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Biomedical subjects

C W Heizmann

Publications and source records attributed to C W Heizmann.

At least 55 records · Page 3Linked to original sources
PubMed2000

Inverse expression of S100A4 and E-cadherin is associated with metastatic potential in gastric cancer.

S100A4 is known to be involved in cancer cell motility by virtue of its ability to activate nonmuscle myosin. E-cadherin has an important role in the homophilic cell-cell adhesion and is called an invasion suppressor gene. In the current study, we investigate the histological type and metastatic potential of gastric cancer from the aspect of the interrelationship of E-cadherin and S100A4 expression. Expression of E-cadherin and S100A4 in gastric cancer cell lines, primary gastric cancers, and their normal counterparts were analyzed by reverse transcription-PCR, Western blot, and immunohistochemical methods. S100A4 protein and E-cadherin were expressed in five of eight gastric cancer cell lines, and inverse expression of the two proteins are found in four cell lines. In the clinical specimens, E-cadherin mRNA expression in differentiated adenocarcinomas (88%, 14 of 16) was significantly more frequent than that in poorly differentiated adenocarcinomas (50%, 22 of 44; P = 0.015). Western blot analysis demonstrates that S100A4 protein expression in poorly differentiated adenocarcinomas was 1.6-fold higher than in well differentiated adenocarcinoma. Immunohistochemically, S100A4 expression was detected in 51 (55%) of 92 primary gastric cancers. Reduced expression of E-cadherin in primary tumors was found in 66 (72%) of 92 tumors. S100A4 expression in the poorly differentiated adenocarcinomas had a strong relation to positive lymph node involvement or peritoneal dissemination. Reduced E-cadherin expression showed a strong relationship with positive serosal involvement and infiltrating type. Tumors classified as a group with reduced E-cadherin and high expression of S100A4 reveal positive peritoneal dissemination, serosal involvement, and infiltrating type in the growth pattern. Furthermore, these tumors showed a strong correlation with the poorly differentiated adenocarcinoma. In contrast, tumors with preserved E-cadherin and low expression of S100A4 have a close relation to the well differentiated adenocarcinoma and a favorable prognosis. By the Cox proportional hazard model, S100A4 and E-cadherin tissue status was judged as an independent prognostic factor. S100A4 and E-cadherin tissue status may be a powerful aid in evaluating metastatic potential or the prognosis of patients with gastric cancer.

Cadherins↗
PubMed2000

Distribution of the Ca2+-binding S100A1 protein at different sarcomere lengths of slow and fast rat skeletal muscles.

The localization of S100A1 in rat soleus (SOL) and extensor digitorum longus (EDL) muscles was studied immunocytochemically at different sarcomere lengths (stretched, relaxed and contracted) at the ultrastructural level. The muscle fibres were contracted by application of 15 mmol/l caffeine. Following aldehyde fixation, dehydration and embedding in Lowicryl HM20 (-35 degrees C) ultrathin sections were incubated with rabbit polyclonal antiserum against S100A1. Goat antirabbit secondary antibodies conjugated with 10 nm gold particles were used to visualize antigen sites. Relative areas of Z-lines, A- and I-bands were estimated from longitudinal sections by the point counting method. The highest densities of the particles were found at the Z-lines. A higher incidence of S100A1 antigen sites in I-bands than in A-bands and a higher density of S100A1 in lateral parts of A-bands (with actin and myosin filaments overlapping) compared with the central area of A-bands are consistent with an interaction of S100A1 with F-actin in skeletal muscles. Antigen sites were also present at M-lines and at distinct locations of the sarcoplasmic reticulum.

Actins↗
PubMed1999

Human S100A11 exhibits differential steady-state RNA levels in various tissues and a distinct subcellular localization.

