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Biomedical subjects

C W Heizmann

Publications and source records attributed to C W Heizmann.

At least 19 recordsLinked to original sources
PubMed1992

Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme.

6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.

Alcohol Oxidoreductases↗
PubMed1992

The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.

Animals↗
PubMed1992

S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart.

Elevated levels of intracellular calcium are a major cause of myocardial dysfunction. To find possible mediators of the deregulated calcium we searched for EF-hand calcium-binding proteins of the S100 family. By PCR technology we identified three members of the S100 protein family (S100 alpha, CACY, and CAPL) in the human heart. We cloned the corresponding cDNAs and examined their expression levels in various human tissues by Northern blot analysis. All three proteins are expressed at high levels in the human heart. Whereas CACY and CAPL mRNAs are expressed ubiquitously, S100 alpha mRNA is restricted to heart, skeletal muscle, and brain. Interestingly, the expression pattern of S100 alpha, CACY, and CAPL in human tissues differs significantly from that in rodent tissues.

Amino Acid Sequence↗
PubMed1992

Development of the olivocerebellar projection in the rat: I. Transient biochemical compartmentation of the inferior olive.

In the present study the early phases of the development of the inferior olive were examined by using immunocytochemical techniques. We observed that, from embryonic day 16 onward, antibodies against the calcium binding proteins parvalbumin and calbindin and the calcitonin gene related peptide stain partially overlapping territories of the inferior olive. This staining delimits a biochemical zonation of the inferior olive which is combinatory and transient. We have previously observed a biochemical parcellation of the cerebellar Purkinje cells which, like that of the inferior olive, is first observed at E16, involves the combined expression of marker proteins and is also transient. In order to know whether the biochemical compartmentations of the cerebellum and inferior olive arise independently, the time course of the development of the olivocerebellar projection was studied by anterograde and retrograde in vitro axonal tracing by using the fluorescent carbocyanine dye DiI. The olivocerebellar axons were found to reach the limit of the cerebellar plate at E16 and to enter it at E17. Even at this age the great majority of the climbing fibers are tightly fasciculated, which minimizes their interactions with the PC clusters. These observations indicate that the topographical heterogeneity of Purkinje cells and inferior olive neurons arise independently. The transient biochemical individualization of subgroups of neurons during development could contribute to recognition mechanisms.

Animals↗
PubMed1992

Development of the olivocerebellar projection in the rat: II. Matching of the developmental compartmentations of the cerebellum and inferior olive through the projection map.

A transient biochemical parcellation has been observed by immunocytochemical methods, during the perinatal development of both the inferior olive and the cerebellum. In the present study, we sought a relationship between this developmental compartmentation and the organization of the olivocerebellar projection. In the inferior olive, a transient parvalbumin immunoreactivity restricted to the dorsal cap of the medial accessory olive is observed around birth. The climbing fiber projection of the dorsal cap was identified in the cerebellum of newborn rats based on its parvalbumin immunoreactivity. The pattern of this projection, restricted to lobules IX and X of the vermis, and to the flocculus, is indistinguishable from that of the adult medial accessory olive, which was previously described from axonal tracing experiments. The parvalbumin immunoreactive climbing fibers were followed between birth and postnatal day 7. In the caudal vermis, Purkinje cell subpopulations can be identified between embryonic day 20 and postnatal day three, on the basis of their differential immunostaining with an antibody directed against a specific peptide, PEP 19. In lobule X, the parvalbumin immunoreactive climbing fibers form two sagittal bands on each side of the midline, one medial and one lateral. The medial parvalbumin immunoreactive climbing fiber band is coextensive with a PEP 19 negative Purkinje cell cluster, indicating a clear relationship between the biochemical parcellations of the cerebellum and inferior olive.

Animals↗
PubMed1992

7-substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency. Mechanism of formation via non-enzymatic transformation from 6-substituted pterins.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1',2'-dihydroxypropyl)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7-Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki approximately 8 microM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.

Biotransformation↗
PubMed1992

6-pyruvoyl tetrahydropterin synthase from salmon liver: amino acid sequence analysis by tandem mass spectrometry.

The most frequent variant of atypical phenylketonuria, an inborn error of metabolism, is characterized by a low activity of the 6-pyruvoyl tetrahydropterin synthase. We purified and characterized this enzyme from salmon liver known to contain high levels. After digestion, peptides were sequenced by tandem mass spectrometry and/or automated Edman microsequence analysis. Both a free amine terminus and an N-acetylated amine terminus were found, indicating the presence of two isoforms. The peptide sequences determined here have a high degree of homology with the protein sequence deduced from cDNA for rat 6-pyruvoyl tetrahydropterin synthase (1), however, the amine termini of these proteins differ significantly.

Alcohol Oxidoreductases↗
PubMed1992

Monoclonal antibody 473 selectively stains a population of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat cerebral cortex.

