Biomedical subjects
Publications and source records attributed to P Madsen.
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The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone. Using RAP affinity chromatography, we have purified a novel approximately 250-kDa brain protein and isolated the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine-based internalization signal and a large external part containing (from the N-terminal): 1) a segment homologous to domains in the yeast vacuolar protein sorting 10 protein, Vps10p, that binds carboxypeptidase Y, 2) five tandemly arranged YWTD repeats and a cluster of 11 class A repeats characteristic of the low density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis but not in several major organs. Both RAP and an antibody against a synthetic peptide derived from a sequence determined in the mature protein detected sorLA-1 in crude human brain extracts. The domain structure suggests that sorLA-1 is an endocytic receptor possibly implicated in the uptake of lipoproteins and of proteases.
Human 2-D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi-bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2-D PAGE reference maps (non-cultured keratinocytes, non-cultured transitional cell carcinomas, MRC-5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2-D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss-Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on-line information is updated regularly.
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) pressures as well as fourteen arterial and venous blood gas variables were followed. Consistent with a reduction of TI during the operation, fluid balance was in excess, and it remained elevated on the first postoperative morning. The HR, MAP and PWP remained stable, while CVP and PAMP decreased. Of the determined variables only TI revealed a meaningful correlation to fluid balance (rho = -0.41; p < 0.01). 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.
A 2985 bp cDNA was isolated from a Lambda Zap Express library and sequenced. The cDNA appeared to represent a previously unknown gene that encodes and acidic 757 amino acid protein containing a bromodomain, several potential sites for phosphorylation by casein kinase-II and small proline-rich segments. The results suggest that the encoded protein might be a novel transcription factor.
Lesional psoriatic epidermis displays a number of phenotypic changes that are distinct from the differentiation program found in normal interfollicular epidermis. In psoriatic epidermis, keratinocytes are hyperproliferative and several differentiation-associated molecules are expressed that are absent in normal skin (e.g., cytokeratins (CK) 6, 16, and 17, and the epidermal proteinase inhibitor SKALP/ elafin). In addition, several molecules which are normally restricted to the stratum granulosum are strongly upregulated in the stratum spinosum (e.g., psoriasis-associated fatty acid binding protein (PA-FABP), psoriasin, involucrin, and transglutaminase). The aim of this study was to develop in vitro culture systems which (a) would allow to study the induction of normal and psoriatic differentiation pathways, and (b) would be amenable for screening of antipsoriatic drugs. Here we have investigated several models for induction of differentiation with respect to the expression of markers for the normal and psoriatic phenotype. Cell cycle parameters and expression levels of CK1, CK10, CK16, SKALP/elafin, transglutaminase, involucrin, psoriasin, and PA-FABP were assessed in these models using flow cytometry, immunocytochemistry, and Northern blot analysis. We observed that induction of differentiation with fetal calf serum resembled the psoriatic phenotype (sustained hyperproliferation; high levels of CK16, SKALP/elafin, transglutaminase, and involucrin; moderate psoriasin expression), whereas differentiation induced by growth factor depletion in a confluent culture resembled the normal differentiation phenotype (low proliferative rate; high expression levels of CK1 and CK10; moderate expression of involucrin and transglutaminase; low expression levels of SKALP/elafin and CK16; absence of psoriasin). We propose that these models can be used to study expression and pharmacological modulation of selected differentiation genes and the coordinated expression of sets of genes associated with epidermal differentiation programs.
The rapid progress in characterizing genes and mRNAs (expressed sequence tags, ESTs) as a result of the Human Genome Project makes it imperative to develop strategies to interface DNA mapping and sequencing data with protein information, as the latter orchestrate most cellular functions. Presently, the only technique able to resolve and record the thousands of proteins present in cells and tissues is two-dimensional (2-D) gel electrophoresis in combination with computer-aided technology to scan the gels, make synthetic images, assign numbers to individual spots as well as to enter qualitative and quantitative information. To date, comprehensive 2-D gel databases containing information about various properties of proteins (cellular localization, identification, regulatory properties, partial amino acid sequences, etc.) have been established (available on the internet: http:@biobase.dk/cgi-bin/celis). What remains is to provide a link between these data and the forthcoming information from the Human Genome Project. We are pursuing two approaches to achieve this goal: (i) microsequencing and mass spectrometry analysis of proteins resolved from 2-D gels and (ii) expression of cDNAs in the vaccinia virus expression system. Using the latter approach we have expressed about 60 cDNAs in human cells under conditions that faithfully reproduce post-translational trimmings and modifications of the proteins. The method, in combination with 2-D gel electrophoresis, allows precise matching of almost any cDNA to its protein product, irrespective of the protein abundance.
