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Biomedical subjects

T E Johansen

Publications and source records attributed to T E Johansen.

At least 19 recordsLinked to original sources
PubMed2000

In vivo protection of nigral dopamine neurons by lentiviral gene transfer of the novel GDNF-family member neublastin/artemin.

The glial cell line-derived neurotrophic factor (GDNF)-family of neurotrophic factors consisted until recently of three members, GDNF, neurturin, and persephin. We describe here the cloning of a new GDNF-family member, neublastin (NBN), identical to artemin (ART), recently published (Baloh et al., 1998). Addition of NBN/ART to cultures of fetal mesencephalic dopamine (DA) neurons increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons by approximately 70%, similar to the maximal effect obtained with GDNF. To investigate the neuroprotective effects in vivo, lentiviral vectors carrying the cDNA for NBN/ART or GDNF were injected into the striatum and ventral midbrain. Three weeks after an intrastriatal 6-hydroxydopamine lesion only about 20% of the nigral DA neurons were left in the control group, while 80-90% of the DA neurons remained in the NBN/ART and GDNF treatment groups, and the striatal TH-immunoreactive innervation was partly spared. We conclude that NBN/ART, similarly to GDNF, is a potent neuroprotective factor for the nigrostriatal DA neurons in vivo.

Amino Acid Sequence↗
PubMed1999

Cellular processing of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.

An important gap in our understanding of the pathogenesis of the amyloidoses is the identification of the cellular events that lead from synthesis of an amyloid precursor protein to its conversion to the amyloid fiber subunit. We address this question by characterizing the effects of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders several alterations in its intracellular trafficking. It forms a stable intracellular dimer that is partially retained in the endoplasmic reticulum and degraded. The bulk of mutant cystatin C that is secreted does not dissociate and is secreted as an inactive dimer. Thus, formation of the stable mutant cystatin C dimer is an early event in the pathogenesis of this disease.

Amino Acid Sequence↗
PubMed1999

Neuroprotection by a novel compound, NS521.

NS521 (1-(1-butyl)-4-(2-oxo-1-benzimidazolinyl)piperidine) belongs to a group of novel benzimidazolones, which exhibit neurotrophic-like activities. In vitro, NS521 rescued neuronal PC12 cells from death induced by serum and nerve growth factor deprivation. The survival effect of NS521 appeared to reflect a delay of the apoptotic process, because the extent of DNA fragmentation was attenuated transiently by NS521. NS521 did not preserve the neurites of the rescued cells, which, otherwise, appeared to be healthy and were able to regenerate when serum and nerve growth factor were added back to the culture. In vivo, NS521 provided significant protection against the delayed loss of hippocampal CA1 neurons in a gerbil model of transient global ischemia. A neuroprotective effect of NS521 in the peripheral nervous system also was observed in rats after transection of the sciatic nerve, where daily treatment with NS521 was found to inhibit retrograde degeneration of the transected nerve. The neuroprotective effect of NS521 is unlikely to be mediated through neurotrophin receptors, such as TrkA, because NS521 did not induce phosphorylation of the 44- and 42-kDa isoforms of mitogen-activated protein kinases (ERK1/2) in PC12 cells.

Animals↗
PubMed1998

[Schistosomiasis of the urinary bladder].

A 12 year old male immigrant from Somalia was admitted to hospital after several years of haematuria and dysuria. Microscopic examination of the urine revealed eggs of the Schistosoma haematobium. Urine culture was negative. Cystoscopy showed a characteristic bilharzial tubercle, and numerous sandy patches were also seen. Mucosal biopsy showed schistosoma eggs, some with calcification. There was squamous cell metaplasia and infiltration of plasma cells and eosinofilic granulocytes. The patient was treated with praziquantel 600 mg x 4 for two days. Three months later no schistosoma eggs were seen in his urine and cystoscopy was negative. In immigrants from countries where bilharzia is endemic, it should be considered a differential diagnosis in patients with haematuria.

Antiplatyhelmintic Agents↗
PubMed1997

Stable expression of the human large-conductance Ca2+-activated K+ channel alpha- and beta-subunits in HEK293 cells.

We have generated HEK293 cell lines stably expressing high levels of either the human BK channel alpha-subunit alone or the BK channel alpha-subunit and beta-subunit together. For co-expression a plasmid with three expression cassettes was constructed. Patch-clamp recordings on inside-out patches from the transfected cells resulted in macroscopic currents reflecting the expression of 200-800 BK channels per patch. No decrease in channel expression could be detected in cells grown for more than 50 passages. The alpha-subunit when expressed alone conducted currents which were sensitive to intracellular Ca2+ in the physiological range. In the presence of the beta-subunit the steady-state activation curves were shifted by -20 to -30 mV and channel deactivation kinetics were slowed. The BK channel opener NS1608 (10 microM) shifted the steady-state activation curves for the alpha-subunit as well as for the alphabeta-subunits by -40 to -50 mV.

Calcium↗
PubMed1997

Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells.

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.

Animals↗
PubMed1997

Peptide binding to MHC class I is determined by individual pockets in the binding groove.

