when compared to uninduced cells . Interestingly, using GST pull-down assays we found that a mutant Htt exon 1 construct with 53 polyglutamine (HD53Q) did not bind to NCoR in a polyglutamine-dependent fashion . These findings suggest that an aberrant NCoR-Htt interaction does not exist in vitro . Expression of the mutant 103Q protein was also found to enhance ligand-dependent activation of TR and retinoic acid receptor . In vitro binding data shows that TR binds to HD53Q in the presence of ligand . Taken together these data suggest that Htt may function as a transcriptional coactivator of nuclear hormone receptors.

Dev Biol, 2003 Jun 15, 258(2), 291 - 306
Positional cloning of a temperature-sensitive mutant emmental reveals a role for sly1 during cell proliferation in zebrafish fin regeneration; Nechiporuk A et al.; Here, we used classical genetics in zebrafish to identify temperature-sensitive mutants in caudal fin regeneration . Gross morphological, histological, and molecular analyses revealed that one of these strains, emmental (emm), failed to form a functional regeneration blastema . Inhibition of emm function by heat treatment during regenerative outgrowth rapidly blocked regeneration . This block was associated with reduced proliferation in the proximal blastema and expansion of the nonproliferative distal blastemal zone . Positional cloning revealed that the emm phenotype is caused by a mutation in the orthologue of yeast sly1, a gene product involved in protein trafficking . sly1 is upregulated in the newly formed blastema as well as during regenerative outgrowth . Thus, sly1 is essential for blastemal organization and proliferation during two stages of fin regeneration.

Methods, 2003 Jul, 30(3), 269 - 76
Apical surface formation in MDCK cells: regulation by the serine/threonine kinase EMK1; Cohen D et al.; It has recently become evident that basic mechanisms for the establishment of cell polarity are conserved between epithelial and nonepithelial systems . The vast catalogue of known gene products involved in various aspects of invertebrate and yeast cell polarity provides a repertoire of candidate proteins that can be tested for their roles in the organization of mammalian epithelia . Here, we describe cell biological approaches to study the development and maintenance of cell polarity in Mardin-Darby canine kidney (MDCK) cells, an established mammalian model cell line for simple epithelia . The assays allowed us to characterize the Caenorhabditis elegans PAR-1 homologue EMK1 as a novel regulator of apical surface formation in epithelial cells.

Surgery, 2003 Jun, 133(6), 678 - 88
Pancreatic elastase is proven to be a mannose-binding protein--implications for the systemic response to pancreatitis; Zhang H et al.; BACKGROUND: Mannose-binding proteins (MBPs) have been isolated from serum, liver, lung, and kidney and are believed to play an important role in first-line host defense during acute phase inflammatory response . Because of the inflammatory nature of pancreatitis, we postulate that the pancreas produces endogenous MBP . METHODS: Pancreatic juice, from both human and rat, was collected by pancreatic duct cannulation and subjected to mannose-Sepharose affinity chromatography to isolate pancreatic MBP (pMBP) . Protein eluates from the mannose-Sepharose column were analyzed using reverse-phase high-performance liquid chromatography, sodium dodeclysulfate-polyacrylamide gel electrophoresis, and, subsequently, by N-terminal protein sequencing . Western blot analysis was used to identify the pMBP, and reverse transcriptionase-polymerase chain reaction was used to examine its mRNA expression . Complement lysis was measured using red blood cells coated with yeast mannan . Tumor necrosis factor (TNF)-alpha mRNA expression in macrophages was measured using RNase protection assay . RESULTS: A 30-kd MBP was isolated from both human and rat pancreatic juice and a rat acinar cell line . Genetic analysis (using RT-PCR with known MBP primers) and protein analysis (using Western blot with a known anti-MBP antibody) suggest that the pMBP is different from any previously described MBP . Protein sequencing analysis of pMBP generated an N-terminus sequence of 12 residues, indicating that pMBP is human pancreatic elastase III . Western blot analysis using an anti-elastase antibody confirms that the pMBP is a pancreatic elastase . Exposure of macrophages to pancreatic elastase resulted in an increased mRNA level of TNF-alpha, a potent proinflammatory cytokine in acute-phase response . Addition of mannan to pancreatic elastase further upregulated the TNF-alpha response . CONCLUSION: We isolated an MBP from the pancreas and identified it as pancreatic elastase . We characterized it as having properties different from that of any previously known MBP . We showed that pMBP or pancreatic elastase is involved in the activation of macrophages, and that this activation is potentiated by mannan . We postulate that the mannose-binding properties of pancreatic elastase identify this enzyme as a candidate catalyst for both pancreatic and systemic inflammation.

J Biol Chem, 2003 Aug 15, 278(33), 30719 - 24 Epub 2003 Jun 07.
Insulin induces the expression of the SHARP-2/Stra13/DEC1 gene via a phosphoinositide 3-kinase pathway; Yamada K et al.; Transcription of the rat fatty acid synthase (FAS) gene in the rat liver can be regulated by feeding a high carbohydrate diet . A carbohydrate response element (ChoRE) located on the rat FAS gene promoter has been identified . Using multiple copies of the ChoRE as the bait in a yeast one-hybrid system, a rat liver cDNA library was screened, and the cDNA of ChoRE-binding proteins was cloned . A positive clone that encodes a basic helix-loop-helix protein, enhancer of split- and hairy-related protein-2 (SHARP-2), was obtained . Northern blot analysis revealed that the levels of SHARP-2 mRNA increase when a high carbohydrate diet is fed to normal rats or when insulin is administered to diabetic rats . In primary cultured rat hepatocytes, insulin rapidly induced an accumulation of SHARP-2 mRNA even in the absence of glucose . A time course for the increase in SHARP-2 mRNA levels indicated that it followed by those of FAS and L-type pyruvate kinase mRNAs and that the initial time course of SHARP-2 mRNA was similar to changes in the levels of glucokinase mRNA and phosphoenolpyruvate carboxykinase mRNA . Although wortmannin, LY294002, and actinomycin D blocked the increase in SHARP-2 mRNA levels by insulin, rapamycin, staurosporine, PD98059, okadaic acid, and 8-bromocyclic AMP had no effect . In addition, nuclear run-on assay revealed that transcription of the rat SHARP-2 gene was induced by insulin . Thus, we conclude that insulin induces the transcription of the rat SHARP-2 gene via a phosphoinositide 3-kinase pathway.

Eukaryot Cell, 2003 Jun, 2(3), 456 - 64
Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima; Belli SI et al.; The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host . If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed . Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall . The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine- and possibly DOPA-protein cross-links in oocyst wall hardening . In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine- and DOPA-mediated cross-linking might be an enzyme-catalyzed event . As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes.

Plant J, 2003 Jun, 34(6), 753 - 67
Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis; Risseeuw EP et al.; Ubiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly specific manner . Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three 'core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts specificity for the ubiquitination of selected target proteins . Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways . In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins . Analysis of the protein interaction profiles involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more specifically with members of the F-box protein family . Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins . Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis . These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.

Biochemistry, 2003 Jun 17, 42(23), 7226 - 37
Identification of the motifs and amino acids in aggrecan G1 and G2 domains involved in product secretion; Kiani C et al.; Members of the large aggregating chondroitin sulfate proteoglycans are characterized by an N-terminal fragment known as G1 domain, which is composed of an immunoglobulin (IgG)-like motif and two tandem repeats (TR) . Previous studies have indicated that the expressed product of aggrecan G1 domain was not secreted . Here we demonstrated that the inability of G1 secretion was associated with the tandem repeats but not the IgG-like motif, and specifically with TR1 of aggrecan . We also demonstrated that the G2 domain, a domain unique to aggrecan, had a similar effect on product secretion . The sequence of TR1 of G1 is highly conserved across species, which suggested similar functions played by these motifs . In a yeast two-hybrid assay, TR1 interacted with the calcium homeostasis endoplasmic reticulum protein . Deletion/mutation experiments indicated that the N-terminal fragment of TR1, in particular, the amino acids H(2)R(4) of this motif were key to its effect on product secretion . However, the N-terminal 55 amino acids were required to exert this function . Taken together, our study suggests a possible molecular mechanism for the function of the tandem repeats in product processing.

J Biol Chem, 2003 Aug 15, 278(33), 31325 - 30 Epub 2003 Jun 06.
A structural basis for half-of-the-sites metal binding revealed in Drosophila melanogaster porphobilinogen synthase; Kundrat L et al.; Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements . Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized . The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding . The pH-dependent activity of DmPBGS is uniquely affected by zinc . A tight binding catalytic zinc binds at 0.5/subunit with a Kd well below microm . A second inhibitory zinc exhibits a Kd of approximately 5 microm and appears to bind at a stoichiometry of 1/subunit . A molecular model of DmPBGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands . Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid . As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc . H10F binds a catalytic zinc at 0.5/subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit . This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS.

Int J Mol Med, 2003 Jul, 12(1), 87 - 93
Cloning and structural characterization of the human histone deacetylase 6 gene; Voelter-Mahlknecht S et al.; Histone deacetylases (HDACs) play a central role in the modification of chromatin structure and thus in the regulation of transcription and cellular differentiation . Based on structural and functional similarities, mammalian histone deacetylases may be grouped into three categories, class I HDACs, which are homologs of the yeast histone deacetylase RPD3, class II HDACs, which share a significant degree of homology with the yeast histone deacetylase HDA1 and class III HDACs which are closely related to the yeast SIR2 protein . We have isolated and characterized the human HDAC6 genomic sequence, which spans a region of 21,923 bp and which has one single genomic locus . Determination of the exon-intron splice junctions established that HDAC6 is encoded by 28 exons ranging in size from 41 bp (exon 5) to 677 bp (exon 24) . Characterization of the 5' flanking genomic region, which precedes the HDAC6 open reading frame, revealed a TATA- and CCAAT-boxless promoter that contains a 1 kb CpG island . The 3,648 bp human HDAC6 mRNA encodes a 1,215 aa protein with a predictive molecular weight of 131.4 kDa . Fluorescence in situ hybridization analysis localized the human HDAC6 gene to the subband border of chromosome Xp11.22-23, a region which is characterized by frequent gains and losses of chromosomal material in several types of cancer and neurological disorders.