In order to analyze the steady-state RNA levels of S100A11 in different tissues, a cDNA fragment of human S100A11 was isolated from a cDNA library. The obtained fragment was labeled and hybridized to RNA isolated from various tissues. The Northern blot analysis revealed that S100A11 RNA levels varied from high in placenta, through intermediate in heart, lung, kidney, and most muscle samples, to barely detectable in brain. An efficient purification method for recombinant S100A11 yielding high quantities was developed. Furthermore, to examine the subcellular localization of this protein, the human polypeptide S100A11 antibodies were raised in rabbit. S100A11 was found to have a localization distinct from other S100 proteins examined, and is mostly localized in the nucleus, with slight variations among different glioblastoma cell types.

Adult↗
PubMed1999

Long-lasting c-fos and NGF mRNA expressions and loss of perikaryal parvalbumin immunoreactivity in the development of epileptogenesis after ethacrynic acid-induced seizure.

A single cerebroventricular injection of ethacrynic acid (EA), a Cl(-)-ATPase inhibitor, induces generalized tonic-clonic convulsions in mice. To clarify whether such convulsive stimulus triggers a long-lasting rearrangement of the neural circuitry culminating in seizure susceptibility, we examined molecular, cellular and behavioral changes following the EA-induced seizure. The expression of immediate early gene c-fos mRNA as an index for cellular activation increased biphasically, with an early transient increase at 60 min and a late prolonged increase on the 10th to 14th day post-EA administration, most remarkably in the hippocampus and pyriform cortex. On the 14th day post-EA seizure, subconvulsive dose of kainic acid (5-17.5 mg/kg) caused severe (stage 5) seizure in 77% of the mice, with 70% mortality. In addition, the expression of nerve growth factor (NGF) also showed biphasic increases with close spatiotemporal correlation with c-fos expression. Moreover, the number of cell somata and the density of axon fibers of parvalbumin (PARV)-positive cells, a subpopulation of GABAergic interneurons, decreased in area dentata, CA1 and CA3 on the 7th and 14th day post-EA seizure. In area dentata and CA1, the density of glutamic acid decarboxylase (GAD)-positive cells also decreased on the 14th day. Thus, the transient EA-induced seizures appear to develop seizure susceptibility by causing damage of a subpopulation of inhibitory interneurons along with increases in the expression of c-fos and NGF in limbic structures.

Animals↗
PubMed1999

Electrospray ionization mass spectrometry: analysis of the Ca2+-binding properties of human recombinant alpha-parvalbumin and nine mutant proteins.

A set of 10 different recombinant human parvalbumins was used to establish a method for the investigation of the Ca2+-binding properties of proteins by electrospray ionization mass spectrometry (ESI-MS). Human PVWT was found to bind 2 mol Ca2+ ions/mol of protein, whereas its mutants (PVE101V, PVD90A, PVE62V, PVD51A, PVD90A,E101V, PVE62V,E101V, PVD51A,D90A, PVD51A,E62V, PVD51A,E62V, D90A,E101V) containing inactivating substitutions in the Ca2+-binding loops bind 0 or 1 Ca2+ ion per protein molecule, depending on the degree of inactivation. These findings fully agree with previously reported results obtained by flow dialysis experiments. The RP-HPLC desalted metal-free proteins were analyzed in 10 mM ammonium acetate at pH 7.0. The experimental conditions were optimized with the recombinant parvalbumin test system before analyzing the Ca2+-binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine alpha-parvalbumins each bind specifically two Ca2+ ions per protein molecule and (ii) both extracted parvalbumins were found to be posttranslationally modified; each protein is acetylated at the N-terminus. Finally, during our investigations of the murine parvalbumin a sequencing error was detected at the C-terminus where the amino acid at position 109 is Ser and not Thr as mentioned in the SwissProt data base (Accession No. P32848). This work demonstrates the great potential of the ESI-MS technique as a sensitive, specific, and rapid method for direct identification and determination of the stoichiometry of Ca2+-binding proteins and other metalloproteins.

Animals↗
PubMed1999

Transcriptional activation of the human S100A2 promoter by wild-type p53.

