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Biomedical subjects

Henrik Vorum

Publications and source records attributed to Henrik Vorum.

29 records · Page 2Linked to original sources
PubMed2004

Heterogeneous nuclear ribonucleoproteins F and H/H' show differential expression in normal and selected cancer tissues.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H/H', containing the quasi-RNA recognition motif (qRRM) domains, are implicated in several steps of pre-mRNA processing and in cellular differentiation. We have compared a set of tissues and found striking differences in their levels of expression as well as in the nuclear versus the cytoplasmic distribution. Generally, hnRNP F is broadly expressed in many tissues with extremely strong expression in the prostate gland while hnRNP H/H' shows a more restricted degree of expression with low expression in some tissues, for example, liver, exocrine acini of the pancreas, thyroid gland and heart. At the cellular level, hnRNP F is, with few exceptions, predominantly expressed in the cytoplasm while hnRNP H/H' is more abundant in the nuclei. A quite pronounced heterogeneous expression pattern is seen in the proximal tubules of the kidney where hnRNP F is present at moderate cytoplasmic levels while hnRNP H/H' is undetectable, whereas both proteins are more evenly expressed in distal tubules and collecting ducts. Generally, tumor tissues reveal a broad expression of hnRNP F in the nuclei as well as in the cytoplasm while hnRNP H/H' is expressed at higher levels in the nuclei than in the cytoplasm. Up-regulation of hnRNP H/H' is found in a few tissues that normally express low cytoplasmic levels of hnRNP H/H', for example, adenocarcinoma of the pancreas, hepatocellular carcinoma and gastric carcinoma. hnRNP F is down-regulated in hepatocellular carcinoma and up-regulated in gastric carcinoma. The present study indicates the important potential role of this subset of hnRNPs on the gene expression in many tissues.

Adenocarcinoma↗
PubMed2004

Functional genomics studied by proteomics.

The human genome contains about 30,000 genes, each creating several transcripts per gene. Transcript structures and expression are studied by high-throughput transcriptomic techniques using microarrays. Generally, transcripts are not directly operating molecules, but are translated into functional proteins, post-translationally modified by proteolysis, glycosylation, phosphorylation, etc., sometimes with great functional impact. Proteins need to be analyzed by proteomic techniques, less suited for high-throughput. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), separating thousands of proteins has developed slowly over the past quarter of a century. This technique is now quite reproducible and suitable for differential proteomics, comparing normal and diseased cells/tissues revealing differentially regulated proteins. 2D-PAGE is combined with protein-identification methods, currently mass spectrometry (MS), which has been significantly improved over the last decade. Other proteomic techniques studying protein-protein interactions are now either established or still being developed, such as peptide or protein arrays, phage display, and the yeast two-hybrid system. The strengths and weaknesses of these techniques are discussed.

Databases, Factual↗
PubMed2004

Proteomic analysis of hyperoxia-induced responses in the human choriocarcinoma cell line JEG-3.

Living cells exposed to changes in the surrounding oxygen tension, have the ability to adapt to the new environment through the regulatory effect of intracellular mediators. In an effort to identify important proteins that may be involved in the hyperoxic response, we performed proteomic analysis on the human choriocarcinoma cell line JEG-3, incubated under high oxygen tension (carbogen, 95% O2/5% CO(2)) or air (21% oxygen/5% CO(2)). We identified 13 protein spots that were significantly down-regulated (p < 0.05) in JEG-3 cells incubated under hyperoxic conditions compared to standard conditions. Ten of these spots were positively identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry as nine different proteins: Villin 2, tublin beta, profilin I, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, peroxiredoxin 1, neuroplypeptide h3, poly(rC)-binding protein 1 and cyclophilin A. These proteins have been implicated in regulating cytoskeletal structure, glycolysis, redox status, signal transduction, transcription and protein folding. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation and motility.

Cell Line, Tumor↗
PubMed2003

Ca2+ binding to complement-type repeat domains 5 and 6 from the low-density lipoprotein receptor-related protein.