Monoclonal antibody (MAb) 473 is shown to outline selectively a subpopulation of GABAergic neurons containing a specific calcium-binding protein parvalbumin (PV) in the adult rat parietal cortex, using preembedding immunocytochemistry at the light microscopic level. About 90% of MAb 473 stained cells in the rat parietal cortex were PV immunoreactive. Thus we compared MAb 473 staining with that of three chemical probes, previously shown to stain selectively a subpopulation of PV-containing GABAergic neurons in this brain region, namely, a lectin, Vicia villosa agglutinin (VVA), with a specific affinity for terminal N-acetyl-galactosamine, MAb 3B3 which is specific for chondroitin sulfate proteoglycan and MAb HNK-1 which is specific for some types of carbohydrate epitope containing a sulfated derivative of glucuronic acid. About 85% of MAb 473 immunoreactive cells were shown to be stained with VVA. Furthermore about 90% of MAb 473 immunoreactive cells were also stained with MAb 3B3. Thus MAb 473 positive cells were almost included into VVA and/or MAb 3B3 positive cells. On the other hand only about 34% of MAb 473 positive cells were HNK-1 positive, whereas about 44% of HNK-1 positive cells were MAb 473 positive. Thus these two MAbs defined different, though partially overlapping, subsets of PV-containing GABAergic neurons in the rat parietal cortex.

Animals↗
PubMed1992

Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis.

In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects of olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) - 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) - 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) - 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70-80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) - 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) - 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.

Animals↗
PubMed1992

Quantitative analysis of neurons and glial cells in the rat somatosensory cortex, with special reference to GABAergic neurons and parvalbumin-containing neurons.

The number of neuronal and glial cells in the rat somatosensory cortex (barrel area) has been estimated by a stereological method, the disector, using pairs of toluidine blue-stained, plastic-embedded 0.5-microns-thick sections, 1.5 microns distant from each other. Chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent semithin sections. Thus we were able to analyze quantitatively number, distribution, and proportion of five cell types: (1) gamma-aminobutyric acid-(GABA)-negative neurons; (2) GABA-like immunoreactive (GABA-LIR) neurons; (3) a specific calcium-binding protein parvalbumin-immunoreactive (PV-IR) neurons, a subpopulation of GABA-LIR neurons; (4) S-100 beta-LIR glial cells (astrocytes); and (5) S-100 beta-negative glial cells (oligodendrocytes and microglia). The densities of total cells, glial cells, and neurons in the rat somatosensory cortex were 85.4 +/- 10(3)/mm3, 30.5 x 10(3)/mm3, and 54.9 x 10(3)/mm3, respectively. Of all neurons 25% and 14% were GABA-LIR and PV-IR, respectively; all PV-IR neurons are GABA-LIR, and thus about 54% of GABA-LIR neurons are PV-positive. The number of total cells under a unit surface area of 1 mm2 through the thickness of the somatosensory cortex was 171.6 x 10(3); the number of neurons and glial cells were 110.2 x 10(3) and 61.4 x 10(3), respectively. There were 27.7 x 10(3) GABA-LIR neurons and 15.0 x 10(3) and 12.7 x 10(3) PV-IR neurons and PV-negative GABA-LIR neurons, respectively. The laminar distribution of each group of cells shows prominent differences, indicating that the cellular composition was different from layer to layer. The density of GABA-LIR neurons was highest in layer IV. The numerical density of PV-IR neurons was 2-4 times higher in layer IV than in layers II/III, V, and VI, whereas that of PV-negative GABA-LIR neurons was almost constant throughout the layers.

Animals↗
PubMed1992

Changes in Ca(2+)-binding proteins in human neurodegenerative disorders.

The cellular distribution of Ca(2+)-binding proteins has been extensively studied during the past decade. These proteins have proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major roles are assumed to be Ca2+ buffering and transport, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca2+ homeostasis, a protective role for Ca(2+)-binding proteins in certain neuron populations has been postulated. As massive neuronal degeneration takes place in several brain diseases of humans, such as Alzheimer's disease, Parkinson's disease and epilepsy, changes in the expression of Ca(2+)-binding proteins have therefore been studied during the course of these diseases. Although the data from these studies are inconsistent, the detection and quantification of Ca(2+)-binding proteins and the neuron populations in which they occur may nevertheless be useful to estimate, for example, the location and extent of brain damage in the various neurological disorders. If future studies advance our knowledge about the physiological functions of these proteins, the neuronal systems in which they are expressed may become important therapeutical targets for preventing neuronal death in an array of neurodegenerative diseases.

Alzheimer Disease↗
PubMed1992

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from human pituitary gland.

6-Pyruvoyl tetrahydropterin synthase, the enzyme that catalyses the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, was purified 3,330-fold from human pituitary gland with an overall recovery of 30%. The native enzyme has a molecular mass of 68 kD and consists of four identical subunits of 16.5 kD. The pH optimum of the enzyme in Tris/HCl buffer is 7.5. The enzyme is dependent on Mg2+ and NADPH and has a Michaelis-Menten constant of 10 microM for its natural substrate, 7,8-dihydroneopterin triphosphate. The isoelectric point of the human enzyme is 4.3-4.6. The human pituitary gland enzyme is heat instable in contrast to the enzymes from human, rat and salmon liver, and Drosophila head. The amino acid composition showed remarkably high content of acidic amino acids Asp and Glu. The N-terminus was found to be blocked.