Science, 2003 Jun 6, 300(5625), 1569 - 74
Equatorial retention of the contractile actin ring by microtubules during cytokinesis; Pardo M et al.; In most eukaryotes cytokinesis is brought about by a contractile actin ring located at the division plane . Here, in fission yeast the actin ring was found to be required to generate late-mitotic microtubular structures located at the division plane, and these in turn maintained the medial position of the actin ring . When these microtubular structures were disrupted, the actin ring migrated away from the cell middle in a membrane traffic-dependent manner, resulting in asymmetrical cell divisions that led to genomic instability . We propose that these microtubular structures contribute to a checkpoint control that retains the equatorial position of the ring when progression through cytokinesis is delayed.

J Biol Chem, 2003 Aug 22, 278(34), 31685 - 90 Epub 2003 Jun 05.
Native human TATA-binding protein simultaneously binds and bends promoter DNA without a slow isomerization step or TFIIB requirement; Masters KM et al.; The association of TATA-binding protein (TBP) with promoter DNA is central to the initiation and regulation of eukaryotic protein synthesis . Our laboratory has previously conducted detailed investigations of this interaction using yeast TBP and seven consensus and variant TATA sequences . We have now investigated this key interaction using human TBP and the TATA sequence from the adenovirus major late promoter (AdMLP) . Recombinant native human protein was used together with fluorescently labeled DNA, allowing real time data acquisition in solution . We find that the wild-type hTBP-DNAAdMLP reaction is characterized by high affinity (Kd < or = 5 nm), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent approximately 100 degrees . These data allow, for the first time, a direct comparison of the reactions of the full-length, native human and yeast TBPs with a consensus promoter, studied under identical conditions . The general reaction characteristics are similar for the human and yeast proteins, although the details differ and the hTBPwt-induced bend is more severe . This directly measured hTBPwt-DNAAdMLP interaction differs fundamentally from a recently published hTBPwt-DNAAdMLP model characterized by low affinity (microM) binding and an unstable complex requiring either a 30-min isomerization or TFIIB to achieve DNA bending . Possible sources of these significant differences are discussed.

J Fam Pract, 2003 Jun, 52(6), 468 - 78
Herbs for serum cholesterol reduction: a systematic view; Thompson Coon JS et al.; OBJECTIVES: To systematically review the clinical evidence for herbal medicinal products in the treatment of hypercholesterolemia . STUDY DESIGN: A systematic review of randomized clinical trials of herbal medicinal products used to lower serum cholesterol . Systematic literature searches were conducted in 6 electronic data-bases . The reference lists of all papers and our files were searched for more relevant publications . Experts in the field and manufacturers of identified herbal medicinal products were contacted for published and unpublished data . No language restrictions were imposed . OUTCOMES MEASURED: All randomized clinical trials of serum cholesterol reduction, in which mono-preparations of herbal medicinal products were administered as supplements to human subjects, were included . RESULTS: Twenty-five randomized clinical trials involving 11 herbal medicinal products were identified . Guggul (Commiphora mukul), fenugreek (Trigonella foenum-graecum), red yeast rice, and artichoke (Cynara scolymus) have been most extensively studied and have demonstrated reductions in total serum cholesterol levels of between10% and 33% . The methodological quality as assessed by the Jadad score was less than 3 (maximum, 5) for 13 of the 25 trials . CONCLUSIONS: Many herbal medicinal products have potential hypocholesterolemic activity and encouraging safety profiles . However, only a limited amount of clinical research exists to support their efficacy . Further research is warranted to establish the value of these extracts in the treatment of hypercholesterolemia.

J Oral Rehabil, 2003 Jul, 30(7), 743 - 8
Oral health in relation to wearing removable dentures provided by dentists, denturists and laboratory technicians; Tuominen R; The aim of this study was to evaluate the oral health of elderly Finnish men wearing removable dentures supplied either by dentists, denturists or laboratory technicians . From a sample of 550 men, 362 subjects were both interviewed and clinically examined . The subjects were asked a range of questions related to their oral health and dentures . Clinical examinations were carried out by one dentist using well-defined criteria and without knowing the information the respective subjects had given in the interview . The 242 denture wearers had a higher frequency (P < 0.001) of mucous membrane lesions (78.7%) than the 120 non-wearers (27.5%) . Differences between the denture providers were small, although subjects with dentures provided by dentists had less often most of the recorded mucous membrane lesions than other denture wearers . Coating of tongue and cheilitis angularis were the most commonly encountered lesions . High levels of yeast growth were observed more frequently (P < 0.02) among subjects who had obtained their dentures from dentists (41.3%) than from either denturists (17.1%) or laboratory technicians (18.2%) . Among dentate subjects, the average number of remaining teeth was higher (P < 0.05) among those who had obtained their dentures from dentists (8.7) than among subjects wearing dentures from denturists (5.9) or laboratory technicians (6.2) . Subjects wearing dentures supplied by dentists had slightly better oral health than other denture wearers . The observed differences can be at least partly explained by dentists' patients having higher number of remaining teeth and also more frequent check-up visits.

Transpl Infect Dis, 2003 Mar, 5(1), 29 - 37
Impaired phagocyte respiratory burst responses to opportunistic fungal pathogens in transplant recipients: in vitro effect of r-metHuG-CSF (Filgrastim); Pursell K et al.; Phagocyte respiratory burst capacity in response to pathogenic fungi and the in vitro effects of granulocyte colony-stimulating factor (G-CSF) were examined in 15 normal volunteers and 39 transplant recipients (4 autologous and 4 allogeneic bone marrow, 3 heart, 10 liver, 8 lung, and 10 kidney) . Chemiluminescence was measured for reaction mixtures of diluted whole blood, opsonized fungi, and luminol, with and without in vitro incubation with r-metHuG-CSF (Filgrastim) . Transplant patients exhibited significantly impaired respiratory burst responses to conidia and yeast compared with controls, but this was reversed with Filgrastim . Responses to hyphae were low for both groups, and G-CSF had little or no effect . There was excellent correlation between responses to fungi and responses to opsonized zymosan . In vitro respiratory burst capacity is impaired in transplant recipients . This may predict susceptibility to invasive fungal infections . G-CSF can reverse impaired phagocyte function and is of potential benefit in the prevention and/or treatment of fungal infection in transplant patients.

J Biol Chem, 2003 Sep 5, 278(36), 34380 - 6 Epub 2003 Jun 04.
Genetic analysis of the myotubularin family of phosphatases in Caenorhabditis elegans; Xue Y et al.; Myotubularins (MTMs) constitute a large family of lipid phosphatases that specifically dephosphorylate phosphatidylinositol (3)P . MTM1 and MTM2 are mutated in X-linked myotubular myopathy and Charcot-Marie-Tooth disease (type 4B), respectively, although the mechanisms whereby MTM dysfunction leads to these diseases is unknown . To gain insight into MTM function, we undertook the study of MTMs in the nematode Caenorhabditis elegans, which possesses representative homologues of the four major subgroups of MTMs identified in mammals . As in mammals, we found that C . elegans MTMs mediate distinct functions . let-512 (vps34) encodes the C . elegans homologue of the yeast and mammalian homologue of the phosphatidylinositol 3-kinase Vps34 . We found that reduction of mtm-6 (F53A2.8) function by RNA inhibition rescued the larval lethality of let-512 (vps34) mutants and that the reduction of mtm-1 (Y110A7A.5) activity by RNA inhibition rescued the endocytosis defect of let-512 animals . Together, these observations provide genetic evidence that MTMs negatively regulate phosphatidylinositol (3)P levels . Analysis of MTM expression patterns using transcriptional green fluorescence protein reporters demonstrated that these two MTMs exhibit mostly non-overlapping expression patterns and that MTM-green fluorescence protein fusion proteins are localized to different subcellular locations . These observations suggest that some of the different functions of MTMs might, in part, be a consequence of unique expression and localization patterns . However, our finding that at least three C . elegans MTMs play essential roles in coelomocyte endocytosis, a process that also requires VPS34, indicates that MTMs do not simply turn off VPS34 but unexpectedly also function as positive regulators of biological processes.

J Biol Chem, 2003 Aug 15, 278(33), 30677 - 85 Epub 2003 Jun 03.
The protein phosphatase-1 (PP1) regulator, nuclear inhibitor of PP1 (NIPP1), interacts with the polycomb group protein, embryonic ectoderm development (EED), and functions as a transcriptional repressor; Jin Q et al.; The nuclear protein NIPP1 (nuclear inhibitor of protein Ser/Thr phosphatase-1) interacts with the splicing factors SAP155 and CDC5L and is involved in a late step of spliceosome assembly . In addition, NIPP1 is an interactor of protein phosphatase-1 and a COOH-terminal NIPP1 fragment displays an RNase E like endoribonuclease activity . A yeast two-hybrid screening resulted in the identification of the Polycomb group protein EED (embryonic ectoderm development), an established transcriptional repressor, as a novel NIPP1 interactor . NIPP1 only interacted with full-length EED, whereas two EED interaction domains were mapped to the central and COOH-terminal thirds of NIPP1 . The NIPP1-EED interaction was potentiated by the binding of (d)G-rich nucleic acids to the central domain of NIPP1 . Nucleic acids also decreased the potency of NIPP1 as an inhibitor of PP1, but they did not prevent the formation of a ternary NIPP1.EED.PP1 complex . EED had no effect on the function of NIPP1 as a splicing factor or as an endoribonuclease . However, similar to EED, NIPP1 acted as a transcriptional repressor of targeted genes and this NIPP1 effect was mediated by the EED interaction domain . Also, the histone deacetylase 2 was present in a complex with NIPP1 . Our data are in accordance with a role for NIPP1 as a DNA-targeting protein for EED and associated chromatin-modifying enzymes.

