Search

The use of real-time plant simulators running in parallel with a nuclear plant to predict the control system behaviour and highlighting unexpected plant behaviour is presented. The study is performed on a 910 MW Generation II Framatome Pressurised Water Reactor model and simulator. By simulating the plant behaviour in real-time whilst comparing it with the real-time transient the plant is following, a complete second set of expected control operations and simulated plant measurements is generated. This enables the calculation of the unknown set of variables introduced into the plant as a fault condition. The benefit of such a system is that plant faults that are too small to detect (especially during transients when the plant operating point is moving around) can be identified as unexpected or faulty plant behaviour. The behaviour of the control system is also continually predicted so the effect of the control system compensating for fault symptoms (which in most cases hides the fault condition) is used to characterise the fault as a control variable acting on the plant. The approach is illustrated by simulating a specific fault, small enough to go undetected for an extended period of time, during a typical transient. This is continually compared with a plant simulation, simulating the same transient without the fault. Using the described methodology, the fault is detected and characterised long before the plant safety is jeopardised or the fault is detected by the conventional protection system.

The Monte Alban state emerged in the Valley of Oaxaca, Mexico, between 300 and 100 BC. Archaeological evidence indicates that this case of state formation was linked to the territorial expansion of the Monte Alban polity. We argue that rulers at Monte Alban adopted an expansionist strategy in order to acquire resources they could use to fund campaigns against rival polities and to bolster their position in a competitive sociopolitical environment. Logistical challenges associated with expansion favored experimentation with new (state) forms of administrative control, while importation of exotic goods contributed to the development of social stratification. Multiple lines of evidence—including excavation data from Tilcajete and Yaasuchi, survey data from the Sola Valley, and a GIS model of potential transportation routes—indicate that the Pacific coast of Oaxaca likely was a target of Monte Alban’s expansion. Our model explains the variable nature of that expansion and its impact on the developmental histories of neighboring regions, as well as the synergistic increase in complexity among interacting societies in Late Formative Oaxaca. Cross-cultural comparison demonstrates that key factors in the formation of the Monte Alban state (e.g., status rivalry, inter-polity conflict, territoriality, militarism, trade) are evident elsewhere in the world.

In addition to normal alternative splicing events (those that take place in untreated cells), there are also those that are inducible in response to environmental stimuli. We previously reported that the alternative splicing of p53-inducible gene 3 (PIG3) exon 4 is modulated in response to UV radiation. Here, we investigate the mechanisms mediating the alternative splicing of this exon. Through the use of various minigene constructs our results reveal that numerous factors influence the normal alternative splicing of PIG3 exon 4, and that these ultimately affect its UV-inducible alternative splicing. Included among these are sequence elements located within exon 4 itself as well as adjacent sequences required for intron definition (the pyrimidine tract, 3'- and 5'-splice sites). Within exon 4, we identified a novel hnRNP A1-dependent exonic splicing silencer (ESS) whose mutation inhibited the alternative splicing of a PIG3 minigene. Although previously implicated in the UV-inducible alternative splicing of other transcripts, RNAi-mediated silencing of hnRNP A1/A2 or hSlu7 did not prevent the UV-enhancement of exon 4 skipping. Overall, our results suggest that numerous factors contribute to the normal alternative splicing of PIG3 exon 4 and that UV-inducible increases in this process require that the splicing of this exon be maintained in a sufficiently weakened state under normal conditions.

