Search PubMed⌕ Search

Biomedical subjects

P Madsen

Publications and source records attributed to P Madsen.

At least 91 records · Page 5Linked to original sources
PubMed1995

Cloning, expression, and chromosome mapping of human galectin-7.

The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a lambda gt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the beta-galactoside interaction are conserved. Galectin-7 was partially externalized to the medium by keratinocytes although it has no typical secretion signal peptide. Immunoblotting as well as immunofluorescence analysis of human tissues with a specific galectin-7 antibody revealed a narrow distribution of the protein which was found mainly in stratified squamous epithelium. The antigen localized to basal keratinocytes, although it was also found, albeit at lower levels, in the suprabasal layers where it concentrated to areas of cell to cell contact. Both, its cellular localization as well as its striking down-regulation in K14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19.

Adult↗
PubMed1995

The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3154 cellular proteins (2224 isoelectric focusing, IEF; and 930 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to post-translational modifications. 1082 polypeptides have been identified (protein name, organelle components, etc.) using a procedure or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies, (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry, (v)vaccinia virus expression of full length cDNAs, and (vi) in vitro transcription/translation of full-length cDNAs. This year, special emphasis has been given to the identification of signal transduction components by using 2-D gel immunoblotting of crude keratinocyte lysates in combination with enhanced chemoluminescence (ECL) detection. Identified proteins are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Ultimately, the aim of the comprehensive database is to gather--through a systematic study of ekeratinocytes--qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Cells, Cultured↗
PubMed1995

Expression of cDNA clones by coupled in vitro transcription/translation and transfection into COS-1 cells: protein mapping in two-dimensional gels.

We present two procedures that can be used to map proteins in two-dimensional gels if the cDNA is at hand. The first procedure, which is illustrated with the expression of cDNAs encoding the fatty acid binding protein 5 (FABP 5), psoriasin and stratifin, makes use of the in vitro transcription/translation assay marketed by Promega. The procedure is simple and allows the mapping of the primary translation product in a very short time. The second method--which faithfully reproduces post-translational modifications--is based on the expression of cDNAs transfected into COS-1 cells using a eukaryotic expression plasmid. This procedure is illustrated with the expression of cDNAs encoding Ha-ras p21 and rab 11, two small GTP-binding proteins known to undergo complex modifications.

14-3-3 Proteins↗
PubMed1995

Refined mapping of the psoriasin gene S100A7 to chromosome 1cen-q21.

Psoriasin is a low molecular weight protein of the S100 family, which is highly upregulated in psoriatic epidermis, and whose function is related to skin inflammatory responses. We have applied a cDNA probe from the corresponding psoriasin gene S100A7 in a refined localisation analysis. S100A7 was mapped physically by human/rodent somatic cell hybrid analysis, and more precisely genetically by multilocus linkage analysis of 40 CEPH (Centre d'Etude du Polymorphisme Humain) families. The resulting 12-point linkage map was supported by odds of at least 1000:1, where S100A7 could be placed with a multipoint lodscore of 27.4 in an approximately 7-cM interval. The order of the 12 loci was as follows (with the best estimates of recombination frequencies given in between): AMY2B-0.091-D1S73(0.039)-D1S11(-0.053)-D1S189(-0 .017)-D1S1252 (-0.017)-D1S13(-0.078)-D1Z5(-0.051)-S100A7(-0.022)- MUC1(-0.026)-SPTA1 (-0.066)-ATP1A2(-0.014)-APOA2. Furthermore, from this map S100A7 could be assigned to the regional position of chromosome 1cen-q21. The linkage information presented should be of great value in association and linkage studies of diseases where psoriasin, or some of the several other very closely linked and functionally related genes, are seen as candidate genes, e.g. in psoriasis.

Calcium-Binding Proteins↗
PubMed1995

Naloxone-provoked vaso-vagal response to head-up tilt in men.