Appl Environ Microbiol, 2003 Jun, 69(6), 3311 - 6
Isolation of a methanogen from deep marine sediments that contain methane hydrates, and description of Methanoculleus submarinus sp . nov; Mikucki JA et al.; We isolated a methanogen from deep in the sediments of the Nankai Trough off the eastern coast of Japan . At the sampling site, the water was 950 m deep and the sediment core was collected at 247 m below the sediment surface . The isolated methanogen was named Nankai-1 . Cells of Nankai-1 were nonmotile and highly irregular coccoids (average diameter, 0.8 to 2 micro m) and grew with hydrogen or formate as a catabolic substrate . Cells required acetate as a carbon source . Yeast extract and peptones were not required but increased the growth rate . The cells were mesophilic, growing most rapidly at 45 degrees C (no growth at </=10 degrees C or >/=55 degrees C) . Cells grew with a maximum specific growth rate of 2.43 day(-1) at 45 degrees C . Cells grew at pH values between 5.0 and 8.7 but did not grow at pH 4.7 or 9.0 . Strain Nankai-1 grew in a wide range of salinities, from 0.1 to 1.5 M Na(+) . The described phenotypic characteristics of this novel isolate were consistent with the in situ environment of the Nankai Trough . This is the first report of a methanogenic isolate from methane hydrate-bearing sediments . Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is most closely related to Methanoculleus marisnigri (99.1% sequence similarity), but DNA hybridization experiments indicated a DNA sequence similarity of only 49% . Strain Nankai-1 was also found to be phenotypically similar to M . marisnigri, but two major phenotypic differences were found: strain Nankai-1 does not require peptones, and it grows fastest at a much higher temperature . We propose a new species, Methanoculleus submarinus, with strain Nankai-1 as the type strain.

FEBS Lett, 2003 Jun 12, 545(1), 39 - 46
Protonmotive pathways and mechanisms in the cytochrome bc1 complex; Hunte C et al.; The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane . In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol . The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur . Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways . In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.

Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 267 - 75
Modulation of YY1 activity by SAP30; Huang NE et al.; Yin Yang 1 (YY1) is a highly conserved and multifunctional transcription factor . The diverse activities of YY1 are regulated and sometimes modified by interaction with various other proteins . By using a yeast two-hybrid screening system, SAP30 was identified as a protein that associates with YY1 and it is able to enhance YY1-mediated repression in a dose-dependent manner . SAP30 is a 30kDa nuclear protein and is a component of the human histone deacetylase complex . In this study, the interaction of SAP30 and YY1 was confirmed both by in vitro and in vivo assays . The interaction domains between YY1 and SAP30 were mapped to the C-terminal segment of YY1 (295-414) and the C-terminal 91 amino acid region of SAP30 . The observation that YY1, SAP30, and HDAC1 form a complex in vivo provides evidence that YY1 also recruits HDAC1 indirectly via its binding to SAP30 . These results describe a novel mechanism for YY1-mediated repression.

Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 198 - 207
Cloning, sequencing, and characterization of the murine nm23-M5 gene during mouse spermatogenesis and spermiogenesis; Hwang KC et al.; Nucleoside diphosphate kinases (NDPKs) are conserved throughout evolution and have been shown to be involved in various biological phenomena . By functional screening in yeast, we identified a new member of the NDPK family, nm23-M5, which encodes a 211-amino acid protein with 86% identity to the human homolog Nm23-H5 . Northern blot analysis revealed that nm23-M5 encodes two transcripts of 0.8 and 0.7kb, which are highly and specifically expressed in adult testis . Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that nm23-M5 transcripts first appear in pachytene spermatocytes and increase in abundance in subsequent stages . However, a low level of nm23-M5 mRNA was detected by RT-PCR in other tissues, such as ovary, brain, heart, and kidney . In situ hybridization studies showed that testicular nm23-M5 transcripts are localized in stage 12 to stage 16 spermatids in the neighboring lumen of seminiferous tubules . This distribution contrasts with that of Nm23-H5 transcripts, which are specifically found in spermatogonia and early spermatocytes . The heterologous expression of nm23-M5 in yeast cells confers protection from cell death induced by Bax, which is due to the generation of reactive oxygen species . Furthermore, overexpression of nm23-M5 in fibroblasts altered the cellular levels of several antioxidant enzymes, particularly glutathione peroxidase 5 . Thus, we believe that the murine nm23-M5 gene plays an important role in late spermiogenesis by elevating the ability of late-stage spermatids to eliminate reactive oxygen species.

Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 79 - 86
MICAL-1 isoforms, novel rab1 interacting proteins; Weide T et al.; Rab1 GTPases participate in regulating the vesicular transport of ER-Golgi compartments . Recently, GM130, p115, and Golgin-84 were identified as effectors of the active conformation of rab1 . Here, we describe a novel protein, MICAL-1b, a splice variant of the MICAL-1a protein . Using the yeast two-hybrid system, we showed that it specifically interacts with rab1 in a nucleotide-dependent manner . The interaction was confirmed by GST pulldown experiments . Cell fractionation revealed that in contrast to the mainly membrane-associated rab1 effector GM130, MICAL-1 displays a predominantly cytosolic localization . We mapped the rab1 interacting domain to the C-terminus of MICAL-1, which also mediates binding to the intermediate filament vimentin . Therefore, the interaction of MICAL-1 and rab1 might provide a link between the Golgi apparatus and the intermediate filament cytoskeleton . We suggest that MICAL-1 isoforms with their multidomain structure are novel rab1 interacting proteins that function as scaffold proteins connecting different components in the cell.

Vet J, 2003 Jul, 166(1), 67 - 78
Evaluation of virucidal activity of three commercial disinfectants and formic acid using bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1; Yilmaz A et al.; A modified version of the test method of the Comite Europeen de Normalisation (CEN) was developed using formic acid and three commercial disinfectants to evaluate virucidal activity against three non-enveloped viruses, bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1 (BAV 1) . Determination of the effects of temperature was carried out at 20 and 10 degrees C . All tests with protein load used bovine serum albumin (BSA) and yeast extract . The investigations were performed in suspension tests and in carrier tests using poplar wood virus carriers . The carrier tests showed that ECBO virus could be inactivated at 20 degrees C with 1% formic acid within a 60 min reaction time . For disinfection of ECBO virus at 10 degrees C within 60 min, a 2% concentration of formic acid was necessary . Formic acid was ineffective against reovirus and bovine adenovirus and cannot be recommended as a reference disinfectant . Inactivation of ECBO virus and adenovirus type 1 using a disinfectant containing aldehydes and alcohols could be achieved, but only at room temperature . The disinfection of reovirus type 1 at room temperature with this product was possible without a protein load . This disinfectant exhibited disinfection ability at 10 degrees C at a concentration of more than 2% or with a longer exposure time . A disinfectant containing aldehydes was effective at room temperature but its effect was reduced in the presence of organic matter . Inactivation at 10 degrees C was found only against adenovirus . The fourth disinfectant, which contained peroxiacetic acid, inactivated all test viruses at a concentration of 0.5% within 15 min independent of temperature and protein load.

J Ethnopharmacol, 2003 Jul, 87(1), 11 - 3
Evaluation of antipyretic potential of Cleome viscosa Linn . (Capparidaceae) extract in rats; Devi BP et al.; The antipyretic activity of a methanol extract of Cleome viscosa Linn . (CVME) was investigated for its potential on normal body temperature and yeast-induced pyrexia in albino rats . The CVME, at doses of 200, 300, and 400mg/kg BW p.o., showed significant reduction in normal body temperature and yeast-provoked elevated temperature in a dose-dependent manner . The effect also extended upto 5h after the drug administration . The anti-pyretic effect of CVME was comparable to that of paracetamol (150mg/kg p.o.), a standard anti-pyretic agent.

Mutat Res, 2003 Jun 19, 527(1-2), 81 - 90
Novel tumorigenic rearrangement, Delta rfp/ret, in a papillary thyroid carcinoma from externally irradiated patient; Saenko V et al.; Molecular analysis of cDNA derived from a papillary thyroid carcinoma (PTC) (follicular variant of papillary thyroid carcinoma on histology) which developed in an externally irradiated patient 4 years after exposure identified a portion of the 5' region, exons 1-3, of the rfp gene juxtaposed upstream of the fragment encoding the tyrosine kinase (TK) domain of the ret gene . The fusion gene, termed Delta rfp/ret, was the result of a balanced chromosomal translocation t(6;10) (p21.3;q11.2) confirmed by interphase FISH painting, with breakpoints occurring in introns 3 and 11 of the rfp and ret genes, respectively . Both Delta rfp/ret and reciprocal ret/rfp chimeric introns had small deletions around breakpoints consistent with presumed misrepair of a radiation-induced double-strand DNA break underlying the rearrangement . No extensive sequence homology was found between the fragments flanking the breakpoints . The fusion protein retained the propensity to form oligomers likely to be mediated by a coiled-coil of the RFP polypeptide as assessed by a yeast two-hybrid system . NIH 3T3 fibroblasts stably transfected with a mammalian expression vector encoding full-length Delta RFP/RET readily gave rise to the tumors in athymic mice suggestive of high transforming potential of the fusion protein . Thus, the Delta rfp/ret rearrangement may be involved in a causative manner in cancerogenesis and provides additional evidence of the role of activated ret oncogene in the development of a subset of papillary thyroid carcinoma.

Curr Opin Cell Biol, 2003 Jun, 15(3), 326 - 31
Coupling transcription, splicing and mRNA export; Reed R; Previous studies have led to the view that mRNAs are transported from the nucleus to the cytoplasm by machinery that is conserved from yeast to humans . Moreover, this machinery is coupled both physically and functionally to the pre-mRNA splicing machinery . During the past year, important new insights into the mechanisms behind this coupling have been made . In addition, recent advances have revealed mechanisms for the co-transcriptional loading of the export machinery on to mRNAs . Finally, a newly identified link between the nuclear exosome and the machineries required for transcription, 3'-end formation and mRNA export suggests that proper mRNP formation is co-transcriptionally monitored.