To characterize a novel splice variant of the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMRalpha), which we discovered in human neutrophils.We used reverse transcriptase polymerase chain reaction to identify, characterize, and examine the expression of a novel splice variant of the GMRalpha transcript. At the protein level, surface plasmon resonance was used to measure the affinity of a recombinant soluble form of the novel GMRalpha protein for GM-CSF ligand. The full-length novel GMRalpha protein was expressed in a recombinant cell culture system, and its expression and localization were examined using Western blotting, I(125) GM-CSF binding assays, flow cytometry, and a soluble GMRalpha enzyme-linked immunosorbent assay.The novel GMRalpha transcript identified herein contains a previously undescribed exon of the GMRalpha gene; this exon derives from an Alu DNA repeat element, and is alternatively spliced in the novel GMRalpha transcript. Inclusion of this 102 nucleotide exon results in translation of a protein product, which we have named Alu-GMRalpha. Alu-GMRalpha is identical to cell surface GMRalpha, but additionally contains a 34 amino-acid insert in the juxtamembrane region of the extracellular domain of GMRalpha. Functionally, the Alu-GMRalpha-specific epitope does not modify the ability of the protein to bind GM-CSF, but rather appears to be preferentially targeted by ectodomain proteases to mediate the release of a third soluble GM-CSF receptor into the extracellular space.This study provides the first example of a cytokine receptor system in which soluble receptors are produced by three distinct mechanisms. Our results highlight the importance of soluble GMRalpha proteins in regulation of GM-CSF signaling.

Prader-Willi syndrome (PWS) is a complex neurobehavioral disorder that results from loss of function of 10 clustered, paternally expressed genes in a 1.5-Mb region of chromosome 15q11-q13. Many of the primary PWS region genes appear to have nuclear RNA regulatory functions, suggesting that multiple genetic pathways could be secondarily affected in PWS. Using a transgenic mouse model of PWS (TgPWS) with an approximately 4-Mb chromosome 7C deletion of paternal origin that models the neonatal phenotype of the human syndrome we compared by oligonucleotide microarrays expression levels of approximately 12,000 genes and ESTs in TgPWS and wild-type brain. Hybridization data were processed with two distinct statistical algorithms and revealed a dramatically reduced expression of 4 imprinted genes within the deletion region in TgPWS mice, with 2 nonimprinted, codeleted genes reduced twofold. However, only 3 genes outside the deletion were significantly altered in TgPWS mouse brain, with approximately 1.5-fold up-regulation of mRNA levels. Remarkably, these genes map to a single chromosome domain (18B3), and by quantitative RT-PCR we show that 8 genes in this domain are up-regulated in TgPWS brain. These 18B3 genes were up-regulated in an equivalent manner in Angelman syndrome mouse (TgAS) brain, which has the same deletion but of maternal origin. Therefore, the trans-regulation of the chromosome 18B3 domain is due to decreased expression of a nonimprinted gene within the TgPWS/AS mouse deletion in mouse chromosome 7C. Most surprisingly, since 48-60% of the genome was screened, it appears that the imprinted mouse PWS loci do not widely regulate mRNA levels of other genes and may regulate RNA structure.

The p53-inducible gene 3 (PIG3) is a transcriptional target of the tumor suppressor protein p53 and is thought to play a role in apoptosis. In this report, we identify a novel alternatively spliced product from the PIG3 gene that we call PIG3AS (PIG3 alternative splice). PIG3AS results from alternative pre-mRNA splicing that skips exon 4 of the five exons included in the PIG3 transcript. The resulting protein product shares its first 206 amino acids with PIG3 but has a unique 42-amino acid C terminus. In unstressed cells and after most DNA damage conditions that induce transcription from the PIG3 gene, production of the PIG3 transcript dominates. However, in response to UV light, pre-mRNA splicing shifts dramatically in favor of PIG3AS. Unlike the PIG3 protein, the PIG3AS protein is rapidly degraded with a short half-life and is stabilized by proteasome inhibition. Our results illustrate the first example of an endogenous, UV-inducible, alternative splicing event and that control of the splicing machinery is involved in the cellular DNA damage response. They also suggest that rapid proteolytic degradation represents a cellular mechanism for uncoupling p53 activity from PIG3 gene activation that is independent of promoter selectivity.

