Search

The initiation of DNA replication involves a minimum of four factors: a specific DNA sequence (origin), an initiator protein which binds to the origin, a helicase that unwinds the origin and a protein that binds single-stranded DNA that stabilizes the unwound origin. In eukaryotic cells, the origin recognition complex (ORC) is the initiator protein and replication protein A (RPA; ref. 3) is the single-stranded DNA-binding protein. However, the helicase has not been identified and the nature of origins remains elusive, except in the case of Saccharomyces cerevisiae. A unique feature of eukaryotic DNA replication is that it occurs at a few-hundred discrete foci. It has thus been proposed that a real origin must contain a specific DNA sequence and must be attached to replication foci. Using Xenopus laevis egg extracts, we have identified and purified a 170-kD protein, focus-forming activity 1 (FFA-1), which is required for the formation of replication foci. Here we report that FFA-1 has DNA-helicase activity. Moreover, it is a homologue of the human Werner syndrome gene product WRN, a protein associated with premature ageing in humans.

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNA I and VA RNA II , that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNA I in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNA II is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNA II in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNA II , and with an unidentified protein, p97, which did bind VA RNA II . Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNA II > VA RNA I > single-stranded RNA. Furthermore, VA RNA II is more effective than VA RNA I as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNA II .

A survey was conducted on the present behavioral characteristics of 187 cases of adult autism in patients over 18 years of age employing Achenbach's Child Behavior Checklist (CBCL). When their behavioral characteristics were evaluated in relation to Present Language Developmental Level (PLDL) and Present Adaptive Level (PAL), it was seen that greater variation in behavior characteristics was seen among those exhibiting increasingly lower PLDL and PAL scores. Behavior characteristics reminiscent of depression were noted even among those exhibiting high PLDL. Behavior pointing to obsession was found in common among almost all cases of autism irrespective of PLDL or PAL. Psychotic symptoms such as hallucinations and delusions were absent in most cases. The results of the present study were indicative not only of the significance of obsessive behavior in autism, but also its significance in terms of delving further into the psychopathology of the disorder.

p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex.

Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210bcr-abl, a chimeric protein with tyrosine kinase activity. Substrates for p210bcr-abl are likely to be involved in the pathogenesis of CML. Here we describe the purification, cDNA cloning, and characterization of a 56-kDa tyrosine phosphorylated protein, p56dok-2 (Dok-2), from p210bcr-abl expressing cells. The human dok-2 cDNA encodes a 412-amino acid protein with a predicted N-terminal pleckstrin homology domain as well as several other features of a signaling molecule, including 13 potential tyrosine phosphorylation sites, six PXXP motifs, and the ability to bind to p120RasGAP. Dok-2 was shown to be 35% identical to p62dok-1, a recently identified RasGAP binding protein from CML cells, and analysis of the expressed sequence tag data base revealed the presence of at least four additional proteins containing a Dok homology sequence motif. Dok mRNAs were primarily expressed in tissues of hematopoietic origin. These findings strongly suggest that a family of Dok-related proteins exists that bind to RasGAP and may mediate the effects of p210bcr-abl in CML.

The human RNA polymerase II and III snRNA promoters have similar enhancers, the distal sequence elements (DSEs), and similar basal promoter elements, the proximal sequence elements (PSEs). The DSE, which contains an octamer motif, binds broadly expressed activator Oct-1. The PSE binds a multiprotein complex referred to as SNAPc or PTF. On DNAs containing both an octamer site and a PSE, Oct-1 and SNAPc bind cooperatively. SNAPc consists of at least four stably associated subunits, SNAP43, SNAP45, SNAP50, and SNAP190. None of the three small subunits, which have all been cloned, can bind to the PSE on their own. Here we report the isolation of cDNAs corresponding to the largest subunit of SNAPc, SNAP190. SNAP190 contains an unusual Myb DNA binding domain consisting of four complete repeats (Ra to Rd) and a half repeat (Rh). A truncated protein consisting of the last two SNAP190 Myb repeats, Rc and Rd, can bind to the PSE, suggesting that the SNAP190 Myb domain contributes to recognition of the PSE by the SNAP complex. SNAP190 is required for snRNA gene transcription by both RNA polymerases II and III and interacts with SNAP45. In addition, SNAP190 interacts with Oct-1. Together, these results suggest that the largest subunit of the SNAP complex is involved in direct recognition of the PSE and is a target for the Oct-1 activator. They also provide an example of a basal transcription factor containing a Myb DNA binding domain.

