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Biomedical subjects

K F Sheu

Publications and source records attributed to K F Sheu.

At least 55 records · Page 3Linked to original sources
PubMed1992

Thiamin and Alzheimer's disease.

Because of clinical and neuropathological overlap between the characteristics of dementia of the Alzheimer type (DAT) and of a human thiamin deficiency syndrome (Wernicke-Korsakoff syndrome), thiamin pyrophosphate (TPP) dependent processes have been studied in DAT brain and other tissues. The activities of 3 TPP-dependent enzymes are reduced in DAT brain: transketolase (TK), the pyruvate dehydrogenase complex (PDHC), and the alpha-ketoglutarate dehydrogenase complex (KGDHC). Quantitatively, the most marked reductions are in KGDHC (to less than 20% of normal). In cultured skin fibroblasts, KGDHC activity is reduced to 50-60% of normal, TK activity to 80-90% of normal, and PDHC is normal. Structural and molecular studies of the DAT and non-DAT enzymes are in process. A lesion of KGDHC may be related to the pathogenesis of DAT. Treatment with large doses of thiamin has not been beneficial, but the data are not totally negative. Further studies of thiamin-dependent mechanisms in DAT seem justified.

Alzheimer Disease↗
PubMed1991

A trial of thiamine in Alzheimer's disease.

Because a previous short-term study demonstrated a statistically significant, but not clinically important, improvement in cognitive test scores during thiamine treatment in patients with dementia of the Alzheimer's type, a 12-month, double-blind, parallel-group study was conducted to examine whether long-term administration of thiamine at 3 g/d might slow the progression of dementia of the Alzheimer's type. Fifteen subjects were enrolled and 10 completed the 1-year study. Data are available for two additional subjects through the first 9 months of study. No significant differences were found between the placebo and thiamine groups at any point during the study. In both groups, overall means for the Mini-Mental State Examination, verbal learning, and naming scores decreased significantly over the 12-month study period. These results do not support the hypothesis that long-term administration of thiamine at 3 g/d might slow the progression of dementia of the Alzheimer's type.

Aged↗
PubMed1991

Immunohistochemical colocalization of amyloid precursor protein with cerebrovascular amyloid of Alzheimer's disease.

Molecular cloning and cDNA sequencing have indicated that the fibril-forming, amyloidogenic beta/A4 peptide of cerebrovasculature and plaque core in AD is encoded as part of a larger precursor, amyloid precursor protein (APP). A panel of antibodies directed against synthetic peptides, which correspond to distinct domains of this putative APP molecule (i.e., amino acid residues 45-62, 587-596, 597-606, 597-638 [beta/A4 peptide], 638-658 and 653-661), were used to probe immunohistochemically serial sections of formalin-fixed, paraffin-embedded Alzheimer's disease (AD) brains for the presence of APP and/or its derivatives. Histochemical staining of adjacent sections with Bielschowsky's silver impregnation and with Congo red or thioflavin S-staining techniques was also done to identify the structures with amyloid deposition. All these antibodies exhibited intense immunoreactivity with amyloidotic cerebral vessels, including meningeal and parenchymal. This observation indicates that the amyloidotic vasculature of AD brain contains, in addition to the fibril-forming beta/A4 protein, nonamyloidogenic APP and/or its derivatives. More importantly, this APP immunoreactivity colocalized with angiopathic amyloid, which is characterized by phenol-resistant, birefringent congophilia. Parallel analyses with a dual SABC/silver impregnation procedure further confirmed that APP and/or its derivatives, including the amyloidogenic beta/A4, colocalized with argentophilic amyloid in the cerebrovasculature of AD.

Alzheimer Disease↗
PubMed1990

Induction of epitopes associated with neurofibrillary tangles in clonal mouse neuroblastoma (S20Y) cells.