Mol Microbiol, 2003 Jun, 48(5), 1209 - 23
Ten families of variant genes encoded in subtelomeric regions of multiple chromosomes of Plasmodium chabaudi, a malaria species that undergoes antigenic variation in the laboratory mouse; Fischer K et al.; The chromosome ends of human malaria parasites harbour many genes encoding proteins that are exported to the surface of infected red cells, often being involved in host-parasite interactions and immune evasion . Unlike other murine malaria parasites Plasmodium chabaudi undergoes antigenic variation during passage in the laboratory mouse and hence is a model suitable for investigation of switching mechanisms . However, little is known about the subtelomeric regions of P . chabaudi chromosomes and its variable antigens . Here we report 80 kb of sequence from an end of one P . chabaudi chromosome . Hybridization of probes spanning this region to two dimensional pulsed field gels of the genome revealed 10 multicopy gene families located exclusively in subtelomeric regions of multiple P . chabaudi chromosomes, interspersed amongst multicopy intergenic regions . Hence all chromosomes share a common subtelomeric structure, presumably playing a similar role in spatial positioning as the P . falciparum Rep20 sequence . Expression in blood stages, domains characteristic of surface antigens and copy numbers between four and several hundred per genome, indicate a functional role in antigenic variation for some of these families . We identify members of the cir family, as well as novel genes, that although clearly homologous to cir have large low complexity regions in the predicted extracellular domains . Although all families have homologues in other rodent Plasmodium species, four were previously not known to be subtelomeric . Six have homologues in human and simian malarias.

Plant J, 2003 Jun, 34(5), 623 - 33
The anticodon and the D-domain sequences are essential determinants for plant cytosolic tRNA(Val) import into mitochondria; Delage L et al.; In higher plants, one-third to one-half of the mitochondrial tRNAs are encoded in the nucleus and are imported into mitochondria . This process appears to be highly specific for some tRNAs, but the factors that interact with tRNAs before and/or during import, as well as the signals present on the tRNAs, still need to be identified . The rare experiments performed so far suggest that, besides the probable implication of aminoacyl-tRNA synthetases, at least one additional import factor and/or structural features shared by imported tRNAs must be involved in plant mitochondrial tRNA import . To look for determinants that direct tRNA import into higher plant mitochondria, we have transformed BY2 tobacco cells with Arabidopsis thaliana cytosolic tRNA(Val)(AAC) carrying various mutations . The nucleotide replacements introduced in this naturally imported tRNA correspond to the anticodon and/or D-domain of the non-imported cytosolic tRNA(Met-e) . Unlike the wild-type tRNA(Val)(AAC), a mutant tRNA(Val) carrying a methionine CAU anticodon that switches the aminoacylation of this tRNA from valine to methionine is not present in the mitochondrial fraction . Furthermore, mutant tRNAs(Val) carrying the D-domain of the tRNA(Met-e), although still efficiently recognized by the valyl-tRNA synthetase, are not imported any more into mitochondria . These data demonstrate that in plants, besides identity elements required for the recognition by the cognate aminoacyl-tRNA synthetase, tRNA molecules contain other determinants that are essential for mitochondrial import selectivity . Indeed, this suggests that the tRNA import mechanism occurring in plant mitochondria may be different from what has been described so far in yeast or in protozoa.

Phys Rev E Stat Nonlin Soft Matter Phys . 2003 May;67(5 Pt 1):051405 . Epub 2003 May 19.
Electrorotation in graded colloidal suspensions; Huang JP et al.; Biological cells can be treated as composites of graded material inclusions . In addition to biomaterials, graded composites are important in more traditional materials science . In this paper, we investigate the electrorotation spectrum of a graded colloidal suspension in an attempt to discuss its dielectric properties . For that, we use the recently obtained differential effective dipole approximation and generalize it for nonspherical particles . We find that variations in the conductivity profile may make the characteristic frequency redshifted and have also an effect on the rotation peak . On the other hand, variations in the dielectric profile may enhance the rotation peak, but do not have any significant effect on the characteristic frequency . In the end, we apply our theory to fit experimental data obtained for yeast cells and find good agreement.

Cell Mol Life Sci, 2003 Apr, 60(4), 701 - 10
The emerging role for sphingolipids in the eukaryotic heat shock response; Jenkins GM; Eukaryotic cells have a highly conserved response to an increase in temperature, termed the heat shock response . Recent research has revealed multiple roles for various sphingolipids in the heat shock responses of both yeast and mammalian cells . Heat stressed or shocked yeast and mammalian cells have an acute activation of serine palmitoyltransferase, resulting in the de novo biosynthesis of sphingolipids . Also, both mammalian and yeast cells were shown to increase ceramide levels upon heat stress or shock . In yeast cells, several functions have emerged for the de novo produced sphingoid bases in terms of the heat stress response . These functions include a role in accumulation of trehalose, a role in the heat-induced transient G0/G1 cell cycle arrest and phytosphingosine activation of a ubiquitin protein degradation pathway . However, in mammalian systems, ceramides have been demonstrated as bioactive lipids . Ceramides produced in response to heat shock were demonstrated to induce the production of c-jun, leading to apoptosis, and to be upstream of dephosphorylation of serine-rich proteins . Increasingly, sphingolipids are emerging as bioactive signaling molecules involved in numerous aspects of the eukaryotic heat shock response.

Cell Mol Life Sci, 2003 Apr, 60(4), 639 - 47
What's new in translation initiation? The first translation determines the fate of mRNA; McKendrick L; The two terms 'translation' and 'protein synthesis' are interchangeable in describing the process whereby the genetic code in the form of messenger RNA (mRNA) is deciphered such that amino acids cognate with the triplet code are joined end to end to form a peptide chain . However, new data suggest that the initial act of translation on newly synthesised mRNA also functions to proofread mRNA for errors . Aberrant mRNAs detected in this way are rapidly degraded before their encoded proteins impede normal cell function . Initiation of surveillance translation appears to differ from that of regular protein synthesis in three ways: (i) composition of the substrate; (ii) temporal and spatial restrictions; (iii) factors used to recruit the ribosome . This review discusses translational aspects of mRNA surveillance, primarily in the context of the mammalian system, although much information has come from studies in yeast and other organisms.

Prep Biochem Biotechnol, 2003 May, 33(2), 101 - 11
Contamination of ribosome inactivating proteins with ribonucleases, separated by affinity chromatography on red sepharose; Wang HX et al.; Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4) . The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity . It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity . It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 923 - 6
Requirement of negative residues, Asp 95 and Asp 105, in S2 on membrane integration of a voltage-dependent K+ channel, KAT1; Sato Y et al.; Voltage-dependent K+ channels consist of a voltage-sensing region and a pore-forming region . Here we have identified the negative residues of the second transmembrane segment in the plant voltage-dependent K+ channel, KAT1, which involves the function of voltage sensing . Point mutations at D95 and D105 but not D89 and D116 failed to complement the K+ uptake deficient properties of the mutant yeast . In vitro translation and translocation experiments showed that the membrane integration of the third and fourth segments involving voltage sensor were impaired by the replacement of D95 or D105 by serine . These data show that both the residues play a crucial role in the membrane topogenesis of the voltage sensor in KAT1.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 704 - 11
Gene cloning and functional analysis of a second delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus; Sakuradani E et al.; We demonstrated that Mortierella alpina 1S-4 has two delta 6-desaturases, which are involved in the desaturation of linoleic acid to gamma-linolenic acid . For one of the two delta 6-desaturases, designated as delta 6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported . In addition, we indicated in this paper that there is an isozyme of the two delta 6-desaturases, designated as delta 6II, in M . alpina 1S-4 . The predicted amino acid sequences of the Mortierella delta 6-desaturases were similar to those of ones from other organisms, i.e . borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast delta 9-desaturase, which has the corresponding domain at the C-terminus . The full-length delta 6II cDNA was expressed in A . oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids . The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quantity of delta 6I RNA was 2.4-, 9-, and 17-fold higher than that of delta 6II RNA on 2, 3, and 4 days in M . alpina 1S-4, respectively . M . alpina 1S-4 is the first fungus to be confirmed to have two functional delta 6-desaturase genes.

Mol Reprod Dev, 2003 Jul, 65(3), 298 - 308
Spermatid nuclear and sperm periaxonemal anomalies in the mouse Ube2b null mutant; Escalier D et al.; Ube2b (yeast Ubc2b/Rad6 homolog) null mice were described previously . Ube2b encodes the murine ubiquitin conjugating enzyme mHR6B . Ube2b(-/-) mice were shown to present male infertility and their sperm head shape anomalies suggested that Ube2b may be involved in the replacement of nuclear proteins during spermatid chromatin condensation . Apoptosis of spermatocytes suggested additional targets of Ube2b during spermatogenesis . Consistently, we found Ube2b transcription in both meiotic and postmeiotic stages by in situ hybridization . Immuno-electron microscopy revealed that transition proteins 1 and 2, protamines 1 and 2, and actin appear normally distributed during morphogenesis of Ube2b(-/-) spermatid heads . Surprisingly, electron microscopy revealed a particular sperm flagellum phenotype characterized by an abnormal distribution of periaxonemal structures . Flagellar anomalies of Ube2b null mice were previously described in infertile men indicating a possible genetic pathway for flagellar periaxonemal assembly in human .

J Biol Chem, 2003 Aug 22, 278(34), 32344 - 51 Epub 2003 Jun 03.
Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3-phosphoinositide-dependent kinase 1; Torbett NE et al.; Protein kinase N 1 (PKN1), which in part resembles yeast protein kinase C, has been shown to be under the control of Rho GTPases and 3-phosphoinositide-dependent kinase 1 (PDK1) . We show here that green fluorescent protein-tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress . PKN1 kinase activity is not necessary for this translocation, and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation . PKN1 translocation is dependent on Rac1 activation, although the GTPase binding HR1abc domain is not sufficient for this recruitment . The PKN1 kinase domain, however, localizes constitutively to this compartment, and we demonstrate that this behavior is selective for PKNs . Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation . It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1-positive compartment upon hyperosmotic stress . Taken together, our findings present a pathway for the selective hyperosmotic-induced Rac1-dependent PKN1 translocation and PDK1-dependent activation.