We report here purification and characterization of chro- mosome condensation protein complexes (termed condensins) containing XCAP-C and XCAP-E, two Xenopus members of the SMC family. Sucrose density gradient centrifugation reveals two major forms of condensins. The 8S form is a heterodimer of XCAP-C and XCAP-E, whereas the 13S form contains three additional subunits. One of them is identified as a homolog of the Drosophila Barren protein whose mutation shows a defect in chromosome segregation. Chromosomal targeting of condensins is mitosis-specific and is independent of topoisomerase IIα. 13S condensin is required for condensation, as demonstrated by immunodepletion and rescue experiments. Our results suggest that the condensin complexes represent the most abundant structural components of mitotic chromosomes and play a central role in driving chromosome condensation.

Characteristic of chronic myelogenous leukemia (CML) is the presence of the chimeric p210(bcr-abl) protein possessing elevated protein tyrosine kinase activity relative to normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and associates with the p120 ras GTPase-activating protein (GAP). We have purified p62(dok) from a hematopoietic cell line expressing p210(bcr-abl). p62(dok) is a novel protein with features of a signaling molecule. Association of p62(dok) with GAP correlates with its tyrosine phosphorylation. p62(dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases.

Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an ≈350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an ≈600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1 , HOS2 , and HOS3 . We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ . These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

Mammalian TFIIIB can be separated into two fractions required for transcription of the adenovirus type 2 VAI gene, which have been designated 0.38M-TFIIIB and 0.48M-TFIIIB. While 0.48M-TFIIIB has not been characterized, 0.38M-TFIIIB corresponds to a TBP-containing complex. We describe here the purification of this complex, which consists of TBP and a closely associated polypeptide of 88 kDa, and the isolation of a cDNA corresponding to the 88-kDa polypeptide. The predicted protein sequence reveals that the 88-kDa polypeptide corresponds to a human homolog of the Saccharomyces cerevisiae BRF protein, a subunit of yeast TFIIIB. Human BRF (hBRF) probably corresponds to TFIIIB90, a protein previously cloned by Wang and Roeder (Proc. Natl. Acad. Sci. USA 92:7026-7030, 1995), although its predicted amino acid sequence differs from that reported for TFIIIB90 over a stretch of 67 amino acids as a result of frameshifts. Immunodepletion of more than 90 to 95% of the hBRF present in a transcription extract severely debilitates transcription from the tRNA-type VAI promoter but does not affect transcription from the TATA box-containing human U6 promoter, suggesting that the 0.38M-TFIIIB complex, and perhaps hBRF as well, is not required for U6 transcription.

A 35-kDa polypeptide belonging to the high mobility group family of proteins was purified from the yeast Saccharomyces cerevisiae on the basis of its association with a DNA helicase activity. Amino acid sequence alignment suggests that this protein, Hmo1p, is related to the HMG1/2 class of chromatin-associated proteins. Consistent with this prediction, the Hmo1 protein immunolocalizes to the nucleus, binds single-stranded DNA, and unwinds DNA in the presence of eukaryotic DNA topoisomerase I. While the purified protein has no DNA helicase activity on its own, immunoprecipitation experiments confirm that Hmo1p associates with a 5' to 3' DNA helicase activity in nuclear extracts. The in vivo role of the protein was investigated by constructing an hmo1 deletion mutant. This strain has a severe growth defect, reduced plasmid stability, and chromatin that is hypersensitive to micrococcal nuclease digestion. Taken together, the data indicate that HMO1 is likely to be the homolog of HMG1/2 in higher cells and that it plays an important role in genome maintenance.

Chromatin assembly factor 1 (CAF-1) assembles nucleosomes in a replication-dependent manner. The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins. There are at least two members of this subfamily in both human (p46 and p48) and yeast cells (Hat2p, a subunit of the B-type H4 acetyltransferase, and Msi1p). Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60. p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and H3 and H4, and promotes DNA replication-dependent chromatin assembly. CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps with, but is distinct from, that reported for newly synthesized H4 isolated from nascent chromatin. Our data suggest that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism.

CDC37 was originally identified as a Start gene in budding yeast and has been shown to be required for association of CDC28 with cyclins. The exact functional mechanism by which CDC37 promotes this association, however, remains unknown. CDK4 is a cyclin D-dependent kinase that controls progression through G1 of the mammalian cell cycle. We have detected a specific association of CDK4 with the molecular chaperon HSP90 and a 44-kDa protein that we identify as mammalian CDC37. A physical interaction between CDC37 and CDK4 suggests that CDC37 may regulate the mammalian cell cycle through a direct effect on CDK4. Association of CDK4 with both CDC37 and HSP90 may also imply a mechanistic link between the functions of CDC37 and HSP90.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.