As in Prader–Willi syndrome (PWS) infants, mouse models of PWS display failure-to-thrive during the neonatal period. In rodents, the hypothalamic neuropeptide, Neuropeptide Y (NPY) and Agouti-related peptide (AgrP) stimulate while α-melanocyte stimulating hormone (α-MSH) inhibits appetite. We hypothesized that altered expression of these neuropeptides in the hypothalamus may underlie the failure-to-thrive in PWS neonatal mice. To test this hypothesis we evaluated mRNA expression of Npy , Agrp , and Pomc by in situ hybridization in the hypothalamic arcuate nucleus (ARC) of 3-day-old female and male PWS neonates. The results showed that Agrp mRNA expression was decreased relative to wild-type (WT) controls in neonates of both sexes, while mRNA expression of Pomc was upregulated in PWS neonates. Since AgrP and the Pomc -derived peptide, α-MSH, are functional antagonists at melanocortin 4 receptors in the hypothalamic regulation of appetitive behavior, these results show that robust anorexigenic melanocortin signaling, may contribute to the failure-to-thrive in PWS neonatal mice.

Precisely how mutant p53 exerts a dominant negative effect over wild type p53 has been an enigma. To understand how wild type and mutant p53 form hetero-oligomers, we studied p53 biogenesis in vitro. We show here that p53 dimers are formed cotranslationally (on the polysome), whereas tetramers are formed posttranslationally (by the dimerization of dimers in solution). Coexpression of wild type and mutant p53 therefore results in 50% of the p53 generated being heterotetramers comprised of a single species: wild type dimer/mutant dimer. Using hot spot mutants of p53 and a variety of natural target sites, we show that all wild type/mutant heterotetramers manifest impaired DNA binding activity. This impairment is not due to the mutant dimeric subunit inhibiting association of the complex with DNA but rather due to the lack of significant contribution (positive cooperativity) from the mutant partner. For all heterotetramers, bias in binding is particularly pronounced against those sequences in genes responsible for apoptosis rather than cell growth arrest. These results explain the molecular basis of p53 dominant negative effect and suggest a functional role in the regulation of p53 tetramerization.

Intronless genes can arise by germline retrotransposition of a cDNA originating as mRNA from an intron-containing source gene. Previously, we described several members of a family of intronless mammalian genes encoding a novel class of zinc-finger proteins, including one that shows imprinted expression and one that escapes X-inactivation. We report here the identification and characterization of the Makorin ring finger protein 1 gene (MKRN1), a highly transcribed, intron-containing source for this family of genes. Phylogenetic analyses clearly indicate that the MKRN1 gene is the ancestral founder of this gene family. We have identified MKRN1 orthologs from human, mouse, wallaby, chicken, fruitfly, and nematode, underscoring the age and conservation of this gene. The MKRN gene family encodes putative ribonucleoproteins with a distinctive array of zinc-finger motifs, including two to four C(3)H zinc-fingers, an unusual Cys/His arrangement that may represent a novel zinc-finger structure, and a highly conserved RING zinc-finger. To date, we have identified nine MKRN family loci distributed throughout the human genome. The human and mouse MKRN1 loci map to a conserved syntenic group near the T-cell receptor beta cluster (TCRB) in chromosome 7q34-q35 and chromosome 6A, respectively. MKRN1 is widely transcribed in mammals, with high levels in murine embryonic nervous system and adult testis. The ancient origin of MKRN1, high degree of conservation, and expression pattern suggest important developmental and functional roles for this gene and its expressed family members.

Summary Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurobehavioral disorders that most often arise from a 4-Mb deletion of chromosome 15q11-q13 during paternal or maternal gametogenesis, respectively. At a de novo frequency of ∼.67–1/10,000 births, these deletions represent a common structural chromosome change in the human genome. To elucidate the mechanism underlying these events, we characterized the regions that contain two proximal breakpoint clusters and a distal cluster. Novel DNA sequences potentially associated with the breakpoints were positionally cloned from YACs within or near these regions. Analyses of rodent-human somatic-cell hybrids, YAC contigs, and FISH of normal or rearranged chromosomes 15 identified duplicated sequences (the END repeats) at or near the breakpoints. The END -repeat units are derived from large genomic duplications of a novel gene ( HERC2 ), many copies of which are transcriptionally active in germline tissues. One of five PWS/AS patients analyzed to date has an identifiable, rearranged HERC2 transcript derived from the deletion event. We postulate that the END repeats flanking 15q11-q13 mediate homologous recombination resulting in deletion. Furthermore, we propose that active transcription of these repeats in male and female germ cells may facilitate the homologous recombination process.