A double-blind paired protocol was used to evaluate, in eight male volunteers, the effects of the endogenous opiate antagonist naloxone (NAL; 0.05 mg.kg-1) on cardiovascular responses to 50 degrees head-up tilt-induced central hypovolaemia. Progressive central hypovolaemia was characterized by a phase of normotensive-tachycardia followed by an episode of hypotensive-bradycardia. The NAL shortened the former from 20 (8-40) to 5 (3-10) min (median and range; P < 0.02). Control head-up tilt increased the means of thoracic electrical impedance [from 35.8 (SEM 2.1) to 40.0 (SEM 1.8) omega; P < 0.01] of heart rate [HR; from 67 (SEM 5) to 96 (SEM 8) beats.min-1, P < 0.02], of total peripheral resistance [TPR; from 25.5 (SEM 3.2) to 50.4 (SEM 10.5)mmHg.min.1-1, P < 0.05] and of mean arterial pressure [MAP; from 96 (SEM 2) to 101 (SEM 2)mmHg, P < 0.02]. Decreases were observed in stroke volume [from 65 (SEM 12) to 38 (SEM 9) ml, P < 0.01], in cardiac output [from 3.7 (SEM 0.7) to 2.5 (SEM 0.5) 1.min-1, P < 0.01], in pulse pressure [from 55 (SEM 4) to 37 (SEM 3)mmHg, P < 0.01] and in central venous oxygen saturation [from 73 (SEM 2) to 59 (SEM 4)%, P < 0.01]. During NAL, mean HR increased from 70 (SEM 3); n.s. compared to control) to only 86 (SEM 9) beats.min-1 (P < 0.02 compared to control) and MAP remained stable.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Arterial diameter during central volume depletion in humans.

The luminal diameter of the radial artery was followed by high frequency ultrasound during 50 degrees head-up tilt-induced central volume depletion in ten healthy subjects of whom six were tilted twice and pretreated with the serotonin receptor antagonist methysergide or placebo following a double-blind randomized design. Eight subjects without active treatment experienced presyncopal symptoms after 16-45 (mean 32 min). Central volume depletion was indicated by an increase in mean thoracic electrical impedance [from 31.5 (SEM 1.6) to 33.4 (SEM 1.7) omega; P < 0.05]. Cardiac output decreased [from 4.1 (SEM 0.3) to 2.2 (SEM 0.3) 1.min-1] and heart rate [HR, from 64 (SEM 3) to 100 (SEM 7) beats.min-1], mean arterial pressure (MAP, from 77 (SEM 4) to 89 (SEM 2) mmHg [10.3 (SEM 0.53 to 11.9 (SEM 0.27) kPa]) and total peripheral resistance (TPR, from 19 (SEM 2) to 34 (SEM 4) mmHg.min.1-1 [2.5 (SEM 0.27) to 4.5 (SEM 0.53) kPa.min.1-1]) increased; but with the appearance of presyncopal symptoms, HR, MAP and TPR were reduced to 65 (SEM 8) beats.min-1, 46 (SEM 4) mmHg [6.1 (SEM 0.53) kPa] and 18 (SEM 3) mmHg.min.1-1 [2.4 (SEM 0.4) kPa.min-1.1-1], respectively (P < 0.05). Vascular resistance was reflected in the arterial diameter which decreased from 2.42 (SEM 0.17) to 2.27 (SEM 0.14) mm during head-up tilt and increased to 2.71 (SEM 0.14) mm with the appearance of presyncopal symptoms (P < 0.05). Methysergide reduced the resting radial (15 +/- 2%) and temporal artery diameters (10 +/- 3%) (P < 0.05); however, it affected neither tilt-tolerance nor the central cardiovascular response to tilt. The results suggested a serotonergic influence on arterial tone at rest, and demonstrated that vessels as large as the radial artery participated in vascular control during central volume depletion independent of such a serotonergic influence.

Adult↗
PubMed1995

Tyrosine metabolism in users of oral contraceptives.

Brain noradrenaline takes part in the regulation of several brain functions. The formation of brain noradrenaline depends on brain tyrosine (Tyr) levels, which associates with the ratio in plasma of Tyr to other large, neutral amino acids (LNAA). Tyr metabolism has been studied in users of the new generation combined oral contraceptives (OC) and comparable controls at the follicular, mid-cycle, and luteal phases of the menstrual cycle. OC users showed significantly increased plasma Tyr transaminase activity, and significantly decreased plasma Tyr and Tyr/LNAA levels at mid-cycle and luteal phase, whereas plasma total 3-methoxy-4-hydroxyphenylglycol (MHPG) was not affected. Following an oral protein load, the area under the curve in plasma of Tyr and Tyr/LNAA in OC users at the luteal phase were 43% and 29%, respectively, of control levels. The results suggest that the decreased Tyr availability to the brain in OC users may result in a substrate-limited reduction of brain noradrenaline formation, which, secondarily, may contribute to disturbances of mood, coping mechanisms, and appetite in susceptible subjects.