Kazuki Hirao, Ryuji Kobayashi, Kenji YabuwakiDepartment of Occupational Therapy, School of Health Science and Social Welfare, Kibi International University, Takahashi City, Okayama, JapanObjective: To determine the association of cognitive judgment and shyness with frequency and quality of flow experience.Design and methods: This was a cross-sectional survey of the relationship between psychological tendency and frequency and quality of flow experience in 68 college students, undertaken in Hiroshima, Japan. The predictors were Shyness Scale scores, measure of ambiguity tolerance scores, and Life Orientation Test scores, and the outcome was the frequency and quality of flow experience.Results: The results of the binary logistic regression analysis indicated that only the measure of ambiguity tolerance (P = 0.02, odds ratio = 1.06, and 95% confidence interval = 1.01–1.11) was a predictor of the quality of flow experience, and only the Shyness Scale (P = 0.007, odds ratio = 0.95, and 95% confidence interval = 0.91–0.98) was a predictor of the frequency of flow experience.Conclusion: The findings suggest that ambiguity tolerance and shyness are associated with the frequency and quality of the flow experience.Keywords: Flow experience, positive psychology, shyness, ambiguity tolerance, life orientation

In normal human fibroblasts, cyclin A-CDK2 exists in a quaternary complex that contains p21 and PCNA. In many transformed cells, p21 disappears, and a substantial fraction of cyclin A-CDK2 complexes with p9ck1/cks2, p19 and p45. To investigate the significance of these rearrangements, we have isolated cDNAs encoding p19 and p45. In vitro reconstitution demonstrated that binding of p19 to cyclin A-CDK2 requires p45. Addition of these proteins to the kinase had no substantial effect on the kinase activity in vitro. Interference with p45 function in vivo by microinjection of antibodies or antisense oligonucleotides prevented entry into S phase in both normal and transformed cells. Cyclin A-CDK2 has previously been identified as a kinase whose activity is essential for S phase. Our results identify p45 as an essential element of this activity. The abundance of p45 is greatly increased in many transformed cells. This could result in changes in cell cycle control that contribute to the process of cellular transformation.

SummaryChromatin assembly factor I (CAF-I) from human cellnuclei is a three-subunit protein complex that assembles histone octamers onto replicating DNA in a cell-free system. Sequences of cDNAs encoding the two largest CAF-I subunits reveal that the p150 protein contains large clusters of charged residues, whereas p60 contains WD repeats. p150 and p60 directly interact and are both required for DNA replication-dependent assembly of nucleosomes. Deletion of the p60-binding domain from the p150 protein prevents chromatin assembly. p150 and p60 form complexes with newly synthesized histories H3 and acetylated H4 in human cell extracts, suggesting that such complexes are intermediates between histone synthesis and assembly onto replicating DNA.

The TATA-box-binding protein TBP exists in the cell complexed with different sets of TBP-associated factors (TAFs). In general, each of these TBP-TAF complexes is dedicated to transcription by a single RNA polymerase. Thus, SL1, TFIID and TFIIIB are required for transcription by polymerases I, II and III, respectively. Here we characterize a fourth TBP-TAF complex called SNAPc. Unlike the other TBP-TAF complexes, SNAPc is implicated in transcription by two types of polymerases; it is required for transcription of both the RNA polymerase II and III small-nuclear RNA genes and binds specifically to the proximal sequence element PSE, a non-TATA-box basal promoter element common to these two types of genes. In addition to TBP, SNAPc is composed of at least three TAFs, SNAP43, SNAP45 and SNAP50. The predicted amino-acid sequence of SNAP43 reveals that it corresponds to a new protein.

The purpose of this study was to develop the Personhood Questionnaire (PQ) to determine the factor structure of "personhood" for elderly healthcare services and identify personhood components other than PQ items.We enrolled 314 healthcare professionals at long-term care facilities in Japan. Participants completed a questionnaire consisting of 17 PQ items. The PQ was designed to assess the degree of need in elderly healthcare services on a 5-point Likert scale (Question 1), and identify personhood components other than PQ items (Question 2). We performed factor analysis for answers to Question 1, and text mining and cluster analysis for answers to Question 2.Factor analysis revealed a four-factor structure. Cronbach's α was 0.87 for the 17 original items and 0.86 for the 15 items after removing two items. Text mining identified 27 personhood components, which were classified into three clusters. The second cluster consisted of non-PQ items.Factor 1 was "forming daily life," factor 2 was "forming career and context," factor 3 was "affecting psychological behavior," and factor 4 was "forming basic attributes." Components of the second cluster require further examination before incorporation into the concept of personhood.• Improving the quality of individualized care, in which "personhood" and dignity of elderly people are respected, is an urgent goal. • The Model of Human Occupation (MOHO), a conceptual practice model for occupational therapy, should be used jointly with the International Classification of Functioning, Disability and Health (ICF) to establish the concept of personhood. • The construction of personhood in elderly health care services consists of a four-factor structure, including "forming daily life," "forming career and context," "affecting psychological behavior," and "forming basic attributes." • This study suggests that provision of high-quality individualized care can be achieved by promoting services that focus on "forming daily life,” which has the highest factor contribution.

