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.