Biomedical subjects

C W Heizmann

Publications and source records attributed to C W Heizmann.

At least 91 records · Page 5Linked to original sources

PubMed1997

Localization of the Ca(2+)-binding S100A1 protein in slow and fast skeletal muscles of the rat.

The distribution of S100A1 in rat soleus and EDL muscles was studied immunocytochemically at the ultrastructural level using immunogold as marker. Antigens were localized mainly in myofibrils at all levels of the sarcomere. Immunogold particles along myofibrils were not uniformly distributed. The highest density of particles was found at Z-lines. An increase in particle density was observed in the middle of half A-bands in EDL and in the middle of half I-bands in the soleus. Antigen sites were also present at M-lines and at distinct locations of the sarcoplasmic reticulum.

Animals↗

PubMed1996

Expression pattern of S100 calcium-binding proteins in human tumors.

The S100 Ca(2+)-binding proteins recently became of major interest because of their differential expression in neoplastic tissues, their involvement in metastatic processes, and the clustered organization of at least 10 S100 genes on human chromosome 1q21, a region frequently rearranged in several tumors. As a first attempt towards a specific and differentiated immunohistochemical classification of human tumors, we produced, purified and characterized a number of human recombinant S100 proteins and raised specific polyclonal antibodies. Their distinct cellular and intracellular localization was examined by immunohistochemical methods in normal and cancerogenic human tissues and cell lines. S100A1 and S100A2 can be detected in a few normal tissues only, whereas S100A4, S100A6, and S100B are expressed at higher levels in cancer tissues. In the future, these S100 antibodies will potentially be of great value in cancer diagnosis and therapy.

Adenocarcinoma↗

PubMed1996

Characterization of the human and mouse cDNAs coding for S100A13, a new member of the S100 protein family.

Here we report the characterization of the human S100A13 cDNA coding for a novel calcium-binding protein belonging to the S100 protein family. The predicted S100A13 protein shows sequence homologies to other S100 proteins between 50.5% (to S100A5) and 59.3% (to S100A12). High mRNA amounts were found in skeletal muscle, heart, kidney, ovary, small intestine and pancreas. Since twelve S100 genes are clustered on human chromosome 1q21, we determined the chromosomal localization of the human S100A13. It co-localizes with S100A1 on the cluster. Furthermore, we characterized the cDNA sequence coding for the mouse homolog of S100A13. Similar to the putative human protein, mouse S100A13 is composed of 98 amino acids displaying a homology of 86.7% compared to human S100A13.

Amino Acid Sequence↗

PubMed1996

Human recombinant alpha-parvalbumin and nine mutants with individually inactivated calcium- and magnesium-binding sites: biochemical and immunological properties.

Human recombinant alpha-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca(2+)-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PVD51A,E62V,D90A,E101V), were expressed and purified. Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K'Ca, of 2.3 x 10(7) M-1 and that Mg2+ competes with a K'Mg.compet. of 4.9 x 10(3) M-1. The three mutants with an inactivated CD site bind 1 Ca2+ with K'Ca, of 2.0 to 2.3 x 10(7) M-1 and K'Mg.compet. of 3.4 to 4.6 x 10(3) M-1, i.e. very similar to those of PVwt. The mutants with an inactivated EF site bind 1 Ca2+ with K'Ca values of 7.9 x 10(6), 4.5 x 10(6) and 3.6 x 10(6) M-1 for PVD91A, PVE102V and PVE101V,D91A, respectively. The K'Mg.compet values of these mutants are about 4-times lower than in PVwt. The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+. After excitation at 259 nm, human PV, which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm. Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+. Specific antibodies against human alpha-parvalbumin were raised in rabbits. Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated.

Animals↗

PubMed1996

Altered expression of the Ca(2+)-binding protein S100A1 in human cardiomyopathy.

The Ca(2+)-binding protein S100A1 displays a tissue-specific expression pattern with highest levels in myocardium and has been shown to interact with SR-proteins regulating the Ca(2+)-induced Ca(2+)-release. We, therefore, hypothesized that changes in S100A1 gene expression might correlate with the pathognomonic finding of altered SR Ca(2+)-transients in human end stage heart failure. To test this hypothesis, we established a specific and sensitive method to analyse S100A1 expression in cardiac tissues by employing hydrophobic interaction-chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) coupled with Electron-Ionisation-Mass-Spectrometry (ESI-MS). Porcine myocardium showed a differential expression of S100A1 with relative protein concentrations of 62 +/- 8% in the right ventricle (RV), 57 +/- 9% in the right atrium (RA), and 25 +/- 15% in the left atrium (LA) as compared to the left ventricle (LV) (100 +/- 10%; P < 0.001). Northern blot analyses confirmed a likewise distribution of porcine S100A1 mRNA implying a regulation on the transcriptional level. Analyses of left ventricular specimen of patients with end stage heart failure (CHF, n = 6; CHD, n = 6) revealed significantly reduced S100A1 protein levels, while integration of S100A1 peaks after RP-HPLC yielded two groups of patients with < 76% (69 +/- 7%, n = 6) and < 35% (23 +/- 12%, n = 6) respectively as compared to controls (100 +/- 8%, n = 3). These data demonstrate for the first time that S100A1 is differentially expressed in myocardium and that in human cardiomyopathy a reduced expression of S100A1 may contribute to a compromised contractility.