SummaryMicrodeletions of a region termed the “imprinting center” (IC) in chromosome 15q11-q13 have been identified in several families with Prader-Willi syndrome (PWS) or Angelman syndrome who show epigenetic inheritance for this region that is consistent with a mutation in the imprinting process. The IC controls resetting of parental imprints in 15q11-q13 during gametogenesis. We have identified a larger series of cases of familial PWS, including one case with a deletion of only 7.5 kb, that narrows the PWS critical region to

Imprinted genes are marked in the germline and retain molecular memory of their parental origin, resulting in allelic expression differences during development. Abnormalities in imprinted inheritance occur in several genetic diseases and cancer, and are exemplified by the diverse genetic defects involving chromosome 15q11-q13 in Prader-Willi (PWS) and Angelman (AS) syndromes. PWS involves loss of function of multiple paternally expressed genes, while mutations in a single gene, UBE3A, which is subject to spatially restricted imprinting, occur in some AS patients. Identification of mutations in the imprinting process in PWS and AS has led to a definition of an imprinting center (IC), involving the promoter (in PWS) or an alternative transcript of the SNRPN gene (in AS). The IC regulates initiation of imprint switching for all genes in a 2 Mb imprinted domain during gametogenesis. Imprinting mutations define a novel mechanism of genetic disease because they have no direct effect in the affected patient but, rather, it is the parental germline effect of an IC mutation that leads to disease in the offspring.

Using single-cell imaging, we investigated developmental changes in the modulation of KCl-evoked Ca2+ entry by various voltage-dependent Ca2+ channels and the coupling of these channels to exocytosis in cultured cerebellar granule neurons. A component of the KCl-evoked Ca2+ elevation sensitive to nifedipine and localized at cell somata, decreases with culture age. A component blocked by 200 nM omega-Agatoxin-IVA increases with age and whilst localized primarily at the cell somata, also becomes evident at the neurites. The change in activity between nifedipine-sensitive Ca2+ channels and omega-Agatoxin-IVA-sensitive Ca2+ channels occurs at 13 days in vitro at cell somata. A component of Ca2+ entry insensitive to nifedipine and 200 nM omega-Agatoxin-IVA is localized primarily at the neurites and is apparent at all ages. Single-cell imaging of exocytosis using FM1-43 destaining indicates that the residual, but not the nifedipine- or omega-Agatoxin-IVA-sensitive components of Ca2+ entry, modulates exocytosis. However cells cultured for 20-26 days develop a component of Ca2+ entry at the neurites which is sensitive to 200 nM omega-Agatoxin-IVA and omega-Conotoxin-MVIIC and which partially controls release. Immunolocalization studies reveal that binding sites for omega-Conotoxin-GVIA are present throughout development, even though this toxin does not inhibit KCl-evoked [Ca2+]c elevations or exocytosis. 300 nM omega-Agatoxin-IVA labels both somata and, at later developmental stages, neurites, consistent with the functional studies.

We have determined the structure, nucleotide sequence, and polymorphisms of the human P gene. Mutations of the P gene result in type II oculocutaneous albinism (OCA2) in humans and pink-eyed dilution (p) in mice. We find that the human P gene is quite large, consisting of 25 exons spanning 250 to 600 kb in chromosome segment 15q11-q13. The P polypeptide appears to define a novel family of small molecule transporters and may be involved in transport of tyrosine, the precursor to melanin synthesis, within the melanocyte. These results provide the basis for analyses of patients with OCA2 and may point toward eventual pharmacologic treatment of this and related disorders of pigmentation.