J Biol Chem, 2003 Aug 15, 278(33), 30669 - 76 Epub 2003 Jun 03.
Rpn5 is a conserved proteasome subunit and required for proper proteasome localization and assembly; Yen HC et al.; Proper function of the 26 S proteasome requires assembly of the regulatory complex, which is composed of the lid and base subcomplexes . We characterized Rpn5, a lid subunit, in fission yeast . We show that Rpn5 associates with the proteasome rpn5 . Deletion (rpn5Delta) exacerbates the growth defects in proteasome mutants, leading to mitotic abnormalities, which correlate with accumulation of polyubiquitinated proteins, such as Cut2/securin . Rpn5 expression is tightly controlled; both overexpression and deletion of rpn5 impair proteasome functions . The proteasome is assembled around the inner nuclear membrane in wild-type cells; however, in rpn5Delta cells, proteasome subunits are improperly assembled and/or localized . In the lid mutants, Rpn5 is mislocalized in the cytosol, while in the base mutants, Rpn5 can enter the nucleus, but is left in the nucleoplasm, and not assembled into the nuclear membrane . These results suggest that Rpn5 is a dosage-dependent proteasome regulator and plays a role in mediating proper proteasome assembly . Moreover, the Rpn5 assembly may be a cooperative process that involves at least two steps: 1) nuclear import and 2) subsequent assembly into the nuclear membrane . The former step requires other components of the lid, while the latter requires the base . Human Rpn5 rescues the phenotypes associated with rpn5Delta and is incorporated into the yeast proteasome, suggesting that Rpn5 functions are highly conserved.

J Biol Chem, 2003 Aug 29, 278(35), 33540 - 9 Epub 2003 Jun 03.
Identification of herpes simplex virus RNAs that interact specifically with regulatory protein ICP27 in vivo; Sokolowski M et al.; Herpes simplex virus type 1 (HSV-1) protein ICP27 has an essential regulatory role during viral replication, in part by post-transcriptional control of gene expression, and has a counterpart in all herpes viruses sequenced so far . Although much is known about the functions of this signature herpesvirus protein, little is known about its RNA binding capabilities; ICP27 interacts with specificity for a subset of intronless HSV-1 RNAs and poly(G), through its RGG box . We performed an in vivo yeast three-hybrid screen of an HSV-1 genomic library, searching for ICP27 interacting RNAs . Comparable with a yeast genomic screen, 24 of 55 single inserts mapped to antisense strands of HSV-1 transcribed regions or non-transcribed regions . The 31 HSV-1 sense RNAs identified were 35 to 225 nucleotides in length and interacted with preferred specificity for ICP27 as compared with an unrelated RNA-binding protein . They map to 10 monocistronic and 10 polycistronic transcripts of all kinetic classes and represent 28 open reading frames encoding predominantly essential viral proteins with roles in viral DNA replication and virion maturation . Several studies show regulatory effects by ICP27 on the majority of these transcripts, consistent with its regulation of the early-late switch in the HSV-1 life cycle . Deletion of the ICP27 RGG box and the ICP27 M15 mutation, both lethal in virus, abolished or severely reduced the ICP27-RNA interactions, indicating their biological relevance . The study facilitates continued study of gene regulation by ICP27 by further defining its interactions with viral RNAs.

Development, 2003 Jul, 130(14), 3283 - 95
Mutations in Arabidopsis condensin genes disrupt embryogenesis, meristem organization and segregation of homologous chromosomes during meiosis; Siddiqui NU et al.; Proper chromatin condensation and sister chromatid resolution are essential for the maintenance of chromosomal integrity during cell division, and is in part mediated by a conserved multisubunit apparatus termed the condensin complex . The core subunits of the complex are members of the SMC2 (Structural Maintenance of Chromosomes) and SMC4 gene families . We have cloned an Arabidopsis gene, AtCAP-E1, which is a functional ortholog of the yeast SMC2 gene . A second, highly homologous SMC2 gene, AtCAPE-2, was identified by the Arabidopsis genome project . SMC2 gene expression in Arabidopsis was correlated with the mitotic activity of tissues, with high level expression observed in meristematic cells . The two genes are differentially expressed with AtCAP-E1 accounting for more than 85% of the total SMC2 transcript pool . The titan3 mutant is the result of a T-DNA insertion into AtCAP-E1, but other than subtle endosperm defects, titan3 is viable and fecund . We identified a T-DNA insertion mutant of AtCAP-E2, which showed no obvious mutant phenotype, indicating that the two genes are functionally redundant . Genetic crosses were employed to examine the consequences of reduced SMC2 levels . Both male and female gametogenesis were compromised in double mutant spores . Embryo lethality was observed for both double homozygous and AtCAP-E1(-/-), AtCAP-E2(+/-) plants; arrest occurred at or before the globular stage and was associated with altered planes of cell division in both the suspensor and the embryo . Down regulation of both genes by antisense technology, as well as in AtCAP-E1(+/-), AtCAP-E2(-/-) plants results in meristem disorganization and fasciation . Our data are consistent with the interpretation that threshold levels of SMC2 proteins are required for normal development and that AtCAP-E2 may have a higher affinity for its target than AtCAP-E1.

J Hum Genet, 2003, 48(6), 315 - 21 Epub 2003 May 29.
Cloning and characterization of a novel gene which encodes a protein interacting with the mitosis-associated kinase-like protein NTKL; Di Y et al.; NTKL is an evolutionarily conserved kinase-like protein . The cell-cycle-dependent centrosomal localization of NTKL suggested that it was involved in centrosome-related cellular function . The mouse NTKL protein is highly homologous with human NTKL . A novel mouse protein was identified as an NTKL-binding protein (NTKL-BP1) by yeast two-hybrid screening, and the full-length cDNA was amplified based on the result of a sequence data analysis cloning strategy . The full-length cDNA sequence of the NTKL-BP1 gene consists of 2,537 bp, which encode 368 amino acids . A database search revealed that homologues of NTKL-BP1 exist in different organisms, including Arabidopsis thaliana, Drosophila melanogaster, Plasmodium falciparum, Geobacter metallireducens, Anopheles gambiae and human . It suggests that NTKL-BP1 is an evolutionarily conserved protein . The expression of NTKL-BP1 was observed in multiple normal mouse tissues . The interaction of the two proteins was confirmed by co-immunoprecipitation . Moreover, immunofluorescent staining indicated that NTKL and NTKL-BP1 were all localized in the cytoplasm.

Plant Cell, 2003 Jun, 15(6), 1281 - 95
The Arabidopsis MALE MEIOCYTE DEATH1 gene encodes a PHD-finger protein that is required for male meiosis; Yang X et al.; In plants, reproductive development requires normal meiosis, which involved several highly coordinated events . Such meiotic events are regulated in a number of ways in yeast and animal systems, including transcriptional and checkpoint control mechanisms . Although a number of mutations that affect different aspects of meiosis have been characterized in plants, very little is known about the regulation of plant meiosis at the molecular level . In particular, no meiosis-specific transcriptional regulators have been identified in plants, and checkpoint control has not been observed during plant meiosis . We report here the isolation and characterization of a new Arabidopsis male-sterile mutant that exhibits meiotic defects . Meiocytes from mutant plants appeared normal up to diakinesis, when they exhibited signs of apoptosis, including defects in chromosome behavior, cytoplasmic shrinkage, and chromatin fragmentation, followed by cell death before cytokinesis . Therefore, the mutant was named male meiocyte death1 (mmd1) . The MMD1 gene was cloned using Dissociation transposon tagging and encodes a plant homeo domain domain-containing protein . MMD1 is expressed preferentially during male meiosis . Our results suggest that MMD1 may be involved in the regulation of gene expression during meiosis and that the mmd1 mutation triggers cell death in male meiocytes.

J Microbiol Methods, 2003 Aug, 54(2), 193 - 201
Metabolic activity in filamentous fungi can be analysed by flow cytometry; Bradner JR et al.; The use of flow cytometry in combination with fluorescent dyes as a technique to rapidly differentiate and enumerate bacterial and yeast cells is well established . We have shown that through the judicial choice of stains, the nondestructive screening and sorting of fungal material is possible . The early stages of growth, from germination through hyphal development of three filamentous fungal species, Penicillium, Phoma and Trichoderma, have been followed using forward- and side-angle scatter on a Becton Dickinson FACSCalibur flow cytometer . By staining isolates with the permeant fluorogenic substrates, dihydroethidium and hexidium iodide metabolic activity in the developing hyphae has been measured . We have been able to demonstrate that there is a 12-13 h window of opportunity during which germination and the early stages of hyphal development of filamentous fungi can be analysed by flow cytometry.

FEBS Lett, 2003 Jun 5, 544(1-3), 93 - 8
Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley; Martens S et al.; Flavone synthases (FNSs) catalyze the oxidation of flavanones to flavones, i.e . the formation of apigenin from (2S)-naringenin . While many plants express a microsomal-type FNS II, the soluble FNS I appears to be confined to a few species of the Apiaceae and was cloned recently from parsley plants . FNS I belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenases characterized by short conserved sequence elements for cofactor binding, and its evolutionary context and mode of action are under investigation . Using a homology-based reverse transcription polymerase chain reaction approach, two additional flavonoid-specific dioxygenases were cloned from immature parsley leaflets, which were identified as flavanone 3beta-hydroxylase (FHT) and flavonol synthase (FLS) after expression in yeast cells . Sequence alignments revealed marginal differences among the parsley FNS I and FHT polypeptides of only 6%, while much less identity (about 29%) was observed with the parsley FLS . Analogous to FNS I, FLS oxidizes the flavonoid gamma-pyrone by introducing a C2, C3 double bond, and (2R,3S)-dihydrokaempferol (cis-dihydrokaempferol) was proposed recently as the most likely intermediate in both FNS I and FLS catalysis . Incubation of either FNS I or FLS with cis-dihydrokaempferol exclusively produced kaempferol and confirmed the assumption that flavonol formation occurs via hydroxylation at C3 followed by dehydratation . However, the lack of apigenin in these incubations ruled out cis-dihydrokaempferol as a free intermediate in FNS I catalysis . Furthermore, neither (+)-trans-dihydrokaempferol nor unnatural (-)-trans-dihydrokaempferol and 2-hydroxynaringenin served as a substrate for FNS I . Overall, the data suggest that FNS I has evolved uniquely in some Apiaceae as a paraphyletic gene from FHT, irrespective of the fact that FNS I and FLS catalyze equivalent desaturation reactions.

