J Biol Chem, 2001 Jul 13, 276(28), 26688 - 93 Epub 2001 May 02. Interaction of the Arabidopsis receptor protein kinase Wak1 with a glycine-rich protein, AtGRP-3; Park AR et al.; The Arabidopsis wall-associated receptor kinase, Wak1, is a member of the Wak family (Wak1-5) that links the plasma membrane to the extracellular matrix . By the yeast two-hybrid screen, we found that a glycine-rich extracellular protein, AtGRP-3, binds to the extracellular domain of Wak1 . Further in vitro binding studies indicated that AtGRP-3 is the only isoform among the six tested AtGRPs that specifically interacts with Waks, and the cysteine-rich carboxyl terminus of AtGRP-3 is essential for its binding to Wak1 . We also show that Wak1 and AtGRP-3 form a complex with a molecular size of approximately 500 kDa in vivo in conjunction with the kinase-associated protein phosphatase, KAPP, that has been shown to interact with a number of plant receptor-like kinases . Binding of AtGRP-3 to Wak1 is shown to be crucial for the integrity of the complex . Wak1 and AtGRP-3 are both induced by salicylic acid treatment . Moreover, exogenously added AtGRP-3 up-regulates the expression of Wak1, AtGRP-3, and PR-1 (for pathogenesis-related) in protoplasts . Taken together, our data suggest that AtGRP-3 regulates Wak1 function through binding to the cell wall domain of Wak1 and that the interaction of Wak1 with AtGRP-3 occurs in a pathogenesis-related process in planta. Plant Cell Physiol, 2001 Apr, 42(4), 404 - 13 A Brassica oleracea gene expressed in a variety-specific manner may encode a novel plant transmembrane receptor; Palmer JE et al.; The species Brassica oleracea includes several agricultural varieties characterized by the proliferation of different types of meristems . Using a combination of subtractive hybridization and PCR (polymerase chain reaction) techniques we have identified several genes which are expressed in the reproductive meristems of the cauliflower curd (B . oleracea var . botrytis) but not in the vegetative meristems of Brussels sprouts (B . oleracea var . gemmifera) axillary buds . One of the cloned genes, termed CCE1 (CAULIFLOWER CURD EXPRESSION 1) shows specific expression in the botrytis variety . Preferential expression takes place in this variety in the meristems of the curd and in the stem throughout the vegetative and reproductive stages of plant growth . CCE1 transcripts are not detected in any of the organs of other B . oleracea varieties analyzed . Based on the nucleotide sequence of a cDNA encompassing the complete coding region, we predict that this gene encodes a transmembrane protein, with three transmembrane domains . The deduced amino acid sequence includes motifs conserved in G-protein-coupled receptors (GPCRs) from yeast and animal species . Our results suggest that the cloned gene encodes a protein belonging to a new, so far unidentified, family of transmembrane receptors in plants . The expression pattern of the gene suggests that the receptor may be involved in the control of meristem development/arrest that takes place in cauliflower. J Biol Chem, 2001 Jul 6, 276(27), 24891 - 900 Epub 2001 May 01. Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70; Gusarova V et al.; Apoprotein B (apoB) is the major protein of liver-derived atherogenic lipoproteins . The net production of apoB can be regulated by presecretory degradation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70 . To further explore the mechanisms of apoB degradation, we have established a cell-free system in which degradation can be faithfully recapitulated . Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated, and ubiquitinylated . Subsequent incubation with rat hepatic cytosol led to proteasome-mediated degradation . To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay . GA increased the recovery of microsomal apoB48 approximately 3-fold and disrupted the interaction between hsp90 and apoB48 . Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation . Finally, apoB48 degradation was reconstituted in vitro using cytosol prepared from wild type yeast . Notably, degradation was attenuated when apoB48-containing microsomes were incubated with cytosol supplemented with GA or with cytosol prepared from yeast strains with mutations in the homologues of mammalian hsp70 and hsp90 . Overall, our data suggest that hsp90 facilitates the interaction between endoplasmic reticulum-associated apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70. Genetics, 2001 May, 158(1), 123 - 32 Minimum requirements for the function of eukaryotic translation initiation factor 2; Erickson FL et al.; Eukaryotic translation initiation factor 2 (eIF2) is a G protein heterotrimer required for GTP-dependent delivery of initiator tRNA to the ribosome . eIF2B, the nucleotide exchange factor for eIF2, is a heteropentamer that, in yeast, is encoded by four essential genes and one nonessential gene . We found that increased levels of wild-type eIF2, in the presence of sufficient levels of initiator tRNA, overcome the requirement for eIF2B in vivo . Consistent with bypassing eIF2B, these conditions also suppress the lethal effect of overexpressing the mammalian tumor suppressor PKR, an eIF2alpha kinase . The effects described are further enhanced in the presence of a mutation in the G protein (gamma) subunit of eIF2, gcd11-K250R, which mimics the function of eIF2B in vitro . Interestingly, the same conditions that bypass eIF2B also overcome the requirement for the normally essential eIF2alpha structural gene (SUI2) . Our results suggest that the eIF2betagamma complex is capable of carrying out the essential function(s) of eIF2 in the absence of eIF2alpha and eIF2B and are consistent with the idea that the latter function primarily to regulate the level of eIF2.GTP.Met-tRNA(i)(Met) ternary complexes in vivo. Bioresour Technol, 2001 Jun, 78(2), 123 - 6 Production of glucose oxidase using Aspergillus niger and corn steep liquor; Kona RP et al.; Glucose oxidase production was optimized using an isolated strain of Aspergillus niger and an economical nutrient source, corn steep liquor (CSL) . The culture produced 580 +/- 30 units/ml of the enzyme using 70 g/l sucrose as the carbon source . Using CSL as the sole nutrient source enzyme synthesis was increased to 640 +/- 36 units/ml . None of the nitrogen sources (nitrates of calcium, sodium, ammonium, potassium and yeast extract, malt extract, and peptone) was beneficial to the enzyme synthesis . Aeration and agitation enhanced enzyme synthesis to 850 +/- 45 units/ml . Glucose oxidase has numerous applications in food industry and clinical fields. Acta Paediatr, 2001 Mar, 90(3), 323 - 7 Malassezia pachydermatis fungaemia in a neonatal intensive care unit; Chryssanthou E et al.; Malassezia pachydermatis, a non-obligatory lipophilic yeast, has occasionally been implicated in nosocomial fungaemias . This study investigated a cluster of eight cases of M . pachydermatis infection and colonization in a neonatal intensive care unit over a 6 mo period . All patients were preterm with very low birthweight and suffered from various underlying diseases . Prolonged use of indwelling catheters and parenteral lipid formulations were important predisposing factors for their infection . All M . pachydermatis strains were susceptible to amphotericin B, fluconazole and itraconazole but resistant against flucytosine . CONCLUSION: Molecular typing by random amplification of polymorphic DNA showed distinct banding profiles for each blood isolate . Since no epidemiological association among the strains could be shown, the reason for this cluster of nosocomial fungaemias remains unclear. Proc Natl Acad Sci U S A, 2001 May 8, 98(10), 5602 - 7 Epub 2001 May 01. Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification; Kurtzman AL et al.; Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis . We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1 . Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro . When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH(2) terminus of the homeodomain . In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli . Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1 . In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker . However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted . Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1 . Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability . These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation. Genes Dev, 2001 May 1, 15(9), 1140 - 51 Sharp, an inducible cofactor that integrates nuclear receptor repression and activation; Shi Y et al.; A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein) . SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2 . In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity . Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors . Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response. J Neurochem, 2001 May, 77(3), 935 - 42 The transcriptional regulator Yin Yang 1 activates the myelin PLP gene; Berndt JA et al.; Inauguration of the myelin program in developing oligodendrocytes requires the activation of those genes that encode the myelin proteins and the enzymes responsible for the synthesis and degradation of myelin lipids . An activator of the most abundantly expressed myelin protein, proteolipid protein (PLP), has been identified in a yeast one-hybrid system . The ubiquitously expressed zinc finger protein Yin Yang 1 (YY1) recognizes the myelin PLP promoter in vitro and in vivo . When overexpressed in an oligodendrocyte cell line, YY1 enhances transcription of the PLP promoter . A truncated version of YY1 that includes only the four zinc finger domains has little effect . The binding site for YY1 in the PLP promoter (site 3) fits the YY1 consensus site and DNA-protein complexes containing site 3 can be supershifted with an antibody directed against YY1 protein . Moreover, oligonucleotides with a mutated version of the PLP promoter site 3 are unable to bind to nuclear proteins or to compete for binding in a gel shift system . Finally, mutation of this site greatly reduces the activity of a 1-kb PLP promoter region in transfected glial cells . Our results suggest that PLP is a target gene for the transcriptional regulator YY1 . This versatile transcription factor and nuclear matrix protein may boost transcription of the PLP gene to meet the demands of actively myelinating oligodendrocytes. J Neurochem, 2001 May, 77(3), 929 - 34 Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations; Kawamata H et al.; alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease . Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease . Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies . In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique . We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1 . A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1 . The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1 . However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1. Int J Biochem Cell Biol, 2001 May, 33(5), 531 - 40 Identification of glycogen phosphorylase and creatine kinase as calpain substrates in skeletal muscle; Purintrapiban J et al.; The goal of this study was to identify calpain substrates in muscle cells . Our hypothesis was that the yeast two-hybrid method could be used to identify novel calpain substrates . To accomplish this, native mu- and m-calpains, as well as a variety of calpain DNA fragments, were expressed in yeast cells and used to screen for binding proteins in a human skeletal muscle cDNA library . Calpain constructs that were used in the screening process included native mu- and m-calpains, a dominant negative (DN) m-calpain (i.e . active site modified), N-terminal truncated DN m-calpain (i.e . autolyzed DN-m-calpain) and, finally, an N- and C-terminal truncated m-calpain (i.e . autolyzed DN-m-calpain lacking a calcium-binding domain) . Yeast cells were transformed using yeast two-hybrid expression vectors containing the different calpain constructs as "baits" . Beta-galactosidase activity was assayed as an index of interaction between calpain and its potential target proteins . From this analysis, four clones (Ca2+-ATPase, novel nebulin-related protein (N-RAP), creatine kinase and glycogen phosphorylase) were recovered . Two of these, creatine kinase and glycogen phosphorylase, were selected for further study . In in-vitro assays, calpain was able to partially digest both proteins, suggesting that both creatine kinase and glycogen phosphorylase are natural calpain substrates. Int J Biochem Cell Biol, 2001 May, 33(5), 483 - 90 Purification and characterization of a potent homodimeric guanine-specific ribonuclease from fresh mushroom (Pleurotus tuber-regium) sclerotia; Wang HX et al.; From the fresh sclerotia of the mushroom Pleurotus tuber-regium, a potent homodimeric ribonuclease exhibiting a molecular weight of 29 kDa in FPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was isolated . The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel, CM-cellulose and Mono S . It manifested strong ribonucleolytic activity toward Poly G, slight activity toward Poly U and Poly A, and minimal activity toward Poly C . Its optimal pH was determined to be 6.5 when yeast transfer RNA was used as substrate . Its ribonucleolytic activity was resistant to heating at 100 degrees C for 30 min but was inhibited by a number of salts . The protein inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 0.09 nM . Three out of the four amino acid residues at the active site (positions 38-41) of P . ostreatus ribonuclease, YNNF, were also found at positions 17-20 in the P . tuber-regum RNase . However, unlike P . ostreatus RNase, no cysteine residues were detected in the N-terminal sequence. Prostaglandins, 2001 Apr, 64(1-4), 143 - 156 Sphingosine-1-Phosphate Phosphatases; Mandala SM; Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets . It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets . Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase) . Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells . By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme . Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family . Type 2 phosphatase activity is Mg(+)-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates . SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate . Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis. Biosci Biotechnol Biochem, 2001 Mar, 65(3), 627 - 33 Characterization and high-level production of D-amino acid oxidase in Candida boidinii; Yurimoto H et al.; D-Amino acid oxidase (DAO, EC 1.4.3.3) from a methylotrophic yeast, Candida boidinii, was produced at a high level under the control of the alcohol oxidase gene promoter in the original host . The enzyme was a peroxisomal and monomeric enzyme, and contained noncovalently-bound FAD as a cofactor . The enzyme was active toward several D-amino acids such as D-Ala, D-Met, and D-Ser . An alcohol oxidase-depleted strain (aod1delta) was found to be a more suitable host for DAO production than the wild-type strain . Several post-translational effects may be responsible for the improvement of the DAO productivity by the aod1delta strain . Finally, an aod1delta strain transformant having multi-copies of an expression plasmid on its chromosome could produce DAO amounting up to 30% of the total soluble proteins. Cell Biochem Biophys, 2000, 32 Spring, 9 - 19 Peroxisomal matrix protein import . Suppression of protein import defects in Hansenula polymorpha pex mutants by overproduction of the PTS1 receptor Pex5p; Kiel JA et al.; In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen . This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome . In addition, it describes recent data that indicate that, in specific pex mutants of the methylotrophic yeast Hansenula polymorpha, defects in matrix protein import can be (partly) rescued by overproduction of the receptor essential for import of these proteins . The implication of these results on the mechanisms of matrix protein import is discussed. Cell Biochem Biophys, 2000, 32 Spring, 187 - 204 Peroxisome proliferator-activated receptors, coactivators, and downstream targets; Qi C et al.; Peroxisomes in liver parenchymal cells proliferate in response to structurally diverse nonmutagenic compounds designated as peroxisome proliferators (PP) . Sustained induction of peroxisome proliferation and peroxisomal fatty acid beta-oxidation system in rats and mice leads to the development of liver tumors . Two mechanistic issues are important for consideration: elucidation of the upstream events responsible for the tissue and species specific induction of the characteristic pleiotropic responses by PPs; and delineation of the downstream events associated with peroxisome proliferation, and their role in the development of liver tumors in species that are sensitive to the induction of peroxisome proliferation . The induction of peroxisome proliferation is mediated by PP-activated receptor alpha (PPAR alpha), a member of a group of transcription factors that regulate the expression of genes associated with lipid metabolism and adipocyte differentiation . Three isotypes of this family of nuclear receptors, namely PPAR alpha, PPAR gamma, and PPAR delta (also called beta), have been identified as products of separate genes . Although PPAR alpha is responsible for the PP-induced pleiotropic responses, PPAR gamma seems to be involved in adipogenesis and differentiation, but the events associated with PPAR gamma do not directly involve peroxisomes and peroxisome proliferation . PPARs heterodimerize with 9-cis retinoic acid receptor (RXR), and bind to PP response element(s) (PPREs) on the target gene promoter to initiate inducible transcriptional activity . Tissue and species responses to PPs depend on pharmacokinetics, relative abundance of PPAR isotypes, nature of PPRE in the upstream regions of target genes, the extent of competition or cross-talk among nuclear transcription factors for PPAR heterodimerization partner retinoid X receptor and the modulating role of coactivators and corepressors on ligand-dependent transcription of PPARs . Using PPAR as bait in the yeast two-hybrid system, the authors recently cloned mouse steroid receptor coactivator-1 (SRC-1) and PPAR-binding protein (PBP), and identified them as PPAR coactivators . Both SRC-1 and PBP contain LXXLL signature motifs, considered necessary and sufficient for the binding of coactivators to nuclear receptors . A multifaceted approach, which includes the identification of additional coactivators that may be responsible for cell specific transcriptional activation of PPAR-mediated target genes, and generation of genetically modified animals (transgenic and gene disrupted), will be necessary to gain more insight into the upstream and downstream targets responsible for the induction of early and delayed PP-induced pleiotropic responses . In this context, it is important to note that mice deficient in fatty acyl-CoA oxidase, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system, revealed that this enzyme is indispensable for the physiological regulation of PPAR alpha, and the absence of this enzyme leads to sustained transcriptional activation of genes regulated by this receptor. Cell Biochem Biophys, 2000, 32 Spring, 155 - 64 Peroxisome biogenesis and molecular defects in peroxisome assembly disorders; Fujiki Y et al.; Peroxisome assembly in mammals requires more than 14 genes . So far, we have isolated seven complementation groups (CGs) of peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24/ZP107, ZP92, ZP105/ZP139, ZP109, ZP110, ZP114 . Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible for peroxisome biogenesis disorders (PBD) such as Zellweger syndrome, of CG-F (the same as CG-X in U.S.A.) and CG-C (the same as CG-IV), respectively . Pex2p is a RING zinc finger membrane protein of peroxisomes and Pex6p is a member of the AAA ATPase family . We likewise isolated PEX12 encoding a peroxisomal integral membrane protein in the RING family, by functional complementation of ZP109, demonstrating PEX12 to be responsible for CG-III PBD . We also cloned PEX1 by screening of human liver cDNA library, using ZP107 . PEX1 mutation was delineated to be the genetic cause of PBD in the most highest incidence group, CG-E (the same as CG-I) . Moreover, we recently found that Pex5p is involved in transport of not only PTS1- but also PTS2-protein, distinct from yeast Pex5p, using PEX5-defective ZP105 and ZP139 . Thus, CHO cell mutants defective in peroxisome biogenesis are indeed shown to be very useful for the studies of peroxisome assembly and delineating pathogenic genes in PBD . Furthermore, we have isolated novel CGs of CHO mutants, ZP119 and ZP126. Cell Biochem Biophys, 2000, 32 Spring, 107 - 16 New aspects of sterol carrier protein 2 (nonspecific lipid-transfer protein) in fusion proteins and in peroxisomes; Bun-ya M et al.; Sterol carrier protein 2 (SCP2) is a 13-kDa peroxisomal protein, identical to nonspecific lipid-transfer protein, and stimulates various steps of cholesterol metabolism in vitro . Although the name is reminiscent of acyl carrier protein, which is involved in fatty acid synthesis, SCP2 does not bind to lipids specifically or stoichiometrically . This protein is expressed either as a small precursor or as a large fusion (termed SCPx) that carries at its C-terminal the complete sequence of SCP2 . SCPx exhibits 3-oxoacyl-CoA thiolase activity, as well as sterol-carrier and lipid-transfer activities . The N- and C-terminal parts of SCPx are similar to the nematode protein P-44 and the yeast protein PXP-18, respectively . P-44, which has no SCP2 sequence, thiolytically cleaved the side chain of bile acid intermediate at a rate comparable to that of SCPx . This, together with the properties of other fusions with SCP2-like sequence, suggests that the SCP2 part of SCPx does not play a direct role in thiolase reaction . PXP-18, located predominantly inside peroxisomes, is similar to SCP2 in primary structure and lipid-transfer activity, and protects peroxisomal acyl-CoA oxidase from thermal denaturation . PXP-18 dimerized at a high temperature, formed an equimolar complex with the oxidase subunit, and released the active enzyme from the complex when the temperature went down . This article attempts to gain insight into the role of SCP2, and to present a model in which PXP-18, a member of the SCP2 family, functions as a molecular chaperone in peroxisomes. Cochrane Database Syst Rev . 2001;(1):CD002845. Oral versus intra-vaginal imidazole and triazole anti-fungal treatment of uncomplicated vulvovaginal candidiasis (thrush); Watson MC et al.; BACKGROUND: Anti-fungals are available for oral and intra-vaginal treatment of vulvovaginal candidiasis (thrush) . OBJECTIVES: The primary objective of this review was to assess the relative effectiveness of oral versus intra-vaginal anti-fungals for the treatment of uncomplicated vulvovaginal candidiasis . The secondary objectives of the review were to assess the cost-effectiveness, safety and patient preference of oral versus intra-vaginal anti-fungals . SEARCH STRATEGY: The following sources were searched: The Cochrane Library (Issue 4, 1999), MEDLINE (January 1985 to May 2000), EMBASE (January 1980 to January 2000) and the Cochrane Collaboration Sexually Transmitted Disease Group Specialised Register of Controlled Trials . The reference lists of retrieved articles were reviewed manually . The manufacturers of anti-fungals available in the UK were contacted . SELECTION CRITERIA: ~Bullet~Randomised controlled trials published in any language . ~Bullet~Trials had to compare at least one oral anti-fungal with one intra-vaginal anti-fungal . ~Bullet~Women (aged 16 years or over) with uncomplicated vulvovaginal candidiasis . ~Bullet~The diagnosis of vulvovaginal candidiasis to be made mycologically (i.e . a positive culture and / or microscopy for yeast) . ~Bullet~Trials were excluded if they solely involved subjects who were HIV positive, immunocompromised, pregnant, breastfeeding or diabetic . ~Bullet~The primary outcome measure was clinical cure . DATA COLLECTION AND ANALYSIS: Duplicate scrutiny was performed of the titles and abstracts of the electronic search results . Full article formats of all selected abstracts were retrieved and independently assessed by two reviewers . Independent duplicate abstraction was performed by four reviewers . Disagreements regarding trial inclusion or data abstraction were resolved by discussion between the reviewers . Odds ratios were pooled using the random effects model . Chi-squared tests with a p-value of less than 0.1 indicated heterogeneity in the results . MAIN RESULTS: Seventeen trials are included