Yeasts

J Cell Physiol, 2003 May, 195(2), 151 - 7
Human brain synembryn interacts with Gsalpha and Gqalpha and is translocated to the plasma membrane in response to isoproterenol and carbachol; Klattenhoff C et al.; Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions . In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha . To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait . We identified a protein member of the synembryn family as one of the interacting proteins . Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis . Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol . This study supports recent findings in C . elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release . Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells .

Nat Cell Biol, 2003 Apr, 5(4), 320 - 9
Ku70 suppresses the apoptotic translocation of Bax to mitochondria; Sawada M et al.; Bax induces mitochondrial-dependent cell death signals in mammalian cells . However, the mechanism of how Bax is kept inactive has remained unclear . Yeast-based functional screening of Bax inhibitors from mammalian cDNA libraries identified Ku70 as a new Bax suppressor . Bax-mediated apoptosis was suppressed by overexpression of Ku70 in mammalian cells, but enhanced by downregulation of Ku70 . We found that Ku70 interacts with Bax, and that the carboxyl terminus of Ku70 and the amino terminus of Bax are required for this interaction . Bax is known to translocate from the cytosol to mitochondria when cells receive apoptotic stimuli . We found that Ku70 blocks the mitochondrial translocation of Bax . These results suggest that in addition to its previously recognized DNA repair activity in the nucleus, Ku70 has a cytoprotective function in the cytosol that controls the localization of Bax.

Am J Dermatopathol, 2003 Apr, 25(2), 152 - 4
Sweet's syndrome-like blastomycosis; Wilkerson A et al.; Cutaneous North American blastomycosis is characterized clinically by verrucous nodules and histologically by pseudoepitheliomatous hyperplasia, intraepidermal neutrophilic microabscesses, and a dermal mixed inflammatory cell infiltrate containing giant cells . We describe a patient who presented clinically with erythematous nodules and plaques on the lower extremities characterized histologically by a diffuse neutrophilic infiltrate, with lack of epidermal hyperplasia . The lesions were clinically and histologically reminiscent of Sweets syndrome . On close microscopic inspection scattered histiocytes and multinucleated giant cells were present in the dermis, and fungal stains demonstrated budding yeast forms consistent with Blastomyces sp.

Mol Cancer Res, 2003 Mar, 1(5), 402 - 9
The absence of SIR2alpha protein has no effect on global gene silencing in mouse embryonic stem cells; McBurney MW et al.; The yeast sir2 gene plays a central role in mediating gene silencing and DNA repair in this organism . The mouse sir2alpha gene is closely related to its yeast homologue and encodes a nuclear protein expressed at particularly high levels in embryonic stem (ES) cells . We used homologous recombination to create ES cells null for sir2alpha and found that these cells did not have elevated levels of acetylated histones and did not ectopically express silent genes . Unlike yeast sir2 mutants, our sir2alpha null ES cells had normal sensitivity to insults such as ionizing radiation and heat shock, and they were able to silence invading retroviruses normally . These sir2alpha null cells were able to differentiate in culture normally . Our results failed to provide evidence that the mammalian SIR2alpha protein plays a role in gene silencing and suggest that the physiological substrate(s) for the SIR2alpha deacetylase may be nuclear proteins other than histones.

Ecotoxicol Environ Saf, 2003 Mar, 54(3), 315 - 22
Potencies of estrogenic compounds in in vitro screening assays and in life cycle tests with zebrafish in vivo; Segner H et al.; The objective of this study was to compare the estrogenic potency of environmental estrogens at two testing tiers: at the initial level of in vitro screening assays, and at the level of definitive fish reproduction tests in vivo . The in vitro tests included a recombinant yeast estrogen receptor (ER) assay, a competitive radioreceptor assay using the hepatic ER of carp (Cyprinus carpio), and assays on vitellogenin induction in cultured hepatocytes of rainbow trout (Oncorhynchus mykiss) and carp . In vivo, full life cycle tests with zebrafish (Danio rerio) were performed, using fertilization success as estrogen-sensitive reproductive endpoint . The test compounds included the natural estrogen 17beta-estradiol (E2) (only applied in the in vitro assays); the synthetic estrogen ethynylestradiol (EE2); and two xenoestrogens, 4-tert-octylphenol (OP) and bisphenol A (BPA) . Among the in vitro assays, differences were observed in the relative ranking of the test substances, and in the absolute sensitivity (EC50 values), although the interassay differences of EC50 values were within one order of magnitude . The in vivo activity of the test compounds was not accurately predicted by the in vitro assays, with respect to neither sensitivity nor ranking . The in vitro assays tended to overestimate the relative potency of the xenoestrogens; i.e . the ratio between the activity of the reference compound, EE2, and that of the test compound . The best prediction of the in vivo fish test results was obtained from the recombinant yeast assay.

Exp Cell Res, 2003 Apr 1, 284(2), 224 - 38
PACE-1, a novel protein that interacts with the C-terminal domain of ezrin; Sullivan A et al.; The ERM proteins (ezrin, radixin, moesin) together with merlin comprise a subgroup of the band 4.1 superfamily . These proteins act as membrane cytoskeletal linker proteins mediating interactions between the cytoplasmic domains of transmembrane proteins and actin . To better understand how the ERM proteins function to regulate these junctional complexes, a yeast 2-hybrid screen was undertaken using ezrin as a bait . We describe here the identification and cloning of a novel protein, PACE-1, which binds to the C-terminal domain of ezrin . Characterization of PACE-1 in human breast cancer cell lines demonstrates it to have two distinct intracellular localizations . A proportion of the protein is associated with the cytoplasmic face of the Golgi apparatus . This distribution is dependent upon the presence of the PACE-1 N-terminal myristoylation consensus sequence but is not dependent on an association with ezrin . In contrast, PACE-1 colocalises with ezrin in the lamellipodia, where ezrin has a role in cell spreading and motility . A notable feature of PACE-1 is the presence of a putative N-terminal kinase domain; however, in biochemical assays PACE-1 was shown to have associated rather than intrinsic kinase activity . Together these data suggest that PACE-1 may play a role in regulating cell adhesion/migration complexes in migrating cells.

FEBS Lett, 2003 Mar 27, 539(1-3), 167 - 73
Identification of three novel Smad binding proteins involved in cell polarity; Warner DR et al.; A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue . Three proteins (Erbin, Par-3, and Dishevelled) were identified that share several similar structural and functional characteristics . Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity . In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein . Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain . Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity . Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity . While transforming growth factor beta (TGFbeta) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFbeta could alter cell polarity via a Smad-dependent signaling mechanism.

J Steroid Biochem Mol Biol, 2002 Dec, 83(1-5), 227 - 33
Stimulation of alkaline phosphatase activity in Ishikawa cells induced by various phytoestrogens and synthetic estrogens; Wober J et al.; Xenoestrogens, phytoestrogens and synthetic estrogens, are able to bind to estrogen receptors, and to mimic estrogenic activities in a cell and tissue specific manner . For the characterization of environmental estrogens mainly mammary derived and yeast based models have been used . The aim of this study was therefore to assess selected natural and synthetic compounds in an endometrial derived model . We measured the relative estrogenic potency of phytoestrogens (genistein, daidzein, coumestrol, some naringenins), synthetic estrogens (bisphenol A, octylphenol, nonylphenol, o,p'-DDT), mycoestrogen (zearalanone) as well as extracts of Cimicifuga racemosa on alkaline phosphatase (AlkP) activity in the endometrial derived adenocarcinoma cell line Ishikawa . We used a modified multiwell plate in vitro bioassay based on the estrogen-specific and dose-dependent enhancement of AlkP activity in this cell line . Estradiol, which induced AlkP at levels as low as 10(-8)M, was used as positive control . Most of the compounds studied showed a clear dose-dependent estrogenic effect . Compared to the vehicle control (ethanol) all phyto- and mycoestrogens, stimulated the AlkP activity 2-4-fold at a concentration of 10(-6)M . The synthetic chemicals bisphenol A and nonylphenol showed an effect at 10(-6)M, octylphenol at 10(-5)M . Effects of o,p'-DTT could not be measured . ICI 182,780, a pure estrogen receptor antagonist, significantly inhibited these effects . The latter result demonstrated the estrogen receptor dependency of this process . In summary, most of the phytoestrogens and industrial chemicals tested, behaved as estrogen receptor agonists in terms of the stimulation of AlkP activity.

J Steroid Biochem Mol Biol, 2002 Dec, 83(1-5), 93 - 9
Transcriptional regulation of aromatase expression in human breast tissue; Chen S et al.; Aromatase (CYP19) is the estrogen synthetase that converts androgen to estrogen . The expression of aromatase in breast cancer cells and surrounding stromal cells is up regulated compared to non-cancerous cells . In situ estrogen synthesis is thought to stimulate breast cancer growth in both an autocrine and a paracrine manner . A complex mechanism is involved in the control of human aromatase expression, in that seven promoters have been identified and found to be utilized in a tissue-selective manner . Increased aromatase expression in breast tumors is, in part, attributed to changes in the transcriptional control of aromatase expression . While promoter I.4 is the main promoter that controls aromatase expression in non-cancer breast tissue, promoters II and I.3 are the dominant promoters that drive aromatase expression in breast cancer tissue . During the last several years, our laboratory performed a series of studies to examine the transcription regulatory mechanism of aromatase expression in breast cancer cells . We functionally characterized promoters II and I.3, and carried out DNase 1 footprinting analysis that identified two regulatory elements, S1 and CREaro . Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that four orphan/nuclear receptors, ERR alpha-1, EAR-2, COUP-TFI and RAR gamma, bind to the S1 element, and that CREB1, Snail (SnaH) and Slug proteins bind to the CREaro element . Studies from this and other laboratories have revealed that in cancer tissue versus normal tissue, several positive regulatory proteins (e.g . ERR alpha-1 and CREB1) are present at higher levels and several negative regulatory proteins (e.g . EAR-2, COUP-TFI, RAR gamma, Snail and Slug proteins) are present at lower levels . This may explain why the activity of promoters II and I.3 is up regulated in cancer tissue . An understanding of the molecular mechanisms of aromatase expression between non-cancerous and cancerous breast tissue, at the transcriptional level, may help in the design of a therapy based on the suppression of aromatase expression in breast cancer tissue.

J Travel Med, 2003 Mar-Apr, 10(2), 128 - 30
A placebo-controlled treatment trial of Blastocystis hominis infection with metronidazole; Nigro L et al.; Blastocystis hominis, previously considered a harmless yeast, is now classified as a protozoan inhabiting the human intestinal tract . The pathogenicity of B . hominis remains controversial and is currently the subject of extensive debate.1- 5 As a result of the uncertainty surrounding the pathogenic role of B . hominis, large-scale treatment trials of B . hominis infection have so far been lacking . In spite of this, several drugs have been reported to be active against the parasite.6-8 The present study was carried out in order to evaluate the efficacy of metronidazole treatment in inducing clinical remission and parasitologic eradication in immunocompetent individuals with B . hominis as the only evident cause of diarrhea.

Mycopathologia, 2002, 155(4), 183 - 9
Allergenic evaluation of Malassezia furfur crude extracts; Gandra RF et al.; Crude extracts of the lipophilic yeast Malassezia furfur were obtained from 2, 6, 10 and 28 day old cultures . The in vitro cultivation periods corresponded, respectively, to the lag phase, middle of the log phase, end of log phase and the decline phase of the growth curve, which was based on viable cell counts obtained with a fluorescent viability test . Biochemical analyses showed that the protein and carbohydrate contents were greater in day 10 extracts . Seventy patients with different allergic manifestations and 30 healthy volunteers were skin prick tested using the extracts . Of these, thirteen (18.57%) patients gave positive responses . SDS PAGE gradient electrophoretic profiles of the preparations indicated that the 28 day extracts contained the greatest number of protein bands with molecular weights ranging mostly between 30 and 94 kDa . Immunoblots incubated with individual patient sera showed that four IgE binding M . furfur allergens of approximately 88, 61, 52 and 39 kDa were present in the 28 day extracts . The components identified could be used for detecting IgE mediated responses to M . furfur among individuals affected with different allergic conditions.

Cell Physiol Biochem, 2003, 13(1), 13 - 20
SGK1 regulation of epithelial sodium transport; Pearce D; Epithelial ion transport is regulated in vertebrates by a variety of hormonal and non-hormonal factors, including mineralocorticoids, insulin, and osmotic shock . SGK1 has been established as an important convergence point for multiple regulators of Na+transport . Unlike most serine-threonine kinases, SGK1 is under dual control: protein levels are controlled through effects on its gene transcription, while its activity is dependent on phosphatidylinositol-3-kinase (PI3K) activity . Aldosterone is the most notable regulator of SGK1 protein level in ion transporting epithelia, while insulin and other activators of the of PI3K are key regulators of its activity . Activated SGK1 regulates a variety of ion transporters, the best characterized of which is the epithelial sodium channel (ENaC) . The apical targeting of ENaC is controlled by the ubiquitin ligase, Nedd4-2, and SGK1 acts, at least in part, through phosphorylation-dependent inhibition of Nedd4-2 . This effect of SGK1 requires physical associations of Nedd4-2 with both SGK1 and ENaC . Moreover, direct physical association between SGK1 and ENaC may also be implicated in the formation of a tertiary complex . Osmotic shock is likely the most important non-hormonal regulator of SGK1 expression, and surprisingly, SGK1 expression can be induced by hypotonic or hypertonic stress in a cell-type dependent fashion . The SGK family represents an ancient arm of the serine-threonine kinase family, present in all eukaryotes that have been examined, including yeast . SGK1 appears to have been implicated in membrane trafficking and possibly in the control of ion transport and cell volume in early single cell eukaryotes . In metazoan epithelia, it seems likely that SGK1 was adapted to the regulation of ion transport in response to hormonal and osmotic signals.

Science, 2003 Mar 21, 299(5614), 1896 - 8
Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant; Sasaki A et al.; Gibberellin (GA) regulates growth and development in plants . We isolated and characterized a rice GA-insensitive dwarf mutant, gid2 . The GID2 gene encodes a putative F-box protein, which interacted with the rice Skp1 homolog in a yeast two-hybrid assay . In gid2, a repressor for GA signaling, SLR1, was highly accumulated in a phosphorylated form and GA increased its concentration, whereas SLR1 was rapidly degraded by GA through ubiquitination in the wild type . We conclude that GID2 is a positive regulator of GA signaling and that regulated degradation of SLR1 is initiated through GA-dependent phosphorylation and finalized by an SCF(GID2)-proteasome pathway.

Protein Sci, 2003 Apr, 12(4), 768 - 75
A peptide model of insulin folding intermediate with one disulfide; Yan H et al.; Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds . Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19 . However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick . To circumvent this difficulty, a model peptide ({A20-B19}PIP) containing the single disulfide A20-B19 was prepared by protein engineering . The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP . The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation . In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred . In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner . By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.

J Biol Chem, 2003 May 30, 278(22), 19619 - 26 Epub 2003 Mar 20.
Reducing the agonist activity of antiandrogens by a dominant-negative androgen receptor coregulator ARA70 in prostate cancer cells; Rahman MM et al.; Although the progression of prostate cancer initially is dependent on androgens, tumor progression to an androgen-independent growth eventually occurs in most of patients treated with androgen ablation and/or antiandrogen therapy . After the initial response, antiandrogens lose their efficacy and eventually act as agonists to promote androgen receptor (AR)-mediated growth of prostate cancer cells . An aberrant regulation of AR activity, presumably by AR coregulators, may contribute to this acquired agonist activity of antiandrogens . Using an in vitro mutagenesis and a double-negative selection in yeast two-hybrid screening, we have identified a dominant-negative AR coregulator ARA70 (dARA70N), which can inhibit AR transcriptional activity by inactivating the normal function of ARA70 in the LNCaP cells . Whereas ARA70 in oligomeric form interacts with AR and enhances its transcriptional activity, dARA70N lacks AR interaction and might retain the ability to form a non-functional heteromer with ARA70 and interrupt AR transcriptional activity without a change in AR protein itself . The addition of dARA70N reduces the agonist activity and rescues the normal function of antiandrogens in prostate cancer cells . RNA-interference-mediated silencing of ARA70 gene further confirms these observations . Taken together, these findings indicate that ARA70 may contribute to the acquired agonist activity of antiandrogens and plays an important role in making prostate cancer cells resistant to androgen ablation and/or antiandrogen therapy . ARA70 may, thus, be a critical target for developing therapeutic agents against AR-mediated progression of prostate cancer.

Cancer Res, 2003 Mar 15, 63(6), 1430 - 7
E1A deregulates the centrosome cycle in a Ran GTPase-dependent manner; De Luca A et al.; By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization . Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines . The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network . The E1A portion responsible for the interaction with Ran is the extreme NH(2)-terminal region (amino acids 1-36), which is also required for the induction of centrosome amplification . In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH(2)-terminal region, does not . In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins functionally related to E1A . These findings suggest a common pathway of these oncoproteins in eliciting virus-induced genomic instability.

Curr Opin Cell Biol, 2003 Apr, 15(2), 206 - 12
Functional genomic maps in Caenorhabditis elegans; Grant BD et al.; The completion of the Caenorhabditis elegans genome sequence was the initial step toward the use of whole-genome analysis in this model organism . Advances in C . elegans genomics include transcript profiling, gene-function screens using RNA-mediated interference, and protein-interaction mapping using the yeast two-hybrid system . Recent reports have employed these methods to gain new insights into diverse biological problems such as tissue-specific gene expression, cell-fate specification, genome organization, the DNA damage response, and early embryonic development . These studies combined genomic approaches to probe complex biological pathways on an unprecedented scale.

Theor Appl Genet, 2003 Mar, 106(5), 923 - 30 Epub 2002 Nov 15.
An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress; Shen YG et al.; We characterize one transcription factor of DRE-binding proteins (TaDREB1) that was isolated from a drought-induced cDNA library of wheat (Triticum aestivum L.) . TaDREB1 contains one conserved EREBP/AP2 domain, and shows similarity with Arabidopsis thaliana DREB family members in both overall amino-acid sequences and the secondary structure arrangement within the DNA-binding motifs . In yeast one-hybrid system, TaDREB1, can specially activate the genes fused with the promoter containing three tandemly repeated copies of the wild-type DRE sequence: TACCGACAT . In different wheat cultivars, the Ta DREB1 gene is induced by low temperature, salinity and drought; and the expression of Wcs120 that contains DRE motifs in its promoter is closely related to the expression of TaDREB1 . These results suggest that TaDREB1 functions as a DRE-binding transcription factor in wheat . We also observed the dwarf phenotype in transgenic rice (T0) overexpressing TaDREB1.

Nature, 2003 Mar 20, 422(6929), 297 - 302
Genetics of gene expression surveyed in maize, mouse and man; Schadt EE et al.; Treating messenger RNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways . Transcript abundances often serve as a surrogate for classical quantitative traits in that the levels of expression are significantly correlated with the classical traits across members of a segregating population . The correlation structure between transcript abundances and classical traits has been used to identify susceptibility loci for complex diseases such as diabetes and allergic asthma . One study recently completed the first comprehensive dissection of transcriptional regulation in budding yeast, giving a detailed glimpse of a genome-wide survey of the genetics of gene expression . Unlike classical quantitative traits, which often represent gross clinical measurements that may be far removed from the biological processes giving rise to them, the genetic linkages associated with transcript abundance affords a closer look at cellular biochemical processes . Here we describe comprehensive genetic screens of mouse, plant and human transcriptomes by considering gene expression values as quantitative traits . We identify a gene expression pattern strongly associated with obesity in a murine cross, and observe two distinct obesity subtypes . Furthermore, we find that these obesity subtypes are under the control of different loci.

J Immunol, 2003 Apr 1, 170(7), 3534 - 43
The B cell coreceptor CD22 associates with AP50, a clathrin-coated pit adapter protein, via tyrosine-dependent interaction; John B et al.; The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor . Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb) . Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation . Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled . In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain . This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr(843) and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50 . Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr(843) or Tyr(863) is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of alpha-adaptin . Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab')(2) blocks ligand-mediated internalization of CD22 . In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.

J Biol Chem, 2003 May 23, 278(21), 18953 - 9 Epub 2003 Mar 19.
Rrn3 becomes inactivated in the process of ribosomal DNA transcription; Hirschler-Laszkiewicz I et al.; The human homologue of yeast Rrn3, a 72-kDa protein, is essential for ribosomal DNA (rDNA) transcription . Although the importance of Rrn3 function in rDNA transcription is well established, its mechanism of action has not been determined . It has been suggested that the phosphorylation of either yeast RNA polymerase I or mammalian Rrn3 regulates the formation of RNA polymerase I.Rrn3 complexes that can interact with the committed template . These and other reported differences would have implications with respect to the mechanism by which Rrn3 functions in transcription . For example, in the yeast rDNA transcription system, Rrn3 might function catalytically, but in the mammalian system it might function stoichiometrically . Thus, we examined the question as to whether Rrn3 functions catalytically or stoichiometrically . We report that mammalian Rrn3 becomes the limiting factor as transcription reactions proceed . Moreover, we demonstrate that Rrn3 is inactivated during the transcription reactions . For example, Rrn3 isolated from a reaction that had undergone transcription cannot activate transcription in a subsequent reaction . We also show that this inactivated Rrn3 not only dissociates from RNA polymerase I, but is not capable of forming a stable complex with RNA polymerase I . Our results indicate that Rrn3 functions stoichiometrically in rDNA transcription and that its ability to associate with RNA polymerase I is lost upon transcription.

J Biol Chem, 2003 May 23, 278(21), 18945 - 52 Epub 2003 Mar 19.
Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase; Meskiene I et al.; Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction . In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes . At present, little is known about the functions and substrates of plant PP2Cs . We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants . In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK . SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity . Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C . A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK . In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen . MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays . Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.

Arch Biochem Biophys, 2003 Apr 1, 412(1), 34 - 41
Leukotriene B4 omega-side chain hydroxylation by CYP4F5 and CYP4F6; Bylund J et al.; Leukotriene B(4) (LTB(4)) is a lipid mediator that plays an important role in inflammation . Metabolism of LTB(4) by cytochrome P450 (CYP) enzymes belonging to the CYP4F subfamily is considered to be of importance for the regulation of inflammation . This study investigates LTB(4) metabolism by recombinant rat CYP4F5 and CYP4F6 expressed in a yeast system and by microsomes isolated from rat organs expressing CYP4F mRNA . CYP4F6 was found to convert LTB(4) into 19-hydoxy- and 18-hydroxy-LTB(4) with an apparent K(m) of 26 microM, and CYP4F5 was found to convert LTB(4) primarily into 18-hydroxy-LTB(4) with an apparent K(m) of 9.7 microM . The rate of formation of 18-hydroxy-LTB(4) by CYP4F5 was surprisingly high . At a substrate concentration of 30 microM, the rate of formation was about 15 nmol/min/mg microsomal protein, approximately 30 times faster than the reaction catalyzed by CYP4F6 . Analysis of LTB(4) metabolism by microsomes isolated from various tissues from the rat suggests that CYP4F5 and CYP4F6 are active in the lung and to some extent in the brain, kidney, and testis . CYP4F5 and CYP4F6, due to their capacities to metabolize LTB(4), may play important roles in modulating inflammatory response in these organs.

Biochem Biophys Res Commun, 2003 Mar 21, 302(4), 904 - 15
ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis; Cui Y et al.; Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus . To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library . Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins . The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo . ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy . Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells . In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups . When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced . When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared . The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis . ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis . The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.

Biochem Biophys Res Commun, 2003 Mar 21, 302(4), 869 - 72
Mammalian Numb is a target protein of Mdm2, ubiquitin ligase; Yogosawa S et al.; Drosophila Numb protein functions as an antagonist against Notch signal . The expression of this protein is asymmetrical in divided cells and thought to be involved in the neural cell differentiation and/or cell fate . Human homologue of Numb (hNumb) was cloned as Mdm2-binding protein by yeast two-hybrid screening . Since Mdm2 is an oncoprotein and has ubiquitin ligase activity toward tumor suppressor p53, we assessed to find out whether Mdm2 ubiquitinylates the hNumb protein . The recombinant hNumb expressed in Sf-9 cells using baculovirus protein expression system bound to Mdm2 in vitro . When hNumb was subjected to in vitro ubiquitinylation assay system, which contains E1, E2, or UbcH5c, and Mdm2, hNumb was ubiquitinylated as efficiently as the p53 protein . However, when the Ring-finger domain mutant of Mdm2 was used in place of wild-type Mdm2, hNumb was not ubiquitinylated . Furthermore, when U2OS cells were co-transfected with hNumb and Mdm2, the hNumb protein was ubiquitinylated and degraded . These data strongly suggest that Mdm2 functions as the ubiquitin ligase toward hNumb and that it induces its degradation in intact cells.

Biochem Biophys Res Commun, 2003 Mar 21, 302(4), 735 - 42
Mortalin-MPD (mevalonate pyrophosphate decarboxylase) interactions and their role in control of cellular proliferation; Wadhwa R et al.; Mortalin (mot-2/GRP75/PBP74/mthsp70) is a member of the hsp70 family of proteins and is differentially distributed in normal and immortal cells . It was shown to be involved in pathways to cell senescence and immortalization . To elucidate its functional aspects, a yeast interactive screen for mortalin (mot-2) binding proteins was performed . Mevalonate pyrophosphate decarboxylase (MPD) was identified as one of the mortalin binding partners . The interactions were confirmed in mammalian cells by two-hybrid assay and in vivo coimmunoprecipitation . MPD is known to furnish prenyl groups required for prenylation, protein modification that is essential for the activity of many proteins including p21(Ras) (Ras) . We have examined the effect of MPD-mot-2 interactions on the level and activity of p21(Ras) and its downstream effectors, p44 and p42 MAP kinases (ERK1/ERK2), in Ras-Raf pathway . An overexpression of mot-2 resulted in reduced level of Ras and phosphorylated ERK2 . These were rescued by co-expression of MPD from an exogenous promoter demonstrating a functional link between mot-2, MPD, and Ras . Ras and its oncogenic forms act as key players in controlling proliferation of normal and cancerous cells . Assigning mot-2 upstream of p21(Ras) offers an important mechanism for influence over cell proliferation.

Biochem Biophys Res Commun, 2003 Mar 21, 302(4), 671 - 8
Inhibition of ubiquitin ligase Siah-1A by disabled-1; Park TJ et al.; Disabled-1 (Dab1) is a cytosolic adaptor protein that plays critical roles in cortical development . However, a detailed mechanism of action has not yet been clearly defined . Through yeast two-hybrid screening, we observed that mouse Siah-1A, an E3 ubiquitin ligase containing a RING finger motif, interacts with Dab1 . Co-immunoprecipitation experiments and in vitro binding experiments both indicated direct interaction between Siah and Dab1 . Steady-state expression of Siah was enhanced by the presence of Dab1 or lactacystin, a representative proteasomal inhibitor . Auto-ubiquitination of Siah was inhibited by the presence of Dab1, suggesting inhibition of Siah activity and subsequent increase of Siah expression by Dab1 . Both Dab1-induced increase of steady-state expression of Deleted in colorectal cancer (DCC), one of the well-known substrates of Siah, and its inhibition by Siah Delta R suggest that Dab1 increases expression of DCC through inhibiting the activity of endogenous Siah . Our results suggest that Dab1 inhibits the activity of Siah.

Biochem J, 2003 Jul 1, 373(Pt 1), 115 - 23
Epitope mapping for the monoclonal antibody that inhibits intramolecular electron transfer in flavocytochrome b2; Le KH et al.; Flavocytochrome b(2) (yeast L-lactate dehydrogenase) carries one FMN and one protohaem IX on each of its four subunits . The prosthetic groups are bound to separate domains, the haem domain (residues 1-99) and the flavin domain (residues 100-485), which interact for electron transfer between lactate-reduced FMN and haem b(2); in vivo, the latter reduces cytochrome c . In the crystal structure, one haem domain out of two is mobile . Previously we have described a monoclonal antibody, raised against the tetramer, that only recognizes the native haem domain and prevents electron transfer between flavin and haem, while having no effect on flavin reduction by the substrate {Miles, Lederer and Le (1998) Biochemistry 37, 3440-3448} . In order to understand the structural basis of the uncoupling between the domains, we proceeded to site-directed mutagenesis, so as to map the epitope on the surface of the haem domain . We analysed the effects of 14 mutations at 12 different positions, located mostly in the domain interface or at its edge; we also analysed the effect of replacing protohaem IX with its dimethyl ester . We used as criteria the antibody-mediated inhibition of cytochrome c reduction by flavocytochrome b(2), competitive ELISA tests and surface plasmon resonance . We have thus defined a minimal epitope surface on the haem domain; it encompasses positions 63, 64, 65, 67, 69 and 70 and one or both haem propionates . When the haem and flavin domains are docked for electron transfer, the 65, 67 and 70 side chains, as well as the haem propionates, are excluded from solvent . The present results thus indicate that, when bound, the antibody acts as a wedge between the domains and constitutes a physical barrier to electron transfer.

Biochem J, 2003 Apr 1, 371(Pt 1), 97 - 108
Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization; Greve K et al.; Numerous, highly conserved RING-H2 domains are found in the model plant Arabidopsis thaliana (thale cress) . To characterize potential RING-H2 protein interactions, the small RING-H2 protein RHA2a was used as bait in a yeast two-hybrid screen . RHA2a interacted with one of the plant-specific NAC {NAM ('no apical meristem'), ATAF1/2, CUC2 ('cup-shaped cotyledons 2')} transcription factors, here named ANAC (abscisic acid-responsive NAC) . The core RING-H2 domain was sufficient for the interaction . The ability of 11 structurally diverse RING-H2 domains to interact with ANAC was then examined . Robust interaction was detected for three of the domains, suggesting multi-specificity for the interaction . The domains that interacted with ANAC contain a glutamic acid residue in a position corresponding to a proline in many RING-H2 domains . Conversion of this glutamic acid residue into proline in RHA2a decreased its ability to interact with ANAC, most likely by changing the interaction surface . This suggested that a short, divergent region in RING-H2 domains modulate interaction specificity . ANAC contains a degenerate bipartite nuclear localization signal (NLS), while RHG1a, also identified as an ANAC interaction partner, contains a basic NLS . Both signals localized beta-glucuronidase reporter fusions to the nucleus . N-terminally truncated RHA2a also directed nuclear localization, apparently dependent on basic amino acids in the RING-H2 domain . Nuclear co-localization of the RING-H2 proteins and ANAC may enable their interaction in vivo to regulate the activity of the ANAC transcription factor.

J Proteome Res, 2002 May-Jun, 1(3), 253 - 61
Use of MEDUSA-based data analysis and capillary HPLC-ion-trap mass spectrometry to examine complex immunoaffinity extracts of RBAp48; Gururaja T et al.; To examine the Jurkat cell interaction partners of RbAp48, we digested entire immunoaffinity extracts with trypsin and identified potential interacting proteins using one- and two-dimensional microcapillary HPLC-ion-trap mass spectrometry . An Oracle-based automated data analysis system (MEDUSA) was used to compare quadruplicate anti-RbAp48 antibody affinity extracts with two sets of quadruplicate control extracts . The anti-RbAp48 extracts contained over 40 difference 1D gel bands . We identified all known proteins of the NuRD/Mi-2 complex including human p66 . Three potential homologues of members of this complex were also found, suggesting that there may be more than one variant of this complex . Eleven proteins associated with RNA binding or pre-mRNA splicing were observed . Four other proteins, including a putative tumor suppressor, were identified, as were 18 ribosomal proteins . There was little overlap with RbAp48-interacting proteins defined by yeast two-hybrid methods . These results demonstrate the analysis of a complex immunoaffinity extract and suggest a more complex cellular role for RbAp48 than previously documented.

J Biol Chem, 2003 Mar 14, 278(11), 9655 - 62
Enhancement of B-MYB transcriptional activity by ZPR9, a novel zinc finger protein; Seong HA et al.; By using the yeast two-hybrid system, the zinc finger protein ZPR9 was identified as one of the B-MYB interacting proteins that associates with the carboxyl-terminal conserved region of B-MYB . ZPR9 was found to form in vivo complexes with B-MYB, as demonstrated by in vivo binding assay and coimmunoprecipitation experiments of the endogenously and exogenously expressed proteins . Deletion analysis revealed that this binding was mediated by all three functional domains, an amino-terminal DNA-binding domain, a transactivation domain, and a carboxyl-terminal conserved region of B-MYB . We show that the interaction of ZPR9 with B-MYB is functional because cotransfection of ZPR9 significantly up-regulates B-MYB transcriptional activity in a dose-dependent manner . In addition, coexpression of ZPR9 with B-MYB caused the accumulation of B-MYB, as well as ZPR9, in the nucleus . Furthermore, constitutive expression of ZPR9 in human neuroblastoma cells induces apoptosis in the presence of retinoic acid . These results strongly suggest that ZPR9 plays an important role in modulation of the transactivation by B-MYB and cellular growth of neuroblastoma cells.

Yi Chuan Xue Bao, 2002, 29(11), 1001 - 4
{Molecular mapping of the fertility restorer gene Rf-4 for WA cytoplasmic male sterility in rice}; Zhang QY et al.; The cytoplasmic male sterility for wild-abortive (CMS-WA) has been wildly used for hybrid rice breeding in China . The fertility restoration of CMS-WA is controlled mainly by two independent and dominant nuclear fertility restoring genes, Rf-3 and Rf-4 . To map the Rf-4 gene with molecular markers, rice YAC clones of RGP, Japan were used to create new molecular marker . YAC contigs located between RFLP markers R1877 and G2155 on chromosome 10 were confirmed by hybridization with 12 RFLP probes . Six YAC clones, Y4630, Y2670, Y4892, Y2111, Y3821 and Y5528 were identified . Chromosome DNAs of the YAC clones were prepared and separated by CHEF . A total of 119 probes were created by sub-cloning of the YAC DNAs . RFLPs were screened between Zhenshan 97A and its near-isogenic lines with Rf-4Rf-4 genotype . Two probes, Y3-8 from Y4892 and Y1-10 from Y4630, were found to be polymorphic . Using F2 populations from crosses between Zhenshan 97A and its near-isogenic lines ZSR11, Y3-8 and Y1-10 were mapped to Rf-4 locus with genetic distances of 0.9 cM and 3.2 cM, respectively.

Plant Physiol, 2003 Mar, 131(3), 1191 - 208
Analysis of the small GTPase gene superfamily of Arabidopsis; Vernoud V et al.; Small GTP-binding proteins regulate diverse processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking . These proteins function as molecular switches that cycle between "active" and "inactive" states, and this cycle is linked to the binding and hydrolysis of GTP . The Arabidopsis genome contains 93 genes that encode small GTP-binding protein homologs . Phylogenetic analysis of these genes shows that plants contain Rab, Rho, Arf, and Ran GTPases, but no Ras GTPases . We have assembled complete lists of these small GTPases families, as well as accessory proteins that control their activity, and review what is known of the functions of individual members of these families in Arabidopsis . We also discuss the possible roles of these GTPases in relation to their similarity to orthologs with known functions and localizations in yeast and/or animal systems.

J Histochem Cytochem, 2003 Apr, 51(4), 549 - 51
Fluorescence in situ hybridization (FISH) on human chromosomes using photoprobe biotin-labeled probes; Weise A et al.; Fluorescence in situ hybridization (FISH) on human chromosomes in meta- and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture . Many different protocols for labeling the DNA probes used for FISH have been published . Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments . Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.

