Human proteasome subunits from 2-dimensional gels identified by partial sequencing.
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Amino Acid Sequence↗
Biomedical subjects
P Kristensen
Publications and source records attributed to P Kristensen.
Purification and characterization of leukotriene A4 hydrolase from human epidermis.
The leukotriene A4 hydrolase is a central enzyme in leukotriene B4 formation. Unlike 5-lipoxygenase, leukotriene A4 hydrolase activity is present in normal human epidermis, where it is likely to be involved in transcellular leukotriene formation. In this study the leukotriene A4 hydrolase was purified from human epidermis and human cultured keratinocytes and compared with leukotriene A4 hydrolase from human neutrophils. To purify leukotriene A4 hydrolase from human epidermis a new non-specific affinity chromatography column, with the leukotriene A4 hydrolase inhibitor bestatin coupled to AH-Sepharose, was introduced. The epidermal leukotriene A4 hydrolase was purified to apparent homogeneity and the molecular weight was determined to be approximately 70,000 Da by SDS-PAGE. The pI was 5.1-5.4 for the epidermal as well as the keratinocyte and neutrophil leukotriene A4 hydrolase, as determined by chromatofocusing. Only minor differences in the amino acid composition were seen between the three enzyme sources. The optimal pH for the hydrolase activity was 7.5-8.5 for the epidermal and neutrophil leukotriene A4 hydrolases. Finally, it was also shown that the epidermal leukotriene A4 hydrolase undergoes suicide inactivation when transforming leukotriene A4 into leukotriene B4. It was concluded that there is a close resemblance between the epidermal leukotriene A4 hydrolase and the hydrolase found in other cell types. Therefore, the human epidermis may be a good model for the in vivo study of transcellular leukotriene formation.
Cells, Cultured↗
Human proteasomes analysed with monoclonal antibodies.
The proteasome or multicatalytic endopeptidase from eukaryotic cells consists of at least 14 subunits that fall into two families, alpha and beta. Subunit-specific monoclonal antibodies against ten different subunits of human proteasomes have been produced, together with an antibody that reacts with a motif (prosbox 1), common to alpha-type subunits. Four of the subunit-specific antibodies were able to precipitate proteasomes. The subunit composition of HeLa-cell proteasomes precipitated with these four different antibodies were identical, as judged from two-dimensional electrophoresis. One of the four antibodies was used to obtain proteasomes from cell lines (HeLa, Daudi, IMR90 and BSC-1) and human tissues (placenta, kidney, and liver). Electrophoretic analysis of these proteasomes, combined with peptide mapping of some subunits, suggests that they all contain 14 types of subunits as their major constituents. However, one subunit was present in two isoelectric isoforms in all cells examined. Two other subunits occurred in two or three isoelectric isoforms in placenta, liver and kidney, but not in the cell cultures. Extracts of human cells (HeLa, IMR90, Daudi and erythrocytes) were analysed by non-denaturing electrophoresis and immunoblotting. All of the 11 subunits detected by antibodies were present in a pair of ATP-stabilized protein complexes, presumed to be the 26 S proteinase, and in a doublet of complexes which migrated more slowly than purified proteasomes. Besides being present in proteasomes, one subunit was also found to occur in the free state in cell extracts.
Amino Acid Sequence↗
Photodetachment study of B- ions: The influence of the first excited boron state.
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Journal Article↗
Laser spectroscopy of the Be- ion: Binding energies of metastable states.
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Journal Article↗
Unchanged balance between levels of mRNA encoding AMPA glutamate receptor subtypes following global cerebral ischemia in the rat.
Transient global ischemia leads to glutamate mediated delayed neuronal death in the CA1 but not in the CA3 region of the rat hippocampus, and changes in AMPA receptor subunit composition has been proposed to cause a difference in excitatory input to the CA1 and CA3 regions. In situ hybridization with riboprobes for AMPA receptor subtype GluR1-4 mRNA was performed on sections from the brain of sham operated and ischemic rats in two models (neck cuff and 4-vessel occlusion combined with hypotension) with identical results: the content of the GluR1-3 mRNA species was down regulated in the hippocampal regions CA1 and CA3 but only weak changes were observed in the dentate gyrus. The down regulation observed in CA1 was non-selective among GluR1-3, i.e. all GluR mRNA species showed approximately the same degree of down regulation. A change in calcium permeability of the AMPA channels mediated by a shift in channel sub-unit composition and corroborating an increased calcium influx is thus not supported by these findings.
Animals↗
Fertility in mice after prenatal exposure to benzo[a]pyrene and inorganic lead.