Adult↗
PubMed1995

Thoracic electrical impedance and fluid balance during aortic surgery.

Indices of fluid balance were evaluated during and after aortic surgery in 16 consecutive patients. Thoracic electrical impedance (TI), heart rate (HR), central venous (CVP), pulmonary artery mean (PAMP), pulmonary wedge (PWP) and mean arterial (MAP) pressure as well as fourteen arterial and venous blood gas variables were followed. Consistent with a reduction of T1 by 4.2 (-5.2 to 9.2) Ohm (median and range) during the operation, fluid balance was in excess of 1.8 (-0.1 to 3.3) 1 when evaporation was not taken into account, and it remained elevated by 1.3 (0.0 to 5.4) 1 on the first postoperative morning. The HR, MAP and PWP remained stable, while CVP and PAMP decreased by 6 (-2 to 13) and 6 (-1 to 22) mmHg, respectively. Of the determined variables only TI revealed a meaningful correlation to fluid balance (rho = -0.41; P < 0.01). Haemoglobin concentrations increased in proportion to the administered packed erythrocytes, while arterial oxygen saturation, pH and base excess decreased in proportion to the excess fluid. The results indicate that while central venous and pulmonary artery mean pressures gave the impression of a volume deficit, the positive fluid balance was mirrored by thoracic electrical impedance, and that even a minor increase of fluid balance may affect pulmonary function in patients subjected to aortic surgery.

Aged↗
PubMed1995

Brain and muscle oxygen saturation during head-up-tilt-induced central hypovolaemia in humans.

Near-infrared spectrophotometry-determined cerebral (ScO2) and muscle oxygen saturations (SmO2) were followed in 15 volunteers during passive 50 degrees head-up-tilt-induced central hypovolaemia, and in nine volunteers during ventilatory manoeuvres affecting arterial carbon dioxide tension. During head-up tilt, mean arterial pressure [MAP, 88 (77-118) to 97 (80-136) mmHg, median and range] and heart rate [HR; 66 (49-77) to 87 (42-132) beats min-1 P < 0.01] increased, but after 22 (1-45) min they declined [to 61 (40-91) mmHg and 69 (38-109) beats min-1, respectively, P = 0.001] and pre-syncopal symptoms developed. Central hypovolaemia was indicated by an increased thoracic electrical impedance, and a decreased cardiac output and central venous oxygen saturation. The arterial oxygen saturation, pulmonal oxygen uptake and skin temperatures remained constant. The ScO2 remained stable at 72 (62-77)% until the pre-syncopal incidence, when it decreased to 62 (31-73)% (P = 0.001), and tilt down made it increase to 75 (36-87)% (P < 0.05) before the recovery value was established. In contrast, SmO2 decreased during tilting [75(70-87) to 65 (53-70)%], and recovered to 70 (53-83)%, P < 0.01) during the hypotensive episode. The end-tidal CO2 tension decreased only during tilt-up. The ScO2 decreased, and SmO2 increased during hyperventilation, and ScO2 increased during breathing of 5% carbon dioxide. Rebreathing from a bag made SmO2 decrease and resulted in a biphasic ScO2 response: it first increased and subsequently decreased. Cardiovascular changes during tilt were not reflected in skin temperature. The ScO2 reflected the maintained autoregulation of cerebral blood flow until the perfusion pressure decreased markedly. In contrast, SmO2 mirrored muscle vasoconstriction early during tilt, and vasodilatation when pre-syncopal symptoms appeared.

Adult↗
PubMed1995

Sympathetic influence on cardiovascular responses to sustained head-up tilt in humans.

