Search

ABSTRACTPrevious research suggests a close relationship between self-reference and emotional valence. The present study investigated potential differences in cognitive resources required for positive vs. negative self-referential processing by examining how self/other-referential processing of positive/negative information affects memory for subsequently presented items. On each encoding trial, participants first judged whether a positive or negative trait adjective described themselves or another person. Then, they were shown a neutral noun and indicated its screen location. Subsequent memory tests showed better memory for self-referenced than other-referenced trait adjectives, and the size of this self-reference effect was not modulated by emotional valence. Although memory for nouns was not affected by preceding positive/negative self/other-referential processing, memory for their associated contextual features was significantly impaired following negative vs. positive self-referential processing. Our findings suggest that negative self-referential processing requires more cognitive resources than positive self-referential processing, thereby leaving relatively less cognitive resources to encode subsequently presented information.

To study the longitudinal dynamics of anti–myelin oligodendrocyte glycoprotein (MOG) autoantibodies in childhood demyelinating diseases.

A cosmid/bacterial artificial chromosome (BAC) contiguous (contig) map of human chromosome (HSA) 19p13.3 has been constructed, and over 50 genes have been localized to the contig. Genes and anonymous ESTs from approximately 4000 kb of human 19p13.3 were placed on the central mouse chromosome 10 map by genetic mapping and pulsed-field gel electrophoresis (PFGE) analysis. A region of approximately 2500 kb of HSA 19p13.3 is collinear to mouse chromosome (MMU) 10. In contrast, the adjacent approximately 1200 kb are inverted. Two genes are located in a 50-kb region after the inversion on MMU 10, followed by a region of homology to mouse chromosome 17. The synteny breakpoint and one of the inversion breakpoints has been localized to sequenced regions in human <5 kb in size. Both breakpoints are rich in simple tandem repeats, including (TCTG)n, (CT)n, and (GTCTCT)n, suggesting that simple repeat sequences may be involved in chromosome breaks during evolution. The overall size of the region in mouse is smaller, although no large regions are missing. Comparing the physical maps to the genetic maps showed that in contrast to the higher-than-average rate of genetic recombination in gene-rich telomeric region on HSA 19p13.3, the average rate of recombination is lower than expected in the homologous mouse region. This might indicate that a hot spot of recombination may have been lost in mouse or gained in human during evolution, or that the position of sequences along the chromosome (telomeric compared to the middle of a chromosome) is important for recombination rates.

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.

AimTo examine the membranous and nuclear distribution of β catenin in the epithelial cells of gut polyps from Peutz-Jeghers syndrome and juvenile polyposis in comparison with other types of polyps and tumours.MethodsImmunohistochemistry for β catenin and proliferation markers was performed on conventional paraffin sections. Immunohistological staining was carried out on Peutz-Jeghers syndrome polyps from four different families, on juvenile polyposis polyps from two different families, on solitary juvenile polyps, and on hyperplastic polyps. The immunohistochemistry was evaluated qualitatively in relation to defined areas of the polyps.ResultsAll polyps from the hamartomatous polyposis syndromes (Peutz-Jeghers syndrome and juvenile polyposis) showed nuclear localisation of β catenin in some epithelial cell nuclei. In Peutz-Jeghers syndrome polyps β catenin positive nuclei were seen at the base of the deep crypt infoldings. In juvenile polyposis polyps and in some solitary juvenile polyps they were found in irregularly distributed cryptal epithelial cells corresponding to the proliferative compartments. Normal mucosa of the gut and hyperplastic polyps of the colon do not show nuclear staining for β catenin.ConclusionsThe dysregulation of cellular β catenin distribution is not only a phenomenon of adenoma formation and adenoma progression in the colon--it is at least focally present in polyps of the hamartomatous type and is related to the proliferation zones of these polyps. The nuclear translocation of β catenin most probably reflects a disturbed β catenin metabolism. In view of the different functions of β catenin during development and cell differentiation, the nuclear translocation of β catenin is likely to be an important factor in enhanced cell proliferation which escapes local control mechanisms.

cDNA clones coding for human S‐protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S‐protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S‐protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of S‐protein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino‐terminal residues 42 are identical with the so‐called somatomedin B peptide suggesting that S‐protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S‐protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways.