Virology, 2003 Feb 15, 306(2), 313 - 23
Regulation of MSV and WDV virion-sense promoters by WDV nonstructural proteins: a role for their retinoblastoma protein-binding motifs; Munoz-Martin A et al.; In this work we demonstrate that wheat dwarf virus (WDV) RepA can activate WDV and maize streak virus (MSV) virion (V)-sense expression in plant tissues . Rep alone does not have any effect on the silent WDV promoter and it represses the basal MSV promoter activity . MSV promoter activation by RepA depends on an intact RepA retinoblastoma protein (RB)-binding domain . Promoter repression by Rep also depends on this domain to some extent . Mutation of the RepA RB-binding domain has no effect on WDV promoter activation . The WDV promoter contains two sites that fit the consensus E2F-binding site . One, WDV1, binds human E2F-1 in one-hybrid assays in yeast . It also binds specifically to maize and wheat proteins in vitro and, when fused to a minimal 35S promoter, it confers responsiveness to RepA only when the RepA RB-binding domain and the WDV1 site are intact . In the whole WDV V-sense promoter context, mutations of this sequence have no effect, suggesting that additional sequences are important for RepA-mediated promoter activation.

Vet Ophthalmol, 2003 Mar, 6(1), 51 - 5
Fungal flora of normal eyes of healthy horses from the State of Rio de Janeiro, Brazil; Rosa M et al.; The conjunctival fungal flora of 32 adult horses with normal eyes (n = 64) from the State of Rio de Janeiro in Brazil was identified in the fall of 2000 using horses of different breeds, both genders and aged 5-19 years old . The culture samples were taken from the conjunctival sac of both eyes with a sterile cotton swab wetted with saline solution, seeded in Sabouraud's dextrose agar with chloramphenicol, and incubated for 5 days at an average temperature of 25 degrees C . The number of fungal colonies per eye varied between 0 and 250 colony forming units (CFUs) . There were often differences in colony types between eyes of the same animal . Filamentous fungi of genera were isolated and identified in the following proportion of the total genera of fungal colonies isolated: Aspergillus (32.2%), Penicillium (25.8%), Scopulariopsis (15.9%), Trichoderma (11.2%), Cladosporium (5.6%), Mucor (2.1%), Syncephalastrum (2.1%), Eurotium (1.7%), Geotrichum (0.9%), Rhizopus (0.9%), Gliomastix (0.4%), Fusarium (0.4%), Staphylotrichum (0.4%) and Verticillium (0.4%) . Yeast genera represented 9% of the total isolates . Over half the horses had at least one normal eye with either Aspergillus, Penicillium, Trichoderma or Scopulariopsis isolated, which is a departure from other studies of the normal horse eye.

J Neurochem, 2003 Apr, 85(1), 282 - 5
Regulation of neuritogenesis and synaptic transmission by msec7-1, a guanine nucleotide exchange factor, in cultured Aplysia neurons; Huh M et al.; msec7-1, a mammalian homologue of yeast sec7p, is known as a GDP/GTP exchange factor (GEF) for the ADP ribosylation factor (ARF) family of small GTPases . Here, we report that msec7-1 overexpression in cultured Aplysia neurons leads to an extensive neuritogenesis in a GEF activity-dependent manner through the modulation of the actin cytoskeleton . Similarly, the overexpression of ARNO, another mammalin GEF, produces extensive neuritogenesis in Aplysia neurons . In addition, msec7-1 overexpression increases the number of varicosities with an altered size and shape in a GEF activity-dependent manner . The overexpression of msec7-1 in pre-synaptic sensory neurons co-cultured with post-synaptic target motor neurons leads to an increase in the amplitude of the excitatory post-synaptic potential through its GEF activity . Our results demonstrate that msec7-1 regulates neuritogenesis and synaptic transmission.

J Clin Lab Anal, 2003, 17(2), 52 - 6
New enzymatic assay for serum urea nitrogen using urea amidolyase; Kimura S et al.; We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species . The method is based on hydrolysis of urea by the enzyme . In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4) . Then in the presence of urea amido-lyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum . The concentra-tion of ammonium ion formed was determined by adding GLDH to produce NADP(+) in the presence of 2-oxoglutarate and NADPH . We then monitored the change of absorbance at 340 nm . The inhibitory effect of calcium ion on this assay was eliminated by adding glyco-letherdiamine-N, N, N', N'-tetraacetic acid to the reaction system . The with-in-assay coefficient of variations (CVs) of the present method were 1.80-3.76% (n = 10) at 2.8-19.0 mmol/L, respectively . The day-to-day CVs were 2.23-4.59% . Analytical recovery was 92-115% . The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammo-nium ion, or calcium ion did not affect this assay system . The correlation be-tween values obtained with the present method (y) and those by another enzy-matic method (x) was 0.997 (y = 1.02x - 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of -0.18 +/- 0.86 mmol/L {(values by reference method - that of present method) +/- SD} using the Bland-Altman technique . J . Clin . Lab . Anal . 17:52-56, 2003 .

Mol Cell Biol, 2003 Apr, 23(7), 2463 - 75
Supramolecular complex formation between Rad6 and proteins of the p53 pathway during DNA damage-induced response; Lyakhovich A et al.; The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis . Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53 . Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes . Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G(2)/M phase of the cell cycle . Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels . Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2 . These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway . Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable . Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination . The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.

J Cell Sci, 2003 Apr 15, 116(Pt 8), 1449 - 62
Chlamydomonas DIP13 and human NA14: a new class of proteins associated with microtubule structures is involved in cell division; Pfannenschmid F et al.; We have cloned and characterized a single copy C . reinhardtii gene containing an open reading frame of 333 nucleotides encoding a 12.7 kDa protein . The novel protein, DIP13, exhibits 60% identity with two mammalian proteins, human NA14 and an unnamed mouse protein . Homologous sequences are also present in several protozoan, trematode and fish genomes, but no homologs have been found in the completed genomes of yeast, Drosophila, C . elegans and A . thaliana . By using a specific antibody we have localized DIP13 to microtubule structures, namely basal bodies, flagellar axonemes and cytoplasmic microtubules . Anti-DIP13 antibody also specifically recognized human NA14 by immunofluorescence and stained basal bodies and flagella of human sperm cells as well as the centrosome of HeLa cells . Expression of the DIP13 open reading frame in antisense orientation in Chlamydomonas resulted in multinucleate, multiflagellate cells, which suggests a role for this protein in ensuring proper cell division . Thus, DIP13/NA14 could represent the founding members of a new class of highly conserved proteins that are associated with microtubule structures.

J Biol Chem, 2003 May 23, 278(21), 19209 - 19 Epub 2003 Mar 14.
MICoA, a novel metastasis-associated protein 1 (MTA1) interacting protein coactivator, regulates estrogen receptor-alpha transactivation functions; Mishra SK et al.; The transcriptional activity of estrogen receptor-alpha (ER-alpha) is modified by coactivators, corepressors, and chromatin remodeling complexes . We have previously shown that the metastasis-associated protein-1 (MTA1), a component of histone deacetylase and nucleosome remodeling complexes, represses ER-driven transcription by recruiting histone deacetylases to the estrogen receptor element (ERE)-containing target gene chromatin in breast cancer cells . Using a yeast two-hybrid screening to clone MTA1-interacting proteins, we identified a previously uncharacterized molecule, which we named as MTA1-interacting coactivator (MICoA) . Our findings suggest that estrogen signaling promotes nuclear translocation of MICoA and that MICoA interacts with MTA1 both in vitro and in vivo . MICoA binds to the C-terminal region of MTA1, whereas MTA1 binds to the N-terminal MICoA containing one nuclear receptor interaction LSRLL motif . We showed that MICoA is an ER coactivator, cooperates with other ER coactivators, stimulates ER-transactivation functions, and associates with the endogenous ER and its target gene promoter chromatin . MTA1 also repressed MICoA-mediated stimulation of ERE-mediated transcription in the presence of ER and ER variants with naturally occurring mutations, such as D351Y and K303R, and that it interfered with the association of MICoA with the ER-target gene chromatin . Because chromatin is a highly dynamic structure and because MTA1 and MICoA could be detected within the same complex, these findings suggest that MTA1 and MICoA might transmodulate functions of each other and any potential deregulation of MTA1 is likely to contribute to the functional inactivation of the ER pathway, presumably by derecruitment of MICoA from ER target promoter chromatin.

J Ethnopharmacol, 2003 Apr, 85(2-3), 221 - 5
The analgesic, antipyretic and anti-inflammatory activity of Diospyros variegata Kruz; Trongsakul S et al.; Pharmacological studies were conducted with the hexane extract of the dry stem of Diospyros variegata Kruz . (Ebenaceae) on experimental animals for evaluating the analgesic, antipyretic and anti-inflammatory activities . In the analgesic test, the hexane extract elicited inhibitory intensity on acetic acid-induced writhing response and on the late phase of formalin test but possessed only a weak effect on the tail-flick response and on the early phase of formalin test . The hexane extract also elicited antipyretic action when tested in yeast-induced hyperthermia in rats . In addition, the hexane extract showed an anti-inflammatory effect when tested in ethyl phenylpropiolate (EPP)- and arachidonic acid (AA)-induced rat ear edema.

Cell Signal, 2003 May, 15(5), 455 - 62
Molecular recognitions in the MAP kinase cascades; Tanoue T et al.; The mitogen-activated protein kinase (MAPK) cascades play a pivotal role in many aspects of cellular functions, and are evolutionarily conserved from yeast to mammals . In mammals, there are four subfamily members in the MAPKs . Each MAPK has its own activators, substrates and inactivators . In order to achieve normal cellular functions, the MAPK cascades should transduce signals with high efficiency and fidelity . However, the molecular basis for the mechanism underlying the specific reactions in the MAPK cascades has not been fully understood . The MAPKs form a globular structure without a distinct domain specific for protein-protein interactions . Recent studies revealed two mechanisms regulating the signalling, the docking interaction and the scaffolding . The docking interaction is achieved through the common docking domain (the CD domain) on MAPKs, and is different from a transient enzyme-substrate interaction through the active centre of the enzymes . Almost all the MAPK-interacting molecules have a conserved motif interacting with the CD domain . The scaffolding usually utilizes a third molecule to tether several components of the MAPK cascades . Both of them are thought to regulate the enzymatic specificity and efficiency.

J Biol Chem, 2003 May 16, 278(20), 18015 - 21 Epub 2003 Mar 12.
Analysis of the interaction of small heat shock proteins with unfolding proteins; Stromer T et al.; The ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding . No obvious substrate specificity has been detected so far . A striking feature of sHsps is that they form large complexes with nonnative proteins . Here, we used several well established model chaperone substrates, including citrate synthase, alpha-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding . The two sHsps differ in their modes of activation . In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding . Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins . For all substrates investigated, complexes of defined size and shape were formed . Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes . The first substrate protein bound seems to determine the complex morphology . Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals.

Blood, 2003 Jul 1, 102(1), 192 - 9 Epub 2003 Mar 13.
GMCSF activates NF-kappaB via direct interaction of the GMCSF receptor with IkappaB kinase beta; Ebner K et al.; Granulocyte-macrophage colony-stimulating factor (GMCSF) has a central role in proliferation and differentiation of hematopoetic cells . Furthermore, it influences the proliferation and migration of endothelial cells . GMCSF elicits these functions by activating a receptor consisting of a ligand-specific alpha-chain and a beta-chain, which is common for GMCSF, interleukin-3 (IL-3), and IL-5 . It is known that various signaling molecules such as Janus kinase 2 or transcription factors of the signal transducer and activator of transcription (STAT) family bind to the common beta-chain and initiate signaling cascades . However, alpha-chain-specific signal transduction adapters have to be postulated given that IL-3, IL-5, and GMCSF induce partly distinct biologic responses . Using a yeast 2-hybrid system, we identified the alpha-chain of the GMCSF receptor (GMRalpha) as putative interaction partner of IkappaB kinase beta, one of the central signaling kinases activating the transcription factor nuclear factor-kappaB (NF-kappaB) . Using endogenous protein levels of endothelial cell extracts, we could verify the interaction by coimmunoprecipitation experiments . Fluorescence resonance energy transfer (FRET) microscopy confirmed the direct interaction of CFP-IKKbeta and YFPGMRalpha in living cells . Functional studies demonstrated GMCSF-dependent activation of IkappaB kinase activity in endothelial cells, degradation of IkappaB, and activation of NF-kappaB . Further biologic studies using GMCSF-dependent TF-1 cells indicated that GMCSF-triggered activation of NF-kappaB is important for cell survival and proliferation.

Obstet Gynecol, 2003 Mar, 101(3), 548 - 56
Incident and persistent vulvovaginal candidiasis among human immunodeficiency virus-infected women: Risk factors and severity; Duerr A et al.; OBJECTIVE: To examine risk factors for vulvovaginal candidiasis among women with or at risk for human immunodeficiency virus (HIV) infection . METHODS: Data were from 856 HIV-infected women and 421 at-risk uninfected women observed semiannually at four study sites from April 1993 through February 1999 . At enrollment women were 15-55 years old and had no acquired immunodeficiency syndrome-defining conditions . Three definitions for vulvovaginal candidiasis of differing severity were constructed using data from vaginal Candida culture and Gram stains scored for yeast and three signs on pelvic examination (vulvovaginal edema, erythema, or discharge): 1) culture or Gram stain positivity plus at least one clinical sign, 2) culture or Gram stain positivity plus at least two clinical signs, and 3) visible yeast on Gram stain plus at least one clinical sign . RESULTS: The prevalence and cumulative incidence of each definition of vulvovaginal candidiasis were greater among HIV-infected women than among women not infected with HIV (P <.01 for all comparisons) . Stratified by status at the preceding visit, vulvovaginal candidiasis was most likely among women with prior vulvovaginal candidiasis, least likely among women without earlier Candida colonization, and intermediately likely among women with preceding subclinical Candida colonization . Among HIV-infected women, lower CD4 count and higher HIV viral load were associated with vulvovaginal candidiasis . Several other factors were independently associated with vulvovaginal candidiasis, with strong associations for diabetes mellitus and pregnancy in particular . Vulvovaginal candidiasis was not more severe among HIV-infected women . CONCLUSION: Vulvovaginal candidiasis occurred with higher incidence and greater persistence, but not greater severity, among HIV-infected women.

Int J Med Microbiol, 2003 Feb, 292(7-8), 527 - 36
Importance of the terminal complement components for immune defence against Candida; Triebel T et al.; Candida activates complement via all three pathways leading to opsonisation and anaphylaxis . The aim of the study was to investigate the influence of the terminal complement system on Candida infections . Thus, fungal cell growth, mitochondrial activity and phagocytosis by polymorphonuclear leukocytes (PMNLs) as well as specific virulence factors, such as release of secreted aspartic protease (Sap) and adherence to epithelial cells, were assessed under the influence of normal or C6/C7-depleted serum . Candida (C.) dubliniensis was used in all experiments as prototype because of its known increased expression of Saps and its strong geno- and phenotypical similarity to the most abundant Candida species C . albicans . Being exposed to sufficient quantities of complement, fungal growth decreased and phagocytosis increased but mitochondrial activities of the yeast increased as well . Concerning the virulence factors, both adhesion and especially Sap release were markedly reduced in the presence of high serum concentrations . Interestingly, at low serum concentrations some opposite effects (an augmented cell growth, a higher Sap release and a stronger adhesion) were observed . In particular, it was shown that the presence of terminal complement factors, and thus the generation of the membrane attack complex, clearly induced a higher fungal mitochondrial activation and has an effect on host defence against yeast cells by augmenting phagocytosis.

EMBO Rep, 2003 Mar, 4(3), 320 - 5
The membrane-tethering protein p115 interacts with GBF1, an ARF guanine-nucleotide-exchange factor; Garcia-Mata R et al.; The membrane-transport factor p115 interacts with diverse components of the membrane-transport machinery . It binds two Golgi matrix proteins, a Rab GTPase, and various members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family . Here, we describe a novel interaction between p115 and Golgi-specific brefeldin-A-resistant factor 1 (GBF1), a guanine-nucleotide exchange factor for ADP ribosylation factor (ARF) . GBF1 was identified in a yeast two-hybrid screen, using full-length p115 as bait . The interaction was confirmed biochemically, using in vitro and in vivo assays . The interacting domains were mapped to the proline-rich region of GBF1 and the head region of p115 . These proteins colocalize extensively in the Golgi and in peripheral vesicular tubular clusters . Mutagenesis analysis indicates that the interaction is not required for targeting GBF1 or p115 to membranes . Expression of the p115-binding (pro-rich) region of GBF1 leads to Golgi disruption, indicating that the interaction between p115 and GBF1 is functionally relevant.

J Virol, 2003 Apr, 77(7), 4149 - 59
Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase; Gao L et al.; To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library . We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M . F . Kleijnen, A . H . Shih, P . Zhou, S . Kumar, R . E . Soccio, N . L . Kedersha, G . Gill, and P . M . Howley, Mol . Cell 6: 409-419, 2000) . In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein . As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol . Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1 . A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B . Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1 . Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

J Virol, 2003 Apr, 77(7), 4113 - 26
Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins; Ward BM et al.; Incorporation of the vaccinia virus A36R protein into the outer membrane of intracellular enveloped virions (IEV) is dependent on expression of the A33R protein . Possible interactions of the 200-amino-acid cytoplasmic domain of the A36R protein with itself or with the cytoplasmic domain of the A33R, A34R, B5R, or F12L IEV membrane protein was investigated by using the yeast two-hybrid system . A strong interaction was detected only between the cytoplasmic domains of the A36R and A33R proteins . Upon further analyses, the interaction site was mapped to residues 91 to 111 of the A36R protein . To investigate the role of the A36R:A33R interaction during viral infection, five recombinant vaccinia viruses containing B5R-GFP as a marker were constructed . Four had the full-length A36R gene replaced with various-length C-terminal truncations of A36R, of which two contained residues 91 to 111 and two were missing this region . The fifth recombinant virus had an A33R gene with most of the 40-amino-acid cytoplasmic tail deleted . Residues 91 to 111 of A36R and the cytoplasmic tail of A33R were required for a strong interaction between the two proteins during viral infection and for maximal amounts of A36R protein on IEV . Mutants lacking these regions of A33R or A36R formed IEV that exhibited only short sporadic intracellular movement, displayed no actin tails, and formed small plaques on cell monolayers equivalent to those of an A36R deletion mutant and smaller than those formed by point mutations that specifically abrogate actin tail formation . The A33R interaction site of the A36R protein is highly conserved among orthopoxviruses and may overlap binding sites for cellular proteins needed for microtubular movement and actin tail formation.

J Virol, 2003 Apr, 77(7), 4043 - 59
Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells; Walters JN et al.; Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid . A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5) . Previously (Z . Hong, M . Beaudet-Miller, J . Durkin, R . Zhang, and A . D . Kwong, J . Virol . 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix . The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction . Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein . This allowed us to focus our efforts on a small region of this large polypeptide . Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine . All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay . From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified . All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system . For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made . The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses . The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented . Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure . Thus, it appears that the L75 residue is important for another essential interaction of VP5 with the capsid shell proteins . The congruence of the data from the previous and present studies demonstrates the key roles of two regions in the N terminus of this large protein that are crucial for this bimolecular interaction . Thus, residues I27, L35, and F39 comprise the first subdomain and residues L58, L65, L67 and L71 comprise a second subdomain of VP5 . These seven hydrophobic residues are important for the interaction of VP5 with the scaffold protein and consequently the formation of an icosahedral shell structure that encloses the viral genome.

J Virol, 2003 Apr, 77(7), 3922 - 8
Evidence that binding of cucumber necrosis virus to vector zoospores involves recognition of oligosaccharides; Kakani K et al.; Despite the importance of vectors in natural dissemination of plant viruses, relatively little is known about the molecular features of viruses and vectors that permit their interaction in nature . Cucumber necrosis virus (CNV) is a small spherical virus whose transmission in nature is facilitated by zoospores of the fungus Olpidium bornovanus . Previous studies have shown that specific regions of the CNV capsid are involved in transmission and that transmission defects in several CNV transmission mutants are due to inefficient attachment of virions to the zoospore surface . In this study, we have undertaken to determine if zoospores contain specific receptors for CNV . We show that in vitro binding of CNV to zoospores is saturable and that vector zoospores bind CNV more efficiently than nonvector zoospores . Further studies show that treatment of zoospores with periodate and trypsin reduces CNV binding, suggesting the involvement of glycoproteins in zoospore attachment . In virus overlay assays, CNV binds to several proteins, whereas CNV transmission mutants either fail to bind or bind at significantly reduced levels . The possible involvement of specific sugars in attachment was investigated by incubating CNV with zoospores in the presence of various sugars . Two mannose derivatives (methyl alpha-D-mannopyranoside and D-mannosamine), as well as three mannose-containing oligosaccharides (mannotriose, alpha3,alpha6-mannopentaose, and yeast mannan) and L-(-)-fucose, all inhibited CNV binding at relatively low concentrations . Taken together, our studies suggest that binding of CNV to zoospores is mediated by specific mannose and/or fucose-containing oligosaccharides . This is the first time sugars have been implicated in transmission of a plant virus.

J Androl, 2003 Mar-Apr, 24(2), 204 - 14
Spermatogenetic expression of RNA-binding motif protein 7, a protein that interacts with splicing factors; Guo TB et al.; We have previously shown that a ubiquitously expressed RNA splicing factor, RNA-binding motif 7 (RBM7), cloned from a testis complementary DNA library, enhances messenger RNA (mRNA) splicing in vitro and is expressed in a cell-restricted fashion . Herein, we detail its mRNA and protein expression in the rodent testis . RNA in situ hybridization shows that Rbm7 expression in rat germ cells closely parallels the entry and progression of meiosis . The expression commences in type B spermatogonia, it rises during the preleptotene stage, peaks in leptotene spermatocytes, and declines afterward, but increases again in stage-associated pachytene spermatocytes . An affinity-purified polyclonal antibody raised against a peptide corresponding to amino acids 202-224 of the mouse RBM7 recognized the predicted 35 kd protein both in testicular lysates and in in vitro translation reactions . Consistent with the in situ hybridization results, RBM7 immunoreactivity was also detected in type B spermatogonia, spanned the entire period of spermatocyte development, and extended to round and early elongated spermatids . Moreover, RBM7 appeared nuclear up to the mid pachytene stage and became cytoplasmic thereafter . Consistent with its role in RNA splicing, yeast 2-hybrid and glutathione S-transferase pull-down assays show that RBM7 interacts with splicing factor 3b subunit 2 (SAP145), and with the splicing regulator, SRp20 . These interactions and the nuclear localization of RBM7 provide insights into its function in pre-mRNA processing in developing spermatocytes during entry into meiosis and progression through the meiotic prophase.

FEBS Lett, 2003 Mar 13, 538(1-3), 117 - 24
Component plane presentation integrated self-organizing map for microarray data analysis; Xiao L et al.; We describe a powerful approach, component plane presentation integrated self-organizing map (SOM), for the analysis of microarray data . This approach allows the display of multi-dimensional SOM outputs of microarray data in multiple sample specific presentations, providing distinct advantages in visual inspection of biological significances of genes clustered in each map unit with respect to each RNA sample . Beneficial potentials of the approach are highlighted by processing microarray data from yeast cells as well as human breast malignancies.

FEBS Lett, 2003 Mar 13, 538(1-3), 53 - 9
Fusion of mitochondria in mammalian cells is dependent on the mitochondrial inner membrane potential and independent of microtubules or actin; Mattenberger Y et al.; Mitochondrial fusion is a poorly characterized process which has mainly been studied in yeast and Drosophila but is thought to occur in all eukaryotes . Until now, there was only indirect evidence to support such a process in mammalian cells . In this study, using a cell fusion system, we found that mitochondrial fusion occurs rapidly in mammalian cells and is completed in less than 24 h . We report that the fusion of mitochondria requires an intact mitochondrial inner membrane potential but is independent of a functional cytoskeleton.

Mol Biol Cell, 2003 Mar, 14(3), 1221 - 39
Importin-alpha mediates the regulated nuclear targeting of serum- and glucocorticoid-inducible protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain; Maiyar AC et al.; The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-alpha nuclear import receptor . In vitro GST pull down assays demonstrated a strong and direct association of importin-alpha with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation . Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex . Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-alpha, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-alpha to import Sgk into nuclei in vitro . Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131-141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-alpha and for nuclear import of full-length Sgk in cultured cells . The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-alpha as well as the PI3-kinase-dependent phosphorylation of Sgk . Our results define a new role importin-alpha in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.

Mol Biol Cell, 2003 Mar, 14(3), 1138 - 48
Obscurin is a ligand for small ankyrin 1 in skeletal muscle; Kontrogianni-Konstantopoulos A et al.; The factors that organize the internal membranes of cells are still poorly understood . We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns . Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR . We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1 . The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain . Binding was confirmed in two in vitro assays . In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates . In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots . Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM . On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein . Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle . Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

Mol Biol Cell, 2003 Mar, 14(3), 871 - 88
Roles of NUDE and NUDF proteins of Aspergillus nidulans: insights from intracellular localization and overexpression effects; Efimov VP; The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway . It binds several proteins, including the NUDE protein . Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes ("comets") that coincide with microtubule ends . Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA . Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain . In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly . Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function . Furthermore, NUDF overproduction totally suppressed deletion of the nudE gene . This implies that the function of NUDE is secondary to that of NUDF . Unexpectedly, NUDF overproduction inhibited one conditional nudA mutant and all tested apsA mutants . An allele-specific interaction between the nudF and nudA genes is consistent with a direct interaction between NUDF and dynein heavy chain . Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between the nudF and apsA genes suggests a role for NUDF at the cell cortex.

Plant J, 2003 Mar, 33(6), 1073 - 86
A novel protein from Brassica napus has a putative KID domain and responds to low temperature; Gao MJ et al.; To identify factors that interact with histone deacetylase (HDAC) in Brassica napus, a yeast two-hybrid library was screened using the Arabidopsis HDA19 as bait . A novel protein, bnKCP1, containing a putative kinase-inducible domain (KID) was found to interact with HDA19 . Southern blot analysis indicated that the bnKCP1 gene belongs to a small gene family of at least three members . Northern blot analysis showed bnKCP1 to be strongly expressed in stems, flowers, roots, and immature siliques, but not in leaf blades of seedlings . The accumulation of bnKCP1 transcript in the leaf blades was induced significantly within 4 h of exposure of B . napus seedlings to cold stress, whereas treatment of leaf blades with inomycin, an ionophore of Ca2+, caused a rapid (30 min) but transient induction of bnKCP1 expression . In contrast to that observed in leaf blades, expression of bnKCP1 in the stems was repressed upon cold treatment . In vitro and in vivo protein-binding assays showed that bnKCP1 interacts with HDA19 via the KID domain, and that S188 is critical for bnKCP1-HDA19 interaction . BnKCP1 also exerted modest transactivation of the lacZ reporter gene in yeast through its N-terminal region . These assays suggest that bnKCP1 may function as a transcription factor, which regulates gene expression through interaction with HDA19.

Plant J, 2003 Mar, 33(6), 957 - 66
Internal telomeric repeats and 'TCP domain' protein-binding sites co-operate to regulate gene expression in Arabidopsis thaliana cycling cells; Tremousaygue D et al.; We have focused our interest on two cis-regulatory elements, named site II motif and telo box, identified within the promoter of plant proliferating cellular nuclear antigen (PCNA) and putatively involved in meristematic expression of the gene . A conserved topological association between site II motifs and telo boxes is observed in the promoter of numerous genes expressed in cycling cells, including several cell cycle-related genes and 153 Arabidopsis genes encoding ribosomal proteins . Meristematic expression of a GUS reporter gene was observed in plants under the control of Arabidopsis site II motif within a minimal promoter . This expression is strongly enhanced by addition of a telo box within this chimaeric promoter . We showed by gel retardation experiments that the site II motif is a target for several DNA-binding activities present in Arabidopsis crude cell extract and can bind a transcription factor, At-TCP20, from the Teosinte branched 1, Cycloidea, PCF (TCP)-domain protein family . In yeast two-hybrid experiments, At-TCP20 appears to be a potential partner of AtPuralpha, which was previously shown to bind telo boxes . An important consequence of this analysis is to reveal new and conserved regulatory processes concerning the regulation of plant ribosomal gene expression in cycling cells . The implication of these observations in plant-specific developmental pathways is discussed.

J Pept Sci, 2003 Feb, 9(2), 120 - 4
Purification and characterization of a new ribonuclease from fruiting bodies of the oyster mushroom Pleurotus ostreatus; Ye XY et al.; A ribonuclease (RNase), possessing an N-terminal sequence disparate from those of ribonucleases from other mushrooms and previously isolated Pleuotus ostreatus RNases, was purified from the fruiting bodies of the edible mushroom Pleurotus ostreatus . The N-terminal sequence of Pleurotus ostreatus RNase did not manifest homology even to a previously reported RNase from the same mushroom . The ribonuclease was adsorbed on CM-Sepharose and Mono S . It exhibited a molecular mass of 12 kDa in both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75 . The ribonuclease displayed an activity of 11490 U/mg on yeast tRNA . The highest ribonuclease activity was exhibited toward poly U, followed by poly A and poly C . No activity was shown toward poly G . The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C . It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 240 nM.

Pharmacogenomics J, 2003, 3(1), 53 - 61
Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation; Xie HJ et al.; The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes . A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles . CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting . 4-Hydroxy-CPA and hydroxy-bupropion were quantified by using HPLC and LC-MS, respectively . 7-Ethoxy-4-trifluoromethyl coumarin O-deethylase activity was measured fluorometrically . The frequencies of CYP2B6*5 and CYP2B6*6 mutant alleles were 9.0 and 16.4%, respectively . CYP2B6 protein expression was detected in 80% of the samples, with a large variation (0.003-2.234, arbitrary units) . There was a high correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation (n=55, r=0.81, P<0.0001) . When based on the CYP2B6 apoprotein levels, the *6 carriers had significantly higher CPA 4-hydroxylation (P<0.05) . CPA 4-hydroxylation also correlated significantly with other CYP2B6-specific reactions (n=20, P<0.0001) . V(max) and K(m) for CPA 4-hydroxylation in recombinant CYP2B6 enzyme were 338 nmol/min/nmol enzyme and 1.4 mM, respectively . CYP2B6 showed much higher in vitro intrinsic clearance than previously observed in recombinant CYP2C19 and CYP2C9 variants in yeast expression system . Our results demonstrate that the polymorphic CYP2B6 is a major enzyme in the bioactivation of CPA . Moreover, we identified a strong impact of CYP2B6*6 on CPA 4-hydroxylation.

Oncogene, 2003 Mar 13, 22(10), 1557 - 67
Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor; Hyland S et al.; Intracellular expression of single-chain antibodies (scFvs) represents a promising approach for selective interference with cellular proto-oncogenes such as the epidermal growth factor receptor (EGFR) . Previously, we have shown that intrabodies targeted to the lumen of the endoplasmic reticulum prevent the transit of EGFR or the related ErbB2 molecule to the cell surface, thereby inactivating their transforming potential . While intramolecular disulfide bridges important for antibody stability are correctly formed during expression in the secretory pathway, scFvs expressed in the reducing environment of the cytosol are often inactive . To overcome this problem and to generate antibody fragments that interact with the intracellular domain of human EGFR in the cytoplasm, here we have chosen a two-step approach combining classical selection of scFvs by phage display with subsequent expression in yeast . After enrichment of EGFR-specific antibody fragments from a combinatorial library by biopanning, a yeast two-hybrid screen was performed using the intracellular domain of EGFR as bait . Screening of 1.5 x 10(5) preselected scFv plasmids under highly stringent conditions yielded 223 colonies that represented at least five independent scFv clones functional in the intracellular milieu of eukaryotic cells . Interaction of selected antibody fragments with the intracellular domain of EGFR was confirmed in GST pull-down and coimmunoprecipitation experiments . Upon cytoplasmic expression in human tumor cells, scFvs colocalized with EGFR at the plasma membrane demonstrating their functionality in vivo.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3374 - 9 Epub 2003 Mar 10.
Sequences in the (A)gamma-delta intergenic region are not required for stage-specific regulation of the human beta-globin gene locus; Gaensler KM et al.; The human beta-globin locus has been extensively studied as a model of tissue and developmental stage-specific gene expression . Structural mapping of naturally occurring mutations, including transfection and transgenic studies, and the recent finding of intergenic transcripts have suggested that there are cis-acting sequence elements in the (A)gamma-delta intergenic region involved in regulating gamma- and beta-globin gene expression . To determine whether previously identified sequences in the (A)gamma-delta intergenic region are required for appropriate developmental expression of the human beta-globin gene cluster, transgenic mice were generated by transfer of yeast artificial chromosomes containing the entire human beta-globin locus . Three different deletions of the (A)gamma-delta intergenic region were introduced, including (i) deletion of the 750-bp (A)gamma 3' regulatory element ((A)gammae), (ii) deletion of 3.2 kb upstream of the delta-globin gene encompassing pyrimidine-rich sequences and the recently described intergenic transcript initiation site, and (iii) deletion of a 12.5-kb fragment encompassing most of the (A)gamma-delta globin intergenic region . Analysis of multiple transgenic lines carrying these deletion constructs demonstrated that the normal stage-specific sequential expression of the epsilon -, gamma-, and beta-globin genes was preserved, despite deletion of these putative regulatory sequences . These studies suggest that regulatory sequences required for activation and silencing of the human beta-globin gene family during ontogeny reside proximally to the genes and immediately 5' to the human gamma- and beta-globin genes.

EMBO J, 2003 Mar 17, 22(6), 1325 - 35
GRIM-19, a death-regulatory gene product, suppresses Stat3 activity via functional interaction; Lufei C et al.; Signal transducer and activator of transcription 3 (Stat3) is a latent cytoplasmic transcription factor that can be activated by cytokines and growth factors . Stat3 plays important roles in cell growth, anti-apoptosis and cell transformation, and is constitutively active in various cancers . We examined its potential regulators by yeast two-hybrid screening . GRIM-19, a gene product related to interferon-beta- and retinoic acid-induced cancer cell death, was identified and demonstrated to interact with Stat3 in various cell types . The interaction is specific for Stat3, but not for Stat1 and Stat5a . The interaction regions in both proteins were mapped, and the cellular localization of the interaction was examined . GRIM-19 itself co-localizes with mitochondrial markers, and forms aggregates at the perinulear region with co-expressed Stat3, which inhibits Stat3 nuclear translocation stimulated by epidermal growth factor (EGF) . GRIM-19 represses Stat3 transcriptional activity and its target gene expression, and also suppresses cell growth in Src-transformed cells and a Stat3-expressing cell line . Our data suggest that GRIM-19 is a novel negative regulator of Stat3.

Trends Cell Biol, 2003 Mar, 13(3), 151 - 6
An array of insights: application of DNA chip technology in the study of cell biology; Panda S et al.; The advent of DNA microarray technology has ushered in an era of systems biology whereby researchers can study the transcriptional behavior of thousands of genes in parallel . Advances in manufacturing techniques and informatics, and the availability of several genome sequences have furthered these capabilities to the point where whole-transcriptome studies can be accomplished in yeast, flies and plants, and soon will be possible in mammals . Concomitant with the expanding ability of the technology has been the development of novel techniques and their application towards the study of cellular biology.

Biochimie, 2002 Dec, 84(12), 1207 - 20
Prohibitin and prohibitone are contained in high-molecular weight complexes and interact with alpha-actinin and annexin A2; Bacher S et al.; The closely related proteins prohibitin (p32) and prohibitone (p37) are evolutionarily conserved with homologues found from cyanobacteria to man . They are thought to be exclusively mitochondrial and have been assigned many-rather different-functions, ranging from a role in lifespan, in mitochondrial inheritance and as chaperones of mitochondrial proteases in yeast . Evidence for a localisation outside of mitochondria has been brought forward in mammalian cells, where they influence cell-cycle progression and are found in association with cell surface receptors . We have employed a yeast two-hybrid screen to identify other interacting proteins and have identified alpha-actinin and annexin A2 as binding partners for prohibitin and prohibitone . Coprecipitation experiments supported the putative binding between prohibitin and prohibitone on the one hand and annexin A2 or alpha-actinin on the other hand in intact cells . Surface plasmon resonance analysis was used to determine relative affinities between prohibitin and alpha-actinin and between prohibitone and annexin A2 and alpha-actinin, respectively . We further show that prohibitin and prohibitone can also form homomeric (preferentially tetrameric) and heteromultimeric complexes, with significant affinities.

