Pigment Cell Res, 2000 Feb, 13(1), 15 - 20 Hermansky-Pudlak syndrome and pale ear: melanosome-making for the millennium; Spritz RA; Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid-lipofuscin lysosomal storage disease . These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles--melanosomes, platelet granules, and lysosomes--suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles . The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified . Functional studies indicate that the HPS protein is part of a high-molecular weight complex involved in the biogenesis of early melanosomes . Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles. Jpn J Cancer Res, 2000 Mar, 91(3), 293 - 300 Mapping of target regions of allelic loss in primary breast cancers to 1-cM intervals on genomic contigs at 6q21 and 6q25.3; Utada Y et al.; Allelic losses on the long arm of human chromosome 6 are frequently observed in cancers of the ovary, prostate, and breast . To identify the locations of putative tumor suppressor genes on 6q, we examined 192 primary breast cancers for patterns of allelic loss at 16 polymorphic microsatellite loci distributed along this chromosome arm . Allelic losses at one or more loci were observed in 105 (55%) of the tumors examined . Detailed deletion mapping with appropriate yeast artificial chromosome (YAC) contigs identified two distinct commonly deleted regions; one was confined to a 1-cM interval at 6q21 flanked by D6S1040 and D6S262 and the other to a 1-cM interval at 6q25.3 flanked by D6S305 and D6S411 . Allelic losses at 6q21 were more frequent in invasive solid tubular and scirrhous carcinomas than in tumors of less aggressive histologic types (P = 0.0006) . Allelic loss at 6q25.3 was associated with loss of progesterone receptor (P = 0.0256) . Our results suggest the presence of two tumor suppressor genes for breast cancer on 6q that are likely to be associated with tumor progression and / or loss of hormonal dependency. FEBS Lett, 2000 Apr 7, 471(1), 17 - 22 An ARID family protein binds to the African swine fever virus encoded ubiquitin conjugating enzyme, UBCv1; Bulimo WD et al.; The NH(2)-terminal end of a protein, named SMCp, which contains an ARID (A/T rich interaction domain) DNA binding domain and is similar to the mammalian SMCY/SMCX proteins and retinoblastoma binding protein 2, was shown to bind the African swine fever virus encoded ubiquitin conjugating enzyme (UBCv1) using the yeast two hybrid system and in in vitro binding assays . Antisera raised against the SMCp protein were used to show that the protein is present in the cell nucleus . Immunofluorescence showed that although UBCv1 is present in the nucleus in most cells, in some cells it is in the cytoplasm, suggesting that it shuttles between the nucleus and cytoplasm . The interaction and co-localisation of UBCv1 with SMCp suggest that SMCp may be a substrate in vivo for the enzyme. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4233 - 8 Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2; Wu X et al.; PTEN is a tumor suppressor gene mutated in human cancers . Although many mutations target the phosphatase domain, others create a truncated protein lacking the C-terminal PDZ-binding motif or a protein that extends beyond the PDZ-binding motif . Using the yeast two-hybrid system, we isolated a membrane-associated guanylate kinase family protein with multiple PDZ domains {AIP-1 (atrophin interacting protein 1), renamed MAGI-2 (membrane associated guanylate kinase inverted-2)} . MAGI-2 contains eight potential protein-protein interaction domains and is localized to tight junctions in the membrane of epithelial cells . PTEN binds to MAGI-2 through an interaction between the PDZ-binding motif of PTEN and the second PDZ domain of MAGI-2 . MAGI-2 enhances the ability of PTEN to suppress Akt activation . Furthermore, certain PTEN mutants have reduced stability, which is restored by adding the minimal PDZ-binding motif back to the truncated protein . We propose that MAGI-2 improves the efficiency of PTEN signaling through assembly of a multiprotein complex at the cell membrane. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4023 - 8 Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression; da Silva Xavier G et al.; Elevated glucose concentrations stimulate the transcription of the pre-proinsulin (PPI), L-type pyruvate kinase (L-PK), and other genes in islet beta cells . In liver cells, pharmacological activation by 5-amino-4-imidazolecarboxamide riboside (AICAR) of AMP-activated protein kinase (AMPK), the mammalian homologue of the yeast SNF1 kinase complex, inhibits the effects of glucose, suggesting a key signaling role for this kinase . Here, we demonstrate that AMPK activity is inhibited by elevated glucose concentrations in MIN6 beta cells and that activation of the enzyme with AICAR prevents the activation of the L-PK gene by elevated glucose . Furthermore, microinjection of antibodies to the alpha2- (catalytic) or beta2-subunits of AMPK complex, but not to the alpha1-subunit or extracellular stimulus-regulated kinase, mimics the effects of elevated glucose on the L-PK and PPI promoter activities as assessed by single-cell imaging of promoter luciferase constructs . In each case, injection of antibodies into the nucleus and cytosol, but not the nucleus alone, was necessary, indicating the importance of either a cytosolic phosphorylation event or the subcellular localization of the alpha2-subunits . Incubation with AICAR diminished, but did not abolish, the effect of glucose on PPI transcription . These data suggest that glucose-induced changes in AMPK activity are necessary and sufficient for the regulation of the L-PK gene by the sugar and also play an important role in the regulation of the PPI promoter. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4017 - 22 The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton; Gallagher AR et al.; Despite the recent positional cloning of the PKD1 and PKD2 genes, which are mutated in the great majority of patients with autosomal-dominant polycystic kidney disease (PKD), the pathogenic mechanism for cyst formation is still unclear . The finding, that the PKD1 and PKD2 proteins interact with each other through their COOH termini, suggests that both proteins are part of the same protein complex or signal transduction pathway . Using a yeast two-hybrid screen with the PKD2 protein, we isolated the PKD2-interacting protein Hax-1 . The specificity of the interaction was demonstrated by the fact that PKD2L, a protein closely related to PKD2, failed to interact with Hax-1 . Immunofluorescence experiments showed that in most cells PKD2 and Hax-1 colocalized in the cell body, but in some cells PKD2 and Hax-1 also were sorted into cellular processes and lamellipodia . Furthermore we demonstrated an association between Hax-1 and the F-actin-binding protein cortactin, which suggests a link between PKD2 and the actin cytoskeleton . We speculate that PKD2 is involved in the formation of cell-matrix contacts, which are dysfunctional without a wild-type PKD2 protein, thus leading to cystic enlargement of tubular structures in the kidney, liver, and pancreas. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3999 - 4004 MIR16, a putative membrane glycerophosphodiester phosphodiesterase, interacts with RGS16; Zheng B et al.; We have identified the protein MIR16 (for Membrane Interacting protein of RGS16) from a yeast two-hybrid screen by using RGS16 as bait . MIR16 shares strong homology with bacterial glycerophosphodiester phosphodiesterases . It interacts with RGS16 and, more weakly, with several other selected RGS proteins . Analysis of deletion mutants showed that the N-terminal region of the RGS domain in RGS16 is required for its interaction with MIR16 . MIR16 is an integral membrane glycoprotein, because it remained associated with membrane fractions after alkaline treatment and because, in some cells, it is sensitive to digestion with endoglycosidase H . By immunofluorescence and immunoelectron microscopy, MIR16 was localized on the plasma membrane in liver and kidney and on intracellular membranes in rat pituitary and cultured pituitary cells . MIR16 represents the only integral membrane protein identified thus far to interact with an RGS domain and, to our knowledge, is the only mammalian glycerophosphodiester phosphodiesterase that has been cloned . The putative enzymatic activity of MIR16 and its interaction with RGS16 suggest that it may play important roles in lipid metabolism and in G protein signaling. Genes Cells, 2000 Mar, 5(3), 191 - 202 NF-kappaB activation through IKK-i-dependent I-TRAF/TANK phosphorylation; Nomura F et al.; BACKGROUND: NF-kappaB is an ubiquitously expressed transcription factor that plays an important role in the immune, anti-apoptotic and inflammatory responses . NF-kappaB is normally sequestered in the cytoplasm by interacting with inhibitory IkappaB molecules . Upon stimulation, IkappaB is phosphorylated and subsequently degraded by the proteasome, allowing NF-kappaB to translocate into the nucleus where they regulate target gene expression . Two kinases, IKK-alpha and IKK-beta, which are responsible for IkappaB phosphorylation were recently identified . We have recently identified a cytokine inducible IKK-i, a kinase related to IKK-alpha and -beta . IKK-i significantly induced NF-kappaB activation upon over-expression, as did IKK-alpha and IKK-beta . Unlike IKK-alpha and IKK-beta, IKK-i phosphorylated Ser36 but not Ser32 in vitro, suggesting that IKK-i activates NF-kappaB by distinct mechanisms from the conventional IKKs . RESULTS: I-TRAF/TANK was isolated as a molecule that interacts specifically with inducible IkappaB kinase (IKK-i) by the yeast two-hybrid screening procedure . The association of IKK-i and I-TRAF is mediated via the interaction between the N-terminal domain of I-TRAF and the C-terminal portion of IKK-i . In vitro kinase assays demonstrate that IKK-i phosphorylates I-TRAF in the middle portion that associates with TRAF2 . Interestingly, TRAF2 is freed from the I-TRAF/TRAF2 complex after I-TRAF phosphorylation . NF-kappaB activation by IKK-i is significantly blocked by coexpression of the N-terminal domain of I-TRAF, dominant negative TRAF2, and dominant negative NIK and IKK-beta . IKK-i over-expression also induced c-Jun N-terminal kinase . These results show that I-TRAF is a substrate of IKK-i . NF-kappaB activation by IKK-i may be mediated through phosphorylation of I-TRAF by IKK-i and subsequent liberation of TRAF2 . CONCLUSION: These results indicate that NF-kappaB activation by IKK-i is mediated through phosphorylation of I-TRAF/TANK by IKK-i and subsequent liberation of TRAF2. Eur J Biochem, 2000 Apr, 267(8), 2156 - 65 A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily; Castillo J et al.; The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described . A cDNA clone of its gene, which was designated psp54, was also isolated . The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54) . p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known . The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins . p16 is also 30% homologous with Nhp2p, a yeast nuclear protein . The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation . Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation) . p16 can be recovered from nuclei cross-linked to histone H3, when the disulfide bridges that occur in vivo are preserved . On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation . We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles . The function of p16 may be related to the protection of chromatin structure against desiccation during seed development. Eur J Biochem, 2000 Apr, 267(8), 2135 - 49 Overexpression of enzymes that repair endogenous damage to DNA; Frosina G; A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin . Efficient repair mechanisms have evolved to cope with this . The main repair pathway involved in repair of endogenous damage is DNA base excision repair . In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine . In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities . In some cases (e.g . O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g . alkyl N-purine glycosylase or DNA polymerase beta) were detrimental and even produced a genome instability phenotype . The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3913 - 8 Identification and characterization of a host protein required for efficient template selection in viral RNA replication; Diez J et al.; Biochemical studies suggest that positive-strand RNA virus replication involves host as well as viral functions . Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of animal and plant positive-strand RNA viruses . Yeast expressing the BMV RNA replication proteins 1a and 2a supports BMV RNA replication and mRNA synthesis . Using the ability of BMV to replicate in yeast, we show that efficient BMV RNA replication requires Lsm1p, a yeast protein related to core RNA splicing factors but shown herein to be cytoplasmic . Haploid yeast with an Lsm1p mutation was defective in an early template selection step in BMV RNA replication, involving the helicase-like replication protein 1a and an internal viral RNA element conserved with tRNAs . Lsm1p dependence of this interaction was suppressed by adding 3' poly(A) to the normally unpolyadenylated BMV RNA . Our results show Lsm1p involvement in a specific step of BMV RNA replication and connections between Lsm1p and poly(A) function, possibly through interaction with factors binding mRNA 5' ends. Plant Physiol, 2000 Apr, 122(4), 1301 - 10 ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation; Huang Y et al.; The modulation of mitogen-activated protein kinase (MAPK) activity regulates many intracellular signaling processes . In animal and yeast cells, MAP kinases are activated via phosphorylation by the dual-specificity kinase MEK (MAP kinase kinase) . Several plant homologs of MEK and MAPK have been identified, but the biochemical events underlying the activation of plant MAPKs remain unknown . We describe the in vitro activation of an Arabidopsis homolog of MAP kinase, ATMPK4 . ATMPK4 was phosphorylated in vitro by an Arabidopsis MEK homolog, AtMEK1 . This phosphorylation occurred principally on threonine (Thr) residues and resulted in elevated ATMPK4 kinase activity . A second Arabidopsis MEK isoform, ATMAP2Kalpha, failed to phosphorylate ATMPK4 in vitro . Tyr dephosphorylation by the Arabidopsis Tyr-specific phosphatase AtPTP1 resulted in an almost complete loss of ATMPK4 activity . Immunoprecipitates of Arabidopsis extracts with anti-ATMPK4 antibodies displayed myelin basic protein kinase activity that was sensitive to treatment with AtPTP1 . These results demonstrate that a plant MEK can phosphorylate and activate MAPK, and that Tyr phosphorylation is critical for the catalytic activity of MAPK in plants . Surprisingly, in contrast to the animal enzymes, AtMEK1 may not be a dual-specificity kinase but, rather, the required Tyr phosphorylation on ATMPK4 may result from autophosphorylation. J Mol Med, 2000, 78(1), 36 - 46 A 500-kb region on chromosome 16p13.1 contains the pseudoxanthoma elasticum locus: high-resolution mapping and genomic structure; Cai L et al.; We have recently mapped the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and cardiovascular system, to chromosome 16p 13.1 . Here we report further data on the fine-mapping and genomic structure of this locus . Haplotype analysis of informative PXE families narrowed the locus to an interval of less than 500 kb located between markers D16B9621 and D16S764 . Three overlapping YAC clones were found to cover this region through YAC-STS content mapping . An overlapping BAC contig was then constructed to cover this interval and the surrounding region . About 80% of this chromosomal region has been fully sequenced using the BAC shotgun technique . Gene content and sequence analysis predicted four genes (MRP1, MRP6, PM5, and a novel transcript) and two pseudogenes (ARA and PKDI) within this interval . By screening a somatic cell hybrid panel we were able to precision-map the breakpoint of Cy185 and the starting point of a chromosomal duplication within 20 kb of BAC A962B4 . The present data further refine the localization of PXE, provide additional physical cloning resources, and will aid in the eventual identification of the genetic defect causing PXE. Trends Biotechnol, 2000 May, 18(5), 218 - 23 Artificial chromosomes: ideal vectors? Brown WR, Mee PJ, Hong Shen M. Artificial chromosomes are DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes . Artificial chromosomes were first assembled in budding yeast and have since been useful in many aspects of yeast genetics . Several attempts have been made at building artificial chromosomes in mammals, although these have been met with limited success . Consequently, mini-chromosomes of defined structure have been developed to address questions regarding mammalian chromosome function and for biotechnological applications . Here we review progress in these areas and consider how it influences plans to build artificial chromosomes in plants and parasites. Plant J, 2000 Feb, 21(3), 311 - 6 Induction of competence for elicitation of defense responses in cucumber hypocotyls requires proteasome activity; Becker J et al.; The epidermal cells of hypocotyls from etiolated cucumber seedlings are not constitutively competent for elicitation of the rapid H2O2 defense response . However, elicitor competence developed while conditioning the surface-abraded seedlings by rotating them in buffer for 4 h . Competence development was greatly potentiated by inducers of systemic acquired resistance and suppressed by specific inhibitors of proteasome activity, clastolactacystin beta-lactone (LAC) and carboxybenzoyl-L-leucyl-L-leucyl-L-leucinal (LLL) . In the freshly abraded seedlings, chitinase gene activation became evident approximately 4 h after elicitor addition . Accumulation of chitinase mRNA was enhanced upon conditioning prior to elicitation and was inhibited by LAC and LLL, indicating that the process which leads to H2O2 elicitation competence is also superimposed on the elicitation of chitinase mRNA . LAC and LLL caused an accumulation of ubiquitin-conjugated proteins and enhanced the expression of a proteasome alpha-subunit, suggesting that proteasome activity was specifically inhibited and that the effect observed on gene expression was not due to impaired gene induction in general . Together, our results suggest that the ubiquitin-proteasome system may play a crucial role in a process which switches the signaling pathway for diverse plant defense responses into a functional state, as is known for many basic cellular processes in both animals and yeast. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 489 - 500 Palaeococcus ferrophilus gen . nov., sp . nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney; Takai K et al.; A novel barophilic, hyperthermophilic archaeon was isolated from a deep-sea hydrothermal vent chimney at the Myojin Knoll in the Ogasawara-Bonin Arc, Japan . The cells were found to be irregular cocci and motile with multiple polar flagella . Growth was observed between 60 and 88 degrees C (opt . 83 degrees C; 30 min doubling time), pH 4.0 and 8.0 (opt . pH 6.0), 20 and 73 g sea salts l-1 (opt . 47 g l-1) and 0.1 and 60 MPa (opt . 30 MPa) . The isolate was a strictly anaerobic chemoorganotroph capable of utilizing proteinaceous substrates such as yeast extract, peptone, tryptone and casein in the presence of elemental sulfur or ferrous iron . The G + C content of the genomic DNA was 53.5 mol% . Phylogenetic analysis based on 16S rDNA sequences indicated that the isolate was a member of an ancient lineage of the Thermococcales that diverged prior to the formation of the two genera Thermococcus and Pyrococcus . On the basis of the physiological and molecular properties of the new isolate, the name Palaeococcus ferrophilus gen . nov., sp . nov . is proposed . The type strain is strain DMJT (= JCM 10417) {corrected}. Mol Cell Biol, 2000 May, 20(9), 3137 - 46 The human SWI-SNF complex protein p270 is an ARID family member with non-sequence-specific DNA binding activity; Dallas PB et al.; p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein . The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain) . The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1 . The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression . Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties . Each binds preferentially to AT-rich sites . In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions. J Nat Prod, 2000 Mar, 63(3), 332 - 8 Antiyeast steroidal saponins from Yucca schidigera (Mohave yucca), a new anti-food-deteriorating agent; Miyakoshi M et al.; A saponin fraction from the stems of Yucca schidigera (Mohave yucca) exhibited potent growth-inhibitory activities against certain food-deteriorating yeasts, film-forming yeasts, and dermatophytic yeasts and fungi . From this fraction, a number of new anti-yeast monodesmosidic spirostanol saponins, named schidigera-saponins A1 (1), A2 (2), A3 (3), B1 (4), C1 (5),C2 (6); 25(R and S) schidigera-saponins D1 (7), D2 (8), E1 (12), F1 (13); and 25(S) schidigera-saponins D3 (9), D4 (10), D5 (11), and F2 (14) were isolated, together with several related known saponins, and the structures were elucidated by spectroscopic methods (see Chart 1) . The relationship between the antiyeast activities and the structures of these saponins is described. Rev Med Chir Soc Med Nat Iasi, 1997 Jul-Dec, 101(3-4), 197 - 201 {Morphological diagnostic problems in a case of peritoneal blastomycosis}; Munteanu G et al.; Blastomycosis is a mycotic disease, caused by a fungal infection . It has a wide spectrum of clinical presentations, and, particularly, can mimic neoplastic disease . Correct diagnosis of the illness requires fungal culture and biopsy . In Romania, mycotic histopathology is insufficiently developed, and morphological tests are recommended to very few people who present this type of pathology . The paper discusses a case of peritoneal blastomycosis found at a patient with an abdominal pseudotumoral mass . The microscopic exam revealed the characteristic histologic features and budding yeast, in specific dyes, typical to the Blastomyces dermatidis (PAS, silver-methenamin) . Authors of this paper hereby intend to draw pathologists' attention on the existence and diagnosis of mycotic lesions, whose number is continuously increasing nowadays. Genomics, 2000 Mar 15, 64(3), 264 - 76 An integrated map of human 6q22.3-q24 including a 3-Mb high-resolution BAC/PAC contig encompassing a QTL for fetal hemoglobin; Game L et al.; Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23 . An initial yeast artificial chromosome contig of 13 clones spanning this region was generated . Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb . We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region . The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC . A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb . About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms . Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig . Genomics, 2000 Mar 15, 64(3), 221 - 9 Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia; McDonell N et al.; The chromosomal abnormality represented by an isodicentric X chromosome {idic(X)(q13)} is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females . A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene . Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region . In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs . Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations . FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML . Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region . Mol Plant Microbe Interact, 2000 Apr, 13(4), 374 - 83 The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis; Takano Y et al.; The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants . Here we report that the C . lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps . CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea . Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant . Surprisingly, in contrast to M . grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination . However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination . Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant . This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi . Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization. J Biomed Sci, 2000 Mar-Apr, 7(2), 160 - 8 Hepatitis B virus core protein interacts with the C-terminal region of actin-binding protein; Huang CJ et al.; Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes . There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis . To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis . A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified . This interaction was further confirmed both in vitro and in vivo . In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein . Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278 . The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs . The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed. J Biomed Sci, 2000 Mar-Apr, 7(2), 152 - 9 Identification of okadaic-acid-induced genes by mRNA differential display in glioma cells; Chin LS et al.; To identify novel genes associated with apoptosis in glioma cells, we treated T98G glioma cells with okadaic acid (OA) . Differential display using 15 random primers was performed on RNA extracted from these cells . Upregulated bands were excised from polyacrylamide gels and cloned . Northern blots were used to confirm RNA expression in T98G cells . 18 RNA fragments corresponding to the untranslated region of genes were identified and sequenced . Three unknown gene fragments were used to screen a fetal brain cDNA library resulting in three complete cDNA sequences . The three sequences corresponded to a human gene homologous to the yeast translation initiation factor Sui-1, a cAMP-regulated phosphoprotein, ARPP-16/19, and a novel gene designated O48 . Transcription of Sui-1 increased in response to all stress factors tested, whereas ARPP only responded to OA . 2-kb and 4-kb O48 RNA species were identified . OA and stress factors increased 2-kb expression while K252a (protein kinase inhibitor) increased 4-kb expression . Differential display is effective for identifying genes associated with apoptosis . Novel genes may be identified by further analysis of the gene fragments identified in this study . The function of O48 is unknown. Curr Opin Genet Dev, 2000 Apr, 10(2), 178 - 86 Think global, act local--how to regulate S phase from individual replication origins; Pasero P et al.; All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved . Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins . Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases . The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins . Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states. Curr Opin Genet Dev, 2000 Apr, 10(2), 193 - 8 mRNA stability in eukaryotes; Mitchell P et al.; During the past two years, the role of the proteins HuR and hnRNP D in regulated mRNA degradation in humans has become clearer, and a putative mRNA deadenylase, DAN or PARN, has been identified . In yeast, the relationship between translation and mRNA turnover is clearer, but the mRNA decapping process has turned out to be unexpectedly complex. Curr Opin Genet Dev, 2000 Apr, 10(2), 151 - 6 Replication and recombination intersect; Marians KJ; A bacterial housekeeping function, which requires both recombination and replication enzymes, has been identified that re-establishes inactivated replication forks under normal growth conditions . Some long-tract gene-conversion events initiated by double-strand breaks in yeast and mammalian cells can be attributed to recombination-directed DNA replication . Double-strand break repair in yeast has been shown to require both leading- and lagging-strand DNA synthesis . These observations suggest that the recombination and replication machinery cooperate to maintain genomic integrity. Carcinogenesis, 2000 Apr, 21(4), 563 - 5 High frequency in esophageal cancers of p53 alterations inactivating the regulation of genes involved in cell cycle and apoptosis; Robert V et al.; Somatic mutations of the tumor suppressor gene p53 have been frequently detected in esophagal cancers, but their biological significance remains to be established . The tumor suppressor activity of p53 results in part from its ability to transactivate genes involved in the cell cycle and apoptosis, such as p21, bax and PIG3, and some p53 mutations may have a differential effect on the transactivation of these target genes . We developed yeast strains in which the activation by wild-type p53 of reporter plasmids containing p53 binding sites present within these target genes induces a change in the color of the colonies (red/white) . Using these strains, we analyzed 56 esophageal cancers from patients residing in Normandy, France, a high incidence geographic area . Forty-seven tumors (84%), scored as mutant with the p21, bax and PIG3 reporter strains and in most of the cases (76%), the percentage of red colonies suggested that both p53 alleles were inactivated . Sequencing analysis allowed the identification of a p53 mutation in each positive sample, and the spectrum of mutations was in agreement with the etiological role of tobacco and alcohol . These results confirm the high frequency of biallelic p53 mutations in esophageal carcinoma and strongly suggest that their biological consequence is the complete alteration of the transactivation of genes involved in the cell cycle and apoptosis, which indicates that p53 alteration is a key event in esophagus carcinogenesis. Xenobiotica, 2000 Mar, 30(3), 219 - 33 Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling; Lightfoot T et al.; 1 . Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme . Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear . 2 . As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6 . 3 . Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position . The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems . 4 . A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable . An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine . 5 . This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6. Protein Sci, 2000 Mar, 9(3), 536 - 43 Cytochrome c folds through a smooth funnel; Panda M et al.; A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding . The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state . A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2 . The results show that the thermodynamic properties of the transition states are very similar . A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues . At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms) . T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein . Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u) . The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic . In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape. J Biol Chem, 2000 Jun 16, 275(24), 18153 - 9 Mammalian mitochondrial ribosomal proteins (4) . Amino acid sequencing, characterization, and identification of corresponding gene sequences; O'Brien TW et al.; Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans . It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations . Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized . MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology . Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins . Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner . Three of them represent "new," i.e . formerly unknown mammalian mitochondrial ribosomal protein classes . Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins . In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined. J Biol Chem, 2000 Jun 23, 275(25), 18724 - 31 Identification and characterization of a novel protein from Sertoli cells, PASS1, that associates with mammalian small stress protein hsp27; Liu C et al.; hsp27 is involved in development of tolerance to stress, possibly by its involvement in molecular chaperoning, maintenance of glutathione status, and/or modulation of microfilament structure and function . We hypothesize that hsp27 function depends on specific association with other proteins . To discover proteins that associate with hsp27, we made a differentiated rat Sertoli cell cDNA expression library and screened it using the yeast two-hybrid system . We obtained a cDNA coding for a novel protein of 428 amino acids that we have named PASS1 (protein associated with small stress proteins 1) . BLAST searches did not reveal major similarity of PASS1 to any known protein, but the cDNA sequence matched several mouse EST clones and shares 34% homology with a Caenorhabditis elegans genomic sequence . In vitro, bacterially expressed glutathione S-transferase-PASS1 fusion protein bound to hsp27, and hsp27 was co-immunoprecipitated with c-Myc-tagged PASS1 overexpressed in several cell lines . The region of PASS1 responsible for association with hsp27 was identified as existing predominantly between amino acids 108 and 208 of PASS1 . Northern hybridization and Western blot analysis demonstrated that PASS1 is expressed in several tissues, with the highest expression occurring in testis, primarily in Sertoli cells . The presence of a 1.4-kilobase PASS1 mRNA in kidney as well as the 1 . 8-kilobase mRNA seen in other tissues suggests that alternate splicing may occur in this organ . Ectopic expression of PASS1 in two cultured cell lines was observed to inhibit the ability of hsp27 to protect cells against heat shock, indicating that PASS1 does interact with hsp27 in the live cell. J Cell Sci, 2000 May, 113 ( Pt 9), 1553 - 64 The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin; Flick MJ et al.; Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus . The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments . Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined . To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners . From this screen, the cytoskeletal protein betaI-spectrin was isolated . Protein interaction assays demonstrate that MLP and betaI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions . betaI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins . The MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin . Confocal microscopy studies also reveal that MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue . Given that beta-spectrin is a known costamere protein, we propose that sarcolemma-associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the beta-spectrin network to the alpha-actinin crosslinked actin filaments of the myofibril. J Cell Sci, 2000 May, 113 ( Pt 9), 1515 - 24 Lysosome-endosome fusion and lysosome biogenesis; Luzio JP et al.; Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle . This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created . Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents . In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation . We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process. Planta, 2000 Feb, 210(3), 371 - 82 Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.) . I . Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator; Hausler RE et al.; The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco . For this purpose, tobacco lines with an antisense repression of the endogenous TPT (alphaTPT) and tobacco lines overexpressing the TPT gene isolated from the C4 plant Flaveria trinervia (FtTPT) were used . The F . trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C3 plants . Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air . Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod . The FtTPT overexpressors incorporated more 14CO2 in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type . There were only small effects on labelling of amino acids and organic acids . The mobilisation of starch was enhanced in alphaTPT lines but decreased in FtTPT overexpressors compared to the wild type . Enzymes involved in starch mobilisation or utilisation, such as alpha-amylase or hexokinase were increased in alphaTPT plants and, in the case of amylases, decreased in FtTPT overexpressors . Moreover, alpha-amylase activity exhibited a pronounced diurnal variation in alphaTPT lines with a maximum activity after 8 h in the light . These changes in starch hydrolytic activities were confirmed by activity staining of native gels . Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity . There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate . In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO2 assimilation . However, in elevated CO2 (1500 microl x l(-1)) and low O2 (2%) the rate of CO2 assimilation was decreased in the alphaTPT lines and was slightly higher in FtTPT lines . This shows that the TPT limits maximum rates of photosynthesis in the wild type. J Eukaryot Microbiol, 2000 Mar-Apr, 47(2), 129 - 38 Centrin protein and genes in Trichomonas vaginalis and close relatives; Brugerolle G et al.; Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina . Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina . Centrin was not demonstrated in the dividing spindle and paradesmosis . Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane . Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules . There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T . vaginalis . Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined . By screening a T . vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found . The sequence comprises the 4 typical EF-hand Ca++-binding domains present in every known centrin . Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity). Curr Opin Oncol, 2000 Mar, 12(2), 143 - 8 Early detection and prevention of lung cancer; Wright GS et al.; Lung cancer remains the leading cause of cancer death in the United States and is one of the world's leading causes of preventable death . Technologic advances have brought new modalities that may be useful for the early detection of lung cancer . However, because of the large number of persons at increased risk for lung cancer, screening is a formidable task . There are several risk factors that can be identified, including potential susceptibility factors, which may aid in pinpointing individuals who need to participate in regular screening programs . Aside from recognized environmental exposures including cigarette smoking, there are a number of genetic and metabolic susceptibility factors that have been examined . These include polymorphisms in the cytochrome p450 enzymes and the metabolizing capability of glutathione s-transferase or acetylation . Additionally, defects in DNA repair and in bleomycin sensitivity assays may also aid in identifying individuals who are at an increased risk for lung cancer . Additional work has been done in the area of characterizing the molecular alterations in the bronchial epithelium in high-risk smokers . This manuscript addresses only selected molecular alterations that have been examined in preneoplastic bronchial epithelium . In addition to mutations in the k-ras oncogene and the p53 gene, which are frequently seen in malignancy, alterations in the p16 gene, microsatellite instability and loss of heterozygocity are also promising potential markers of preneoplasia . The hnRNP A2/B1 gene also shows some promising increased expression in preneoplasia . Lung cancer prevention has made some strides . A number of trials with molecular and morphologic intermediate endpoints have been conducted and have suggested that some of the molecular alterations and morphologic alterations are reversible . However, the rate of spontaneous regression of these lesions is, as yet, uncharacterized . Two recent large studies, the beta-carotene and retinol efficacy trial (CARET) trial conducted in the United States and the Alpha-Tocopherol Beta Carotene (ATBC) trial conducted in Finland, both demonstrated an unexpected increased risk for lung cancer associated with beta-carotene supplementation . The EUROSCAN trial evaluation of vitamin A and N-acetylcystine also showed no benefit to supplementation in reducing risk for lung cancer . Results from the Intergroup study of 1 3-cis-retinoic acid are pending, and plans are underway for an Intergroup trial studying high selenium yeast to reduce lung cancer risk . Hopefully, the combination of identifying markers of increased risk among the numerous current and former smokers will identify high-risk populations to participate in future trials of promising agents that may lead to reduction in incidence and mortality of the leading cause of cancer death. J Biol Chem, 2000 Jun 30, 275(26), 20052 - 60 Crystal structure of a conformation-selective casein kinase-1 inhibitor; Mashhoon N et al.; Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis . Unlike most protein kinases, they appear to function as constitutively active enzymes . As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes . To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening . Here we report the crystal structure of 3-{(2,4,6-trimethoxyphenyl) methylidenyl}-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution . The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations . We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor. J Biol Chem, 2000 Jun 30, 275(26), 19866 - 76 Protein-arginine methyltransferase I, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3; Tang J et al.; Arginine methylation is a common post-translation modification found in many proteins . Protein-arginine methyltransferase I (PRMT1) contributes >90% of type I protein-arginine methyltransferase activity in cells and tissues . To expand our knowledge on the regulation and role of PRMT1 in cells, we used the yeast two-hybrid system to identify proteins that interact with PRMT1 . One of the interacting proteins we cloned is interleukin enhancer-binding factor 3 (ILF3), also known as M phase phosphoprotein 4 . ILF3 is closely related to nuclear factor 90 (NF90) . Using an immunofluorescence analysis, we determined that ILF3 and PRMT1 co-localize in the nucleus . Moreover, PRMT1 and ILF3 co-precipitate in immunoprecipitation assays and can be isolated together in "pull-down" experiments using recombinant fusion proteins . ILF3 is a robust substrate for methylation by PRMT1 and can modulate PRMT1 activity in in vitro methylation assays . Deletion studies demonstrated that the COOH-terminal region of ILF3, which is rich in arginine, glycine, and serine, is responsible for the strong interaction between PRMT1 and ILF3 and is the site of ILF3 methylation by PRMT1 . Although ILF3 and NF90 are highly similar, they differ in their carboxyl-terminal regions . Because of this difference, NF90 does not interact with PRMT1, is a much poorer substrate than ILF3 for PRMT1-dependent methylation, and does not modulate PRMT1 enzyme activity. Biochem J, 2000 Apr 15, 347(Pt 2), 501 - 9 The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells; Warner AJ et al.; Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined . Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously . It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells . In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR . Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR . We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src-homology 2 (SH2) domain as tyrosine-1175 of KDR . We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells . Furthermore, we demonstrate that in the two-hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1. Cancer Res, 2000 Mar 15, 60(6), 1690 - 7 A 700-kb physical map of a region of 16q23.2 homozygously deleted in multiple cancers and spanning the common fragile site FRA16D; Paige AJ et al.; We have identified a >600-kb region at 16q23.2 that is homozygously deleted from malignant ovarian ascites using representational difference analysis . Overlapping homozygous deletions were also observed in the colon carcinoma cell line HCT116 and a xenograft established from the small cell lung cancer cell line WX330 . This region coincides with that described previously by others as showing loss of heterozygosity in prostate and breast cancers (C . Li et al., Genes Chromosomes Cancer, 24: 175-182, 1999; A . Latil et al., Cancer Res., 57: 1058-1062, 1997; K . Driouch et al., Genes Chromosomes Cancer, 19: 185-191, 1997; A . Iida et al., Br . J . Cancer, 75: 264-267, 1997) . In addition, the minimally deleted region spans the common fragile site FRA16D . We have constructed a 700-kb physical map encompassing the deleted region . By fluorescence in situ hybridization of aphidicolin-induced metaphase chromosomes, we have preliminary data to suggest that P1-derived bacterial artificial chromosome clones from the contig lie on both sides of FRA16D . This is confirmed by extensive fluorescence in situ hybridization analysis of the region reported in the accompanying article (M . Mangelsdorf et al., Cancer Res., 60: 1683-1689, 2000) and is consistent with an involvement of this common fragile site in the loss of 16q23.2 material in various cancer types . The minimally deleted region of approximately 210 kb has been characterized using our own markers and public domain markers . Eleven distinct expressed sequences mapped to the region, providing a basis for identifying the predicted tumor suppressor gene in this region. Cancer Res, 2000 Mar 15, 60(6), 1683 - 9 Chromosomal fragile site FRA16D and DNA instability in cancer; Mangelsdorf M et al.; It has been proposed that common aphidicolin-inducible fragile sites, in general, predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer . Although this appears to be the case for the fragile site FRA3B and may be the case for FRA7G, it is not yet clear whether this association is a general property of this class of fragile site . The major aim of the present study was to determine whether the FRA16D chromosomal fragile site locus has a role to play in predisposing DNA sequences within and adjacent to the fragile site to DNA instability (such as deletion or translocation), which could lead to or be associated with neoplasia . We report the localization of FRA16D within a contig of cloned DNA and demonstrate that this fragile site coincides with a region of homozygous deletion in a gastric adenocarcinoma cell line and is bracketed by translocation breakpoints in multiple myeloma, as reported previously (Chesi, M., et al., Blood, 91: 4457-4463, 1998) . Therefore, given similar findings at the FRA3B and FRA7G fragile sites, it is likely that common aphidicolin-inducible fragile sites exhibit the general property of localized DNA instability in cancer cells. Cancer Res, 2000 Mar 15, 60(6), 1531 - 5 Potential role of BRCA2 in a mitotic checkpoint after phosphorylation by hBUBR1; Futamura M et al.; BRCA2, a gene responsible for inherited susceptibility to breast cancer in a number of families, is thought to be critical for replication and repair of DNA during S-phase . To elucidate the physiological functions of BRCA2, we used a yeast two-hybrid system to screen for proteins that could associate with BRCA2 . Here we report interaction of BRCA2 with a mitotic checkpoint protein, hBUBR1, and its phosphorylation by hBUBR1 in vitro . After cotransfection of BRCA2 and hBUBR1 expression vectors into the COS7 cell line, both proteins were stained together in the nuclei of cells whose spindle fibers were disrupted, but not in undamaged cells . Treatment with vincristine, which disrupts microtubules, significantly increased expression of both hBUBR1 and BRCA2 in the MCF7 cells . The results suggest that BRCA2 protein might be involved in a mitotic checkpoint in vivo after it has been phosphorylated by hBUBR1. Nat Biotechnol, 2000 Apr, 18(4), 393 - 7 Large-scale functional analysis using peptide or protein arrays; Emili AQ et al.; The array format for analyzing peptide and protein function offers an attractive experimental alternative to traditional library screens . Powerful new approaches have recently been described, ranging from synthetic peptide arrays to whole proteins expressed in living cells . Comprehensive sets of purified peptides and proteins permit high-throughput screening for discrete biochemical properties, whereas formats involving living cells facilitate large-scale genetic screening for novel biological activities . In the past year, three major genome-scale studies using yeast as a model organism have investigated different aspects of protein function, including biochemical activities, gene disruption phenotypes, and protein-protein interactions . Such studies show that protein arrays can be used to examine in parallel the functions of thousands of proteins previously known only by their DNA sequence. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 1 - 16 The plant plasma membrane H(+)-ATPase: structure, function and regulation; Morsomme P et al.; The proton-pumping ATPase (H(+)-ATPase) of the plant plasma membrane generates the proton motive force across the plasma membrane that is necessary to activate most of the ion and metabolite transport . In recent years, important progress has been made concerning the identification and organization of H(+)-ATPase genes, their expression, and also the kinetics and regulation of individual H(+)-ATPase isoforms . At the gene level, it is now clear that H(+)-ATPase is encoded by a family of approximately 10 genes . Expression, monitored by in situ techniques, has revealed a specific distribution pattern for each gene; however, this seems to differ between species . In the near future, we can expect regulatory aspects of gene expression to be elucidated . Already the expression of individual plant H(+)-ATPases in yeast has shown them to have distinct enzymatic properties . It has also allowed regulatory aspects of this enzyme to be studied through random and site-directed mutagenesis, notably its carboxy-terminal region . Studies performed with both plant and yeast material have converged towards deciphering the way phosphorylation and binding of regulatory 14-3-3 proteins intervene in the modification of H(+)-ATPase activity . The production of high quantities of individual functional H(+)-ATPases in yeast constitutes an important step towards crystallization studies to derive structural information . Understanding the specific roles of H(+)-ATPase isoforms in whole plant physiology is another challenge that has been approached recently through the phenotypic analysis of the first transgenic plants in which the expression of single H(+)-ATPases has been up- or down-regulated . In conclusion, the progress made recently concerning the H(+)-ATPase family, at both the gene and protein level, has come to a point where we can now expect a more integrated investigation of the expression, function and regulation of individual H(+)-ATPases in the whole plant context. J Biol Chem, 2000 May 26, 275(21), 16167 - 73 The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits; Hironaka K et al.; Glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays a crucial role in cerebellum-dependent motor learning . Although GluRdelta2 belongs to an ionotropic GluR family, little is known about its pharmacological features and downstream signaling cascade . To study molecular mechanisms underlying GluRdelta2-dependent motor learning, we employed yeast two-hybrid screening to isolate GluRdelta2-interacting molecules and identified protein-tyrosine phosphatase PTPMEG . PTPMEG is a family member of band 4.1 domain-containing protein-tyrosine phosphatases and is expressed prominently in brain . Here, we showed by in situ hybridization analysis that the PTPMEG mRNA was enriched in mouse thalamus and Purkinje cells . We also showed that PTPMEG interacted with GluRdelta2 as well as with N-methyl-d-aspartate receptor GluRepsilon1 in cultured cells and in brain . PTPMEG bound to the putative C-terminal PDZ target sequence of GluRdelta2 and GluRepsilon1 via its PDZ domain . Examination of the effect of PTPMEG on tyrosine phosphorylation of GluRepsilon1 unexpectedly revealed that PTPMEG enhanced Fyn-mediated tyrosine phosphorylation of GluRepsilon1 in its PTPase activity-dependent manner . Thus, we conclude that PTPMEG associates directly with GluRdelta2 and GluRepsilon1 . Moreover, our data suggest that PTPMEG plays a role in signaling downstream of the GluRs and/or in regulation of their activities through tyrosine dephosphorylation. J Biol Chem, 2000 Jun 30, 275(26), 20033 - 44 The RIM/NIM family of neuronal C2 domain proteins . Interactions with Rab3 and a new class of Src homology 3 domain proteins; Wang Y et al.; RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins . RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles . We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain . Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C-terminal C(2) domains that are separated by long alternatively spliced sequences . Unexpectedly, the 3'-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2 . NIM2 is composed of a unique N-terminal sequence followed by the C-terminal part of RIM2 . Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected . To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium-triggered exocytosis in PC12 cells . NIM3 induced a dramatic increase in calcium-evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s . To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1 . Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified . RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction . In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C(2) domains, N-terminal to the beginning of NIMs . RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs . With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3. J Biol Chem, 2000 Jun 2, 275(22), 16802 - 9 The autoimmune regulator protein has transcriptional transactivating properties and interacts with the common coactivator CREB-binding protein; Pitkanen J et al.; Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, caused by mutations in the autoimmune regulator (AIRE) gene, is an autosomal recessive autoimmune disease characterized by the breakdown of tolerance to organ-specific antigens . The 545 amino acid protein encoded by AIRE contains several structural motifs suggestive of a transcriptional regulator and bears similarity to cellular proteins involved in transcriptional control . We show here that AIRE fused to a heterologous DNA binding domain activates transcription from a reporter promoter, and the activation seen requires the full-length protein or more than one activation domain . At the structural level AIRE forms homodimers through the NH(2)-terminal domain, and molecular modeling for this domain suggests a four-helix bundle structure . In agreement, we show that the common transcriptional coactivator CREB-binding protein (CBP) interacts with AIRE in vitro and in yeast nuclei through the CH1 and CH3 conserved domains . We suggest that the transcriptional transactivation properties of AIRE together with its interaction with CBP might be important in its function as disease-causing mutations almost totally abolish the activation effect. J Biol Chem, 2000 Jun 16, 275(24), 18225 - 33 Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA; MacDonald JI et al.; We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA . Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays . Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent . By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684) . By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding . Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules. J Biol Chem, 2000 Jun 2, 275(22), 16752 - 7 BSAP (Pax5)-importin alpha 1 (Rch1) interaction identifies a nuclear localization sequence; Kovac CR et al.; BSAP (Pax5) is an essential transcription factor for early B cell and central nervous system development . In later B cell development, BSAP has been implicated in the regulation of 3' Ig enhancers and a number of B cell-specific genes . Previous studies have suggested a role for BSAP-interacting proteins in the regulation of the function of BSAP . Using the yeast two-hybrid system, we identified importin alpha1 (Rch1) as a BSAP-interacting protein . Importin alpha proteins have been shown to escort proteins into the nucleus through interaction with a nuclear localization signal (NLS), composed of short stretches of basic amino acids . A predicted NLS in BSAP (NKRKRDE, located at amino acids 195-201 in the central domain) was confirmed to be essential for interaction with importin alpha1 by the yeast two-hybrid assay . Physical interaction between BSAP and importin alpha1 was detected in vitro by a glutathione S-transferase (GST) pulldown assay . The NLS sequence in BSAP conferred nuclear localization to green fluorescent protein (GFP)-BSAP fusion proteins . Although the N-terminal paired (DNA-binding) domain of BSAP also conferred nuclear localization when coupled to green fluorescent protein, this domain did not bind to importin alpha1 in the yeast two-hybrid assay . The NLS sequence in the central domain of BSAP binds to the C-terminal 98-amino acid fragment of importin alpha1. J Biol Chem, 2000 May 12, 275(19), 14767 - 76 Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei . Sequence conservation and implications for 2'-O-ribose methylation of rRNA; Dunbar DA et al.; We report the identification of 17 box C/D fibrillarin-associated small nucleolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei . To systematically isolate and characterize these snoRNAs, the T . brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was cloned and polyclonal antibodies to the recombinant fibrillarin protein were generated in rabbits . Immunoprecipitations from T . brucei extracts with the anti-fibrillarin antibodies indicated that this trypanosomatid has at least 30 fibrillarin-associated snoRNAs . We have sequenced seventeen of them and designated them TBR for T . brucei RNA 1-17 . All of them bear conserved box C, D, C', and D' elements, a hallmark of fibrillarin-associated snoRNAs in eukaryotes . Fourteen of them are novel T . brucei snoRNAs . Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2'-O-ribose methylation . Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences . Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and vertebrate methylation guide snoRNAs . Our results indicate that T . brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2'-O-ribose methylation of rRNA and that the mechanism for targeting the nucleotide to be methylated at the fifth nucleotide upstream of box D or D' originated in early eukaryotes. J Biol Chem, 2000 May 26, 275(21), 15851 - 60 Gamma1- and gamma2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells; Piluso G et al.; Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies . At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound . These are a family of cytoplasmic peripheral membrane proteins . Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa . Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins . The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids . The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues . We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25 . Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins . We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections . Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system. J Biol Chem, 2000 Jun 2, 275(22), 17173 - 9 The SCAN domain mediates selective oligomerization; Schumacher C et al.; The SCAN domain is described as a highly conserved, leucine-rich motif of approximately 60 amino acids found at the amino-terminal end of zinc finger transcription factors . Although no specific biological function has been attributed to the SCAN domain, its predicted amphipathic secondary structure led to the suggestion that this domain may mediate protein-protein associations . A yeast two-hybrid screen identified members of two SCAN domain protein families that interact with the SCAN domain of the zinc finger protein ZNF202 . The interacting ZNF191 protein represents the family of SCAN domain-containing zinc finger proteins, whereas the novel SDP1 protein establishes a new family of genes that encode an isolated SCAN domain . Isolated SCAN domain proteins may form asymmetric homodimers in solution . Biochemical binding studies confirmed the associations of ZNF191 and SDP1 with ZNF202 and established the SCAN domain as a selective hetero- and homotypic oligomerization domain . SCAN mediated protein associations might therefore represent a new regulatory mechanism of transcriptional activity. J Biol Chem, 2000 May 26, 275(21), 15645 - 51 p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting directly with the N-terminal A/B domains; Kobayashi Y et al.; Estrogen receptor (ER) alpha and beta mediate estrogen actions in target cells through transcriptional control of target gene expression . For 17beta-estradiol-induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required . Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown . To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/CREB binding protein, steroid receptor coactivator-1/transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein- 205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function . We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ERalpha and human ERbeta . Direct interactions of p300 with the A/B domains of ERalpha and ERbeta were observed in an in vitro glutathione S-transferase pull-down assay in accordance with the interactions in yeast and mammalian two-hybrid assays . Furthermore, mutations in the p300 binding sites (56-72 amino acids in ERalpha and 62-72 amino acids in ERbeta) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ERalpha and ERbeta . Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ERalpha and ERbeta. J Biol Chem, 2000 May 19, 275(20), 14791 - 4 Insertional mutation of the murine kisimo locus caused a defect in spermatogenesis; Yanaka N et al.; Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products . An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules . We cloned the disrupted locus of kisimo and identified a novel testis-specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse . The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-1epsilon, suggesting that THEG protein functions as a regulatory factor in protein assembly . Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility. J Biol Chem, 2000 May 5, 275(18), 13167 - 70 Physical and functional interaction of rabphilin-11 with mammalian Sec13 protein . Implication in vesicle trafficking; Mammoto A et al.; Rab11a small G protein (Rab11p) is implicated in vesicle trafficking, especially vesicle recycling . We have previously isolated a downstream effector of Rab11p, named rabphilin-11 . We found here that rabphilin-11 directly bound the mammalian counterpart of yeast Sec13 protein (mSec13p) in cell-free and intact cell systems . Yeast Sec13p is involved as a component of coat proteins II in the Sar1p-induced vesicle formation from the endoplasmic reticulum, but the precise role of mSec13p is unknown . The interaction of rabphilin-11 with mSec13p was enhanced by GTP-Rab11p . Rabphilin-11 localized on the vesicles in perinuclear regions and along microtubules oriented toward the plasma membrane, whereas mSec13p partly colocalized with rabphilin-11 in the perinuclear regions, most presumably the Golgi complex . Disruption of the rabphilin-11-mSec13p interaction by overexpression of the mSec13p-binding region of rabphilin-11 impaired vesicle trafficking . These results indicate that the rabphilin-11-mSec13p interaction is implicated in vesicle trafficking. Lett Appl Microbiol, 2000 Mar, 30(3), 183 - 7 Medium-size droplets of methyl ricinoleate are reduced by cell-surface activity in the gamma-decalactone production by Yarrowia lipolytica; Wache Y et al.; Size of methyl ricinoleate droplets during biotransformation into gamma-decalactone by Yarrowia lipolytica was measured in both homogenized and non-homogenized media . In non-homogenized but shaken medium, droplets had an average volume surface diameter d32 of 2.5 microm whereas it was 0.7 microm