Mech Dev, 2003 May, 120(5), 629 - 37
Tob proteins enhance inhibitory Smad-receptor interactions to repress BMP signaling; Yoshida Y et al.; Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts . Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7 . A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob . Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors . Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos . Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells . Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.

Genomics, 2003 Jun, 81(6), 579 - 87
Identification of a novel protein interacting with laforin, the EPM2a progressive myoclonus epilepsy gene product; Ianzano L et al.; We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy . The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system . The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs . Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated . The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed . The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3' untranslated regions, maps to human chromosome 3p22.1 . The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs . Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found . The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.

Fitoterapia, 2003 Jun, 74(4), 345 - 9
Anti-inflammatory, analgesic and antipyretic properties of Clitoria ternatea root; Devi BP et al.; Clitoria ternatea roots methanol extract when given by oral route to rats was found to inhibit both the rat paw oedema caused by carrageenin and vascular permeability induced by acetic acid in rats . Moreover, the extract exhibited a significant inhibition in yeast-induced pyrexia in rats . In the acetic acid-induced writhing response, the extract markedly reduced the number of writhings at doses of 200 and 400 mg/kg (p.o.) in mice.

Curr Biol, 2003 May 27, 13(11), 942 - 6
Human POT1 facilitates telomere elongation by telomerase; Colgin LM et al.; Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang . Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure . Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA . In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified . Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation {10} . Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand . We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length . Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line . All three variants lengthened telomeres, and splice variant 1 was the most effective . hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced . These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.

Curr Biol, 2003 May 27, 13(11), 922 - 32
C . elegans PAT-6/actopaxin plays a critical role in the assembly of integrin adhesion complexes in vivo; Lin X et al.; BACKGROUND: The novel focal adhesion protein actopaxin includes tandem unconventional calponin homology (CH) domains and a less well-conserved N-terminal stretch . Dominant-negative studies have implicated actopaxin in focal adhesion formation . RESULTS: PAT-6/actopaxin, the sole actopaxin homolog in C . elegans, is located in body wall muscle attachments that are in vivo homologs of focal adhesions . We show using pat-6 protein null alleles that PAT-6/actopaxin has critical nonredundant roles during attachment maturation . It is required to recruit UNC-89 and myofilaments to newly forming attachments, and also to reposition the attachments so that they form the highly ordered array of dense body and M line attachments that are characteristic of mature muscle cells . PAT-6/actopaxin is not required for the deposition of UNC-52/perlecan in the basal lamina, nor for the initiation of attachment assembly, including the clustering of integrin into foci and the recruitment of attachment proteins PAT-4/ILK, UNC-112, and DEB-1/vinculin from the cytosol . PAT-6/actopaxin, PAT-4/ILK, and UNC-112 are each required for the same steps during attachment assembly in vivo, consistent with the notion that they work together in multiprotein complex . Supporting this idea, PAT-4/ILK can simultaneously bind to PAT-6/actopaxin and UNC-112, forming a ternary complex, in yeast three-hybrid assays . Finally, we show that both calponin homology domains are required for PAT-6/actopaxin's critical functions during attachment assembly in vivo . CONCLUSIONS: We show directly by loss-of-function genetics that PAT-6/actopaxin plays essential roles during the maturation of integrin-mediated muscle attachments in vivo.

Biochem J, 2003 Sep 1, 374(Pt 2), 359 - 67
Regulation of alternative splicing of Gtf2ird1 and its impact on slow muscle promoter activity; Tay ES et al.; A human MusTRD (muscle TFII-I repeat domain (RD)-containing protein) isoform was originally identified in a yeast one-hybrid screen as a protein that binds the slow fibre-specific enhancer of the muscle gene troponin I slow {O'Mahoney, Guven, Lin, Joya, Robinson, Wade and Hardeman (1998) Mol . Cell . Biol . 18, 6641-6652} . MusTRD shares homology with the general transcription factor TFII-I by the presence of diagnostic I-RDs {Roy (2001) Gene 274, 1-13} . The human gene encoding MusTRD, GTF2IRD1 ( WBSCR11 / GTF3 ), was subsequently located on chromosome 7q11.23, a region deleted in the neurodegenerative disease, Williams-Beuren Syndrome {Osborne, Campbell, Daradich, Scherer, Tsui, Franke, Peoples, Francke, Voit, Kramer et al . (1999) Genomics 57, 279-284; Franke, Peoples and Francke (1999) Cytogenet . Cell . Genet . 86, 296-304; Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur . J . Hum . Genet . 7, 737-747} . The haploinsufficiency of MusTRD has been implicated in the myopathic aspect of this disease, which manifests itself in symptoms such as lowered resistance to fatigue, kyphoscoliosis, an abnormal gait and joint contractures {Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur . J . Hum . Genet . 7, 737-747} . Here, we report the identification of 11 isoforms of MusTRD in mouse skeletal muscles . These isoforms were isolated from a mouse skeletal muscle cDNA library and reverse transcription-PCR on RNA from various adult and embryonic muscles . The variability in these isoforms arises from alternative splicing of a combination of four cassettes and two mutually exclusive exons, all in the 3' region of the primary transcript of Gtf2ird1, the homologous mouse gene . The expression of some of these isoforms is differentially regulated spatially, suggesting individual regulation of the expression of these isoforms . Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters . This indicates that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.

Biochem J, 2003 Aug 15, 374(Pt 1), 261 - 8
XB51 isoforms mediate Alzheimer's beta-amyloid peptide production by X11L (X11-like protein)-dependent and -independent mechanisms; Sumioka A et al.; XB51 (derived from X11-like binding protein of clone number 51) was isolated by yeast two-hybrid cDNA screening using the N-terminal domain of X11L (X11-like protein) as a bait . X11L is a neuron-specific adaptor protein that is known to down-regulate APP (beta-amyloid precursor protein) metabolism by associating with the cytoplasmic domain of APP, but the detailed mechanisms are still unknown . Thus the X11L-associated protein XB51 is believed to regulate APP metabolism by modifying X11L function through its interaction with X11L . Here we report that the hXB51 (human XB51 ) gene can yield two transcripts, one with exon 9 spliced out (resulting in the hXB51beta isoform) and the other containing exon 9 (yielding the hXB51alpha isoform) . hXB51alpha binds to X11L to form a tripartite complex composed of hXB51alpha, X11L and APP . Complex-formation results in blocking X11L's suppression of Abeta (beta-amyloid) generation from APP . hXB51beta associates with X11L and inhibits its interaction with APP . However, hXB51beta suppresses Abeta generation and secretion in an X11L-independent manner . Thus the hXB51 isoforms regulate Abeta generation differently, either enhancing it by modifying the association of X11L with APP or suppressing it in an X11L-independent manner . These observations advance our understanding of the molecular mechanisms regulating intracellular Abeta production and the pathogenesis of Alzheimer's disease.

Biochemistry, 2003 Jun 10, 42(22), 6827 - 39
Equilibrium folding of the core histones: the H3-H4 tetramer is less stable than the H2A-H2B dimer; Banks DD et al.; To compare the stability of structurally related dimers and to aid in understanding the thermodynamics of nucleosome assembly, the equilibrium stabilities of the recombinant wild-type H3-H4 tetramer and H2A-H2B dimer have been determined by guanidinium-induced denaturation, using fluorescence and circular dichroism spectroscopies . The unfolding of the tetramer and dimer are highly reversible . The unfolding of the H2A-H2B dimer is a two-state process, with no detected equilibrium intermediates . The H3-H4 tetramer is unstable at moderate ionic strengths (mu approximately 0.2 M) . TMAO (trimethylamine-N-oxide) was used to stabilize the tetramer; the stability of the H2A-H2B dimer was determined under the same solvent conditions . The equilibrium unfolding of H3-H4 was best described by a three-state mechanism, with well-folded H3-H4 dimers as a populated intermediate . When compared to H2A-H2B, the H3-H3 tetramer interface and the H3-H4 histone fold are strikingly less stable . The free energy of unfolding, in the absence of denaturant, for the H3-H4 and H2A-H2B dimers are 12.4 and 21.0 kcal mol(-)(1), respectively, in 1 M TMAO . It is postulated that the difference in stability between the histone dimers, which contain the same fold, is the result of unfavorable tertiary interactions, most likely the partial to complete burial of three salt bridges and burial of a charged hydrogen bond . Given the conservation of these buried interactions in histones from yeast to mammals, it is speculated that the H3-H4 tetramer has evolved to be unstable, and this instability may relate to its role in nucleosome dynamics.