S100A2, a calcium binding protein of the EF-hand family, was recently identified to be inducible by etoposide, a p53 activator. A potential p53 binding site was identified in the promoter of the S100A2 gene, which binds to purified p53 as well as p53 in nuclear extract activated by etoposide. Transactivation assays using the promoter driven luciferase reporters revealed that the S100A2 promoter was transcriptionally activated by wild-type p53, but not by p53 mutants, in a dose-dependent as well as a p53 binding site-dependent manner. The p53-induced transactivation of the S100A2 promoter was enhanced by etoposide and blocked by a dominant negative p53 mutant. Furthermore, endogenous S100A2 mRNA expression is induced by etoposide in p53 positive, but not in p53 negative cells. Thus, p53 appears to positively regulate S100A2 expression.

Binding Sites↗
PubMed1999

Distribution of a specific calcium-binding protein of the S100 protein family, S100A6 (calcyclin), in subpopulations of neurons and glial cells of the adult rat nervous system.

S100A6 (calcyclin) is a member of the large S100 Ca2+-binding protein family, considered to activate several processes along the calcium signal transduction pathway including the regulation of cell growth, proliferation, secretion, and exocytosis. In the present study, the distribution of S100A6 in the rat nervous system was examined by immunohistochemistry with a goat antiserum against recombinant human S100A6, which recognizes the rat S100A6 homologue. The main S100A6-immunoreactive elements were 1) neuronal somata and dendrites in some specific regions of the limbic system (e.g., the basolateral amygdaloid nucleus, ventral tip of the CA1-subicular border region, entorhinal cortex, and parasubiculum), most of which were identified as a subpopulation of pyramidal cells; 2) olfactory receptor cells and olfactory nerve fibers and terminals in the olfactory bulb; 3) some tracts of the hindbrain and spinal cord (e.g., the spinal trigeminal tract, solitary tract, dorsal root fibers, and the tract of Lissauer) and their terminals (e.g., the principal sensory trigeminal nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, marginal zone, substantia gelatinosa, and proper sensory nucleus of the dorsal horn), as well as some sensory neurons of their origins in the dorsal root and trigeminal ganglia; 4) a subpopulation of astrocytes in the white matter (e.g., the corpus callosum, cingulum, external capsule, internal capsule, and fimbria of the hippocampus) and around the ventricles; 5) some ependymal cells, especially around the central canal; and 6) Schwann cells. These results will improve our understanding of the diverse function of Ca2+-binding proteins in the CNS.

Animals↗
PubMed1999

Demonstration of heterodimer formation between S100B and S100A6 in the yeast two-hybrid system and human melanoma.

S100B (S100beta) and S100A6 (calcyclin) are two 10-kDa Ca2+- and Zn2+-binding proteins coexpressed in melanoma and cell-cycle regulated. These proteins are members of the S100 subfamily and are thought to exert their function through interaction with intracellular target proteins. In order to search for potential target proteins interacting with S100B, we used a yeast two-hybrid strategy with human S100B as bait to screen a human brain cDNA library. The fusion proteins interacting with the S100B bait were identified as S100B, S100A1, and S100A6. This indicates the potential of S100B to form homodimers and heterodimers with other members of the S100 subfamily. By Northern and Western blotting, S100B and S100A6 were shown to be expressed at high levels in a panel of human melanoma cell lines. S100B and S100A6 were coimmunoprecipitated from melanoma cell lysates in the presence of 100 microM Zn2+. Confocal microscopy demonstrated that both proteins were distributed throughout the cytoplasm and concentrated in the nucleus. The demonstration of an association and colocalization of S100B and S100A6 in melanoma supports the possibility that an S100B/S100A6 heterodimer plays a functional role in these cells.

Animals↗
PubMed1999

Immunohistochemical localisation of pterin-4alpha-carbinolamine dehydratase in rat peripheral organs.