Sympathetic beta-adrenergic influences on cardiovascular responses to 50 degrees head-up tilt were evaluated with metoprolol (beta 1-blockade; 0.29 mg kg-1) and propranolol (beta 1 and beta 2-blockade; 0.28 mg kg-1) in eight males. A normotensive-tachycardic phase was followed by a hypotensive-bradycardic episode associated with presyncopal symptoms after 23 +/- 3 min (control, mean +/- SE). Head-up tilt made thoracic electrical impedance (3.0 +/- 1.0 omega), mean arterial pressure (MAP, 86 +/- 4-93 +/- 4 mmHg), heart rate (HR, 63 +/- 3-99 +/- 10 beats min-1) and total peripheral resistance (TPR, 15 +/- 1-28 +/- 4 mmHg min L-1) increase, while central venous oxygen saturation (74 +/- 2-58 +/- 4%), cardiac output (5.7 +/- 0.1-3.1 +/- 0.3 L min-1), stroke volume (95 +/- 6-41 +/- 5 mL) and pulse pressure (55 +/- 4-49 +/- 4 mmHg) decreased (P < 0.05). Central venous pressure decreased during head-up tilt (7 +/- 2-0 +/- 1 mmHg), but it remained stable during the sustained tilt. At the appearance of presyncopal symptoms MAP (49 +/- 3 mmHg), HR (66 +/- 4 beats min-1) and TPR (15 +/- 3 mmHg min L-1) decreased (P < 0.05). Neither metoprolol or propranolol changed tilt tolerance or cardiovascular variables, except for HR that remained at 57 +/- 2 (metoprolol) and 55 +/- 3 beats min-1 (propranolol), and MAP that remained at 87 +/- 5 mmHg during the first phase with metoprolol. In conclusion, sympathetic activation was crucial for the heart rate elevation during normotensive head-up tilt, but not for tilt tolerance or for the associated hypotension and bradycardia.

Adrenergic beta-1 Receptor Antagonists↗
PubMed1994

Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family.

We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa sequence contains 9 Trp residues, some of which are localized in repeats and that characterise the protein as a member of the WD-40 family, a group of proteins having 40-aa repeats containing Trp and Asp [Duronio et al., Proteins 13 (1992) 41-56; Van der Voorn and Ploegh, FEBS Lett. 307 (1992) 131-134]. The protein contains a nuclear targeting signal (KKKGK), and fractionation of transformed human amnion cells (AMA) in karyoplasts and cytoplasts confirmed that it is predominantly localized in the nucleus. Database searching indicated that IEF SSP 9502 is a putative human homologue of the Saccharomyces cerevisiae periodic Trp protein, PWP1, a polypeptide that may play a regulatory role in cell growth and/or transcription.

Amino Acid Sequence↗
PubMed1994

Stimulus-evoked EMG monitoring during transpedicular lumbosacral spine instrumentation. Initial clinical results.

OBJECTIVES: The authors developed and evaluated an electrophysiologic method for minimizing the risk of nerve root trauma associated with the placement of pedicle screws during transpedicular lumbosacral fixation in humans. SUMMARY OF BACKGROUND DATA: Various methods have been evaluated to reduce the high complication rates associated with lumbosacral transpedicular fixation, but none are without significant limitations or drawbacks. Using a pig model, we previously developed a technique for assessing, by electrophysiologic means, the potential risk associated with placement of a screw at a given site. In this report, the authors describe their experience with this technique in patients. METHODS: Electromyogram (EMG) was monitored from eight lower extremity muscles bilaterally. Square-wave electrical shocks (200 microseconds, 7 mA) were delivered through the instruments used to form and evaluate each pedicle hole, and through the screw itself if placement was deemed safe. Provided that the instruments used (e.g., awl, tap, probe) do not exit from bone in any direction below the entry point, the resistance of bone to the 7 mA stimulus intensity is high enough such that no nerve roots are stimulated, and the EMG traces remain flat. Conversely if EMG is evoked, it warns of a potential perforation in a pedicle wall or in the anterior body. RESULTS: The authors evaluated this technique in 18 patients in whom a total of 102 screws were placed. Based on results of electrophysiologic and palpatory evaluation, 68% of the screws were placed in a satisfactory manner. Electrophysiologic evidence of a perforation, which could not be confirmed by palpation or visualization, was seen in another 13% of this total. The remaining 19% of screw placements involved sites where a defect was missed originally by palpation alone, but was located based on electrophysiological testing combined with palpation and visualization (11%), and where a perforation was initially palpated (8%). There was no postoperative morbidity associated with malpositioned screws. CONCLUSIONS: Our results indicate that the technique is sensitive and, based on early clinical results, reliable in the detection of perforations in pedicle screw placement. Moreover, the method is inexpensive, rapid, and easily implemented into a standard intraoperative monitoring protocol.

