Nat Struct Biol, 2002 Oct, 9(10), 725 - 8 L-A virus at 3.4 A resolution reveals particle architecture and mRNA decapping mechanism; Naitow H et al.; The structure of the yeast L-A virus was determined by X-ray crystallography at 3.4 A resolution . The L-A dsRNA virus is 400 A in diameter and contains a single protein shell of 60 asymmetric dimers of the coat protein, a feature common among the inner protein shells of dsRNA viruses and probably related to their unique mode of transcription and replication . The two identical subunits in each dimer are in non-equivalent environments and show substantially different conformations in specific surface regions . The L-A virus decaps cellular mRNA to efficiently translate its own uncapped mRNA . Our structure reveals a trench at the active site of the decapping reaction and suggests a role for nearby residues in the reaction. J Cell Sci, 2002 Oct 15, 115(Pt 20), 3909 - 22 Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase beta interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis; Taverna E et al.; Neuronal calcium sensor 1 (NCS-1) belongs to a family of EF-hand calcium-binding proteins and is mainly expressed in neurons and neuroendocrine cells, where it causes facilitation of neurotransmitter release through unknown mechanisms . The yeast homologue of NCS-1 has been demonstrated to interact with and regulate the activity of yeast phosphatidylinositol 4-OH kinase beta (PI4Kbeta) . However, in neurons and neurosecretory cells NCS-1 has not unequivocally been shown to interact with PI4Kbeta . Here we have compared the subcellular distribution of NCS-1 and PI4Kbeta and investigated whether they are capable of forming complexes . In neurons, both proteins are widely distributed and are present in perikarya and, to a lesser extent, in nerve terminals . A consistent portion of NCS-1 and PIK4beta is cytosolic, whereas a portion of both proteins appears to be associated with the membranes of the endoplasmic reticulum and the Golgi complex . Very small amounts of NCS-1 and PI4Kbeta are present in synaptic vesicles . Our results further demonstrate that in neurosecretory cells, endogenous NCS-1 and PIK4beta interact to form a complex that can be immunoisolated from membrane as well as from cytosolic fractions . Moreover, both proteins can be recruited to membranes when cells are treated with nucleotide receptor agonists known to increase polyphosphoinositide turnover and concomitantly induce exocytosis of secretory vesicles . Finally, in PC12 cells overexpressing NCS-1, the amount of PI4Kbeta associated with the membranes is increased concomitantly with the increased levels of NCS-1 detected in the same membrane fractions . Together, these findings demonstrate that mammalian NCS-1 and PI4Kbeta interact under physiological conditions, which suggest a possible role for NCS-1 in the translocation of PI4Kbeta to target membranes. J Biol Chem, 2002 Nov 22, 277(47), 45428 - 34 Epub 2002 Sep 18. Rapostlin is a novel effector of Rnd2 GTPase inducing neurite branching; Fujita H et al.; Rho family GTPases are central regulators of neuronal morphology . Recently, Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been identified as new members of Rho family GTPases . Of these, Rnd2 is specifically expressed in neurons in brain; however, the signaling pathways of Rnd2 are not known . Here we have performed a yeast two-hybrid screen using Rnd2 as a bait and identified a novel Rnd2-effector protein, expressed predominantly in brain . We named it Rapostlin (apostle of Rnd2) . Rapostlin has two functional domains, Fer-CIP4 homology (FCH) domain at the amino terminus and SH3 (Src homology 3) domain at the carboxyl terminus . In in vitro binding assays, Rapostlin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner, and the Rnd2-binding domain of Rapostlin is localized between FCH and SH3 domains . Rapostlin directly binds to microtubules, and the amino-terminal region containing the FCH domain of Rapostlin is essential for this interaction . In PC12 cells, Rapostlin induces neurite branching in response to Rnd2, and at least the amino-terminal region of Rapostlin is necessary for this activity . Therefore, Rapostlin is the first effector of Rnd2, regulating neurite branch formation. J Biol Chem, 2002 Nov 22, 277(47), 45338 - 46 Epub 2002 Sep 18. High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis; Okumoto S et al.; Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns . A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR . Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis . Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap . AAP6 may thus function in uptake of amino acids from xylem . Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds . Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds . The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable . Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity . AAP1, -6 and -8 are the closest AAP paralogs . Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged . Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy. J Biol Chem, 2002 Nov 22, 277(47), 45558 - 65 Epub 2002 Sep 18. Aurora-A kinase interacting protein (AIP), a novel negative regulator of human Aurora-A kinase; Kiat LS et al.; Aurora kinases have evolved as a new family of mitotic centrosome- and microtubule-associated kinases that regulate the structure and function of centrosomes and spindle . One of its members, Aurora-A, is a potential oncogene . Overexpression of Aurora-A is also implicated in defective centrosome duplication and segregation, leading to aneuploidy and tumorigenesis in various cancer cell types . However, the regulatory pathways for mammalian Aurora-A are not well understood . Exploiting the lethal phenotype associated with the overexpression of Aurora-A in yeast, we performed a dosage suppressor screen in yeast and report here the identification of a novel negative regulator of Aurora-A, named AIP (Aurora-A kinase Interacting Protein) . AIP is a ubiquitously expressed nuclear protein that interacts specifically with human Aurora-A in vivo . Ectopic expression of AIP with Aurora-A in NIH 3T3 and COS cells results in the down-regulation of ectopically expressed Aurora-A protein levels, and this down-regulation is demonstrated to be the result of destabilization of Aurora-A through a proteasome-dependent protein degradation pathway . A noninteracting deletion mutant of AIP does not down-regulate Aurora-A protein, suggesting that the interaction is important for the protein degradation . AIP could therefore be a potential useful target gene for anti-tumor drugs. Biosens Bioelectron, 2002 Oct, 17(10), 851 - 7 Genetic modification of glucose oxidase for improving performance of an amperometric glucose biosensor; Chen LQ et al.; Glucose oxidase (GOD) was genetically modified by adding a poly-lysine chain at the C-terminal with a peptide linker inserted between the enzyme and poly-lysine chain . The poly-lysine chain was added in order to anchor more electron transfer mediator, ferrocenecarboxylic acid, to GOD for the purpose of improving sensitivity and stability of glucose biosensors . The modified GOD had similar K(m) and K(cat) to those of the wild type enzyme . After interacted with the electron transfer mediator, the modified enzyme retained 90.01% of its native activity, while the commercial GOD and the wild type GOD (Aspergillus niger) retained only 22.43 and 22.17%, respectively . Screen-printed electrodes coated with the modified GOD, wild type yeast-derived GOD or the commercial GOD were tested in glucose solution of different concentrations . Experimental results showed that the biosensor based on the modified GOD gave the largest signal among the three . In addition, the linear range of the biosensor prepared by the modified GOD could extend to 45 mM, while they were about 20 mM for the biosensors based on the wild type yeast-derived enzyme and the commercial enzyme. Toxicol Lett, 2002 Sep 5, 135(1-2), 111 - 23 No androgenic/anti-androgenic effects of bisphenol-A in Hershberger assay using immature castrated rats; Kim HS et al.; Several studies have demonstrated that bisphenol A (BPA) exhibited weak estrogenic activity in the 3-day uterotrophic assay using ovariectomized (OVX) and immature rats (Toxicol . Lett . 115 (2000) 231; Regul . Toxicol . Pharmacol . 32 (2000) 118; J . Toxicol . Sci . 26 (2001) 111) and BPA also possessed anti-androgenic activity in in vitro yeast based assays (J . Endocrinol . 158 (1998) 327) . To investigate anti-androgenic effects of BPA . a rodent Hershberger assay was carried out using immature Sprague-Dawley male rats . An androgen agonist, testosterone (0.4 mg/kg per day), was administered for 7 consecutive days by subcutaneous (s.c.) injection as a positive control . Additionally, a pure androgen antagonist, flutamide (1, 5 . 10 mg/kg per day . oral) was co-administered with testosterone (0.4 mg/kg per day s.c.) . BPA was also administered orally with or without testosterone (0.4 mg/kg per day, s.c.) for 7 consecutive days . In the testosterone treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles (LABC) weights were significantly increased compared with control . However . flulamide dose-dependently inhibited the testosterone-induced re-growth of seminal vesicles, ventral prostate, and LABC, with a significant decrease at flutamide 1.0 mg/kg and above (P<0.05) . Serum LH levels were also significantly increased (5 mg/kg and above, P<0.05), but no changes in serum testosterone levels . In contrast, BPA had no effects on the re-growth of seminal vesicles, ventral prostate and LABC induced by testosterone, and no significant differences were observed in serum LH and testosterone levels . In summary, the Hershberger assay could be a sensitive method for detecting androgenic or anti-androgenic chemicals, but BPA did not exhibit any androgenic or anti-androgenic activities in Hershberger assay. Exp Parasitol, 2002 May, 101(1), 13 - 24 Giardia lamblia: identification and characterization of Rab and GDI proteins in a genome survey of the ER to Golgi endomembrane system; Langford TD et al.; To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia, we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins . G . lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation . Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells . Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles . GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI's key role in Rab cycling between organelles within the cell. Mol Cells, 2002 Aug 31, 14(1), 9 - 15 Two transactivation domains of hypoxia-inducible factor-1alpha regulated by the MEK-1/p42/p44 MAPK pathway; Lee E et al.; At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress . A common transactivator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes . HIF-1 is a heterodimeric transactivator that is composed of alpha and beta subunits . HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the alpha subunit, whereas the HIF-1beta subunit is expressed constitutively . Our previous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1alpha . In this paper, we dissected the transactivation domain of HIF-1alpha in more detail, and tested the correlation between specific domains of HIF-1alpha and specific signaling pathways . We designed several fusion proteins that contain deletion mutants of HIF-1alpha that is linked to the DNA binding domain of the yeast protein Gal4 . By using the Gal4-driven reporter system, we tested the transactivation activities of the Gal4/HIF-1alpha fusion proteins in Hep3B cells . Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1alpha . We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1alpha . Treatment of PD98059, a MEK-1 inhibitor, blocked the hypoxia-induced transactivation abilities of both the 522-649 and 650-822 amino acids of the C-terminal half of HIF-1alpha . This implies that the MEK-1/p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains. J Ind Microbiol Biotechnol, 2002 Sep, 29(3), 145 - 8 Differential expression of polygalacturonase-encoding genes from Penicillium griseoroseum in different carbon sources; Ribon AO et al.; A second polygalacturonase-encoding gene (pgg2) of Penicillium griseoroseum was cloned and consists of an opening reading frame of 1107 bp after removal of two introns . The gene encodes a protein of 369 amino acids with a predicted molecular mass of 38.3 kDa . The deduced protein sequence exhibited high homology with other fungal endopolygalacturonases . A polymerase chain reaction (PCR)-based strategy was used to study the expression patterns of pgg1 and pgg2 genes under different culture conditions and our results show that both genes are regulated by the carbon source at the transcriptional level . The pgg1 transcript was detected at 76 h of fungal growth in pectin while the pgg2 transcript was also induced by sucrose . The addition of yeast extract to glucose medium abolished the repressive effect of glucose, suggesting that the transcription of these genes is controlled by different mechanisms. Plant Cell, 1995 Jun, 7(6), 667 - 676 Expression and Regulation of aERD2, a Gene Encoding the KDEL Receptor Homolog in Plants, and Other Genes Encoding Proteins Involved in ER-Golgi Vesicular Trafficking; Bar-Peled M et al.; aERD2 and aSAR1 of Arabidopsis are functional homologs of yeast genes encoding proteins essential for endoplasmic reticulum (ER)-to-Golgi transport . The regulation of these secretory pathway genes in yeast, mammals, and plants is not known . High levels of expression of aERD2 and aSAR1 were observed in roots, flowers, and inflorescence stems, with the highest levels being detected in roots . The aSAR1 transcript levels were highest in young leaves and declined during leaf maturation . Low levels of aERD2 were detected in both young and fully mature leaves when compared with roots . In situ hybridization showed that trichomes accumulate more aERD2 transcript as the leaf expands, whereas aSAR1 is expressed equally in all leaf cell types . Treating plants with tunicamycin, a drug that blocks N-glycosylation in the ER, or with cold shock, known to block secretory protein transport, led to a marked accumulation of aERD2 and aSAR1 transcripts . The Arabidopsis ARF gene, which encodes a GTPase probably involved in Golgi vesicle traffic, was not affected by these treatments . This study is an essential first step toward understanding the regulation of genes that encode proteins involved in vesicular trafficking. Plant Cell, 1995 Oct, 7(10), 1545 - 1554 Phloem Loading by the PmSUC2 Sucrose Carrier from Plantago major Occurs into Companion Cells; Stadler R et al.; High levels of mRNA for the sucrose-H+ symporter PmSUC2 have been found in the vascular bundles of petioles from Plantago major . The possible role of PmSUC2 in phloem loading was studied with antiserum raised against the recombinant PmSUC2 protein . This antiserum labeled a single 35-kD protein band in detergent extracts of P . major vascular bundles . It showed no cross-reaction with the P . major sucrose carrier PmSUC1, which was tested with detergent extracts from plasma membranes of transgenic yeast strains containing either the P . major sucrose transporter PmSUC1 or PmSUC2 . The antiserum was used to determine the site of PmSUC2 expression in leaves, petioles, and roots of P . major . In cross-sections and longitudinal sections, the PmSUC2 protein was found in only one single cell type . These cells were identified as companion cells because they are nucleated, contain a dense cytoplasm, and are always adjacent to a sieve element . The labeled cells had the same longitudinal extension as did their sister sieve elements and always ended next to the sieve plates, which were characterized by specific staining . PmSUC2 mRNA and PmSUC2 protein were also detected in P . major roots . The function of PmSUC2 in the different organs and its role in phloem loading are discussed. J Ethnopharmacol, 2002 Oct, 82(2-3), 191 - 5 Pharmacological evidence favouring the folkloric use of Diospyros mespiliformis Hochst in the relief of pain and fever; Adzu B et al.; The methanol extract of Diospyros mespiliformis was evaluated for its claimed folkloric usage in the relief of pain and fever . Antipyretic, analgesic and anti-inflammatory effects of the extract were evaluated in rats and mice . Studies were carried out on yeast-induced pyrexia in rats, acetic acid-induced writhing in mice, formalin test and egg albumin-induced anti-inflammatory activity in rats . The extract (50 and 100 mg/kg i.p.) gave a potent antipyretic effect for 100 mg/kg and significant activity (P<0.05) against all the analgesic and anti-inflammatory models used . The LD(50) of the extract was estimated to be 513.80+/-33.92 mg/kg i.p . in mice . These results provide support for the use of the plant in relieving pain and fever. J Ethnopharmacol, 2002 Oct, 82(2-3), 89 - 95 Estrogenic effects of ethanol and ether extracts of propolis; Song YS et al.; Propolis obtained from honeybee hives has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, or immunomodulatory agent . The potential estrogenic activity of propolis was investigated in vitro using the MCF-7 human breast cancer cell proliferation, human estrogen receptor (hER) binding and yeast-based steroid receptor transcription, and in vivo using the immature rat uterotrophic effect . Treatments with ethanol extract of propolis (EEP) and ether extract of propolis (REP) enhanced MCF-7 cell proliferation in concentrations ranging from 0.8 to 4 microg/ml . Both EEP and REP competed for binding of {3H}17beta-estradiol to the hER with IC(50) values of 9.14 and 9.72 microg/ml, respectively . In yeast estrogen receptor transcription assay, both EEP and REP were found to be estrogenic with EC(50) values of 9.48, and 8.55 microg/ml, respectively . Animals treated with EEP or REP for 4 days (500-1000 mg/kg per day, s.c.) exhibited significant dose-dependent increases in uterine wet weight . However, in the yeast androgen and progesterone receptor transcription assays, either EEP or REP was found not to be active . The results suggest that propolis produces estrogenic effects through activation of estrogen receptors. Mol Biol Rep, 2002, 29(1-2), 67 - 71 Control analysis of DNA microarray expression data; Curtis RK et al.; DNA microarrays produce large amounts of data . Complex changes in gene expression are revealed; sometimes thousands of mRNAs change between experiments . Here we apply modular regulation analysis to microarray data to reveal and quantify the mRNA changes that are important for cellular responses . The mRNAs are sorted into clusters . How strongly a perturbation alters each cluster is multiplied by how strongly each cluster affects an output, to obtain coefficients that describe how much of the change in the output is transmitted through each mRNA cluster . An example published dataset is analysed to reveal that the response ('relative fitness') of yeast to 2-deoxy-D-glucose is not transmitted by a single mRNA cluster, but instead many clusters contribute to the overall response . The method is applicable to microarray, transcriptome, proteome and metabolome data. Plant Cell, 1996 May, 8(5), 793 - 803 Systemic Acquired Resistance Mediated by the Ectopic Expression of Invertase: Possible Hexose Sensing in the Secretory Pathway; Herbers K et al.; Systemic acquired resistance (SAR) has been reported to be associated with lesion-mimic mutants . Tobacco plants expressing vacuolar and apoplastic yeast-derived invertase (vaclnv and cwlnv, respectively) develop spontaneous necrotic lesions similar to hypersensitive responses caused by avirulent pathogens . Therefore, SAR and metabolic alterations leading to the activation of defense-related responses were studied in these plants . Defense-related gene transcripts, callose content, peroxidase activities, and levels of salicylic acid were found to be elevated . The defense reactions were accompanied by increased resistance toward potato virus Y and were measured as decreased viral spreading and reduced multiplication in systemic leaves of the transgenic plants . Interestingly, the accumulation of pathogenesis-related (PR) protein transcripts (PR-Q) and repression of photosynthetic gene transcripts (chlorophyll a/b binding protein) were inversely correlated and required the same threshold level of hexoses for induction and repression . Expression of a cytosolic yeast-derived invertase in transgenic tobacco plants with equally increased levels of sugars neither displayed SAR responses nor showed decreased levels of photosynthetic genes . It is suggested that hexose sensing in the secretory pathway is essential for mediating the activation of defense-related genes as well as repression of photosynthetic genes in vaclnv and cwlnv plants. J Biol Chem, 2002 Nov 29, 277(48), 45811 - 20 Epub 2002 Sep 16. Enhancement of alpha -helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro . Implications for vaccine design; Joyce JG et al.; The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5 . Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope . Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for alpha-helical conformation . We have examined DP178 in the context of a model for optimized alpha-helices and show that the native sequence conforms poorly to the model . Solution conformation of DP178 was studied by circular dichroism and NMR spectroscopy and found to be predominantly random, consistent with previous reports . NMR mapping was used to show that the low percentage of alpha-helix present was localized to residues Glu(662) through Asn(671), a region encompassing the 2F5 epitope . Using NH(2)-terminal extensions derived from either GP41 or the yeast GCN4 leucine zipper dimerization domain, we designed peptide analogs in which the average helicity is significantly increased compared with DP178 and show that these peptides exhibit both a modest increase in affinity for 2F5 using a novel competitive solution-based binding assay and an increased ability to inhibit viral entry in a single-cycle infectivity model . Selected peptides were conjugated to carrier protein and used for guinea pig immunizations . High peptide-specific titers were achieved using these immunogens, but the resulting sera were incapable of viral neutralization . We discuss these findings in terms of structural and immunological considerations as to the utility of a 2F5-like response. Cardiovasc Res, 2002 Oct, 56(1), 93 - 103 Cardiac-enriched LIM domain protein fhl2 is required to generate I(Ks) in a heterologous system; Kupershmidt S et al.; OBJECTIVE: Co-expression of the KvLQT1 and minK potassium channel subunits is required to recapitulate I(Ks), the slow component of the cardiac delayed rectifier current, and mutations in either gene cause the congenital Long QT syndrome . It is becoming increasingly well-recognized that multiprotein channel complexes containing proteins capable of modulating channel function assemble at the plasma membrane . Thus, the aim of our study was to identify proteins involved in I(Ks) modulation . METHODS AND RESULTS: Using a yeast-two-hybrid screen with the intracytoplasmic C-terminus of minK as bait, we identified the cardiac-enriched four-and-a-half LIM domain-containing protein (fhl2) as a potential minK partner . We show interaction between the two proteins in GST pulldown assays and demonstrate overlapping subcellular localization using immunocytochemistry of transfected cells supporting a potential interaction . At the functional level, expression of KvLQT1and minK in HEK cells, which lack endogenous fhl2 protein, generated I(Ks) only when