This study was performed to investigate the relationship between mental state (especially flow experience [Flow Experience Checklist of Ishimura]) health-related quality of life (Medical Outcome Study 8-Item Short-Form Health Survey), and stress (salivary amylase concentrations). The subjects were 119 healthy elderly persons living in rural areas who participated in a "meeting for the elderly" at a nursing home in Kagawa Prefecture, Japan. The subjects were classified into apathetic, anxious, relaxed, and flow groups. It was found that physical health was significantly better when performing important daily activities in the group who experienced flow and the relaxed group than in the group that was in an apathetic state. However, no significant relationship was observed between the degree of flow experience and stress. The present findings suggested that interventions which make the activities of daily life either "high-challenge, high-skill" situations or "low-challenge, high-skill" situations could have a positive influence on the physical health of community-living elderly Japanese. A longitudinal study should be performed in the future.

Katanin p60 is a microtubule-severing protein and is involved in microtubule cytoskeleton organization in both mitotic and non-mitotic processes. Its role in cancer metastasis is unknown.Differential protein profiles of bone marrow aspirates were analyzed by chromatography, electrophoresis, and mass spectrometry. Expression of katanin p60 in primary and metastatic prostate cancer was examined by immunohistochemistry. Cellular function of katanin p60 was further examined in prostate cell lines.In a proteomic profiling of bone marrow aspirates from men with prostate cancer, we found that katanin p60 was one of the proteins differentially expressed in bone metastasis samples. Immunohistochemical staining showed that katanin p60 was expressed in the basal cells in normal human prostate glands. In prostatic adenocarcinomas, in which the basal cells were absent, katanin p60 was expressed in the prostate cancer cells. In the specimens from bone metastasis, katanin p60 was detectable in the metastatic cancer cells. Strikingly, some of the metastatic cancer cells also co-expressed basal cell biomarkers including the tumor suppressor p53-homologous protein p63 and the high molecular weight cytokeratins, suggesting that the metastatic prostate cancer cells may have a basal cell-like phenotype. Moreover, overexpression of katanin p60 inhibited prostate cancer cell proliferation but enhanced cell migration activity.Katanin p60 was aberrantly expressed during prostate cancer progression. Its expression in the metastatic cells in bone was associated with the re-emergence of a basal cell-like phenotype. The elevated katanin p60 expression may contribute to cancer cell metastasis via a stimulatory effect on cell motility.

The induction of M phase in eukaryotic cell cycles requires robust activation of Cdc2/cyclin B by Cdc25, which itself is robustly activated by serine/threonine phosphorylations. Although multiple protein kinases that directly activate Cdc25C have been identified, whether the combination of different primary phosphorylations of Cdc25C is sufficient to fully activate Cdc25C has not been determined. By analyzing the GST-Cdc25C phosphorylating activity in Xenopus egg extracts, we previously defined roles of MAPK and Cdc2/cyclin B in partially activating Cdc25C and predicted the presence of another major Cdc25C-activating kinase. In this study, we demonstrate that this missing kinase is RSK2, which phosphorylates three sites in Cdc25C and also partially activates Cdc25C. However, the phosphorylations catalyzed by MAPK, Cdc2, and RSK2 fail to fully activate Cdc25C, suggesting that additional biochemical events are required to fully activate this key cell cycle regulator.

Summary Objective The purpose of this study was to investigate the influence of core exercises on upper extremity function relative to skilled motor behavior and postural sway. Method We examined the effects of core exercises on the skilled motor behavior and postural sway of 40 healthy students who were assigned randomly to the core exercise group or the control group. Independent variable is extent of exposure to core exercise and dependent variables are skilled motor behavior and postural sway. A Purdue pegboard which measures skilled motor behavior and a stabilometer which measures postural sway were used to evaluate the influence of core exercises. Pre-intervention and post-intervention skilled motor behavior and postural sway were compared between the core exercise group and control group using the Wilcoxon rank sum test; a significance level of α = 0.05 was considered statistically significant. Also, we investigated the application of core exercises in a clinical setting for one patient with cerebral vascular disease. Results The post intervention skilled motor behavior ( p = 0.04) and postural sway, LNG ( p = 0.05), LNG/TIME ( p = 0.04) and X LNG ( p = 0.02) were significantly higher in the core exercise group than control group. In the case report, there were good results; function of the upper extremity improved after doing the exercises. There were positive changes in some daily living activities. Discussion Core exercises are likely to enhance trunk stabilization to improve upper extremity function. It is possible for core exercises to be adapted for patients.