in the review, reporting 19 oral versus intra-vaginal anti-fungal comparisons . No statistically significant differences were shown between oral and intra-vaginal anti-fungal treatment for clinical cure at short term (OR 1.00 (95% CI, 0.72 to 1.40)) and long term (OR 1.03 (95% CI, 0.72 to 1.49)) follow-up . No statistically significant differences for mycological cure were observed between oral and intra-vaginal treatment at short term (OR 1.20(95% CI, 0.87 to 1.65)) or long term follow-up (OR 1.30 (95% CI, 0.99 to 1.71)) . Two trials each reported one withdrawal from treatment due to an adverse reaction . Treatment preference data were poorly reported . REVIEWER'S CONCLUSIONS: No differences exist in terms of the relative effectiveness (measured as clinical and mycological cure) of anti-fungals administered by the oral and intra-vaginal routes for the treatment of uncomplicated vaginal candidiasis . No definitive conclusion can be made regarding the relative safety of oral and intra-vaginal anti-fungals for uncomplicated vaginal candidiasis . The oral route of administration is the preferred route for anti-fungals for the treatment of vulvovaginal candidiasis . The decision to prescribe or recommend the purchase of an anti-fungal for oral or intra-vaginal administration should take into consideration: safety, cost and treatment preference . Unless there is a previous history of adverse reaction to one route of administration or contraindications: if women are purchasing their own treatment, they should be given full information about the characteristics and costs of treatment to make their own decision . If health services are paying the treatment cost, decision-makers should consider whether the higher cost of oral anti-fungal administration is worth the gain in convenience, if this is the patient's preference. J Cell Sci, 2001 May, 114(Pt 10), 1875 - 82 Nuclear dispositions of subtelomeric and pericentromeric chromosomal domains during meiosis in asynaptic mutants of rye (Secale cereale L.); Mikhailova EI et al.; The nuclear dispositions of subtelomeric and pericentromeric domains in pollen mother cells (PMCs) were tracked during meiosis in wildtype and two asynaptic mutants of rye (Secale cereale L.) by means of fluorescence in situ hybridization (FISH) . Homozygotes for sy1 and sy9 non-allelic mutations form axial elements during leptotene of male meiosis, but fail to form synaptonemal complexes . Consequently, recombination is severely impaired, and high univalency is observed at metaphase I . Simultaneous FISH with pSc200 subtelomeric tandem repeat and CCS1 centromeric sequence revealed that at pre-meiotic interphase the two domains are in a bipolar Rabl orientation in both the PMCs and tapetal cells . At the onset of meiotic prophase, the subtelomeric regions in PMCs of wildtype and sy9 cluster into a typical bouquet conformation . The timing of this event in rye is comparable with that in wheat, and is earlier than that observed in other organisms, such as maize, yeast and mammals . This arrangement is retained until later in leptotene and zygotene when the pericentromeric domains disperse and the subtelomeric clusters fragment . The mutant phenotype of sy9 manifests itself during leptotene to zygotene, when the pericentromeric regions become distinctly more distended than in wildtype, and largely fail to pair during zygotene . This indicates that difference in the nature or timing of chromosome condensation in this region is the cause or consequence of asynapsis . By contrast, sy1 fails to form comparable aggregates of subtelomeric regions at leptotene in only half of the nuclei studied . Instead, two to five aggregates are formed that fail to disperse at later stages of meiotic prophase . In addition, the pericentromeric regions disperse prematurely at leptotene and do not associate in pairs at any subsequent stage . It is supposed that the sy1 mutation could disrupt the nuclear disposition of centromeres and telomeres at the end of pre-meiotic interphase, which could cause, or contribute to, its asynaptic phenotype. Biochemistry, 2001 Feb 20, 40(7), 2186 - 93 Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c . Optical multichannel detection; Szundi I et al.; Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye . Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase . Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm . On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved . A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined . The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1) . A significant fraction of the triplet returns back to the ground state on a similar time scale . The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c . Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1) . On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized . The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism. Mol Endocrinol, 2001 May, 15(5), 681 - 94 Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules; Vitt UA et al.; The cystine knot three-dimensional structure is found in many extracellular molecules and is conserved among divergent species . The identification of proteins with a cystine knot structure is difficult by commonly used pairwise alignments because the sequence homology among these proteins is low . Taking advantage of complete genome sequences in diverse organisms, we used a complementary approach of pattern searches and pairwise alignments to screen the predicted protein sequences of five model species (human, fly, worm, slime mold, and yeast) and retrieved proteins with low sequence homology but containing a typical cystine knot signature . Sequence comparison between proteins known to have a cystine knot three-dimensional structure (transforming growth factor-beta, glycoprotein hormone, and platelet-derived growth factor subfamily members) identified new crucial amino acid residues (two hydrophilic amino acid residues flanking cysteine 5 of the cystine knot) . In addition to the well known members of the cystine knot superfamily, novel subfamilies of proteins (mucins, norrie disease protein, von Willebrand factor, bone morphogenetic protein antagonists, and slit-like proteins) were identified as putative cystine knot-containing proteins . Phylogenetic analysis revealed the ancient evolution of these proteins and the relationship between hormones {e.g . transforming growth factor-beta (TGFbeta)} and extracellular matrix proteins (e.g . mucins) . They are absent in the unicellular yeast genome but present in nematode, fly, and higher species, indicating that the cystine knot structure evolved in extracellular signaling molecules of multicellular organisms . All data retrieved by this study can be viewed at http://hormone.stanford.edu/. DNA Seq, 2000, 11(5), 405 - 17 cDNA sequence and genomic structure of the rat RET proto-oncogene; Matera I et al.; The RET proto-oncogene, a member of the Receptor Tyrosine Kinase family, plays a crucial role during the development of the excretory system and the enteric nervous system, as demonstrated by in vivo animal studies and by its involvement in the pathogenesis of several human neurocristopathies like Hirschsprung disease and Multiple Endocrine Neoplasia type 2 . Using a multistep RT-PCR approach we have isolated and sequenced the cDNA of the whole rat RET proto-oncogene, reporting the deduced amino acid sequence in comparison with the human and mouse counterparts . Moreover, two different isoforms (RET9 and RET51) have been confirmed in the rat, while a third RET isoform demonstrated in human (RET43) has not resulted to be conserved in this species . Finally, we have determined the genomic structure of the rat RET proto-oncogene comparing the exon-intron boundaries and intron sizes with the known structure of the human homologous gene . Our findings will facilitate the molecular study of appropriate rat models of RET related human diseases. Biochem Biophys Res Commun, 2001 May 4, 283(2), 430 - 6 Expression of p40/Epstein-Barr virus nuclear antigen 1 binding protein 2; Henning D et al.; Nucleolar protein p40/EBP2 is a proliferation-associated antigen that interacts with Epstein-Barr virus nuclear antigen 1 (EBNA1) to maintain the Epstein-Barr virus (EBV) episomes . The yeast p40/EBP2 functions in the processing of 27S-A into 27S-B ribosomal RNA . The present study reports high evolutionary conservation of the cDNA-derived amino acid sequences of p40/EBP2 from frog, chicken, pig, rat, mouse, bovine, and human . p40/EBP2 is ubiquitously expressed in human tissues . It is highly expressed in myelogenous leukemia K-562 compared to other cell lines tested . The human p40/EBP2 gene is located in chromosome 1 with nine exons and eight introns . The minimal promoter region resides 300 nucleotides upstream of a putative ATG initiation codon preceded by a pyrimidine-rich region . These two regions contain eight Sp1 and four c-Ets-1 putative binding sites . Analysis of the p40/EBP2 gene and its promoter region will facilitate studies on the regulation of its expression in EBV-infected and noninfected cells. Nihon Shinkei Seishin Yakurigaku Zasshi, 2000 Nov, 20(5), 203 - 12 {BMAL1 and circadian rhythm}; Ikeda M; hBmal1 cDNA has been cloned from a human brain cDNA library and revealed that the cDNA encoded a novel bHLH-PAS transcription factor . The rBmal1 mRNA expression was found not only at high levels in the suprachiasmatic nuclei (SCN), but also in the pyriform cortex, hippocampus, and cerebellum . Furthermore, this expression was found in the eyes, pineal body, and peripheral organs, such as the muscle, liver, and heart . Bmal1 mRNA levels reveal a circadian rhythm in the rat SCN, with the highest expression levels at ZT18 . The yeast two-hybrid screening have demonstrated that CLOCK forms a heterodimer with BMAL1 . CLOCK . BMAL1 heterodimer functions as a positive transcription factor through binding to the E-box in the promoter region of the mPer1 . Nuclear translocation is promoted by dimerization among PERs, or between PERs and CRYs, and the complexes inhibit the transcription of their own genes . This feedback loop is used in the clock output system driving the transcription of arginine vasopressin peptide and serotonin N-acetyl-transferase by binding the CLOCK-BMAL1 dimer to an E-box in their promoters. J Biol Chem, 2001 Aug 10, 276(32), 30031 - 5 Epub 2001 Apr 26. The Bloom's syndrome protein (BLM) interacts with MLH1 but is not required for DNA mismatch repair; Langland G et al.; Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers . Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations . BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family . To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase . The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction . Cell extracts deficient in BLM were competent for DNA mismatch repair . These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair. Cancer Res, 2001 May 1, 61(9), 3578 - 80 Loss of heterozygosity events impeding breast cancer metastasis contain the MTA1 gene; Martin MD et al.; Breast cancer mortality is seldom attributable to the primary tumor, but rather to the presence of systemic (metastatic) disease . Axillary lymph node dissection can identify the presence of metastatic breast cancer cells and serves as a marker for systemic disease . Previous work in our laboratory determined that rates of loss of heterozygosity (LOH) of a 1.6-Mb region of chromosome 14q 31.2 is much higher in axillary lymph node-negative primary breast tumors than in axillary lymph node-positive primary breast tumors (P . O'Connell et al., J . NATL: Cancer INST:, 91: 1391-1397, 1999.) . This unusual observation suggests that, whereas the LOH of this region promotes primary breast cancer formation, some gene(s) mapping to this 1.6-Mb region is rate-limiting for breast cancer metastasis . Thus, if primary breast cancers delete this region, their ability to metastasize decreases . To identify this gene(s), we have physically mapped this area of chromosome 14q, confirmed the position of two known genes and 13 other expressed sequence tags into this 1.6-Mb region . One of these, the metastasis-associated 1 (MTA1) gene, previously identified as a metastasis-promoting gene (Y . Toh et al., J . BIOL: CHEM:, 269: 22958-22963, 1994.), mapped to the center of our 1.6-Mb target region . Thus, MTA1 represents a strong candidate for this breast cancer metastasis-promoting gene. Int J Biol Macromol, 2001 Jun 12, 28(5), 343 - 9 Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, alpha-chymotrypsinogen A, and alcohol dehydrogenase; Matsue S et al.; Effects of water activity (aW) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and alpha-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives . With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change . The parameter alpha, determined from the activity coefficient of water, representing the deviation of aW from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and alpha-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant aW . Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested. J Anim Sci, 2001 Apr, 79(4), 956 - 66 Prolonged feeding of high dietary levels of organic and inorganic selenium to gilts from 25 kg body weight through one parity; Kim YY et al.; An experiment evaluated the selenosis effects from feeding high dietary Se levels of organic or inorganic Se sources to growing gilts with the dietary treatments continued through a reproductive cycle . A total of 88 gilts were allotted at 25 kg BW to two replicates in a 2 x 4 factorial arrangement in a randomized complete block design . Inorganic Se (sodium selenite) or organic (Se-enriched yeast) Se were added to diets at 0.3, 3, 7, or 10 ppm Se . At 105 kg BW, four gilts per treatment were killed and livers collected for Se analysis . At 8 mo of age, three gilts from each treatment group were bred and fed their treatment diet, with subsequent reproductive performance and selenosis effects evaluated . Serum collected at various intervals in gilts, sows, and progeny measured glutathione peroxidase activity and Se concentrations . Sow colostrum and milk was analyzed for their Se concentrations . Three pigs per treatment were killed before colostrum consumption and at weaning (14 d) and tissue collected for Se analysis . Gilt gains (P < 0.01) and feed intakes (P < 0.05) declined during the grower period as dietary Se level increased for both Se sources . Serum and liver Se concentrations increased as dietary Se level increased and was higher when organic Se was fed (P < 0.01) . Sows fed dietary Se levels at > 7 ppm had lower gestation weights (P < 0.05) and lower lactation feed intakes (P < 0.05) . As Se level increased, sows fed organic Se had a lower number of live pigs born (P < 0.05) and weaned fewer pigs (P < 0.05) with lower litter gains (P < 0.05) than did sows fed inorganic Se . Colostrum and milk Se concentrations increased as dietary Se levels increased particularly when organic Se was fed (P < 0.01) . Neonatal and weanling pig tissue Se and serum Se concentrations increased as dietary Se level increased and when organic Se was fed, resulting in interaction responses (P < 0.01) . Pigs nursing sows fed > 7 ppm inorganic Se had hoof separation and alopecia, with the severity being greater when sows were fed the inorganic Se source . These results suggest that both the organic and inorganic Se sources were toxic when fed at 7 to 10 ppm for a prolonged period, but organic Se seemed to express the selenotic effects more on reproductive performance, whereas inorganic Se was more detrimental during lactation. J Anim Sci, 2001 Apr, 79(4), 949 - 55 Effect of dietary selenium source, level, and pig hair color on various selenium indices; Kim YY et al.; The first experiment evaluated the effects of feeding various levels of Se, two Se sources, and hair color on selenosis responses in growing-finishing pigs . The study conducted in two replicates was a 2 x 6 x 2 factorial arrangement in a split-plot design . Sodium selenite and Se-enriched yeast added at 0.3, 1, 3, 5, 7, and 10 ppm Se served as the main plot and pig hair color as the subplot . A total of 96 crossbred pigs were allotted and fed their treatment diets for a 12-wk period . White and dark (red or black) hair samples were collected from the dorsal-midline at the 4-, 8-, and 12-wk periods from one pig of each hair color from each treatment pen . Lower pig weights (P < 0.10) and daily gains (P < 0.05) occurred as dietary Se level increased when pigs were fed either Se source . Selenosis responses were somewhat more severe, when the inorganic Se source was fed . Alopecia and hoof separation were encountered after the 8-wk period when pigs were fed inorganic rather than organic Se . Plasma Se increased as dietary level increased (P < 0.01), when organic Se was provided (P < 0.01), and was higher (P < 0.05) when pigs were white-haired . A time x hair color x dietary Se level interaction (P < 0.05) occurred, in which hair Se concentration was higher in dark- than in white-colored pigs and increased as dietary Se level increased as the experiment progressed . The correlation coefficient between dietary Se level and hair Se concentration averaged 0.90 (P < 0.01) . Cysteine was the amino acid in the highest concentration in hair, but this and other amino acids were not affected by Se level, Se source, or hair color . A second experiment was a 3 x 6 factorial arrangement in a split-plot design with three 9-mo-old gilts from each of the Yorkshire, Duroc, and Hampshire breeds to determine whether hair Se concentration differed by body location and breed . Hair samples were collected from the shoulder, back, rump, front-leg, belly, and hind-leg areas . Hair Se concentration was higher in red- and white-haired pigs and lower in black-haired gilts (P < 0.01) . Higher hair Se concentrations (P < 0.05) occurred from the lower than from the upper body areas . Our results suggest that selenosis occurs at dietary levels > 5 ppm and that white-haired pigs exhibit alopecia sooner than dark-haired pigs . No difference in hair Se concentration occurred when diets were < 1 ppm Se, but as dietary Se level increased dark-haired pigs retained more Se in their hair than white-haired pigs. J Anim Sci, 2001 Apr, 79(4), 942 - 8 Comparative effects of high dietary levels of organic and inorganic selenium on selenium toxicity of growing-finishing pigs; Kim YY et al.; This experiment evaluated the effect of high dietary Se levels using organic or inorganic Se on the selenosis responses in growing-finishing swine . A 2 x 4 factorial arrangement of treatments in a randomized complete block design was conducted in two replicates . Sodium selenite or Se-enriched yeast was added at 5, 10, 15, or 20 ppm Se to corn-soybean meal diets . A basal diet without added Se was a ninth treatment group . Ninety crossbred barrows initially averaging 24.7 kg BW were allotted at five pigs per pen . Pigs were bled at 3-wk intervals and plasma Se, glutathione peroxidase (GSH-Px) activity, glutamic oxalacetic transaminase (PGOT), hemoglobin, packed cell volume, and blood cell Se concentration were measured . After 12 wk, pigs were killed and various tissues and bile were collected for Se analyses . Pig body weights, daily gains, and feed intakes were similar for both Se sources when provided at < or = 5 ppm Se, but each measurement declined in a different manner for each Se source as the dietary Se level increased . The decline was more rapid when the inorganic rather than organic Se source was fed, resulting in interaction responses (P < 0.01) . Hair loss (alopecia) and separation of the hoof at the coronary band site occurred at > or = 10 ppm inorganic Se but at > or = 15 ppm organic Se level . Plasma GSH-Px activity increased (P < 0.01) when high dietary Se levels of either Se source was fed . Plasma and blood cell Se increased at each period as dietary Se level increased (P < 0.01) and was greater when organic Se was provided (P < 0.05) . Blood cell Se concentration reached a plateau when inorganic Se, but not when organic Se, was fed and increased as the experiment progressed . This resulted in a three-way interaction (P < 0.01) . Plasma GOT activity at the 12-wk period was elevated when inorganic Se was provided at > or = 15 ppm Se but not when organic Se was fed, resulting in an interaction (P < 0.05) . Tissue Se concentrations increased as dietary Se level increased and when organic Se was provided, resulting in interaction responses (P < 0.05) . Bile was a yellow color when the basal diet was fed but was dark brown at > 10 ppm inorganic Se and at 20 ppm when organic Se was provided . Bile Se increased as dietary Se level increased (P < 0.01) . These results suggest that dietary Se from inorganic or organic sources was toxic at > or = 5 ppm Se, but subsequent selenosis effects were more severe and occurred sooner when sodium selenite was the Se source. Virus Genes, 2001 Mar, 22(2), 159 - 65 The product of ORF III in cauliflower mosaic virus interacts with the viral coat protein through its C-terminal proline rich domain; Leclerc D et al.; Using the yeast two-hybrid system, we show that the ORF III product of cauliflower mosaic virus (pIII) interacts through its C-terminus with the viral coat protein . The last five amino acids of pIII were essential for the interaction and virus infectivity . Deletion of the last three amino acids or the mutation F129A decreased the strength of the interaction by 90% . We further show that pIII is closely associated with virus particles found in the inclusion bodies of infected plants but not in viral particles released from the inclusion bodies by urea treatment. Prostaglandins Other Lipid Mediat, 2001 Apr, 64(1-4), 143 - 56 Sphingosine-1-phosphate phosphatases; Mandala SM; Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets . It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets . Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase) . Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells . By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme . Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family . Type 2 phosphatase activity is Mg+-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates . SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate . Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis. Acta Pharmacol Sin, 2000 Mar, 21(3), 265 - 70 Nonhistone protein purified from porcine kidney acts as a suicide substrate inhibitor on furin-like enzyme; Fei H et al.; AIM: To search and purify a naturally occurring protein inhibitor of the furin-like enzyme from the porcine kidney . METHODS: Recombinant kexin, a furin-like enzyme, from the yeast secretion expression was used as a target enzyme . The inhibitor component was extracted and purified from the acetone powder of porcine kidney . The inhibitory activity was monitored using a fluorogenic peptide substrate Boc-Arg-Val-Arg-MCA at spectrofluorimeter . RESULTS: The purified inhibitor component is a basic protein with an isoelectric point over 9.5 . Its partial N-terminal sequence of 22 residues was determined, showing a high homology with nonhistone chromosomal protein HMG-17 in which there are four sites composed of dibasic residues, susceptible to be cleaved by the furin-like enzyme . This nonhistone protein could strongly compete with the fluorogenic substrate . However, this nonhistone protein would be degraded as a substrate by kexin if it was incubated with the enzyme for long time before adding the fluorogenic substrate, and subsequently lost its temporary inhibitory activity . CONCLUSION: The nonhistone protein isolated from the porcine kidney functioned as a suicide substrate inhibitor for the furin-like enzyme. J Biol Chem, 2001 Aug 3, 276(31), 29410 - 9 Epub 2001 Apr 25. Kidney androgen-regulated protein interacts with cyclophilin B and reduces cyclosporine A-mediated toxicity in proximal tubule cells; Cebrian C et al.; The gene for kidney androgen-regulated protein (KAP) is the most abundant and specific gene expressed in mouse kidney proximal tubule cells, where it is tightly regulated by steroid and thyroid hormones in different tubule segments . Despite the cell-specific expression, strict regulatory mechanisms, and relative abundance, nothing is known of the function of its encoded protein, which does not exhibit known structural or functional domains, or homologies with other sequences in the data bases . We raised monoclonal antibodies against KAP, which specifically recognize a protein with an apparent molecular mass of 20 kDa in crude kidney homogenates, the distribution and regulation of which parallel that of its mRNA . To gain insight into its function, we performed a yeast two hybrid screen and determined that KAP specifically interacts with cyclophilin B . Furthermore, cyclosporine A (CsA)-treated mice exhibited a significant decrease in KAP levels, and tetracycline-controlled overexpression of KAP in stably transfected proximal tubule cells significantly decreased the toxic effects of CsA . Taken together, these results indicate a functional relationship among KAP-, cyclophilin B-, and CsA-mediated nephrotoxicity and suggest an important role of KAP in renal physiology, providing new data on the molecular mechanisms implied in the toxic effects of CsA. J Biol Chem, 2001 Jul 20, 276(29), 27480 - 7 Epub 2001 Apr 25. Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport; Zhang T et al.; The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion . The functional role of its mammalian homologue (Ykt6) has not been established . Using antibodies specific for mammalian Ykt6, it is revealed that it is found mainly in Golgi-enriched membranes . Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex . Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus . Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites . Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage . ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6 . In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport . Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies . It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex. Carcinogenesis, 2001 May, 22(5), 693 - 700 DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) induces apoptosis via caspase-9 in primary cultured rat hepatocytes; Shiotani B et al.; The mechanism of cytotoxicity induced by the DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido{4,3-b} indole (Trp-P-1) was investigated in primary cultured rat hepatocytes . Cytotoxicity was caused by intact Trp-P-1 and not by metabolically activated derivatives prepared using a recombinant yeast strain AH22/pAMR2 expressing rat cytochrome P450 1A1, and not by metabolically activated derivatives . We also found internucleosomal DNA fragmentation 6 h after treatment with 30 microM Trp-P-1, indicating that the cytotoxicity was due to the induction of apoptosis . After treatment with Trp-P-1, c-Myc protein level increased in a time-dependent manner and p53 protein also increased transiently with a subsequent increase in Bax protein level . This apoptotic pathway required the activation of caspase-9 as an initiator after leakage of cytochrome c into the cytosol from mitochondria and the activation of caspase-3 and -7 as executioners, but not caspase-1, -6 or -8 as measured using the corresponding peptide inhibitors and substrates or western blotting . The activated caspases in turn cleaved poly(ADP-ribose) polymerase as an intracellular substrate . Furthermore, we detected NUC18-like endonuclease activity during apoptosis induced by Trp-P-1 . These findings suggest that this apoptosis may have a role against heterocyclic amine-type carcinogens in normal cells. Thromb Res, 2001 Apr 15, 102(2), 177 - 85 The interaction of the calcium- and integrin-binding protein (CIBP) with the coagulation factor VIII; Fang X et al.; The gene encoding the C-terminal part of A1-domain of human blood coagulation factor VIII (FVIII), a 110-amino acid fragment from Ala(227) to Arg(336), namely A1(Delta1-226), was cloned and used as a 'bait' to screen a protein, which might interact with this region by using the yeast two-hybrid system . A gene coding for a related protein of FVIII named calcium- and integrin-binding protein (CIBP) was isolated from the normal human liver cDNA library . The results were confirmed by using the mammalian two-hybrid system and coimmunoprecipitation . The gene coding for CIBP was constructed by polymerase chain reaction (PCR) and then cotransfected with the B-domain-deleted FVIII gene into mammalian cell lines using the expression vector of FVIII for transient or stable expression . The culture supernatant was collected and analyzed both by enzyme-linked immunosorbent assay (ELISA) for FVIII antigen level and by one-stage method for procoagulant activity . Coexpressed with CIBP, the antigen level of FVIII in the mammalian cell line baby hamster kidney (BHK) cells increased up to about 170% and its bioactivity rose accordingly. Eur J Biochem, 2001 May, 268(9), 2725 - 32 N-Terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8; Ito K et al.; We have previously cloned cDNAs encoding the N-terminally extended class III human ubiquitin-conjugating enzymes (E2s), UBE2E2 and UBE2E3, the biological functions of which are not known . In this study, we performed yeast two-hybrid screening for protein(s) interacting with UBE2E2, and two RING-finger proteins, ARA54 and RNF8, were identified . Both ARA54, a ligand-dependent androgen receptor coactivator, and RNF8 interacted with class III E2s (UBE2E2, UbcH6, and UBE2E3), but not with other E2s (UbcH5, UbcH7, UbcH10, hCdc34, and hBendless) in the yeast two-hybrid assay . The use of various deletion mutants of UBE2E2 and RING-finger proteins and two RING point mutants, ARA54 C(220)S and RNF8 C(403)S, in which the RING structure is disrupted, showed that the UBC domain of UBE2E2 and the RING domain of these RING-finger proteins were involved in this association . Wild-type ARA54 and RNF8, expressed in insect Sf9 cells, catalyzed E2-dependent autoubiquitination in vitro, whereas the point mutated proteins showed markedly reduced activity . Ubiquitination of wild-type ARA54 and RNF8, expressed in COS-7 cells, was also observed, and a proteasome inhibitor, MG132, prevented the degradation of these wild-type proteins, but was much less effective in protecting the RING mutants . Transfection of COS-7 cells with a green fluorescent protein chimera showed that RNF8 was localized in the nucleus, and ARA54 in both the cytoplasm and nucleus . Our results suggest that ARA54 and RNF8 possibly act as Ub-ligases (E3) in the ubiquitination of certain nuclear protein(s). Eur J Biochem, 2001 May, 268(9), 2609 - 15 Carbohydrate binding properties of banana (Musa acuminata) lectin I . Novel recognition of internal alpha1,3-linked glucosyl residues; Mo H et al.; Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars . Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan) . The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran . Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans . This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin . It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch. Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 5258 - 63 Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A; Lin R et al.; We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors . Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes . The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments . Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A . In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes . Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane . These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors . Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton. Genes Chromosomes Cancer, 2001 Jun, 31(2), 125 - 33 Identification of recurrent chromosomal rearrangements and the unique relationship between low-level amplification and translocation in glioblastoma; Kubota H et al.; To elucidate the structural abnormalities and the relationship between chromosome structural disorders and DNA copy number aberrations in tumor cells, we applied the techniques of spectral karyotyping (SKY), comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH), using yeast artificial chromosome (YAC) probes for nine human glioblastoma cell lines . One striking finding was that independently derived cell lines had the same recurrent marker chromosomes . Seven recurrent chromosomes were detected by these cytogenetic methods . In particular, cell lines U251, SNB-19, and U373-MG showed very similar karyotypes . It is also interesting that regions of DNA amplification were found translocated and/or inserted at a high rate (91.7%) . In all, there were 12 amplified loci in five of the nine cell lines . These amplified chromosomal bands were scattered on the chromosomes, including the normal chromosome, with one exception (7q32-qter in U373-MG) . FISH with YAC clones mapping to these chromosomal regions as DNA probes often showed DNA probe signals not only at original chromosomal sites but also in translocated or inserted segments . This form of DNA amplification was characterized by low-level increases (four- to 10-fold) and by translocation or insertion of the relevant chromosomal locus . These studies shed light on typical derivative chromosomes and the relationship between DNA amplification and chromosomal translocation in glioblastoma . Biol Reprod, 2001 May, 64(5), 1315 - 9 Cloning and functional expression of an E box-binding protein from rat granulosa cells; Yamada K et al.; Ovarian granulosa cells undergo cell growth and cytodifferentiation during follicular maturation . In a number of tissues, the gene expression that is responsible for the cytodifferentiation is largely dependent on E box(es) located upstream of the responsible genes . In this study, we report on the cloning of cDNA(s) encoding E box (5'-CACGTG-3')-binding protein from a rat granulosa cell cDNA library using a yeast one-hybrid system . When multiple E box sequences were used as target, we obtained a positive clone that encodes the rat homologue of upstream stimulatory factor 2 (USF2) . An analysis of the nucleotide sequence and its deduced amino acid sequence reveals that rat USF2 protein consists of 346 amino acid residues and belongs to the basic helix-loop-helix/leucine zipper protein family . Northern blot analysis shows that rat USF2 mRNA exists as multiple forms between 1.6 and 2.2 kilobases . The size of the cloned insert was identical to that of the transcript of maximal length . Electrophoretic mobility shift assays showed that in vitro-translated rat USF2 specifically binds to the E box . In addition, cotransfection experiments with luciferase-reporter constructs in HepG2 cells reveal that the overexpression of rat USF2 leads to an increase of luciferase activity in the E box sequence-dependent manner . Thus, we report molecular cloning, expression, and functional characterization of full-length rat USF2 cDNA. Genes Cells, 2001 Apr, 6(4), 303 - 12 Regulation of gene expression, cellular localization, and in vivo function of Caenorhabditis elegans DNA topoisomerase I; Lee MH et al.; BACKGROUND: DNA topoisomerase I is dispensable in yeast, but is essential during the embryogenesis of Drosophila and mouse . In order to determine functions of the enzyme in the development of Caenorhabditis elegans, phenotypes resulting from the deficiency were observed and correlated with the expression of the gene . RESULTS: The transcriptional regulation of the C . elegans DNA topoisomerase I gene was investigated by mRNA localization and reporter gene expression in C . elegans . The mRNA was expressed in the gonad and in the early embryos, followed by a rapid decrease in its level during the late embryonic stage . A reporter gene expression induced by the 5'-upstream DNA sequence appeared at the comma stage of embryos, continued through the L1 larval stage, and began to decrease gradually afterwards . The DNA topoisomerase I protein was immuno-localized in the nuclei of meiotic gonad cells and interphase embryonic cells, and unexpectedly in centrosomes of mitotic embryonic cells . Double-stranded RNA interference of DNA topoisomerase I gene expression resulted in pleiotropic phenotypes showing abnormal gonadogenesis, oocyte development and embryogenesis . CONCLUSION: These phenotypes, along with expressional regulations, demonstrate that DNA topoisomerase I plays important roles in rapidly growing germ cells and embryonic cells. Genomics, 2001 Apr 15, 73(2), 238 - 41 Human ITCH is a coregulator of the hematopoietic transcription factor NF-E2; Chen X et al.; We have cloned a new protein that interacts with the hematopoietic DNA-binding transcription factor, p45/NF-E2, by screening a human erythroleukemia cell cDNA library with the yeast two-hybrid approach . Predicted peptide sequence and chromosomal mapping identified the cloned molecule to be the product of the human ortholog of the mouse Itch gene, which has been implicated previously in the regulation of growth and differentiation of erythroid and lymphoid cells . Transfection experiments indicate that this human ITCH protein can act as a transcriptional corepressor of p45/NF-E2 . Our data provide novel insights into the functional roles of the mammalian ITCH proteins in the development of hematopoietic cell lineages . J Parasitol, 2001 Apr, 87(2), 457 - 60 Effect of iron on the virulence of Trichomonas vaginalis; Ryu JS et al.; The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice . Iron-supplemented and iron-depleted Diamond's trypticase-yeast extract-maltose (TYM) media were prepared by adding 360 microM of ferrous sulfate and 100 microM of 2,2'-dipyridyl . Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology . In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium . In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2'-dipyridyl the virulence of T . vaginalis is reduced. Dig Dis Sci, 2001 Mar, 46(3), 661 - 7 Lamina propria lymphocytes produce interferon-gamma and develop suppressor activity in response to lactoglobulin; Ebert EC et al.; This study examines the in vitro response of human lamina propria lymphocytes (LPLs) to food antigens . LPLs were obtained from jejunum of healthy individuals undergoing gastric bypass operations for morbid obesity . Proliferation was assayed by {3H}thymidine incorporation and cytokine production by ELISA . LPLs proliferated in response to keyhole limpet hemocyanin, ovalbumin, lactoglobulin, and phytohemagglutinin (PHA), but not to yeast . The responses to lactoglobulin and PHA were inhibited by the CTLA4/Fc chimera and by MAbs against CD2, CD58, CD80, and CD86, indicating stimulation of CD28+ LPLs with antigen-presenting cells through activation of the CD2 pathway . Besides producing IL-2, IL-10, and TNF-alpha, LPLs synthesized large amounts of IFN-gamma (100 ng/ml) with lactoglobulin, a process dependent upon CD80/CD86, CD40/CD40L, and IL-12 . After a three-day culture with lactoglobulin, ovalbumin, or PHA, LPLs developed suppressor activity that reduced proliferation of naive LPLs to these same stimuli . In summary, LPLs first respond to lactoglobulin by proliferation and IFN-gamma production, then by development of antigen nonspecific suppression. Eur J Clin Pharmacol, 2001 Mar, 56(12), 881 - 8 Cytochrome P450 isoforms involved in melatonin metabolism in human liver microsomes; Facciola G et al.; OBJECTIVE: The present study was carried out to identify the cytochrome P450 enzyme(s) involved in the 6-hydroxylation and O-demethylation of melatonin . METHODS: The formation kinetics of 6-hydroxymelatonin and N-acetylserotonin were determined using human liver microsomes and cDNA yeast-expressed human enzymes (CYP1A2, 2C9 and 2C19) over the substrate concentration range 1-1000 microM . Selective inhibitors and substrates of various cytochrome P450 enzymes were also employed . RESULTS: Fluvoxamine was a potent inhibitor of 6-hydroxymelatonin formation, giving 50 +/- 5% and 69 +/- 9% inhibition at concentrations of 1 microM and 10 microM, respectively, after incubation with 50 microM melatonin . Furafylline, sulphaphenazole and omeprazole used at low and high concentrations substantially inhibited both metabolic pathways . cDNA yeast-expressed CYP1A2, CYP2C9 and CYP2C19 catalysed the formation of the two metabolites, confirming the data obtained with specific inhibitors and substrates . CONCLUSIONS: Our results strongly suggest that 6-hydroxylation, the main metabolic pathway of melatonin, is mediated mainly, but not exclusively, by CYP1A2, the high-affinity enzyme involved in melatonin metabolism, confirming the observation that a single oral dose of fluvoxamine increases nocturnal serum melatonin levels in healthy subjects . Furthermore, the results indicate that there is a potential for interaction with drugs metabolised by CYP1A2 both at physiological levels and after oral administration of melatonin, while CYP2C19 and CYP2C9 are assumed to be less important. J Biol Chem, 2001 Jun 29, 276(26), 24232 - 41 Epub 2001 Apr 20. Dysbindin, a novel coiled-coil-containing protein that interacts with the dystrobrevins in muscle and brain; Benson MA et al.; The dystrophin-associated protein complex (DPC) is required for the maintenance of muscle integrity during the mechanical stresses of contraction and relaxation . In addition to providing a membrane scaffold, members of the DPC such as the alpha-dystrobrevin protein family are thought to play an important role in intracellular signal transduction . To gain additional insights into the function of the DPC, we performed a yeast two-hybrid screen for dystrobrevin-interacting proteins . Here we describe the identification of a dysbindin, a novel dystrobrevin-binding protein . Dysbindin is an evolutionary conserved 40-kDa coiled-coil-containing protein that binds to alpha- and beta-dystrobrevin in muscle and brain . Dystrophin and alpha-dystrobrevin are co-immunoprecipitated with dysbindin, indicating that dysbindin is DPC-associated in muscle . Dysbindin co-localizes with alpha-dystrobrevin at the sarcolemma and is up-regulated in dystrophin-deficient muscle . In the brain, dysbindin is found primarily in axon bundles and especially in certain axon terminals, notably mossy fiber synaptic terminals in the cerebellum and hippocampus . These findings have implications for the molecular pathology of Duchenne muscular dystrophy and may provide an alternative route for anchoring dystrobrevin and the DPC to the muscle membrane. Oncogene, 2001 Feb 8, 20(6), 726 - 38 A translationally regulated Tousled kinase phosphorylates histone H3 and confers radioresistance when overexpressed; Li Y et al.; The gene Tousled of Arabidopsis Thaliana encodes a protein kinase which, when mutated, results in abnormal flower development . From a library of mRNAs that are translationally upregulated by overexpression of the translation initiation factor 4E, we identified a mammalian Tousled Like kinase (TLK1B) . The human TLK1B mRNA contains a 5'UTR 1088-nt-long with two upstream AUG codons, and was found to be very inhibitory for translation . The TLK1B protein localizes almost exclusively to the nuclei . TLK1B overexpression in mammalian cells rendered them more resistant to ionizing radiation (IR) . Purified TLK1B phosphorylated histone H3 at S(10) with high specificity both in a mix of core histones and in isolated chromatin, suggesting that histone H3 is a physiological substrate for TLK1B . Moreover, overexpression of TLK1B in transfected cells resulted in a higher degree of H3 phosphorylation . Expression of TLK1B in a yeast strain that harbors a temperature-sensitive mutation of the major H3 kinase, Ipl1, complemented the growth defect; restored normal levels of histone H3 phosphorylation; and increased their resistance to IR . Phosphorylation of H3 has been linked to the activation of the immediate-early genes upon mitogenic stimulation, and to chromatin condensation during mitotic/meiotic events . A possible role for TLK1B in radioprotection is discussed. Oncogene, 2001 Jan 25, 20(4), 484 - 9 p15(PAF), a novel PCNA associated factor with increased expression in tumor tissues; Yu P et al.; Proliferating cell nuclear antigen (PCNA) is an essential protein in both DNA replication and DNA damage repair . A novel 15 kD protein, p15(PAF), was identified as a PCNA-associated factor in a yeast two-hybrid screen using PCNA as the bait . p15(PAF) is localized primarily in the nucleus . p15(PAF) shares the conserved PCNA binding motif with several other PCNA binding proteins including CDK inhibitor p21 . Overexpression of p15(PAF) competes with p21-PCNA binding . Mutation of this motif in p15(PAF) abolished its PCNA-binding activity . Notably, p15(PAF) expression in several types of tumor tissues was significantly increased, especially in esophageal tumors . Like PCNA, p15(PAF) may possess prognostic significance in a broad array of human cancers. Oncogene, 2001 Jan 25, 20(4), 411 - 9 The ENL moiety of the childhood leukemia-associated MLL-ENL oncoprotein recruits human Polycomb 3; Garcia-Cuellar MP et al.; The translocation t(11;19) is frequently found in acute leukemia in infants . This event truncates the proto-oncogene MLL and fuses the 5' end of MLL in frame with the ENL gene . ENL contributes a crucial protein-protein interaction domain to the resulting oncoprotein MLL-ENL . Here we show by yeast two-hybrid assays, GST-pull-down experiments and in a far western blot analysis that this domain is necessary and sufficient to recruit a novel member of the human Polycomb protein family (hPc3) . hPc3 RNA was detected throughout the human hematopoietic system . Similar to other Polycomb proteins hPc3 acts as a transcriptional repressor . The ENL-hPc3 interaction was verified by mutual co-precipitation of the proteins from cell extracts . ENL and hPc3 tagged with fluorescent proteins co-localized in living cells in a nuclear dot pattern . An internal region of hPc3 was responsible for binding to ENL . Finally, hPc3 binds to the C-terminus of AF9, another common MLL fusion partner . The recruitment of a repressive function by ENL opens up a new insight into a possible mechanism of leukemogenesis by the fusion protein MLL-ENL. Oncogene, 2001 Jan 18, 20(3), 346 - 57 Identification of a novel interaction between integrin beta1 and 14-3-3beta; Han DC et al.; Integrins are cell surface receptors for extracellular matrix, which play important roles in a variety of biological processes . 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in regulation of various cellular functions . Here, we report identification of an interaction between the integrin beta1 cytoplasmic domain and 14-3-3beta by using the yeast two-hybrid screen . Like several other proteins, the integrin beta1 cytoplasmic domain associated with 14-3-3beta by a non-phosphoserine mechanism . The 14-3-3beta/integrin beta1 interaction was confirmed by in vitro binding assays as well as co-precipitation in vivo . Furthermore, we found that 14-3-3beta co-localized with integrin beta1 during the early stage of cell spreading on fibronectin, suggesting a potential role of the 14-3-3beta/integrin beta1 interaction in the regulation of cell adhesion . Using tetracycline-regulated expression system, we showed that overexpression of 14-3-3beta stimulated cell spreading and migration on fibronectin but not on poly-L-lysine . However, the induced expression of 14-3-3beta did not affect tyrosine phosphorylation of FAK or its substrates, p130(cas) and paxillin, suggesting that 14-3-3beta regulated integrin-mediated cell spreading and migration by FAK-independent mechanisms . Taken together, these results identify an interaction between integrin and 14-3-3 proteins and suggest a potentially novel cellular function for 14-3-3 proteins in the regulation of integrin-mediated cell adhesion and signaling events. Eur J Hum Genet, 2001 Apr, 9(4), 307 - 10 Saturating density of STSs (1/6 kb) in a 1.1 Mb region on 3q28-q29: a valuable resource for cloning of disease genes; Alexander C et al.; We have fine mapped 29 ESTs of Genemap'99 to YACs and radiation hybrids covering 8 cM of the chromosomal region of 3q28-q29 . Focusing on the genetic interval of approximately 1 Mb between markers D3S3669 and D3S3562 we established a sequence-ready PAC contig which covers the OPA1 locus containing the gene causing autosomal dominant optic atrophy (ADOA; OMIM*165500) . The fidelity of the contig was increased by the generation of 181 PAC end sequences, 84 of which resulted in PCR-able STSs . Sequence content evaluation of the PAC ends by BLAST analysis identified two novel ESTs localising to the OPA1 crucial interval. Eur J Hum Genet, 2001 Feb, 9(2), 121 - 9 A high-resolution integrated map spanning the SDHD gene at 11q23: a 1.1-Mb BAC contig, a partial transcript map and 15 new repeat polymorphisms in a tumour-suppressor region; Baysal BE et al.; Chromosomal region 11q22-q23 is a frequent target for deletion during the development of many solid tumour types, including breast, ovary, cervix, stomach, bladder carcinomas and melanoma . One of the most commonly deleted subregions contains the SDHD gene, which encodes the small subunit of cytochrome b (cybS) in mitochondrial complex II (succinate-ubiquinone oxidoreductase) . Germline mutations in SDHD cause hereditary paraganglioma type 1 (PGL1), and suggest a tumour suppressor role for cybS . We present a high-resolution physical map