Breast Cancer Res Treat, 2003 May, 79(1), 37 - 46
Comparison of p53 mutational status with mRNA and protein expression in a panel of 24 human breast carcinoma cell lines; Concin N et al.; We analyzed the p53 mutational status, mRNA and protein expression in 24 human breast carcinoma cell lines . Following measurement of their DNA content with flow cytometry, we ascertained the copy numbers of the centromere of chromosome 17 (cen17) and p53 with fluorescence in situ hybridization (FISH) . A functional yeast assay (FASAY) was used to screen for inactivating mutations . Positive results were subsequently verified by DNA sequencing . Finally, we assessed the mRNA expression with a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay and the protein expression with immunocytochemical staining, western blot, and quantitative flow cytometry . The DNA content of the cell lines ranged from 0.85 to 2.58 . Nine cell lines had concordant copy numbers (between two and four) of p53 and cen17, whereas 12 had more, and three less cen17 than p53 copies . The FASAY was successful in all but one cell line and revealed the presence of mutated alleles in 16 of them, 13 cell lines expressed only the mutated, and three both the mutated and the wild-type alleles . The mutations were comprised of 11 missense, two nonsense, and three frameshift mutations . Immunocytochemical staining, western blot and quantitative flow cytometry yielded comparable p53 protein expression results . However, both the mRNA and the protein expression levels varied considerably in the different cell lines and no consistent pattern with regard to the respective p53 mutational status became evident . The results obtained in these breast carcinoma cell lines indicate that no clear-cut linear relationship exists between the p53 mutational status and the extent of its respective mRNA and protein expression . Therefore, direct DNA analyses and functional assays remain the only methods for the reliable detection of p53 mutations.

J Biol Chem, 2003 Sep 5, 278(36), 34309 - 19 Epub 2003 May 30.
Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes . Possible link between Nop56p and the nucleolar protein treacle responsible for Treacher Collins syndrome; Hayano T et al.; Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation . Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing . However, its precise function remains elusive, especially in higher eukaryotes . Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes . Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown . Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells . Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes . The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction . In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.

J Biol Chem, 2003 Aug 15, 278(33), 30478 - 86 Epub 2003 May 30.
ARP-1/COUP-TF II determines hepatoma phenotype by acting as both a transcriptional repressor of microsomal triglyceride transfer protein and an inducer of CYP7A1; Kang S et al.; L35 and FAO cells were derived as single cell isolates from H35 cells . Whereas L35 cells do not express microsomal triglyceride transfer protein (MTP), which regulates lipoprotein secretion, they express CYP7A1, which regulates bile acid synthesis from cholesterol . FAO cells display the opposite phenotype (i.e . expression of MTP but not CYP7A1) . We examined the molecular basis of the transcriptional inactivation of the MTP gene in L35 cells . Nested deletion and mutagenesis studies show that a conserved DR1 element within the 135-bp proximal MTP promoter is responsible for differential expression by L35 and FAO cells . Yeast one-hybrid screening identified apolipoprotein A1 regulatory protein-1/chicken ovalbumin upstream promoter transcription factor II (ARP-1/COUP-TFII) and retinoid X receptor (RXRalpha) as the protein factors that can bind to the conserved DR1 element . Nuclear extracts from L35 cells contained 2-fold more ARP-1/COUP-TFII and 50% less RXRalpha than those from FAO cells . Immunologic studies show that in L35 cells, ARP-1/COUP-TFII is bound to the DR1 element, whereas in FAO cells, a complex containing RXRalpha is bound to the DR1 element . Co-transfection studies show that ARP-1/COUP-TFII repressed MTP promoter activity by approximately 70% in FAO hepatoma cells, whereas RXRalpha and its ligand 9-cis-retinoic acid increased MTP promoter activity by 6-fold in L35 cells . The combined data suggest that in the context of the MTP promoter, ARP-1/COUP-TFII (repressor) and a complex containing RXRalpha (inducer) compete for the DR1 element . Analysis of the CYP7A1 promoter revealed that it is approximately 5-fold more active in L35 cells than in FAO cells . Co-transfection of an ARP-1/COUP-TFII expression vector showed that it enhances CYP7A1 promoter activity by 6-fold in FAO cells . These combined findings indicate that ARP-1/COUP-TFII acts as both a transcriptional repressor (of MTP) and as a transcription activator (of CYP7A1) . This dual function of ARP-1/COUP-TFII may play an important role in determining the metabolic phenotype of individual liver cells.

BMC Mol Biol . 2003 May 30;4(1):7.
Interactions within the mammalian DNA methyltransferase family; Margot JB et al.; BACKGROUND: In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b . Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation . Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases . Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity . RESULTS: The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system . We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b . This interaction was confirmed by immunoprecipitation and was localized by deletion mapping . Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b . CONCLUSIONS: The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain . Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif . Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

Oncogene, 2003 May 29, 22(22), 3417 - 23
ZNF198 protein, involved in rearrangement in myeloproliferative disease, forms complexes with the DNA repair-associated HHR6A/6B and RAD18 proteins; Kunapuli P et al.; A highly specific t(8;13)(p11;q12) translocation has been consistently identified in bone marrow cells from patients with an atypical myeloproliferative disease that is associated with peripheral blood eosinophila and T- or B-cell leukemias . In all patients analysed to date, the translocation event results in a chimeric gene in which the atypical zinc-finger domain of ZNF198 is fused to the N-terminal end of the catalytic domain of the FGFR1 receptor tyrosine kinase . To understand more about the consequences of this rearrangement we have investigated the normal function of the ZNF198 gene . Using yeast two-hybrid analysis we identified HHR6 as a protein binding partner and confirmed this using immunoprecipitation studies . The ZNF198/FGFR1 fusion protein also binds to HHR6 . We demonstrate here that the human RAD18 is also present in the ZNF198/HHR6 protein complex, although it does not coimmunoprecipitate with the fusion kinase . Cells expressing the fusion kinase gene show a marked increased sensitivity to UVB irradiation, suggesting that it acts in a dominant-negative way to affect DNA repair . These observations support the idea that ZNF198, through its interaction with HHR6 and RAD18, may be involved in the DNA repair process.

Oncogene, 2003 May 29, 22(22), 3395 - 406
The mixed lineage leukemia fusion partner AF9 binds specific isoforms of the BCL-6 corepressor; Srinivasan RS et al.; The mixed lineage leukemia (MLL) gene at chromosome band 11q23 is commonly involved in reciprocal translocations that are detected in acute leukemias . Evidence suggests that the resulting MLL fusion genes contribute to leukemogenesis . AF9 is a common MLL fusion partner in acute myeloid leukemia . The AF9 protein functions as a transcriptional activator in artificial reporter gene assays and a structurally related protein in yeast, ANC1/TFG3, is a component of the SWI/SNF complex . Apart from these observations, little is known about the biologic function of AF9 in mammals . We have found that a recently described transcriptional repressor, BCL-6 corepressor (BCoR), interacts with the carboxy-terminus of AF9 . The interaction of AF9 with BCoR has been confirmed by independent in vitro and in vivo protein-binding studies . The BCoR gene is expressed as several alternatively spliced transcripts . AF9 only binds BCoR isoforms that contain a unique 34 aa sequence located in the mid-portion of the protein . In artificial reporter gene assays, a BCoR isoform that binds AF9 efficiently suppresses AF9 transcriptional activity, while a nonbinding isoform does not . These results indicate that different isoforms of BCoR have unique biologic properties and that cell function may be partly determined by the different isoforms that are present within the cell.

EMBO Rep, 2003 Jun, 4(6), 616 - 22
Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron; Mouaikel J et al.; The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5' cap . Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs . Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein . Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies . The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy . These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.

EMBO Rep, 2003 Jun, 4(6), 571 - 4
IQGAP proteins are integral components of cytoskeletal regulation; Briggs MW et al.; IQGAP1 is a scaffolding protein that binds to a diverse array of signalling and structural molecules . By interacting with its target proteins, human IQGAP1 participates in multiple cellular functions, including Ca(2+)/calmodulin signalling, cytoskeletal architecture, CDC42 and Rac signalling, E-cadherin-mediated cell-cell adhesion and beta-catenin-mediated transcription . Yeast IQGAP homologues are important regulators of cellular morphogenesis because they are required for budding and cytokinesis . Here we discuss the structure and function of IQGAP1 as a member of the family of IQGAP proteins and summarize the current knowledge about IQGAP1 and IQGAP2 . Collectively, these data reveal that IQGAP1 is a fundamental regulator of cytoskeletal function.

J Biol Chem, 2003 Aug 22, 278(34), 32115 - 23 Epub 2003 May 28.
Kinectin anchors the translation elongation factor-1 delta to the endoplasmic reticulum; Ong LL et al.; Kinectin has been proposed to be a membrane anchor for kinesin on intracellular organelles . A kinectin isoform that lacks a major portion of the kinesin-binding domain does not bind kinesin but interacts with another resident of the endoplasmic reticulum, the translation elongation factor-1 delta (EF-1 delta) . This was shown by yeast two-hybrid analysis and a number of in vitro and in vivo assays . EF-1 delta provides the guanine nucleotide exchange activities on EF-1 alpha during elongation step of protein synthesis . The minimal EF-1 delta-binding domain on kinectin resides within a conserved region present in all the kinectin isoforms . Overexpression of the kinectin fragments in vivo disrupted the intracellular localization of EF-1 delta proteins . This report provides evidence of an alternative kinectin function as the membrane anchor for EF-1 delta on the endoplasmic reticulum and provides clues to the EF-1 complex assembly and anchorage on the endoplasmic reticulum.

J Biol Chem, 2003 Aug 15, 278(33), 30975 - 84 Epub 2003 May 28.
The Rho guanine nucleotide exchange factor Lsc homo-oligomerizes and is negatively regulated through domains in its carboxyl terminus that are absent in novel splenic isoforms; Eisenhaure TM et al.; Rho GTPases control fundamental cellular processes, including cytoskeletal reorganization and transcription . Rho guanine nucleotide exchange factors (GEFs) compose a large (>65) and diverse family of related proteins that activate Rho GTPases . Lsc/p115-RhoGEF is a Rho-specific GEF required for normal B and T lymphocyte function . Despite its essential role in lymphocytes, Lsc/p115-RhoGEF signaling in vivo is not well understood . To define Lsc/p115-RhoGEF signaling pathways in vivo, we set out to identify proteins that interact with regulatory regions of Lsc . The 146-amino acid C terminus of Lsc contains a predicted coiled-coil domain, and we demonstrated that deletion of this C terminus confers a gain of function in vivo . Surprisingly, a yeast two-hybrid screen for proteins that interact with this regulatory C terminus isolated a larger C-terminal fragment of Lsc itself . Co-immunoprecipitation experiments in mammalian cells demonstrated that Lsc specifically homo-oligomerizes and that the coiled-coil domain in the C terminus is required for homo-oligomerization . Mutagenesis experiments revealed that homo-oligomerization and negative regulation are distinct functions of the C terminus . Two novel isoforms of Lsc found in the spleen lack portions of this C terminus, including the coiled-coil domain . Importantly, the C termini of both isoforms confer a gain of function and eliminate homo-oligomerization . These results define two important features of Lsc signaling . First, Lsc homo-oligomerizes and is negatively regulated through domains in its C terminus; and second, functionally distinct isoforms of Lsc lacking these domains are present in the spleen.