Comparison of protein profiles of sera acquired before and after preoperative chemotherapy for breast cancer may reveal tumor markers that could be used to monitor tumor response. In this study, we analyzed pre- and post-chemotherapy protein profiles of sera from 39 HER2-postive breast cancer patients (n=78 samples) who received 6 months of preoperative chemotherapy using LC-MALDI-TOF/MS technology. We detected qualitative and quantitative differences in pair-wise comparison of pre- and post chemotherapy samples that were different in patients who achieved pathological complete response (pCR, n=21) compared with those with residual disease (n=18). We identified 2329 and 3152 peaks as differentially expressed in the pre-chemotherapy samples of the responders and non-responders. Comparison of matching pre- and post-chemotherapy samples identified 34 (32 decreased, two increased) and 304 peaks (157 decreased, 147 increased) that significantly changed (p

Effects of ammonia (NH 3 ) on the growth rate and the surface morphology of GaAs were studied in chloride vapor phase epitaxy (C-VPE). We found that the NH 3 addition increases the growth rate at the substrate temperatures of 640–680°C, but does not at higher temperatures in a conventional C-VPE method. For a single flat temperature zone (SFT) C-VPE method, an increase of the growth rate due to the NH 3 addition was not observed in any growth temperature range. These results indicate that the NH 3 addition enhances only the desorption of GaCl 3 , which is the rate determining step of conventional C-VPE at low temperatures. In addition to that, we found that the NH 3 addition greatly suppressed the formation of hillocks which were often observed on the GaAs surface grown on (001)just-oriented substrates. A simple model of the mechanism will be proposed.

A follow-up survey was conducted on 201 young adults with autism who were 18 or older (mean age, 21.5). All had participated previously in intensive therapeutic camping or had therapeutic involvement at medical consultation agencies to evaluate their outcome. Their social outcome was better than that previously reported in Japan. Although 31.5% had shown marked deterioration during adolescence, 43.2% had shown marked improvement during that period. Possible factors contributing to these results are discussed.

We have identified an activity that is required for transcription of downstream promoter element (DPE)–containing core promoters in vitro. The purified factor was found to be the Drosophila homolog of the transcriptional repressor known as NC2 or Dr1-Drap1. Purified recombinant dNC2 activates DPE-driven promoters and represses TATA-driven promoters. A mutant version of dNC2 can activate DPE promoters but is unable to repress TATA promoters. Thus, the activation and repression functions are distinct. These studies reveal that NC2 (Dr1-Drap1) is a bifunctional basal transcription factor that differentially regulates gene transcription through DPE or TATA box motifs.

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0°C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.

Low temperature growth of GaAs and AlAs was studied by direct reaction between CaCl 3 , AlCl 3 and undecomposed AsH 3 , using a hybrid furnace equipped with both infrared lamp heating and resistive heating. When solid arsenic (As 4 ) was used as the arsenic source, the growth of GaAs and AlAs was obtained at substrate temperatures of 500–650°C and above 650°C, respectively. On the other hand, a much lower temperature ( ∼ 300°C) growth of GaAs could be obtained when undecomposed AsH 3 was used. The growth of AlAs, however, was obtained at only above 500°C, even for the direct reaction between AlCl 3 and undecomposed AsH 3 . This is due to the fact that a higher temperature is necessary to reduce the AlCl 3 compared with GaCl 3 . The difference of the growth temperatures between GaAs and AlAs, and between growth with As 4 vapor and with undecomposed AsH 3 , could be qualitatively explained by the thermodynamic analysis.