spanning SDHD, covered by 19 YACs and 20 BACs . An approximate 1.1-Mb gene-rich region around SDHD is spanned by a complete BAC contig . Twenty-six new STSs are developed from the BAC clone ends . In addition to the discovery and characterisation of 15 new simple tandem repeat polymorphisms, we provide integrated positional information for 33 ESTs and known genes, including KIAA1391, POU2AF1 (OBF1), PPP2R1B, CRYAB, HSPB2, DLAT, IL-18, PTPS, KIAA0781 and KAIA4591, which is mapped by NotI site cloning . We describe full-length transcript sequence for PPP2R1B, encoding the protein phosphatase 2A regulatory subunit A beta isoform . We also discover a processed pseudogene for USA-CYP, a cyclophilin associated with U4/U6 snRPNs, and a novel gene, DDP2, encoding a mitochondrial protein similar to the X-linked deafness-dystonia protein, which is juxtaposed 5'-to-5' to SDHD . This map will help assess this gene-rich region in PGL and in other common tumours. Cell Death Differ, 2001 Feb, 8(2), 137 - 43 Baculoviruses and apoptosis: the good, the bad, and the ugly; Clem RJ; Since 1991, when a baculovirus was first shown to inhibit apoptosis of its host insect cells, considerable contributions to our knowledge of apoptosis have arisen from the study of these viruses and the anti-apoptotic genes they encode . Baculovirus anti-apoptotic genes include p35, which encodes the most broadly acting caspase inhibitor protein known, and iap (inhibitor of apoptosis) genes, which were the first members of an evolutionarily conserved gene family involved in regulation of apoptosis and cytokinesis in organisms ranging from yeast to humans . Baculoviruses also provide an ideal system to study the effects of an apoptotic response on viral pathogenesis in an animal host . In this review, I discuss a number of interesting recent developments in the areas of apoptotic regulation by baculoviruses and the effects of apoptosis on baculovirus replication and pathogenesis. Bull Cancer, 2001 Mar, 88(3), 269 - 76 {Transcriptome analysis in cancerology: bioinformatics aspects}; Claverie JM; Recent technological advances (e.g . various DNA arrays and chips) allow the measurement of expression level (mRNA abundance) for thousand of genes simultaneously, over multiple conditions or time . Initially developed and tested on model systems such as yeast or in vitro cell line cultures, these techniques have recently begun to be applied to the analysis of human cancers . Initial results are promising, and large-scale gene expression profiling is now expected to become a clinical tool for better tumour identification, prognosis, and optimal treatment design . It is thus important that clinicians become familiar with the theoretical principles underlying the interpretation of gene expression profiles as used in three different contexts: gene discovery, tumour class prediction, and molecular diagnosis . This is the purpose of the present article. J Agric Food Chem, 2000 Dec, 48(12), 6196 - 9 3-methylthiopropionaldehyde as precursor of dimethyl trisulfide in aged beers; Gijs L et al.; Hop S-methylcysteine sulfoxide has previously been postulated as the precursor of dimethyl trisulfide (DMTS) in beers . The present data point to 3-methylthiopropionaldehyde, the Strecker aldehyde issued from methionine, as another potential precursor in aged beers . Spiking either fresh beer or wort before boiling leads in all cases to higher levels of DMTS after storage . Moreover, special malts with a high level of 3-methylthiopropionaldehyde also favor polysulfide synthesis . A higher pH should increase this onion-like off-flavor, whereas a low pH is unfortunately known to enhance the cardboard flavor of aged beers . 3-methylthiopropanol, issued from yeast reducing activity, can be considered as an additional DMTS source during aging. Virology, 2001 Apr 25, 283(1), 110 - 20 HBV X protein targets HIV Tat-binding protein 1; Barak O et al.; The HBV X protein (HBx) is implicated in infection and development of hepatocellular carcinoma . HBx has a pleiotropic effect on cells, suggesting multiple targets in the virus-host cell interaction . We employed the cytoplasmic-based two-hybrid screen and identified the HIV Tat-binding protein 1 (Tbp1) as a novel HBx interacting protein . Tbp1 interacts in vivo with HBx both in yeast and in animal cells . This interaction maps to the functionally important ATP-binding motif of Tbp1 . Furthermore, HBx and Tbp1 interaction is functionally significant and regulates HBV transcription . Tbp1 homologues, such as Sug1, are known members of the proteasome 19S regulatory cap particle and have also been implicated in transcription coactivation . Remarkably, Tbp1 and Sug1 interact with multiple viral effector proteins including HIV Tat, SV40 large T antigen, and adenovirus E1A, establishing these proteins as important targets of the viral oncogenes . Virology, 2001 Apr 25, 283(1), 7 - 18 Rack1 binds HIV-1 Nef and can act as a Nef-protein kinase C adaptor; Gallina A et al.; Nef proteins of primate immunodeficiency viruses exert pleiotropic effects, such as enhanced endocytosis of CD4 and MHC-I cell surface molecules, perturbation of signal transduction cascades, and virion infectivity enhancement . Nef function intersects that of a number of cell kinases, including C kinases (PKCs) and Src-family kinases . Here the interaction of HIV-1 Nef with Rack1 (receptor for activated C kinase 1) is reported . Nef binds the Rack1 C-terminal moiety in a yeast two-hybrid system and in cell-free pull-down assays and copurifies with in vitro translated Rack1 . Nef and Rack1 partially colocalize on the trans-Golgi network and plasma membranes . The presence of Rack1 doubles Nef phosphorylation by PKCs in vitro . Our data agree with the idea that Rack1 acts as a Nef intracellular docking site, bringing Nef and PKCs together . Other signal transduction or endocytosis proteins, in particular Src-like kinases, might meet Nef by intermediation of the Rack1 adaptor . J Am Acad Dermatol, 2001 May, 44(5), 795 - 802 Pruritus vulvae in prepubertal children; Paek SC et al.; BACKGROUND: Vulvar pruritus is a common complaint in females of all ages . However, little has been published on pruritus vulvae in children as a primary symptom . OBJECTIVE: Our purpose is to review the causes and treatments of vulvar pruritus in prepubertal girls and to retrospectively evaluate the causes and outcomes of the premenarchal children we studied . METHODS: The records of 44 premenarchal girls with vulvar pruritus were reviewed, and follow-up interviews were performed by telephone . RESULTS: Thirty-three patients (75%) had nonspecific pruritus . Lichen sclerosus, bacterial infections, yeast infection, and pinworm infestation were seen in a minority of patients . At follow-up, pruritus had cleared in 15 patients, been alleviated in 13, and remained the same in 4 . CONCLUSION: Most of our patients had nonspecific pruritus, which was alleviated by or cleared with better hygiene and avoidance of irritants . In prepubertal girls, poor hygiene and irritants such as soap are major contributors to pruritus vulvae . All patients may benefit from following hygienic measures and irritant precautions in addition to specific therapy directed at underlying causes. J Biol Chem, 2001 Jun 29, 276(26), 23275 - 81 Epub 2001 Apr 18. CCAAT/enhancer-binding protein beta mediates interferon-gamma-induced p48 (ISGF3-gamma ) gene transcription in human monocytic cells; Xiao W et al.; Previous studies have identified a novel interferon-stimulated response element-like element, termed gamma-interferon-activating transcription element, within the interferon-stimulating gene factor-3gamma (p48) promoter region that is bound by novel transcription factors in response to stimulation with interferons (IFNs) (Weihua, X., Kolla, V., and Kalvakolanu, D . V . (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 103-108) . In the present study, we have identified CCAAT/enhancer-binding protein beta (C/EBP-beta) as one of the gamma-interferon-activating transcription element cognate transcription factors by screening a human monophage-derived cDNA library in a yeast one-hybrid system . Electrophoretic mobility shift assay studies suggest that C/EBP-beta dynamically regulates p48 gene expression upon IFN-gamma stimulation by undergoing changes in its heterodimerization partners . Transient transfection studies demonstrate that overexpression of C/EBP-beta strongly enhanced IFN-gamma-induced transcription from the p48 promoter . However, deletion mutants of C/EBP-beta that lack the N-terminal transactivation domain were unable to stimulate the p48 promoter . Western blotting revealed that C/EBP-beta is induced by IFN-gamma stimulation in THP-1-derived macrophages . Collectively, these results suggest that C/EBP-beta plays an important role in the human IFN-gamma signaling pathway by transcriptional regulation of p48 gene expression, an essential component in the IFN signaling pathway. Vaccine, 2001 Apr 30, 19(23-24), 3154 - 63 Physicochemical and immunological characterization of hepatitis B virus envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein; Yamada T et al.; The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region . Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired . We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells {J Biol Chem 267 (1992) 1953} . In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized . By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle . By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm . The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol . When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies . The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles . Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment . These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine. Int J Biochem Cell Biol, 2001 Mar, 33(3), 287 - 92 Isolation of a small chitinase-like antifungal protein from Panax notoginseng (sanchi ginseng) roots; Lam SK et al.; An antifungal protein, with a molecular weight of 15 kDa and an N-terminal sequence analogous to those of chitinases, was first isolated from the Chinese medicinal material Panax notoginseng, using cation exchange chromatography and affinity chromatography . The protein was adsorbed on CM-cellulose, Affi-gel Blue Gel and Mono S . It exerted antifungal activity against Coprinus comatus, Fusarium oxysporum and Mycosphaerella arachidicola but not against Rhizoctonia solani . The protein was devoid of ribonuclease activity against yeast tRNA. Gene, 2001 Apr 4, 267(1), 23 - 30 Gene structure and enzymatic activity of mouse eosinophil-associated ribonuclease 2; McDevitt AL et al.; Mouse eosinophil-associated ribonuclease-2 (mEAR-2) is one of a cluster of genes identified in the genome of the mouse Mus musculus that are highly divergent orthologs of the primate ribonucleases, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) . Northern analysis revealed expression of genes hybridizing to mEAR-2 in mouse lung, liver and spleen tissues . We obtained full-length cDNA by hybridization screening of mouse eosinophil and lung cDNA libraries and by rapid amplification of cDNA ends (RACE) from liver, spleen and lung RNA . Using these methods we have isolated the 195 base pair (bp) 3' untranslated region (UTR) that includes a typical polyadenylation signal preceding a poly A tail and the 5' UTR which includes 63-71 bp and three distinct transcriptional start sites . Using unidirectional PCR we isolated a 361-bp 5' promoter region and delineated the intronic / exonic boundaries which include a non-coding exon 1, a single intron, and a coding exon 2, a structure that is typical of genes of the RNase A superfamily . Consensus sites for PU.1 and EoTF, both active as intronic enhancer elements of the gene encoding EDN, are also present in the intron of the gene encoding mEAR-2 . The catalytic activity of recombinant baculovirus-derived mEAR-2 is similar to that of rhEDN from this source, with catalytic constants k(cat)/K(m)=5.6x10(6) M(-1) s(-1) and 10.5x10(6) M(-1) s(-1), respectively, against a standard yeast tRNA substrate . Sequence analysis of the non-coding regions and enzymatic characterization of the gene product provide further evidence indicating that mEAR-2 is a structural and functional ortholog of primate EDNs and ECPs. Mol Plant Microbe Interact, 2001 Apr, 14(4), 536 - 44 