Experimental evidence suggests that inorganic lead and benzo[a]pyrene (BaP) suppress the development of primordial oocytes during fetal life. We examined the single and combined effects of prenatal exposure to BaP and moderate doses of lead. The fertility and ovarian morphology of F1 female NMRI mice in four treatment groups (nine mice per group) were investigated: control; lead (F0 given 1 g PbCl2/L in drinking water until mating); BaP (10 mg/kg body weight daily by oral intubation on days 7-16 of F0 pregnancy); and combined lead and BaP. F1 groups exposed prenatally to BaP either alone or in combination with inorganic lead showed markedly reduced fertility with few ovarian follicles compared to controls, whereas the group exposed to lead only had measures comparable to the controls. Mice exposed to both lead and BaP had a significantly longer gestation period (days to litter) compared to mice exposed only to BaP, lead, or controls. There is a nonsignificant indication that the compounds together further reduce number of offspring, number of litters, and litter size. These results suggest that lead and BaP have synergistic effects on impairment of fertility. The possibility of synergism may be of human relevance as inorganic lead and BaP are ubiquitous environmental pollutants.
Animals↗
The natural course of peptic ulcer disease and its predictors.
BACKGROUND: Little is known about today's natural course of peptic ulcer disease (PUD). METHODS: A follow-up study based on a structured telephone interview was attempted in 728 patients with an endoscopic diagnosis of peptic ulcer in 1980-84. RESULTS: Seven patients (1%) died because of PUD during the 8- to 10-year follow-up period. Of the 441 interviewed patients 15.2% had experienced no further clinical manifestations of PUD, 10.9% had had bleeding and 0.7% perforation, and 17.5% had been operated on. The operated patients reported fewer symptoms (p < 0.01) during the last 2 weeks before the interview than those not operated on. On an average the unoperated patients had had symptoms and had used histamine-2-receptor antagonists (H2RA) 12 and 10 weeks per year, respectively. Long-term treatment with H2RA was reported by 18%. More than one-third (36%) of the unoperated patients stated that the symptoms had had a significant negative impact on their lives. Age at onset of disease and index ulcer, family history, use of anti-inflammatory drugs and alcohol, bleeding, and another chronic disease were found to be significant predictors of the course. CONCLUSION: In more than one-third of the patients with PUD the course is still burdened with many symptoms and complications.
Adolescent↗
A fast, economical and efficient method for DNA purification by use of a homemade bead column.
A fast, efficient and economical method for purification of DNA from various sources by the use of silica particles packed in homemade bead columns is described. The method is a further development of the method devised by Carter and Milton (1), improved by the introduction of a column operated with a vacuum system. The method is highly suitable for multiple sample handling in the preparation of DNA for sequencing and other purposes. Protocols are devised for plasmid minipreparation, gel elution and purification of DNA from solutions e.g. enzyme reactions.
DNA, Bacterial↗
Human proteasome subunits from 2-dimensional gels identified by partial sequencing.
Human proteasomes consist of 14 major subunits which can be resolved by two-dimensional polyacrylamide gel electrophoresis. Tryptic peptides from the subunits were sequenced. This allowed identification of all proteins in two-dimensional electrophoresis gels with proteasome subunits whose sequences are known from cDNA. The results also precisely define the specificity of a panel of subunit-specific monoclonal antibodies. A new proteasome subunit (Z) was discovered. In contrast, the putative subunit MECL-1 could not be detected in human placenta proteasomes.
Amino Acid Sequence↗
Effects of typical and atypical neuroleptics on Fos protein expression in the rat forebrain.
The effect of the atypical neuroleptics clozapine and risperidone, the potential atypical neuroleptics sertindole and NNC 22-0031 as well as the prototypical neuroleptic haloperidol on Fos protein expression in the rat forebrain was examined by use of immunohistochemistry. Fos protein expression in the limbic medial prefrontal cortex (PFC) relative to Fos protein expression in the non-limbic dorsolateral striatum (DLSt) was more pronounced for the atypical and potential atypical neuroleptics than observed with the prototypical neuroleptic, indicating that atypical and prototypical neuroleptics can be differentiated on the basis of their ability to induce Fos protein in these regions.
Animals↗
[Chemical occupational environment of artists (painting, graphic, sculpture].
Visual artists are exposed to chemical irritants with a potential for reactions of the skin and mucous membranes. Such reactions are probably prevalent, and the professional and health consequences may be serious for persons with a low tolerance. Exposure to organic solvents may be high in silk screen printing and reinforced polyester casting. However, the risk of toxic encephalopathy requires exposure for several years. Lead and other heavy metals, plastic monomers and carcinogenic agents are used in several techniques. The health risk from these chemical agents is considered to be small, except among artists using special techniques and work routines. Changes in daily work routines seem to be most important way of reducing the occurrence of health effects among artists.
Brain↗
Expression of uPA and its receptor by both neoplastic and stromal cells during xenograft invasion.
Recent studies have shown that molecules involved in generation and regulation of extracellular proteolytic activity are often expressed by non-malignant stromal cells during human cancer invasion. We have studied the expression of the urokinase-type plasminogen activator and the urokinase-type plasminogen activator cell-surface receptor in xenografts of human MDA-MB-231 mammary carcinoma cells growing invasively in nude mice. Northern analysis showed the presence of both human and mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA in tumor extracts. By in situ hybridization, mRNA for human urokinase-type plasminogen activator and its receptor was detected in virtually all the cancer cells, while mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA was expressed by tumor-infiltrating fibroblast-like and macrophage-like cells. In invasive areas the cells expressing the 2 murine mRNAs were either the same or located immediately adjacent to each other. This model system has several advantages for studies of the mechanism by which cancer cells induce or recruit stromal cells to produce molecules involved in proteolysis.