The bifunctional protein PCD/DCoH is both a pterin-4alpha-carbinolamine dehydratase (PCD) involved in the recycling of tetrahydrobiopterin (BH4) and a dimerisation cofactor (DCoH) of the hepatic nuclear factor 1alpha (HNF-1alpha). An antiserum raised against rat PCD/DCoH was used to localise the protein in peripheral organs. In liver, all the hepatocytes but not the other cell types are immunoreactive. In kidney, the protein is prevalent in the proximal and distal convoluted tubules. In adrenals, all the cells of the medulla are labelled. Positive nerve cells occur in myenteric ganglia of the whole gastrointestinal tract and in the intestinal submucous ganglia. Many positive endocrine cells are present in the epithelium. The immunoreactivity is either cytoplasmic (hepatocytes, convoluted tubules of the kidney and part of the gastrointestinal endocrine cells) or prominently nuclear (kidney collecting tubules, adrenals, intestinal neural plexuses and part of the gastrointestinal endocrine cells). Our results show that PCD/DCoH is present in cells expressing enzymes that use BH4 as a cofactor and/or HNF-1alpha. In addition, PCD/DCoH is present in other cells, for example, neurons in the submucosal plexus. This fact and the prominent nuclear immunoreactivity found in all the positive cells derived from the neural crests argue in favour of a new, still unknown function for the protein.

Adrenal Cortex↗
PubMed1999

Subcellular distribution of S100 proteins in tumor cells and their relocation in response to calcium activation.

S100 proteins, a subgroup of the EF-hand Ca2+-binding protein family, regulate a variety of cellular processes via interaction with different target proteins. Several pathological disorders, including cancer, are linked to altered Ca2+ homeostasis and might involve the multifunctional S100 proteins, which are expressed in a cell- and tissue-specific manner. The present work demonstrates a distinct intracellular localization of S100A6, S100A4, and S100A2 in two tumor cell lines derived from metastatic epithelial breast adenocarcinoma (MDA-MB231) and cervical carcinoma (HeLa). Treatment of the cells by thapsigargin, the ionophore A23187, or cyclic ADP-ribose, to increase [Ca2+]i via different pathways, led to relocation of S100A6 and S100A4 but only partially of the nuclear S100A2, as demonstrated by confocal laser scanning microscopy. These findings support the hypothesis that S100 proteins could play a crucial role in the regulation of Ca2+ homeostasis in cancer cells.

Adenosine Diphosphate Ribose↗
PubMed1999

Overexpression of pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 in human colon cancer.

Pterin-4a-carbinolamine dehydratase (PCD) is a bifunctional protein also known as DCoH (dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)). PCD/DCoH modulates the DNA binding specificity of HNF1, thus acting on its transcriptional activity. In addition, it participates in the recycling of tetrahydrobiopterin (BH(4)), an essential cofactor of several metabolic reactions. We investigated colorectal tumors and colorectal tumor cell lines as compared to normal colon samples in search of a potential differential expression of PCD/DCoH. Immunohistochemistry was conducted on 20 human colorectal tumors and 20 normal samples using a specific polyclonal antibody. Immunoblotting and RT-PCR analysis for PCD/DCoH and HNF1 were also performed on both human tissues and CACO-2 and HT-29 cell lines. All of the 20 tumors and both colon cancer cell lines presented a strong and widespread immunoreactivity for PCD/DCoH, contrasting with the absence of expression in the normal epithelia. We thus report the massive overexpression of PCD/DCoH in colon tumors, which is in striking contrast with the absence of staining in normal counterparts. The sharp contrast in the expression of a modulator of transcriptional activity between tumoral and normal cells may have a physiopathological role. PCD/DCoH could potentially be a new marker of malignant colon cells in vivo.

Adenocarcinoma↗
PubMed1999

Ca2+-binding S100 proteins in the central nervous system.

A number of neurodegenerative disorders have been attributed to abberrations of intracellular Ca2+ homeostasis regulated by Ca2+-binding proteins. This chapter will focus on the S100B and S100A6 proteins, which are highly expressed in the central nervous system. Their protein structures, localizations, and association with brain pathology as well as their potential use in clinical diagnostics will be discussed.

Animals↗
PubMed1999

Supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas are characterized by a differential expression of S100 proteins.