Accumulation of paired helical filaments (PHF) in neurofibrillary tangles is a key neuropathological hallmark in Alzheimer's disease (AD). To date, PHF have been found primarily in humans. Cultured murine cholinergic neuroblastoma (S20Y) cells, following exposure to a serum-free medium or a differentiation medium, developed immunoreactivity to anti-PHF antibodies, and to the Alz-50 by immunocytochemical and immunoblot analyses. Electron microscopic examination revealed abundant fascicles of 10-nm filaments coursing tortuously amongst organelles, such as mitochondria, endoplasmic reticulum and dense-core vesicles, in perikarya and in neuritic extensions. However, subcellular structures identical or similar to PHF could not be found in these non-human cells. This convenient cell culture model may prove to be useful for studying certain aspects of the mechanisms underlying the abnormal cytoskeletal alterations which are characteristic of AD and related neurodegenerative disorders.

Alzheimer Disease↗
PubMed1990

Expression in cultured human neuroblastoma cells of epitopes associated with affected neurons in Alzheimer's disease.

Of three human neuroblastoma lines tested, IMR32K (and IMR32 parental line) was the only cell line that, after its exposure to a differentiation medium, consistently developed materials recognized immunocytochemically by a panel of antibodies against paired helical filaments (PHF). Ultrastructurally, these cells accumulated, at their perikarya and neuritic extensions, spatially discrete arrays of fibrils, which occasionally occurred in twisted pairs. When these fibrillar structures appeared as paired helices, they exhibited dimensions and configurations reminiscent of PHF found in affected Alzheimer neurons, although less compact. Immunoelectron microscope examinations of the fibrillar structures in these neuroblastoma cells with one of these anti-PHF immunoprobes revealed that only subsets of fibrillar structures that appeared thickened or aggregated to form bundles were selectively immunolabeled. Cultures of these immortal neuroblastoma lines may provide a convenient model for studying aspects of PHF formation that are hard to examine in Alzheimer brain obtained at autopsy.

Alzheimer Disease↗
PubMed1990

The gene for the alpha polypeptide of pyruvate dehydrogenase is X-linked in humans.

The chromosomal location of the gene for the alpha polypeptide of the pyruvate dehydrogenase (alpha E1), a major component of the pyruvate dehydrogenase complex, was determined by using a cloned cDNA for alpha E1. This 1-kb cDNA was isolated from a human liver lambda gt11 expression library with specific antibodies and included the coding (from amino acid 144 to the carboxy terminus) and the 3' untranslated regions. Southern blot analysis of the DNA from a panel of rodent-human hybrid cells showed that the absence or the presence of the major EcoRI fragment that hybridized with this cDNA probe was concordant with the presence of the Xq24-p22 region of the human X chromosome. The result of in situ hybridization with human metaphase chromosomes further mapped the alpha E1 gene to the Xp arm.

Antibodies↗
PubMed1989

Deficiency of pyruvate dehydrogenase complex (PDHC) in Leigh's disease fibroblasts: an abnormality in lipoamide dehydrogenase affecting PDHC activation.

Several groups have reported abnormalities of the pyruvate dehydrogenase complex (PDHC) in cultured cells or other tissues from patients with Leigh's disease (subacute necrotizing encephalomyelopathy). We therefore undertook studies to elucidate the molecular basis of the defect of PDHC in cultured skin fibroblasts from two patients with Leigh's disease. The deficit of total PDHC activity in homogenates of Leigh's disease fibroblasts could be restored by adding exogenous lipoamide dehydrogenase (LAD, E3), the third component of PDHC. The LAD in these Leigh's disease cells had a markedly reduced ability (less than 20% of normal LAD) to reconstitute with other PDHC components to form active enzyme complex. A polyclonal antibody to pig heart LAD inhibited LAD activity in control cells more efficiently than in Leigh's disease cells. Other mitochondrial enzyme activities and growth of these two Leigh's disease cells appeared normal. These results suggest that the deficiency of PDHC in these two patients with Leigh's disease was due to a structural abnormality of the LAD component of PDHC.

Brain Diseases, Metabolic↗
PubMed1988

Studies on suppressor factors produced by T-cell hybridomas. I. Characterization of antigen-specific suppressor factors.