Animals↗
Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-gamma for acquirement of the functional diversity responsible for antigen processing.
Proteasomes catalyze the non-lysosomal, ATP-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-gamma) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also almost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomal subunit highly homologous to LMP7, and that subunit Y is identical to the LMP2-related proteasomal subunit delta. Thus, IFN-gamma appears to induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing 'immuno-proteasomes' with the functional diversity responsible for processing of endogenous antigens.
Amino Acid Sequence↗
Clusters of anophthalmia ... or in Norway.
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Anophthalmos↗
Human epidermis transforms exogenous leukotriene A4 into peptide leukotrienes: possible role in transcellular metabolism.
Leukotriene B4 formation can take place by cell interaction between keratinocytes and neutrophils. Thus, keratinocytes without proven 5-lipoxygenase activity can transform neutrophil-derived leukotriene A4 into leukotriene B4. The purpose of the present study was to investigate whether human epidermis is able to transform leukotriene A4 sequentially into the peptide leukotrienes (LTC4, LTD4 and LTE4). Epidermis isolated using the suction blister technique or keratomed skin specimens were incubated with either neutrophils or exogenously added leukotriene A4. Peptide leukotrienes were determined by integrated optical density after RP-HPLC separation, and the identity of leukotrine C4 was confirmed by (1) the retention time similarity with authentic leukotriene C4; (2) the UV spectrum determined with an on-line diode array detector; and (3) conversion by gamma-glutamyl transpeptidase of the peak coeluting with authentic leukotriene C4 into a new peak coeluting with authentic leukotriene D4. The results of this study showed that while human epidermis cannot form detectable amounts of peptide leukotrienes by itself, it can transform exogenous leukotriene A4 into peptide leukotrienes. Furthermore, coincubation of human epidermis and neutrophils resulted in a marked increase (90%) in peptide leukotriene formation when compared with neutrophils alone, indicating that human epidermis can transform neutrophil-derived leukotriene A4 into peptide leukotrienes. These results indicate that human skin contains leukotriene C4 synthase activity capable of producing significant amounts of leukotriene C4 from leukotriene A4, and that the keratinocytes may play a more active role in peptide leukotriene formation in the skin than previously thought.(ABSTRACT TRUNCATED AT 250 WORDS)
Biotransformation↗
Characterization of two alternatively spliced forms of a metabotropic glutamate receptor in the central nervous system of the rat.
Amplification of complementary DNA by the polymerase chain reaction and anti-peptide antibodies were used to characterize the expression of two alternatively spliced forms of a metabotropic glutamate receptor (mGluR1 alpha and mGluR1 beta) in the central nervous system of the rat. Polymerase chain reaction analysis showed that mGluR1 alpha was the predominate of the two forms in the cerebellum, diencephalon, mesencephalon, olfactory bulb and brainstem, while mGluR1 beta was the major form present in the hippocampus. Approximately equal amounts of the two receptors were expressed in the cerebral cortex, septum and striatum. Immunochemical analyses of the two receptors were conducted in the rat cerebellum and hippocampus. An mGluR1 alpha-specific antibody labelled a protein with a relative molecular weight of 146,000 on immunoblots of the hippocampus and cerebellum. Immunoblot analysis of the developmental expression of mGluR1 alpha in the hippocampus and cerebellum demonstrated that in both structures, the levels of mGluR1 alpha were at or near their maximum levels in the adult brain. In contrast, two mGluR1 beta-specific antibodies failed to detect mGluR1 beta on immunoblots of brain tissue, thus precluding an immunocytochemical analysis of this receptor. Although low levels of a higher-molecular weight protein, possibly a dimeric form of mGluR1 beta were seen with one of the mGluR1 beta-specific antibodies, we hypothesize that some of the mGluR1 beta present in brain tissue may undergo proteolytic cleavage of the carboxy terminus. Immunocytochemical analysis of mGluR1 alpha showed that very high levels of this receptor were expressed in Purkinje cell bodies and dendrites. In the granule cell layer, some Golgi neurons were immunostained. The granule cells were not labelled. In the hippocampus, mGluR1 alpha immunoreactivity was present in interneurons of the stratum oriens and the dentate hilar region. Double-labelling studies demonstrated that these interneurons were also immunopositive for the neuropeptide somatostatin. The presence of mGluR1 alpha in cells of the hippocampus that are associated with the release of somatostatin, suggest that this receptor could play a role in regulating hippocampal excitability in both normal and epileptic tissues.
Alternative Splicing↗
Changes in metabotropic glutamate receptor mRNA levels following global ischemia: increase of a putative presynaptic subtype (mGluR4) in highly vulnerable rat brain areas.
Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin-stimulated cyclic AMP formation. The mGluR4 subtypes may represent L-2-amino-4-phosphonobutyrate-sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1-mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre- and postsynaptically located metabotropic glutamate receptors.
Animals↗