EMBO J, 2003 Jun 2, 22(11), 2852 - 9
The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment; Daughenbaugh KF et al.; The positive-strand RNA genomes of caliciviruses are not capped, but are instead covalently linked at their 5' ends to a viral protein called VPg . The lack of a cap structure typical of eukaryotic mRNA and absence of an internal ribosomal entry site suggest that VPg may function in translation initiation on calicivirus RNA . This hypothesis was tested by analyzing binding of Norwalk virus VPg to translation initiation factors . The eIF3d subunit of eIF3 was identified as a binding partner of VPg by yeast two-hybrid analysis . VPg bound to purified mammalian eIF3 and to eIF3 in mammalian cell lysates . To test the effects of the VPg- eIF3 interaction on translation, VPg was added to cell-free translation reactions programmed with either capped reporter RNA, an RNA containing an EMCV internal ribosomal entry site (IRES) or an RNA with a cricket paralysis virus IRES . VPg inhibited translation of all reporter RNAs in a dose-dependent manner . Together, the data suggest that VPg may play a role in initiating translation on calicivirus RNA through unique protein-protein interactions with the translation machinery.

EMBO J, 2003 Jun 2, 22(11), 2841 - 51
Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates; Trotta CR et al.; 60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other . We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP . Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3 . Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation . When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation . The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei . These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export . We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.

EMBO J, 2003 Jun 2, 22(11), 2764 - 75
Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly; Sakai A et al.; Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively . They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiled-coil with the terminal globular domains and the central hinge . Condensin and cohesin holo-complexes contain three and two non-SMC subunits, respectively . In this study, DNA interaction with cohesin and condensin complexes purified from fission yeast was investigated . The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holo-condensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21-bound heterotrimer complexes . One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity . In addition, the coiled-coil and hinge region of another SMC are needed . Atomic force microscopy discloses the molecular events of DNA reannealing . SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step . SMC is eliminated from the completed double-stranded DNA . The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non-SMC heterotrimeric complex.

Proj Inf Perspect, 2000 Mar, (29), 14 - 5
Treating and preventing fungal infections naturally; Crossover interference in humans; Departments of Mathematics and Biology, Indiana University, Bloomington, IN 47405, USA . ehouswor@indiana.edu

Crossing-over between homologous chromosomes facilitates proper disjunction of chromosomes during meiosis I . In many organisms, gene functions that are essential to crossing-over also facilitate the intimate chromosome pairing called "synapsis." Many organisms--including budding yeast, humans, zebrafish, Drosophila, and Arabidopsis--regulate the distribution of crossovers, so that, most of the time, each chromosome bundle gets at least one crossover while the mean number of crossovers per chromosome remains modest . This regulation is obtained through crossover interference . Recent evidence suggests that the organisms that use recombination functions to achieve synapsis have two classes of crossovers, only one of which is subject to interference . We statistically test this two-pathway hypothesis in the CEPH data and find evidence to support the two-pathway hypothesis in humans.

Nucleic Acids Res, 2003 Jun 1, 31(11), 2915 - 25
A new member of the MCM protein family encoded by the human MCM8 gene, located contrapodal to GCD10 at chromosome band 20p12.3-13; Johnson EM et al.; The MCM8 protein from HeLa cells, a new member of the MCM family, co-isolates through several steps with MCM6 and MCM7, and MCM8 co-immunoprecipitates with MCM4, MCM6 and MCM7, proteins reportedly forming a helicase complex involved in initiation of DNA replication . MCM8 mRNA is expressed in placenta, lung and liver, but is also significantly expressed in adult heart, a tissue with a low percentage of proliferating cells . The MCM8 gene, consisting of 19 exons, is located contrapodal to a gene, consisting of 11 exons, encoding a homolog of the yeast GCD10 gene product . The region between these two transcription units, comprising as few as 62 bp, is TATA-less and highly GC-rich, containing multiple CpG units . MCM8 expression is altered in certain forms of neoplasia . In a case of choriocarcinoma MCM8 mRNA is aberrant, leading to expression of a protein lacking 16 amino acids . In several cases of colon adenocarcinoma MCM8 expression is greatly reduced relative to matched non-cancerous tissue . The potential helicase domain of MCM8 is different from those of other MCM proteins in that it is more homologous to canonical ATP-binding domains of other known helicases . Results suggest that MCM8 may interact with other MCM proteins to alter the function of the replicative MCM protein complex.

J Cell Sci, 2003 Jul 15, 116(Pt 14), 2929 - 36 Epub 2003 May 27.
BTG2 antiproliferative protein interacts with the human CCR4 complex existing in vivo in three cell-cycle-regulated forms; Morel AP et al.; The yeast CCR4-NOT complex exists in two forms (1.0 and 1.9 MDa) that share several common subunits, including yCCR4, yCAF1 and five NOT proteins (NOT1-5) . Here, we report that different complexes containing mammalian homologs of CCR4-NOT subunits exist in mammalian cells, with estimated sizes of approximately 1.9 MDa, approximately 1 MDa and approximately 650 kDa, and that BTG2, a member of a protein family with antiproliferative functions, can associate with these complexes . Immunoprecipitation and gel filtration experiments established that BTG2 interacts in vivo with hCCR4 protein via hCAF1 and hPOP2 . Moreover, we show that hCCR4, as well as hCAF1 and BTG2, modulate the transcription regulation mediated by ERalpha . Finally, we demonstrate that the cellular localization of hCAF1 and the cell content in hCAF1-containing complexes change as cells progress from quiescence to S phase . These findings suggest that the different regulatory pathways in which hCAF1 is involved, notably transcription regulation and mRNA turnover, may occur through distinct CCR4 complexes in the course of cell-cycle progression.

J Biol Chem, 2003 Aug 22, 278(34), 31564 - 73 Epub 2003 May 27.
Two novel proteins that are linked to insulin-like growth factor (IGF-I) receptors by the Grb10 adapter and modulate IGF-I signaling; Giovannone B et al.; Grb10 is a protein that binds to the intracellular domains of activated tyrosine kinase receptors, including insulin-like growth factor (IGF-I) and insulin receptors . This occurs through the interaction of two C-terminal Grb10 motifs (BPS and Src homology domains) with receptor phosphotyrosine residues . Published data from transfection/overexpression studies support both positive and negative regulatory effects of Grb10, thus leaving its physiological role unclear . Because Grb10 has the structure of an adapter protein, the objective of this study was to determine whether Grb10 links other proteins to IGF-I receptors and thus modulates IGF-I signaling . Using yeast two-hybrid screening, the N terminus of Grb10 was shown to interact with two novel proteins, designated GIGYF1 (Grb10 interacting GYF protein 1) and GIGYF2 . Mutation analysis indicates that a 17-amino acid sequence in GIGYF1 and GIGYF2, homologous to the GYF domain described previously, binds to tandem proline-rich regions in the N terminus of Grb10 . In IGF-I receptor-expressing R+ fibroblasts, there is detectable binding of a Myc-tagged fragment of GIGYF1 to Grb10 in the basal state . Stimulation with IGF-I results in increased binding of GIGYF1 to Grb10 and transient binding of both Grb10 and GIGYF1 to IGF-I receptors, presumably via the adapter function of Grb10 . At later time points, GIGYF1 dissociates, but Grb10 remains linked to IGF-I receptors . Overexpression of the Grb10 binding fragment of GIGYF1 in R+ cells results in a significant increase in IGF-I-stimulated receptor tyrosine phosphorylation . In conclusion, we have identified two members of a novel protein family, which become transiently linked to IGF-I receptors by the Grb10 adapter protein following IGF-I stimulation . Grb10 and GIGYFs may act cooperatively to regulate receptor signaling.

J Biol Chem, 2003 Aug 8, 278(32), 29560 - 70 Epub 2003 May 27.
The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1; Kruljac-Letunic A et al.; Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton . In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait . A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1 . Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1 . Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells . Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation . We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.

J Cell Biol, 2003 May 26, 161(4), 671 - 2
Is HP1 an RNA detector that functions both in repression and activation?
Kellum R.
Heterochromatin is defined as regions of compact chromatin that persist throughout the cell cycle (Heitz, 1928) . The earliest cytological observations of heterochromatin were followed by ribonucleotide labeling experiments that showed it to be transcriptionally inert relative to the more typical euchromatic regions that decondense during interphase . Genetic studies of rearrangements that place euchromatic genes next to blocks of heterochromatin also pointed out the repressive nature of heterochromatin (Grigliatti, 1991; and references therein) . The discovery of the heterochromatin-enriched protein heterochromatin protein 1 (HP1)**Abbreviation used in this paper: HP1, heterochromatin protein 1 . by Elgin and co-workers in the mid-1980s suggested that the distinct cytological features of this chromatin may be related to its unique nucleoprotein composition (James and Elgin, 1986; James et al., 1989) . HP1 immunostaining on polytene chromosomes from Drosophila larval salivary glands was used to show enrichment of the protein in pericentric heterochromatin . Since that initial discovery, HP1 homologues have been found in species ranging from fission yeast to humans where it is associated with gene silencing (Eissenberg and Elgin, 2000; and references therein) . A number of euchromatic sites of localization were also reported in this original study . It has been generally assumed that these sites might constitute euchromatic sites of transcriptional repression by HP1 . Indeed, several genes located at one of these sites (cytological region 31) have increased transcript levels in mutants for HP1 (Hwang et al., 2001).

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