Antigen-specific and antigen-nonspecific suppressor T cells were generated when spleen cells prepared from C57BL/6J (H-2b) were incubated with trinitrophenylated polyacrylamide beads (TNP-PAA) in vitro. T hybridomas were prepared by fusion of spleen cells cultured with TNP-PAA for 4 days and the thymoma cell line BW5147. More than 100 hybridomas were generated, and 15 of them suppressed the anti-TNP PFC response of fresh spleen cells cultured with TNP-PAA. The suppression was antigen specific with three of these five hybridoma supernatants tested. Hybridomas that caused antigen-specific suppression secrete factors which bring about suppression of the anti-TNP PFC response by spleen cells cultured with TNP-PAA. These hybridoma supernatants which cause antigen-specific suppression typically depressed the anti-TNP PFC response by 60% while depressing anti-SRBC PFC response by only 10%. The antigen-specific suppressor factors were bound to a TNP-BGG column but not to a BGG column. The suppressor factors, purified by affinity chromatography on a TNP-BGG column, were bound to anti-I-Jb antibody.

Acrylic Resins↗
PubMed1988

Reduced activities of thiamine-dependent enzymes in the brains and peripheral tissues of patients with Alzheimer's disease.

A report of cell loss in the nucleus basalis of Meynert in patients with Wernicke-Korsakoff disease prompted the examination of thiamine pyrophosphate (TPP)-dependent enzymes in the brain and peripheral tissues of patients with Alzheimer's disease. In these brains, the activities of the 2-ketoglutarate dehydrogenase complex were reduced more than 75% and those of transketolase more than 45%. Decreases occurred in histologically damaged and in relatively undamaged areas. Small but statistically significant abnormalities of transketolase, but not of 2-ketoglutarate dehydrogenase complex, were identified in red blood cells and cultured fibroblasts. Previous studies have shown deficiencies in the brain and variable effects in peripheral tissues on another TPP-dependent enzyme--the pyruvate dehydrogenase complex. Activities of TPP-dependent enzymes appear to be deficient in the brain and perhaps in some peripheral tissues in patients with Alzheimer's disease.

Aged↗
PubMed1988

Studies of transketolase abnormality in Alzheimer's disease.

The partially purified transketolase from each of eight well-nourished patients with Alzheimer's disease contained significantly less heat-stable component with a significantly longer half-life of heat inactivation than that from eight controls. Immunochemical studies utilizing antibodies to the purified human liver transketolase did not distinguish between red blood cell transketolases of patients with Alzheimer's disease and those of controls. However, three brains from patients with Alzheimer's disease that were deficient in transketolase activity lacked a 69-kilodalton form on immunoblots. Subtle structural abnormalities of transketolase appear to occur in a high proportion of patients with Alzheimer's disease.

Alzheimer Disease↗
PubMed1988

Pyruvate dehydrogenase complex is inhibited in calcium-loaded cerebrocortical mitochondria.

An impairment of mitochondrial functions as a result of Ca-loading may be one of the significant events that lead to neuronal death after an ischemic insult. To assess the metabolic consequences of excess Ca on brain mitochondria, pyruvate oxidation was studied in isolated cerebrocortical mitochondria loaded with Ca in vitro. The flux of pyruvate dehydrogenase complex (PDHC) [( 1-14C]pyruvate decarboxylation) was inhibited as the mitochondria accumulated excess Ca under the conditions tested: the inhibition in state 3 (i.e., in the presence of added ADP) was greater than in state 4 (i.e., in the absence of added adenine nucleotides). In state 4, the inhibition of the PDHC flux was accompanied by a similar reduction of the in situ activity of PDHC, indicating a change in PDHC phosphorylation. In state 3, the inhibition of the PDHC flux was greater than the corresponding decrease of the in situ PDHC activity. Thus, mechanisms other than the phosphorylation of PDHC might also contribute to the inhibition of pyruvate oxidation. Measurement of PDHC enzymatic activity in vitro indicated that PDHC, similar to alpha-ketoglutarate dehydrogenase complex, was inhibited by millimolar levels of Ca. This observation suggests that PDHC may also be inhibited non-covalently in Ca-loaded mitochondria in a manner similar to that of alpha-ketoglutarate dehydrogenase complex.

Animals↗
PubMed1988

Mitochondrial enzymes in hereditary ataxias.