fhl2 was co-expressed . By contrast, in CHO-K1 cells, which express fhl2 endogenously, I(Ks) was suppressed by anti-fhl2 antisense which did not affect the currents generated by KvLQT1alone . CONCLUSION: These data indicate that at least in heterologous cells, the generation of I(Ks) requires fhl2 as an additional protein component. Mol Plant Microbe Interact, 2002 Sep, 15(9), 907 - 21 Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi; Gotesson A et al.; Polygalacturonases (PGs) are secreted by fungal pathogens during saprophytic and parasitic growth, and their degradation of pectin in the plant cell wall is believed to play a major role in tissue invasion and maceration . In this study, PG activity was demonstrated in culture filtrates of the oomycete plant pathogen, Phytophthora cinnamomi . A P . cinnamomi pg gene fragment amplified using degenerate primers based on conserved regions in fungal and plant PGs was used to isolate 17 complete P . cinnamomi pg genes and pseudogenes from a genomic library and partial sequence for another two genes . Gel blotting of genomic DNA indicated that there may be even more pg genes in the P . cinnamomi genome . P . cinnamomi pg gene sequences were expressed in PG-deficient yeast and found to confer PG activity, thereby confirming their functional identity . The predicted mature P . cinnamomi PGs fall into subgroups that exhibit large differences in the extent of N-glycosylation, isoelectric points, and N- and C-terminal structure . Evidence for birth-and-death and reticulate evolution in the P . cinnamomi pg gene family was obtained, and some codons for surface exposed residues in the P . cinnamomi PGs were shown to have been subject to diversifying selection . Contrary to accepted phylogenies for other proteins, phylogenetic analysis of the P . cinnamomi PGs revealed a closer relationship with PGs from true fungi than with those from plants. Vet Clin North Am Food Anim Pract, 2002 Jul, 18(2), 233 - 52 Brewing by-products: their use as animal feeds; Westendorf ML et al.; Brewers grains, a by-product of beer production, are often used as a livestock feed . Because brewers grains provide protein, fiber, and energy, they can be useful in a variety of diets . Protein in brewers grains can meet a significant portion of supplemental protein requirements; in addition, they provide fiber and needed bulk in the diets of ruminants and horses . Brewers grains and other brewers by-products have also been fed to pigs, sheep, and poultry . Currently, the primary market for wet brewers grains is as a dairy cattle feed; however, some may be fed to beef cattle in feedlots . Brewers grains have historically been marketed wet or dry, but wet brewers grains currently make up the majority of the marketed product . Brewers grains provide protein, energy, and fiber in livestock diets, but product variability can influence their utilization and necessitate a testing program to determine nutrient content. Nucleic Acids Res, 2002 Sep 15, 30(18), 4040 - 50 Identification of mRNA decapping activities and an ARE-regulated 3' to 5' exonuclease activity in trypanosome extracts; Milone J et al.; mRNA turnover is a regulated process that contributes to the steady state level of cytoplasmic mRNA . The amount of each mRNA determines, to a large extent, the amount of protein produced by that particular transcript . In trypanosomes, there is little transcriptional regulation; therefore, differential mRNA stability significantly contributes to mRNA levels in each stage of the parasite life cycle . To investigate the enzymatic activities that contribute to mRNA turnover, we developed a cell-free system for mRNA turnover using the trypanosome Leptomonas seymouri . We identified a decapping activity that removed m(7)GDP from mRNAs that contain an m(7)GpppN cap at their 5' end . In yeast, the release of m(7)GDP by the pyrophosphatase Dcp1p/Dcp2p is a rate-limiting step in mRNA turnover . A secondary enzymatic activity, similar to the human cap scavenger activity, was identified in the trypanosome extracts . Both the human and trypanosome scavenger activities generate m(7)GMP from short capped RNA and are inhibited by addition in trans of m(7)GpppG . A third enzymatic activity uncovered in the parasite extracts functioned as a 3' to 5' exonuclease . Importantly, this exonuclease activity was stimulated by an AU-rich element present in the RNA . In summary, the cell-free system has defined several RNA turnover steps that likely contribute to regulated mRNA decay in trypanosomes. Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 13154 - 9 Epub 2002 Sep 16. The membrane trafficking protein calpactin forms a complex with bluetongue virus protein NS3 and mediates virus release; Beaton AR et al.; Bluetongue virus, an arbovirus of the Orbivirus genus, infects and replicates in both insect and mammalian cells . However, the cytopathic effect (cpe) on each host is very different . Mammalian cells show substantial cpe, most likely a result of the mechanism of virus release, whereas insect cells show little cpe and appear to release virus without cell lysis . Expression analysis of each infected cell type shows one protein, the nonstructural (NS) protein NS3, to be differentially expressed in the different cell types, suggesting it may act in the virus egress pathway . The molecular basis of such an interaction, however, has never been clear . Here, by using yeast two-hybrid analysis, we show that NS3 interacts with a cellular protein p11 (calpactin light chain), part of the annexin II complex that is involved in exocytosis . We map the NS3 region of interaction with p11 to a 13-residue peptide found at the N terminus of the protein and show it effectively competes with p36 (annexin II heavy chain) for p11 ligand binding . Further, we show that the C-terminal domain of NS3 interacts with VP2, the outermost protein of the fully assembled virus particle, suggesting that NS3 forms a bridging molecule that draws assembled virus into contact with the cellular export machinery . Our data describe the first host protein involvement in orbivirus egress and provide new insights into understanding arbovirus interactions with their hosts. J Biol Chem, 2002 Nov 22, 277(47), 44613 - 22 Epub 2002 Sep 13. The Kruppel-like factor Zf9 and proteins in the Sp1 family regulate the expression of HSP47, a collagen-specific molecular chaperone; Yasuda K et al.; In several cells and tissues the synthesis of HSP47, a collagen-specific molecular chaperone in the endoplasmic reticulum, is closely correlated with the synthesis of collagen . We previously reported that the Sp1 binding site at -210 bp in the promoter region and the first and second introns are required for the tissue-specific expression of HSP47 in transgenic mice (Hirata, H., Yamamura, I., Yasuda, K., Kobayashi, A., Tada, N., Suzuki, M., Hirayoshi, K., Hosokawa, N., and Nagata, K . (1999) J . Biol . Chem . 274, 35703-35710) . Here, we analyze how these introns influence the transcriptional regulation of the hsp47 gene in BALB/c 3T3 cells, which produce high levels of HSP47 . In vitro promoter analysis using a luciferase reporter and gel mobility shift analysis revealed that two cis-acting elements in the first and second introns, BS5-B and EP7-D, respectively, are required for the activation of hsp47 in BALB/c 3T3 cells . Several members of the Kruppel-like factor (KLF) family of proteins were identified as BS5-B-binding proteins by yeast one-hybrid analysis using these elements as baits . One of these proteins, KLF-6/Zf9, binds to the BS5-B element and activates expression of the reporter construct when transfected into cells . Chromatin immunoprecipitation assay analysis revealed that the endogenous KLF-6/Zf9 binds the BS5-B elements that contain the CACCC motif, which is a consensus recognition sequence for other proteins in the KLF family . We also showed that BS5-B and EP7-D are bound by two members of the Sp1 family, Sp2 and Sp3 . These results suggest that at least three sequences are required for the constitutive expression of hsp47 in BALB/c 3T3 cells: the -210 bp Sp1 binding site, the BS5-B element in the first intron, and the EP7-D element in the second intron . We suggest that KLF proteins regulate the transcription of hsp47 by binding the BS5-B element in cooperation with Sp2 and/or Sp3. J Biol Chem, 2002 Nov 22, 277(47), 45611 - 8 Epub 2002 Sep 15. Human papilloma virus 16 E6 oncoprotein inhibits retinoic X receptor-mediated transactivation by targeting human ADA3 coactivator; Zeng M et al.; The expression of human papillomavirus (HPV) E6 oncoprotein is causally linked to high-risk HPV-associated human cancers . We have recently isolated hADA3, the human homologue of yeast transcriptional co-activator yADA3, as a novel E6 target . Human ADA3 binds to the high-risk (cancer-associated) but not the low-risk HPV E6 proteins and to immortalization-competent but not to immortalization-defective HPV16 E6 mutants, suggesting a role for the perturbation of hADA3 function in E6-mediated oncogenesis . We demonstrate here that hADA3 directly binds to the retinoic X receptor (RXR)alpha in vitro and in vivo . Using chromatin immunoprecipitation, we show that hADA3 is part of activator complexes bound to the native RXR response elements within the promoter of the cyclin-dependent kinase inhibitor gene p21 . We show that hADA3 enhances the RXR(alpha)-mediated sequence-specific transactivation of retinoid target genes, cellular retinoic acid-binding protein II and p21 . Significantly, we demonstrate that E6 inhibits the RXR(alpha)-mediated transactivation of target genes, implying that perturbation of RXR-mediated transactivation by E6 could contribute to HPV oncogenesis. J Biol Chem, 2002 Nov 1, 277(44), 41307 - 10 Epub 2002 Sep 13. Molecular and functional interaction of the ATP-binding cassette transporter A1 with Fas-associated death domain protein; Buechler C et al.; ATP-binding cassette transporter A1 (ABCA1) is a major regulator of cellular cholesterol and phospholipid homeostasis . Its function has not been fully characterized and may depend on the association with additional proteins . To identify ABCA1-interacting proteins a human liver yeast two-hybrid library was screened with the 144 C-terminal amino acids of ABCA1 . Fas-associated death domain protein (FADD) was identified to bind to ABCA1, and this interaction was confirmed by pull-down assays and co-immunoprecipitations . Recombinant expression of a dominant negative form of FADD or the C terminus of ABCA1 in the human hepatoma cell line HepG2 markedly reduced the transfer of phospholipids to apoA-I . This indicates that the binding of additional proteins, one of them being full-length FADD, is required for ABCA1 function . The association of FADD with ABCA1 provides an unexpected link between high density lipoprotein metabolism and an adaptor molecule mainly described in death receptor signal transduction. J Cell Biol, 2002 Sep 16, 158(6), 1039 - 49 Epub 2002 Sep 16. Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis; Le Stunff H et al.; Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger . Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast . Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin . Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane . Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis . Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis . Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide . Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of {3H}palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide . Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(4), 325 - 334 A 4.3 Mb YAC Contig in Human Xp11.2: Long-Range Restriction Mapping and Identification of CpG Islands; Wei Y et al.; The Xp11.2 region o the human X chromosome contains genes involved in a number of inherited diseased, with at least one locus that escapes X chromosome inactivation, as well as abnormal methylation polymorphism . We isolated a series of yeast artificial chromosome (YAC) clones by hybridization screening with DNA probes localized within this region and assembled them into a 4.3 Mb contig spanning from Xp11.21 to Xp11.23 by a combination of Alu-PCR fingerprinting, STS-PCR and DNA probe cross hybridization . On the basis of these overlapping YAC clones we have constructed the long-range restriction map of this interval and placed exactly some DNA markers . Four CpG-dense regions between ARAF1 and OATL2 were identified based on the long-range restriction mapping, which indicated the distribution of genes within this interval . It should assist in the future nucleic acid sequence analysis and novel gene identification in this region. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(6), 616 - 623 The DNA Sequence and Structural Characteristic of The 5'-Nontranscribed Spacer of Silkworm Attacus ricini rDNA; He ML et al.; We have found that the SacII-EcoRI fragment in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA is a nuclear scaffold-associated region (SRA) and showed the function as the ARS element in yeast . This paper reports the sequence of this NTS region and the various characteristic potential functional motifs as analyzed by computer . It is 1 025 bp long and AT-Rich . With 9 bent DNA motifs, 10 T-boxes, 5 A-boxes motifs, 13 topoisomerase II as well as 15 ARS consensus sequences . In addition, there are several dozens of inverted repeats and ATTA/TAAT, ATTTA/TAAAT, ATATTT/AAATAT motifs commonly believed to be the binding sites of many homeodomain proteins . These motifs, concentrated in the SAR region, may play very important role in the regulation of gene transcription and replication at the chromatin level. Plant Physiol, 1994 Aug, 105(4), 1427 - 1432 Licodione Synthase, a Cytochrome P450 Monooxygenase Catalyzing 2-Hydroxylation of 5-Deoxyflavanone, in Cultured Glycyrrhiza echinata L . Cells; Otani K et al.; Cultured Glycyrrhiza echinata L . (Leguminosae) cells produce a retrochalcone echinatin (4,4{prime}-dihydroxy-2-methoxychalcone) and its biosynthetic intermediate licodione {1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione, a dibenzoylmethane (keto form) or its enol tautomer ({beta}-hydroxychalcone)}, when treated with elicitor-active substances, e.g . yeast extract . A microsomal fraction (160,000g pellet) prepared from yeast extract-induced suspension cultures of G . echinata catalyzed the formation of licodione from (2S)-liquiritigenin (7,4{prime}-dihydroxyflavanone) in the presence of NADPH and air . This licodione synthase activity was shown to be dependent on cytochrome P450 by its microsomal localization, requirement of NAD(P)H and O2 for activity, and inhibition by typical cytochrome P450 inhibitors . Licodione synthase activity transiently increased in the cells after treatment with yeast extract . When (2S)-naringenin (5,7,4{prime}-trihydroxyflavanone) and NADPH were incubated with the same microsomal preparation, a polar compound, which further converted into apigenin (5,7,4{prime}-trihydroxyflavone) when treated with acid, was produced . The reaction mechanism of licodione synthase is likely to be 2-hydroxylation of the flavanone molecule and subsequent hemiacetal opening and is possibly the same as the previously suggested mechanism of flavone synthase II from soybean and, furthermore, closely related to isoflavone synthase from Pueraria lobata. Plant Physiol, 1994 May, 105(1), 69 - 79 Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases); Laquel P et al.; DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis . Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits . Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD . Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD . After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed . In the absence of DNA polymerases, the purified primase synthesizes long RNA products . The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes . Lowering the primase concentration in the assay also favored short RNA primer synthesis . Several properties of the wheat DNA primase using oligoadenylate {oligo(rA)}-primed or unprimed polythymidilate templates were studied . The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration . Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM . We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer . Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants. Traffic, 2002 Oct, 3(10), 740 - 51 Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and Hgs, an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4; Katz M et al.; Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation . By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation . Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes . Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation . We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis . Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events. Plant Physiol, 1995 Nov, 109(3), 1115 - 1123 Purification and Partial Characterization of Tomato Extensin Peroxidase; Brownleader MD et al.; Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall . The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases . We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill . and Lycopersicon peruvianum L . {Mill.}) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography . Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin . The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined . Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked . We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato . A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo . A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense. Plant Physiol, 1995 Sep, 109(1), 277 - 284 Changes in Protein Isoprenylation during the Growth of Suspension-Cultured Tobacco Cells; Morehead TA et al.; Isoprenylation facilitates the association of proteins with intracellular membranes and/or other proteins . In mammalian and yeast cells, isoprenylated proteins are involved in signal transduction, cell division, organization of the cytoskeleton, and vesicular transport . Recently, protein isoprenylation has been demonstrated in higher plants, but little is currently known about the functions of isoprenylated plant proteins . We report that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (lovastatin) or prenyl:protein transferases (perilly alcohol) severely impair the growth of cultured tobacco (Nicotiana tabacum) cells but only when added within the first 2 d following transfer to fresh medium, before any increase in culture volume is detectable . This "window" of sensitivity to inhibitors of protein isoprenylation correlates temporally with an increase in {14C}mevalonate incorporation into tobacco cell proteins in vitro . We have also observed a marked increase in farnesyl:protein transferase activity at this early time in the growth of tobacco cultures . In contrast, type I geranylgeranyl:protein transferase activity does not change significantly during culture growth . Although these events coincide with the replication of DNA, I {mu}M lovastatin-treated cells are capable of DNA synthesis, suggesting that lovastatin-induced cell growth arrest is not due to inhibition of DNA replication . Together, these data support the hypothesis that protein isoprenylation is necessary for the early stages of growth of tobacco cultures. Plant Physiol, 1995 Jun, 108(2), 813 - 819 Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants; Roberts G et al.; We investigated the nature of the complex ATP activation kinetics of plant H+-ATPases . To this aim we analyzed that activation in three isolated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis thaliana . The isoforms were obtained by heterologous expression in endoplasmic reticulum of yeast . ATP stimulation was always with low affinity (K0.5 between 500 and 1800 {mu}M) . In addition, the curves were not Michaelian and displayed positive cooperativity . Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine exhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kinetics with negative cooperativity, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule . Under this condition the ATP activation kinetics was Michaelian or had a slight negative cooperativity and the K0.5ATP was reduced 3-fold . These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativity and kinetics competence of the E(ATP) and E(ATP)2 complexes . Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of the enzyme molecule. Plant Physiol, 1995 Feb, 107(2), 491 - 500 Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells; Okpodu CM et al.; Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture cells (C.M . Okpodu, W . Gross, W.F . Boss {1990} Plant Physiol 93: S-63) . We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography . The Mr of the enzyme was estimated to be 83,000 by gel filtration . PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots . The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate . Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8 . The enzyme required divalent cations . At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+ . Calcium from 0.01 {mu}M to 1 mM had no effect on the enzyme activity . The Km of the enzyme for ATP was estimated to be between 400 and 463 {mu}M . The enzyme was inhibited by adenosine (100 {mu}M); however, ADP (up to 100 {mu}M) had no effect on the activity . The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast. J Biol Chem, 2002 Nov 22, 277(47), 45091 - 8 Epub 2002 Sep 12. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1; Le Rouzic E et al.; The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein . Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA . Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex . An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells . This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein . Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope . This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA. Plant Physiol, 2002 Sep, 130(1), 58 - 67 Differential expression of a metallothionein gene during the presymbiotic versus the symbiotic phase of an arbuscular mycorrhizal fungus; Lanfranco L et al.; A full-length cDNA encoding a metallothionein (MT)-like polypeptide, designated GmarMT1, was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita (BEG34) . The GmarMT1 gene is composed of two exons separated by an 81-bp intron . It codes for a 65-amino acid polypeptide comprising a plant type 1 MT-like N-terminal domain and a C-terminal domain that is most closely related to an as-yet-uncharacterized fungal MT . As revealed by heterologous complementation assays in yeast, GmarMT1 encodes a functional polypeptide capable of conferring increased tolerance against Cd and Cu . The GmarMT1 RNA is expressed in both presymbiotic spores and symbiotic mycelia, even in the absence of metal exposure, but is significantly less abundant in the latter stage . An opposite pattern was observed upon Cu exposure, which up-regulated GmarMT1 expression in symbiotic mycelia but not in germinated spores . Together, these data provide the first evidence, to our knowledge, for the occurrence in an arbuscular mycorrhizal fungus of a structurally novel MT that is modulated in a metal and life cycle stage-dependent manner and may afford protection against heavy metals (and other types of stress) to both partners of the endomycorrhizal symbiosis. Plant Physiol, 1996 Dec, 112(4), 1659 - 1668 Solubilization, Partial Purification, and Characterization of a Binding Site for a Glycopeptide Elicitor from Microsomal Membranes of Tomato Cells; Fath A et al.; Purified glycopeptides derived from yeast invertase act as highly potent elicitors in suspension-cultured tomato (Lycopersicon esculentum {L.