Adult↗
PubMed1994

Identification, molecular cloning, expression and chromosome mapping of a family of transformation upregulated hnRNP-K proteins derived by alternative splicing.

Acidic nuclear proteins (M(r) between 64,000 and 66,000; pI 4.9 to 5.5) that are highly upregulated in transformed cells and that belong to the hnRNP-K family have been identified using a monoclonal antibody (mAB B4B6) that distinguish between quiescent and proliferating human keratinocytes. The family, which is composed of four major proteins (hnRNPs-K A, B, C and D) and their modified forms, is present in similar overall levels in quiescent and proliferating normal keratinocytes although clear differences were observed in the levels of some of the individual variants. Immunofluorescence staining of proliferating normal keratinocytes with mAB B4B6 showed that about 40% of the keratinocytes, corresponding mainly to G1 and to half of the cells in S-phase, reacted with the antibody depicting a dotted, nucleoplasmic staining that excluded the nucleolus. Only 3 to 4% of the quiescent keratinocytes reacted with the antibody while simian virus 40 (SV40) transformed keratinocytes (K14) stained constitutively throughout the cell cycle. Using mAB B4B6 as a probe we cloned a cDNA coding for one member of the family (hnRNP-K B) and this was used to screen for additional family members. Sequencing of the positive clones revealed four different cDNAs, all resulting from alternative splicing of a common primary transcript of a gene that mapped to chromosome 9. Expression of the cDNAs in the vaccinia virus system confirmed their identity as hnRNPs-K A, B, C and D and showed that their modified forms are phosphorylated. All four hnRNPs bound poly(rC) on NorthWestern blots, although the more acidic of the phosphorylated forms, did so at a much reduced level. hnRNP-K has been implicated in pre-mRNA metabolism of transcripts containing cytidine-rich sequences and our results point towards a role during cell cycle progression.

Alternative Splicing↗
PubMed1994

The human keratinocyte two-dimensional protein database (update 1994): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3087 cellular proteins (2168 isoelectric focusing, IEF; and 919 none-quilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to posttranslational modifications, 890 polypeptides have been identified (protein name, organelle components, etc.) using one or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry and (v) vaccinia virus expression of full length cDNAs. These are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore, we list 239 microsequenced proteins recorded in the database. We also report a database of proteins recovered from the medium of noncultured, unfractionated keratinocytes. This database lists 398 polypeptides (309 IEF; 89 NEPHGE) of which 76 have been identified. The aim of the comprehensive databases is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and, ultimately, to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Amino Acid Sequence↗
PubMed1994

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis.

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified human mononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia.

Apoptosis↗
PubMed1994

Psoriasin binds calcium and is upregulated by calcium to levels that resemble those observed in normal skin.

Recently, we described a small molecular weight protein termed psoriasin that showed sequence similarity with the S100 calcium-binding proteins and that is highly upregulated in psoriatic epidermis as well as in primary human keratinocytes undergoing abnormal differentiation. Here we present evidence showing that natural and recombinant psoriasin binds calcium, as judged by the calcium overlay assay, and that it contains all the sequence features characteristic of the S100 family. Furthermore, [35S]-methionine labeling experiments showed that psoriasin synthesis is upregulated by 2 mM Ca++ (ratio Ca++/control at 88 h = 2.56) to levels that resemble those observed in unfractionated keratinocyte populations obtained from normal skin.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2.

We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from the previously reported cDNA, where it was suggested that translation might initiate at a CTG codon instead of at an ATG codon. Using the vaccinia virus (Vv) expression system, we demonstrate that translation starts at the conventional ATG start codon and not at the CTG codon. The protein is synthesized as a pro-protein of 282 amino acids (aa) that is post-translationally processed by removal of the initial 73 aa to a mature protein of 209 aa.

Amino Acid Sequence↗