The levels of expression of the S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B proteins were immunohistochemically assayed and quantitatively determined in a series of 95 astrocytic tumors including 26 World Health Organization (WHO) grade I (pilocytic astrocytomas), 23 WHO grade II (astrocytomas), 25 WHO grade III (anaplastic astrocytomas) and 21 WHO grade IV (glioblastomas) cases. The level of the immunohistochemical expression of the S100 proteins was quantitatively determined in the solid tumor tissue (tumor mass). In addition twenty blood vessel walls and their corresponding perivascular tumor astrocytes were also immunohistochemically assayed for 10 cases chosen at random from each of the four histopathological groups. The data showed modifications in the level of S100A3 protein expression; these modifications clearly identified the pilocytic astrocytomas from WHO grade II-IV astrocytic tumors as a distinct biological group. Modifications in the level of S100A6 protein expression enabled a clear distinction to be made between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumors. Very significant modifications occurred in the level of S100A1 protein expression (and, to a lesser extent, in their of the S100A4 and S100B proteins) in relation to the increasing levels of malignancy. While the S100A5 protein was significantly expressed in all the astrocytic tumors (but without any significant modifications in the levels of malignancy), the S100A2 protein was never expressed in these tumors. These data thus indicate that several S100 proteins play major biological roles in human astrocytic tumors.

Adolescent↗
PubMed1998

The identification and differential expression of calcium-binding proteins associated with ocular melanoma.

Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

Adult↗
PubMed1998

Clustered organization of S100 genes in human and mouse.

S100 Ca2+-binding proteins became of major interest because of their differential expression in tissues and their association with human diseases. Earlier studies showed that 13 S100 genes are located as a cluster on human chromosome 1q21. Since a number of mouse S 100 genes, such as S100A4 and S100A6, have been localized to a syntenic region on mouse chromosome 3, we investigated if the S100 gene cluster exists in mouse and is structurally conserved during evolution. First we identified the cDNA sequences of mouse S100A1, S100A3 and S100A5. Then we isolated a 490 kb mouse YAC clone which gives a specific signal by FISH most likely on chromosome 3. Hybridization studies with different mouse S100 cDNAs revealed that eight mouse S100 genes are arranged in a clustered organization similar to that in human. The linkage relationships between the genes S100A8-S100A9 and S100A3-S100A4-S100A5-S100A6 were conserved during divergence of human and mouse about 70 million years ago. However, the separation of the mouse S100 genes S100A1 and S100A13 in comparison to the human linkage group suggests rearrangement processes between human and mouse. Our data demonstrate that the S100 gene cluster is structurally conserved during evolution. Further studies on the genomic organization of the S100 genes including various species could generate new insights into gene regulatory processes and phylogenetic relationships.

Amino Acid Sequence↗
PubMed1998

Probing the structure of the human Ca2+- and Zn2+-binding protein S100A3: spectroscopic investigations of its transition metal ion complexes, and three-dimensional structural model.

A large-scale procedure was developed for the anaerobic purification of the human recombinant Ca2+- and Zn2+-binding protein S100A3 for spectroscopic studies. S100A3 eluted as a non-covalently bound dimer (20.8 kDa). It contained 7.5+/-0.1 free thiol groups/monomer, and bound Ca2+ with a Kd of approximately 4 mM, which corresponds to a tenfold increase in affinity compared to the aerobically purified protein. The transition metal ions Co2+, Zn2+ and Cd2+ were used as spectroscopic probes to investigate the role of the 10 cysteine residues per monomer S100A3 in metal binding. Spectrophotometric titrations suggest the formation of dinuclear thiolate-bridged clusters consisting of a Me2+(S(Cys))4 and a Me2+(S(Cys))3(N(His)) site as described for zinc finger proteins. A three-dimensional structural model of S100A3 was proposed on the basis of the NMR structure of the structurally related rabbit S100A6 protein, and taking into account the structural influence of cysteine residues.

Amino Acid Sequence↗
PubMed1998

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins.

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.

Animals↗