AIDS Educ Prev, 2003 Feb, 15(1), 93 - 108
Responses of male inmates to primary partner requests for condom use: effects of message content and domestic violence history; Neighbors CJ et al.; Many women at high risk for HIV infection face resistance and, in some cases, violence as a response to their requests for condom use . The current study investigated how domestically violent and nonviolent men reacted to various condom negotiation approaches . Ten different scenarios, in which the partner provides a justification for a condom request or the context suggests one, were presented to 84 male inmates selected at random from a county jail . As predicted, condom scenarios factored into groupings with content suggestive of high and low relationship threat . Of the justifications presented, yeast infections generated more favorable responses than standard HIV prevention messages . The riskiest condom negotiation scenario was one that suggested infidelity on the part of the woman . Level of male violence severity in the relationship predicted more coercive responses to suggestions of a woman's infidelity . The results suggest that creative strategies that do not call into question the fidelity or commitment of either partner may be more effective in getting men to use condoms and/or to not react violently.

Hematol Oncol Clin North Am, 2003 Feb, 17(1), 313 - 41
Heparin, low-molecular-weight heparins, and heparin pentasaccharide: basic and clinical differentiation; Hoppensteadt D et al.; As a result of advanced technology, dramatic developments in the area of new anticoagulant and antithrombotic drugs appear to have made a profound impact on the use of LMWHs . Furthermore, because porcine mucosal heparin is used for the preparation of these agents, it is likely that alternative drugs with comparable pharmacologic and clinical efficacy are sought . Antithrombin drugs such as argatroban and hirudin are already approved for alternative management of heparin-compromised patients . Their efficacy in other indications is less superior . The development of specific anti-Xa drugs is slow . Although these agents may inhibit factor Xa and thrombin generation, none of them are capable of mimicking the polytherapeutic effects of LMWHs and thus can only be given in drug combinations . Synthetic and recombinant protein-derived anti-tissue factor agents have also been developed . These drugs only inhibit the tissue factor-mediated process and are limited in their therapeutic spectrum . Plasma-derived and recombinant serine protease inhibitors (serpins) are also available for the management of thrombotic and inflammatory disorders, but these agents cannot be given subcutaneously . Furthermore, because they are proteins, antibodies to these agents are generated . Nucleic acid derivatives (natural and synthetic aptomers) are developed for intravenous administration, but they are relatively weak antithrombotic agents . Dermatans, heparans, and chondroitin sulfates represent nonheparin GAGs, and, in mono-compositional and polycompositional form, these drugs are mainly used for the intravenous management of DVT prophylaxis . They can be given to patients who are heparin compromised . Synthetic heparinomimetics include heparin consensus-binding oligosaccharides and synthetic oligosaccharides with non-serpin affinity . In addition, binding oligosaccharides are conjugated with antithrombin agents to mimic the anti-Xa/anti-IIa activities of heparin . Biotechnology using bacterial and yeast cultures, aqua cultures for marine products, and plant carbohydrates have been the focus of developing heparin analogues . Development of these agents is in the early phase; however, it is likely that this approach may provide a reasonable alternative to LMWHs . Despite these developments, it is unlikely that any of these drugs will have a profound impact on the use of LMWHs in the near future . Unfractionated heparin and LMWHs collectively represent an important group of polypharmacologic drugs without which the management of thrombosis and vascular disorders would not be possible . The continual development of LMWHs in expanded indications did not comprise the use of unfractionated heparin in surgical and interventional cardiovascular indications . Ever since their introduction in the 1980s, the use of LMWHs has continually increased . This is primarily because of expanded indications and growing awareness among the clinicians . It is likely that once an antidote is developed and additional information is available on the mechanism of action of LMWHs, these drugs may gradualty be used for surgery patients . Despite these developments, it is likely that unfractionated heparin will continue to be used for specific indications . Drug combinations with heparins may necessitate dose adjustments, but it is unclear whether unilateral reduction of heparins will be optimal . The coming years will provide useful clinical and applied data on the improved use of unfractionated heparin . LMWHs, and pentasaccharide in the management of thrombotic and cardiovascular disorders . In addition, use of these drugs will be extended to many conditions, including cancer, inflammation, sepsis, and autoimmune diseases . Polytherapeutic approaches emphasizing LMWHs as primary and secondary drugs will also have an impact on the management of thrombotic and nonthrombotic disorders . Ultra-LMWHs and synthetic heparinomimetics, such as fondaparinux, that exhibit a narrow pharmacologic spectrum will only be useful in specific indications and in combination with other drugs.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3369 - 73 Epub 2003 Mar 07.
Conditional tradeoffs between aging and organismal performance of Indy long-lived mutant flies; Marden JH et al.; Alterations that extend the life span of animals and yeast typically involve decreases in metabolic rate, growth, physical activity, and/or early-life fecundity . This negative correlation between life span and the ability to assimilate and process energy, to move, grow, and reproduce, raises questions about the potential utility of life span extension . Tradeoffs between early-life fitness and longevity are central to theories of the evolution of aging, which suggests there is necessarily a price to be paid for reducing the rate of aging . It is not yet clear whether life span can be extended without undesirable effects on metabolism and fecundity . Here, we report that the long-lived Indy mutation in Drosophila causes a decrease in the slope of the mortality curve consistent with a slowing in the rate of aging without a concomitant reduction in resting metabolic rate, flight velocity, or age-specific fecundity under normal rearing conditions . However, Indy mutants on a decreased-calorie diet have reduced fecundity, suggesting that a tradeoff between longevity and this aspect of performance is conditional, i.e., the tradeoff can occur in a stressful environment while being absent in a more favorable environment . These results provide evidence that there do exist mechanisms, albeit conditional, that can extend life span without significant reduction in fecundity, metabolic rate, or locomotion.

Glycobiology, 2003 May, 13(5), 401 - 10 Epub 2003 Jan 22.
Characterization of carbohydrate recognition by langerin, a C-type lectin of Langerhans cells; Stambach NS et al.; Langerin is a type II transmembrane cell surface receptor found on Langerhans cells . The extracellular domain of langerin consists of a neck region containing a series of heptad repeats and a C-terminal C-type carbohydrate-recognition domain (CRD) . A role for langerin in processing of glycoprotein antigens has been proposed, but until now there has been little study of the langerin protein . In this study, analytical ultracentrifugation and circular dichroism spectroscopy of recombinant soluble fragments of human langerin have been used to show that the extracellular region of this receptor exists as a stable trimer held together by a coiled coil of alpha-helices formed by the neck region . The langerin CRD shows specificity for mannose, GlcNAc, and fucose, but only the trimeric extracellular domain fragment binds to glycoprotein ligands . Langerin extracellular domain binds mammalian high mannose oligosaccharides, as well mannose-containing structures on yeast invertase but does not bind complex glycan structures . Full-length langerin stably expressed in rat fibroblast transfectants mediates efficient uptake and degradation of a mannosylated neoglycoprotein ligand . pH-dependent ligand release appears to involve interactions between the CRDs or between the CRDs and the neck region in the trimer . The results are consistent with a role for langerin in internalization of both self and nonself glycoprotein antigens.

Glycobiology, 2003 Apr, 13(4), 285 - 94 Epub 2003 Jan 03.
GALT deficiency causes UDP-hexose deficit in human galactosemic cells; Lai K et al.; Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency . We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose . Control fibroblasts accumulated little Gal-1-P and continued to grow . The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose . The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium . When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively . Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively . Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM) . We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax . The Ki of the reaction was 0.47 mM . Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine . We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses . Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.

Protein Pept Lett, 2003 Feb, 10(1), 35 - 42
Inhibition of human napsin A; Cronshaw RF et al.; The newly-discovered human aspartic proteinase, napsin A was not susceptible to protein inhibitors from potato, squash or yeast but was weakly inhibited by the 17 kDa polypeptide from Ascaris lumbricoides and potently by isovaleryl and lactoyl-pepstatins . A series of synthetic inhibitors was also investigated which contained in the P(1)-P(1)' positions the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues and in which the residues occupying P(4)-P(3)' were varied systematically . On this basis, the active site of napsin A can be readily distinguished from other human aspartic proteinases.

Endocrine, 2002 Dec, 19(3), 293 - 300
Invertase, maltase, lactase, and peroxidase activities in duodenum of BB rats; Courtois P et al.; The development of immune-mediated diabetes in BB rats may involve a defect of the gastrointestinal tract (GI), as suggested by increased gut permeability . This study aimed at measuring invertase, maltase, lactase, and peroxidase activities in the duodenum of diabetesprone BioBreeding (BBdp) rats and control BioBreeding rats (BBc) given free access to NIH-07 diet up to the time of killing at 60 66 d of age . After washing the entire small intestine, the duodenal mucosa was scraped off in the first 5-cm segment from the pylorus and frozen in distilled water . Invertase, maltase, and lactase activities were measured by monitoring the conversion of {U-(14)C}sucrose, {U-(14)C}maltose, and {D-{1-(14)C}glucose} lactose to radioactive hexoses, which were phosphorylated in the presence of adenosine triphosphatase and yeast hexokinase and then separated from their precursor by ion-exchange chromatography . Peroxidase activity was measured by a spectrophotometric procedure . In the BBdp rats, the activity of invertase, maltase, and lactase averaged, respectively, 70.2 +/- 4.4, 81.2 +/- 4.3, and 75.7 +/- 4.1% (n = 16 and p < 0.001 in all cases) of the control values found in BBc rats of the same sex . Inversely, after exclusion of two female BBc rats with abnormally high plasma D-glucose concentration, the activity of peroxidase in the BBdp rats averaged 157.4 +/- 20.0% (n = 16; p < 0.02) of the mean control value recorded in BBc rats of the same sex (100.0 +/- 9.3%; n = 14) . These findings are compatible with the view that a proinflammatory state of the GI associated with compromise function may precede the occurrence of pancreatic insulitis in BBdp rats and, possibly, human subjects with type 1 diabetes.

J Biol Chem, 2003 May 30, 278(22), 20029 - 36 Epub 2003 Mar 06.
The C-terminal kinase domain of the p34cdc2-related PITSLRE protein kinase (p110C) associates with p21-activated kinase 1 and inhibits its activity during anoikis; Chen S et al.; The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases . During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C) . The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells . In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells . To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C . The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis . The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1 . Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed . Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C . Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis . Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.

Vet Rec, 2003 Feb 8, 152(6), 172 - 5
Pathology of sporotrichosis in 10 cats in Rio de Janeiro; Schubach TM et al.; Ten cats with sporotrichosis were examined clinically and pathologically . They were in very poor general condition, and had widespread ulcerated cutaneous lesions and respiratory signs . Gross internal abnormalities were found only in the lungs and lymph nodes . Histologically, an inflammatory infiltrate and yeast-like structures were observed in the skin, lungs, liver and lymph nodes . The spleen was congested and contained fungal elements . No microscopical changes were observed in the pancreas, kidneys and heart . Sporothix schenckii was isolated from all the skin samples and nasal swabs obtained in vivo, and from all the samples of lung, liver, spleen, lymph nodes, heart and kidney taken postmortem.

J Ir Dent Assoc, 2002, 48(4), 132 - 8
Control of denture plaque and mucosal inflammation in denture wearers; Khasawneh S et al.; The aims of this study were to collect data about the popular methods and materials used for cleaning dentures among complete and partial denture wearers in Jordan and to discuss the relationship between denture base plaque and mucosal inflammation under dentures . A questionnaire consisting of six questions regarding denture hygiene practices and cleaning products was completed by 321 patients who attended two prosthodontic clinics for replacement or adjustment of their dentures . Following careful oral examination and examination of dentures, the relationship between denture hygiene and inflammation under the denture base was investigated . In this study there were 615 dentures and 321 patients . The mean age of patients was 65 years and it ranged from 18 to 100 years (s.d . = 10.1) . The mean age of their dentures was 7.3 years (s.d . = 5.6) ranging from one to 27 years . The most popular method of cleaning dentures was brushing . Ninety-four (29 per cent) of the denture wearers had denture stomatitis . There was a statistically significant relationship between poor denture hygiene and denture stomatitis (P = 0.0001) . There was also a significant relationship between continuous wearing of the dentures, day and night, and denture stomatitis (P < 0.0001) . The presence of bacterial and yeast plaque on the fitting surface of the denture base appeared to be of critical importance for development and maintenance of denture stomatitis . It is necessary, therefore, that dentists should give instructions to denture wearers on how to clean their denture surfaces properly so as to maintain good hygiene and prevent denture stomatitis.

Mutagenesis, 2003 Mar, 18(2), 105 - 12
Induction of endoreduplication by topoisomerase II catalytic inhibitors; Cortes F et al.; The striking phenomenon of endoreduplication has long attracted attention from cytogeneticists and researchers into cell cycle enzymology and dynamics alike . Because of the variety of agents able to induce endoreduplication and the various cell types where it has been described, until now no clear or unique mechanism of induction of this phenomenon, rare in animals but otherwise quite common in plants, has been proposed . Recent years, however, have witnessed the unfolding of a number of essential physiological roles for DNA topoisomerase II, with special emphasis on its major role in mitotic chromosome segregation after DNA replication . In spite of the lack of mammalian mutants defective in topoisomerase II as compared with yeast, experiments with inhibitors of the enzyme have supported the hypothesis that this crucial untangling of daughter DNA molecules by passing an intact helix through a transient double-stranded break carried out by the enzyme, when it fails, leads to aberrant mitosis that results in endoreduplication, polyploidy and eventually cell death . Anticancer drugs that interfere with topoisomerase II can be classified into two groups . The classical poisons act by stabilizing the enzyme in the so-called cleavable complex and result in DNA damage, which represents a problem in the study of endoreduplication . The true catalytic inhibitors, which are not cleavable complex stabilizers, allow us to use doses efficient in the induction of endoreduplication while eliminating high levels of DNA and chromosome damage . This review will discuss the basic and applied aspects of this as yet scarcely explored field.

J Biol Chem, 2003 May 9, 278(19), 17299 - 306 Epub 2003 Mar 05.
HSF-1 interacts with Ral-binding protein 1 in a stress-responsive, multiprotein complex with HSP90 in vivo; Hu Y et al.; Heat shock factor 1 (HSF1) regulates the rapid and transient expression of heat shock genes in response to stress . The transcriptional activity of HSF1 is tightly controlled, and under physiological growth conditions, the HSF1 monomer is in a heterocomplex with the molecular chaperone HSP90 . Through unknown mechanisms, transcriptionally repressed HSF1.HSP90 heterocomplexes dissociate following stress, which triggers HSF1 activation and heat shock gene transcription . Using a yeast two-hybrid screening system, we have identified Ral-binding protein 1 (RalBP1) as an additional HSF1-interacting protein . We show that RalBP1 and HSF1 interact in vivo, and transient cotransfection of HSF1 and RalBP1 into hsf1(-/-) mouse embryo fibroblasts represses HSP70 expression . Furthermore, transient cotransfection of HSF1 and the constitutively active form of RalA (RalA23V), an upstream activator of the RalBP1 signaling pathway, increases the heat-inducible expression of HSP70, whereas the dominant negative form (RalA28N) suppresses HSP70 expression . We further find that alpha-tubulin and HSP90 are also present in the RalBP1.HSF1 heterocomplexes in unstressed cells . Upon heat shock, the Ral signaling pathway is activated, and the resulting RalGTP binds RalBP1 . Concurrently, HSF1 is activated, leaves the RalBP1 x HSF1 x HSP90 x alpha-tubulin heterocomplexes, and translocates into the nucleus, where it then activates transcription . In conclusion, these observations reveal that the RalGTP signal transduction pathway is critical for activation of the stress-responsive HSF1 and perhaps HSP90 molecular chaperone system.

Development, 2003 Apr, 130(8), 1493 - 504
HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis; Bollman KM et al.; Loss-of-function mutations of HASTY (HST) affect many different processes in Arabidopsis development . In addition to reducing the size of both roots and lateral organs of the shoot, hst mutations affect the size of the shoot apical meristem, accelerate vegetative phase change, delay floral induction under short days, adaxialize leaves and carpels, disrupt the phyllotaxis of the inflorescence, and reduce fertility . Double mutant analysis suggests that HST acts in parallel to SQUINT in the regulation of phase change and in parallel to KANADI in the regulation of leaf polarity . Positional cloning demonstrated that HST is the Arabidopsis ortholog of the importin beta-like nucleocytoplasmic transport receptors exportin 5 in mammals and MSN5 in yeast . Consistent with a potential role in nucleocytoplasmic transport, we found that HST interacts with RAN1 in a yeast two-hybrid assay and that a HST-GUS fusion protein is located at the periphery of the nucleus . HST is one of at least 17 members of the importin-beta family in Arabidopsis and is the first member of this family shown to have an essential function in plants . The hst loss-of-function phenotype suggests that this protein regulates the nucleocytoplasmic transport of molecules involved in several different morphogenetic pathways, as well as molecules generally required for root and shoot growth.

Virology, 2003 Feb 1, 306(1), 87 - 99
Requirement of E6AP and the features of human papillomavirus E6 necessary to support degradation of p53; Cooper B et al.; E6 oncoproteins from human papillomavirus type 16 (16E6) and Bovine Papillomavirus type 1 (BE6) bind to leucine rich peptides (called charged leucine, LXXLL, or signature peptides) found on target cellular proteins . BE6 and 16E6 both bind the product of the UBE3A gene called E6AP on a charged leucine peptide, LQELL . E6AP is an E3 ubiquitin ligase that together with 16E6 interacts with p53 to target p53 degradation . Although both BE6 and 16E6 bind the LQELL peptide of E6AP, only 16E6 acts as an adapter to then bring p53 to E6AP . In order to determine how E6 proteins function as adapters, 16E6, p53, and E6AP were expressed in yeast, and were shown to form a tri-molecular complex . 16E6 mutants were selected that retained interactions with E6AP yet were defective for interaction with p53 . Such 16E6 mutations were typically within the amino-terminus of 16E6 . Through the use of E6AP null cells, transfected E6AP was shown to be necessary and sufficient for the degradation of p53 in the presence of 16E6 . However, the interaction of 16E6 with E6AP was complex . While BE6 interacts only with the LQELL motif of E6AP, an intact LQELL motif is not necessary either for interaction of 16E6 with E6AP or for p53 degradation . In addition, 16E6 mutants that fail to bind the LQELL motif of E6AP can support p53 degradation . These results indicate that 16E6 may have multiple modes of interaction with E6AP and that assembly of p53 containing complexes for targeted degradation by E6AP may occur in more than one way . These results have implications for potential targeting of the interaction of 16E6 and E6AP in the therapy of HPV-induced cancer.

Virology, 2003 Feb 1, 306(1), 51 - 9
Modulation of interferon expression by hepatitis C virus NS5A protein and human homeodomain protein PTX1; Ghosh AK et al.; Hepatitis C virus (HCV) NS5A protein transcriptionally modulates a number of cellular genes . Since there is no evidence of binding of NS5A protein to DNA, it is likely to exert its activity in concert with cellular factor(s) . In this study, we have identified a specific interaction of HCV NS5A with homeodomain protein PTX1 of human origin by a yeast two-hybrid interacting cloning system . The authenticity of this interaction was verified by mammalian two-hybrid assay, in vivo co-immunoprecipitation analysis, and from a colocalization study . Recently, murine PTX1 (mPTX1) has been shown to repress virus-induced murine interferonA4 promoter activity . Interferon-a alone or together with ribavirin is the only available therapy for HCV-infected patients . Therefore, we examined whether coexpression of NS5A and human PTX1 (hPTX1) proteins modulate human IFN-a promoter activity . An in vitro reporter assay by transfection of HepG2 cells with NS5A suggested an activation of IFN-a promoter to approximately 20-fold upon Newcastle disease virus (NDV) infection . Under similar experimental conditions, hPTX1-activated IFN-a prompter to approximately sevenfold, unlike mPTX1 . However, cotransfection of NS5A and hPTX1 displayed a lower interferon promoter activity, probably for physical association between these two proteins . Subsequent study demonstrated that activation of IFN promoter by NS5A is associated with an increased expression of IRF-3 . Further analysis revealed that ectopic expression of NS5A in HepG2 cells enhances endogenous IFN-a secretion and MxA expression upon induction with NDV . However, exogenous expression of hPTX1 did not significantly alter NS5A-mediated function in the stable transfectants . Taken together, these results suggested that the level of endogenous hPTX1 is not sufficient to block the function of NS5A for augmentation of virus-mediated IFN activity in HepG2 cells.

Fungal Genet Biol, 2003 Mar, 38(2), 187 - 97
Analysis of expressed sequence tags from Gibberella zeae (anamorph Fusarium graminearum); Trail F et al.; Gibberella zeae is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world . To better understand fungal development and pathogenicity, we have generated 7996 ESTs from three cDNA libraries . Two libraries were generated from carbon-(C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P) . In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression . To assign putative function to cDNAs, sequences were initially assembled using StackPack . The estimated total number of genes identified from the three EST databases was 2110: 1088 contigs and 1022 singleton sequences . These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank nonredundant database using BLASTX . Based on presumptive gene function identified by this process, we found that the two starved cultures had similar, but not identical, patterns of gene expression, whereas the developmental cultures were distinct in their pattern of expression . Of the three libraries, the perithecium library had the greatest percentage (46%) of ESTS falling into the "unclassified" category . Homologues of some known fungal virulence or pathogenicity factors were found primarily in the N- and C-libraries . Comparisons also were made with ESTs from the related fungi, Neurospora crassa and Magnaporthe grisea and the genomic sequence of N . crassa.

Mol Cell, 2003 Feb, 11(2), 303 - 13
The telomere protein Taz1 is required to prevent and repair genomic DNA breaks; Miller KM et al.; One fundamental function of telomeres is to prevent the ends of chromosomes from being sensed and treated as DNA damage . Here we present evidence for additional roles of telomeres in promoting proper chromosome segregation and DNA repair . We find that the fission yeast telomere protein Taz1p is required for cell cycle progression at 20 degrees C, a temperature at which taz1Delta cells exhibit a G(2)/M DNA damage checkpoint delay, chromosome missegregation, and DNA double-strand breaks (DSBs) . Spindle assembly checkpoint components and a checkpoint-independent function of Rad3p are required for taz1Delta cells to survive at 20 degrees C . Disruption of topoisomerase II activity suppresses the cold sensitivity of taz1Delta cells, suggesting a scenario in which telomeric entanglement is the primary defect . Furthermore, hypersensitivity to treatments that induce DSBs suggests that Taz1p is involved in DSB repair . Our observations imply roles for Taz1p-containing telomeres in preventing and repairing DNA breaks throughout the genome.

Curr Biol, 2003 Mar 4, 13(5), 437 - 41
The c-Myc Oncoprotein Interacts with Bcr; Mahon GM et al.; Bcr is a multifunctional protein that is the fusion partner for Abl (p210 Bcr-Abl) in Philadelphia chromosome positive leukemias . We have identified c-Myc as a binding partner for Bcr in both yeast and mammalian cells . We are also able to observe interactions between natively expressed c-Myc and Bcr in leukemic cell lines . Although Bcr and Max have overlapping binding sites on c-Myc, Bcr cannot interact with Max, or with the c-Myc.Max heterodimer . Bcr expression blocks activation of c-Myc-responsive genes, as well as the transformed phenotype induced by coexpression of c-Myc and H-Ras, and this finding suggests that one function of Bcr is to limit the activity of c-Myc . However, Bcr does not block c-Myc function by preventing its nuclear localization . Interestingly, increased Bcr dosage in COS-7 and K-562 cells correlates with a reduction in c-Myc protein levels, suggesting that Bcr may in fact be limiting c-Myc activity by regulating its stability . These data indicate that Bcr is a novel regulator of c-Myc function whose disrupted expression may contribute to the high level of c-Myc protein that is observed in Bcr-Abl transformed cells.

Curr Biol, 2003 Mar 4, 13(5), 384 - 93
Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion; Kinley AW et al.; BACKGROUND: Modulation of actin cytoskeleton assembly is an integral step in many cellular events . A key regulator of actin polymerization is Arp2/3 complex . Cortactin, an F-actin binding protein that localizes to membrane ruffles, is an activator of Arp2/3 complex . RESULTS: A yeast two-hybrid screen revealed the interaction of the cortactin Src homology 3 (SH3) domain with a peptide fragment derived from a cDNA encoding a region of WASp-Interacting Protein (WIP) . GST-cortactin interacted with WIP in an SH3-dependent manner . The subcellular localization of cortactin and WIP coincided at the cell periphery . WIP increased the efficiency of cortactin-mediated Arp2/3 complex activation of actin polymerization in a concentration-dependent manner . Lastly, coexpression of cortactin and WIP stimulated membrane protrusions . CONCLUSIONS: WIP, a protein involved in filopodia formation, binds to both actin monomers and cortactin . Thus, recruitment of actin monomers to a cortactin-activated Arp2/3 complex likely leads to the observed increase in cortactin activation of Arp2/3 complex by WIP . These data suggest that a cortactin-WIP complex functions in regulating actin-based structures at the cell periphery.

Genome Biol . 2003;4(3):R18 . Epub 2003 Feb 17.
Feminizing chicks: a model for avian sex determination based on titration of Hint enzyme activity and the predicted structure of an Asw-Hint heterodimer; Pace HC et al.; BACKGROUND: In birds and some lizards, females are heterogametic with a ZW karyotype, while males are ZZ homogametes . The molecular basis for sexual differentiation in birds is unknown: arguments exist for doses of Z masculinizing chicks and for W information feminizing . ASW was identified as a tandemly repeated gene conserved on avian W chromosomes that is expressed in early female development and appears to be an inactive form of avian Z-encoded HINT . Hint is a dimeric enzyme that hydrolyzes AMP linked to lysine, whose enzyme activity is required for regulation of the Cdk7 homologous Kin28 kinase in yeast . Of 16 residues most conserved across all life forms for AMP interactions, 15 are sexually dimorphic in birds, that is, altered in the female-specific Asw protein . Genomic and expression data suggest that Asw may feminize chicks, dominantly interfering with Hint function by heterodimerization . RESULTS: We consider whether positive cooperativity could explain how Hint heterodimerization with an inert enzyme might reduce specific activity by more than 50% and provide data sufficient to reject this model . Instead, we hypothesize that Asw carries a signal for mislocalization and/or proteolysis, and/or dominantly suppresses the remaining Hint active site to function as a dominant negative . CONCLUSIONS: Molecular modeling suggests that Asw and Hint can heterodimerize and that Gln 127, an Asw-specific alteration for Trp123, dominantly interferes with the Hint active site . An extra dose of HINT in ZZW chicks, and thus more Hint homodimer, may partially overcome the feminizing influence of ASW and lead to the observed intersexual characteristics of ZZW triploids.

J Bone Miner Res, 2003 Mar, 18(3), 466 - 72
Functional domains for amelogenin revealed by compound genetic defects; Paine ML et al.; We have previously used the yeast two-hybrid assay and multiple in vitro methodologies to show that amelogenin undergoes self-assembly involving two domains (A and B) . Using transgenic animals, we show that unique enamel phenotypes result from disruptions to either the A- or B-domain, supporting the role of amelogenin in influencing enamel structural organization . By crossbreeding, animals bearing two defective amelogenin gene products have a more extreme enamel phenotype than the sum of the defects evident in the individual parental lines . At the nanoscale level, the forming matrix shows alteration in the size of the amelogenin nanospheres . At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization caused by perturbed organization of the precursor organic matrix . These studies reflect the critical dependency of amelogenin self-assembly to form a highly organized enamel organic matrix, and that amelogenins engineered to be defective in self-assembly produce compound defects in the structural organization of enamel.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 158 - 60
Nuclear localization of senescence marker protein-30, SMP30, in cultured mouse hepatocytes and its similarity to RNA polymerase; Ishigami A et al.; Senescence marker protein-30 (SMP30), expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity . By immunohistochemistry and western blotting, we found that SMP30 was in both the nuclei and cytoplasm of cultured mouse hepatocytes . By a homology search, we found that a domain of the SMP30 sequence 51 amino acid residues long was 60-66% similar to bacterial and yeast RNA polymerases.

Oncogene, 2003 Mar 6, 22(9), 1371 - 80
The parathyroid hormone-responsive B1 gene is interrupted by a t(1;7)(q42;p15) breakpoint associated with Wilms' tumour; Vernon EG et al.; Wilms' tumour (WT) has a diverse and complex molecular aetiology, with several different loci identified by cytogenetic and molecular analyses . One such locus is on chromosome 7p, where cytogenetic abnormalities and loss of heterozygosity (LOH) indicate the presence of a Wilms' tumour suppressor gene . In order to isolate a candidate gene for this locus, we have characterized the breakpoint regions at a novel constitutional chromosome translocation (t(1;7)(q42;p15)), found in a child with WT and skeletal abnormalities . We identified two genes that were interrupted by the translocation: the parathyroid hormone-responsive B1 gene (PTH-B1) at 7p and obscurin at 1q . With no evidence for LOH at 1q42, we focused on the characterization of PTH-B1 . We detected novel alternately spliced isoforms of PTH-B1, which were expressed in a wide range of adult and foetal tissues . Importantly, expression of two isoforms were disrupted in the WT of the t(1;7) patient . We also identified an additional splice isoform expressed only in 7p LOH tumours . The disruption of PTH-B1 by the t(1;7), together with aberrant splicing in sporadic WTs, suggests that PTH-B1 is a candidate for the 7p Wilms' tumour suppressor gene.

J Biol Chem, 2003 May 9, 278(19), 17210 - 7 Epub 2003 Mar 04.
p47phox participates in activation of RelA in endothelial cells; Gu Y et al.; Activation of endothelial cell NF-kappaB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response . In this context, oxidants have been associated with NF-kappaB activation, although the molecular source and mechanism of targeting have remained obscure . We found that RelA, essential for NF-kappaB activation by IL-1, was associated with the NADPH oxidase adapter protein p47(phox) in yeast two-hybrid, coprecipitation, and in vitro binding studies . RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions . Overexpression of p47(phox) synergized with IL-1beta in the activation of an artificial kappaB-luciferase reporter and specifically augmented IL-1beta-induced RelA transactivation activity . p47(phox) overexpression also greatly increased IL-1beta-stimulated RelA phosphorylation, whereas it had no effect on I-kappaB degradation or on RelA nuclear translocation or kappaB binding . The tandem SH3 domains of p47(phox) were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains . The RelA-PR peptide blocked interaction of p47(phox) and RelA, and ectopic expression of RelA-PR abrogated IL-1beta-induced transactivation of the NF-kappaB-dependent E-selectin promoter . Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1beta-induced E-selectin promoter activation, suggesting that p47(phox) facilitates NF-kappaB activation through linkage with the NADPH oxidase . IL-1beta rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1beta signaling pathway leading to RelA activation.

Genetics, 2003 Feb, 163(2), 677 - 84
Trichothecene nonproducer Gibberella species have both functional and nonfunctional 3-O-acetyltransferase genes; Kimura M et al.; The trichothecene 3-O-acetyltransferase gene (FgTri101) required for trichothecene production by Fusarium graminearum is located between the phosphate permease gene (pho5) and the UTP-ammonia ligase gene (ura7) . We have cloned and sequenced the pho5-to-ura7 regions from three trichothecene nonproducing Fusarium (i.e., F . oxysporum, F . moniliforme, and Fusarium species IFO 7772) that belong to the teleomorph genus Gibberella . BLASTX analysis of these sequences revealed portions of predicted polypeptides with high similarities to the TRI101 polypeptide . While FspTri101 (Fusarium species Tri101) coded for a functional 3-O-acetyltransferase, FoTri101 (F . oxysporum Tri101) and FmTri101 (F . moniliforme Tri101) were pseudogenes . Nevertheless, F . oxysporum and F . moniliforme were able to acetylate C-3 of trichothecenes, indicating that these nonproducers possess another as yet unidentified 3-O-acetyltransferase gene . By means of cDNA expression cloning using fission yeast, we isolated the responsible FoTri201 gene from F . oxysporum; on the basis of this sequence, FmTri201 has been cloned from F . moniliforme by PCR techniques . Both Tri201 showed only a limited level of nucleotide sequence similarity to FgTri101 and FspTri101 . The existence of Tri101 in a trichothecene nonproducer suggests that this gene existed in the fungal genome before the divergence of producers from nonproducers in the evolution of Fusarium species.

Biochim Biophys Acta, 2003 Mar 17, 1603(2), 113 - 28
Autophagy: a barrier or an adaptive response to cancer; Ogier-Denis E et al.; Macroautophagy or autophagy is a degradative pathway terminating in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles . The recent discovery of the molecular controls of autophagy that are common to eukaryotic cells from yeast to human suggests that the role of autophagy in cell functioning is far beyond its nonselective degradative capacity . The involvement of proteins with properties of tumor suppressor and oncogenic properties at different steps of the pathway implies that autophagy must be considered in tumor progression . Autophagy as a stress response mechanism protects cancer cells from low nutrient supply or therapeutic insults . Autophagy is also involved in the elimination of cancer cells by triggering a non-apoptotic cell death program, suggesting a negative role in tumor development . These two aspects of autophagy will be discussed in this review .

Mech Ageing Dev, 2003 Jan, 124(1), 125 - 32
Inverse Genomics as a powerful tool to identify novel targets for the treatment of neurodegenerative diseases; Rhoades K et al.; Toward the prevention of neurodegeneration we have used Immusol's Inverse Genomics platform to identify gene targets involved in neuronal cell death . Inverse genomics uses a combinatorial library of unique hairpin ribozymes with randomized substrate binding sequences to cleave unique RNA transcripts, thereby decreasing translation of the encoded proteins . Using the SK-N-MC neuroblastoma cell line a cell based survival selection assay was designed with C2-ceramide or TNFalpha as an induction signal for apoptosis . SK-N-MC cells were stably transduced with a ribozyme vector library, and then exposed to 20 microM C2-ceramide or 50 ng/ml TNFalpha to induce cell death . Surviving cells were harvested, their DNA isolated, and the ribozymes rescued by PCR for re-introduction into fresh cells . After several rounds of selection and ribozyme rescue we have identified individual ribozymes that protect neuronal cells from C2-ceramide induced apoptosis . Three of the cellular targets of these ribozyme sequence tags have been validated . Microarray analysis and yeast two-hybrid screens have also been used to gain insight into the pathways involved by identifying additional players involved in these pathways . These target genes may also serve as therapeutic targets for development of drugs for Alzheimer and Parkinson's diseases.

Gene Expr Patterns, 2002 Dec, 2(3-4), 289 - 96
Identification and expression of Ima, a novel Ral-interacting Drosophila protein; Beller M et al.; We report the identification of Ima, a novel Drosophila MAGUK-like protein, which contains two WW and four PDZ protein interaction domains and interacts with the small GTPase dRal in the yeast two-hybrid system and pull-down assays . The gene is expressed in distinct spatiotemporal patterns throughout embryonic development . Overexpression of Ima interferes with normal Drosophila development, indicating that the gene functions in a tissue specific manner.

J Am Chem Soc, 2003 Mar 12, 125(10), 2913 - 22
Structural model for an alkaline form of ferricytochrome C; Assfalg M et al.; An (15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy . The mutations were planned to produce a cytochrome c with a single conformer . Despite suboptimal conditions for the collection of spectra (i.e., pH approximately equal to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism . 76% of amino acids and 49% of protons were assigned successfully . The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions . Lys73 was assigned as the axial ligand together with His18 . In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 +/- 0.2 A . Most of the native cytochrome c conformation is maintained at high pH . The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact . Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent . Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.