Animals↗

PubMed1996

Chromosomal localization, genomic structure and characterization of the human gene and a retropseudogene for 6-pyruvoyltetrahydropterin synthase.

Autosomal recessive mutations in the 6-pyruvoyltetrahydropterin synthase (PTPS) gene are the most common reason for hyperphenylalaninemia due to tetrahydrobiopterin deficiency. We used the previously isolated PTPS cDNA as a probe and identified the human gene, PTS, located on chromosome 11q22.3-q23.3, and a retropseudogene, PTS-P1, assigned to 9p12-p13 (symbols approved by the human genome nomenclature committee). PTS-P1 has 74% similarity to the 3' portion of PTPS cDNA. The PTS gene spans about 8 kb and consists of 6 exons, as revealed by DNA-sequence analysis. This gene structure differs from that published previously which was reported to contain only two exons [Ashida, A., Owada, M. & Hatakeyama, K. (1994) Genomics 24,408-410]. By means of intron-specific primers, we amplified exon 3 from genomic DNA of a PTPS-deficient patient and found a mutation in the 3' acceptor splice site, which is responsible for skipping of exon 3.

Alcohol Oxidoreductases↗

PubMed1996

Retrovirus-mediated gene transfer of 6-pyruvoyl-tetrahydropterin synthase corrects tetrahydrobiopterin deficiency in fibroblasts from hyperphenylalaninemic patients.

Tetrahydrobiopterin (BH4) deficiency, a variant form of hyperphenylalaninemia with progressive neurological dysfunction, is primarily caused by autosomal recessive mutations in the gene encoding the 6-pyruvoyl-tetrahydropterin synthase (PTPS). PTPS is a biosynthetic enzyme for the BH4 co-factor, and its deficiency is associated with a malfunction of the phenylalanine catabolism in the liver and a lack of biogenic amine neurotransmitters dopamine and serotonin in the brain. We have previously isolated the wild-type PTPS cDNA and identified several mutations responsible for a decreased enzyme in patients. This study reports the in vitro correction of BH4 deficiency by using retrovirus mediated transfer of the PTPS cDNA into primary fibroblast cultures established from different patients. The Bing packaging cell line was used for amphotropic virus production. Following PTPS gene transfer, stimulation with cytokines restored biosynthesis of BH4 in originally defective cells to values comparable to those of heterozygous fibroblasts from clinically healthy subjects. These results not only provide a direct proof that the mutations in PTPS were causative for the mutant phenotype, but they are also the first step toward gene therapy as a potential alternative approach to treat BH4 deficiency.

Alcohol Oxidoreductases↗

PubMed1996

Amino acid sequence determination of human S100A12 (P6, calgranulin C, CGRP, CAAF1) by tandem mass spectrometry.

S100A12 has been isolated from human neutrophils. The molecular weight and the amino acid sequence of S100A12 was determined by electrospray-mass spectrometry, tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A12 with all known human S100 proteins revealed that S100A12 is the most divergent of the S100 proteins.

Amino Acid Sequence↗

PubMed1996

Tetrahydrobiopterin loading test in xanthine dehydrogenase and molybdenum cofactor deficiencies.

The objectives of this study were to find additional diagnostic information for the evaluation of xanthine dehydrogenase deficiency and molybdenum cofactor deficiency. Patients were given an oral loading test with 10 mg/kg 5,6,7,8-tetrahydrobiopterin. Urine excretion of pterin and isoxanthopterin was measured by HPLC. Control subjects had a fairly constant ratio of urinary pterin/isoxanthopterin before (0.57-5.32) and after (0.55-4.55) 5,6,7,8-tetrahydrobiopterin loading. These ratios were increased to 33 and 22 in a patient with hereditary xanthinuria and to 570 and 8030 in a patient with molybdenum cofactor deficiency. Obligate heterozygotes had an entirely normal test result. Evidence was obtained for the in vivo involvement of xanthine dehydrogenase in the conversion of pterin to isoxanthopterin. This test could be a sensitive marker for the establishment of residual enzyme activity.