Alcohol Oxidoreductases↗
PubMed1992

Atypical (mild) forms of dihydropteridine reductase deficiency: neurochemical evaluation and mutation detection.

We investigated two patients with an atypical (mild) form of dihydropteridine reductase (DHPR) deficiency. Both responded to the loading test with tetrahydrobiopterin; their plasma phenylalanine levels were lowered from 278 mumol/L to 85 and 48 mumol/L and from 460 mumol/L to 97 and 36 mumol/L after 4 and 8 h, respectively. In one of the patients, a combined loading test with phenylalanine followed by tetrahydrobiopterin was also carried out and showed a profile typical for DHPR deficiency. The phenylalanine hydroxylation rate was calculated to be 43 and 87%, 4 and 8 h after cofactor administration, respectively. Diagnosis was confirmed by the absence of DHPR activity in the patient's erythrocytes. In cultured fibroblasts, residual activity of 4 and 10%, respectively, was found. Excretion of urinary pterins was essentially normal, and the biopterin to neopterin ratio in cerebrospinal fluid was increased. Although in both patients cerebrospinal fluid homovanillic acid was found to be normal, and 5-hydroxyindoleacetic acid was substantially reduced, there was no sign of neurologic alterations until the age of 2 y. However, one of the patients recently developed deceleration of head growth, whereas psychomotor development continued to be normal for age. Using the chemical cleavage method on the amplified cDNA, mismatches of T to G at nucleotide 659 and of G to A at nucleotide 475, respectively, were identified. These results also demonstrate that screening for tetrahydrobiopterin deficiency by urinary pterin analysis alone can miss some newborns with mild DHPR deficiency and that all children with tetrahydrobiopterin defects need full neurochemical evaluation together with analysis of the enzyme activity.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Calcium-binding proteins: basic concepts and clinical implications.

Calcium ions exert their effects in part via interactions with a wide variety of intracellular calcium-binding proteins. One class of these proteins shares a common calcium-binding motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of new members of this family of EF-hand proteins, which now has over 200 members. A few of these proteins are present in all cells, whereas the vast majority are expressed in a tissue-specific fashion. The physiological function of a few of these proteins is known to be achieved via a calcium-dependent interaction with other proteins, thereby regulating their activity. Some members, like parvalbumin, calbindin, and calretinin, proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major role is assumed to be buffering, transport of Ca2+, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca2+ homeostasis, a protective role for Ca(2+)-binding proteins in certain neuron populations has been postulated. Another protein family are the annexins, members of which interact with phospholipids and cellular membranes in a calcium-dependent manner. In some cases members of the annexin family were even found to interact with EF-hand proteins. Certain annexins have been suggested to be involved in anti-inflammatory response, inhibition of blood coagulation, membrane trafficking or cytoskeletal organization, but several of these functions have been questioned recently. The elucidation of the interactions and functions of the majority of these proteins remains a challenging task for the coming years.

Animals↗
PubMed1991

Different populations of parvalbumin- and calbindin-D28k-immunoreactive neurons contain GABA and accumulate 3H-D-aspartate in the dorsal horn of the rat spinal cord.

The colocalization of parvalbumin (PV), calbindin-D28k (CaBP), GABA immunoreactivities, and the ability to accumulate 3H-D-aspartate selectively were investigated in neurons of laminae I-IV of the dorsal horn of the rat spinal cord. Following injection of 3H-D-aspartate into the basal dorsal horn (laminae IV-VI), perikarya selectively accumulating 3H-D-aspartate were detected in araldite embedded semithin sections by autoradiography, and consecutive semithin sections were treated to reveal PV, CaBP and GABA by postembedding immunocytochemistry. Perikarya accumulating 3H-D-aspartate were found exclusively in laminae I-III, and no labelled somata were found in deeper layers or in the intermediolateral column although the labelled amino acid clearly spread to these regions. More than half of the labelled cells were localized in lamina II. In this layer, 16.4% of 3H-D-aspartate-labelled perikarya were also stained for CaBP. In contrast to CaBP, PV or GABA was never detected in neurons accumulating 3H-D-aspartate. A high proportion of PV-immunoreactive perikarya were also stained for GABA in laminae II and III (70.0% and 61.2% respectively). However, the majority of CaBP-immunoreactive perikarya were GABA-negative. GABA-immunoreactivity was found in less than 2% of the total population of cells stained for CaBP in laminae I-IV. A significant proportion of the GABA-negative but PV-immunoreactive neurons also showed CaBP-immunoreactivity in laminae II and IV. These results show that out of the two calcium-binding proteins, CaBP is a characteristic protein of a small subpopulation of neurons using excitatory amino acids and PV is a characteristic protein of a subpopulation of neurons utilizing GABA as a transmitter. However, both proteins are present in additional subgroups of neurons, and neuronal populations using inhibitory or excitatory amino acid transmitters are heterogeneous with regard to their content of calcium-binding proteins in the dorsal horn of the rat spinal cord.

Animals↗