Signal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines; Wang X et al.; Secretions from the esophageal gland cells of plant-parasitic nematodes play critical roles in the nematode-parasitic cycle . A novel method to isolate cDNA encoding putative nematode secretory proteins was developed that utilizes mRNA for reverse transcription-polymerase chain reaction derived from microaspiration of the esophageal gland cell contents of parasitic stages of the soybean cyst nematode Heterodera glycines . The resulting H . glycines gland cell cDNA was cloned into the pRK18 vector, and plasmid DNA was transformed into a mutated yeast host for specific selection of cDNA inserts that encode proteins with functional signal peptides . Of the 223 cDNA clones recovered from selection in yeast, 97% of the clones encoded a predicted signal peptide . Fourteen unique cDNA clones hybridized to genomic DNA of H . glycines on Southern blots and, among them, nine cDNA clones encoded putative extracellular proteins, as predicted by PSORT II computer analysis . Four cDNA clones hybridized to transcripts within the dorsal esophageal gland cell of parasitic stages of H . glycines, and in situ hybridization within H . glycines was not detected for eight cDNA clones . The protocol provides a direct means to isolate potential plant-parasitic nematode esophageal gland secretory protein genes. Pharmacotherapy, 2001 Apr, 21(4), 481 - 7 Evaluation of the antihyperlipidemic properties of dietary supplements; Caron MF et al.; We reviewed the published literature regarding the antihyperlipidemic effects of dietary supplements . A search of MEDLINE database, EMBASE Drugs and Pharmacology database, and the Internet was performed, and pertinent studies were identified and evaluated . References from published articles and tertiary references were used to gather additional data . Published trials indicate that red yeast rice, tocotrienols, gugulipid, garlic, and soy protein all have antihypercholesterolemic effects . These supplements, as well as omega-3 fatty acids, also have antihypertriglyceridemic effects . In clinical trials none of the agents led to a reduction in low-density lipoproteins greater than 25%, suggesting modest efficacy . When recommending these supplements, clinicians should keep in mind that their long-term safety is not established and patients should be monitored closely. J Cell Biol, 2001 Apr 16, 153(2), 295 - 305 OSP/claudin-11 forms a complex with a novel member of the tetraspanin super family and beta1 integrin and regulates proliferation and migration of oligodendrocytes; Tiwari-Woodruff SK et al.; Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths . TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways . We have identified an OSP/claudin-11-associated protein (OAP)1, using a yeast two-hybrid screen . OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes . OAP-1, OSP/claudin-11, and beta1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry . Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line . Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes . These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair. J Biol Chem, 2001 Jun 29, 276(26), 24153 - 9 Epub 2001 Apr 17. Molecular determinants of the interaction between the inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) and cGMP kinase Ibeta; Ammendola A et al.; Cyclic GMP-dependent protein kinase I (cGKI) affects the inositol 1,4,5-trisphosphate (InsP(3))-dependent release of intracellular calcium by phosphorylation of IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate) . IRAG is present in a macromolecular complex with the InsP(3) receptor type I (InsP(3)RI) and cGKIbeta . The specificity of the interaction between these three proteins was investigated by using the yeast two-hybrid system and by co-precipitation of expressed proteins . The amino-terminal region containing the leucine zipper (amino acids 1-53) of cGKIbeta but not that of cGKIalpha or cGKII interacted with the sequence between amino acids 152 and 184 of IRAG in vitro and in vivo most likely through electrostatic interaction . cGKIbeta did not interact with the InsP(3)RI, but co-precipitated the InsP(3)RI in the presence of IRAG indicating that IRAG bound to the InsP(3)RI and to cGKIbeta . cGKIbeta phosphorylated up to four serines in IRAG . Mutation of these four serines to alanine showed that cGKIbeta-dependent phosphorylation of Ser(696) is necessary to decrease calcium release from InsP(3)-sensitive stores . These results show that cGMP induced reduction of cytosolic calcium concentrations requires cGKIbeta and phosphorylation of Ser(696) of IRAG. Clin Cancer Res, 2001 Apr, 7(4), 901 - 8 Frequent deletions and mutations of the beta-catenin gene are associated with overexpression of cyclin D1 and fibronectin and poorly differentiated histology in childhood hepatoblastoma; Takayasu H et al.; Hepatoblastoma (HBL) is the most common malignant liver tumor in young children . Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, we searched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, all of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation . However, no loss of function mutation of the APC gene was detected by the yeast functional assay . Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR . The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmic accumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene . The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed . Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor . Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo. J Cell Sci, 2001 May, 114(Pt 9), 1743 - 56 Self-assembly and binding of a sorting nexin to sorting endosomes; Kurten RC et al.; The fate of endocytosed membrane proteins and luminal contents is determined by a materials processing system in sorting endosomes . Endosomal retention is a mechanism that traps specific proteins within this compartment, and thereby prevents their recycling . We report that a sorting nexin SNX1, a candidate endosomal retention protein, self-assembles in vitro and in vivo, and has this property in common with its yeast homologue Vps5p . A comparison of SNX1 expressed in bacterial and in mammalian systems and analyzed by size-exclusion chromatography indicates that in cytosol SNX1 tetramers are part of a larger complex with additional proteins . An endosomal retention function would require that SNX1 bind to endosomal membranes, yet the complexes that we analyzed were largely soluble and little SNX1 was found in pellet fractions . Using green fluorescent protein fusions, endocytic compartment markers and fluorescence recovery after photobleaching, we found that there is an equilibrium between free cytoplasmic and early/sorting endosome-bound pools of green fluorescent protein-SNX1 . Fluorescence resonance energy transfer indicated that spectral variants of green fluorescent protein-SNX1 were oligomeric in vivo . In cell extracts, these green fluorescent protein-SNX1 oligomers corresponded to tetrameric and larger complexes of green fluorescent protein-SNX1 . Using video microscopy, we observed small vesicle docking and tubule budding from large green fluorescent protein-SNX1 coated endosomes, which are features consistent with their role as sorting endosomes . http://www.biologists.com/JCS/movies/jcs2058.html Cytogenet Cell Genet, 2001, 92(1-2), 134 - 8 Cloning, organisation, chromosomal localization and expression analysis of the mouse Prkag1 gene; Shamsadin R et al.; The mammalian 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of alpha-, beta- and gamma-subunits . The alpha-subunit is the catalytic subunit . The non-catalytic subunits AMPK-beta and AMPK-gamma form, together with the catalytic AMPK-alpha, the active kinase complex in mammals and its homologue in yeast . The gene for AMPK-gamma-1 has been designated recently as PRKAG1 . We have isolated mouse Prkag1 cDNA from testis (1623 nt) coding for 330 aa and we have shown its ubiquitous expression as a 1.8-kb transcript . A comparison between mouse, rat and human PRKAG1 cDNA and protein sequences shows that the gene is highly conserved among these species with a homology of 96% at the protein level . Southern blot analysis indicates that there is more than one gene for PRKAG in the mouse genome . Prkag1 contains 12 exons with short introns . Analysis of 50 interspecific backcross mice mapped the mouse gene to the distal region of chromosome 15. Cytogenet Cell Genet, 2001, 92(1-2), 59 - 62 Improvement of FISH mapping resolution on combed DNA molecules by iterative constrained deconvolution: a quantitative study; Monier K et al.; Image restoration approaches, such as digital deconvolution, are becoming widely used for improving the quality of microscopic images . However, no quantification of the gain in resolution of fluorescence images is available . We show that, after iterative constrained deconvolution, fluorescent cosmid signals appear to be 25% smaller, and 1.2-kb fragment signals on combed molecules faithfully display the expected length. Mol Pharmacol, 2001 May, 59(5), 1249 - 55 Synthetic phytoceramides induce apoptosis with higher potency than ceramides; Hwang O et al.; Ceramides are naturally occurring compounds recognized to mediate apoptosis . N-acylsphingosines, containing a double bond at carbons 4 and 5 of their sphingoid backbone, are thought to be the active form, because N-acylsphinganines with completely saturated sphingoid are inactive . In the present study, we synthesized a series of N-acyl-4D-ribo-phytosphingosines (phytoceramides) that contain a hydroxyl group at carbon 4 and investigated structure-cytotoxicity relationship of the presumed functional groups in ceramides . N-Acetylphytoceramide (PCer2) and N-hexanoylphytoceramide (PCer6) were found to be more cytotoxic than ceramides as determined by released lactate dehydrogenase activity and morphological criteria . This was not caused by intracellular conversion of phytoceramides to ceramides, because no N-hexanoylsphingosine was formed after incubation of cell lysate with PCer6 . Among phytoceramides having acyl chains two to eight carbons long, the cytotoxicity was highest with five or six carbons . The carbonyl group of the amide bond did not seem to be critical, because substitution of the oxygen with sulfur did not influence the cytotoxicity . The phytoceramide-induced cell death was observed to be apoptotic in nature with the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling and propidium iodide staining . Because phytoceramides can be readily synthesized from yeast sources, they may present a potential and economical alternative to ceramide in future studies and therapies. J Biol Chem, 2001 Jul 13, 276(28), 26332 - 9 Epub 2001 Apr 16. HSP-CBF is an NF-Y-dependent coactivator of the heat shock promoters CCAAT boxes; Imbriano C et al.; The cellular response to toxic stimuli is elicited through the expression of heat shock proteins, a transcriptional process that relies upon conserved DNA elements in the promoters: the Heat Shock Elements, activated by the heat shock factors, and the CCAAT boxes . The identity of the CCAAT activator(s) is unclear because two distinct entities, NF-Y and HSP-CBF, have been implicated in the HSP70 system . The former is a conserved ubiquitous trimer containing histone-like subunits, the latter a 110-kDa protein with an acidic N-terminal . We analyzed two CCAAT-containing promoters, HSP70 and HSP40, with recombinant NF-Y and HSP-CBF using electrophoretic mobility shift assay, protein-protein interactions, transfections and chromatin immunoprecipitation assays (ChIP) assays . Both recognize a common DNA-binding protein in nuclear extracts, identified in vitro and in vivo as NF-Y . Both CCAAT boxes show high affinity for recombinant NF-Y but not for HSP-CBF . However, HSP-CBF does activate HSP70 and HSP40 transcription under basal and heat shocked conditions; for doing so, it requires an intact NF-Y trimer as judged by cotransfections with a diagnostic NF-YA dominant negative vector . HSP-CBF interacts in solution and on DNA with the NF-Y trimer through an evolutionary conserved region . In yeast two-hybrid assays HSP-CBF interacts with NF-YB . These data implicate HSP-CBF as a non-DNA binding coactivator of heat shock genes that act on a DNA-bound NF-Y. EMBO Rep, 2001 Apr, 2(4), 336 - 41 The Golgi matrix protein GM130: a