Here we describe a direct approach for two-dimensional (2-D) gel mapping of proteins that are modified by post-translational isoprenylation in mammalian cells. Briefly, transformed human amnion cells (AMA) and transfected COS-1 cells were metabolically labeled with either [3H]farnesyl-pyrophosphate or [3H]geranylgeranyl-pyrophosphate following treatment with lovastatin, which blocks the synthesis of mevalonic acid. The proteins were then separated by 2-D gel electrophoresis and electrotransferred to nitrocellulose filters. The membranes were immersed in dimethyl ether, containing 10% of 2,5-diphenyloxazole prior to fluorography. Over 40 [3H]farnesyl-labeled proteins and over 25 [3H]geranylgeranylated proteins were identified on the 2-D autoradiograms. Several [3H]farnesyl-labeled proteins exhibited the same coordinates (M(r) and pI) as their [3H]geranylgeranylated counterparts, raising the possibility that they may be substrates for both farnesyl and geranylgeranyl transferase(s). The approach offers high resolution of both farnesylated and geranylgeranylated proteins and it may serve as a powerful tool for the identification of hitherto unknown prenylated proteins as well as for the determination of prenylated protein levels, type of isoprenoid modification, and possible changes in protein prenyltransferase activity.
Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca(2+)-binding proteins, and that is highly up-regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH2-terminus. The protein was purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion-exchange chromatography and characterized by two-dimensional (2-D) gel electrophoresis and NH2-terminal sequencing. The ability of rh psoriasin to bind Ca2+, Zn2+, and Mg2+ was determined by dialysis experiments. We found that rh psoriasin may bind at least seven molecules of Ca2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3-1.6 x 10(4) M-1. This indicates that psoriasin may cooperatively bind several molecules of Ca2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca2+ is relatively high. At least eight molecules of Zn2+ were bound in KCl and four in NaCl, with an affinity just below 1 x 10(4) M-1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations. Mg2+ and Ca2+ are bound anti-cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.
There is some evidence that intake of sea food rich in n-3 polyunsaturated fatty acids (PUFA) may protect against coronary heart disease (CHD). Thus, even very low daily amounts of n-3 PUFA below 0.5 g have been reported to reduce the incidence of CHD, although it is unknown by what mechanisms this may occur. Monocytes are of major importance in atherogenesis, and we therefore studied the effect of a daily supplement with 0.65 g of n-3 PUFA for 12 weeks on monocyte function in a randomized placebo-controlled trial of 32 healthy subjects. No effect of dietary n-3 PUFA could be demonstrated on monocyte chemotaxis, on chemiluminescence, or on formation of cytokines and leukotriene B4 from activated monocytes.
Inflammatory skin disorders such as psoriasis show a preferential epidermal infiltration of neutrophils and T lymphocytes. This observation raises a question as to which factors determine the appearance and composition of leukocyte tissue infiltrations. Previously, we described a low molecular mass calcium-binding protein (psoriasin, molecular mass 11,457 Da, pI 6.77) belonging to the S1OO family that is highly upregulated in psoriatic keratinocytes and whose expression patterns implied a role in the inflammatory response. Here we report that human psoriasin is a potent and selective chemotactic inflammatory protein for CD4+ T lymphocytes and neutrophils at concentrations of about 10(-11) M. Psoriasin is not structurally related to the alpha or the beta chemokine subfamilies or to lymphotactin, a member of a newly described class of chemokines. Thus, we have observed a chemotactic protein outside the chemokine subfamilies that could be an important new inflammatory mediator.
The effect of hypovolaemic shock on subcutaneous oxygen and carbon dioxide tensions was studied in man. Subcutaneous oxygen (Psc,O2) and carbon dioxide (Psc,CO2) tensions were monitored, during 50 degrees head-up tilt (anti-Trendelenburg's position)-induced central hypovolaemia, in two females and eight males, using a silicone tonometer on the lateral upper aspect of the right arm. All cardiovascular variables remained stable at rest. Incremental tilting to 50 degrees increased heart rate (HR) and mean arterial pressure (MAP) (p < 0.01), while stroke volume (SV), cardiac output (CO) and central venous saturation (SvO2) decreased (p < 0.05). Presyncopal symptoms appeared after 28 (8-48) min (mean and range) as HR decreased from 82 (63-108) to 52 (36-70) beats min-1 (p < 0.05), MAP from 88 (61-106) to 46 (37-54) mmHg and SvO2 from 0.68 (0.56-0.76) to 0.58 (0.39-0.70) (p < 0.01). On return of the tilt table to the horizontal position HR, MAP, SV, CO and SvO2 immediately re-established resting values. The Psc,O2 was 83 (72-102) mmHg at rest and during tilting it increased to 89 (82-111) mmHg followed by a decrease to 72 (58-97) mmHg (p < 0.01) at the appearance of presyncopal symptoms. Psc,O2 returned to the pretilt level over 45 (30-60) min of recovery. Arterial oxygen, carbon dioxide and Psc,CO2 did not change significantly. Subcutaneous oxygen tension decreases during marked central hypovolaemia and it returns slowly to the resting level during recovery. Psc,O2 is a more sensitive marker of impaired tissue oxygenation than arterial oxygen pressure.