The human neutrophil and monocyte-derived serine protease homologues neutrophil elastase (NE), proteinase 3 (PR3), and azurocidin (AZU) are involved in a variety of immune defense reactions. NE and PR3 assist in the destruction of phagocytosed microorganisms, cleave the important connective-tissue protein elastin, and generate chemotactic activities by forming alpha 1-proteinase inhibitor complexes and elastin peptides. AZU is cytotoxic to certain microorganisms and chemotactic for monocytes. All three proteins are produced and packaged into azurophil granules in large quantities during neutrophil differentiation. We have isolated several cosmid clones each of which contains the functional genes for AZU, PR3, and NE in this order. The PR3 gene is separated by 8 kilobases from the 3' end of the AZU gene and by 3 kilobases from the 5' end of the NE gene. We report a physical map of the gene cluster, its location on chromosome 19pter, and the exon-intron organization of the AZU and PR3 genes. Our fluorescence in situ hybridization studies disprove the previous chromosomal assignment of the human NE gene to 11q14. The five exons of AZU and PR3 are organized like those of NE and other granule-associated serine proteases of hematopoietic cells. NE, PR3, and AZU are coordinately downregulated in the premonocytic cell line U937 during induced terminal differentiation. The cluster-like physical organization of these genes and concerted regulation during hematopoietic differentiation suggests that they are located in a developmentally activated chromatin domain promoting high-level, cell-specific expression in the monocyte-myelocyte lineage.

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.

To define the cis-acting elements involved in the regulation of the murine vitronectin (Vn) gene in inflammation, the 5'-flanking region was isolated, fused to the luciferase reporter gene, and the basal and interleukin 6 (IL-6)-stimulated transcriptional activity was tested in transfection experiments using Hep3B cells. Treatment with IL-6 induced this construct by more than 20-fold, whereas the corresponding 5'-flanking region of the human Vn gene was not stimulated. Transfection studies using murine Vn constructs with serial 5'-deletions revealed that two sequences were important in the IL-6 response, and specific mutations in both sequences abolished the response. A 2-base pair mutation converted the human sequence to that of a murine IL-6 responsive element and partially conveyed IL-6 inducibility. In contrast, transforming growth factor beta stimulated the human construct and the endogenous Vn gene in human Hep3B cells in a dose-dependent manner, whereas the murine construct was not responsive. The transforming growth factor beta responsive region was localized to a 30-base pair fragment with little homology to the murine sequence. These studies reveal that the structural basis for the differential regulation of the human and murine Vn genes resides in the differences in promoter sequence.

Complement C6 is one of five plasma proteins that are incorporated into the lytic terminal complement complex on lipid membranes (C5b-9m) upon activation of the complement cascade. Oligonucleotide probes derived from partial amino acid sequences of purified C6 were used to isolate cDNA clones from a human liver cDNA library. The complete polypeptide structure of mature C6 deduced from the cDNA sequence consists of 913 amino acid residues preceded by a typical 21-residue signal peptide. C6 is most similar in structure to complement C7, sharing 33.5% identical residues with C7 including all 56 cysteine residues. The low density lipoprotein receptor class A and B modules, the thrombospondin type I module at the carboxyl terminus, and the two short consensus repeat modules are arranged in the same way as in C7. In contrast to C7 and other terminal complement proteins, the thrombospondin type I module at the amino terminus occurs as a tandem repeat in C6. The last tandem repeat at the carboxyl terminus of C6 and C7 has been identified as a new distinct module (factor I module), which is closely related to a segment in the heavy chain of complement control factor I. Binding studies with filter-bound C6 fragments generated by proteolysis showed that the C5b-binding domain of C6 was located in the 34-kDa carboxyl terminal fragment consisting of two short consensus repeats and two factor I modules.