Administration, Oral↗

PubMed1996

Electron microscopic serial-sectioning/reconstruction study of parvalbumin-containing neurons in the external plexiform layer of the rat olfactory bulb.

Neurons containing a calcium-binding protein parvalbumin in the external plexiform layer of the rat olfactory bulb were identified light microscopically with the pre-embedding immunocytochemistry and were subsequently analysed with the electron microscopic serial-sectioning and three-dimensional reconstructions. In the present study we chose several different types of parvalbumin-immunoreactive neurons identified light microscopically as Van Gehuchten cell type, superficial short-axon cell type and multipolar cell type. Parvalbumin-immunoreactive somata were similar to one another in their ultrastructural characteristics, showing nuclear indentations, moderately developed Golgi apparatus and abundant mitochondria; these structural features appeared to resemble those of the short axon cells around the glomeruli and in the granule cell layer reported in previous electron microscopic studies. All neurons analysed in the present study made symmetrical synapses on to dendrites and somata of presumed mitral/tufted cells and received asymmetrical synapses from them, and occasionally formed reciprocal synapses with them. On the parvalbumin-immunoreactive processes, the asymmetrical synapses nearly equalled the symmetrical ones in number and about 30-50% of them were identified as reciprocal pairs. In contrast, no presynaptic sites were observed on parvalbumin-immunoreactive somata, and thick portions (more than approximately 2 microns in diameter) of the proximal dendrites, where they were occasionally postsynaptic in some asymmetrical and symmetrical synapses from parvalbumin-immunonegative profiles. Characteristically, parvalbumin-immunoreactive process frequently make direct contacts with one another; processes regarded light microscopically as arising from a soma or a dendrite or parvalbumin-immunoreactive neurons were sometimes revealed to be separate but directly contacting processes with electron microscopic examinations. Although puncta adherentia were occasionally observed between these contact sites, so far neither gap junctions nor chemical synapses were observed. Until now, it has been believed that in the external plexiform layer only granule cells form reciprocal synapses with mitral/tufted cells. However, the present study clearly demonstrates that interneurons different from granule cells, namely GABAergic neurons containing a calcium-binding protein parvalbumin, also make reciprocal synapses with mitral/tufted cells in the external plexiform layer. Therefore, neuronal processes making reciprocal synapses with mitral/tufted cells in the external plexiform layer cannot be determined a priori as granule cell processes.

Animals↗

PubMed1996

Direct evidence of an extensive GABAergic innervation of the spinal dorsal horn by fibres descending from the rostral ventromedial medulla.

A long line of studies emphasizes the contribution of serotonergic fibres descending from the rostral ventromedial medulla in the control of spinal nociceptive information processing. A growing body of evidence, however, suggests that the relative contribution of serotonin to the mediation of spinal neuronal activity from the rostral ventromedial medulla may require re-evaluation. It has recently been substantiated that, in addition to the serotonergic fibres, the spinal dorsal horn receives an abundant non-serotonergic projection from the rostral ventromedial medulla. Furthermore, stimulation in the rostral ventromedial medulla could result in a powerful inhibition of nociceptive spinothalamic tract cells without any detectable serotonin release in the dorsal horn. After labelling raphe-spinal axons and axon terminals in the rat by iontophoretic injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin into the central region of the rostral ventromedial medulla (nucleus raphe magnus) and revealing GABA and glycine immunoreactivities of the labelled raphe-spinal terminals and their postsynaptic targets by postembedding immunocytochemical methods, here we demonstrate an extensive GABAergic projection from the rostral ventromedial medulla to the spinal dorsal horn. We show that the majority of the labelled raphe-spinal terminals in laminae I-IIo and IV-V contain GABA and some of the GABA-immunoreactive terminals are also immunoreactive for glycine. We also disclose that GABA-immunoreactive raphe-spinal terminals establish synaptic contacts primarily with GABA- and glycine-negative, presumably excitatory, spinal neurons, including Calbindin-D28k- as well as parvalbumin-immunoreactive cells in both laminae I-IIo and IV-V. The results suggest that volleys in fibres descending from the rostral ventromedial medulla may evoke GABA release from raphe-spinal terminals, and the released GABA, in some cases probably acting together with glycine, might play a crucial, as yet mostly unidentified, role in the inhibition of nociceptive information processing in the dorsal horn of the spinal cord.

Animals↗

PubMed1996

Ultrastructural localization of alpha-parvalbumin in the epiphyseal plate cartilage and bone of growing rats.