BACKGROUND: The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca2+ ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca2+ ion as a necessity for correct folding and stabilization of this protein module. Additional Ca2+ binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca2+ cage within CR-domains from this family of receptors that function in endocytosis and signalling. RESULTS: We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca2+ ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca2+, and show that CR56 binds 2 Ca2+ ions with an average affinity of KD = 10.6 microM, and there is no indication of additional Ca2+ binding sites within this receptor fragment. CONCLUSIONS: Both CR-domains of CR56 bind a single Ca2+ ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.

Amino Acid Sequence↗
PubMed2003

Essential role of the apolipoprotein E receptor-2 in sperm development.

The apolipoprotein (apo) E receptor-2 (apoER2) is a member of the low density lipoprotein receptor gene family and an important regulator of neuronal migration. It acts as a receptor for the signaling factor Reelin and provides positional cues to neurons that migrate to their proper position in the developing brain. Besides brain formation defects, apoER2-deficient mice also exhibit male infertility. The role of the receptor in male reproduction, however, remained unclear. Here we demonstrate that apoER2 is highly expressed in the initial segment of the epididymis, where it affects the functional expression of clusterin and phospholipid hydroperoxide glutathione peroxidase (PHGPx), two proteins required for sperm maturation. Reduced PHGPx expression in apoER2 knockout mice results in the inability of the sperm to regulate the cell volume and in abnormal sperm morphology and immotility. Because insufficient expression of PHGPx is a major cause of infertility in men, these findings not only highlight an important new function for apoER2 that is unrelated to neuronal migration, but they also suggest a possible role for apoER2 in human infertility.

Animals↗
PubMed2003

Renal Na,K-ATPase structure from cryo-electron microscopy of two-dimensional crystals.

The molecular structure of Na,K-ATPase was determined by electron crystallography from two-dimensional crystals induced in purified membranes isolated from the outer medulla of pig kidney. The P2 type unit cell contains two protomers in the E(2) conformation, each of them with a size of 65 x 75 x 150 A(3). The alpha, beta, and gamma subunits in the membrane crystals were demonstrated in the crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as to the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains. One of these gives rise to an elongated projection onto the membrane plane, while the putative nucleotide binding and phosphorylation domains form compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit. Ten helices from the catalytic a subunit correspond to two groups of distinct densities in the transmembrane region. The structure of the lipid bilayer spanning part also suggests positions for the transmembrane helices from the beta and gamma subunits. The overall structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy is similar to the X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmic domains.

Animals↗
PubMed2003

Immunocytochemical localization of Na,K-ATPase gamma subunit and CHIF in inner medulla of rat kidney.

The gamma subunit of Na,K-ATPase and CHIF both belong to the FXYD single-membrane-spanning protein family and have been suggested to have regulatory functions in kidney tubules. CHIF is known to be present in the collecting duct, and gamma has been demonstrated in several segments of the rat kidney tubule, but never clearly in the inner medullary collecting duct (IMCD). Here, we demonstrate the cellular and subcellular localization of the gamma subunit and CHIF in the IMCD in inner medulla by using Western blotting, laser-scanning confocal immunofluorescence, and immunoelectron microscopy. In the initial quarter of the IMCD (next to the outer medulla), antibodies against the C-terminal of gamma as well as splice variant gammaa labeled the basolateral surface of intercalated cells (ICs), while principal cells (PCs) remained unlabeled. In the middle segment of the IMCD, all PCs exhibited distinct basolateral staining for the gammaC-terminal as well as gammaa and CHIF. Immunoelectron microscopy showed that the gammaC-terminal and CHIF were associated with the inner leaflet of the basolateral plasma membrane in the labeled cells. Immunoblotting demonstrated the presence of both the gammaC-terminal and gammaa in inner medullary tissue. However, splice variant gammab was not detected in inner medulla by immunocytochemistry or immunoblotting. The present observations demonstrate that the Na,K-ATPase gamma subunit and CHIF are strategically located in the inner medulla to participate in the fine-tuning of urine ion composition through the regulation of the Na,K-ATPase activity in the IMCD.

Animals↗
PubMed2002

Endonuclein is a cell cycle regulated WD-repeat protein that is up-regulated in adenocarcinoma of the pancreas.