} Mill) cells, inducing ethylene biosynthesis and phenylalanine ammonia lyase half-maximally at concentrations of 1 to 5 nM . We previously demonstrated the presence of a high-affinity binding site that specifically recognized these glycopeptides in cells and microsomal membranes of tomato (C.W . Basse, A . Fath, T . Boller {1993} J Biol Chem 268: 14724-14731) . This elicitor-binding site was solubilized in an active form from the microsomal membranes using the neutral detergents n-dodecylmaltoside and n-dodecanoylsucrose and purified 67-fold in a single step by anion-exchange chromatography . Ligand saturation studies and competition experiments with unlabeled glycopeptides and glycans demonstrated that the detergent-solubilized elicitor-binding site retained the high affinity (Kd approximately 1-4 nM) and selectivity of the membrane-bound form . The binding site was found to have a high affinity for N-linked glycans with nine mannosyl residues from fungal glycoproteins, whereas it did not recognize the typical mammalian glycans with nine mannosyl residues, demonstrating further its high selectivity. Plant Physiol, 1996 Dec, 112(4), 1491 - 1497 In Vitro Prenylation of the Small GTPase Rac13 of Cotton; Trainin T et al.; Previous work (D.P . Delmer, J . Pear, A . Andrawis, D . Stalker {1995} Mol Gen Genet 248: 43-51) has identified a gene in cotton (Gossypium hirsutum), Rac13, that encodes a small, signal-transducing GTPase and shows high expression in the fiber at the time of transition from primary to secondary wall synthesis . Since Rac13 may be important in signal transduction pathway(s), regulating the onset of fiber secondary wall synthesis, we continue to characterize Rac13 by determining its ability to undergo posttranslational modification . In animals Rac proteins contain the C-terminal consensus sequence CaaL (where "a" can be any aliphatic residue), which is a site for geranylgeranylation (B.T . Kinsella, R.A . Erdman, W.A . Maltese {1994} J Biol Chem 266: 9786-9794) . We have identified activities in developing cotton fibers that resemble in specificity the geranylgeranyl- and farnesyltransferases of animals and yeast . In addition, using prenyltransferases from rabbit reticulocytes, we show that Rac13, having a C-terminal sequence of CAFL, can serve as an in vitro substrate for geranylgeranylation but not farnesylation . However, the presence of the uncommon penultimate F residue appears to slow the rate of prenylation considerably compared with other acceptors. Plant Physiol, 1996 Apr, 110(4), 1349 - 1359 Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif; Yalovsky S et al.; Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity . Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates . Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins . Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes . However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source . This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants . Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate . This modification required a conserved cysteine-cysteine motif . A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog . The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the {alpha} subunit of yeast Rab GGTase (bet4 ts) . These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity. J Biol Chem, 2002 Dec 6, 277(49), 47331 - 7 Epub 2002 Sep 10. Binding of the concave surface of the Sds22 superhelix to the alpha 4/alpha 5/alpha 6-triangle of protein phosphatase-1; Ceulemans H et al.; Functional studies of the protein phosphatase-1 (PP1) regulator Sds22 suggest that it is indirectly and/or directly involved in one of the most ancient functions of PP1, i.e . reversing phosphorylation by the Aurora-related protein kinases . We predict that the conserved portion of Sds22 folds into a curved superhelix and demonstrate that mutation to alanine of any of eight residues (Asp(148), Phe(170), Glu(192), Phe(214), Asp(280), Glu(300), Trp(302), or Tyr(327)) at the concave surface of this superhelix thwarts the interaction with PP1 . Furthermore, we show that all mammalian isoforms of PP1 have the potential to bind Sds22 . Interaction studies with truncated versions of PP1 and with chimeric proteins comprising fragments of PP1 and the yeast PP1-like protein phosphatase Ppz1 suggest that the site(s) required for the binding of Sds22 reside between residues 43 and 173 of PP1gamma(1) . Within this region, a major interaction site was mapped to a triangular region delineated by the alpha4-, alpha5-, and alpha6-helices . Our data also show that well known regulatory binding sites of PP1, such as the RVXF-binding channel, the beta12/beta13-loop, and the acidic groove, are not essential for the interaction with Sds22. J Biol Chem, 2002 Nov 22, 277(47), 45267 - 75 Epub 2002 Sep 10. Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH; Courbard JR et al.; In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR) . Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied . By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C . elegans counterpart Cel-WWP1 . Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases . They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions . We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments . We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation . In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination . Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling . In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling. Curr Biol, 2002 Sep 3, 12(17), 1519 - 23 Functional analysis of the tubulin-folding cofactor C in Arabidopsis thaliana; Kirik V et al.; The biogenesis of microtubules comprises several steps, including the correct folding of alpha- and beta-tubulin and heterodimer formation . In vitro studies and the genetic analysis in yeast revealed that, after translation, alpha- and beta-tubulin are processed by several chaperonins and microtubule-folding cofactors (TFCs) to produce assembly-competent alpha-/beta-tubulin heterodimers . One of the TFCs, TFC-C, does not exist in yeast, and a potential function of TFC-C is thus based only on the biochemical analysis . In this study and in a very recently published study by Steinborn and coworkers, the analysis of the Arabidopsis porcino (por) mutant has shown that TFC-C is important for microtubule function in vivo . The predicted POR protein shares weak amino acid similarity with the human TFC-C (hTFC-C) . Our finding that hTFC-C under the control of the ubiquitously expressed 35S promoter can rescue the por mutant phenotype shows that the POR gene encodes the Arabidopsis ortholog of hTFC-C . The analysis of plants carrying a GFP:POR fusion construct showed that POR protein is localized in the cytoplasm and is not associated with microtubules . While, in por mutants, microtubule density was indistinguishable from wild-type, their organization was affected. Curr Biol, 2002 Sep 3, 12(17), 1448 - 61 TOR deficiency in C . elegans causes developmental arrest and intestinal atrophy by inhibition of mRNA translation; Long X et al.; BACKGROUND: TOR is a phosphatidylinositol kinase (PIK)-related kinase that controls cell growth and proliferation in response to nutritional cues . We describe a C . elegans TOR homolog (CeTOR) and phenotypes associated with CeTOR deficiency . These phenotypes are compared with the response to starvation and the inactivation of a variety of putative TOR targets.RESULTS: Whether caused by mutation or RNA interference, TOR deficiency results in developmental arrest at mid-to-late L3, which is accompanied by marked gonadal degeneration and a pronounced intestinal cell phenotype . A population of refractile, autofluorescent intestinal vesicles, which take up the lysosomal dye Neutral Red, increases dramatically in size, while the number of normal intestinal vesicles and the intestinal cytoplasmic volume decrease progressively . This is accompanied by an increase in the gut lumen size and a compromise in the intestine's ability to digest and absorb nutrients . CeTOR-deficient larvae exhibit no significant dauer characteristics, but share some features with starved L3 larvae . Notably, however, starved larvae do not have severe intestinal atrophy . Inactivation of C . elegans p70S6K or TAP42 homologs does not reproduce CeTOR deficiency phenotypes, nor does inactivation of C . elegans TIP41, a putative negative regulator of CeTOR function, rescue CeTOR deficiency . In contrast, inactivating the C . elegans eIF-4G homolog and eIF-2 subunits results in developmental arrest accompanied by the appearance of large, refractile intestinal vesicles and severe intestinal atrophy resembling that of CeTOR deficiency.CONCLUSIONS: The developmental arrest and intestinal phenotypes of CeTOR deficiency are due to an inhibition of global mRNA translation . Thus, TOR is a major upstream regulator of overall mRNA translation in C . elegans, as in yeast. Int Rev Cytol, 2002, 220, 93 - 144 Endocytosis and the cytoskeleton; Qualmann B et al.; In this review we describe the potential roles of the actin cytoskeleton in receptor-mediated endocytosis in mammalian cells and summarize the efforts of recent years in establishing a relationship between these two cellular functions . With molecules such as dynamin, syndapin, HIP1R, Abp1, synaptojanin, N-WASP, intersectin, and cortactin a set of molecular links is now available and it is likely that their further characterization will reveal the basic principles of a functional interconnection between the membrane cytoskeleton and the vesicle-budding machinery . We will therefore discuss proteins involved in endocytic clathrin coat formation and accessory factors to control and regulate coated vesicle formation but we will also focus on actin cytoskeletal components such as the Arp2/3 complex, spectrin, profilin, and motor proteins involved in actin dynamics and organization . Additionally, we will discuss how phosphoinositides, such as PI(4,5)P2, small GTPases thought to control the actin cytoskeleton, such as Rho, Rac, and Cdc42, or membrane trafficking, such as Rab GTPases and ARF proteins, and different kinases may participate in the functional connection of actin and endocytosis . We will compare the concepts and different molecular mechanisms involved in mammalian cells with yeast as well as with specialized cells, such as epithelial cells and neurons, because different model organisms often offer complementary advantages for further studies in this thriving field of current cell biological research. Plant Physiol, 1997 Mar, 113(3), 841 - 852 The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus; Bogre L et al.; To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs . The cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured . The cdc2Ms kinase is activated at the G1/S transition when phosphate-starved cells from the G0 phase re-enter the cell cycle and remain active as cells transit the S, G2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa . In contrast, when cdc2Ms kinase was purified by binding to p13suc1, it was active only in the G2 and M phases . In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus . By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible . Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added . Furthermore, purified CDK inhibitors, such as the mouse p27kip1 and the yeast p40sic1, blocked the purified plant CDK activity. J Neurosci, 2002 Sep 15, 22(18), 7991 - 8001 Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels; Wen H et al.; Calmodulin (CaM) was identified as a KCNQ2 and KCNQ3 potassium channel-binding protein, using a yeast two-hybrid screen . CaM is tethered constitutively to the channel, in the absence or presence of Ca2+, in transfected cells and also coimmunoprecipitates with KCNQ2/3 from mouse brain . The structural elements critical for CaM binding to KCNQ2 lie in two conserved motifs in the proximal half of the channel C-terminal domain . Truncations and point mutations in these two motifs disrupt the interaction . The first CaM-binding motif has a sequence that conforms partially to the consensus IQ motif, but both wild-type CaM and a Ca2+-insensitive CaM mutant bind to KCNQ2 . The voltage-dependent activation of the KCNQ2/3 channel also shows no Ca2+ sensitivity, nor is it affected by overexpression of the Ca2+-insensitive CaM mutant . On the other hand, KCNQ2 mutants deficient in CaM binding are unable to generate detectable currents when coexpressed with KCNQ3 in CHO cells, although they are expressed and targeted to the cell membrane and retain the ability to assemble with KCNQ3 . A fusion protein containing both of the KCNQ2 CaM-binding motifs competes with the full-length KCNQ2 channel for CaM binding and decreases KCNQ2/3 current density in CHO cells . The correlation of CaM binding with channel function suggests that CaM is an auxiliary subunit of the KCNQ2/3 channel. J Neurosci, 2002 Sep 15, 22(18), 7879 - 91 The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner; Dhavan R et al.; Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission . Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease . Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified . To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen . In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39 . CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other . The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium . Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect . The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning. J Neurosci, 2002 Sep 15, 22(18), 7862 - 72 Caspase cleavage of mutant huntingtin precedes neurodegeneration in Huntington's disease; Wellington CL et al.; Huntington's disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites . Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo . Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration . Using antibodies that detect only caspase-cleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552 . We detect caspase-cleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote . Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration . These results suggest that caspase cleavage of htt may be a normal physiological event . However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract . Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD. J Biol Chem, 2002 Nov 22, 277(47), 45655 - 61 Epub 2002 Sep 09. EphB1 associates with Grb7 and regulates cell migration; Han DC et al.; EphB1 is a member of the Eph family of receptor tyrosine kinases that play important roles in diverse biological processes including nervous system development, angiogenesis, and neural synapsis formation and maturation . Grb7 is an adaptor molecule implicated in the regulation of cell migration . Here we report identification of an interaction between Grb7 and the cytoplasmic domain of EphB1 by using Grb7 as a "bait" in a yeast two-hybrid screening . Co-immunoprecipitation was used to confirm the interaction of Grb7 with the cytoplasmic domain of EphB1 as well as the full-length receptor in intact cells . This interaction is mediated by the SH2 domain of Grb7 and requires tyrosine autophosphorylation of EphB1 . Furthermore, Tyr-928 of EphB1 was identified as the primary binding site for Grb7 . Stimulation of endogenous EphB1 in embryonal carcinoma P19 cells with its ligand ephrinB1 increased its association with Grb7, which is consistent with a role for the autophosphorylation of EphB1 . We also found that EphB1 could phosphorylate Grb7 and mutation of either Tyr-928 or Tyr-594 to Phe decreased this activity . Finally, we show that EphB1 could stimulate fibroblast motility on extracellular matrix in a kinase-dependent manner, which also correlated with its association with Grb7 . Consistent with this, co-expression of Grb7 with EphB1 further enhanced cell motility, whereas co-expression of the Grb7 SH2 domain abolished EphB1-stimulated cell migration . Together, our results identified a novel interaction between EphB1 with the adaptor molecule Grb7 and suggested that this interaction may play a role in the regulation of cell migration by EphB1. J Microbiol Methods, 2002 Nov, 51(3), 411 - 6 Media for cultivation of indoor streptomycetes; Suutari M et al.; The growth of 10 indoor Streptomyces spp . isolates on nutritionally complex and selective 26 media revealed that the mycelium production had a tendency to increase in order: starch-casein < glycerol-arginine < glucose-tryptone, and NH(4)NO(3) < Na-caseinate-asparagine . Yeast extract increased mycelium biosynthesis, but not always the growth rate . The strains belonging to streptomycetes most common environmental isolates produced visible mycelium in 5 days on all media . Fungal Genet Biol, 2002 Oct, 37(1), 39 - 48 Expression of a Pho85 cyclin-dependent kinase is repressed during the dimorphic transition in Sporothrix schenckii; de Jesus-Berrios M et al.; Sporothrix schenckii is a pathogenic fungus that undergoes a dimorphic transition from yeast to mycelium in response to environmental conditions such as cell density, temperature, and calcium . We identified a homolog of the Pho85 cyclin-dependent kinase (Cdk) that mediates cellular responses to environmental conditions in other organisms . By Western blot, three proteins containing the PSTAIRE motif, which characterize the cyclin-dependent protein kinases, were identified in S . schenckii . The gene encoding a Pho85 homolog, PhoSs, was identified and sequenced . The phoSs gene consists of 990bp, contains one intron, and encodes a protein of 306 amino acids . The S . schenckii Pho85 homolog shares features with Cdks, including the PSTAIRE motif, an ATP binding domain, and a serine-threonine kinase domain . By quantitative competitive RT-PCR, expression of the phoSs gene was found to decrease 30-fold during the yeast to mycelium transition . The addition of extracellular calcium accelerated the dimorphic transition and restored phoSs expression . These findings suggest PhoSs may participate in the control of the yeast to mycelium transition in S . schenckii. J Dairy Res, 2002 May, 69(2), 281 - 92 Influence of selected factors on browning of Camembert cheese; Carreira A et al.; Experimental Camembert cheeses were made to investigate the effects on browning of the following factors: inoculation with Yarrowia lipolytica, the use of Penicillium candidum strains with different proteolytic activity, the addition of tyrosine, and the addition of Mn2+ thus leading to 16 different variants of cheese . Two physical colour parameters were used to describe browning, depending on the location in the cheeses: a whiteness index for the outside browning (mould mycelium), and a brownness index for the inside browning (surface of the cheese body) . Mn2+ promoted a significant increase of browning at both locations, whereas Yar . lipolytica had the opposite effect . Outside browning was significantly more intense when using the Pen . candidum strain with higher proteolytic activity . A significant interaction was found between Yar . lipolytica and Pen . candidum . The yeast had no effect in combination with a low proteolytic strain of Pen . candidum, but significantly reduced proteolysis and browning in combination with a high proteolytic strain of Pen . candidum . We further confirmed that both strains of Pen . candidum were able to produce brown pigments from tyrosine and thus both are presumably responsible for the browning activity in this type of cheese. Biol Chem, 2002 Jun, 383(6), 873 - 92 DNA double-strand break repair by homologous recombination; van den Bosch M et al.; The induction of double-strand breaks (DSBs) in DNA by exposure to DNA damaging agents, or as intermediates in normal cellular processes, constitutes a severe threat for the integrity of the genome . If not properly repaired, DSBs may result in chromosomal aberrations, which, in turn, can lead to cell death or to uncontrolled cell growth . To maintain the integrity of the genome, multiple pathways for the repair of DSBs have evolved during evolution: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) . HR has the potential to lead to accurate repair of DSBs, whereas NHEJ and SSA are essentially mutagenic . In yeast, DSBs are primarily repaired via high-fidelity repair of DSBs mediated by HR, whereas in higher eukaryotes, both HR and NHEJ are important . In this review, we focus on the functional conservation of HR from fungi to mammals and on the role of the individual proteins in this process. J Nutr, 2002 Sep, 132(9), 2494 - 505 Effects on the human serum lipoprotein profile of beta-glucan, soy protein and isoflavones, plant sterols and stanols, garlic and tocotrienols; Kerckhoffs DA et al.; The effects of beta-glucan, soy protein, isoflavones, plant sterols and stanols, garlic and tocotrienols on serum lipoproteins have been of great interest the last decade . From a critical review of the literature, it appeared that recent studies found positive as well as no effects of beta-glucan from oats on serum LDL cholesterol concentrations . These conflicting results may suggest that the cholesterol-lowering activity of products rich in oat beta-glucan depends on factors, such as its viscosity in the gastrointestinal tract, the food matrix and/or food processing . The effects of beta-glucan from barley or yeast on the lipoprotein profile are promising, but more human trials are needed to further substantiate these effects . It is still not clear whether the claimed hypocholesterolemic effects of soy can be attributed solely to the isoflavones . Several studies found no changes in serum LDL cholesterol concentrations after consumption of isolated soy isoflavones (without soy protein), indicating that a combination of soy protein and isoflavones may be needed for eliciting a cholesterol-lowering effect of soy . Therefore, the exact (combination of) active ingredients in soy products need to be identified . The daily consumption of 2-3 g of plant sterols or stanols reduces LDL cholesterol concentrations by 9-14% . It has been demonstrated that functional foods enriched with plant sterols and stanols are effective in various population groups, and in combination with cholesterol-lowering diets or drugs . Whether garlic or garlic preparations can be used as a lipid-lowering agent is still uncertain . It is important to characterize the active components in garlic and their bioavailability after ingestion . It is not very likely that tocotrienols from palm oil or rice bran oil have favorable effects on the human serum lipoprotein profile. Mol Biol Cell, 2002 Sep, 13(9), 3268 - 80 A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport; Hattula K et al.; The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells . We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules . To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators . We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5 . Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8 . Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains . Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures . Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions . The C-terminal region of Rabin8 plays an essential role in this transport . We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures. Mol Biol Cell, 2002 Sep, 13(9), 3235 - 45 Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex; Takahashi M et al.; Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC) . In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus . However, mammalian protein that anchors gamma-TuRC remains to be elucidated . A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region . This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin . The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3 . Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo . These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes . Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition . These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC. J Biol Chem, 2002 Nov 15, 277(46), 44013 - 20 Epub 2002 Sep 06. Inhibitor-2 regulates protein phosphatase-1 complexed with NimA-related kinase to induce centrosome separation; Eto M et al.; Centrosome separation is regulated by balance of in situ protein kinase/phosphatase activities during the cell cycle . The mammalian NimA-related kinase Nek2 forms a complex with the catalytic subunit of protein phosphatase-1 (PP1C) . This complex is located at centrosomes and has been implicated in regulation of the cycle of duplication and separation . Inhibitor-2 (Inh2) is an inhibitor protein specific for PP1C, and its expression level fluctuates during the cell cycle . Here we report cellular regulation of the Nek2.PP1C complex by Inh2 . PP1C-binding segments of Nek2 were isolated by yeast two-hybrid screening using Inh2 bait . Inh2 indirectly associates with Nek2 via PP1C, which binds to both proteins, forming a bridged heterotrimeric complex . Double Ala mutation of the PP1C-binding site (KVHF) in Nek2 eliminated both PP1C and Inh2 interactions in both a yeast conjugation assay and an in vitro binding assay . The kinase activity of Nek2.PP1C was enhanced 2-fold by addition of recombinant Inh2, with EC(50) = 10 nm . Immunofluorescence showed concentration of endogenous Inh2 at centrosomes and in a region surrounding the centrosomes . Transient expression of wild-type Inh2 increased by 5-fold dispersed/split centrosomes in fibroblasts, mimicking the phenotype produced by overexpression of Nek2 . Deletion of the Inh2 C-terminal domain yielded Inh2-(1-118), which failed to interact with or activate the Nek2.PP1C complex, suggesting that the C-terminal region of Inh2 is required for regulation of the Nek2.PP1C complex . Thus, Inh2 can enhance the kinase activity of the Nek2.PP1C complex via inhibition of phosphatase activity to initiate centrosome separation. Trends Cell Biol, 2002 Sep, 12(9), 407 - 9 Location, location, location: translational control in development and neurobiology; Mendez R et al.; Control of gene expression by regulating mRNA translation is essential for proper growth and development in species ranging from yeast to mammals . Recently, translational regulation has also been implicated in neuronal function, where long-lasting changes in synaptic strength and the formation of memory require new protein synthesis . Crucial to both developmental and neurobiological function, translational control allows for the asymmetric distribution of gene products and a rapid and local response to stimulation--as discussed at a recent Serono Foundation EMBO Workshop held in Mallorca, Spain. FEBS Lett, 2002 Sep 11, 527(1-3), 176 - 80 Characterization of the spermidine synthase-related gene family in Arabidopsis thaliana; Hanzawa Y et al.; The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms . Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes . Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3 . Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase-deficient mutants, but the enzyme activity of SPDS3 remained to be determined . RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells. FEBS Lett, 2002 Sep 11, 527(1-3), 71 - 5 The COOH-terminus of parathyroid hormone-related protein (PTHrP) interacts with beta-arrestin 1B; Conlan LA et al.; Parathyroid hormone-related protein (PTHrP) has a diverse range of proposed biological activities participating in both extracellular and intracellular signaling . In order to identify candidate protein effectors, yeast two-hybrid screens were conducted using mature human PTHrP (residues 1-141) and the COOH-terminus (residues 107-141) . Both PTHrP baits interacted with a beta-arrestin 1B fragment, an important component of G-protein-coupled receptor desensitization and MAPK signaling . Co-immunoprecipitation, in vitro binding assays and colocalization experiments confirmed this interaction in human cells and this required residues 122-141 of PTHrP . These findings suggest that beta-arrestin 1 acts as an effector for a novel function of PTHrP in cytoplasm. Biochem Biophys Res Commun, 2002 Sep 13, 297(1), 148 - 53 HIPK2 associates with RanBPM; Wang Y et al.; Using the yeast two-hybrid system, we have identified the Ran-binding protein (RanBPM) as an interaction partner of homeodomain-interacting protein kinase 2 (HIPK2) . RanBPM has been described as a centrosomal protein through which Ran regulates the centrosomal function . HIPK2 is mainly a nuclear protein, which among other functions represses transcription mediated by homeodomain containing transcription factors . Here, we show that overexpressed wildtype HIPK2 and a kinase defective mutant of HIPK2 directly interact with RanBPM in the nucleus of mammalian cells . Overexpressed wildtype RanBPM and a kinase defective mutant of HIPK2 co-localise with HIPK2 in defined nuclear structures . A carboxy- and an amino-terminal deletion of HIPK2 do not seem to be able to bind to RanBPM. Biochem Biophys Res Commun, 2002 Sep 13, 297(1), 17 - 23 A new ERK2 binding protein, Naf1, attenuates the EGF/ERK2 nuclear signaling; Zhang S et al.; Extracellular signal regulated kinase1/2 (ERK1/2), an important factor in signal transduction, controls cell growth, differentiation, and death . To elucidate the details of the mechanism of ERK1/2 signaling in human cells, we isolated Nef-associated factor 1 alpha (Naf1 alpha) by a yeast two-hybrid system, which bound to human ERK2 . The binding was confirmed by a pull-down assay in vitro and immunoprecipitation in vivo . Upon EGF treatment, Naf1 alpha was phosphorylated by the EGF/MEK/ERK2 signal transduction pathway . To identify the role of Naf1 alpha in the ERK2 signaling, Naf1 alpha-expressing Saos-2 cells were analyzed for ERK2 nuclear translocation and activation of its downstream target . Overexpression of Naf1 alpha suppressed ERK2 entering into the nucleus and inhibited the ERK2-dependent Elk1-driven luciferase transcription, suggesting Naf1 alpha to be an attenuator of activated ERK2 signaling. Biochem Biophys Res Commun, 2002 Sep 13, 297(1), 1 - 5 Gene targeting by homologous recombination: a powerful addition to the genetic arsenal for Drosophila geneticists; Rong YS; A series of recent publications have firmly established the notion that Drosophila researchers now have a general method to subject genes for targeted modification by homologous recombination (HR) {Science 288 (2000) 2013; Genetics 157 (3) (2001) 1307; Genes Dev . 16 (12) (2002) 1568; Genetics 161 (2002) 1125-1136} . This method allows one to knockout essentially any gene starting with the DNA sequence of the gene . It has greatly enhanced studies of gene function as demonstrated by over 20 years of gene targeting practice in yeast and mouse . Here, I discuss the basic targeting methodology for eukaryotic organisms . I compare the Drosophila method with the traditional targeting scheme in yeast and mouse mainly to show that the targeting mechanism as well as many aspects of the experimental design remain unchanged, and that the Drosophila scheme differs only in the way in which the donor molecule for targeting is generated . I propose that the Drosophila method can be readily adapted in other organisms without culturable stem cells, since the mechanism for in vivo donor generation in Drosophila is likely to be functional in a variety of different organisms. Cell Struct Funct, 2002 Jun, 27(3), 145 - 55 Evidence of a novel dipeptidyl aminopeptidase in mammalian GH(3) cells: new insights into the processing of peptide hormone precursors; Cheong KH et al.; We investigated whether yeast signals could regulate hormone processing in mammalian cells . Chmeric genes coding for the prepro region of yeast alpha-factor and the functional hormone region of anglerfish somatostatin was expressed in rat pituitary GH(3) cells . The nascent prepro-alpha-factor-somatostatin peptides disappeared from cells with a half-life of 30 min, and about 20% of unprocessed precursors remained intracellular after a 2 h chase period . Disappearance of propeptide was insensitive to lysosomotropic agents, but was inhibited at 15 degrees C or 20 degrees C, suggesting that the hybrid propeptides were not degraded in the secretory pathway to the trans Golgi network or in lysosomes . It appeared that while most unprocessed precursors were constitutively secreted into the medium, a small portion were processed at their paired dibasic sites by prohormone-processing enzymes located in trans Golgi network/secretory vesicles, resulting in the production of mature somatostatin peptides . To test this hypothesis, we investigated the processing pattern of two different hybrid precursors: the 52-1 hybrid precursor, which has a Glu-Ala spacer between the prepro region of alpha-factor and somatostatin, and the 58-1 hybrid precursor, which lacks the Glu-Ala spacer . Processing of metabolically labeled hybrid propeptides to smaller somatostatin peptides was assessed by HPLC . When pulse-labeled cells were chased for up to 2 h, 68% of the initially synthesized propeptides were secreted constitutively . About 22% of somatostatin-related products were proteolytically processed to mature somatostatin, of which 38.7% were detected intracellularly after 2 h . From N-terminal peptide sequence determination of somatostatin-related products in GH(3)-52 and GH(3)-58 cells, we found that both hybrid precursors were accurately cleaved at their dibasic amino acid sites . Notably, we also observed that the Glu-Ala spacer sequence was removed from 52-1 hybrid precursors . The latter result strongly suggests that a novel dipeptidyl aminopeptidase activity - a yeast STE13-like enzyme - is present in the post-trans Golgi network compartment of GH(3) cells . The data from these studies indicate that mechanisms which control protein secretion are conserved between yeast and mammalian cells. Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 166 - 73 A concerted, alternating sites mechanism of ubiquinol oxidation by the dimeric cytochrome bc(1) complex; Trumpower BL; A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism . A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation . The potential of the Rieske iron-sulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c(1) . A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol-cytochrome c reductase kinetics of the bc(1) complex include both a pH dependence and a dependence on Rieske iron-sulfur protein midpoint potential.An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc(1) complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex . One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound . An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time . This provides a raison d'etre for the dimeric structure of the enzyme, in that bc(1) activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme. Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 44 - 7 Peroxisomes: surprisingly versatile organelles; Veenhuis M et al.; Peroxisome development is a dynamic process that is not yet completely understood . We use the methylotrophic yeast Hansenula polymorpha as model in our studies on peroxisome homeostasis . Cells of this species may contain different types of peroxisomes that differ in protein composition and capacity to incorporate matrix proteins . This protein import machinery is highly flexible and can accommodate unfolded and complex folded proteins. Nat Biotechnol, 2002 Oct, 20(10), 1041 - 4 Epub 2002 Sep 03. A light-switchable gene promoter system; Shimizu-Sato S et al.; Regulatable transgene systems providing easily controlled, conditional induction or repression of expression are indispensable tools in biomedical and agricultural research and biotechnology . Several such systems have been developed for eukaryotes . Most of these rely on the administration of either exogenous chemicals or heat shock . Despite the general success of many of these systems, the potential for problems, such as toxic, unintended, or pleiotropic effects of the inducing chemical or treatment, can impose limitations on their use . We have developed a promoter system that can be induced, rapidly and reversibly, by short pulses of light . This system is based on the known red light-induced binding of the plant photoreceptor phytochrome to the protein PIF3 and the reversal of this binding by far-red light . We show here that yeast cells expressing two chimeric proteins, a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion, are induced by red light to express selectable or "scorable" marker genes containing promoters with a GAL4 DNA-binding site, and that this induction is rapidly abrogated by subsequent far-red light . We further show that the extent of induction can be controlled precisely by titration of the number of photons delivered to the cells by the light pulse . Thus, this system has the potential to provide rapid, noninvasive, switchable control of the expression of a desired gene to a preselected level in any suitable cell by simple exposure to a light signal. Mod Pathol, 2002 Sep, 15(9), 939 - 43 Lymphadenopathy due to Penicillium marneffei infection: diagnosis by fine needle aspiration cytology; Chaiwun B et al.; Penicillium marneffei is an opportunistic fungal infection that usually causes disseminated disease, mainly in immunocompromised individuals, especially those with HIV infection . Untreated cases are usually fatal . Diagnosis is traditionally made by biopsy and/or culture; successful diagnosis by fine needle aspiration (FNA) has only been reported once . We present eight cases of HIV-infected patients with lymphadenopathy caused by P . marneffei infection, in which the diagnosis was made by FNA . In all cases, intracellular and extracellular yeast forms were visualized, and the characteristic cross-septation of P . marneffei was highlighted by GMS staining . All diagnoses were confirmed by culture . Anti-fungal treatment for P . marneffei was initiated, resulting in marked clinical improvement . We conclude that a diagnosis of lymphadenopathy caused by P . marneffei can reliably be made by FNA . The diagnosis is more rapid than biopsy or culture, allowing rapid institution of therapy, particularly important in immunocompromised patients . In all our cases, not only were lymphoma and other causes of lymphadenopathy ruled out, but also the necessity for an open surgical biopsy was obviated . This can be especially beneficial to patients (e.g., three in our study) in which lymphadenopathy is confined to deep intra-abdominal nodes. Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12079 - 84 Epub 2002 Sep 06. Cytoplasmic poly(A) polymerases mediate cellular responses to S phase arrest; Read RL et al.; The S-M checkpoint delays mitosis until DNA replication is complete; cells defective in this checkpoint lose viability when DNA replication is inhibited . This inviability can be suppressed in fission yeast by overexpression of Cid1 or the related protein Cid13 . Fission yeast contain six cid1/cid13-like genes, whereas budding yeast has just two, TRF4 and TRF5 . Trf4 and Trf5 were recently reported to comprise an essential DNA polymerase activity required for the establishment of sister chromatid cohesion . In contrast, we find that Cid1 is not a DNA polymerase but instead uses RNA substrates and has poly(A) polymerase activity . Unlike the previously characterized yeast poly(A) polymerase, which is a nuclear enzyme, Cid1 and Cid13 are constitutively cytoplasmic . Cid1 has a degree of substrate specificity in vitro, consistent with the notion that it targets a subset of cytoplasmic mRNAs for polyadenylation in vivo, hence increasing their stability and/or efficiency of translation . Preferred Cid1 targets presumably include mRNAs encoding components of the S-M checkpoint, whereas Cid13 targets are likely to be involved in dNTP metabolism . Cytoplasmic polyadenylation is known to be an important regulatory mechanism during early development in animals . Our findings in yeast suggest that this level of gene regulation is of more general significance in eukaryotic cells. Gynecol Oncol, 2002 Sep, 86(3), 288 - 96 Bax, Bcl-2, and p53 expression in endometrial cancer; Sakuragi N et al.; BACKGROUND: It has not been fully clarified whether alteration of Bax and other apoptosis-relating proteins of Bcl-2 and p53 is involved in endometrial carcinogenesis . METHODS: A total of 56 frozen tissues, which included 14 normal endometria, 13 endometrial hyperplasias (10 without atypia and 3 with atypia), and 29 endometrial carcinomas, were examined for the expression of Bax, Bcl-2, and p53 using immunohistochemistry . For Bax-negative cases, PCR-direct sequencing was performed for the bax gene . For cases with p53 overexpression, mutational analysis was performed for the p53 gene using a yeast functional assay and sequencing . RESULTS: Both Bax and Bcl-2 were distinctly expressed in the normal proliferative phase endometrium . A decreased Bcl-2/Bax ratio in the secretory phase endometrial gland cells due to suppressed Bcl-2 expression was observed . Bax expression was positive in all 13 endometrial hyperplasias, while it was absent in 6 of 29 endometrial carcinomas (20.7%) . Negative Bax expression in endometrial carcinoma was not related to tumor stage, histologic subtype, or other histopathologic prognostic factors . Bax expression showed no relationship to either p53 overexpression or Bcl-2 expression . In the DNA of 6 Bax-negative cases, we found a frameshift insertion mutation at codon 58 (AAG to CAAG) in the BH3 domain despite the absence of mutation in the (G)8 tract, suggesting that this codon may be another preferred target for bax mutation other than the (G)8 tract . Mutational analysis was available for 7 of 10 cases with p53 overexpression, in which 5 cases were found to have a missense mutation and 2 cases had no mutation of the p53 gene . At least 10 of 29 (34.5%) cases of endometrial carcinoma were associated with sequence-verified mutation in the bax gene and/or p53 gene . CONCLUSIONS: The bax gene frameshift mutation appears to cause a loss of Bax expression in endometrial carcinoma . Codon 58 may be a preferred target of bax gene mutation in endometrial carcinomas . The bax gene mutation seems to occur in the early stage of the genesis of a subset of endometrial carcinomas. Science, 2002 Sep 27, 297(5590), 2232 - 7 Epub 2002 Sep 05. Establishment and maintenance of a heterochromatin domain; Hall IM et al.; The higher-order assembly of chromatin imposes structural organization on the genetic information of eukaryotes and is thought to be largely determined by posttranslational modification of histone tails . Here, we study a 20-kilobase silent domain at the mating-type region of fission yeast as a model for heterochromatin formation . We find that, although histone H3 methylated at lysine 9 (H3 Lys9) directly recruits heterochromatin protein Swi6/HP1, the critical determinant for H3 Lys9 methylation to spread in cis and to be inherited through mitosis and meiosis is Swi6 itself . We demonstrate that a centromere-homologous repeat (cenH) present at the silent mating-type region is sufficient for heterochromatin formation at an ectopic site, and that its repressive capacity is mediated by components of the RNA interference (RNAi) machinery . Moreover, cenH and the RNAi machinery cooperate to nucleate heterochromatin assembly at the endogenous mat locus but are dispensable for its subsequent inheritance . This work defines sequential requirements for the initiation and propagation of regional heterochromatic domains. Mol Cell Biol, 2002 Oct, 22(19), 6681 - 8 The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice; Zhang P et al.; The GCN2 eIF2alpha kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth . GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids . To investigate the function of mammalian GCN2, we have generated a Gcn2(-/-) knockout strain of mice . Gcn2(-/-) mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions . However, prenatal and neonatal mortalities are significantly increased in Gcn2(-/-) mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation . Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice . Cultured embryonic stem cells derived from Gcn2(-/-) mice failed to show the normal induction of eIF2alpha phosphorylation in cells deprived of leucine . To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted . Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2alpha and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2(-/-) mice. Plant Cell, 2002 Sep, 14(9), 2277 - 87 A cysteine-rich extracellular protein, LAT52, interacts with the extracellular domain of the pollen receptor kinase LePRK2; Tang W et al.; Pollen germination and pollen tube growth are thought to require extracellular cues, but how these cues are perceived and transduced remains largely unknown . Pollen receptor kinases are plausible candidates for this role; they might bind extracellular ligands and thereby mediate cytoplasmic events required for pollen germination and pollen tube growth . To search for pollen-expressed ligands for pollen receptor kinases, we used the extracellular domains of three pollen-specific receptor kinases of tomato (LePRK1, LePRK2, and LePRK3) as baits in a yeast two-hybrid screen . We identified numerous secreted or plasma membrane-bound candidate ligands . One of these, the Cys-rich protein LAT52, was known to be essential during pollen hydration and pollen tube growth . We used in vivo coimmunoprecipitation to demonstrate that LAT52 was capable of forming a complex with LePRK2 in pollen and to show that the extracellular domain of LePRK2 was sufficient for the interaction . Soluble LAT52 can exist in differently sized forms, but only the larger form can interact with LePRK2 . We propose that LAT52 might be a ligand for LePRK2. Plant Cell, 2002 Sep, 14(9), 2265 - 76 The Arabidopsis TUBULIN-FOLDING COFACTOR A gene is involved in the control of the alpha/beta-tubulin monomer balance; Kirik V et al.; The control of the stoichiometric balance of alpha- and beta-tubulin is important during microtubule biogenesis . This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast . Here, we show that the TFC A gene is important in vivo in plants . The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene . Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A . kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched . Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants . The kis phenotype was rescued by overexpression of an alpha-tubulin, indicating that KIS is involved in the control of the correct balance of alpha- and beta-tubulin monomers. Plant Cell, 2002 Sep, 14(9), 2251 - 64 Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5' ADH1 matrix attachment region; Brouwer C et al.; Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix . Thus, they may define discrete gene-containing chromatin loops in vivo . We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants . Three MAR elements, two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro . In callus, two of the MAR elements (Adh1 5' MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression . In transgenic pl