J Cell Sci, 2003 Apr 1, 116(Pt 7), 1359 - 66
The lipoma preferred partner LPP interacts with alpha-actinin; Li B et al.; The lipoma preferred partner LPP is a member of the zyxin family of proteins . In this paper, we demonstrate that the structural similarities observed between zyxin and LPP also extend to their interaction capabilities . Similar to zyxin, LPP was found to bind to alpha-actinin in vitro . This interaction was confirmed in yeast and mammalian cells . Studies utilizing the three-hybrid system further indicated that zyxin and LPP compete for the same binding site in alpha-actinin . This site was mapped to the central rod of alpha-actinin, which contains spectrin-like repeats 2 and 3 . In the case of LPP, a conserved motif present at the N-terminus was shown to be responsible for the interaction . Constructs lacking this motif did not bind to alpha-actinin in the yeast two-hybrid system and were not able to recruit alpha-actinin to an ectopic site in mammalian cells . Quantitative data obtained with the two-hybrid and the three-hybrid system suggest that LPP has a lower affinity for alpha-actinin than zyxin . It is likely that this difference leads to slightly different roles played by LPP and zyxin during the assembly and disassembly of focal adhesions.

J Cell Sci, 2003 Apr 1, 116(Pt 7), 1219 - 33
Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains; Hatzfeld M et al.; p0071, a member of the armadillo protein family, is most closely related to p120(ctn) and the plakophilins 1-3 . Whereas plakophilins are desmosomal plaque proteins, p120(ctn) localizes to adherens junctions and interacts with classical cadherins . In contrast, p0071 has been described as a protein with dual localization in adherens junctions and desmosomes depending on the cell type examined . Here we have analyzed the localization of p0071 and its domains in detail . Although by sequence analysis, p0071 is more closely related to the adherens junction proteins p120(ctn), ARVCF and delta-catenin, endogenous p0071 associated preferentially with desmosomes in MCF-7 epithelial cells . Overexpressed p0071 localized along cell borders and overlapped only partially with desmosomal markers but colocalized with non-desmosomal cadherins and recruited cadherins to the membrane . The head domain of p0071 was sufficient for desmosomal targeting, whereas the arm repeat domain associated with adherens junctions and enhanced membrane association of classical cadherins . The tail domain localized preferentially to the nucleus and associated with desmosomes . To examine the mechanism underlying this dual localization more closely we determined binding partners of p0071 by using yeast-two-hybrid and mom-targeting assays . These approaches show that the head domain interacted with desmosomal proteins desmocollin 3a and desmoplakin, whereas the armadillo repeat domain binds to non-desmosomal cadherins . Head and armadillo repeat domains both interacted with plakoglobin by binding to different sites . Our data suggest that, in addition to plakoglobin, p0071 is the second armadillo protein present in both types of adhesive junctions and may play a role in regulating crosstalk between adherens junctions and desmosomes.

Plant Cell, 2003 Mar, 15(3), 790 - 800
AtDUR3 encodes a new type of high-affinity urea/H+ symporter in Arabidopsis; Liu LH et al.; Urea is the major nitrogen form supplied as fertilizer in agricultural plant production but also an important nitrogen metabolite in plants . We report the cloning and functional characterization of AtDUR3, a high-affinity urea transporter in plants . AtDUR3 contains 14 putative transmembrane-spanning domains and represents an individual member in Arabidopsis that belongs to a superfamily of sodium-solute symporters . Heterologous expression in urea uptake-defective yeast as well as two-electrode voltage clamp and uptake studies using (14)C-labeled urea in AtDUR3-expressing oocytes demonstrated that AtDUR3 mediates urea transport . In both heterologous systems, urea transport was stimulated at low pH . In oocytes, inward currents indicated that urea is cotransported with protons . By contrast, a supply of Na(+) ions could not stimulate urea transport . Transport of (14)C-labeled urea by AtDUR3 in oocytes exhibited saturation kinetics with a K(m) of approximately 3 micro M . AtDUR3 was expressed in shoots and roots and upregulated during early germination and under nitrogen deficiency in roots . We propose a role of AtDUR3 in urea uptake by plant cells at low external urea concentrations.

Plant Cell, 2003 Mar, 15(3), 626 - 38
Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression; Vlachonasios KE et al.; We previously identified Arabidopsis genes homologous with the yeast ADA2 and GCN5 genes that encode components of the ADA and SAGA histone acetyltransferase complexes . In this report, we explore the biological roles of the Arabidopsis ADA2b and GCN5 genes . T-DNA insertion mutations in ADA2b and GCN5 were found to have pleiotropic effects on plant growth and development, including dwarf size, aberrant root development, and short petals and stamens in flowers . Approximately 5% of the 8200 genes assayed by DNA microarray analysis showed changes of expression in the mutants, three-fourths of which were upregulated and only half of which were altered similarly in the two mutant strains . In cold acclimation experiments, C-repeat binding factors (CBFs) were induced in the mutants as in wild-type plants, but subsequent transcription of cold-regulated (COR) genes was reduced in both mutants . Remarkably, nonacclimated ada2b-1 (but not gcn5-1) mutant plants were more freezing tolerant than nonacclimated wild-type plants, suggesting that ADA2b may directly or indirectly repress a freezing tolerance mechanism that does not require the expression of CBF or COR genes . We conclude that the Arabidopsis ADA2b and GCN5 proteins have both similar and distinct functions in plant growth, development, and gene expression and may be components of both a common coactivator complex and separate complexes with distinct biological activities.

J Biol Chem, 2003 May 9, 278(19), 16791 - 6 Epub 2003 Mar 03.
Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins; Seeger M et al.; Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome . We show that both proteins bind to different regions of the proteasome subunit Mts4 . The binding site for Pus1 was mapped to a cluster of repetitive sequences also found in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4 . The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed.

J Dermatol Sci, 2003 Feb, 31(1), 43 - 51
Gene expression of Sh3d19, a novel adaptor protein with five Src homology 3 domains, in anagen mouse hair follicles; Shimomura Y et al.; BACKGROUND: Sh3yl1, which contains one Src homology (SH) 3 domain, has been previously identified from mouse skin and considered to play an important role in hair follicle formation by interacting with other proteins . OBJECTIVE: We performed this study to identify proteins capable of associating with Sh3yl1 . METHODS: We screened a mouse skin cDNA library using the SH3 domain of Sh3yl1 as bait in the yeast two-hybrid system . RESULTS: We identified a 420-amino acid-long protein containing a proline-rich stretch and five carboxyl-terminal SH3 domains, which we have termed Sh3d19 . We confirmed the interactions between Sh3yl1 and Sh3d19 by in vitro binding assays . Northern blot analysis showed that Sh3d19 transcripts in mouse skin were expressed in accordance with the hair cycle . Furthermore, RNA in situ hybridization studies demonstrated that its transcripts were detected predominantly in the medulla cells at the level corresponding to the keratogenous zone of the hair follicles during the mid and late anagen phases . CONCLUSION: Sh3d19 is a novel adaptor protein that may be involved in the development of medulla cells during the anagen phase.

Biochem Biophys Res Commun, 2003 Mar 14, 302(3), 526 - 33
Characterization of zetin 1/rBSPRY, a novel binding partner of 14-3-3 proteins; Birkenfeld J et al.; 14-3-3 proteins are ubiquitously expressed proteins which serve as central adaptors in different signal transduction cascades . In this study, yeast two-hybrid screening of a rat brain cDNA library identified a novel gene product termed zetin 1/rBSPRY that interacts with 14-3-3 zeta . The zetin 1/rBSPRY gene is ubiquitously expressed in a variety of rat tissues, with highest expression being found in testis . In adult brain, high levels of zetin 1/rBSPRY mRNA were observed in the hippocampus, cerebral cortex, and piriform cortex . Biochemical studies confirmed zetin 1/rBSPRY to interact with 14-3-3 zeta . Transient co-transfection in COS 7 cells caused a partial redistribution of zetin 1/rBSPRY into 14-3-3 zeta enriched submembranous foci at leading edges . Our results suggest a role for zetin 1/rBSPRY-14-3-3 interactions at specialized submembrane domains.

Hum Gene Ther, 2003 Jan 20, 14(2), 117 - 27
Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors; Wang WJ et al.; The first large randomized phase III trial in gene therapy demonstrated no improvement in the survival of patients injected with packaging cells that produced conventional replication-defective retroviral vectors carrying the herpes simplex virus thymidine kinase gene, a disappointing result that was attributed to extremely poor levels of transduction efficiency . To circumvent this problem, we have developed a modified replication-competent retrovirus (RCR) that is capable of transducing human glioma cell lines A-172, U-87, T-98G, U-373, and U-138 and rat glioma cell lines C6 and 9L, over multiple infection cycles in vitro, resulting in a tremendous enhancement in transduction efficiency over conventional replication-defective retroviral vectors at the same dose . Whereas the transduction efficiency of conventional retroviral vectors injected into preestablished subcutaneous U-87 tumors at a dose of 1.0 x 10(5) transducing units (TU) was only 0.2% at 6 weeks postinjection, the same dose of RCR vector resulted in up to 97.2% transduction . When RCR vectors at a dose of 1.0 x 10(4) TU were injected into preestablished intracranial U-87 tumors, transduction efficiency at 2 and 3 weeks was 74 and 98.1%, respectively . Notably, however, intracranial injection of RCR vectors did not result in detectable infection of normal brain cells . Furthermore, using a sensitive polymerase chain reaction assay, no detectable RCR signal could be observed in any extracerebral tissues, including lung, liver, kidney, upper gastrointestinal tract (esophagus and stomach), lower gastrointestinal tract (colon and small intestine), skin, spleen, and bone marrow . Treatment of U-87 intracranial gliomas with RCR vectors carrying the yeast cytosine deaminase suicide gene followed by 5-fluorocytosine prodrug administration resulted in 100% survival over a 60-day follow-up period, compared with 0% survival of control groups receiving vector alone or prodrug alone . Our results demonstrate that RCR vectors can achieve therapeutically significant levels of transduction in malignant human gliomas, and that RCR vector spread after intratumoral injection is restricted to the tumor itself.

Pediatr Res, 2003 Apr, 53(4), 546 - 53 Epub 2003 Feb 20.
Functional attenuation of UFD1l, a 22q11.2 deletion syndrome candidate gene, leads to cardiac outflow septation defects in chicken embryos; Yamagishi C et al.; Microdeletion of chromosome 22q11.2 is commonly associated with congenital cardiovascular defects that involve development of cranial neural crest cells (NCC) that emigrate through the pharyngeal arches . UFD1l is one of several candidate genes for 22q11.2 deletion syndrome (22q11DS) . UFD1l encodes a protein whose yeast counterpart is involved in a ubiquitin-dependent proteolytic degradation pathway; however, the role of UFD1L in NCC development remains unknown . Mouse embryos that lack Ufd1l die before organogenesis . We have therefore studied the function of Ufd1l in the chick system . Chick Ufd1l encoded a 307-amino acid protein that was highly conserved with mouse and human UFD1L . Chick Ufd1l was expressed in the developing neural tube, NCC, and mesenchyme of the head and pharyngeal arch structures, as well as in the conotruncal region (cardiac outflow tract), consistent with the clinical features of 22q11DS . To determine loss-of-function effects of chick Ufd1l in NCC, we infected cardiac NCC with a retrovirus expressing antisense Ufd1l transcripts in chick embryos before their migration . Morphologic analysis of infected embryos at a later developmental stage demonstrated that functional attenuation of chick Ufd1l in cardiac NCC resulted in an increased incidence of conotruncal septation defects . These data suggest that Ufd1l may play a role in cardiac NCC during conotruncal septation.

J Nutr, 2003 Mar, 133(3), 668 - 72
Oxidative folding of interleukin-2 is impaired in flavin-deficient jurkat cells, causing intracellular accumulation of interleukin-2 and increased expression of stress response genes; Camporeale G et al.; Secretory proteins such as interleukin (IL)-2 undergo oxidative folding (disulfide formation) in the endoplasmic reticulum (ER) before secretion . Studies in yeast have suggested that oxidative folding depends on the flavoprotein Ero1p; unfolded proteins accumulate in the ER, triggering cellular stress response . Here, human lymphoid cells (Jurkat cells) were used to model effects of cellular flavin supply on secretion of IL-2 (containing one disulfide bond) and cellular stress response . Cells were cultured in media containing 0.85, 3.1, 12.6 or 300.6 nmol/L riboflavin for 5 wk, representing severely deficient, moderately deficient, physiologic and pharmacologic plasma concentrations in humans, respectively . Transport rates of riboflavin were increased in severely and moderately deficient cells compared with cells cultured in physiologic medium; this increase was not sufficient to prevent intracellular depletion of riboflavin, as judged by glutathione reductase activity and intracellular concentrations of glutathione . Intracellular accumulation of IL-2 was greater in severely deficient cells than in other groups . Nevertheless, severely deficient cells secreted normal amounts of IL-2 into the extracellular space, mediated by increased transcriptional activity of the IL-2 gene . Riboflavin-deficient cells responded to intracellular accumulation of IL-2 with increased expression of genes encoding ubiquitin-activating enzyme E1 and X box-binding protein, consistent with cellular stress . These findings are consistent with the hypothesis that flavin deficiency may cause cellular stress by accumulation of unfolded proteins in human cells.

Mol Cell Biol, 2003 Mar, 23(6), 1968 - 82
Dominant negative dimerization of a mutant homeodomain protein in Axenfeld-Rieger syndrome; Saadi I et al.; Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein . We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain . By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA . Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region . Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73) . In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity . To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R) . The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif . Like K88E, K88R formed relatively stronger dimers with WT . As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity . These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.

Mol Cancer Res, 2003 Feb, 1(4), 247 - 61
DJBP: a novel DJ-1-binding protein, negatively regulates the androgen receptor by recruiting histone deacetylase complex, and DJ-1 antagonizes this inhibition by abrogation of this complex; Niki T et al.; DJ-1 was identified by us as a novel oncogene that transforms mouse NIH3T3 cells in cooperation with ras . We later identified PIAS (protein inhibitor of activated STAT)xalpha as a DJ-1-binding protein, and found that DJ-1 restored androgen receptor (AR) transcription activity that was repressed by PIASxalpha . To further characterize the function of DJ-1, we cloned cDNA encoding a novel DJ-1-binding protein, DJBP, by a yeast two-hybrid system . DJBP mRNA was found to be specifically expressed in the testis . In addition to the binding of DJBP to the COOH-terminal region of DJ-1, DJBP was also found to bind in vitro and in vivo to the DNA-binding domain of the AR in a testosterone-dependent manner and to be colocalized with DJ-1 or AR in the nucleus . Furthermore, a co-immunoprecipitation assay showed that the formation of a ternary complex between DJ-1, DJBP, and AR occurred in cells in which DJ-1 bound to the AR via DJBP . It was found that DJBP repressed a testosterone-dependent AR transactivation activity in monkey Cos1 cells by recruiting histone deacetylase (HDAC) complex, including HDAC1 and mSin3, and that DJ-1 partially restored its repressed activity by abrogating DJBP-HDAC complex . These results suggest that AR is positively regulated by DJ-1, which antagonizes the function of negative regulators, including DJBP.

Bioinformatics, 2003 Mar 1, 19(4), 474 - 82
Clustering of time-course gene expression data using a mixed-effects model with B-splines; Luan Y et al.; MOTIVATION: Time-course gene expression data are often measured to study dynamic biological systems and gene regulatory networks . To account for time dependency of the gene expression measurements over time and the noisy nature of the microarray data, the mixed-effects model using B-splines was introduced . This paper further explores such mixed-effects model in analyzing the time-course gene expression data and in performing clustering of genes in a mixture model framework . RESULTS: After fitting the mixture model in the framework of the mixed-effects model using an EM algorithm, we obtained the smooth mean gene expression curve for each cluster . For each gene, we obtained the best linear unbiased smooth estimate of its gene expression trajectory over time, combining data from that gene and other genes in the same cluster . Simulated data indicate that the methods can effectively cluster noisy curves into clusters differing in either the shapes of the curves or the times to the peaks of the curves . We further demonstrate the proposed method by clustering the yeast genes based on their cell cycle gene expression data and the human genes based on the temporal transcriptional response of fibroblasts to serum . Clear periodic patterns and varying times to peaks are observed for different clusters of the cell-cycle regulated genes . Results of the analysis of the human fibroblasts data show seven distinct transcriptional response profiles with biological relevance . AVAILABILITY: Matlab programs are available on request from the authors.

Bioinformatics, 2003 Mar 1, 19(4), 459 - 66
Comparisons and validation of statistical clustering techniques for microarray gene expression data; Datta S et al.; MOTIVATION: With the advent of microarray chip technology, large data sets are emerging containing the simultaneous expression levels of thousands of genes at various time points during a biological process . Biologists are attempting to group genes based on the temporal pattern of their expression levels . While the use of hierarchical clustering (UPGMA) with correlation 'distance' has been the most common in the microarray studies, there are many more choices of clustering algorithms in pattern recognition and statistics literature . At the moment there do not seem to be any clear-cut guidelines regarding the choice of a clustering algorithm to be used for grouping genes based on their expression profiles . RESULTS: In this paper, we consider six clustering algorithms (of various flavors!) and evaluate their performances on a well-known publicly available microarray data set on sporulation of budding yeast and on two simulated data sets . Among other things, we formulate three reasonable validation strategies that can be used with any clustering algorithm when temporal observations or replications are present . We evaluate each of these six clustering methods with these validation measures . While the 'best' method is dependent on the exact validation strategy and the number of clusters to be used, overall Diana appears to be a solid performer . Interestingly, the performance of correlation-based hierarchical clustering and model-based clustering (another method that has been advocated by a number of researchers) appear to be on opposite extremes, depending on what validation measure one employs . Next it is shown that the group means produced by Diana are the closest and those produced by UPGMA are the farthest from a model profile based on a set of hand-picked genes . Availability: S+ codes for the partial least squares based clustering are available from the authors upon request . All other clustering methods considered have S+ implementation in the library MASS . S+ codes for calculating the validation measures are available from the authors upon request . The sporulation data set is publicly available at http://cmgm.stanford.edu/pbrown/sporulation

Environ Health Perspect, 2003 Mar, 111(3), 329 - 34
Estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes; Kitamura S et al.; In this study we examined estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes . Trans-1,2-diphenylcyclobutane (TCB), cis-1,2-diphenylcyclobutane (CCB), 1,3-diphenylpropane, 2,4-diphenyl-1-butene, 2,4,6-triphenyl-1-hexene, and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin were negative in the yeast estrogen screening assay and estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7 . However, TCB exhibited estrogenic activity after incubation with liver microsomes of phenobarbital-treated rats in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) . Minor activity was observed when liver microsomes of untreated or 3-methylcholanthrene-treated rats were used instead of those from phenobarbital-treated rats . CCB, 1,3-diphenylpropane, and 2,4-diphenyl-1-butene also exhibited estrogenic activity after metabolic activation by liver microsomes, but the activity was lower than that of TCB . 2,4,6-Triphenyl-1-hexene and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin did not show estrogenic activity after such incubation . When TCB was incubated with liver microsomes of phenobarbital-treated rats in the presence of NADPH, three metabolites were detected by high-performance liquid chromatography (HPLC) . One metabolite isolated by HPLC exhibited a significant estrogenic activity . The active metabolite was identified as trans-1-(4-hydroxyphenyl)-2-phenylcyclobutane by mass and nuclear magnetic resonance spectral analysis . These results suggest that the estrogenic activity of TCB was caused by the formation of the 4-hydroxylated metabolite.

Science, 2003 Feb 28, 299(5611), 1355 - 9
Aging and genome maintenance: lessons from the mouse?
Hasty P, Campisi J, Hoeijmakers J, van Steeg H, Vijg J.
Recent progress in the science of aging is driven largely by the use of model systems, ranging from yeast and nematodes to mice . These models have revealed conservation in genetic pathways that balance energy production and its damaging by-products with pathways that preserve somatic maintenance . Maintaining genome integrity has emerged as a major factor in longevity and cell viability . Here we discuss the use of mouse models with defects in genome maintenance for understanding the molecular basis of aging in humans.

Science, 2003 Feb 28, 299(5611), 1342 - 6
Evolutionary medicine: from dwarf model systems to healthy centenarians?
Longo VD, Finch CE.
Restriction of the number of calories consumed extends longevity in many organisms . In rodents, caloric restriction decreases the levels of plasma glucose and insulin-like growth factor I (IGF-1) and postpones or attenuates cancer, immunosenescence, and inflammation without irreversible side effects . In organisms ranging from yeast to mice, mutations in glucose or IGF-I-like signaling pathways extend life-span but also cause glycogen or fat accumulation and dwarfism . This information suggests a new category of drugs that could prevent or postpone diseases of aging with few adverse effects.

Plant Cell Physiol, 2003 Feb, 44(2), 206 - 11
Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants; Suenaga A et al.; To characterize ammonium transport pathways in rice, two cDNAs with high homology to MEP/AMT2-type ammonium transporters, OsAMT2;1 and OsAMT3;1, were isolated . Expression of OsAMT2;1 in an ammonium-uptake-defective yeast mutant showed that this gene encodes functional ammonium transporters . OsAMT2;1 was constitutively expressed in both roots and shoots irrespective of the supply of inorganic nitrogen to the medium, whereas OsAMT3;1 expression was relatively weak . A database search with the amino acid sequence of OsAMT2;1 showed that there are 10 putative OsAMT genes in rice, i.e . three each for OsAMT1, OsAMT2 and OsAMT3, respectively, and one for OsAMT4.

J Biol Chem, 2003 May 2, 278(18), 15886 - 96 Epub 2003 Feb 27.
Profile-based data base scanning for animal L-type lectins and characterization of VIPL, a novel VIP36-like endoplasmic reticulum protein; Nufer O et al.; Consensus profiles were established to screen data bases for novel animal L-type lectins . The profiles were generated from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the entire carbohydrate recognition domain of these proteins . The search revealed numerous orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast, as well as the sequence of a novel family member related to VIP36, named VIPL for VIP36-like . Sequence analysis suggests that VIPL is a ubiquitously expressed protein and appeared earlier in evolution than VIP36 . The cDNA of VIPL was cloned and expressed in cell culture . VIPL is a high-mannose type I membrane glycoprotein with similar domain organization as VIP36 . Unlike VIP36 and ERGIC-53 that are predominantly associated with postendoplasmic reticulum (ER) membranes and cycle in the early secretory pathway, VIPL is a non-cycling resident protein of the ER . Mutagenesis experiments indicate that ER retention of VIPL involves a RKR di-arginine signal . Overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL-receptor and the overall morphology of the early secretory pathway . The results suggest that VIPL may function as a regulator of ERGIC-53.

Circ Res, 2003 Apr 4, 92(6), 630 - 6 Epub 2003 Feb 27.
The antiinflammatory endothelial tyrosine kinase Tie2 interacts with a novel nuclear factor-kappaB inhibitor ABIN-2; Hughes DP et al.; Tie2 is a receptor tyrosine kinase expressed predominantly in endothelial cells and is essential for blood vessel formation and maintenance . The receptor has potent antiinflammatory effects on endothelial cells, suppressing vascular endothelial growth factor- and tumor necrosis factor-induced expression of leukocyte adhesion molecules and procoagulant tissue factor and inhibiting vascular leakage . To delineate the signaling pathways utilized by Tie2, we performed yeast two-hybrid screening of a human endothelial cell cDNA library and identified a novel protein interacting with the intracellular domain of the receptor . This protein was found to be human A20 binding inhibitor of NF-kappaB activation-2, ABIN-2, an inhibitor of NF-kappaB-mediated inflammatory gene expression . Coexpression of Tie2 and ABIN-2 in CHO cells confirmed the interaction occurs in mammalian cells . In contrast, Tie1 did not interact with ABIN-2 in the yeast two-hybrid system or mammalian cells . Deletion analysis identified the Tie2 binding motif to be encompassed between residues 171 and 272 in ABIN-2 . Interaction was dependent on Tie2 autophosphorylation but ABIN-2 was not tyrosine phosphorylated by Tie2 . Furthermore, in endothelial cells the interaction was stimulated by the Tie2 ligand angiopoietin-1 . Expression of ABIN-2 deletion mutants in endothelial cells suppressed the ability of angiopoietin-1 to inhibit phorbol ester-stimulated NF-kappaB-dependent reporter gene activity . These findings provide the first direct link between Tie2 and a key regulator of inflammatory responses in endothelial cells . Interaction between Tie2 and ABIN-2 may be important in the vascular protective antiinflammatory actions of Tie2.

Plant J, 2003 Feb, 33(4), 707 - 17
A Mak-like kinase is a repressor of GAMYB in barley aleurone; Woodger FJ et al.; GAMYB is a gibberellin (GA)-regulated activator of hydrolase gene expression in the aleurone layer of germinating cereal grains . Although it is clear that GAMYB expression is regulated by GA, more remains to be understood about how this transcription factor operates within the GA-response pathway . In order to isolate new components from the GA-response pathway, barley aleurone libraries were screened for GAMYB-binding proteins using a recently developed yeast two-hybrid system, which is compatible with the use of transcription factors as baits . We isolated a new member of the emerging Mak-subgroup of cdc2- and MAP kinase-related protein kinases . We have termed this GAMYB-binding protein KGM (for kinase associated with GAMYB) . Transient expression of KGM specifically repressed alpha-amylase promoter activity at the level of GAMYB function but a mutation designed to de-stabilise the activation loop of KGM alleviated this repression . We propose that KGM is a negative regulator of GAMYB function in aleurone that may prevent precocious hydrolase gene expression.

Plant J, 2003 Mar, 33(5), 939 - 48
Cell cycle-dependent association of Arabidopsis actin-related proteins AtARP4 and AtARP7 with the nucleus; Kandasamy MK et al.; Arabidopsis encodes at least eight actin-related proteins (ARPs) most of which have orthologs in other distant organisms . To gain insight into the role of ARPs in plants, we have examined the spatial expression and subcellular distribution of two highly divergent Arabidopsis ARPs, AtARP4 and AtARP7 . AtARP4 is a homolog of human BAF53 and yeast Arp4, and AtARP7 is a novel, ancient and plant-specific actin-related protein that is not distinctly related to any known ARPs from other kingdoms . Analysis of both these proteins with AtARP4- and AtARP7-specific antibodies revealed that they were most abundant in young meristematic and floral tissues, but were expressed constitutively in all organs and cell types irrespective of their developmental stage . Immunofluorescence studies showed that both AtARP4 and AtARP7 were localized predominantly to the nucleus during interphase . In mitotic cells lacking a nuclear envelope (e.g . metaphase, anaphase, and early telophase stages), these ARPs were excluded from the condensed chromosomes and dispersed throughout the cytoplasm . In contrast, a putative Arabidopsis histone H2B protein remained associated with the interphase nuclei as well as chromosomes throughout the cell cycle . Based on our results and data on the yeast ortholog of AtARP4, these two nuclear plant ARPs may be involved in the modulation of chromatin structure and transcriptional regulation mainly in interphase cells.

Plant J, 2003 Mar, 33(5), 825 - 40
Interaction of NtCDPK1 calcium-dependent protein kinase with NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum; Lee SS et al.; Using a yeast two-hybrid system, we identified NtRpn3, a regulatory subunit of 26S proteasome, as an interacting protein of NtCDPK1 calcium-dependent protein kinase in Nicotiana tabacum . Rpn3 in yeast is an essential protein involved in proteolysis of cell cycle regulatory proteins, and the carrot homolog of Rpn3 was previously isolated as a nuclear antigen that is mainly expressed in the meristem . NtCDPK1 physically interacts with NtRpn3 in vitro in a Ca2+-independent manner and phosphorylates NtRpn3 in a Ca2+-dependent manner with Mg2+ as a cofactor . NtCDPK1 and NtRpn3 are co-localized in the nucleus, nuclear periphery, and around plasma membrane in vivo . Both NtCDPK1 and AtRpn3, an NtRpn3 homolog of Arabidopsis, are mainly expressed in the rapidly proliferating tissues including shoot and root meristems, and developing floral buds . Virus-induced gene silencing of either NtRpn3 or NtCDPK1 resulted in the phenotypes of abnormal cell morphology and premature cell death in newly emerged leaves . Finally, NtCDPK1 interacts with NtRpn3 in vivo as shown by co-immunoprecipitation . Based on these results, we propose that NtCDPK1 and NtRpn3 are interacting in a common signal transduction pathway possibly for regulation of cell division, differentiation, and cell death in tobacco.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2284 - 9 Epub 2003 Feb 26.
The p53-inducible TSAP6 gene product regulates apoptosis and the cell cycle and interacts with Nix and the Myt1 kinase; Passer BJ et al.; The p53 tumor suppressor protein plays a crucial role in tumorigenesis by controlling cell-cycle progression and apoptosis . We have previously described a transcript designated tumor suppressor activated pathway-6 (TSAP6) that is up-regulated in the p53-inducible cell line, LTR6 . Cloning of the murine and human full-length TSAP6 cDNA revealed that it encodes a 488-aa protein with five to six transmembrane domains . This gene is the murine and human homologue of the recently published rat pHyde . Antibodies raised against murine and human TSAP6 recognize a 50- to 55-kDa band induced by p53 . Analysis of the TSAP6 promoter identified a functional p53-responsive element . Functional studies demonstrated that TSAP6 antisense cDNA diminished levels of the 50- to 55-kDa protein and decreased significantly the levels of p53-induced apoptosis . Furthermore, TSAP6 small interfering RNA inhibited apoptosis in TSAP6-overexpressing cells . Yeast two-hybrid analysis followed by GST/in vitro-transcribed/translated pull-down assays and in vivo coimmunoprecipitations revealed that TSAP6 associated with Nix, a proapoptotic Bcl-2-related protein and the Myt1 kinase, a negative regulator of the G(2)/M transition . Moreover, TSAP6 enhanced the susceptibility of cells to apoptosis and cooperated with Nix to exacerbate this effect . Cell-cycle studies indicated that TSAP6 could augment Myt1 activity . Overall, these data suggest that TSAP6 may act downstream to p53 to interface apoptosis and cell-cycle progression.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2322 - 7 Epub 2003 Feb 26.
Interaction of FK506-binding protein 13 with brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1): effects of FK506; Padilla PI et al.; BIG1 and BIG2 are brefeldin A-inhibited guanine nucleotide-exchange proteins that activate ADP-ribosylation factors (ARFs), critical components of vesicular trafficking pathways . These proteins can exist in macromolecular complexes and move between Golgi membranes and cytosol . In the BIG1 molecule, a centrally located Sec7 domain is responsible for ARF activation, but functions of other regions are largely unknown . Yeast two-hybrid screens of a human placenta cDNA library with BIG1 cDNA constructs revealed specific interaction of the N-terminal region (amino acids 1-331) with FK506-binding protein 13 (FKBP13) . The association was confirmed by immunoprecipitation of both endogenous BIG1 and FKBP13 from Jurkat T cells with antibodies against either one . Binding of BIG1, BIG2, and ARF to cell membranes in vitro was increased by guanosine 5'-{gamma-thio}triphosphate, and further increases were induced by FK506 . Incubation of Jurkat T cells with FK506 increased binding of BIG1, BIG2, and ARF to Golgi and other membranes in a time- and concentration-dependent manner, without effects on clathrin or gamma-adaptin binding . Binding of BIG1, BIG2, and ARF to membranes was also increased by L-732,531, an agonist structurally related to FK506, but was not increased by a related antagonist, L-685,818, nor by cyclosporin A or rapamycin . These findings are consistent with a role for FKBP13 and FK506 in vesicular trafficking, influencing ARF activity through their guanine nucleotide-exchange proteins.

EMBO J, 2003 Mar 3, 22(5), 1168 - 79
HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo; Zhang Y et al.; Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes . Reversible acetylation on the epsilon-amino group of alpha-tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics . Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently . Here we report that beta-tubulin interacts with histone deacetylase-6 (HDAC-6) in a yeast two-hybrid assay and in vitro . We find that HDAC-6 is a micro tubule-associated protein capable of deacetylating alpha-tubulin in vivo and in vitro . HDAC-6's microtubule binding and deacetylation functions both depend on the hdac domains . Overexpression of HDAC-6 in mammalian cells leads to tubulin hypoacetylation . In contrast, inhibition of HDAC-6 function by two independent mechanisms--pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC-6 in embryonic stem cells)--leads to hyperacetylation of tubulin and microtubules . Taken together, our data provide evidence that HDAC-6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non-histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors.

EMBO J, 2003 Mar 3, 22(5), 1125 - 33
Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6; Garrard SM et al.; Cdc42 is a small GTPase that is required for cell polarity establishment in eukaryotes as diverse as budding yeast and mammals . Par6 is also implicated in metazoan cell polarity establishment and asymmetric cell divisions . Cdc42.GTP interacts with proteins that contain a conserved sequence called a CRIB motif . Uniquely, Par6 possesses a semi-CRIB motif that is not sufficient for binding to Cdc42 . An adjacent PDZ domain is also necessary and is required for biological effects of Par6 . Here we report the crystal structure of a complex between Cdc42 and the Par6 GTPase-binding domain . The semi-CRIB motif forms a beta-strand that inserts between the four strands of Cdc42 and the three strands of the PDZ domain to form a continuous eight-stranded sheet . Cdc42 induces a conformational change in Par6, detectable by fluorescence resonance energy transfer spectroscopy . Nuclear magnetic resonance studies indicate that the semi-CRIB motif of Par6 is at least partially structured by the PDZ domain . The structure highlights a novel role for a PDZ domain as a structural scaffold.

EMBO J, 2003 Mar 3, 22(5), 1075 - 87
Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint; Yamaguchi S et al.; Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast . We have found that the spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells . Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites . A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage . The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function . The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function . Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.

EMBO J, 2003 Mar 3, 22(5), 1036 - 46
A role for N-glycanase in the cytosolic turnover of glycoproteins; Hirsch C et al.; Successful maturation determines the intracellular fate of secretory and membrane proteins in the endoplasmic reticulum (ER) . Failure of proteins to fold or assemble properly can lead to their retention in the ER and redirects them to the cytosol for degradation by the proteasome . Proteasome inhibitors can yield deglycosylated cytoplasmic intermediates that are the result of an N-glycanase activity, believed to act prior to destruction of these substrates by the proteasome . A gene encoding a yeast peptide:N-glycanase, PNG1, has been cloned, but this N-glycanase and its mammalian homolog were reported to be incapable of deglycosylating full-length glycoproteins . We show that both the yeast PNG1 enzyme and its mammalian homolog display N-glycanase activity towards intact glycoproteins . As substrates, cytosolic PNGase activity prefers proteins containing high-mannose over those bearing complex type oligosaccharides . Importantly, PNG1 discriminates between non-native and folded glycoproteins, consistent with a role for N-glycanase in cytoplasmic turnover of glycoproteins.

Virus Res, 2003 Mar, 92(1), 37 - 45
Non-covalent interaction between nucleocapsid protein of Tula hantavirus and small ubiquitin-related modifier-1, SUMO-1; Kaukinen P et al.; To find cellular binding counterparts for the nucleocapsid protein (N) of Tula hantavirus (TULV), two cDNA libraries were screened using yeast two-hybrid systems based on LexA and Gal4 transcription factors . Five cDNA clones encoding SUMO-1 (Small Ubiquitin-related MOdifier, also known as sentrin) were selected in the LexA system . Confocal microscopy revealed that, in infected cells, TULV N protein and SUMO-1 colocalize at the perinuclear area providing further evidence for interaction between the two proteins . Neither endogenous nor transiently expressed SUMO-1 was found to be covalently linked to the N protein . Additional evidence that the interaction is non-covalent was obtained in immunoprecipitation experiments: N protein-specific antibodies precipitated SUMO-1 from TULV-infected Vero E6 cell lysate . By using a pepscan assay, two basic amino acid stretches in the N-terminal part of SUMO-1 were shown to be involved in the interaction.