Tumor associated monocytes/macrophages (MO/MA) are known contributors to the immune-inflammatory cell environment of advanced epithelial ovarian carcinoma (EOC). The secreted proteome of ascitic MO/MA was examined as an aid to the discovery of novel proteins in EOC that are likely to have biological relevance in the inflammatory pathways of EOC. Ascitic fluid MO/MA were isolated from EOC patients, grown short-term in serum-free media. MO/MA supernatants were analyzed for secreted proteins by HPLC fractionation followed by LC-tandem mass spectrometric analysis. The 14-3-3 zeta adaptor protein was identified in supernatants of three of three EOC patients but not in supernatants of buffy coat monocytes isolated from normal donors or the established monocyte cell line THP1. Moreover, 14-3-3 zeta was identified in ascitic fluids in eight of eight chemotherapy-naive patients by both immunoblot and mass spectrometric analysis. Immunofluorescent staining for 14-3-3 zeta demonstrated expression of the protein on ascitic and peritumoral macrophages in EOC patients. 14-3-3 zeta was also expressed on endothelial cells in the peritumoral stroma and partially on tumor cells. Uptake of 14-3-3 zeta was observed in EOC cell lines co-cultured with the recombinant protein expressed in E. coli. It is demonstrated for the first time that the important adaptor protein 14-3-3 zeta is common to the secretome of ascitic MO/MA and the ascites of advanced EOC patients.

The lungs are a common site of serious infection in both healthy and immunocompromised subjects, and the most likely route of delivery of a bioterror agent. Since the airway epithelium shows great structural plasticity in response to inflammatory stimuli, we hypothesized it might also show functional plasticity.To test the inducibility of lung defenses against bacterial challenge.Mice were treated with an aerosolized lysate of ultraviolet-killed nontypeable (unencapsulated) Haemophilus influenzae (NTHi), then challenged with a lethal dose of live Streptococcus pneumoniae (Spn) delivered by aerosol.Treatment with the NTHi lysate induced complete protection against challenge with a lethal dose of Spn if treatment preceded challenge by 4 to 24 hours. Lesser levels of protection occurred at shorter (83% at 2 h) and longer (83% at 48-72 h) intervals between treatment and challenge. There was also some protection when treatment was given 2 hours after challenge (survival increased from 14 to 57%), but not 24 hours after challenge. Protection did not depend on recruited neutrophils or resident mast cells and alveolar macrophages. Protection was specific to the airway route of infection, correlated in magnitude and time with rapid bacterial killing within the lungs, and was associated with increases of multiple antimicrobial polypeptides in lung lining fluid.We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.

GaAs was grown by mixing either AsH 3 or arsenic vapor (As 4 or As 2 ) from As metal with GaCl 3 supplied by He or H 2 gas. When the carrier gas of GaCl 3 was He, GaAs was grown when AsH 3 gas was used. Gas etching of the substrate occurred if As metal was used as the As source. When the carrier gas of GaCl 3 was H 2 , however, GaAs was grown by using either AsH 3 or As metal, but dependence of the growth rate on the substrate position was different. These results indicate that AsH 3 is not completely decomposed into As 4 or As 2 but directly reacts with GaCl 3 and GaCl, while the arsenic vapor (As 4 or As 2 ) can react only with GaCl but not with GaCl 3 .

Programmed cell death (PCD) is involved in a variety of biologic events. Based on the morphologic appearance of the cells, there are two types of PCD as follows: apoptotic (type I) and autophagic (type II). However, the molecular machinery that determines the type of PCD is poorly defined. The purpose of this study was to show whether the presence of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1), a modulator of apoptosis, determines which type of PCD the cell undergoes. Treatment with C(2)-ceramide was associated with both the cleavage of caspase-3 and poly(ADP-ribose) polymerase and the degradation of autophagy-related Beclin 1 and Atg5 proteins, without a change in the cyclin-CDK activity, which culminated in apoptosis in p21(+/+) mouse embryonic fibroblasts (MEFs). On the other hand, C(2)-ceramide did not cleave caspase-3 or poly(ADP-ribose) polymerase and kept Beclin 1 and Atg5 proteins stable in p21(-/-) MEFs, events that this time culminated in autophagy. When expression of the p21 protein was inhibited by small interfering RNA or when the overexpression of Beclin 1 or Atg5 was induced, autophagy rather than apoptosis was initiated in the p21(+/+) MEFs treated with C(2)-ceramide. In contrast, the exogenous expression of p21 or the silencing of Beclin 1 and Atg5 with small interfering RNA increased the number of apoptotic cells and decreased the number of autophagic cells among C(2)-ceramide-treated p21(-/-) MEFs. gamma-Irradiation, which endogenously generates ceramide, induced a similar tendency in these MEFs. These results suggest that p21 plays an essential role in determining the type of cell death, positively for apoptosis and negatively for autophagy.