Prader-Willi (PWS) and Angelman (AS) syndromes illustrate a disease paradigm of genomic imprinting, an epigenetic modification of DNA that results in parent-of-origin specific expression during embryogenesis and in the adult. From genetic data, at least two imprinted genes may be required for the classical PWS phenotype, whereas AS probably involves a single imprinted gene, and rare familial forms of both disorders involve imprinting mutations. In addition, the nonimprinted P gene is associated with pigmentation disorders in PWS, AS and oculocutaneous albinism. Identification of new genes, delineation of small deletions in unique patients, and direct screening for imprinted sequences, should soon identify candidate genes for PWS and AS. The mechanism of imprinting involves DNA methylation and replication timing, and appears to include multiple imprinted genes within a large imprinted domain. Imprinting of these genes may be regulated in cis, by an imprinting control element (ICE). Future studies can be expected to unravel the gene identities and imprinting mechanisms involved in these fascinating disorders; ultimately it may be possible to reactivate imprinted gene expression as a therapeutic approach.

Partial cDNAs of the human TYRO3 gene, encoding a putative receptor tyrosine kinase, and its processed pseudogene (TYRO3P) were cloned from human teratocarcinoma cell, bone marrow and melanocyte cDNA libraries. The tyrosine kinase homologous domains of TYRO3 and TYRO3P were sequenced and compared with each other and with the mouse TYRO3 gene. Abundant levels of the 4.2-kb TYRO3 mRNA were detected in human brain, and lower levels in other human tissues. TYRO3 and TYRO3P were both assigned to human chromosome 15q14-q25 by analysis of DNAs from somatic cell hybrids.

The Prader-Willi and Angelman syndromes are now well established as the paradigm of genomic imprinting in human disease. Over the past year, much has been learnt about the mechanisms by which these syndromes arise and molecular diagnostics for the majority of patients are now available. Mouse models for aspects of the syndromes have been established, and the first association between a gene, located in chromosome 15, at 15q11-q13, and a phenotype (albinism) has been proven. Large parts of the critical regions have been cloned and at least six genes identified. Three genes or DNA sequences may be imprinted: two of these demonstrate DNA-methylation imprints and one is functionally imprinted in mouse. While the molecular mechanism of imprinting is not yet understood, it is beginning to yield its secrets to DNA methylation, replication, and chromatin structure studies of the phenomenon.

It is my honor to introduce the 2002 Curt Stern Awardee, Dr. James R. Lupski, for his ground-breaking work in the initial identification and subsequent characterization of genomic disorders. Jim first coined the term “genomic disorders” to refer to those genetic diseases in which the basis is a chromosome structural alteration due to homologous recombination between flanking low-copy repeat (LCR) sequences (Lupski 1998). These LCRs, also called “duplicons” to distinguish them from dispersed, common repetitive elements and short tandem repeat elements in the genome (Eichler 1998), are region specific and derived by local genomic duplications that have been evolutionarily fixed. In most instances, the phenotypic consequences of genomic disorders arise due to deletion or duplication of one or more dosage-sensitive genes within the chromosome region undergoing rearrangement, and Jim’s work has illustrated this for several genomic disorders associated with chromosome 17p subregions. Clearly, the significance of this group of disorders is illuminated by the recognition that duplicons comprise ∼5%–10% of human genome sequence distributed in a nonrandom pattern within all human chromosomes (Bailey et al. 2002) and that there are dozens of different genetic diseases that are genomic disorders (table 1; Lupski 1998; Ji et al. 2000; Emanuel and Shaikh 2001; Stankiewicz and Lupski 2002). Indeed, at least 1 of every 1,000 births is a new mutation due to this recombination mechanism (Ji et al. 2000; Shaffer and Lupski 2000). With completion of the human genome sequence and with future directed analysis of genes surrounded by duplicons and their potential involvement in developmental disorders and psychiatric conditions, these numbers are likely to increase further. Nevertheless, the current wealth of knowledge began with just one disease and the identification of one small duplicon (or LCR) barely 1 decade ago, and it is for his major role in the key discovery of the chromosome 17p12 duplication in Charcot-Marie-Tooth syndrome type 1A (CMT1A) (Lupski et al. 1991; Pentao et al. 1992), as well as for his subsequent outstanding work in the field of genomic disorders, that Jim is being recognized with this award. Table 1 Genomic Disorders James R. Lupski, M.D., Ph.D., is the Cullen Professor of Molecular and Human Genetics and Professor of Pediatrics at Baylor College of Medicine in Houston, Texas, where he has built a distinguished career since 1986. In his award presentation, Dr. Lupski will elaborate upon the paths that led him and his colleagues to their discoveries in genomic disorders (Lupski 2003). True to the goals of the award, the 2002 Curt Stern Award is presented to Dr. James R. Lupski for his insight and body of work illuminating the importance of genomic disorders and the mechanisms underlying homologous recombination events due to genomic architecture that explain many human diseases.