Raised plasma neutrophil elastase has been reported to be an early and effective marker of infection in neonates. In order to assess the feasibility of implementing a rapid assay on capillary plasma, repeated heel prick samples were taken in a series of 79 neonates with clinically suspected bacterial infection, comparing the elastase response with C-reactive protein (CRP) and with the band:mature neutrophil ratio. Rises in elastase were in agreement with rises in CRP, but were more frequently manifest at onset of symptoms, showing elastase to be a more rapid marker of infection as previously described. However, 23% of elastase measurements either failed due to haemolysis or coagulation or yielded inexplicably high values. Comparison of simultaneous bilateral heel prick values confirmed unacceptable variation, unlike comparison of paired venous samples or venous and capillary samples from adults, indicating that capillary blood sampling in neonates produces an unpredictable release of neutrophil elastase. Thus, in newborn infants samples for determination of elastase should be taken by venous punctures or from indwelling catheters.
PURPOSE: To report a single biomarker, psoriasin (Mr 11.0 kd, pI 6.2), a calcium binding protein which is expressed largely by stratified squamous epithelia and is externalized to the urine of bladder squamous cell carcinoma (SCC) bearing patients. MATERIALS AND METHODS: Protein expression profiles of SCCs obtained immediately after surgery were analyzed by two-dimensional gel electrophoresis and Coomassie blue staining. Protein identity was determined by microsequencing and immunoblotting. Protein expression in cryosections was studied by immunofluorescence. RESULTS: Four patients with SCC were identified from 100 samples of patients with suspected transitional cell carcinoma (TCC). The protein profiles of the 4 SCCs (56-1, grade III, T4; 181-1, grade I, T3; 219-1, grade III, T3 and 239-1, grade not determined, T2-4) resembled that of keratinocytes, suggesting that these cells express an early developmental pattern of gene expression. Besides expressing markers characteristic of keratinizing stratified squamous epithelia, the SCCs exhibited psoriasin, a protein externalized to the medium by keratinocytes. Immunohistochemistry of 3 of the SCCs with psoriasin antibodies showed that the positive cells were confined chiefly to the "squamous pearls." The presence of psoriasin in the urine of the 4 SCC patients was demonstrated by two-dimensional gel immunoblotting. Similar analysis of 43 urines from patients with bladder tumors other than SCC revealed 7 positives, some of which may reflect squamous differentiation. Analysis of the urine of 13 control individuals (12 males matched by age and a 42-year-old female) revealed 2 positives. Immunoblotting of the SCC patients' serum proteins with psoriasin antibodies failed to reveal the protein. CONCLUSION: The results point towards psoriasin, alone or as part of a biomarker profile, as a potential marker for the noninvasive follow-up of patients with SCC.
PURPOSE: To identify and describe the normal intradural vessels detected on MR angiograms of the thoracolumbar spine. METHODS: Six adult subjects who had clinical evidence of myelopathy, yet normal findings at spinal digital subtraction angiography (DSA), were also studied without and with contrast-enhanced MR imaging and three-dimensional time-of-flight, single-slab MR angiography. Sagittal and coronal subvolume (targeted) maximum intensity projection images were compared with arterial and venous phase DSA images. Angiographic images were then compared with postmortem, formalin-fixed cord specimens. RESULTS: Recognizable intradural vessels were detected only on contrast-enhanced MR angiograms. These vessels corresponded to the posterior and/or anterior median (midline) veins and the great medullary veins. The median veins had variable but mild tortuosity. The medullary veins, which extended from the median veins and coronal venous plexus on the cord surface to the epidural venous plexus, were relatively straight and usually located at T-12 or L-1. The anterior spinal artery could partially contribute to the anterior midline vascular signal. CONCLUSION: The intradural vessels identified on contrast-enhanced MR angiograms are primarily veins, and these are usually the largest vessels on or near the cord surface. The limited number and minimal tortuosity of these veins may serve as a baseline for the examination of patients with clinically suspected arteriovenous malformation or fistula.
We report a novel approach for identifying monomeric GTP-binding proteins that is based on probing cDNA expression libraries with [alpha-32P]GTP. In short, a nitrocellulose replica from a plated cDNA expression library is treated with 2% SDS to block the GTP-binding activity of various G proteins expressed by E. coli, thus allowing the direct identification of positive clones. Using this procedure we have cloned several small GTP-binding proteins from human keratinocytes including the human homolog of rheb, a novel member of the ras-related GTP-binding proteins. Human rheb cDNA shares 90% identity with the rat counterpart and it is highly upregulated in transformed human cells of various origin. Northern analysis showed that human rheb is ubiquitously expressed, with the highest levels observed in skeletal and cardiac muscle, and not in brain, as it is the case for rat rheb. The human RHEB gene was mapped to chromosome 10q11.
Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.
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