J Gen Virol, 2003 Mar, 84(Pt 3), 665 - 76
Kaposi's sarcoma-associated herpesvirus K8 protein interacts with hSNF5; Hwang S et al.; Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus related to Epstein-Barr virus (EBV) and herpesvirus saimiri . KSHV open reading frame K8 encodes a basic region-leucine zipper protein of 237 aa that homodimerizes . K8 shows significant similarity to the EBV immediate-early protein Zta, a key regulator of EBV reactivation and replication . In this study, a carboxyl-terminal deletion mutant of K8, K8(1-115), that had strong transactivating properties was found . Screening using transcriptionally inactive K8(1-75) showed that K8 interacts and co-localizes with hSNF5, a cellular chromatin-remodelling factor, both in vivo and in vitro . This interaction requires aa 48-183 of hSNF5 and 1-75 of K8 . In a yeast expression system, the ability of K8 and K8(1-115) to activate transcription requires the presence of SNF5, the yeast homologue of hSNF5 . These data suggest a mechanism by which the SWI-SNF complex is recruited to specific genes . They also suggest that K8 functions as a transcriptional activator under specific conditions and that its transactivation activity requires its interaction with the cellular chromatin remodelling factor hSNF5.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2712 - 7 Epub 2003 Feb 25.
Cdc42-interacting protein 4 binds to huntingtin: neuropathologic and biological evidence for a role in Huntington's disease; Holbert S et al.; Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt) . Pathogenesis in HD seems to involve the formation of neuronal intranuclear inclusions and the abnormal regulation of transcription and signal transduction . To identify previously uncharacterized htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library . We found a predicted SH3 domain protein (K08E3.3b) that interacts with N-terminal htt in two-hybrid tests . A human homolog of K08E3.3b is the Cdc42-interacting protein 4 (CIP4), a protein involved in Cdc42 and Wiskott-Aldrich syndrome protein-dependent signal transduction . CIP4 interacted in vitro with full-length htt from lymphoblastoid cells . Neuronal CIP4 immunoreactivity increased with neuropathological severity in the neostriatum of HD patients and partially colocalized to ubiquitin-positive aggregates . Marked CIP4 overexpression also was observed in Western blot from human HD brain striatum . The overexpression of CIP4 induced the death of striatal neurons . Our data suggest that CIP4 accumulation and cellular toxicity may have a role in HD pathogenesis.

J Biol Chem, 2003 May 9, 278(19), 17491 - 9 Epub 2003 Feb 25.
Perlecan protein core interacts with extracellular matrix protein 1 (ECM1), a glycoprotein involved in bone formation and angiogenesis; Mongiat M et al.; The goal of this study was to discover novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes, and to examine new interactions through which perlecan may influence cell behavior . We employed the yeast two-hybrid system and used perlecan domain V as bait to screen a human keratinocyte cDNA library . Among the strongest interacting clones, we isolated a approximately 1.6-kb cDNA insert that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis . The sequencing of the clone revealed the existence of a novel splice variant that we name ECM1c . The interaction was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells, and the specific binding site within each molecule was identified employing various deletion mutants . The C terminus of ECM1 interacted specifically with the epidermal growth factor-like modules flanking the LG2 subdomain of perlecan domain V . Perlecan and ECM1 were also co-expressed by a variety of normal and transformed cells, and immunohistochemical studies showed a partial expression overlap, particularly around dermal blood vessels and adnexal epithelia . ECM1 has been shown to regulate endochondral bone formation, stimulate the proliferation of endothelial cells, and induce angiogenesis . Similarly, perlecan plays an important role in chondrogenesis and skeletal development, as well as harboring pro- and anti-angiogenic activities . Thus, a physiological interaction could also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 847 - 53
Relationships between respiration and susceptibility to azole antifungals in Candida glabrata; Brun S et al.; Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly . Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species . In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C . glabrata were evaluated . Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide . Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles . Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed . This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar . This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy . Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency . Finally, this mutant exhibited cross-resistance to all the azoles tested . In conclusion, blockage of respiration in C . glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA . This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.

Chem Biol Interact, 2003 Feb 1, 143-144, 469 - 80
The critical role of the N-terminus of 11beta-hydroxysteroid dehydrogenase type 1, as being encoded by exon 1, for enzyme stabilization and activity; Blum A et al.; 11beta-Hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders, including insulin resistance and obesity . Because 11beta-HSD 1 is a membrane protein with a very hydrophobic character, it is difficult to purify it in an active state . Not much is known about the topological and structural determinants of 11beta-HSD 1, although the elucidation of the structure of 11beta-HSD 1 would be a great advantage in identifying specific 11beta-HSD 1 inhibitors . Bacterial expression of full-length or truncated 11beta-HSD 1 forms only led to insoluble proteins or to low amounts of enzyme, not sufficient for crystallization . Recently, we reported that the solubility of 11beta-HSD 1 could be increased by substitution of hydrophobic amino acid residues with arginine without affecting activity . Unfortunately, these truncated and soluble forms of 11beta-HSD 1 exhibited an unstable activity that declined very rapidly . So far, the proteins obtained were not suitable for crystallization . To obtain 11beta-HSD 1 in an active and soluble state, in the present investigation we focused on the amino acid sequence encoded by the first exon . Using bacterial and yeast expression systems, we found that this N-terminal peptide could be divided into two parts that have functions other than to anchor 11beta-HSD 1 into the ER membrane . The first hydrophobic part, consisting of amino acid residues 1-15, represents the membrane spanning domain and anchors 11beta-HSD 1 in the ER membrane . The second hydrophilic part of the peptide, consisting of amino acid residues 16-30, plays a crucial role in stabilizing the catalytic domain of 11beta-HSD 1 and in addition, acts as a spacer to keep the catalytic domain of 11beta-HSD 1 into the lumen of the ER . Evidently, we found that the hydrophilic amino acids 24-30 determine 11beta-HSD 1 enzyme activity . Combined, all information obtained should help to design an optimal 11beta-HSD 1 enzyme in the near future with all desired attributes: soluble, active and easy to obtain and purify in sufficient amounts . This soluble and active 11beta-HSD 1 form should be the basis for our ongoing project, which is the determination of the three dimensional structure of 11beta-HSD 1.

Chem Biol Interact, 2003 Feb 1, 143-144, 255 - 61
Multiplicity of eukaryotic ADH and other MDR forms; Jornvall H et al.; Eukaryotic genomes code for at least eight medium-chain dehydrogenases/reductases (MDR) enzyme families of two types, with and without Zn(2+) at the active site . Four families have Zn(2+): 'Dimeric alcohol dehydrogenases (ADHs)' (including liver ADHs), 'Tetrameric ADHs' (including the yeast ADHs), 'Cinnamyl ADHs' and 'Polyol DHs' . In the human genome, there are minimally 23 MDR genes, but the list is still growing from further interpretations . Of these, seven genes on chromosome 4 (and three pseudogenes) represent the ADH classes in the gene order IV, Igamma, Ibeta, Ialpha, V, II and III . The lineages leading to human ADH establish five levels of divergence, with nodes at the MDR/short-chain dehydrogenases/reductases (SDR), dimer/tetramer, class III/non-III, further class, and intraclass levels of divergence . These multiplicities allow conclusions on pathways of function for ADHs and suggest this activity to have two roles in addition to its function in metabolism, one of a basic defence nature, the other of regulatory value in higher eukaryotes.

Pac Symp Biocomput . 2003;:140-51.
Assessment of the reliability of protein-protein interactions and protein function prediction; Deng M et al.; As more and more high-throughput protein-protein interaction data are collected, the task of estimating the reliability of different data sets becomes increasingly important . In this paper, we present our study of two groups of protein-protein interaction data, the physical interaction data and the protein complex data, and estimate the reliability of these data sets using three different measurements: (1) the distribution of gene expression correlation coefficients, (2) the reliability based on gene expression correlation coefficients, and (3) the accuracy of protein function predictions . We develop a maximum likelihood method to estimate the reliability of protein interaction data sets according to the distribution of correlation coefficients of gene expression profiles of putative interacting protein pairs . The results of the three measurements are consistent with each other . The MIPS protein complex data have the highest mean gene expression correlation coefficients (0.256) and the highest accuracy in predicting protein functions (70% sensitivity and specificity), while Ito's Yeast two-hybrid data have the lowest mean (0.041) and the lowest accuracy (15% sensitivity and specificity) . Uetz's data are more reliable than Ito's data in all three measurements, and the TAP protein complex data are more reliable than the HMS-PCI data in all three measurements as well . The complex data sets generally perform better in function predictions than do the physical interaction data sets . Proteins in complexes are shown to be more highly correlated in gene expression . The results confirm that the components of a protein complex can be assigned to functions that the complex carries out within a cell . There are three interaction data sets different from the above two groups: the genetic interaction data, the in-silico data and the syn-express data . Their capability of predicting protein functions generally falls between that of the Y2H data and that of the MIPS protein complex data . The supplementary information is available at the following Web site: http://www-hto.usc.edu/-msms/AssessInteraction/.

J Biol Chem, 2003 May 2, 278(18), 15550 - 7 Epub 2003 Feb 24.
A conserved calcineurin-binding motif in human T lymphotropic virus type 1 p12I functions to modulate nuclear factor of activated T cell activation; Kim SJ et al.; The PXIXIT calcineurin binding motif or highly related sequences are found in a variety of calcineurin-binding proteins in yeast, mammalian cells, and viruses . The accessory protein p12(I) encoded in the HTLV-1 pX ORF I promotes T cell activation during the early stages of HTLV-1 infection by activating nuclear factor of activated T cells (NFAT) through calcium release from the endoplasmic reticulum . We identified in p12(I), a conserved motif, which is highly homologous with the PXIXIT calcineurin-binding motif of NFAT . Both immunoprecipitation and calmodulin agarose bead pull-down assays indicated that wild type p12(I) and mutants of p12(I) that contained the motif-bound calcineurin . In addition, an alanine substitution p12(I) mutant (p12(I) AXAXAA) had greatly reduced binding affinity for calcineurin . We then tested whether p12(I) binding to calcineurin affected NFAT activity . p12(I) competed with NFAT for calcineurin binding in calmodulin bead pull-down experiments . Furthermore, the p12(I) AXAXAA mutant enhanced NFAT nuclear translocation compared with wild type p12(I) and increased NFAT transcriptional activity 2-fold greater than wild type p12(I) . Similar to NFAT, endogenous calcineurin phosphatase activity was increased in Jurkat T cells expressing p12(I) independent of its calcineurin binding property . Thus, the reduced binding of p12(I) to calcineurin allows enhanced nuclear translocation and transcription mediated by NFAT . Herein, we are the first to identify a retroviral protein that binds calcineurin . Our data suggest that HTLV-1 p12(I) modulates NFAT activation to promote early virus infection of T lymphocytes, providing a novel mechanism for retrovirus-mediated cell activation.

Genes Dev, 2003 Feb 15, 17(4), 443 - 8
Identification of genes that protect the C . elegans genome against mutations by genome-wide RNAi; Pothof J et al.; An RNA interference (RNAi)-based genome-wide screen was performed to detect genes that contribute to genome stability in somatic cells of Caenorhabditis elegans . We identified 61 such genes; these also affect spontaneous mutagenesis in the germ line . Their sequence suggests a role in DNA repair and/or replication, in chromatin remodeling, or in cell cycle control; there are also many novel genes that are highly conserved from yeast to human . Because known mutator genes are causally involved in many hereditary and sporadic human cancers, it is likely that some of these new mutators are equally relevant in cancer etiology.

J Clin Virol, 2003 Feb, 26(2), 143 - 52
The role of Vif during HIV-1 infection: interaction with novel host cellular factors; Lake JA et al.; BACKGROUND: Current research suggests that human immunodeficiency virus type-1 (HIV-1) virion infectivity factor (Vif) acts during viral assembly in producer cells to ensure infectivity in target cells but the exact mechanism of action has not been defined . Vif interacts with Gag, viral protease and RNA and these interactions are proposed to be important for correct particle assembly and stability of the reverse transcription complex . OBJECTIVES: The existence of cells that are either permissive or non-permissive for replication of Vif deficient viruses suggests the involvement of host cellular factors in its function . Current research suggests an association of Vif with the intermediate filament protein, vimentin, and the tyrosine kinase, Hck, but the significance of these associations remains to be defined . More recently HP68, a cellular ATP binding protein, has been shown to be important for capsid formation and an interaction between Vif and HP68 has been shown . Our aim was to further identify host cellular factors involved in Vif function . STUDY DESIGN: We have employed the yeast 2-hybrid system to identify cellular proteins which interact with HIV-1 Vif . Sixteen clones were isolated from a high stringency yeast-2-hybrid screen of a human leucocyte cDNA library with Vif derived from the T-cell tropic HIV-1 strain NL4.3 . Of these, 8 clones were confirmed as specifically binding Vif, fully sequenced and identified via GenBank homology searches . RESULTS: Thus far 3 of these clones, spermine/spermidine N1-acetyltransferase, Triad 3 and a novel gene which we have termed 'novel Vif binding protein', have been characterised and represent attractive candidates for mediating Vif action during HIV replication . CONCLUSIONS: Through identification and characterisation of cellular factors interacting with HIV-1 Vif we hope to unravel the mechanism of action of Vif which may ultimately aid therapeutic design .

Ai Zheng, 2003 Feb, 22(2), 123 - 7
{Expression of BRD7-interacting proteins,BRD2 and BRD3, in nasopharyngeal carcinoma tissues}; Zhou M et al.; BACKGROUND & OBJECTIVE: BRD7 is a novel gene tightly associated with nasopharyngeal carcinoma(NPC) cloned by cDNA representational difference analysis (cDNA RDA) . Two proteins,BRD2 and BRD3, including bromodomain and interacting with BRD7 protein had been screened from human fetal brain cDNA library by yeast two-hybrid system . This study was designed to further identify the interactions of BRD2 and BRD3 with BRD7 respectively and to investigate the expression and action pattern of BRD2 and BRD3 in NPC . METHODS: BRD2 and BRD3 genes were respectively co-transformed to yeast Y187 with BRD7 gene, then the yeast cotransformers were blotted to nylon membrane . And then the expression of report gene Lac Z by beta-Gal was determined and the interactions of BRD2 and BRD3 proteins with BRD7 protein were identified . Besides,reverse transcription-polymerase chain reaction (RT- PCR) was used to examine the expression of BRD2 and BRD3 genes in normal nasopharyngeal epithelium and NPC biopsies, and to detect the effect of re-expression of BRD7 gene on the expression of BRD2 and BRD3 genes in HNE1 stably transfected BRD7 gene . RESULTS: The yeast transformers showed all blue by yeast two-hybrid system, which further identified that BRD2 and BRD3 proteins could respectively interact with BRD7 protein . Down-expression or loss of BRD2 and BRD3 genes were detectable in NPC biopsies . The expression levels of BRD2 and BRD3 were increasing with the re-expression of BRD7 gene in HNE1 stably transfected with BRD7 . CONCLUSION: BRD7 protein could respectively interact with proteins, BRD2 and BRD3, and BRD7 could up-regulate the expression levels of BRD2 and BRD3 genes in mRNA level to some extent . Each of these three homolog proteins might be capable of forming heteromers with the others, which play important roles in the suppression of growth of NPC cells.

J Neurosci, 2003 Feb 15, 23(4), 1310 - 9
Novel espin actin-bundling proteins are localized to Purkinje cell dendritic spines and bind the Src homology 3 adapter protein insulin receptor substrate p53; Sekerkova G et al.; We identified a group of actin-binding-bundling proteins that are expressed in cerebellar Purkinje cells (PCs) but are not detected in other neurons of the CNS . These proteins are novel isoforms of the actin-bundling protein espin that arise through the use of a unique site for transcriptional initiation and differential splicing . Light and electron microscopic localization studies demonstrated that these espin isoforms are enriched in the dendritic spines of PCs . They were detected in the head and neck and in association with the postsynaptic density (PSD) of dendritic spines in synaptic contact with parallel or climbing fibers . They were also highly enriched in PSD fractions isolated from cerebellum . The PC espins efficiently bound and bundled actin filaments in vitro, and these activities were not inhibited by Ca2+ . When expressed in transfected neuronal cell lines, the PC espins colocalized with actin filaments and elicited the formation of coarse cytoplasmic actin bundles . The insulin receptor substrate p53 (IRSp53), an Src homology 3 (SH3) adapter protein and regulator of the actin cytoskeleton, was identified as an espin-binding protein in yeast two-hybrid screens . Cotransfection studies and pull-down assays showed that this interaction was direct and required the N-terminal proline-rich peptide of the PC espins . Thus, the PC espins exhibit the properties of modular actin-bundling proteins with the potential to influence the organization and dynamics of the actin cytoskeleton in PC dendritic spines and to participate in multiprotein complexes involving SH3 domain-containing proteins, such as IRSp53.

Annu Rev Biophys Biomol Struct, 2003, 32, 135 - 59 Epub 2003 Feb 11.
New insight into site-specific recombination from Flp recombinase-DNA structures; Chen Y et al.; The lamba integrase, or tyrosine-based family of site-specific recombinases, plays an important role in a variety of biological processes by inserting, excising, and inverting DNA segments . Flp, encoded by the yeast 2-mum plasmid, is the best-characterized eukaryotic member of this family and is responsible for maintaining the copy number of this plasmid . Over the past several years, structural and biochemical studies have shed light on the details of a common catalytic scheme utilized by these enzymes with interesting variations under different biological contexts . The emergence of new Flp structures and solution data provides insights not only into its unique mechanism of active site assembly and activity regulation but also into the specific contributions of certain protein residues to catalysis.

Ann Oncol, 2003 Mar, 14(3), 406 - 13
Locally advanced/inflammatory breast cancers treated with intensive epirubicin-based neoadjuvant chemotherapy: are there molecular markers in the primary tumour that predict for 5-year clinical outcome?
Bonnefoi H, Diebold-Berger S, Therasse P, Hamilton A, van de Vijver M, MacGrogan G, Shepherd L, Amaral N, Duval C, Drijkoningen R, Larsimont D, Piccart M.
BACKGROUND: Locally advanced and/or inflammatory breast cancer (LABC) is a heterogeneous disease . Molecular markers may help to understand this heterogeneity . This paper reports the results of a study assessing the potential prognostic or predictive value of HER-2, p53, cyclinD1, MIB1, ER and PgR expression by immunohistochemistry from patients included in an EORTC-NCIC-SAKK trial . PATIENTS AND METHODS: A total of 448 patients with a cytological or histological diagnosis of LABC were randomised into a trial comparing two anthracycline-based neoadjuvant regimens . Chemotherapy was followed by standard locoregional therapy . Survival was comparable in both arms . We collected and analysed centrally paraffin-embedded tumour specimens from 187 (72.5%) of 258 patients that had a histological diagnosis . RESULTS: Of the patients included in this molecular marker study 114 relapsed and 91 died . In the multivariate analysis p53 positivity was associated with a shorter progression-free survival {hazard ratio (HR) = 1.96; 95% CI 1.33-2.91; P = 0.0008) and a shorter overall survival (HR = 1.98; 95% CI 1.28-3.06; P = 0.002) . PgR positivity predicted for a longer overall survival (HR = 0.54; 95% CI 0.35-0.83; P = 0.0045) . CONCLUSIONS: p53 was an independent factor predicting for survival . In order to clarify whether p53 is a pure prognostic and/or a predictive factor, a phase III trial is being conducted (EORTC 10994/BIG 00-01 study) using functional assay in yeast from frozen tumour samples.

Biochem J, 2003 Jun 1, 372(Pt 2), 595 - 602
Caenorhabditis elegans pseudouridine synthase 1 activity in vivo: tRNA is a substrate, but not U2 small nuclear RNA; Patton JR et al.; The formation of pseudouridine (Psi) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes . Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice . In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA) . In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained . The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases . The locations of most modified nucleotides on small RNAs in C . elegans are not known, and the positions of Psi were determined on four tRNAs and U2 snRNA . The uridine at position 27 of tRNA(Val) (AAC), a putative Pus1p-modification site, was converted into Psi in the wild-type worms, but the tRNA(Val) (AAC) from mutant worms lacked the modification . Psi formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p . The absence of Pus1p in C . elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Psi at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions . This result was unexpected, given the known dual specificity of yeast Pus1p.

Curr Top Microbiol Immunol, 2003, 274, 143 - 69
The SWI/SNF family of ATP-dependent chromatin remodelers: similar mechanisms for diverse functions; Wang W; The SWI/SNF family of complexes utilizes the energy of ATP hydrolysis to remodel chromatin structures, thereby allowing transcription factors to gain access to DNA . Recent studies suggest that these remodelers also participate in other DNA metabolic reactions such as replication and viral integration, and even in control of cell growth and tumor suppression . The SWI/SNF remodelers can be classified into at least two distinct subfamilies: one includes human BAF (also known as hSWI/SNF-A) and yeast SWI/SNF; the other comprises human PBAF (hSWI/SNF-B) and yeast RSC . Although both types of complexes have similar subunit composition and chromatin remodeling activity in vitro, they cannot replace each other during transcription mediated by specific activators . Thus, each remodeler probably works with a specific set of activators during gene activation . The availability of distinct types of remodelers can allow cells to regulate expression of a specific group of genes by modulating the activity of corresponding remodelers.

J Biomed Sci, 2003 Mar-Apr, 10(2), 242 - 52
An LKB1-interacting protein negatively regulates TNFalpha-induced NF-kappaB activation; Liu WK et al.; The Peutz-Jeghers syndrome (PJS) is a hereditary disorder that predisposes an individual to benign and malignant tumors in multiple organ systems . Recently, the locus responsible for PJS was mapped genetically to the LKB1 gene, with a subsequent investigation proving that it is responsible for most cases of PJS . LKB1 encodes a nuclear serine/threonine protein kinase, and potential tumor-suppressing activity has been attributed to LKB1 kinase . However, how LKB1 exerts its tumor-suppressing function remains to be determined . In this report, we describe the identification of a putative human LKB1-interacting protein, FLIP1, using the yeast two-hybrid system . Two regions of the LKB1 sequence have been determined to be crucial for the interaction with FLIP1 . FLIP1 encodes a protein of 429 amino acids with a predicted molecular weight of 47 kd . In contrast to LKB1, which is mainly nuclear, FLIP1 is a cytoplasmic protein, and its expression is ubiquitous in all human tissues examined to date . Interestingly, deletion of the 195 N- terminal amino acids allows FLIP1 to enter the nucleus, suggesting the presence of a regulatory mechanism through its N-terminus for nuclear entry . In addition, we found that ectopic expression of FLIP1 selectively blocks cytokine-induced NF-kappaB activation . The involvement of FLIP1 in the regulation of NF-kappaB activity may shed new light on the role of LKB1 in tumor suppression .

Nucleic Acids Res . 2003 Mar 1;31(5):e23.
De novo production of diverse intracellular antibody libraries; Tanaka T et al.; Many therapeutic targets are intracellular proteins and molecules designed to interact with them must effectively bind to their target inside the cell . Intracellular antibodies (intrabodies) recognise and bind to proteins in cells and various methods have been developed to produce such molecules . Intracellular antibody capture (IAC) is based on a genetic screening approach and is a facile methodology with which effective intracellular antibodies can be obtained . During the development of the IAC technology, consensus immunoglobulin variable frameworks were identified which can form the basis of intrabody libraries for direct screening . In this paper, we describe the de novo synthesis of intrabody libraries based on the IAC consensus sequence . The procedure comprises in vitro production of a single antibody gene fragment from oligonucleotides and diversification of CDRs of the immunoglobulin variable domain by mutagenic PCR . Completely de novo intrabody libraries can be rapidly generated in vitro by these approaches . As an example, a single immunoglobulin VH domain intrabody library was screened directly in yeast with an oncogenic BCR-ABL antigen bait and distinct antigen binders were isolated illustrating the functional utility of the library . This second generation IAC approach (IAC2) has many practical advantages, in particular the ability to isolate intrabodies by direct genetic selection, which obviates the need for in vitro production of antigen for pre-selection of antibody fragments.

Biochimie, 2002 Nov, 84(11), 1143 - 50
Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig; Poggiali P et al.; A de novo SAC was constructed by making use of YAC technology and a humanized yeast strain . The construct (about 310 kb) contained pig centromeric DNA and the Neo gene . The construct was introduced into a pig cell line by yeast-mammalian cell fusion and G418 resistant clones were obtained . One clone was characterized by FISH and shown to contain an episomally located microchromosome containing YAC, Neo and pig centromere sequences . FISH analysis over time showed that the SAC was mitotically stable for at least 34 generations in the absence of selection . The size of the SAC was determined by confocal microscopy of the SAC and shown to be approximately 7 Mb, which is about 25-fold greater than the size of the original YAC . From its behavior in pulsed field gel electrophoresis, FISH analysis of stretched DNA fibers, and its appearance under scanning confocal microscopy, it was concluded that the SAC is a circularized and multimerized derivative of the original YAC . Possible applications as vectors for animal transgenesis are discussed.

BMC Genet . 2003 Feb 05;4(1):4.
Genomic organization of ATOX1, a human copper chaperone; Liu PC et al.; BACKGROUND: Copper is an essential trace element that plays a critical role in the survival of all living organisms . Menkes disease and occipital horn syndrome (OHS) are allelic disorders of copper transport caused by defects in a X-linked gene (ATP7A) that encodes a P-type ATPase that transports copper across cellular membranes, including the trans-Golgi network . Genetic studies in yeast recently revealed a new family of cytoplasmic proteins called copper chaperones which bind copper ions and deliver them to specific cellular pathways . Biochemical studies of the human homolog of one copper chaperone, ATOX1, indicate direct interaction with the Menkes/OHS protein . Although no disease-associated mutations have been reported in ATOX1, mice with disruption of the ATOX1 locus demonstrate perinatal mortality similar to that observed in the brindled mice (Mobr), a mouse model of Menkes disease . The cDNA sequence for ATOX1 is known, and the genomic organization has not been reported . RESULTS: We determined the genomic structure of ATOX1 . The gene contains 4 exons spanning a genomic distance of approximately 16 kb . The translation start codon is located in the 3' end of exon 1 and the termination codon in exon 3 . We developed a PCR-based assay to amplify the coding regions and splice junctions from genomic DNA . We screened for ATOX1 mutations in two patients with classical Menkes disease phenotypes and one individual with occipital horn syndrome who had no alterations detected in ATP7A, as well as an adult female with chronic anemia, low serum copper and evidence of mild dopamine-beta-hydroxylase deficiency and no alterations in the ATOX1 coding or splice junction sequences were found . CONCLUSIONS: In this study, we characterized the genomic structure of the human copper chaperone ATOX1 to facilitate screening of this gene from genomic DNA in patients whose clinical or biochemical phenotypes suggest impaired copper transport.

Eur J Immunol, 2003 Jan, 33(1), 172 - 82
Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies; Chen D et al.; Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases . To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions . The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases . Lymphoid tissues as well as CD4(+) T cells develop normally . Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2 . CD11c(+) dendritic cells express high levels of DR and DQ . Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb . These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile . Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection . Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC) . Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells . Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions . Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.

Gene, 2003 Feb 13, 305(1), 91 - 100
SF4 and SFRS14, two related putative splicing factors on human chromosome 19p13.11; Sampson ND et al.; The splicing of nascent mRNA precursors is an essential step for the expression of all intron-containing eukaryotic genes . Removal of intron sequences from nascent transcripts is mediated by the spliceosome, a large multicomponent complex . We describe here the identification of two genes encoding related, putative splicing factors on human chromosome 19p13.11, SF4 (splicing factor 4) and SFRS14 (splicing factor arginine/serine-rich 14) . Both genes encode proteins containing a SURP motif; this domain is found in several splicing proteins including Drosophila alternative splicing regulator, suppressor-of-white-apricot (SWAP) and the yeast splicing factor, prp21p . In addition, SF4 and SFRS14 contain a G-patch domain at their C-termini, a motif present in a large number of eukaryotic RNA-binding proteins . SFRS14 also contains an N-terminal region that is rich in arginine/serine residues, suggesting SFRS14 is a novel member of the SR-related family of pre-mRNA processing factors . We have also identified the mouse orthologues of SF4 and SFRS14, based on conserved domain organization and high sequence similarity . Interestingly, SFRS14 undergoes alternative 3'-end processing events that are conserved between human and mouse, suggesting a functional significance.

Gene, 2003 Feb 13, 305(1), 71 - 8
Analysis of the flanking regions of the human alpha-lactalbumin gene responsible for position-effect independent expression; Fujiwara Y et al.; Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the alpha-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA . To identify elements that confer a position effect, we compared the yLALBA and bLA sequences . yLALBA was chimeric . A common 32 kb region was identified and the total nucleotide sequence was determined . We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes . The -6 to +9 kb region is considered to be necessary for position-independent expression . Transgenic rats lacking the -3.4 to -0.85 kb region had a severe position effect . This 2.5 kb region contains two DNaseI hypersensitive sites at -1.0 and -2.8 kb . The 2.5 kb region is proposed to be a locus control region of the human alpha-lactalbumin gene.

Gene, 2003 Feb 13, 305(1), 47 - 59
Comparative sequence and expression analyses of four mammalian VPS4 genes; Beyer A et al.; The VPS4 gene is a member of the AAA-family; it codes for an ATPase which is involved in lysosomal/endosomal membrane trafficking . VPS4 genes are present in virtually all eukaryotes . Exhaustive data mining of all available genomic databases from completely or partially sequenced organisms revealed the existence of up to three paralogues, VPS4a, -b, and -c . Whereas in the genome of lower eukaryotes like yeast only one VPS4 representative is present, we found that mammals harbour two paralogues, VPS4a and VPS4b . Most interestingly, the Fugu fish contains a third VPS4 paralogue (VPS4c) . Sequence comparison of the three VPS4 paralogues indicates that the Fugu VPS4c displays sequence features intermediate between VPS4a and VPS4b . Using complete mammalian VPS4a and VPS4b cDNA clones as probes, genomic clones of both VPS4 paralogues in human and mouse were identified and sequenced . The chromosomal loci of all four VPS4 genes were determined by independent methods . A BLAST search of the human genome database with the human VPS4A sequence yielded a double match, most likely due to a faulty assembly of sequence contigs in the human draft sequence . Fluorescent in situ hybridization and radiation hybrid analyses demonstrated that human and mouse VPS4A/a and VPS4B/b are located on syntenic chromosomal regions . Northern blot and semi-quantitative reverse transcription analyses showed that mouse VPS4a and VPS4b are differentially expressed in different organs, suggesting that the two paralogues have developed different functional properties since their divergence . To investigate the subcellular distribution of the murine VPS4 paralogues, we transiently expressed various fluorescent VPS4 fusion proteins in mouse 3T3 cells . All tested VPS4 fusion proteins were found in the cytosol . Expression of dominant-negative mutant VPS4 fusion proteins led to their concentration in the perinuclear region . Co-expression of VPS4a-GFP and VPS4b-dsRed fusion proteins revealed a partial co-localization that was most prominent with mutant VPS4a and VPS4b proteins . A physical interaction between the mouse paralogues was also supported by two-hybrid analyses.

Biochem Biophys Res Commun, 2003 Feb 28, 302(1), 1 - 5
Hypothesis: a glycoprotein-degradation complex formed by protein-protein interaction involves cytoplasmic peptide:N-glycanase; Suzuki T et al.; A cytoplasmic peptide:N-glycanase has been implicated in the proteasomal degradation of newly synthesized misfolded glycoproteins that are exported from the endoplasmic reticulum to the cytosol . Recently, the gene encoding this enzyme (Png1p) was identified in yeast and shown to bind to the 26S proteasome through its interaction with a component of the DNA repair system, Rad23p . Moreover, a mouse homologue of Png1p (mPng1p), which has an extended N-terminal domain, was found to bind not only to the Rad23 protein, but also to various proteins related to the ubiquitin/proteasome pathway . An extended N-terminus of mPng1p, which is not found in yeast, contains a potential site of protein-protein interaction called the PUB/PUG domain . The PUB/PUG domain is predicted to be helix-rich and is found in various proteins that may be involved in the ubiquitin/proteasome-related pathway . This review will discuss the consequence of the deglycosylation reaction by peptide:N-glycanase in cellular processes . In addition, the potential importance of the PUB/PUG domain for the formation of a putative "glycoprotein-degradation complex" will be discussed.

Curr Biol, 2003 Feb 18, 13(4), 350 - 7
Genome-wide analyses of steroid- and radiation-triggered programmed cell death in Drosophila; Lee CY et al.; Apoptosis and autophagy are two forms of programmed cell death that play important roles in the removal of unneeded and abnormal cells during animal development . While these two forms of programmed cell death are morphologically distinct, recent studies indicate that apoptotic and autophagic cell death utilize some common regulatory mechanisms . To identify genes that are associated with apoptotic and autophagic cell death, we monitored changes in gene transcription by using microarrays representing nearly the entire Drosophila genome . Analyses of steroid-triggered autophagic cell death identified 932 gene transcripts that changed 5-fold or greater in RNA level . In contrast, radiation-activated apoptosis resulted in 34 gene transcripts that exhibited a similar magnitude of change . Analyses of these data enabled us to identify genes that are common and unique to steroid- and radiation-induced cell death . Mutants that prevent autophagic cell death exhibit altered levels of gene transcription, including genes encoding caspases, non-caspase proteases, and proteins that are similar to yeast autophagy proteins . This study also identifies numerous novel genes as candidate cell death regulators and suggests new links between apoptosis and autophagic cell death.

Br J Cancer, 2003 Feb 24, 88(4), 579 - 85
hAG-2 and hAG-3, human homologues of genes involved in differentiation, are associated with oestrogen receptor-positive breast tumours and interact with metastasis gene C4.4a and dystroglycan; Fletcher GC et al.; hAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development . Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21 . hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines . In this study, we have used real-time quantitative RT - PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues . Tumour expression of both genes correlated with OR (hAG2, P=0.0002; hAG-3, P=0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P=0.003) . Yeast two-hybrid cloning identified metastasis-associated GPI-anchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning . hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.

Mayo Clin Womens Healthsource, 2003 Feb, 7(2), 4 - 5
Vaginal problems? Treat yourself to the right solution; Identification of human dehydrodolichyl diphosphate synthase gene; Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, Katahira 2-1-1, Aoba-ku, 980-8577, Sendai, JapanWe isolated a cDNA encoding human dehydrodolichyl diphosphate (Dedol-PP) synthase and expressed the gene in a yeast mutant strain SNH23-7D, which is deficient in Dedol-PP synthase activity . The identity of the cloned enzyme was confirmed by functional complementation of SNH23-7D strain together with in vitro Dedol-PP synthase activity assay . Northern blot analysis indicated that testis and kidney expressed Dedol-PP synthase mRNA at high levels.

Biochim Biophys Acta, 2003 Feb 20, 1625(3), 269 - 79
Molecular cloning, differential expression, and functional characterization of a family of class I ubiquitin-conjugating enzyme (E2) genes in cotton (Gossypium); Zhang XD et al.; Two cDNAs and their corresponding genes (GhUBC1 and GhUBC2) encoding ubiquitin-conjugating enzymes (E2s) have been cloned and characterized from allotetraploid cotton Gossypium hirsutum ((AD)(1) genome) . Three additional E2 genes (GaUBC1, GtUBC2, and GrUBC2) have also been identified from diploid cottons Gossypium arboreum (A(2) genome), Gossypium thurberi (D(1) genome), and Gossypium raimondii (D(5) genome), respectively . The derived amino acid sequences of the five closely related cotton E2s are 77-79% identical to yeast ScUBC4 and ScUBC5 . The GhUBC1/2 gene family is composed of two members, and genomic origin analysis indicates that GhUBC1 and 2 are individually present in the A and D subgenomes of G . hirsutum . The transcript levels of GhUBC1/2 increased significantly in leaves and flowers at senescence, suggesting that GhUBC1/2 may play a role in the degradation of target proteins that function in the delay of the senescence program . Correlated with high auxin content and auxin-associated effects, GhUBC1/2 are also highly expressed in the youngest leaves, the apical part of lateral roots, and elongating fibers . Genetic complementation experiments revealed that GhUBC1 and 2 can substitute for the function of ScUBC4 and 5 required for the selective degradation of abnormal and short-lived proteins in a yeast ubc4ubc5 double mutant.