The objective of this study was to assess the effect of pre-analytical processing on proteomic analysis of saliva and to identify salivary biomarkers for potential clinical applications. Saliva samples from five healthy individuals and three head and neck squamous carcinoma (HNSC) patients were initially depleted of major protein constituents. Saliva from healthy subjects was divided and processed by three different methods prior to liquid chromatography and tandem mass spectrometry technique (LC-MS/MS) analysis. The results showed marked differences amongst the methods. The SDS-PAGE separation and in-gel digestion method yielded the highest number of proteins that included the majority of those identified by the other two methods. The in gel-digestion method was used in the LC-MS/MS analysis of saliva from three HNSC patients and the results were compared with those from healthy subjects. Our analysis identified two proteins, alpha-1-B-glycoprotein and complement factor B proteins, to be present in patients but not in normal specimens. Paradoxically, cystatin S, parotid secretory factor, and poly-4-hydrolase beta-subunit proteins were detected in most normal salivas but not in patient specimens. Subsequent analysis of complement factor B by Western blotting showed strong immunoreactive bands of complement factor B in HNSC patients' and negative or weakly positive in normal saliva samples. We conclude that: 1) initial saliva processing affects protein analysis, 2) in-gel digestion followed by LC-MS/MS detects the most saliva proteins, 3) certain proteins are differentially found in patient and normal salivas and 4) a small set of proteins can be targeted for future validation for clinical investigation.

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation is poorly understood. We have demonstrated that AKT, which is activated downstream from epidermal growth factor receptor signaling, phosphorylates beta-catenin at Ser552 in vitro and in vivo. AKT-mediated phosphorylation of beta-catenin causes its disassociation from cell-cell contacts and accumulation in both the cytosol and the nucleus and enhances its interaction with 14-3-3zeta via a binding motif containing Ser552. Phosphorylation of beta-catenin by AKT increases its transcriptional activity and promotes tumor cell invasion, indicating that AKT-dependent regulation of beta-catenin plays a critical role in tumor invasion and development.

The objective of this study was to assess the effect of pre-analytical processing on proteomic analysis of saliva and to identify salivary biomarkers for potential clinical applications. Saliva samples from five healthy individuals and three head and neck squamous carcinoma (HNSC) patients were initially depleted of major protein constituents. Saliva from healthy subjects was divided and processed by three different methods prior to liquid chromatography and tandem mass spectrometry technique (LC-MS/MS) analysis. The results showed marked differences amongst the methods. The SDS-PAGE separation and in-gel digestion method yielded the highest number of proteins that included the majority of those identified by the other two methods. The in gel-digestion method was used in the LC-MS/MS analysis of saliva from three HNSC patients and the results were compared with those from healthy subjects. Our analysis identified two proteins, alpha-1-B-glycoprotein and complement factor B proteins, to be present in patients but not in normal specimens. Paradoxically, cystatin S, parotid secretory factor, and poly-4-hydrolase beta-subunit proteins were detected in most normal salivas but not in patient specimens. Subsequent analysis of complement factor B by Western blotting showed strong immunoreactive bands of complement factor B in HNSC patients' and negative or weakly positive in normal saliva samples. We conclude that: 1) initial saliva processing affects protein analysis, 2) in-gel digestion followed by LC-MS/MS detects the most saliva proteins, 3) certain proteins are differentially found in patient and normal salivas and 4) a small set of proteins can be targeted for future validation for clinical investigation.

The purpose of this study is to examine frontal lobe function of non-dementia Parkinson's disease (PD) patients. We examined the neuropsychological and behavioral evaluation (Stroop reaction time: SRT). Target image (20%) and non-target image (80%) were presented to PD patients, healthy elderly controls, and healthy young subjects at random. We instructed subjects that the button should pressed on the target image. This study was designed to investigate the mental set under three conditions. The Stroop test, Word Fluency Test (WFT), and Geriatric Depression Scale (GDS) were used to assess cognitive function. The SRT of PD were congruent condition and incongruent condition increased compared with simple condition. The delay of a similar SRT was seen in the healthy elderly control and young groups. However, the SRT of PD patients was slower under incongruent condition than under congruent condition. It appears that PD spent time on judgment in under incongruent condition because the meaning of the character did not correspond to the color of the character. Because, there were no significant between PD and healthy elderly subjects in Stroop test, WFT, and GDS, frontal lobe function had a partial deficit. The SRT of PD was increased by the partial deficit in information processing ability in addition to movement dysfunction.