Cytoplasmic granules of cytolytic T lymphocytes contain at least six related serine esterases (granzymes) that are released together with perforin, a pore-forming protein related to complement component C9, during target-cell lysis. Polyclonal antibodies were used to isolate a large number of cDNA clones from an expression library derived from cytolytic-T-cell mRNA. Three distinct full-length cDNA clones coding for granzymes D, E, and F were identified by restriction site mapping and nucleotide sequencing. The three deduced amino acid sequences are highly similar to one another (between 72% and 90% amino acid identities) and to the sequences of granzymes B and C, cathepsin G, and rat mast-cell proteases I and II (between 43% and 57% amino acid identities). Cysteine residues capable of forming intramolecular disulfide bonds are conserved, as are the catalytic-site residues characteristic of serine proteases. Comparison of the cDNA-derived protein sequences with the amino termini of the isolated granzymes provides evidence that they are stored in a fully processed, activated form after removal of the signal peptide and two additional residues (propeptide) at the amino terminus. Immunoelectron microscopic studies demonstrated that granzymes D, E, and F are present in the same morphologically distinct cytoplasmic granules in which perforin has been found previously.

A component of soluble terminal complement complexes was identified and affinity-purified to homogeneity by using a monoclonal antibody previously developed against the soluble C5b-9 complex. The protein, which we have designated complement cytolysis inhibitor (CLI), has a molecular mass of 70 kDa and consists of two nonidentical, disulfide-linked subunits of 35 kDa. Partial amino acid sequences determined for the amino-termini of the two subunits were identical with those of a recently characterized serum protein called SP-40,40. An almost full-length cDNA clone of 1651 base pairs was isolated from a human liver cDNA library by using long synthetic oligonucleotides as probes. The encoded amino acid sequence of CLI consists of 427 amino acid residues preceded by a 21-residue-long typical signal peptide and shows an overall 75.6% amino acid sequence homology to sulfated glycoprotein 2 (SGP-2), a major Sertoli cell-derived protein of rat testis fluid. As in SGP-2, proteolytic processing between residues 206 and 207 yields the two disulfide-linked subunits of plasma CLI. CLI and SGP-2 were shown to be orthologous single-copy genes in humans and rats by Southern blotting experiments. In addition, CLI was immunologically identified in human seminal plasma. Functional studies with purified terminal complement components showed that CLI suppresses the cytolytic potential of nascent C5b-7 complexes at physiological blood plasma concentrations (approximately 50 micrograms/ml). Its presence on the surface of mature sperm cells and its relative abundance in seminal plasma (approximately 250 micrograms/ml) suggest that CLI protects sperm cells and epithelial tissues against complement attack in the male reproductive tract.

This study examined the effect of the pro-inflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) on the induction of MHC class I–related genes in functionally mature brain neurons derived from cultures of dissociated rat hippocampal tissue. Patch clamp electrophysiology combined with single cell RT-PCR demonstrated that ∼50% of the untreated neurons contained mRNA for MHC class I heavy chains, while, with few exceptions, the cells failed to transcribe β2-microglobulin and TAP1/TAP2 gene transcripts. No constitutive expression of MHC class I protein was detectable by confocal laser microscopy on the surface of neurons. All neurons transcribed the α-chain of the interferon-type II receptor (binding IFN-γ) along with the p55 receptor for TNF-α. Sustained exposure to IFN-γ resulted in transcription of β2microglobulin and TAP1/TAP2 genes and MHC class I surface expression in a minor part of the neurons, but did not alter their electrophysiological activities as assessed by whole cell electrophysiology. Suppression of neuronal electric activity by the sodium channel blocker tetrodotoxin drastically increased to almost 100% IFN-γ-mediated induction of MHC class I chains, of both TAP transporters, and of membrane expression of MHC class I protein. The effect of tetrodotoxin is at least partly reverted by the neurotransmitter glutamate. In contrast to IFN-γ, treatment with TNF-α did neither upregulate TAP1/TAP2 nor β2microglobulin gene expression, but induced MHC class I heavy chain gene transcription in all neurons. Consequently, no MHC class I molecules were detectable on the membranes of TNF-α-treated neurons.