The distribution of the calcium-binding protein, alpha-parvalbumin, in the epiphyseal plate cartilage and bone of growing rats was examined by electron microscope immunocytochemistry of undecalcified samples. Parvalbumin immunoreactivity, as revealed by gold particles, increased with maturation of chondrocytes and was maximal in the zone of calcification. It was found in the cytoplasm of chondrocytes, osteoblasts and osteocytes, corroborating light microscope observations. The immunolabeling was associated with amorphous electron-dense material in the cytoplasm and not bound to membranes. There was moderate parvalbumin immunolabeling over the dense chromatin in the nuclei of chondrocytes and bone cells, but none in the cell processes of mature and hypertrophic chondrocytes, in the matrix vesicles themselves, or in the cell processes of osteoblasts. However, there was parvalbumin immunoreactivity in the cell processes of the osteocytes of compact cortical bone. The uncalcified and calcified matrix of the epiphyseal cartilage, the osteoid, and the fully mineralized cortical bone were devoid of parvalbumin immunoreactivity. Thus, immunoreactive parvalbumin is confined to the cell bodies of chondrocytes and osteoblasts, and is unlikely to be directly involved in mineral deposition. The maximal parvalbumin immunoreactivity in the last terminal chondrocytes of the zone of calcification suggests that the protein is involved in buffering intracellular Ca2+, preventing the stimulation of degenerative processes by high intracellular calcium. The parvalbumin immunoreactivity in the cell processes of osteocytes of compact cortical bone seems to indicate that this calcium-binding protein may be involved in the regulation of Ca2+ fluxes and hence in calcium homeostasis in bone.

Animals↗

PubMed1996

Alpha-parvalbumin reduces depolarization-induced elevations of cytosolic free calcium in human neuroblastoma cells.

We investigated whether the expression of human alpha-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca2+]i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM. Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K+. During this period, [Ca2+]i was monitored by video microfluorimetry using the Ca2+ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca2+]i. The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca2+]i than non-parvalbumin-expressing cells. Resting [Ca2+]i did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca2+]i in neuroblastoma cells can be suppressed by parvalbumin.

Animals↗

PubMed1996

Characterization of the human S100A12 (calgranulin C, p6, CAAF1, CGRP) gene, a new member of the S100 gene cluster on chromosome 1q21.

Here, we report the characterization of a human cDNA coding for the recently published amino acid sequence of a calcium-binding S100 protein, S100A12 (CGRP, calgranulin C, CAAF1, p6). The exon/intron structure of the S100A12 gene is similar to most other S100 genes. It is composed of three exons which are divided by two introns of 900 bp and 400 bp. The protein is encoded by sequences in exons 2 and 3, with exon 2 coding for the N-terminal 45 amino acids and exon 3 coding for the C-terminal 46 amino acids. So far, ten S100 genes are known to be located on human chromosome 1q21 in a clustered organization. Hence, we investigated whether S100A11 (S100C, calgizzarin) and S100A12 are also localized in the S100 gene cluster. We found both genes within the cluster, with S100A11 being close to S100A10 and S100A12 between the genes S100A8 and S100A9. Therefore, the S100 gene cluster now is composed of 12 differentially expressed family members.

Amino Acid Sequence↗

PubMed1996

Dense GABAergic input on somata of parvalbumin-immunoreactive GABAergic neurons in the hippocampus of the mouse.

GABAergic neurons in the hippocampus proper are greatly diverse in their morphological and physiological features. In the present study we examined whether or not they are also diverse regarding the density of GABAergic input on their somata. GABAergic neurons were immunocytochemically identified with antibodies against glutamic acid decarboxylase (GAD), and the densities of GAD-immunoreactive (GAD-IR) boutons that abutted on GAD-IR somata were estimated by conventional light microscopic, combined light and electron microscopic, and confocal laser scanning microscopic analyses. GAD-IR somata were apparently diverse regarding the density of GABAergic input on them, and those surrounded by higher densities of GAD-IR boutons were distributed mainly in the strata pyramidale and oriens of the CA3 and CA1 regions and could be correlated to a parvalbumin (PV)-IR subpopulation of GABAergic neurons. Quantitative analysis clearly revealed the statistically significant difference between PV-positive and PV-negative GAD-IR neurons in the densities of their somatic GAD-IR boutons. Particularly, most of PV-IR neurons in the CA3 stratum pyramidale as well as some in other layers are characterized by an exceedingly high density of perisomatic GAD-IR boutons. Furthermore, the majority of GAD-IR boutons on PV-IR somata in the stratum pyramidale were also PV-IR. Bilateral transection of the fimbria-fornix, which was supposed to remove GABAergic afferents from the septum, had only partial effects on the densities of PV-IR boutons on PV-IR somata, indicating these PV-IR boutons mainly originated from intrinsic PV-IR neurons. These observations indicate the dense mutual connection between PV-IR GABAergic neurons through perisomatic synaptic contacts, particularly in the stratum pyramidale.