Type B leukemogenic virus (TBLV), a mouse mammary tumor virus (MMTV) variant, often induces T-cell leukemias and lymphomas by c-mycactivation following viral DNA integration. Transfection assays using a c-mycreporter plasmid indicated that the TBLV long terminal repeat (LTR) enhancer is necessary for T-cell-specific increases in basal reporter activity. The sequence requirements for this effect were studied using mutations of the 62-bp enhancer region in an MMTV LTR reporter vector. Deletion of a nuclear factor A-binding site dramatically reduced reporter activity in Jurkat T cells. However, a 41-bp enhancer missing the RUNX1 site still retained minimal enhancer function. DNA affinity purification using a TBLV enhancer oligomer containing the RUNX1 binding site followed by mass spectrometry resulted in the identification of ALY. Subsequent experiments focused on the reconstitution of enhancer activity in epithelial cells. ALY overexpression synergized with RUNX1B on TBLV enhancer activity, and synergism required the RUNX1B-binding site. A predicted c-Myb binding site in the enhancer was confirmed after c-myboverexpression elevated TBLV LTR reporter activity, and overexpression of c-Myb and RUNX1B together showed additive effects on reporter gene levels. ALY also synergized with c-Myb, and coimmunoprecipitation experiments demonstrated an interaction between ALY and c-Myb. These experiments suggest a central role for ALY in T-cell enhancer function and oncogene activation.

International audience

Histone acetylation is important in regulating DNA accessibility. Multifunctional Sin3 proteins bind histone deacetylases (HDACs) to assemble silencing complexes that selectively target chromatin. We show that, in fission yeast, an essential HDAC, Clr6, exists in two distinct Sin3 core complexes. Complex I contains an essential Sin3 homolog, Pst1, and other factors, and predominantly targets gene promoters. Complex II contains a nonessential Sin3 homolog, Pst2, and several conserved proteins. It preferentially targets transcribed chromosomal regions and centromere cores. Defects in complex II abrogate global protective functions of chromatin, causing increased accessibility of DNA to genotoxic agents and widespread antisense transcripts that are processed by the exosome. Notably, the two Clr6 complexes differentially repress forward and reverse centromeric repeat transcripts, suggesting that these complexes regulate transcription in heterochromatin and euchromatin in similar manners, including suppression of spurious transcripts from cryptic start sites.

The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with alpha-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of alpha-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly.

The CUL4-ROC1 E3 ligase complex regulates genome stability, replication and cell cycle progression. A novel WD40 domain-containing protein, L2DTL/CDT2 and PCNA were identified as proteins associated with CUL4/DDB1 complexes. Inactivation of CUL4A, L2DTL, PCNA, DDB1 or ROC1 induced p53 stabilization and growth arrest. L2DTL, PCNA and DDB1/CUL4A complexes were found to physically interact with p53 tumor suppressor and its regulator MDM2/HDM2. The isolated CUL4A complexes display potent and robust polyubiquitination activity towards p53 and this activity is dependent on L2DTL, PCNA, DDB1, ROC1 and MDM2/HDM2. We also found that the interaction between p53 and CUL4 complex is regulated by DNA damage. Our data further showed that MDM2/HDM2 is rapidly proteolyzed in response to UV irradiation and this process is regulated by CUL4/DDB1 and PCNA. Our studies demonstrate that PCNA, L2DTL and the DDB1-CUL4A complex play critical and differential roles in regulating the protein stability of p53 and MDM2/HDM2 in unstressed and stressed cells.

Critical roles played by some protein kinases in neoplastic transformation and progression provide a rationale for developing selective, small-molecule kinase inhibitors as antineoplastic drugs. Protein kinase Cepsilon (PKCepsilon) is a rational target for cancer therapy, because it is oncogenic and prometastatic in transgenic mouse models. PKCepsilon is activated by sn-1,2-diacylglycerol (DAG). Attempts to develop selective PKCepsilon inhibitors that block activation by DAG or compete with ATP have not yet met with success, suggesting a need for new strategies. We previously reported that cystamine and a metabolic cystine precursor inactivate PKCepsilon in cells in a thiol-reversible manner. In this report, we first determined that PKCepsilon became resistant to inactivation by disulfides when Cys452 was replaced with alanine by site-specific mutagenesis of human PKCepsilon or a constitutively active PKCepsilon mutant. These results showed that the disulfides inactivated PKCepsilon by thiol-disulfide exchange, either upon Cys452 S-thiolation or by rearrangement to an intra-protein disulfide. Mass spectrometric analysis of peptide digests of cystamine-inactivated, carbamidomethylated PKCepsilon detected a peptide S-cysteaminylated at Cys452, indicating that Cys452 S-cysteaminylation is a stable modification. Furthermore, PKCepsilon inactivation by N-ethylmaleimide was Cys452 dependent, providing corroborative evidence that PKCepsilon inhibitors can be designed by targeting Cys452 with small molecules that stably modify the residue. Cys452 is an active site residue that is conserved in only 11 human protein kinase genes. Therefore, the PKCepsilon-inactivating Cys452 switch is a rational target for the design of antineoplastic drugs that selectively inhibit PKCepsilon.