H-2Kb and HLA-A2 are MHC4 class I molecules with a similar overall structure. Important differences between these two class I molecules reside in the structure of the individual pockets in the antigenic-peptide-binding groove. H-2Kb, which has a deep C pocket, binds specifically peptides with a tyrosine or a phenylalanine at position 5. In contrast, HLA-A2 has a shallow C pocket, which cannot accommodate large side chains at position 5. Site-directed mutagenesis was used to generate a chimera between the murine H-2Kb and the human HLA-A2 [H-2Kb/HLA-A2(C')]. The structure of this chimera is similar to H-2Kb except for the region around the deep C pocket, where residues at positions 9, 97 and 99 were substituted with those bulkier residues from HLA-A2. Peptide binding between this chimera and H-2Kb-binding peptides [VSV (52-59), OVA (257-264), and MCMV pp89 (168-176)], revealed that the deep C pocket of H-2Kb was crucial for high-affinity binding. While a peptide, VSV (52-59), was found to bind with severalfold lower 'affinity' to H-2Kb/HLA-A2(C') than to the wild-type H-2Kb, a VSV analogue with the tyrosine in position 5 (Tyr5) substituted with an alanine was found to bind with a similar 'affinity' to both MHC class I molecules. Computer-aided modelling of the H-2Kb/HLA-A2(C') complex indicates that the VSV (52-59) peptide probably binds to the chimeric MHC molecule with the peptide side chain of anchor residue Tyr5 pointing away from the groove. These results confirm a role of the individual pockets in determining peptide-binding affinity and specificity and suggest that this may be accomplished by changes in side-chain orientation.

Animals↗
PubMed1996

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Amino Acid Sequence↗
PubMed1996

Modulation of the Ca(2+)-dependent K+ channel, hslo, by the substituted diphenylurea NS 1608, paxilline and internal Ca2+.

The high-conductance Ca(2+)-activated K channel (BK channel) is not only regulated by a number of physiological stimuli, but it is also sensitive to pharmacological modulation. We have stably expressed the alpha-subunit of the human BK channel, hslo, in HEK 293 cells and studied by patch-clamp technique how its gating is modulated by the channel activator NS 1608, by the selective channel blocker paxilline, as well as by changes in [Ca2+]i and Vm. The cells expressed 200-800 hslo channels per patch. The channel activity was determined by tail current analysis, and the activation curves were fitted to single Boltzmann functions, from which a gating charge for the hslo channel of 1.2 elementary charges was deduced. The hslo channel was very sensitive to changes in [Ca2+]i within the physiological range, whereas Ca(2+)-independent openings were seen at Ca2+ concentrations of 15 nM or below. NS 1608 shifted the hslo channel activation curve towards negative membrane potentials with an EC50 of 2.1 microM and a maximal shift of -74 mV. The channels activated by NS 1608 were sensitive to block by paxilline, but the two molecules apparently did not interact within the same site, since paxilline reduced the size of the tail current at all voltages, whereas NS 1608 shifted the activation curve along the voltage axis. Further, the effect of paxilline was Ca(2+)-sensitive, whereas NS 1608 elicited identical effects in the presence of either < 0.5 nM or 500 nM [Ca2+]i. NS 1608 hyperpolarized the cells by -50 to -70 mV, and paxilline depolarized them towards 0 mV. In addition to the effects on the steady state current NS 1608 also significantly influenced the non-stationary channel kinetics. In the presence of NS 1608 the time constants for deactivation of tail currents were more than tripled at all potentials. We have shown, that NS 1608 modulates steady-state BK currents and channel gating kinetics through a Ca(2+)-independent interaction with the alpha-subunit of the channel.

Calcium↗
PubMed1995

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line.

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with Kd of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with Kd 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with Kd 0.42 was identified as rat alpha-CGRP as was the peak with Kd 0.53. The peak with Kd 0.68 was identified as 19-37 rat alpha-CGRP and the peak with Kd 0.85 as 28-37 rat alpha-CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated alpha-CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat beta-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.

Amino Acid Sequence↗
PubMed1994

Analysis of the structure of empty and peptide-loaded major histocompatibility complex molecules at the cell surface.

We compared the conformation of empty and peptide-loaded class I major histocompatibility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the alpha 2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to beta 2-microglobulin. No FRET was found between Fab fragments bound to empty H-2Kb, but FRET was detected when empty H-2Kb molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2Kb molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.

Animals↗
PubMed1994

[Contents and updating of disaster plans].

The disaster contingency plans of 62 hospitals with surgical, acute emergency functions were evaluated. Most plans were incomplete with regard to continued and extended activity or evacuation of the entire hospital in the event of war. Only 13% had adequate plans for internal accidents or situations with reduced of lost supply of vital resources such as water, medical gases or electricity. Plans for serious accidents in peacetime were to some degree deficient with regard to important aspects of the organization of the hospital in an emergency situation. Efforts to improve this crucial planning work are discussed.

Accidents↗
PubMed1994

[Form and readability of disaster plans].

We have evaluated the disaster contingency plans for 62 out of 69 Norwegian hospitals with surgical, acute emergency functions. Page volume varied from 2-115, mean 33 pages. Only nine hospitals had pocket size printed documents (A5). At the rest of the hospitals the plans had an A4-format and consisted of photocopied pages. Illustrations were few and of low quality. Too much text contained general information with no practical relevant instruction. Readability varied for different groups of health workers. Doctors received the best instructions for their tasks. Hospitals should seek professional assistance to edit, illustrate and produce the plans, and a certain degree of standardization of the plans should be aimed at. National health authorities should encourage the hospitals in this work.

Disaster Planning↗
PubMed1994

[Urinary bladder diverticula].

We describe two patients with urinary tract infection and obstructive voiding problems which lasted for respectively 21 months and five years before a bladder diverticulum was diagnosed by cystography and cystoscopy. Both patients were operated on by open diverticulectomy. The post-operative courses were uneventful and both patients were relieved of urinary symptoms after surgery. The article includes a review of the literature on bladder diverticula.

Adult↗