The transcript encoding endonuclein, the human homolog of yeast PWP1, was previously found up-regulated in pancreatic cancer tissue. By immunohistochemistry we detected a ubiquitous presence in several tissues examined: skin, liver, thyroid gland, heart muscle, neurons, kidney, bladder, pancreas, adrenal gland, ovary, uterus, testis and prostate gland. We especially noticed that normal pancreatic exocrine cells exhibited low protein levels while pancreatic adenocarcinoma cells revealed high levels. We found a heterogeneous subcellular distribution, especially with varying nuclear levels. In proliferating cells endonuclein protein expression and localization was cell cycle dependent, with increasing levels and nuclear focusing during the interphase toward mitosis. Ultrastructural analysis revealed ER and nuclear localization. Endonuclein contains five WD-repeats, indicating a putative role in crucial regulatory activities in the nucleus as well as in the ER.

Adenocarcinoma↗
PubMed2000

Immunoelectron microscopic localization of the electrogenic Na/HCO(3) cotransporter in rat and ambystoma kidney.

Immunofluorescence analysis has revealed that electrogenic Na(+)/HCO(3)(-) (NBC1) is expressed in the proximal tubule of rat kidney and in the proximal and distal tubules of the salamander AMBYSTOMA: tigrinum kidney. The present study was undertaken to define the detailed subcellular localization of the NBC1 in rat and AMBYSTOMA: kidney using high-resolution immunoelectron microscopy. For this purpose, two rabbit polyclonal antibodies raised against amino acids 928 to 1035 and amino acids 1021 to 1035 of the C-terminus of rat kidney (rkNBC1) were developed. The affinity-purified antibodies revealed a strong band of approximately 140 kD in immunoblots of membranes from rat kidney cortex but no signal in membranes isolated from outer and inner medulla. Deglycosylation reduced the apparent molecular weight to approximately 120 kD, corresponding to the predicted molecular weight. A similar but weaker band was also present in membranes isolated from the lateral part of Ambystoma: kidney. In rat kidney, immunohistochemistry confirmed the presence of rkNBC1 in convoluted segments of the proximal tubules. In ultrathin cryosections or Lowicryl HM20 sections from rat kidney cortex, distinct immunogold labeling was associated with the basolateral plasma membrane of segments S1 and S2 of proximal tubules, whereas in S3 no labeling was observed. The labeling density was similar at the basal and lateral plasma membrane and was specifically associated with the inner surface of the membrane consistent with the internal position of the C-terminus of the transporter. In contrast, rkNBC1 was absent from the apical plasma membrane and not observed in intracellular vesicles, including those closely associated with basolateral plasma membrane. In Ambystoma: kidney, a weak labeling was present in the basolateral membrane of the proximal tubule and stronger labeling was observed in the late distal segment. The results demonstrate that rkNBC1 is expressed only in segment S1 and segment S2 of rat proximal tubule as well as Ambystoma: proximal and late distal tubule and that rkNBC1 is present in both basal and lateral plasma membranes and absent in intracellular vesicles of the apical plasma membrane.

Ambystoma↗
PubMed2000

Receptor-associated protein is important for normal processing of megalin in kidney proximal tubules.

The receptor-associated protein (RAP) has been identified as a chaperone regulating the expression and processing of the LDL receptor-related protein. RAP also binds to the related 600-kD multiligand endocytic receptor megalin expressed in many absorptive epithelia including renal proximal tubule. The present study examines the effect of RAP gene disruption on megalin expression and subcellular distribution in the proximal tubule as well as the effect on tubular protein reabsorption. It is shown that RAP is important for the normal expression and function of megalin. Megalin expression was reduced to approximately 23% estimated by immunoblotting and supported by immunocytochemistry and by the amount of megalin recovered by RAP affinity chromatography. Light- and electron microscope immunocytochemistry as well as analyses on separated membrane fractions showed significant changes in the subcellular distribution of megalin. A significant reduction in the normal brush border labeling was observed in association with increased labeling of rough endoplasmic reticulum and the smooth paramembranous endoplasmic reticulum along the basolateral membranes. RAP deficiency was associated with changes in urinary protein composition, enabling the identification of alpha-amylase as a new ligand for megalin. In addition, an increased excretion of vitamin D-binding protein, a recently identified ligand to megalin, was observed supporting changes in tubular protein reabsorption. The present data show that RAP is of crucial importance for normal processing and function of megalin, suggesting a chaperone-like function of this protein in the kidney proximal tubule.

Animals↗