Exp Hematol, 2003 Feb, 31(2), 150 - 8
X-linked thrombocytopenia caused by a mutation in the Wiskott-Aldrich syndrome (WAS) gene that disrupts interaction with the WAS protein (WASP)-interacting protein (WIP); Luthi JN et al.; OBJECTIVE: We studied two adult brothers with severe congenital thrombocytopenia in order to determine the genetic etiology of their inherited disorder . Despite the absence of eczema or immunodeficiency, a mutation of the Wiskott-Aldrich syndrome (WAS) gene was suspected because of the presence of microthrombocytes . MATERIALS AND METHODS: Peripheral blood was obtained for characterization of hematopoietic cells and megakaryocyte progenitors . The coding region of the WAS gene was fully sequenced, and expression of the Wiskott-Aldrich syndrome protein, WASP, was evaluated by immunoblotting . The ability of WASP to physically associate with the WASP-interacting protein, WIP, was tested by yeast and mammalian two-hybrid techniques . RESULTS: In addition to thrombocytopenia, our investigation revealed an increased frequency of peripheral megakaryocyte progenitors (CFU-Mk) and incomplete cytoplasmic maturation by electron microscopy . Sequencing the WAS gene revealed a single base mutation, resulting in substitution of proline for arginine 138 (i.e., Arg138Pro) . Immunoblotting demonstrated reduced expression of the mutant WAS protein, and we showed that the Arg138Pro mutation significantly, but incompletely, disrupts WASP-WIP interaction . CONCLUSIONS: In this pedigree, X-linked thrombocytopenia is caused by a rare mutation in the fourth exon of the WAS gene . WASP levels are reduced in lymphocyte cell lines derived from the affected individuals . Furthermore, the mutation significantly but incompletely disrupts WASP-WIP interaction, whereas substitution of alanine or glutamic acid residues at the same position does not . This raises the possibility that protein-protein interaction and WASP stability are related properties.

Brain Res Mol Brain Res, 2003 Feb 20, 110(2), 298 - 304
Sensory neuron proteins interact with the intracellular domains of sodium channel NaV1.8; Malik-Hall M et al.; Voltage-gated sodium channels initiate and propagate action potentials in excitable cells . The tetrodotoxin-resistant Na(+) channel (Na(V)1.8/SNS) is expressed in damage-sensing neurons (nociceptors) and plays an important role in pain pathways . Expression of high levels of functional Na(V)1.8 in heterologous cells has proved problematic, even in the presence of known sodium channel accessory beta-subunits . This suggests that other regulatory proteins are required for normal levels of Na(V)1.8 expression . Here we report the use of a yeast two-hybrid system and a rat dorsal root ganglion cDNA library to identify 28 different clones encoding proteins which interact with intracellular domains of Na(V)1.8 . Many clones are expressed at high levels in small diameter DRG neurons as judged by in situ hybridization . Interacting proteins include cytoplasmic elements and linker proteins (e.g . beta-actin and moesin), enzymes (e.g . inositol polyphosphate 5-phosphatase and TAO2 thousand and one protein kinase), channels and membrane-associated proteins (voltage-dependent anion channel VDAC3V and tetraspanin), as well as motor proteins (dynein intermediate and light chain) and transcripts encoding previously undescribed proteins . Immunoprecipitation (pull-down) assays confirm that some of the proteins interact with, and may hence regulate, Na(V)1.8 in vivo.

Dev Genes Evol, 2003 Feb, 213(1), 50 - 2 Epub 2002 Dec 06.
The homeobox genes of Encephalitozoon cuniculi (Microsporidia) reveal a putative mating-type locus; Burglin TR; Homeobox genes have been found in animals, fungi, and plants . Recently, the complete genomic sequence of Encephalitozoon cuniculi has become available and it was shown that this Microsporida species is related to fungi . Given this close relatedness to fungi, I have searched the genome of E . cuniculifor homeobox genes . There are 12 homeobox genes as well as one STE12 orthologue . The large number of homeobox genes when compared to the annotations, which only list one, suggests that possibly other smaller genes may have been overlooked in the published analysis and E . cuniculi may have more than 1,997 genes . Sequence and phylogenetic analyses show that the E . cuniculi homeobox genes also fall into two distinct groups, with two TALE and ten typical homeobox genes being present . Like in the mating-type locus of yeast and other fungi, one TALE and one typical homeobox are found in close proximity of each other on the chromosome, suggesting that Microsporidia also contain a mating-type locus.

Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 991 - 9
The interaction between ADAM 22 and 14-3-3zeta: regulation of cell adhesion and spreading; Zhu P et al.; The ADAM family consists of a number of transmembrane proteins that contain disintegrin-like and metalloproteinase-like domains . Therefore, ADAMs potentially have cell adhesion and protease activities . 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in the regulation of various cellular functions . Here we report the identification of a novel interaction between the ADAM 22 cytoplasmic tail and the 14-3-3zeta isoform by a yeast two-hybrid screen . The interaction between the ADAM 22 cytoplasmic tail and 14-3-3zeta was confirmed by an in vitro protein pull-down assay as well as by co-immunoprecipitation, and the binding sites were mapped to the 28 amino acid residues of the C-terminus of the ADAM 22 cytoplasmic tail . Furthermore, we found that overexpression of the ADAM 22 cytoplasmic tail in human SGH44 cells inhibited cell adhesion and spreading and that deletion or mutation of the binding site for 14-3-3zeta within the ADAM 22 cytoplasmic tail abolished the ability of the overexpressed cytoplasmic tail to alter cell adhesion and spreading . Taken together, these results for the first time demonstrate an association between ADAM 22 and a 14-3-3 protein and suggest a potential role for the 14-3-3zeta/ADAM 22 association in the regulation of cell adhesion and related signaling events.

Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 941 - 7
The Drosophila SSL gene is expressed in larvae, pupae, and adults, exhibits sexual dimorphism, and mimics properties of the beta subunit of casein kinase II; Karandikar U et al.; Drosophila melanogaster casein kinase II (CKII) is composed of catalytic alpha and regulatory beta subunits that generate the alpha2beta2 holoenzyme . A two-hybrid screen of a Drosophila embryo library using CKIIalpha as bait has resulted in the isolation of multiple cDNAs encoding SSL, a CKIIbeta-like polypeptide . We demonstrate that CKIIbeta, beta', and SSL exhibit robust and comparable interaction with CKIIalpha . Residues in SSL that mediate interaction with CKIIalpha appear similar to those in CKIIbeta, and SSL forms homodimers and heterodimers with CKIIbeta or beta' as well . We have tested all known Drosophila CKIIbeta-like proteins for rescue of the ion-homeostasis defect of yeast lacking beta subunits and find that CKIIbeta and SSL complement, beta' has marginal function, and Stellate appears non-functional . We have used real-time RT-PCR to assess developmental expression, and find that CKIIbeta is robust and ubiquitous, whereas SSL is restricted to males (third-instar-larvae, pupae, and adults), but is nondetectable in females of the corresponding stages . These results indicate that SSL expression encompasses a greater developmental window than that previously suggested and may confer distinct functions to CKII in a sex-specific manner.

Biochem J, 2003 May 15, 372(Pt 1), 87 - 96
Vertebrate tankyrase domain structure and sterile alpha motif (SAM)-mediated multimerization; De Rycker M et al.; Tankyrases 1 and 2 are two highly related poly(ADP-ribose) polymerases that interact with a variety of cytoplasmic and nuclear proteins . Both proteins have been implicated in telomere length regulation, insulin signalling and centrosome function . To learn more about their mode of action, we have isolated the chicken tankyrase homologues and examined their interaction partners and subcellular location . Cross-species sequence comparison indicated that tankyrase domain structure is highly conserved and supports division of the ankyrin domain into five subdomains, which are each separated by a highly conserved LLEAAR/K motif . Glutathione S-transferase pull-down experiments demonstrated that the ankyrin domains of both proteins interact with chicken telomere repeat factor 1 (TRF1) . Analysis of total cellular and nuclear proteins revealed that cells contain approximately twice as much tankyrase 1 as tankyrase 2 . Although > or = 90% of each protein is present in the cytoplasm, both tankyrase 1 and 2 were detected in the nucleus . The nuclear location together with its ability to interact with TRF1, point to tankyrase 2 having a telomeric function . Yeast two-hybrid and cross-linking experiments show that both tankyrases can multimerize through their sterile-alpha motif domains . These results indicate that tankyrases may be master scaffolding proteins, capable of regulating assembly of large protein complexes.

Mol Genet Genomics, 2003 Feb, 268(5), 598 - 606 Epub 2003 Jan 15.
Analysis of the petunia MADS-box transcription factor family; Immink RG et al.; Transcription factors are key regulators of plant development . One of the major groups of transcription factors is the MADS-box family, of which at least 80 members are encoded in the Arabidopsis genome . In this study, 23 members of the petunia MADS-box transcription factor family were investigated by Northern hybridisation, phylogenetic and yeast two-hybrid analyses . Many of the genes characterised appeared to have one or more close relatives that shared similar expression patterns . Comparison of the binding interactions of these proteins revealed that some show similar interaction patterns, and hence are likely to be functionally redundant . From an evolutionary point of view, their coding genes are probably derived from a recent duplication event . Furthermore, protein-protein interaction patterns, in combination with expression patterns and phylogenetic classification, appear to offer good criteria for the identification of functional homologues . Based on comparison of such data between petunia and Arabidopsis, functions can be predicted for several MADS-box transcription factors in both species.

Mol Biol Cell, 2003 Feb, 14(2), 685 - 97
In vitro and in vivo interactions of DNA ligase IV with a subunit of the condensin complex; Przewloka MR et al.; Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair . Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability . To identify protein-protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods . These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E . The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV . Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes . Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation . We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.

Mol Biol Cell, 2003 Feb, 14(2), 600 - 10
Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes; Griffis ER et al.; The vertebrate nuclear pore is an enormous structure that spans the double membrane of the nuclear envelope . In yeast, most nucleoporins are found symmetrically on both the nuclear and cytoplasmic sides of the structure . However, in vertebrates most nucleoporins have been localized exclusively to one side of the nuclear pore . Herein, we show, by immunofluorescence and immunoelectron microscopy, that Nup98 is found on both sides of the pore complex . Additionally, we find that the pore-targeting domain of Nup98 interacts directly with the cytoplasmic nucleoporin Nup88, a component of the Nup214, Nup88, Nup62 subcomplex . Nup98 was previously described to interact with the nuclear-oriented Nup160, 133, 107, 96 complex through direct binding to Nup96 . Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96 . Autoproteolytic cleavage of the Nup98 C terminus is required for both of these binding interactions . When cleavage is blocked by a point mutation, a minimal eight amino acids downstream of the cleavage site is sufficient to prevent most binding to either Nup96 or Nup88 . Thus, Nup98 interacts with both faces of the nuclear pore, a localization in keeping with its previously described nucleocytoplasmic shuttling activity.

Mol Biol Cell, 2003 Feb, 14(2), 361 - 9
The AtC-VPS protein complex is localized to the tonoplast and the prevacuolar compartment in arabidopsis; Rojo E et al.; Plant cells contain several types of vacuoles with specialized functions . Although the biogenesis of these organelles is well understood at the morphological level, the machinery involved in plant vacuole formation is largely unknown . We have recently identified an Arabidopsis mutant, vcl1, that is deficient in vacuolar formation . VCL1 is homologous to a protein that regulates membrane fusion at the tonoplast in yeast . On the basis of these observations, VCL1 is predicted to play a direct role in vacuolar biogenesis and vesicular trafficking to the vacuole in plants . In this work, we show that VCL1 forms a complex with AtVPS11 and AtVPS33 in vivo . These two proteins are homologues of proteins that have a well-characterized role in membrane fusion at the tonoplast in yeast . VCL1, AtVPS11, and AtVPS33 are membrane-associated and cofractionate with tonoplast and denser endomembrane markers in subcellular fractionation experiments . Consistent with this, VCL1, AtVPS11, and AtVPS33 are found on the tonoplast and the prevacuolar compartment (PVC) by immunoelectron microscopy . We also show that a VCL1-containing complex includes SYP2-type syntaxins and is most likely involved in membrane fusion on both the PVC and tonoplast in vivo . VCL1, AtVPS11, and AtVPS33 are the first components of the vacuolar biogenesis machinery to be identified in plants.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2556 - 61 Epub 2003 Feb 14.
Ends-out, or replacement, gene targeting in Drosophila; Gong WJ et al.; Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting . Both have been used for targeted mutagenesis, but require donors of differing design . Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus . Although ends-in targeting has been successful in Drosophila, an attempt at ends-out targeting failed . To test whether ends-out targeting could be used in Drosophila, we applied two strategies for ends-out gene replacement at the endogenous yellow (y) locus in Drosophila . First, a mutant allele was rescued by replacement with an 8-kb y(+) DNA fragment at a rate of approximately 1/800 gametes . Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of approximately 1/380 gametes . The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor . When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting.

Mycoses, 2003 Feb, 46(1-2), 7 - 11
Identification of clinically relevant Trichosporon species; Middelhoven WJ; A dichotomous identification key to pathogenic species of the basisiomycetous genus Trichosporon Behrend is provided . It is based on growth tests with carbon sources not traditionally used in yeast taxonomy, viz . uric acid, ethylamine, L-4-hydroxyproline, tyramine and L-phenylalanine as sources of carbon and nitrogen, and polygalacturonate, quinate, 4-ethylphenol, 2,3-dihydroxybenzoate and orcinol as sole carbon sources . Of the standard growth tests, assimilation of L-rhamnose and the maximum growth temperature proved to be useful . In addition to medically relevant species, other species able to grow at 37 degrees C were treated as well.

Phytomedicine, 2002 Dec, 9(8), 731 - 3
Evaluation of anti-pyretic potential of Ficus racemosa bark; Rao RB et al.; A study was carried out to evaluate the anti-pyretic effect of a methanol extract of stem bark of Ficus racemosa Linn . (MEFR) on normal body temperature and yeast-induced pyrexia in albino rats . A yeast suspension (10 ml/kg body wt.) increased rectal temperature 19 h after subcutaneous injection . The MEFR, at doses of 100, 200 and 300 mg/kg body wt . p.o., showed significant dose-dependent reduction in normal body temperature and yeast-provoked elevated temperature . The effect extended up to 5 h after drug administration . The anti-pyretic effect of MEFR was comparable to that of paracetamol (150 mg/kg body wt., p.o.), a standard anti-pyretic agent.

Phytomedicine, 2002 Dec, 9(8), 727 - 30
Evaluation of antipyretic activity of leaf extracts of Mallotus peltatus (Geist) Muell . arg . var acuminatus: a folk medicine; Chattopadhyay D et al.; A study was carried out to evaluate the anti-pyretic potential of the methanol extract of Mallotus peltatus (Geist) Muell . Arg . var acuminatus leaf, a folk medicine of Onge tribes of Bay Islands, on normal body temperature and yeast-induced pyrexia in Wister albino rats . The leaf extract at oral doses of 100, 200 and 300 mg kg(-1), p.o., showed significant reduction in normal body temperature and yeast-provoked elevated temperature in a dose-dependent manner and the anti-pyretic effect was comparable to that of standard anti-pyretic agent paracetamol (150 mg kg(-1), p.o.) . The effect also extended up to 5 hours after the drug administration.

Cell Biol Educ, 2002 Spring, 1(1), 43 - 62
Using the two-hybrid screen in the classroom laboratory; Odom DP et al.; The National Science Foundation and others have made compelling arguments that research be incorporated into the learning of undergraduates . In response to these arguments, a two-hybrid research project was incorporated into a molecular biology course that contained both a lecture section and a laboratory section . The course was designed around specific goals for educational outcomes, including introducing research to a wide range of students, teaching students experimental design and data analysis, and enhancing understanding of course material . Additional goals included teaching students to search genomic databases, to access scientific articles, and to write a paper in scientific format . Graded events tested these goals, and a student evaluation indicated student perception of the project . According to our analysis of the data, the yeast two-hybrid screen was a success: several novel clones were identified; students met expectations on graded lab reports, the poster session, and the final paper; and evaluations indicated that students had achieved the outlined goals . Students indicated on the evaluations that the research project increased their interest in research and greatly improved understanding of the course material . Finally, several students in the course intend to submit the findings of the research project to an undergraduate research journal.

Plant Physiol, 2003 Feb, 131(2), 443 - 53
Genomic and proteomic analysis of mitochondrial carrier proteins in Arabidopsis; Millar AH et al.; Plant mitochondria maintain metabolic communication with the cytosol through a family of carrier proteins . In Arabidopsis, a subset of 45 putative genes encoding members of this family have been identified based on generalized mitochondrial carrier features . No gene clusters are apparent and few of the predicted protein products have mitochondrial targeting sequences recognized by bioinformatic predictors . Only nine genes are currently represented by more than 10 expressed sequence tags at The Institute for Genomic Research . Analyses of public microarray experiments reveal differential expression profiles of the more highly expressed members of this gene family in different plant organs and in response to plant hormone application and environmental stresses . A comparison of this Arabidopsis carrier subset (45) to the yeast gene family (35) reveals 10 orthologous groups between the two species . Recent surveys of the Arabidopsis mitochondrial proteome by two-dimensional gel separations have not identified any of these carrier proteins, presumably because of their hydrophobicity and basicity . Isolating integral membrane proteins from Arabidopsis mitochondria, using one-dimensional electrophoresis for protein separation and tandem mass spectrometry-based sequencing of doubly charged peptides, we have unequivocally identified specific carrier gene products located in mitochondria . This approach has identified six of the nine carriers represented highly in expressed sequence tag databases: adenine nucleotide translocator (At3g8580 and At5g13490), dicarboxylate/tricarboxylate carrier (At5g19760), phosphate carrier (At5g14040), uncoupling protein (At3g54110), and a carrier gene of unknown function (At4g01100) . Overall, the combined transcript and protein expression data indicates that only a small subset of the carrier family of genes provide the majority of carrier proteins of Arabidopsis mitochondria.

J Biol Chem, 2003 Apr 25, 278(17), 15239 - 45 Epub 2003 Feb 13.
Identification of phospholipid scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE)); Kametaka S et al.; beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) is an integral membrane aspartic proteinase responsible for beta-site processing of APP, and its cytoplasmic region composed of 24 amino acid residues has been shown to be involved in the endosomal localization of BACE . With the yeast two-hybrid screening, we found that the cytoplasmic domain of phospholipid scramblase 1 (PLSCR1), a type II integral membrane protein, interacts with the cytoplasmic region of BACE . In cultured cells, BACE and PLSCR1 were colocalized in the Golgi area and in endosomal compartments, whereas they were co-redistributed in late endosome-derived multivesicular bodies when treated with U18666A, suggesting that both proteins share a common trafficking pathway in cells . Co-immunoprecipitation analysis showed that both proteins form a protein complex at an endogenous expression level in the human neuroblastoma SH-SY5Ycells, and the dileucine residue of the BACE tail is also revealed to be essential for the physical interaction with PLSCR1 in vitro and in vivo . Moreover, both BACE and PLSCR1 were localized in a low buoyant lipid microdomain in SH-SY5Y cells . The dileucine-defective BACE mutant was also fractionated into the lipid microdomain, but much less stably than wild-type BACE . Taken together, our current study suggests the functional involvement of PLSCR1 in the intracellular distribution of BACE and/or recruitment of BACE into the detergent-insoluble lipid raft.

Dev Cell, 2003 Feb, 4(2), 144 - 6
IRE1: a role in UPREgulation of ER degradation; Hampton RY; The unfolded protein response entered the mechanistic realm with the discovery of IRE1 as the key signal transducer in yeast . Although also found in mammals, it appeared to function in assisting the work of other players . The featured studies indicate a separate role for IRE1, and highlight the flexibility that bigger eukaryotes possess in this critical pathway.

Oncogene, 2003 Feb 13, 22(6), 819 - 25
Interactions of the DNA mismatch repair proteins MLH1 and MSH2 with c-MYC and MAX; Partlin MM et al.; MSH2 and MLH1 have a central role in correcting mismatches in DNA occurring during DNA replication and have been implicated in the engagement of apoptosis induced by a number of cytotoxic anticancer agents . The function of MLH1 is not clearly defined, although it is required for mismatch repair (MMR) and engagement of apoptosis after certain types of DNA damage . In order to identify other partners of MLH1 that may be involved in signalling MMR or apoptosis, we used human MLH1 in yeast two-hybrid screens of normal human breast and ovarian cDNA libraries . As well as known partners of MLH1 such as PMS1, MLH3 and MBD4, we identified the carboxy terminus of the human c-MYC proto-oncogene as an interacting sequence . We demonstrate, both in vitro by yeast two-hybrid and GST-fusion pull-down experiments, as well as in vivo by coimmunoprecipitation from human tumour cell extracts, that MLH1 interacts with the c-MYC protein . We further demonstrate that the heterodimeric partner of c-MYC, MAX, interacts with a different MMR protein, MSH2, both in vitro and in vivo . Using an inducible c-MYC-ER fusion gene, we show that elevated c-MYC expression leads to an increased HGPRT mutation rate of Rat1 cells and an increase in the number of frameshift mutants at the HGPRT locus . The effect on HGPRT mutation rate is small (2-3-fold), but is consistent with deregulated c-MYC expression partially inhibiting MMR activity.

Cytogenet Genome Res, 2002, 98(1), 29 - 37
Physical map of the chromosome 6q22 region containing the oculodentodigital dysplasia locus: analysis of thirteen candidate genes and identification of novel ESTs and DNA polymorphisms; Boyadjiev SA et al.; Oculodentodigital dysplasia (ODDD) is an autosomal dominant condition with congenital anomalies of the craniofacial and limb regions and neurodegeneration . Genetic anticipation for the dysmorphic and neurologic features has been inferred in a few families . Our previous linkage studies have refined the ODDD candidate region to chromosome 6q22-->q23 . In an attempt to clone the ODDD gene, we created a yeast artificial chromosome contig with 31 redundant clones spanning the region and identified and ordered candidate genes and markers . Fluorescent IN SITU hybridization mapped two of these YAC clones to chromosome 6q22.2 telomeric to a known 6q21 fragile site, excluding it as a possible cause of the suggested anticipation . We performed mutation analysis on thirteen candidate genes - GRIK2, HDAC2, COL10A1, PTD013, KPNA5, PIST, ROS1, BRD7, PLN, HSF2, PKIB, FABP7, and HEY2 . Although no mutations were found, we identified 44 polymorphisms, including 28 single nucleotide polymorphisms . Direct cDNA selection was performed and fifty-five clones were found to contain sequences that were not previously reported as known genes or ESTs . These clones and polymorphisms will assist in the further characterization of this region and identification of disease genes .

J Virol, 2003 Mar, 77(5), 3339 - 44
Defects in virion production caused by mutations affecting the C-terminal portion of the Moloney murine leukemia virus capsid protein; Wang MQ et al.; The capsid (CA) domain of the Moloney murine leukemia virus (Mo-MuLV) Gag protein has a unique carboxy terminus with a highly charged arginine-rich sequence . Mutant viruses harboring arginine-to-alanine mutations affecting this region of CA displayed significant defects in virion release, and the few viral particles produced were noninfectious . The interaction between the mutant Gag precursors was affected, as judged by the yeast two-hybrid assay . The results suggest that the unique carboxy terminus of CA in the Mo-MuLV plays an important role in Gag-Gag association during virion production.

Eur Biophys J, 2003 Feb, 31(8), 586 - 94 Epub 2002 Oct 03.
Are there temperature-dependent structural transitions in the "intrinsically unstructured" protein prothymosin alpha?
Gast K, Zirwer D, Damaschun G.
Prothymosin alpha, a typical member of the class of the so-called "intrinsically unstructured" proteins, adopts a random-chain conformation under physiological environmental conditions . An apparent formation of ordered secondary structure and a moderate compaction are observed upon the change from neutral to acid pH at room temperature . We have addressed the question of whether there are temperature-dependent changes of the conformational state of prothymosin alpha at low pH using circular dichroism spectroscopy and static and dynamic light scattering . In contrast to previous investigations, we did not observe a heat-induced conformational transition . For comparison, we have also carried out the same experimental procedures with acid-unfolded phosphoglycerate kinase from yeast . In this case we observed a weak compaction and a slight apparent increase in ordered secondary structure with increasing temperature, probably caused by the higher average hydrophobicity as compared to prothymosin alpha . In the absence of a clear structural transition, we deduce the observed effects result mainly from a progressive redistribution in the population of phi-psi angles of the polypeptide backbone when the temperature is increased . Furthermore, the paper should demonstrate the difficulties in distinguishing between such a progressive change amongst a continuum of states within the ensemble of unfolded conformations from the formation of authentic stable secondary structures in highly unfolded proteins . This problem is not solved presently and convincing evidence can only be supplied by the combination of various experimental techniques.

Theor Appl Genet, 2002 Feb, 104(2-3), 308 - 314
Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene; Yamada T et al.; A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance . A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH) . Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis . Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 micro mol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment . These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment . The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants . These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment . Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced . Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides . These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone . This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato.

Cell, 2003 Feb 7, 112(3), 286 - 7
Better chemistry for better survival, through regulation; Cox MM; In this issue of Cell, report on new aspects of the regulation of yeast ribonucleotide reductase, the mechanism by which dNTP levels are increased following DNA damage, and the consequences of the metabolic changes.

Anim Genet, 2003 Feb, 34(1), 59 - 61
Cloning, characterization and chromosomal localization of the Sus scrofa SLC31A1 gene; Harboe TL et al.; Copper is an essential element necessary for normal function of numerous enzymes in all living organisms . Uptake of copper into the cell is thought to occur through the membrane protein, SLC31A1 (CTR1), which has been described in a variety of species including yeast, human and mouse . In this study, we present cloning, gene structure, chromosomal localization and expression pattern of the Sus scrofa SLC31A1 gene, which encodes a 189 amino acid protein . The (SSC) SLC31A1 gene is organized in four exons and spans an approximately 2.3 kb genomic region . We have localized the gene to chromosome 1q28-q2.13 using a somatic cell hybrid panel . This region shows conservation of synteny with human chromosome 9, where the human SLC31A1 (CTR1) gene has been localized . Expression studies suggest that SLC31A1 mRNA is transcribed in all tissues examined.

J Biol Chem, 2003 Apr 18, 278(16), 13912 - 8 Epub 2003 Feb 10.
Inherent instability of plasminogen activator inhibitor type 2 mRNA is regulated by tristetraprolin; Yu H et al.; Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level . At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR) . For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR . In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE . This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein . UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro . Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR . The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript . The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits . This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.

Biochemistry, 2003 Feb 18, 42(6), 1470 - 7
Structural basis for a lethal mutation in U6 RNA; Sashital DG et al.; U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal . The U80G mutation at the essential magnesium binding site of the U6 3' intramolecular stem-loop region (ISL) is lethal in yeast . To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy . The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67 x G80), and disrupts a protonated C67 x A79 wobble pair that forms in the wild-type structure . Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs . The thermodynamic stability of the U80G mutant is significantly increased (Delta Delta G(fold) = -3.6 +/- 1.9 kcal/mol), consistent with formation of the Watson-Crick pair . Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization . This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4.

Vestn Khir Im I I Grek, 2002, 161(4), 85 - 90
{Immunopathogenesis of severe wounds and traumas: possibilities of immune correction}; Lebedev VF et al.; The authors describe the present-day views on the nature of immune dysfunctions in severe traumas . Based on personal clinical experiences and literature data the authors discuss the role of immune dysfunctions in pathogenesis of the traumatic disease . Special attention is given to the role of the immune system in the development of the life-threatening condition: polyorganic insufficiency whose formation mainly results from disorganization and functional failure of the system of immune reactivity . Clinical investigations have shown high effectiveness of early administration for severe wounds and traumas of a new means of immunocorrection--yeast recombinant interleukin-2 of man (preparation Roncoleukin) . The administration of this immunocorrector in complex schemes of intensive therapy of the victims was shown to prevent the development of severe pyo-septic pathology and perfectly change the course of the traumatic disease.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 2001 Mar, 21(3), 203 - 5
{Effect of guizhi decoction on adenyl cyclase and phosphodiesterase in hypothalamus of rats models of fever and hypothermia}; Qi Y et al.; OBJECTIVE: To observe the effect of Guizhi Decoction (GZD) on adenyl cyclase (AC) and phosphodiesterase (PDE) activities in hypothalamus of rat models of fever and hypothermia . METHODS: The AC and PDE activities in hypothalamus were determined using radio-isotope method . RESULTS: GZD could lower the AC activity in yeast induced fever rat model (P < 0.05), but cause rise of AC activity in aminopyrine induced hypothermia model (P < 0.05) . No significant influence of GZD on PDE activity was shown in both fever or hypothermia models . CONCLUSION: The bi-directional thermo-regulation effect of GZD might be partially due to influence on AC activity.

Cell Struct Funct, 2002 Dec, 27(6), 431 - 41
Diversity of signaling controls of macroautophagy in mammalian cells; Petiot A et al.; Macroautophagy is a major lysosomal catabolic process conserved from yeast to human . The formation of autophagic vacuoles is stimulated by a variety of intracellular and extracellular stress situations including amino acid starvation, aggregation of misfolded proteins, and accumulation of damaged organelles . Several signaling pathways control the formation of autophagic vacuoles . As some of them are engaged in the control of protein synthesis or cell survival this suggests that macroautophagy is intimately associated with the execution of cell proliferation and cell death programs . Whether or not these different signaling pathways converge to a unique point to trigger the formation of autophagic vacuole remains an open question.

Blood, 2003 Jun 1, 101(11), 4492 - 9 Epub 2003 Feb 06.
Regulation of T-cell receptor D beta 1 promoter by KLF5 through reiterated GC-rich motifs; Yang XO et al.; Rearrangement of T-cell receptor (TCR) and immunoglobulin genes by a common V(D)J recombination machinery is regulated by developmentally specific chromatin changes at the target locus, a process associated with transcription . At the TCRbeta locus, the Ebeta enhancer and the Dbeta1 promoter regulate germline transcription originating near the TCR Dbeta1 gene segment . The Dbeta1 promoter contains 3 GC-rich motifs that bind a common set of nuclear proteins from pro-T-cell lines . Mutations that diminish the binding of nuclear proteins also diminish the activity of the Dbeta1 promoter in transcriptional reporter assays . Using a yeast one-hybrid approach, 3 Kruppel-like factors-KLF3, KLF5, and KLF6-and a novel zinc finger protein were identified in a thymus library, all of which bound the GC-rich motif in a sequence-specific manner . Of these genes, KLF5 mRNA was expressed in a restricted manner in lymphoid cells and tissues, with highest expression in pro-T-cell lines and Rag-deficient thymocytes . Antibody supershift studies and chromatin immunoprecipitation assay confirmed that KLF5 bound the Dbeta1 promoter . In reporter gene assays, KLF5 but not KLF6 efficiently transactivated the Dbeta1 promoter, whereas a dominant-negative KLF5 construct inhibited reporter expression . These data suggest that reiterated GC motifs contribute to germline TCRbeta transcription through binding of KLF5 and other Kruppel family members and that restricted expression of KLF5 may contribute to lineage-specific regulation of germline TCRbeta transcription.

Am J Physiol Lung Cell Mol Physiol, 2003 Jun, 284(6), L1027 - 36 Epub 2003 Feb 07.
Transcription factor USF2 is developmentally regulated in fetal lung and acts together with USF1 to induce SP-A gene expression; Gao E et al.; Expression of the pulmonary surfactant protein A (SP-A) gene is lung specific, developmentally regulated, and enhanced by hormones and factors that increase cAMP . We previously identified two E-box-like enhancers termed distal binding element (DBE) and proximal binding element (PBE) in the 5'-flanking region of the rabbit (r) SP-A gene that are essential for cAMP induction of rSP-A promoter activity (Gao E, Alcorn JL, and Mendelson CR . J Biol Chem 268: 19697-19709, 1993) . We also found that DBE and PBE serve as binding sites for the basic helix-loop-helix-leucine zipper transcription factor, upstream stimulatory factor-1 (USF1) (Gao E, Wang Y, Alcorn JL, and Mendelson CR . J Biol Chem 272: 23398-23406, 1997) . In the present study, PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert encoding the structurally related rabbit upstream stimulatory factor-2 (rUSF2) was isolated . The levels of rUSF2 mRNA reach peak levels in fetal rabbit lung at 28 days of gestation, in concert with the time of maximal induction of SP-A gene transcription . In yeast two-hybrid analysis, rUSF2 was found to preferentially form heterodimers, compared with homodimers, with rUSF1 . Binding complexes of nuclear proteins isolated from fetal rabbit lung type II cells with the DBE and PBE were supershifted by anti-rUSF2 antibodies . Binding activity was enriched in nuclear proteins from type II cells compared with fibroblasts . Overexpression of rUSF2 in transfected lung A549 cells increased rSP-A promoter activity and acted synergistically with rUSF1 . We suggest that heterodimers of USF2 and USF1 bound to two E-box elements in the SP-A gene 5'-flanking region serve a key role in developmental and hormonal regulation of SP-A gene expression in pulmonary type II cells.

J Ethnopharmacol, 2003 Mar, 85(1), 151 - 6
Anti-inflammatory and antipyretic properties of Clerodendrum petasites S . Moore; Panthong A et al.; The methanol extract from Clerodendrum petasites S . Moore (CP extract) was assessed for anti-inflammatory and antipyretic activities on the experimental animal models . It was found that CP extract possessed moderate inhibitory activity on acute phase of inflammation in a dose-related manner as seen in ethyl phenylpropiolate-induced ear edema (ED(50)=2.34 mg/ear) as well as carrageenin-induced hind paw edema (ED(30)=420.41 mg/kg) in rats . However, CP extract did not elicit any inhibitory effect on arachidonic acid-induced hind paw edema in rats . In subchronic inflammatory model, CP extract provoked a significant reduction of transudation but had no effect on proliferative phase when tested in cotton pellet-induced granuloma model . CP extract also reduced the alkaline phosphatase activity in serum of rats in this animal model . Moreover, CP extract possessed an excellent antipyretic effect when tested in yeast-induced hyperthermic rats . It is postulated that the anti-inflammatory and antipyretic effects of CP extract are caused by the inhibition of the prostaglandin synthesis . Anyhow, CP extract did not possess any analgesic activity in acetic acid-induced writhing response in mice . The results obtained show that C . petasites has beneficial properties since it possesses potent antipyretic and moderate anti-inflammatory activities without ulcerogenic effect.

Mol Plant Microbe Interact, 2003 Feb, 16(2), 132 - 40
TIP, a novel host factor linking callose degradation with the cell-to-cell movement of Potato virus X; Fridborg I et al.; The cell-to-cell movement of Potato virus X (PVX) requires four virus-encoded proteins, the triple gene block (TGB) proteins (TGB25K, TGB12K, and TGB8K) and the coat protein . TGB12K increases the plasmodesmal size exclusion limit (SEL) and may, therefore, interact directly with components of the cell wall or with plant proteins associated with bringing about this change . A yeast two-hybrid screen using TGB12K as bait identified three TGB12K-interacting proteins (TIP1, TIP2, and TIP3) . All three TIPs interacted specifically with TGB12K but not with TGB25K or TGB8K . Similarly, all three TIPs interacted with beta-1,3-glucanase, the enzyme that may regulate plasmodesmal SEL through callose degradation . Sequence analyses revealed that the TIPs encode very similar proteins and that TIP1 corresponds to the tobacco ankyrin repeat-containing protein HBP1 . A TIP1::GFP fusion protein localized to the cytoplasm . Coexpression of this fusion protein with TGB12K induced cellular changes manifested as deposits of additional cytoplasm at the cell periphery . This work reports a direct link between a viral movement protein required to increase plasmodesmal SEL and a host factor that has been implicated as a key regulator of plasmodesmal SEL . We propose that the TIPs are susceptibility factors that modulate the plasmodesmal SEL.