Autologous Blood Transfusion (ABT) has become the standard of transfusion practice in elective surgery and should complement techniques that are aimed to conserve blood and to minimise the use of homolgous blood eg appropriate preoperative assessment, the use of DDAVP (desmopressin) or Trasylol to decrease surgical blood loss, normovolaemic haemcdilution with collection of autologous blood, surgical attention to haemostasis and the application of blood salvage techniques. Predeposit autologous programs can contribute 10% to the total blood usage and can and should be used for 50% of elective surgical cases. Controversies as to ‘high risk’ donors eg elderly, pregnant, children or cardiac surgery patients have been dispelled and residual controversies pertaining to pretransfusion testing and crossmatching, the redirection of unused ABT units and the use of directed donors are being clarified and resolved. ‘Newer’ aspects of blood transfusion practice such as the evolution of change in the criteria for blood administration ie transfusion triggers, the increasing use of whole blood in massive blood transfusion situations, the development of transfusion control or surveillance teams at the hospital level and the increasing medical involvement and clinical input at the user level are all developing and are progressing in parallel with the evolution of transfusion medicine as a speciality in its own right.

The normal coordinate analyses of hydrazine and hydrazine- d 4 have been performed employing both general valence and modified Urey-Bradley force fields. The PED confirms the assignment of D urig et al. [1] for hydrazine but suggests revision of the assignment for the 938 cm −1 band of hydrazine- d 4 . The i.r. spectra of some transition metal complexes, MCl 2 ·2N 2 H 4 (M  Co, Ni, Zn, Cd) and MCl 2 ·2N 2 D 4 (M  Zn, Cd) and the Raman spectra of MCl 2 ·2N 2 H 4 (M  Zn, Cd), have been measured. The band shifts of the coordinated hydrazine are interpreted in terms of the results of the force constant calculations and heavy isotope frequency shifts. The NN stretching frequency in bidentate hydrazine complexes is re-assigned to the band in the 1150–1170 cm −1 region.

Complexes of pyrazole (pz), 3-methylpyrazole (3MePz) and 3,5-dimethylpyrazole (DMP) with copper(II) salts can be crystallized from aqueous and ethanolic solutions. With pyrazole and 3-methylpyrazole, tetragonal complexes of the type Cu X 2 L 4 (X = Cl, Br, 1 2 So 4 , NO 3 ) ; and CuX2L2 (X = Cl, Br) are formed. 3,5-Dimethylpyrazole forms CuX2(DMP)4 (X = Br, NO3); CuCl2(DMP)2, CuSO4(DMP)2·2H2O, CuCl2(DMP) and the copper(I) complex CuBr(DMP). The electronic spectra of the complexes are reported.

The activities of TiO2, V2O3, Cr2O3, MnO, Fe2O3, Co3O4, NiO, and ZnO towards the hydrogenation of ethylene were studied. The catalysts were identified by X-ray powder crystallography, and as a criterion of the absence of metal the magnetic susceptibility of each sample was determined. An activity pattern was found which closely resembled that found previously in the hydrogen-deuterium exchange reaction, and the disproportionation and dehydrogenation of cyclohexene, catalyzed by the same oxides.

The i.r. and Raman spectra of (PCl 4 [Ti 2 Cl 10 ] have been measured and partial assignments made. The thirty fundamental vibrational modes for a general M 2 X 10 , D 2 h system are illustrated.