in homogenized and shaken medium . But as soon as yeast cells were inoculated, both diameters became similar at about 0.7 microm and did not vary significantly until the end of the culture . The growth of Y . lipolytica in both media was very similar except for the lag phase which was lowered in homogenized medium conditions. Physiol Rev, 2000 Apr, 80(2), 593 - 614 Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm; Hsu SY et al.; Apoptosis is an essential physiological process by which multicellular organisms eliminate superfluous cells . An expanding family of Bcl-2 proteins plays a pivotal role in the decision step of apoptosis, and the differential expression of Bcl-2 members and their binding proteins allows the regulation of apoptosis in a tissue-specific manner mediated by diverse extra- and intracellular signals . The Bcl-2 proteins can be divided into three subgroups: 1) antiapoptotic proteins with multiple Bcl-2 homology (BH) domains and a transmembrane region, 2) proapoptotic proteins with the same structure but missing the BH4 domain, and 3) proapoptotic ligands with only the BH3 domain . In the mammalian ovary, a high rate of follicular cell apoptosis continues during reproductive life . With the use of the yeast two-hybrid system, the characterization of ovarian Bcl-2 genes serves as a paradigm to understand apoptosis regulation in a tissue-specific manner . We identified Mcl-1 as the main ovarian antiapoptotic Bcl-2 protein, the novel Bok (Bcl-2-related ovarian killer) as the proapoptotic protein, as well as BOD (Bcl-2-related ovarian death agonist) and BAD as the proapoptotic ligands . The activity of the proapoptotic ligand BAD is regulated by upstream follicle survival factors through its binding to constitutively expressed 14-3-3 or hormone-induced P11 . In contrast, the channel-forming Mcl-1 and Bok regulate cytochrome c release and, together with the recently discovered Diva/Boo, control downstream apoptosis-activating factor (Apaf)-1 homologs and caspases . Elucidation of the role of Bcl-2 members and their interacting proteins in the tissue-specific regulation of apoptosis could facilitate an understanding of normal physiology and allow the development of new therapeutic approaches for pathological states. J Clin Microbiol, 2000 Apr, 38(4), 1599 - 608 Evaluation of phenotypic markers for selection and identification of Candida dubliniensis; Tintelnot K et al.; Candida dubliniensis is often associated with C . albicans in cultures . Easy-to-perform selective isolation procedures for these closely related species do not exist . Therefore, we evaluated previously described discriminatory phenotypic markers for C . dubliniensis . A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida . Dark green colonies described as being indicative of C . dubliniensis and other green colonies, 170 in total, were isolated . Chlamydospore formation, intracellular beta-D-glucosidase activity, ability to grow at 42 degrees C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization . Sequencing of the 5' end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for C . dubliniensis . C . dubliniensis was found in 34% of yeast-colonized HIV-infected patients . The color of the colonies on CHROMagar Candida proved to be insufficient for selecting C . dubliniensis, since only 30 of 53 proven C . dubliniensis isolates showed a dark green color in primary cultures . The described typical chlamydospore formation can give only some indication of C . dubliniensis . The assimilation pattern proved to be insufficient to discriminate C . dubliniensis from C . albicans . All C . dubliniensis strains showed no or highly restricted growth at 42 degrees C and a lack of beta-D-glucosidase activity . Unfortunately, atypical C . albicans strains can also exhibit these phenotypic traits . FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species. J Cell Biol, 2000 Apr 3, 149(1), 209 - 22 The function of plakophilin 1 in desmosome assembly and actin filament organization; Hatzfeld M et al.; Plakophilin 1, a member of the armadillo multigene family, is a protein with dual localization in the nucleus and in desmosomes . To elucidate its role in desmosome assembly and regulation, we have analyzed its localization and binding partners in vivo . When overexpressed in HaCaT keratinocytes, plakophilin 1 localized to the nucleus and to desmosomes, and dramatically enhanced the recruitment of desmosomal proteins to the plasma membrane . This effect was mediated by plakophilin 1's head domain, which interacted with desmoglein 1, desmoplakin, and keratins in the yeast two-hybrid system . Overexpression of the armadillo repeat domain induced a striking dominant negative phenotype with the formation of filopodia and long cellular protrusions, where plakophilin 1 colocalized with actin filaments . This phenotype was strictly dependent on a conserved motif in the center of the armadillo repeat domain . Our results demonstrate that plakophilin 1 contains two functionally distinct domains: the head domain, which could play a role in organizing the desmosomal plaque in suprabasal cells, and the armadillo repeat domain, which might be involved in regulating the dynamics of the actin cytoskeleton. Microbiology, 2000 Mar, 146 ( Pt 3), 695 - 9 L-{U-14C} lactate binding to a 43 kDa protein in plasma membranes of Candida utilis; Geros H et al.; To identify the putative lactate transporter protein of Candida utilis, plasma membranes from cells grown either on lactic acid (presence of lactate proton symport) or glucose (absence of lactate proton symport) were incubated with L-{U-14C}lactic acid and the membrane proteins were then separated by SDS-PAGE . A well-defined peak of radioactivity occurred in the lane of the gel containing plasma membrane proteins from lactic-acid-grown cells but not from glucose-grown cells . Binding was inhibited by unlabelled pyruvate and lactate, whereas succinate and citrate were not inhibitory . The monocarboxylate transporter inhibitor of animal cells, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, competitively inhibited the lactate proton symport in the whole yeast and also inhibited lactate binding to proteins of isolated plasma membranes . The polypeptide pattern of plasma membranes from lactic-acid-grown cells revealed a 43 kDa polypeptide associated with the peak of labelled lactate . Altogether the results suggest that this polypeptide is either the lactate transporter or a component of it. Br Med Bull, 1999, 55(3), 544 - 55 Disorders of copper transport; Cox DW; Copper is an essential component of a number of important enzymes . Efficient systems have developed for providing sufficient copper for essential functions, while eliminating excess to avoid tissue toxicity . Copper transport is disrupted in two human diseases: Wilson disease and Menkes disease . Both have defects in copper transporting membrane proteins . Many other proteins are involved in copper transport . Some of these proteins have been identified through a study of the similar copper pathway in yeast . This suggests other copper transport diseases are yet to be discovered . Molecular diagnosis holds promise for reliable diagnosis of patients . Testing of flanking markers is a reliable way to detect presymptomatic sibs of a definite patient. Med Mycol, 2000 Feb, 38(1), 81 - 3 Initial case of Candida dubliniensis infection from Asia: non-mucosal infection; Kamei K et al.; A yeast, repeatedly isolated from a post-surgical abdominal infection of a 75-year-old Japanese man, was genotyped as Candida dubliniensis . This is the first reported case in Asia of this recently described fungus. Med Mycol, 2000 Feb, 38(1), 27 - 30 Isolation of Trichosporon asahii from environmental materials; Sugita T et al.; Trichosporon asahii is the most clinically important pathogenic yeast in the genus Trichosporon, as this species causes both deep-seated infection and summer-type hypersensitivity pneumonitis . We isolated 29 T . asahii colonies from environmental samples using the polymerase chain reaction (PCR) and dichloran rose bengal chloramphenicol (DRBC) medium . Our results suggest that T . asahii is common in nature. Med Mycol, 2000 Feb, 38(1), 15 - 22 Monitoring internalization of Histoplasma capsulatum by mammalian cell lines using a fluorometric microplate assay; Scott AJ et al.; We have developed a fluorometric microtiter plate assay to quantify the internalization of Histoplasma capsulatum yeasts by macrophages . The assay utilizes the fluorescent dye Calcofluor White to label the yeast cell wall and the vital dye trypan blue, which does not enter viable macrophages, to quench fluorescence of extracellular labeled yeasts . Murine RAW 264.7 cells showed more efficient internalization of strain G217B yeasts than human U937 cells . Both cell lines exhibited a dependence upon actin, and, to a lesser degree, microtubules, in G217B uptake. J Med Virol, 2000 May, 61(1), 29 - 36 Lack of clinical evidence for involvement of hepatitis C virus interferon-alpha sensitivity-determining region variability in RNA-dependent protein kinase-mediated cellular antiviral responses; Mihm S et al.; The hepatitis C virus (HCV) interferon-alpha (IFN-alpha) sensitivity-determining region (ISDR) has been shown to suppress double-stranded RNA-dependent protein kinase (PKR) activity in vitro in a yeast PKR expression system . Since variability of ISDR was shown to correlate with nonresponsiveness to IFN-alpha therapy in chronically HCV-infected patients, it has been suggested that prototype ISDR might be a viral inhibitor of cellular PKR . The present study evaluates the biological significance of ISDR variability in situ, relating it to PKR-mediated cellular antiviral responses within the liver . ISDR variability was determined in patients chronically infected with HCV genotypes 1a, 1b, and 3a by direct sequencing using liver-derived RNA preparations as starting material . As surrogate parameters for PKR-mediated cellular responses, hepatic endogenous IFN-alpha gene expression as well as MxA expression were analysed by a competitive, quantitative reverse transcription-polymerase chain reaction technique . Irrespectively of intra- or intergenotypic ISDR amino acid substitutions, ISDR variability was found not to correlate with endogenous hepatic IFN-alpha or with hepatic MxA gene expression . The data suggest that at least two prominent PKR-mediated cellular responses might be largely unaffected by HCV ISDR variability . FEBS Lett, 2000 Mar 31, 470(3), 360 - 4 Isolation of Ich-1S (caspase-2S)-binding protein that partially inhibits caspase activity; Ito A et al.; Members of the caspase family are essential executors of apoptosis . Caspase-2 has two messenger RNAs generated by alternative splicing, which encode caspase-2L and caspase-2S . Although caspase-2L induces apoptosis, caspase-2S also has the ability to antagonize cell death . Experiments in caspase-2-deficient mice showed that caspase-2 functions to delay cell death in some neuronal populations, suggesting that caspase-2S dominantly acts for cell survival in the brain . However, the mechanism of caspase-2S-mediated anti-apoptotic effect is still unclear . Here, we isolated a protein that interacts with caspase-2S, designated as Ich-1S (caspase-2S)-binding protein (ISBP), by yeast two-hybrid screening using full-length caspase-2S cDNA as a bait . ISBP is identical to the recently isolated calcium and integrin-binding protein, and a small molecule calcium-binding protein with two EF-hand motifs of its C-terminus . In vitro transcribed and translated ISBP interacts specifically with glutathione-S-transferase-fused caspase-2S . Moreover, the interaction between ISBP and caspase-2S was observed in cultured cells . Northern blot analysis indicated that ISBP may be a ubiquitous protein . Interestingly, ISBP can partially inhibit the processing of pro-caspase-2L induced by anti-Fas antibody-treated Jurkat cytosolic lysates . These results suggested that ISBP may be the mediator for the survival function of caspase-2S. FEBS Lett, 2000 Mar 31, 470(3), 239 - 43 The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome; Savino C et al.; The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented . The fold typical of other plant toxins is conserved, despite some differences in the loop regions . The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate . Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications . A contact surface inside the C-terminal region of saporin has been identified . Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition. FEBS Lett, 2000 Mar 31, 470(3), 227 - 31 Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner; Menon RP et al.; We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble beta-galactoside-binding protein, galectin-3 . We utilised as bait murine full-length galectin-3 to screen a murine 3T3 cDNA library . Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology . In various assays in vitro the novel protein was shown to bind galectin-3 in a carbohydrate-independent manner . The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins . Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein. J Med Genet, 2000 Apr, 37(4), 250 - 5 Mutation screening in Rett syndrome patients; Xiang F et al.; Rett syndrome (RTT) was first described in 1966 . Its biological and genetic foundations were not clear until recently when Amir et al reported that mutations in the MECP2 gene were detected in around 50% of RTT patients . In this study, we have screened the MECP2 gene for mutations in our RTT material, including nine familial cases (19 Rett girls) and 59 sporadic cases . A total of 27 sporadic RTT patients were found to have mutations in the MECP2 gene, but no mutations were identified in our RTT families . In order to address the possibility of further X chromosomal or autosomal genetic factors in RTT, we evaluated six candidate genes for RTT selected on clinical, pathological, and genetic grounds: UBE1 (human ubiquitin activating enzyme E1, located in chromosome Xp11.23), UBE2I (ubiquitin conjugating enzyme E2I, homologous to yeast UBC9, chromosome 16p13.3), GdX (ubiquitin-like protein, chromosome Xq28), SOX3 (SRY related HMG box gene 3, chromosome Xq26-q27), GABRA3 (gamma-aminobutyric acid type A receptor alpha3 subunit, chromosome Xq28), and CDR2 (cerebellar degeneration related autoantigen 2, chromosome 16p12-p13.1) . No mutations were detected in the coding regions of these six genes in 10 affected subjects and, therefore, alterations in the amino acid sequences of the encoded proteins can be excluded as having a causative role in RTT . Furthermore, gene expression of MECP2, GdX, GABRA3, and L1CAM (L1 cell adhesion molecule) was also investigated by in situ hybridisation . No gross differences were observed in neurones of several brain regions between normal controls and Rett patients. Structure Fold Des, 2000 Mar 15, 8(3), 329 - 38 Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha; Conti E et al.; BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs) . The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif . Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor . RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution . Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide . The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues . CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them . The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS . In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region . Versatility in the specific recognition of NLSs relies on the modular. Anal Chem, 2000 Mar 15, 72(6), 1334 - 41 DNA sensing on a DNA probe-modified electrode using ferrocenylnaphthalene diimide as the electrochemically active ligand; Takenaka S et al.; Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA . On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing . A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode . Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s . The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode . Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level . The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol. Anal Chem, 2000 Mar 15, 72(6), 1163 - 8 Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer; Borchers C et al.; The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction . It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags) . Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity . Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search . This uncertainty, however, is overcome by MS/MS analysis of the peptides . Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein . This procedure is thus, highly sensitive, accurate, and rapid . In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling . Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1. Anal Chem, 2000 Mar 15, 72(6), 1112 - 8 Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching; Goodlett DR et al.; A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described . Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm . The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits . Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues . Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis . The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra . The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database . As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping. Prog Cell Cycle Res, 2000, 4, 107 - 14 Cell cycle regulation by the Cdc25 phosphatase family; Nilsson I et al.; Activation of cyclin-dependent kinases in higher eukaryotic cells can be achieved through dephosphorylation by members of the Cdc25 phosphatase family, Cdc25A, Cdc25B and Cdc25C . Cdc25A plays an important role at the G1/S-phase transition . Cdc25B undergoes activation during S-phase and plays a role in activating the mitotic kinase Cdk1/cyclin B in the cytoplasm . Active Cdk1/cyclin B then phosphorylates and activates Cdc25C leading to a positive feedback mechanism and to entry into mitosis . Cdc25A and B are potential human oncogenes . In addition, Cdc25 is a main player of the G2 arrest caused by DNA damage or in the presence of unreplicated DNA. Trends Plant Sci, 2000 Apr, 5(4), 160 - 7 Pre-mRNA splicing in higher plants; Lorkovic ZJ et al.; Most plant mRNAs are synthesized as precursors containing one or more intervening sequences (introns) that are removed during the process of splicing . The basic mechanism of spliceosome assembly and intron excision is similar in all eukaryotes . However, the recognition of introns in plants has some unique features, which distinguishes it from the reactions in vertebrates and yeast . Recent progress has occurred in characterizing the splicing signals in plant pre-mRNAs, in identifying the mutants affected in splicing and in discovering new examples of alternatively spliced mRNAs . In combination with information provided by the Arabidopsis genome-sequencing project, these studies are contributing to a better understanding of the splicing process and its role in the regulation of gene expression in plants. Exp Cell Res, 2000 Apr 10, 256(1), 168 - 78 Crk-associated substrate p130(Cas) interacts with nephrocystin and both proteins localize to cell-cell contacts of polarized epithelial cells; Donaldson JC et al.; Crk-associated substrate (p130(Cas), Cas) is a docking protein first recognized as having elevated phosphotyrosine content in mammalian cells transformed by v-Src and v-Crk oncoproteins . Subsequent studies have implicated Cas in the control of normal cell behavior through its roles in integrin-mediated signal transduction and organization of the actin cytoskeleton at sites of cell adhesion . In this study, we sought to gain new insight into normal Cas function by identifying previously unrecognized interacting proteins . A yeast two-hybrid screen using the C-terminal region of Cas as a bait identified the Src homology 3 (SH3) domain of the mouse "nephrocystin" protein-orthologous to a human protein whose loss of function leads to the cystic kidney disease familial juvenile nephronophthisis . The putative full-length mouse and partial canine nephrocystin sequences were deduced from cDNA clones . Additional studies using epitope-tagged mouse nephrocystin indicated that nephrocystin and Cas can interact in mammalian cells and revealed that both proteins prominently localize at or near sites of cell-cell contact in polarized Madin-Darby canine kidney epithelial cells . Our findings provide novel insight into the normal cellular activities regulated by both Cas and nephrocystin, and raise the possibility that these proteins have a related function in polarized epithelial cells . Protein Sci, 2000 Jan, 9(1), 129 - 37 Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases; Raman CS et al.; The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39 . The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early . The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)) . N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation . A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter . Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c . This study illustrates that surface formation can be coupled to early events in protein folding . Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding. Protein Sci, 2000 Jan, 9(1), 83 - 94 Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB; Bateman KS et al.; Turkey ovomucoid third domain (OMTKY3) is a cano