Zhong Yao Cai, 2000 Oct, 23(10), 630 - 2
{Pharmacological studies of trilex on treatment of pharyngitis}; Jiang J et al.; Trilex consisted of three species: Ilex latifolia, Ilex asprella and Ilex rotunda . Trilex had the antipyretic effects, decreased the rat body temperature about 0.8 degree C on yeast induced hyperthermia . Trilex could obviously enhance the threshold of hot plate induced pain in mice . The maximum threshold increased 43.8% . Trilex could also inhibit acetic acid induced inflammatory ooze in mice abdominal cavity . The inhibitory rate was 34.8% . These results showed that trilex had antipyretic, analgesic and anti-inflammatory effects . It could be used to treat acute and chromic pharyngitis.

J Biol Chem, 2003 May 2, 278(18), 16289 - 96 Epub 2003 Feb 05.
Mouse GGN1 and GGN3, two germ cell-specific proteins from the single gene Ggn, interact with mouse POG and play a role in spermatogenesis; Lu B et al.; The germ cell-deficient (gcd) mutation is a recessive transgenic insertional mutation leading to a deficiency of primordial germ cells (PGCs) . We have recently shown that the gene underlying this mutation is Pog, which is necessary for normal proliferation of PGCs . Here we show that Pog is also involved in spermatogenesis in that meiosis is impaired in Pog-deficient mice . Yeast two-hybrid screening revealed that POG interacted with GGN1 and GGN3, two proteins formed by alternate splicing of the same gene, gametogenetin (Ggn) . Ggn had more than 10 different splice variants giving rise to three proteins, GGN1, GGN2, and GGN3 . The three proteins had different subcellular localizations, with GGN1, GGN2, and GGN3 localized along the nuclear membrane, in the cytoplasm, and in the nucleus/nucleoli respectively . The expression of Ggn was confined to late pachytene spermatocytes and round spermatids, a time window concomitant with the occurrence of meiosis . Mouse Ggn and Pog were both expressed in primary spermatocytes . Co-expression of POG with GGN1 or GGN3 in HeLa cells changed the localization of POG to the perinuclear localization or the nucleoli, respectively . Our data showed that in addition to functioning in proliferation of primordial germ cells, POG also functioned in spermatogenesis . Two spatial and temporal regulated proteins, GGN1 and GGN3, interacted with POG, regulated the localization of POG, and played a role in spermatogenesis.

Curr Biol, 2003 Feb 4, 13(3), 241 - 6
Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16; Memedula S et al.; Eukaryotic transcription initiation requires the complex dynamics of hundreds of proteins, many of which are found in large multisubunit complexes . Recent experiments have suggested stepwise recruitment of preassembled complexes, including chromatin remodeling, general transcription factor, mediator, and polymerase complexes, in which the actual order of recruitment may vary for different promoters . How do these complexes access target sequences contained within tightly condensed chromatin? While chromatin remodeling activities may facilitate the accessibility of large transcription and polymerase complexes to promoters, it is not known how they themselves are targeted within condensed chromatin . Gene activation in the context of condensed chromatin does occur . A yeast acidic activator, Gal4, can overcome heterochromatin gene silencing in Drosophila, and the addition of LCRs (locus control regions) to transgenes overcomes position effect silencing, even within centromeric chromatin . Here, we directly visualize the recruitment of HAT and SWI/SNF components after tethering the VP16 acidic activation domain within condensed chromatin . A recruitment delay of tens to hundreds of minutes for catalytic HAT subunits and SWI/SNF subunits, relative to other HAT and SWI/SNF components, suggests sequential recruitment/assembly of chromatin remodeling complexes within condensed chromatin.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 90 - 7
Improving glucose and glutamine metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme; Elias CB et al.; Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology . The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells . The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway . One of the key enzymes controlling this flux is pyruvate carboxylase . Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth . In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme . In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia . Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells . The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined . The results obtained and their impact on the process development for protein and viral vector production are discussed.

Mycoses, 2002 Oct, 45(8), 282 - 6
Comparison of seven Blastomyces dermatitidis antigens for the detection of antibodies in humans with occupationally acquired blastomycosis; Roomiany PL et al.; Yeast phase lysate antigens, prepared from seven isolates of Blastomyces dermatitidis (dogs, T-58, M-98; human, Le; soil, S; sea lion, SL; polar bear, PB; cat, C) were assayed by enzyme-linked immunosorbent assay (ELISA) to detect antibodies in human sera . The sera were from individuals potentially exposed to B . dermatitidis while working on a prairie dog relocation project in Colorado . All antigens exhibited greatest reactivity with three specimens in two separate assays . Absorbance values ranged from 0.370 (M-98) to 0.427 (LE) (Trial 1) and from 0.579 (M-98) to 0.714 (PB) (Trial 2) with serum 2; from 0.368 (C) to 0.453 (LE) (Trial 1) and from 0.565 (SL) to 0.694 (S) (Trial 2) with serum 3 and from 0.392 (M-98) to 0.506 (LE) (Trial 1) and from 0.557 (SL) to 0.758 (T-58) (Trial 2) with serum 6 . The greatest reactivity was observed with sera from two persons (2 and 3 from the same individual and serum 6) who became ill with symptoms of pulmonary disease and sought medical care . Both patients were subsequently diagnosed with blastomycosis by microscopic and culture methods . The study indicated that all of the lysate antigens, regardless of source, could be used to reliably diagnose blastomycosis.

P R Health Sci J, 2002 Dec, 21(4), 323 - 8
Effects of a high molecular mass Convolvulus arvensis extract on tumor growth and angiogenesis; Meng XL et al.; BACKGROUND: Plant materials represent promising sources of anti-cancer agents . We developed and tested a novel extract from the ubiquitous plant Convolvulus arvensis . MATERIALS AND METHODS: Convolvulus arvensis components were extracted in boiling water, and small molecules were removed by high-pressure filtration . The extract's biological activity was assessed by measuring its effects on S-180 fibrosarcoma growth in Kun Ming mice and on heparin-induced angiogenesis in chick embryos . We also examined the extract's effects on lymphocytes ex vivo and tumor cell growth in vitro . RESULTS: The extract (primarily proteins and polysaccharides) inhibited tumor growth in a dose-dependent fashion when administered orally . At the highest dose tested, 200 mg/kg/day, tumor growth was inhibited by roughly seventy percent . Subcutaneous or intraperitoneal administration at 50 mg/kg/day also inhibited tumor growth by over seventy percent . The extract's acute LD50 in Kun Ming mice was 500 mg/kg/day when injected, indicating that tumor growth inhibition occurred at non-toxic doses . It inhibited angiogenesis in chick embryos, improved lymphocyte survival ex vivo, and enhanced yeast phagocytosis, but did not kill tumor cells in culture . CONCLUSION: High molecular mass extract deserves further study as an anti-cancer agent.

Neuropediatrics, 2002 Dec, 33(6), 320 - 3
Refractory photosensitive epilepsy associated with a complex rearrangement of chromosome 2; Van Esch H et al.; We describe the relevant clinical and therapeutic parameters in a single patient with a complex chromosome 2 abnormality presenting with refractory myoclonic photosensitive epilepsy . FISH technology using yeast artificial chromosomes (YACs) was employed to determine breakage points, microdeletions and inversions on the affected chromosome . In this patient with refractory photosensitive epilepsy, 12 breakpoints and one small inversion were identified on the abnormal chromosome 2 . Our data can be used in further genetic studies on the exact location and identification of photosensitivity genes.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1627 - 32 Epub 2003 Feb 05.
Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2); Li H et al.; Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately 200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A . The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP . To explore possible functions of the N-terminal region of BIG2 (1-832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RI alpha . Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RI alpha, as well as of the endogenous proteins in cytosol of cultured HepG2 cells . Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA . Residues 27-48 (domain A) interacted with RI alpha and RI beta, 284-301 (domain B) interacted with RII alpha and RII beta, and 517-538 (domain C) interacted with RI alpha, RII alpha, and RII beta . Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits . Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin . All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1609 - 14 Epub 2003 Feb 05.
Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex; Parsons XH et al.; Sir2 is an NAD-dependent histone deacetylase required for transcriptional silencing . To study the mechanism of Sir2 function, we examined the biochemical properties of purified recombinant Drosophila Sir2 (dSir2) . First, we performed histone deacetylation assays and found that dSir2 deacetylates a broad range of acetylated lysine residues . We then carried out in vitro transcription experiments and observed that dSir2 does not repress transcription with either naked DNA templates or chromatin assembled from native (and mostly unacetylated) histones . It was possible, however, that repression by dSir2 requires an acetylated histone substrate . We therefore tested the transcriptional effects of dSir2 with native histones that were hyperacetylated by treatment with acetic anhydride . Assembly of the hyperacetylated histones onto DNA yields a soluble histone-DNA complex that differs from canonical nucleosomal chromatin . With this hyperacetylated histone-DNA complex, we observed potent (50- to 100-fold) NAD-dependent transcriptional repression by purified dSir2 . In contrast, repression by dSir2 was not observed in parallel experiments in which histones were hyperpropionylated with propionic anhydride . We also found that dSir2 mediates the formation of a nuclease-resistant fast-sedimenting histone-DNA complex in an NAD-dependent manner . Unlike dSir2, the dHDAC1 deacetylase does not strongly repress transcription or generate a nuclease-resistant histone-DNA complex . Furthermore, with yeast Sir2, the transcriptional repression we observe correlates with deacetylation activity in vitro and silencing activity in vivo . These findings suggest that deacetylation by Sir2 causes a conformational change or rearrangement of histones into a transcriptionally repressive chromatin structure.

J Cell Sci, 2003 Mar 1, 116(Pt 5), 897 - 906
TES is a novel focal adhesion protein with a role in cell spreading; Coutts AS et al.; Previously, we identified TES as a novel candidate tumour suppressor gene that mapped to human chromosome 7q31.1 . In this report we demonstrate that the TES protein is localised at focal adhesions, actin stress fibres and areas of cell-cell contact . TES has three C-terminal LIM domains that appear to be important for focal adhesion targeting . Additionally, the N-terminal region is important for targeting TES to actin stress fibres . Yeast two-hybrid and biochemical analyses yielded interactions with several focal adhesion and/or cytoskeletal proteins including mena, zyxin and talin . The fact that TES localises to regions of cell adhesion suggests that it functions in events related to cell motility and adhesion . In support of this, we demonstrate that fibroblasts stably overexpressing TES have an increased ability to spread on fibronectin.

J Cell Sci, 2003 Mar 1, 116(Pt 5), 791 - 801
The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth; Bouquin T et al.; The lue1 mutant was previously isolated in a bio-imaging screen for Arabidopsis mutants exhibiting inappropriate regulation of an AtGA20ox1 promoter-luciferase reporter fusion . Here we show that lue1 is allelic to fra2, bot1 and erh3, and encodes a truncated katanin-like microtubule-severing protein (AtKSS) . Complementation of lue1 with the wild-type AtKSS gene restored both wild-type stature and luciferase reporter levels . Hormonal responses of lue1 to ethylene and gibberellins revealed inappropriate cortical microtubule reorientation during cell growth . Moreover, a fusion between the AtKSS protein and GFP decorated cortical microtubules . A yeast two-hybrid screen with AtKSS as the bait identified proteins related to those involved in microtubule processing, including a katanin p80 subunit and a kinesin ortholog . These results indicate that AtKSS is involved in microtubule dynamics in response to plant hormones.

J Biol Chem, 2003 Apr 18, 278(16), 13795 - 802 Epub 2003 Feb 05.
Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha; Lim ST et al.; Syndecan-4 is a transmembrane heparan sulfate proteoglycan that acts as a coreceptor with integrins in focal adhesion formation . The central region of syndecan-4 cytoplasmic domain (4V; LGKKPIYKK) binds phosphatidylinositol 4,5-bisphosphate, and together they regulate protein kinase C alpha (PKC alpha) activity . Syndecan 4V peptide directly potentiates PKC alpha activity, leading to "superactivation" of the enzyme, apparently through an interaction with its catalytic domain . We now have performed yeast two-hybrid and in vitro binding assays to determine the interaction sites between 4V and PKC alpha . Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly . A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKC alpha, indicating that tyrosine 192 in the syndecan-4 cytoplasmic domain might be critical for this interaction . Further assays identified a novel interaction site in the C terminus of the catalytic domain of PKC alpha (amino acid sequence 513-672) . This encompasses the autophosphorylation sites, which are implicated in activation and stability . Yeast two-hybrid data were confirmed by in vitro binding and coimmunoprecipitation assays . The interaction of syndecan-4 with PKC alpha appears unique since PKC delta and epsilon did not interact with 4V in yeast two-hybrid assays or coimmunoprecipitate with syndecan-4 . Finally, overexpression of syndecan-4 in rat embryo fibroblast cells, but not expression of the YF mutant, increased PKC alpha localization to focal adhesions . The data support a mechanism where syndecan-4 binds PKC alpha and localizes it to focal adhesions, whose assembly may be regulated by the kinase.

Appl Environ Microbiol, 2003 Feb, 69(2), 796 - 804
Characterization of the initial reactions during the cometabolic oxidation of methyl tert-butyl ether by propane-grown Mycobacterium vaccae JOB5; Smith CA et al.; The initial reactions in the cometabolic oxidation of the gasoline oxygenate, methyl tert-butyl ether (MTBE), by Mycobacterium vaccae JOB5 have been characterized . Two products, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), rapidly accumulated extracellularly when propane-grown cells were incubated with MTBE . Lower rates of TBF and TBA production from MTBE were also observed with cells grown on 1- or 2-propanol, while neither product was generated from MTBE by cells grown on casein-yeast extract-dextrose broth . Kinetic studies with propane-grown cells demonstrated that TBF is the dominant (> or = 80%) initial product of MTBE oxidation and that TBA accumulates from further biotic and abiotic hydrolysis of TBF . Our results suggest that the biotic hydrolysis of TBF is catalyzed by a heat-stable esterase with activity toward several other tert-butyl esters . Propane-grown cells also oxidized TBA, but no further oxidation products were detected . Like the oxidation of MTBE, TBA oxidation was fully inhibited by acetylene, an inactivator of short-chain alkane monooxygenase in M . vaccae JOB5 . Oxidation of both MTBE and TBA was also inhibited by propane (K(i) = 3.3 to 4.4 microM) . Values for K(s) of 1.36 and 1.18 mM and for V(max) of 24.4 and 10.4 nmol min(-1) mg of protein(-1) were derived for MTBE and TBA, respectively . We conclude that the initial steps in the pathway of MTBE oxidation by M . vaccae JOB5 involve two reactions catalyzed by the same monooxygenase (MTBE and TBA oxidation) that are temporally separated by an esterase-catalyzed hydrolysis of TBF to TBA . These results that suggest the initial reactions in MTBE oxidation by M . vaccae JOB5 are the same as those that we have previously characterized in gaseous alkane-utilizing fungi.

AIHA J (Fairfax, Va), 2003 Jan-Feb, 64(1), 102 - 7
Influence of storage on the fungal concentration determination of impinger and filter samples; Lin WH et al.; The effects of storage on concentrations of airborne fungal samples were evaluated in a laboratory test chamber . Spores of Penicillium citrinum and cells of Candida famata var . flareri were aerosolized by a Pitt-3 generator and a Collison nebulizer, respectively . The evaluated sampling methods were AGI-30 impingers, Nuclepore filters, and gelatin filters . The effect of storage time was determined as the ratio, C(t)/C(0), where C(t) and C(0) were the total counts or colony concentrations of the simultaneously collected samples stored for t and 0 hr, respectively . In addition, the effect of storage temperature was investigated by storing AGI-30 and filter samples at 25 and 4 degrees C . The results demonstrated that the culturability of Penicillium spores in the impinger samples decreased as storage time increased . In contrast, the Candida yeast cells could survive and reproduce more cells in the impinger fluid . Moreover, it was observed that refrigeration of the impinger samples could inhibit the growth rate of collected cells . Therefore, it was suggested that the impinger samples should be refrigerated and processed as soon as possible to avoid the loss of spore culturability or the increase of yeast cells . Regarding the filtration methods, the effect of storage time and temperature for Penicillium spores was demonstrated to be insignificant . However, Candida yeast recovery from filters was found to decrease as storage time increased.

Planta, 2003 Feb, 216(4), 561 - 70 Epub 2002 Oct 10.
Regulation of biosynthesis and intracellular localization of rice and tobacco homologues of nucleosome assembly protein 1; Dong A et al.; The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes . However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity . Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.) . Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers . During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition . These results are consistent with a role for plant NAP1-like proteins in cell division . In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone . Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized . It is thus likely that different plant NAP1-like proteins have distinct functions in vivo . Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.

Mol Endocrinol, 2003 May, 17(5), 845 - 59 Epub 2003 Jan 30.
Dissociation of steroid receptor coactivator 1 and nuclear receptor corepressor recruitment to the human glucocorticoid receptor by modification of the ligand-receptor interface: the role of tyrosine 735; Stevens A et al.; Within the human glucocorticoid receptor (GR) steroid binding pocket, tyrosine 735 makes hydrophobic contact with the steroid D ring . Substitution of tyrosine735 selectively impairs glucocorticoid transactivation but not transrepression . We now show, using both mammalian two-hybrid and glutathione-S-transferase pull downs, that such substitutions reduce interaction with steroid receptor coactivator 1, both basally and in response to agonist binding . Using a yeast two-hybrid screen we identified one of the three nuclear receptor interacting domains (NCoR-N1) of nuclear receptor corepressor (NCoR) as interacting with the GR C terminus in an RU486-specific manner . This was confirmed in mammalian two-hybrid experiments, and so we used the NCoR-N1 peptide to probe the GR C-terminal conformation . Substitution of Tyr735phe, Tyr735val, and Tyr735 ser, which impaired steroid receptor coactivator 1 (SRC1) interaction, enhanced NCoR-N1 recruitment, basally and after RU486 . RU486 did not direct SRC1 recruitment to any of the GR constructs, and dexamethasone did not allow NCoR-N1 recruitment . Using a glutathione-S-transferase pull-down approach, the NCoR-N1 peptide was found to bind the full-length GR constitutively, and no further induction was seen with RU486, but it was reduced by dexamethasone . As both SRC1 and NCoR are predicted to recognize a common hydrophobic cleft in the GR, it seems that changes favorable to one interaction are detrimental to the other, thus identifying a molecular switch.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1535 - 40 Epub 2003 Feb 04.
Mapping the binding interface between human eukaryotic initiation factors 1A and 5B: a new interaction between old partners; Marintchev A et al.; The translation initiation factors (IFs) IF1/eIF1A and IF2e/IF5B have been conserved throughout all kingdoms . Although the central roles of the bacterial factors IF1 and IF2 were established long ago, the importance of their eukaryotic homologs, eukaryotic IFs (eIFs) eIF1A and eIF5B, has only recently become evident . The translation machinery in eukaryotes is more complex and accordingly, eIF1A and eIF5B seem to have acquired a number of new functions while also retaining many of the roles of bacterial IF1 and IF2 . IF1 and IF2 have been shown to interact on the ribosome but no binding has been detected for the free factors . In contrast, yeast eIF1A and eIF5B have been reported to interact in the absence of ribosomes . Here, we have identified the binding interface between human eIF1A and the C-terminal domain of eIF5B by using solution NMR . That interaction interface involves the C termini of the two proteins, which are not present in bacterial IF1 and IF2 . The interaction is, therefore, unique to eukaryotes . A structural model for the interaction of eIF1A and eIF5B in the context of the ribosome is presented . We propose that eIF1A and eIF5B simultaneously interact at two sites that are >50 A apart: through their C termini as reported here, and through an interface previously identified in bacterial IF1 and IF2 . The binding between the C termini of eIF1A and eIF5B has implications for eukaryote-specific mechanisms of recruitment and release of translation IFs from the ribosome.

J Natl Cancer Inst, 2003 Feb 5, 95(3), 237 - 41
Effects of dietary selenium supplementation on DNA damage and apoptosis in canine prostate; Waters DJ et al.; The trace mineral selenium inhibits cancer development in a variety of experimental animal models . We used an in vivo canine model to evaluate the effects of dietary selenium supplementation on DNA damage in prostate tissue and on apoptosis in prostate epithelial cells . Sexually intact elderly male beagle dogs were randomly assigned to receive an unsupplemented diet (control group) or diets that were supplemented with selenium (treatment group), either as selenomethionine or as high-selenium yeast at 3 micro g/kg or 6 micro g/kg body weight per day for 7 months . The extent of DNA damage in prostate cells and in peripheral blood lymphocytes, as determined by the alkaline comet assay, was lower among the selenium-supplemented dogs than among the control dogs (prostate P<.001; peripheral blood lymphocytes P =.003; analysis of variance) but was not associated with the activity of the antioxidant enzyme glutathione peroxidase in plasma . The median number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive (i.e., apoptotic) prostate epithelial cells was 3.7 (interquartile range = 1.1-7.6) for the selenium-supplemented dogs and 1.7 (interquartile range = 0.2-2.8) for the control dogs ( P =.04, Mann-Whitney U test) . These data suggest that dietary selenium supplementation decreases DNA damage and increases epithelial cell apoptosis within the aging canine prostate.

Am J Med Genet A, 2003 Mar 1, 117(2), 122 - 6
Molecular characterization of a 12q22-q24 deletion associated with congenital deafness: confirmation and refinement of the DFNA25 locus; Petek E et al.; The DFNA25 locus for autosomal dominant nonsyndromic hereditary hearing loss has been mapped to 12q21-q24 by linkage analysis . A de novo deletion in a six-year-old boy with congenital hearing loss as well as mental and motor retardation now provides independent confirmation of this genetic localization and narrows the critical interval to 13 cM in the 12q22-q24.1 region . Mapping of the deletion was performed using fluorescence in situ hybridization (FISH) analysis with region-specific yeast artificial chromosome (YAC) clones . Ten YACs 929_e_4, 959_c_3, 746_h_7, 817_h_10, 886_a_6, 916_h_9, 969_c_12, 747_e_2, 812_h_12, and 959_f_8 were absent from one chromosome 12 from the patient . Molecular analyses of eight polymorphic markers helped to narrow down the breakpoints and demonstrated that the derivative chromosome 12 is of paternal origin . Several known genes including ATP2A2, UBE3B, and VR-OAC that map in the 12q22-q24.1 region are included in the deletion . These results provide evidence that haploinsufficiency for a gene or genes in 12q22-q24.1 is associated with autosomal dominant deafness .

Horm Res, 2003, 59 Suppl 1, 85 - 93
Nuclear receptors and co-regulators in adrenal tumors; Shibata H et al.; We have reported that excessive steroid hormone production in adrenal cortical tumors results from the disordered expression and activity of specific steroidogenic enzymes . Since no genetic mutations in these steroidogenic enzymes have as yet been identified, disordered expression at the transcription level may be crucial for excessive hormone production in adrenocortical tumors . Nuclear receptors SF-1 and COUP-TF/DAX-1 have been shown to activate and repress, respectively, the transcription of CYP17 gene in a mutually exclusive manner in Y-1 cells . Interestingly, the expression of COUP-TF and DAX-1 is significantly decreased in the cortisol-producing adenomas, in which CYP17 is overexpressed . Conversely, DAX-1 is highly expressed in deoxycorticosterone-producing adenomas, where CYP17 expression is almost absent . These expression profiles indicate the possibility that COUP-TF and DAX-1 play important roles in the transcriptional repression of CYP17 in adrenal tumors . To clarify the mechanisms of COUP-TF-mediated repression, we therefore screened for COUP-TF-interacting proteins using a yeast two-hybrid system from a cortisol-producing adenoma cDNA library . We then cloned a novel COUP-TF-interacting protein-1 (CIP-1), which interacts with COUP-TFI, COUP-TFII, and SF-1 . Functionally, CIP-1 can act as a transcriptional co-repressor for COUP-TF repression activity . CIP-1 expression profiles parallel those of COUP-TFI in steroidogenic tissues, strongly suggesting that, together, COUP-TFI and CIP-1 play an important role in steroidogenesis .

Plant Cell, 2003 Feb, 15(2), 533 - 43
The roles of auxin response factor domains in auxin-responsive transcription; Tiwari SB et al.; Auxin response factors (ARFs) are transcription factors that bind to TGTCTC auxin response elements in promoters of early auxin response genes . ARFs have a conserved N-terminal DNA binding domain (DBD) and in most cases a conserved C-terminal dimerization domain (CTD) . The ARF CTD is related in amino acid sequence to motifs III and IV found in Aux/IAA proteins . Just C terminal to the DBD, ARFs contain a nonconserved region referred to as the middle region (MR), which has been proposed to function as a transcriptional repression or activation domain . Results with transfected protoplasts reported here show that ARFs with Q-rich MRs function as activators, whereas most, if not all other ARFs, function as repressors . ARF DBDs alone are sufficient to recruit ARFs to their DNA target sites, and auxin does not influence this recruitment . ARF MRs alone function as activation or repression domains when targeted to reporter genes via a yeast Gal4 DBD, and auxin does not influence the potency of activation or repression . ARF CTDs, along with a Q-rich MR, are required for an auxin response whether the MRs plus CTDs are recruited to a promoter by an ARF DBD or by a Gal4 DBD . The auxin response is mediated by the recruitment of Aux/IAA proteins to promoters that contain a DNA binding protein with a Q-rich MR and an attached CTD.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 2008 - 13 Epub 2003 Feb 03.
Chemosensory regulation of developmental gene expression in Myxococcus xanthus; Kirby JR et al.; The delta-proteobacterium Myxococcus xanthus coordinates its motility during aggregation and fruiting body formation . While searching for chemotaxis genes in M . xanthus, we identified a third chemotaxis-like gene cluster, the che3 cluster, encoding homologs to two methyl-accepting chemotaxis proteins (MCPs), a CheW, a hybrid CheA, a CheB, a CheR, but no CheY . Mutations in mcp3A, mcp3B, and cheA3 did not show obvious defects in motility or chemotaxis but did affect the timing of entry into development . Mutations in these genes caused early aggregation of starving cells, even at low cell densities . Furthermore, these mutants showed pronounced overexpression of the developmentally regulated Tn5lac fusions Omega4403, Omega4411, and Omega4521 as well as overexpression of mbhA and tps, markers for peripheral rods and aggregating cells, respectively . Divergently transcribed from the che3 promoter region is another gene, crdA (chemosensory regulator of development), predicted to encode a transcriptional activator of sigma(54)-dependent promoters . To test the hypothesis that CrdA functions as the cognate response regulator for the histidine kinase CheA3, CrdA and CheA3 were assayed and found to interact strongly in the yeast two-hybrid system . Mutant analysis showed that crdA cells were delayed in development (12-24 h) and delayed in MbhA production relative to the wild type . An mcp3BcrdA double mutant displayed the crdA phenotype, indicating that crdA is epistatic to mcp3B . We conclude that the Che3 chemotaxis-like system functions to control developmental gene expression by regulating a sigma(54) transcriptional activator, CrdA.

Genome Res, 2003 Feb, 13(2), 238 - 43
Coexpression of neighboring genes in Caenorhabditis elegans is mostly due to operons and duplicate genes; Lercher MJ et al.; In many eukaryotic species, gene order is not random . In humans, flies, and yeast, there is clustering of coexpressed genes that cannot be explained as a trivial consequence of tandem duplication . In the worm genome this is taken a step further with many genes being organized into operons . Here we analyze the relationship between gene location and expression in Caenorhabditis elegans and find evidence for at least three different processes resulting in local expression similarity . Not surprisingly, the strongest effect comes from genes organized in operons . However, coexpression within operons is not perfect, and is influenced by some distance-dependent regulation . Beyond operons, there is a relationship between physical distance, expression similarity, and sequence similarity, acting over several megabases . This is consistent with a model of tandem duplicate genes diverging over time in sequence and expression pattern, while moving apart owing to chromosomal rearrangements . However, at a very local level, nonduplicate genes on opposite strands (hence not in operons) show similar expression patterns . This suggests that such genes may share regulatory elements or be regulated at the level of chromatin structure . The central importance of tandem duplicate genes in these patterns renders the worm genome different from both yeast and human.

Hum Mol Genet, 2003 Feb 15, 12(4), 365 - 73
Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor; Mitton KP et al.; NRL (neural retina leucine zipper) is a basic motif leucine zipper transcription factor of the Maf-subfamily . Multiple phosphorylated isoforms of NRL are detected specifically in rod photoreceptors . NRL regulates the expression of several rod-specific genes, including rhodopsin and cGMP phosphodiesterase beta-subunit, in synergy with other transcription factors (e.g . the homeodomain protein CRX) . Missense mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa, whereas the loss of its function leads to rodless retina in Nrl-knockout mice that exhibit enhanced S-cone function . To further elucidate the molecular mechanism(s) underlying NRL-mediated transcriptional regulation, we used yeast two-hybrid screening to isolate NRL-interacting proteins in the retina and report the identification of Flt3-interacting zinc-finger protein, Fiz1 . Interaction of Fiz1 and NRL-leucine zipper was validated by GST pulldown assays and co-immunoprecipitation from bovine retinal nuclear extracts . Fiz1 suppressed NRL- but not CRX-mediated transactivation of rhodopsin promoter activity in transiently transfected CV1 cells . The mRNA and the protein for both Fiz1 and its only other known interacting protein Flt3, a receptor tyrosine kinase, are expressed in the retina . Our results indicate potential cross-talk among signaling pathways in the retina and suggest that the function of NRL is modulated by its interaction with specific repressor proteins.

Biochem Biophys Res Commun, 2003 Feb 7, 301(2), 324 - 9
Effect of cisplatin treatment on speckled distribution of a serine/arginine-rich nuclear protein CROP/Luc7A; Umehara H et al.; The C-half of cisplatin resistance-associated overexpressed protein (CROP), an SR-related protein, comprises domains rich in arginine and glutamate residues (RE domain), and is rich in arginine and serine residues (RS domain) . We analyzed the role of the individual domains of CROP in cellular localization, subnuclear localization, and protein-protein interaction . CROP fused with green fluorescent protein, GFP-CROP, localized exclusively to the nucleus and showed a speckled intranuclear distribution . The yeast two-hybrid system revealed that CROP interacted with SF2/ASF, an SR protein involved in RNA splicing, as well as CROP itself . The RE and RS domains were necessary for both the intranuclear speckled distribution and the protein-protein interaction . CROP was phosphorylated by mSRPK1, mSRPK2, and Clk1 in vitro, and when cells were treated with cisplatin the subnuclear distribution of GFP-CROP was changed . These results suggest that cisplatin affects RNA splicing by changing the subnuclear distribution of SR proteins including CROP.

Biochem Biophys Res Commun, 2003 Feb 7, 301(2), 287 - 92
Self-interaction of heterochromatin protein 1 is required for direct binding to histone methyltransferase, SUV39H1; Yamamoto K et al.; Heterochromatin protein 1 (HP1) binds to the nucleosome via a methylated lysine residue 9 of histone H3 which is catalyzed by a histone methyltransferase such as SUV39H1 . Although co-localization of HP1 and SUV39H1 has been evident in immunostaining and immunoprecipitation experiments, direct protein-protein interactions have remained to be characterized . We examined interactions between mouse HP1 alpha (mHP1 alpha) and SUV39H1 in yeast and in vitro . A yeast two-hybrid and a glutathione S-transferase pull-down study indicated that the chromo shadow domain of mHP1 alpha directly interacts with the N-terminal 39 amino acid stretch of SUV39H1 . The IY165/168EE mutation in the chromo shadow domain of mHP1 alpha abrogated a self-interaction and this mutant did not interact with SUV39H1 . The 13-mer peptide containing a consensus sequence for binding to the dimer surface formed by the chromo shadow domains inhibited interaction between mHP1 alpha and SUV39H1 . It seems that self-interaction through the chromo shadow domain of HP1 is crucial for recruitment of SUV39H1 onto nucleosomes.

Biochem Biophys Res Commun, 2003 Feb 14, 301(3), 704 - 10
Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: functional analysis by RNA interference; Mise-Omata S et al.; Mammalian actin binding protein 1 (mAbp1, also called SH3P7/Hip55) is structurally and functionally related to yeast Abp1 and to cortactin, both of which have been implicated in endocytotic processes . mAbp1 associates through its SH3 domain with dynamin, a large GTPase essential for vesicle fission . To clarify the function of mAbp1, we specifically knocked down its expression in human embryonic kidney 293T cells, using RNA interference (RNAi) . Co-transfection of a short interfering RNA (siRNA) together with a plasmid coding for a surface marker, followed by purification of transfected cells, enabled us to obtain a cell population having up to 90% inhibition of mAbp1 expression . In mAbp1-knocked down cells, transferrin (Tf) receptor endocytosis was significantly inhibited and intracellular distribution of the early endosomal compartment was modified . In contrast, in these cells actin and microtubule filaments appeared normal, and formation of lamellipodia induced by active Rac was not inhibited . This study provides definitive evidence that mAbp1 is indispensable for receptor-mediated endocytosis.

Toxicon, 2003 Feb, 41(2), 217 - 27
Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca; Assakura MT et al.; Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs) . Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca . The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin . The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains . In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites . The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen . Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like+cysteine-rich domains but not the metalloproteinase domain . These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin.

J Lipid Res, 2003 Mar, 44(3), 441 - 9 Epub 2003 Jan 16.
New perspectives on the regulation of intermembrane glycerophospholipid traffic; Voelker DR; In eukaryotes, phosphatidylserine (PtdSer) can serve as a precursor of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho), which are the major cellular phospholipids . PtdSer synthesis originates in the endoplasmic reticulum (ER) and its subdomain named the mitochondria-associated membrane (MAM) . PtdSer is transported to the mitochondria in mammalian cells and yeast, and decarboxylated by PtdSer decarboxylase 1 (Psd1p) to form PtdEtn . A second decarboxylase, Psd2p, is also found in yeast in the Golgi-vacuole . PtdEtn produced by Psd1p and Psd2p can be transported to the ER, where it is methylated to form PtdCho . Organelle-specific metabolism of the aminoglycerophospholipids is a powerful tool for experimentally following lipid traffic that is now enabling identification of new proteins involved in the regulation of this process . Genetic and biochemical experiments demonstrate that transport of PtdSer between the MAM and mitochondria is regulated by protein ubiquitination, which affects events at both membranes . Similar analyses of PtdSer transport to the locus of Psd2p now indicate that a membrane-bound phosphatidylinositol transfer protein and the C2 domain of Psd2p are both required on the acceptor membrane for efficient transport of PtdSer . Collectively, these recent findings indicate that novel multiprotein assemblies on both donor and acceptor membranes participate in interorganelle phospholipid transport.