Animals↗

PubMed1996

The S100 family of EF-hand calcium-binding proteins: functions and pathology.

Calcium lons as second messengers control many biological processes, at least in part, via interaction with a large number of Ca(2+)-binding proteins. One class of these proteins shares a common Ca(2+)-binding motif, the EF-hand, Here, we describe some functional aspects of EF-hand proteins, which have been found recently in different cellular compartments. Novel links between EF-hand proteins, particularly S100 proteins, and specific diseases are now emerging.

Calcium-Binding Proteins↗

PubMed1996

Immunohistochemical evaluation of the Ca(2+)-binding S-100 proteins S-100A1, S-100A2, S-100A4, S-100A6 and S-100B in salivary gland tumors.

The Ca(2+)-binding S-100 proteins are involved in the regulation of a number of cellular processes and an altered expression has been reported in several neoplastic tissues. Tissue specimens of normal salivary glands (n = 23), pleomorphic adenomas (n = 60), basal cell adenomas (n = 6), canalicular ademomas (n = 2), myoepitheliomas (n = 2), adenoid cystic carcinomas (n = 26) and adenocarcinomas NOS (n = 11) were evaluated for the expression of S-100A1, S-100A2, A-100A4, S-100A6 and S-100B by using highly specific polyclonal and monoclonal antibodies generated against the recombinant human protein. In normal salivary glands, the ductal cells showed mild to intense immunoreactivity for S-100A1, S-100A2, S-100A4 and S-100A6, while S-100B was observed in nerve fibers in the connective tissue. The normal myoepithelial cells were unreactive. In pleomorphic adenoma, the luminal tumor cells of the duct-like structures showed moderate to intense immunoreactivity for S-100A2, while reactivity for S-100A1, S-100A4 and S-100A6 was relatively weak. The non-luminal cells, also termed neoplastic myoepithelial cells, showed immunoreactivity for S-100B, while tumor cells in the solid, myxoid and chondroid areas were immunoreactive for S-100A1, S-100A4, S-100A6 and S-100B. The non-luminally located tumor cells in basal cell adenomas and canalicular adenomas, and numerous tumor cells in clusters in myoepitheliomas were intensely reactive for S-100A2. In adenoid cystic carcinomas and in adenocarcinomas not otherwise specified, the luminal cells forming the tubular or cribriform structures were markedly positive for S-100A2 and/or S-100A6. Squamous metaplastic cells in salivary tumors showed intense immunoreactivity for S-100A2. The results of the present study suggest that the majority of the tumor cells in salivary neoplasms, despite the most heterogeneous tumor cell differentiation, express S-100 proteins more heterogeneously than the normal glandular ducts. The salivary ducts in normal glands, the luminal tumor cells and squamous metaplastic cells in the neoplastic lesions were intensely immunoreactive for S-100A2 as compared to S-100A1, S-100A4 or S-100A6. In contrast, the non-luminal tumor cells showed a rather heterogeneous expression of the S-100 proteins.

Adenocarcinoma↗

PubMed1995

Structural and functional consequences of mutations in 6-pyruvoyltetrahydropterin synthase causing hyperphenylalaninemia in humans. Phosphorylation is a requirement for in vivo activity.

Four naturally occurring mutants with single amino acid alterations in human 6-pyruvoyltetrahydropterin synthase (PTPS) were overexpressed and characterized in vitro. The corresponding DNA mutations were found in patients with hyperphenylalaninemia and monoamine neurotransmitter insufficiency due to lack of the tetrahydrobiopterin biosynthetic enzyme PTPS. To predict the structure of the mutant enzymes, computer modeling was performed based on the solved three-dimensional structure of the homohexameric rat enzyme. One mutant (delta V57) is incorrectly folded and thus unstable in vitro and in vivo, while a second mutant (P87L) has substantial activity but enhanced sensitivity to local unfolding. Two other mutants, R16C and R25Q, form stable homomultimers and exhibit significant activity in vitro but no activity in COS-1 cells. In vivo 32P labeling showed that wild-type PTPS, P87L, and R25Q are phosphorylated, while R16C is not modified. This strongly suggests that the serine 19 within the consensus sequence for various kinases, RXXS, is the site of modification. Our results demonstrate that PTPS undergoes protein phosphorylation and requires additional, not yet identified post-translational modification(s) for its in vivo function.

Alcohol Oxidoreductases↗