J Gen Virol, 2003 Feb, 84(Pt 2), 319 - 27
Physical interaction of Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) with human oestrogen-related receptor 1 (hERR1): hERR1 interacts with a conserved domain of EBNA-LP that is critical for EBV-induced B-cell immortalization; Igarashi M et al.; Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and plays a critical role in EBV-induced transformation . To identify the cellular proteins associating with EBNA-LP, we performed a yeast two-hybrid screen using EBNA-LP cDNA containing a single W1W2 domain as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes . Our results were as follows . (i) A cDNA in the positive yeast colony was found to encode a cellular protein, human oestrogen-related receptor 1 (hERR1), which is a constitutive transcriptional activator of the various types of oestrogen response elements . (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to hERR1 specifically formed complexes with EBNA-LPs containing one (EBNA-LPR1), two (EBNA-LPR2) or four W1W2 repeats (EBNA-LPR4) transiently expressed in COS-7 cells . Reciprocally, GST fused to EBNA-LPR1 or EBNA-LPR2 pulled down hERR1 transiently expressed in COS-7 cells . (iii) Mutational analyses of EBNA-LP revealed that the Y2 domain of EBNA-LP is responsible for the interaction with hERR1 and two leucines in the Y2 domain (Leu-78 and -82), which are conserved among a subset of primate gammaherpesviruses, are interactive sites for hERR1 . So far, it has been reported that the only domain of EBNA-LP critical for EBV-induced transformation is the Y1Y2 domain . Potential roles of hERR1 in EBV-induced transformation are discussed.

Nucleic Acids Res, 2003 Feb 1, 31(3), 1033 - 7
Identification of pseudogenes in the Drosophila melanogaster genome; Harrison PM et al.; Pseudogenes are copies of genes that cannot produce a protein . They can be detected from disruptions to their apparent coding sequence, caused by frameshifts and premature stop codons . They are classed as either processed pseudogenes (made by reverse transcription from an mRNA) or duplicated pseudogenes, arising from duplication in the genomic DNA and subsequent disablement . Historically, there is anecdotal evidence that the fruit fly (Drosophila melanogaster) has few pseudogenes . Investigators have linked this to a high deletion rate of genomic DNA, for which there is evidence from genetic experiments on genome size . Here, we apply a homology-based pipeline that was developed previously to identify pseudogenes in other eukaryotic genomes, to the fruit fly, so as to derive the first complete survey of its pseudogene population . We find approximately 100 pseudogenes, with at least a sixth of these as candidate processed pseudogenes . This gives a much lower proportion of pseudogenes (compared with the size of the proteome) than in the genomes of other eukaryotes for which data are available (human, nematode and budding yeast) . Closest matching proteins to Drosophila pseudogenes are significantly longer than the average protein in its proteome (up to approximately 60% more than the average protein's length), in contrast to the situation in the three other eukaryotic genomes . This may be due to the persistence of fragments of longer genes . In the fly pseudogene population, we found most pseudogenes for serine proteases (which are more abundant in the Drosophila lineage compared with the other eukaryotes), immunoglobulin-motif-containing proteins and cytochromes P450 . Data on the sequences and positions of the putative pseudogenes are available at: The detection of a small number of pseudogenes in the Drosophila genome and the higher mean length for the closest matching proteins to pseudogenes (possibly because remnants of genes encoding longer proteins are more likely to persist) are further evidence for a high deletion rate of genomic DNA in the fruit fly . The data are useful for molecular evolution study in Drosophila.

Nucleic Acids Res, 2003 Feb 1, 31(3), 799 - 804
The pre-ribosomal network; Milkereit P et al.; Recent achievements in yeast functional proteomics have significantly advanced our knowledge about ribosome biogenesis . Here, we present a program developed to integrate data from various proteome analyses with cell biological data on components present in the ribosome producing factories . This program allows users to attribute factors to certain complexes and to specific steps of ribosome biogenesis . Thus, it helps to gain novel insights into the complex network involved in maturation of ribosomal subunits . The database can be accessed at the URL http://www.pre-ribosome.de.

Biochem Biophys Res Commun, 2003 Jan 24, 300(4), 927 - 31
Plexin-A1 and plexin-B1 specifically interact at their cytoplasmic domains; Usui H et al.; Semaphorin 3A (Sema3A) is a member of semaphorins and functions as an axonal repulsive guidance molecule . Neuropilin-1 and plexin-As form receptor complexes for Sema3A and plexin-As are thought to initiate the intracellular signaling cascade . However, the molecule by which plexin-As transduce their signal is not well understood . We searched molecules that interact with intracellular domains of plexin-A1 by yeast two-hybrid screening and identified a 349 amino acid fragment of plexin-B1 as a plexin-A1 interacting protein . We, then, cloned mouse plexin-B1 and confirmed their interaction in a mammalian expression system . Plexin-B1 physically associated with plexin-A1, but not with plexin-A2 or A3 . Northern blot analysis showed the expression of both plexin-A1 and B1 in adult brain . We propose that plexin-A1 and B1 interact in the adult brain and transduce Sema3A signaling in cooperation.

Biochem Biophys Res Commun, 2003 Jan 24, 300(4), 862 - 7
Characterization of KIBRA, a novel WW domain-containing protein; Kremerskothen J et al.; In a yeast two hybrid screen with the human isoform of Dendrin (KIAA0749), a putative modulator of the postsynaptic cytoskeleton, we isolated a cDNA coding for a novel protein, KIBRA, possessing two amino-terminal WW domains, an internal C2-like domain and a carboxy-terminal glutamic acid-rich stretch . Northern blot analysis revealed that the expression of KIBRA mRNA was predominately found in kidney and brain . In vitro interaction studies revealed that the first KIBRA WW domain binds specifically to PPxY motifs . Transient transfection of monkey kidney cells with constructs encoding Myc-tagged KIBRA displayed a cytoplasmic localization and a perinuclear enrichment of the protein.

Biochem Biophys Res Commun, 2003 Jan 24, 300(4), 824 - 31
Genomic occurrence of microsatellites containing integral and non-integral repeat numbers; Hrabcova I et al.; We calculated occurrences of all dinucleotide and trinucleotide microsatellites in the human, mouse, and yeast genomes . The microsatellites were considered separately not only according to the repeated dinucleotide or trinucleotide and the microsatellite length but also according to the starting/terminal nucleotide . The analysis showed that dramatically non-equal amounts occurred in the human genome of microsatellites that differed only by the terminal nucleotides . For example, the 23-mer (TTG)(7)TT occurs 635 times in the human genome whereas (GTT)(7)GT is present only three times in the human genome though the two 23-mers share a 22 nucleotide sequence . The dramatically non-equal occurrences of microsatellites differing only by the terminal nucleotides are observed for most dinucleotide and trinucleotide microsatellites and in all analyzed genomes . We suppose that the strikingly non-equal genomic occurrences of these closely related microsatellites originate from conformational properties of DNA.

Clin Chim Acta, 2003 Feb, 328(1-2), 179 - 84
Enzymatic assay for determination of bicarbonate ion in plasma using urea amidolyase; Kimura S et al.; BACKGROUND: One of the important buffering systems to maintain blood pH is carbonic acid-bicarbonate . Together with other clinical tests, the measurement of bicarbonate ion concentrations is widely used for the diagnosis of the acid-base balance . We developed a kinetic assay for measurement of bicarbonate ion in plasma using urea amidolyase (EC 3.5.1.45) from yeast species . We evaluated the analytical performance of the present enzymatic method and examined the relationship between bicarbonate ion concentrations by present method and with ABL 520 blood gas system . METHODS: Urea amidolyase catalyzes the reaction of bicarbonate ion with urea to rise to allophanate . We eliminated endogenous ammonium ion by the use of glutamate dehydrogenase (EC 1.4.1.4), and then monitored the production of ammonium ion in the presence of urea amidolyase, urea, ATP, potassium, and magnesium ions . Ammonium ion was produced proportional to the bicarbonate ion concentration and was determined by adding glutamate dehydrogenase to produce NADP(+) in the presence of 2-oxoglutarate and NADPH, and the change of absorbance at 340 nm was monitored . RESULTS: The within-assay and day-to-day assay coefficient variations (CVs) of the present method were 1.3-2.8% and 3.1-5.4%, respectively . The analytical recoveries were 90-110% . The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, hydrogen phosphate, dihydrogen phosphate, ammonium, or calcium ion did not affect this assay . The correlation coefficient between the values obtained by present method (y) and Radiometer ABL 520 blood gas system (x) was 0.983 (y = 1.029x-0.737 mmol/l, Sy/x = 0.764, n = 100), with a mean difference of 0.03 +/- 0.77 mmol/l {(values by reference method-that of present method) +/- S.D.} using the Bland-Altman technique .

Traffic, 2003 Feb, 4(2), 97 - 112
CD2AP/CMS regulates endosome morphology and traffic to the degradative pathway through its interaction with Rab4 and c-Cbl; Cormont M et al.; The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes . To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4-Q67L, the GTP-bound form of Rab4 . We cloned an ubiquitous 80-kDa protein, identical to CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4-Q67L in the yeast two-hybrid system and in vitro . CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments . None of the known markers of early endosomes {Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11} colocalized with the CD2AP/CMS-positive vesicles . However, coexpression of Rab4-Q67L with CD2AP/CMS induces a significant enlargement of EEA1-positive early endosomes . Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes . Coexpression of c-Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes . Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c-Cbl . The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c-Cbl inhibits ligand-induced PDGF receptor degradation . We propose that CD2AP/CMS, through interactions with Rab4 and c-Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.

Environ Toxicol Chem, 2003 Feb, 22(2), 329 - 35
In vitro and in vivo estrogenicity and toxicity of o-, m-, and p-dichlorobenzene; Versonnen BJ et al.; The estrogenicity of o-, m-, and p-dichlorobenzene (DCB) was evaluated with a yeast estrogen screen (YES) and zebrafish (Danio rerio) vitellogenin (VTG) assays . With the YES, p-DCB and m-DCB were found to be estrogenic in a concentration-responsive manner . The relative potency measured with the YES (relative to 17beta-estradiol) was 2.2 x 10(-7) for p-DCB and 1.04 x 10(-8) for m-DCB . Following acute toxicity tests with the zebrafish, plasma VTG production was measured to examine the in vivo estrogenic activity of the three compounds after a 14-d exposure . Adult zebrafish were exposed to different concentrations of o-, m- and p-DCB, ranging from 0.1 to 32 mg/L; ethynylestradiol ({EE2}; 5 ng/L, 10 ng/L, 50 ng/L, and 100 ng/L) was used as a positive control . After exposure, blood samples were taken and protein electrophoresis was performed to determine the relative VTG content . Gonadosomatic indices (GSI) and condition factors (CF) were also calculated . Elevated VTG levels and decreased female GSIs were found in fish exposed to > or = 5 ng EE2/L and in fish exposed to > or = 10 mg p-DCB/L . Low GSIs coincided with high levels of VTG in the blood of female zebrafish . This relation was not only found in fish exposed to EE2 but also in controls and fish exposed to DCB . Therefore, a direct or indirect effect of VTG on the GSI is suggested rather than a direct toxic effect of the tested compounds on the gonads.

Genet Couns, 2002, 13(4), 417 - 25
Small terminal 10q26 deletion in a male patient with Noonan-like stigmata: diagnosis by cytogenetic and FISH analysis; Lukusa T et al.; A male patient is reported with terminal 10q26 deletion, developmental retardation, special behaviour, and multiple clinical anomalies including hypotonia, short stature of postnatal onset, short webbed neck, craniofacial dysmorphism, pectus excavatum with widely spaced small nipples, cryptorchidism with scrotal hypoplasia, limb and musculoskeletal anomalies . The facial dysmorphism mainly consisted of trigonocephaly, a long, triangular and asymmetrical face, hypertelorism with pseudoepicanthus, broad nasal bridge, high-arched palate, retrognathia, low-set dysplastic auricles and, on ophthalmologic examination, strabismus, astigmatism and myopia . Some of these clinical stigmata were suggesting the diagnosis of Noonan syndrome . The extremities showed special features including shortening of proximal limbs, brachydactyly with syndactyly of toes II-III and left fingers III-IV, hypoplastic toenails and joint abnormalities . A diastasis of abdominal muscles was noted and, on X-rays a thoracic scoliosis and bilateral coxa valga were evidenced . Analyses of G- and T-banded chromosomes complemented by FISH analyses using different subtelomere probes detected a terminal 10q26 deletion . Subsequent FISH studies using different probes of the 10q26 region were performed in an attempt to closely define the breakpoint and the extent of the deletion and, thereby, to allow karyotype/phenotype comparison between this patient and a previously reported case with an apparently similar 10q26 deletion.

Genet Couns, 2002, 13(4), 411 - 6
Partial proximal trisomy 10q syndrome: a new case; Nucaro A et al.; We report a case of partial proximal trisomy of the long arm of chromosome 10 confirmed by fluorescence in situ hibridization (FISH) performed with whole chromosome 10 specific painting and specific yac clones . The phenotypic findings, compared to those found in other published cases with the same karyotype, support the recognition of a distinctive partial proximal trisomy 10q syndrome (10q11-->q22).

Anal Sci, 2003 Jan, 19(1), 33 - 7
Simultaneous measurement of the migration velocity and adsorption force of micro-particles using an electromagnetophoretic force under a high magnetic field; Iiguni Y et al.; A simultaneous measurement technique for determining the migration velocity of a micrometer-sized particle in a capillary and the adsorption force to the inner surface of the capillary has been proposed . This technique is based on an electromagnetophoretic force being exerted on a micro-particle in an electrolyte solution, which is governed mainly by the electromagnetic buoyancy, when a homogeneous magnetic field is applied at a right angle to the electric current through the medium . By the electromagnetic buoyancy, micro-particles such as polystyrene, carbon and yeast were migrated perpendicular to the direction of the electric current and reached a fused-silica wall . A switching of the current direction could desorb the particle from the wall, and allowed to calculate the detaching force from the desorbing current . The migration velocity normalized to the size in the magnetic field of 10 T was increased in the order of yeast, carbon and polystyrene, while reflecting the decreasing order of the apparent conductivity of the particles . The desorption force could be measured up to 1 nN with a sensitivity of pN . The observed interaction forces of polystyrene and carbon were in the range of 250-600 pN with large deviations.

Mol Psychiatry, 2003 Jan, 8(1), 83 - 9
A novel CpG-associated brain-expressed candidate gene for chromosome 18q-linked bipolar disorder; Goossens D et al.; We previously identified 18q21-q22 as a candidate region for bipolar (BP) disorder and constructed a yeast artificial chromosome (YAC) contig map . Here we identified three potential CpG islands using CCG/CGG YAC fragmentation . Analysis of available genomic sequences using bioinformatic tools identified an exon of 3639 bp downstream of a CpG island of 1.2 kb containing a putative transcription initiation site . The exon contained an open reading frame coding for 1212 amino acids with significant homology to the SART-2 protein; weaker homology was found with a series of sulphotransferases . Alignment of cDNA sequences of corresponding ESTs and RT-PCR sequencing predicted a transcript of 9.5 kb which was confirmed by Northern blot analysis . The transcript was expressed in different brain areas as well as in multiple other peripheral tissues . We performed an extensive mutation analysis in 113 BP patients . A total of nine single nucleotide polymorphisms (SNPs) were identified . Five SNPs predicted an amino acid change, of which two were present in BP patients but not in 163 control individuals.

J Biol Chem, 2003 Apr 4, 278(14), 12022 - 8 Epub 2003 Jan 28.
Tyrphostin A23 inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the AP-2 adaptor complex; Banbury DN et al.; Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins . Many such motifs conform to the consensus YXXPhi, where Phi represents a bulky hydrophobic residue . This motif interacts with the medium chain (mu) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation . The YXXPhi motif is similar to motifs in which the tyrosine residue is phosphorylated by tyrosine kinases . Tyrphostins (structural analogs of tyrosine) are inhibitors of tyrosine kinases and function by binding to the active sites of the enzymes . We previously showed that, in vitro and in yeast two-hybrid interaction assays, some tyrphostins can inhibit the interaction between YXXPhi motifs and the mu2 subunit of the AP-2 adaptor complex (Crump, C., Williams, J . L., Stephens, D . J., and Banting, G . (1998) J . Biol . Chem . 273, 28073-28077) . A23 is such a tyrphostin . We now show that molecular modeling of tyrphostin A23 into the tyrosine-binding pocket in mu2 provides a structural explanation for A23 being able to inhibit the interaction between YXXPhi motifs and mu2 . Furthermore, we show that A23 inhibited the internalization of (125)I-transferrin in Heb7a cells without having any discernible effect on the morphology of compartments of the endocytic pathway . Control tyrphostins, active as inhibitors of tyrosine kinase activity, but incapable of inhibiting the YXXPhi motif/mu2 interaction, did not inhibit endocytosis . These data are consistent with A23 inhibition of the YXXPhi motif/mu2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.

Mol Cell Biol, 2003 Feb, 23(4), 1181 - 95
batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor; Faucheux M et al.; Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development . We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced . However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes . The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family . We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene . The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay . Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element . This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences . Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

Am J Med Genet B Neuropsychiatr Genet, 2003 Feb, 117(1), 23 - 32
Genetic refinement and physical mapping of a 2.3 Mb probable disease region associated with a bipolar affective disorder susceptibility locus on chromosome 4q35; Badenhop RF et al.; A susceptibility locus for bipolar affective disorder has been mapped to chromosome 4q35 in a large multigenerational pedigree . We have expanded this analysis to include 55 pedigrees (674 individuals, 214 affecteds) . The evidence for linkage to 4q35 was strengthened in this larger cohort, with a maximum two-point LOD score of 3.2 for marker D4S1652 . Several other markers in the region gave LOD scores greater than 1.5 . Non-parametric analysis provided additional support for linkage to the 4q35 region . To further refine this region, haplotype analysis was carried out in 16 of the 55 pedigrees that showed evidence of linkage . As there is no evidence for an ancestral haplotype, nor a one-to-one correspondence between the disease and putative disease haplotype, we undertook an analysis based on pedigree-specific, identical-by-descent allele-sharing in order to define a probable disease region . This analysis indicated that the percentage sharing of alleles, identical-by-descent, in affecteds of all linked pedigrees increases from 60% at the centromeric markers to 75% for markers at the telomere . Maximal allele sharing occurred between markers D4S3051 and 4qTEL13 with this 24 cM region defining a probable disease region . We have constructed a physical map of the 4q35 interval consisting of a YAC contig and BAC clones . Based on this map the probable disease region between D4S3051 and 4qTEL13 corresponds to only 2.3 Mb . This region is very gene poor with only three known genes indicated from the YAC/BAC map . The small number of genes will facilitate systematic screening for variations associated with bipolar disorder .

Oncogene, 2003 Jan 30, 22(4), 627 - 31
Establishment of latrunculin-A resistance in HeLa cells by expression of R183A D184A mutant beta-actin; Fujita M et al.; Actin plays central roles in cell motility through formation of the actin cytoskeleton . Recently, the very intriguing possibility that actin also contributes to processes in the cell nucleus has been emerging . To dissect its dynamics and functions, several actin-disrupting drugs have been widely and effectively employed . Among them, latrunculin-A has proved particularly useful, supplanting the classical drug cytochalasin-D . One reason is that latrunculin-A appears to bind only to actin monomers impairing the nucleotide exchange, the mode being simpler than with cytochalasin . This property may be especially crucial when studying actin functions as a monomer, as suggested for nuclear actin . Very importantly, actin mutations that cause cells to become resistant to the effects of latrunculin-A have been identified in budding yeast . However, it remains controversial as to whether all of the various phenotypes observed with latrunculin in mammalian cells more complicated than yeast are truly the consequence of its specific actions against actin . Here, we show that the expression of R183A D184A mutant beta-actin specifically confers resistance to the effects of latrunculin-A on actin cytoskeleton formation and cell growth in HeLa cells . The established system provides a strong tool to address the various functions of actin in mammalian cells.

Mol Endocrinol, 2003 Apr, 17(4), 507 - 19 Epub 2003 Jan 23.
Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) gene transcription is regulated by wnt4 in the female developing gonad; Mizusaki H et al.; Dax-1 {dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (NR0B1)} is an orphan nuclear receptor acting as a suppressor of Ad4 binding protein/steroidogenic factor 1 {Ad4BP/SF-1 (NR5A1)} and as an anti-Sry factor in the process of gonadal sex differentiation . The roles of these nuclear receptors in the differentiation of the gonads and the adrenal cortex have been established through studies of the mutant phenotype in both mice and humans . However, the mechanisms underlying transcriptional regulation of these genes remain largely unknown . Here, we examined the relationship between Dax-1 gene transcription and the Wnt4 pathway . Reporter gene analysis revealed that Dax-1 gene transcription was activated by beta-catenin, a key signal-transducing protein in the Wnt pathway, acting in synergy with Ad4BP/SF-1 . Interaction between beta-catenin and Ad4BP/SF-1 was observed using yeast two-hybrid and in vitro pull-down assays . The region of Ad4BP/SF-1 essential for this interaction consists of an acidic amino acid cluster, which resides in the first helix of the ligand-binding domain . Mutation of the amino acid cluster impaired transcriptional activation of Dax-1 as well as interaction of Ad4BP/SF-1 with beta-catenin . These results were supported by in vivo observations using Wnt4 gene-disrupted mice, in which Dax-1 gene expression was decreased significantly in sexually differentiating female gonads . We thus conclude that Wnt4 signaling mediates the increased expression of Dax-1 as the ovary becomes sexually differentiated.

J Biol Chem, 2003 Apr 4, 278(14), 11962 - 9 Epub 2003 Jan 27.
Identification of a phosphothionate analogue of lysophosphatidic acid (LPA) as a selective agonist of the LPA3 receptor; Hasegawa Y et al.; Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors . Most cell lines in culture express one or more LPA receptors, making it difficult to assign a response to specific LPA receptors . Dissection of the signaling properties of LPA has been hampered by lack of LPA receptor subtype-specific agonists and antagonists . The present study characterizes an ester-linked thiophosphate derivative (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT) of LPA . OMPT is a functional LPA analogue with potent mitogenic activity in fibroblasts . In contrast to LPA, OMPT does not couple to the pheromone response through the LPA(1) receptor in yeast cells . OMPT induces intracellular calcium increases efficiently in LPA(3) receptor-expressing Sf9 cells but poorly in LPA(2) receptor-expressing cells . Guanosine 5'-O-(3-{(35)S}thio)triphosphate binding assays in mammalian cells showed that LPA exhibits agonistic activity on all three LPA receptor subtypes, whereas OMPT has a potent agonistic effect only on the LPA(3) receptor . In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA(3) but not the LPA(1) or LPA(2) receptors . Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA(1)/LPA(3) receptor-selective antagonist VPC12249 . These results establish that OMPT is an LPA(3)-selective agonist . OMPT binding to the LPA(3) receptor in mammalian cells is sufficient to elicit multiple responses, including activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases . Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.

J Exp Bot, 2003 Feb, 54(383), 669 - 80
Arabidopsis mutants sensitive to gamma radiation include the homologue of the human repair gene ERCC1; Hefner E et al.; Mutants sensitive to ionizing radiation in yeast and mammals include an assortment of DNA repair genes . The majority of these DNA repair genes are involved in the repair of DNA double-strand breaks . In this study a forward genetic screen is used to identify gamma-sensitive mutants of Arabidopsis thaliana . The gamma-plantlet screen used here also reveals two general mutant classes based on size of cotyledons and hypocotyls . One of the mutants discovered is a homologue of the mammalian nucleotide excision repair gene ERCC1.

EMBO J, 2003 Feb 3, 22(3), 641 - 50
SMG-5, required for C.elegans nonsense-mediated mRNA decay, associates with SMG-2 and protein phosphatase 2A; Anders KR et al.; mRNAs that contain premature stop codons are degraded selectively and rapidly in eukaryotes, a phenomenon termed 'nonsense-mediated mRNA decay' (NMD) . We report here molecular analysis of smg-5, which encodes a novel protein required for NMD in Caenorhabditis elegans . Using a combination of immunoprecipitation and yeast two-hybrid assays, we identified a series of protein-protein interactions involving SMG-5 . SMG-5 interacts with at least four proteins: (i) SMG-7, a previously identified protein required for NMD; (ii) SMG-2, a phosphorylated protein required for NMD in worms, yeasts and mammals; (iii) PR65, the structural subunit of protein phosphatase 2A (PP2A); and (iv) PP2A(C), the catalytic subunit of PP2A . Previous work demonstrated that both SMG-5 and SMG-7 are required for efficient dephosphorylation of SMG-2 . Our results suggest that PP2A is the SMG-2 phosphatase, and the role of SMG-5 is to direct PP2A to its SMG-2 substrate . We discuss cycles of SMG-2 phosphorylation and their roles in NMD.

EMBO J, 2003 Feb 3, 22(3), 621 - 32
Plant dicistronic tRNA-snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z; Kruszka K et al.; Small nucleolar RNAs (snoRNAs) guiding modifications of ribosomal RNAs and other RNAs display diverse modes of gene organization and expression depending on the eukaryotic system: in animals most are intron encoded, in yeast many are monocistronic genes and in plants most are polycistronic (independent or intronic) genes . Here we report an unprecedented organization: plant dicistronic tRNA-snoRNA genes . In Arabidopsis thaliana we identified a gene family encoding 12 novel box C/D snoRNAs (snoR43) located just downstream from tRNA(Gly) genes . We confirmed that they are transcribed, probably from the tRNA gene promoter, producing dicistronic tRNA(Gly)-snoR43 precursors . Using transgenic lines expressing a tagged tRNA-snoR43.1 gene we show that the dicistronic precursor is accurately processed to both snoR43.1 and tRNA(Gly) . In addition, we show that a recombinant RNase Z, the plant tRNA 3' processing enzyme, efficiently cleaves the dicistronic precursor in vitro releasing the snoR43.1 from the tRNA(Gly) . Finally, we describe a similar case in rice implicating a tRNA(Met-e) expressed in fusion with a novel C/D snoRNA, showing that this mode of snoRNA expression is found in distant plant species.

J HIV Ther, 2002 May, 7(2), 40 - 5
Improving HIV-specific immune responses in HIV-infected patients; Hardy GA et al.; Deficiencies in potentially highly protective HIV-1-specific immune responses have led to interventions with immunotherapeutic strategies such as post-exposure vaccination . The application of exogenous antigen to the HIV-infected individual by therapeutic vaccination might be expected to induce renewed virus-specific effector T-cell and neutralising-antibody responses . However, the nature of HIV-1 immunopathogenicity precludes this approach: high levels of viral turnover, persistent expression and presentation of HIV-1 antigen to the immune system, T-cell hyperactivation and exhaustion and clonal T-cell anergy all successfully counter any effect of exogenous antigen . Even with the use of highly active antiretroviral therapy (HAART), when HIV-1 activity is profoundly restricted, the induction of immune responses remains problematic . Different vaccine strategies are currently being tested, including a whole killed virus preparation (Remune), a yeast virus-like particle (p24 VLP), whole antigen preparations (VaxSyn), canarypox immunogens (ALVAC) and DNA plasmids . Therapeutic vaccine strategies currently in the earliest stages of development include adenovirus vectors and a topical DNA preparation, DermaVir.

J Cell Biochem Suppl, 2002, 39, 231 - 8
In vivo molecular imaging; Gillies RJ; The relatively young field of molecular imaging is focused on the visualization of molecular phenotypes in whole organisms . This is achieved using imaging systems based on radionuclides, nuclear magnetic resonance, ultrasound, or the visible-IR region of the optical spectrum . Molecularly defined contrast in these modalities is generated by exogenous probes of the endogenous proteome, or through transgenes . Examples of exogenous probes include those that are transported and trapped (glucose, nucleoside analogs), those directed against extracellular receptors (somatostatin, opioid, melanotropin), and those activated by extracellular proteases . Transgenes that have been used in molecular imaging include the above receptors, non-mammalian enzymes that trap pro-drugs (HSV-tk, yeast CD), and optical reporter proteins (luciferase, fluorescent proteins) . Cutting edge technologies in this field include in vivo assays for protein-protein interactions, and in vivo assays for mRNA expression patterns . The number of degrees of freedom in designing new agents is daunting, and advancements in this field will require a significant participation from molecular and cellular biochemists .

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 981 - 5 Epub 2003 Jan 27.
Depletion of a single nucleoporin, Nup107, prevents the assembly of a subset of nucleoporins into the nuclear pore complex; Boehmer T et al.; The nuclear pore complex (NPC) is a protein assembly that contains several distinct subcomplexes . The mammalian nucleoporin (Nup)-107 is part of a hetero-oligomeric complex, that also contains Nup160, Nup133, Nup96, and the mammalian homolog of yeast Sec13p . We used transfection of HeLa cells with small interfering RNAs to specifically deplete mRNA for Nup107 . In a domino effect, Nup107 depletion caused codepletion of a subset of other Nups on their protein but not on their mRNA level . Among the affected Nups was a member of the Nup107 subcomplex, Nup133, whereas two other tested members of this complex, Nup96 and Sec13, were unaffected and assembled into Nup107Nup133-deficient NPCs . We also tested several phenylalanine-glycine repeat-containing Nups that serve as docking sites for karyopherins . Some of these, such as Nup358, Nup214 on the cytoplasmic, and Nup153 on the nucleoplasmic side of the NPC, failed to assemble into Nup107Nup133-depleted NPCs, whereas p62, a Nup at the center of the NPC, was unaffected . Interestingly, the filamentous, NPC-associated protein Tpr also failed to assemble into the NPCs of Nup107-depleted cells . These data indicate that Nup107 functions as a keystone Nup that is required for the assembly of a subset of Nups into the NPC . Despite the depletion of Nup107 and the accompanying effects on other Nups, there was no significant effect on the growth rate of these cells and only a partial inhibition of mRNA export . These data indicate redundancy of Nups in the function of the mammalian NPC.

Learn Mem, 2003 Jan-Feb, 10(1), 40 - 3
Using an aplysia two-hybrid system to examine the interactions between transcription factors involved in long-term facilitation in the nervous system of aplysia; Choi JH et al.; Interactions between ApCREB1a, ApCREB2, and ApC/EBP have been studied using conventional methods such as the yeast two-hybrid system . However, it is unclear whether these memory-related transcription factors actually interact in the native environment in neurons . To clarify this question, we have developed an Aplysia two-hybrid system and found, consistent with previous studies that ApCREB2 interacts with ApCREB1a and ApC/EBP, and that ApC/EBP forms homodimers . We have also found that ApCREB1a and ApC/EBP do not interact . Therefore, our study shows that formerly described interactions between the proteins actually occur in the Aplysia neurons and that interactions between these transcription factors are specific.

Haematologica, 2003 Jan, 88(1), 31 - 8
Cytogenetic and molecular delineation of a region of chromosome 3q commonly gained in marginal zone B-cell lymphoma; Gazzo S et al.; BACKGROUND AND OBJECTIVES: Whole or partial trisomy 3 represents the most recurrent chromosomal abnormality occurring in marginal zone B-cell lymphoma (MZBCL), a distinct subtype of B-cell non-Hodgkin's lymphoma (NHL) . By conventional cytogenetic analysis, unbalanced translocations involving chromosome 3 and leading to a partial trisomy 3q were identified in a series of 14 MZBCL patients . Fluorescent in situ hybridization (FISH) experiments were then performed to characterize the breakpoints further and to delineate the extent of the 3q gained region more accurately . DESIGN AND METHODS: We studied 14 cases of MZBCL combining cytogenetics and FISH techniques using specific probes for the long arm of chromosome 3, including the chromosome 3 a satellite probe, a representative panel of yeast artificial chromosome (YAC) clones mapping the chromosomal 3q region (3q11.2 to 3q23) and the chromosome 3 subtelomeric (3q29) probe . RESULTS: In the 14 cases, additional chromosome 3q material was found to be involved in different unbalanced translocations with chromosomes 1, 6, 7, 8, 11, 13, 14, 15, 17, 19 and 21, leading to a derivative chromosome . None of the chromosomal abnormality juxtaposed the 3q regions with the heavy and/or light k and l immunoglobulin gene loci . Eight different breakpoints distributed between the 3q11.2 and the 3q13.32 regions were identified and a common 3q13.32 3q29 overrepresented region was delineated . INTERPRETATION AND CONCLUSIONS: These results suggest that this critical region may be of importance in the pathogenesis of MZBCL and support the hypothesis that a gene dosage effect rather than a specific gene disruption may be involved in the development of this disease.

Cancer Genet Cytogenet, 2003 Jan 1, 140(1), 49 - 54
Detection of t(11;18)(q21;q21) in marginal zone lymphoma of mucosa-associated lymphocytic tissue type on paraffin-embedded tissue sections by using fluorescence in situ hybridization; Nomura K et al.; A chromosomal t(11;18)(q21;q21) recurrently found in marginal zone lymphomas of mucosa-associated lymphocytic tissue (MALT) type is one of the key determinants of their treatment and outcome because t(11;18)-positive cases are resistant to Helicobacter pylori eradication therapy . To detect t(11;18) on paraffin-embedded tissue sections, we established a method of dual-color fluorescence in situ hybridization (D-FISH) using DNA probes directly labeled with SpectrumGreen and SpectrumOrange (tissue-FISH {T-FISH}) . T-FISH detected the t(11;18) as colocalized signals between the band-specific yeast artificial chromosome (YAC) clones y966e4 (11q21) and y943b8 (18q21) on the der(11)t(11;18) . The t(11;18) was detected in four of 22 patients with marginal zone lymphoma of MALT type in 72%-78% of the cells . In both patients studied, the percentage of t(11;18)-positive cells was much higher in tissue-FISH analysis than in interphase-FISH: 74% versus 30% in patient 1 and 75% versus 31% in patient 4 . None of the 12 patients with diffuse large B-cell lymphoma of the stomach showed the t(11;18) . Tissue-FISH is a powerful tool for the diagnosis of marginal zone lymphoma of MALT type because it can be applied not only to small specimens obtained from endoscopic biopsy samples but also to archival materials, hence facilitating the delineation of subtypes in marginal zone lymphoma of MALT type.

Cancer Genet Cytogenet, 2003 Jan 1, 140(1), 31 - 6
Interphase fluorescence in situ hybridization analysis of del(11)(q23) and del(17)(p13) in chronic lymphocytic leukemia . a study of 40 early-onset patients; Doneda L et al.; Although B-cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in Western countries, little is known about its underlying molecular abnormalities and their prognostic significance, particularly for use in early therapeutic interventions in young patients . As TP53 tumor suppressor gene abnormalities and 11q23 deletions are reported to be prognostically adverse in hematologic malignancies, we used interphase fluorescence in situ hybridization to analyze their incidence and prognostic significance in young B-CLL patients . Bone marrow samples from 40 untreated B-CLL patients at diagnosis were studied using five yeast artificial chromosome clones from the 11q23.1 approximately q23.3 chromosomal region and a probe specific for the 17p13.1 locus . Twenty-three patients (58%) carried 11q deletions . Interestingly, 16 of 17 patients (94%) who showed early disease progression exhibited this chromosomal abnormality, suggesting that 11q deletions may help to identify more aggressive disease in early stage patients . In contrast, monoallelic TP53 deletions were found in all of the patients . The TP53 and 11q deletions were only present in a proportion of the clonal B-cells, which suggests that they are secondary events in B-CLL.

Cancer Metastasis Rev, 2002, 21(3-4), 217 - 30
Micronutrients in cancer chemoprevention; Greenwald P et al.; The selection of micronutrients, defined as essential and nonessential dietary components consumed in minute quantities, for testing in clinical chemoprevention trials is based on the totality of evidence arising from epidemiologic, in vitro, animal, and clinical studies . Those micronutrients that surface with chemopreventive potential, in terms of high efficacy and low toxicity, in early-phase clinical studies are then candidates for large-scale, randomized clinical chemoprevention trials with cancer endpoints . Micronutrients currently being examined in National Cancer Institute (NCI)-sponsored phase I, II, or III chemoprevention trials for prostate, breast, and colon cancers include isoflavones, lycopene, selenized yeast, selenomethionine, selenium, vitamin E, perillyl alcohol, folic acid, vitamin D, calcium, and curcumin . The response to micronutrients may vary not only in magnitude but also in direction . This variation and response likely depend on individual genetic polymorphisms and/or interactions among dietary components that influence absorption, metabolism, or site of action . Research priorities include investigation of possible molecular targets for micronutrients and whether genetic and epigenetic events dictate direction and magnitude of the response.

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