As a test of the hypothesis that mitochondrial abnormalities are common in patients with hereditary ataxias, the activities of two mitochondrial enzymes were studied in platelets from an unselected series of patients. For the group of ataxics, the activity of the pyruvate dehydrogenase complex (PDHC) was 68% of the control (P less than 0.01) and that of glutamate dehydrogenase (GDH) was 81% of the control (P less than 0.05). Of the ataxics studied, 30% had activities of either or both mitochondrial enzymes more than 2 SD below the control mean. Immunoblots of PDHC revealed antibody cross-reacting material in platelets and fibroblasts very similar to those in human brain and appeared normal in platelets from patients with ataxias. Immunoblots of GDH showed a single antibody cross-reacting material in brain but at least two species in normal fibroblasts and platelets. The pathophysiology of hereditary ataxias may often involve mitochondrial damage associated with secondary decreases in the activities of mitochondrial enzymes.

Antibodies↗
PubMed1987

Glial glutamate dehydrogenase: ultrastructural localization and regional distribution in relation to the mitochondrial enzyme, cytochrome oxidase.

Glutamate dehydrogenase (GDH) is primarily a mitochondrial enzyme involved in the metabolism of glutamate. We have recently shown by light microscopic immunocytochemistry that, within detergent-permeabilized brain tissue, GDH is enriched in glial cells, particularly in regions utilizing L-glutamate as a neurotransmitter. In this study, we used immunogold labeling to quantitatively establish that the form of the enzyme recognized by the presently used GDH antiserum is associated primarily with a subpopulation of mitochondria in ultrathin, plastic-embedded sections of the rat cortex and striatum. Permeabilization with detergents was omitted in these studies, so as to preserve the ultrastructure. As expected, labeled mitochondria occurred both in neurons and glia. Furthermore, light microscopic comparisons of the regional distributions of peroxidase immunoreactivity for GDH and a histochemical reaction product for a second mitochondrial enzyme, cytochrome oxidase (CO), were used to demonstrate that high levels of GDH in glia of glutamate-receptive areas do not necessarily reflect the areas' demand for elevated oxidative metabolism. While all regions showing intense labeling for glial GDH also exhibited high levels of CO activity, many additional regions showing high levels of CO activity contained no detectable immunoreactivity for glial GDH. These light-microscopic comparisons reveal that the energy requirements are not the only factors accounting for the regional heterogeneity of the enzyme. We conclude that glial mitochondria are heterogeneous with respect to their GDH content and that GDH is enriched in areas exhibiting chronically active glutamatergic transmission.

Animals↗
PubMed1987

The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebrocortical mitochondria.

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxybutyrate on the flux of PDHC (as determined by [1-14C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-14C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-14C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861-1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.

3-Hydroxybutyric Acid↗
PubMed1987

Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate transmission.

The principally mitochondrial enzyme glutamate dehydrogenase (GDH) exhibited low-intensity, uniform immunoreactivity in neurons and intense heterogeneous labeling of glial cells of rat brain. Simultaneous peroxidase labeling for GDH and immunoautoradiography for glial fibrillary acidic protein (GFAP) confirmed the astrocytic localization of the enzyme. Immunoreactivity in astrocytes, but not in neurons, required the presence of Triton X-100 as a solubilizing agent. Most of the intensely labeled glial processes were localized to regions previously reported as containing moderate to high densities of binding sites for the excitatory amino acids, L-glutamate or L-aspartate, and glutamatergic fibers. These included several forebrain regions, such as the superficial layers of the rostral neocortex, dorsal neostriatum, nucleus accumbens, septohippocampal nucleus, intralaminar thalamic nuclei, and external capsules. However, the central gray of the midbrain, the nuclei of the reticular formation, brain stem regions projecting to the cerebellum, and cranial nuclei of the trigeminal and vagal nerves also exhibited intense glial labeling for GDH, even though some of these regions are known to receive only weak glutamatergic projections. A second factor determining the distribution of GDH appeared to be neuronal activity, as assessed by correspondence with reported high densities of cytochrome oxidase. We conclude that GDH enriched in glial populations exists in a subcellular compartment distinct from that of neurons and may serve as one of the enzymes involved in glutamatergic transmission. Deficiencies of glial GDH and the consequent cytotoxic effects of high levels of excitatory amino acids may contribute to a number of neurodegenerative disorders.

Animals↗
PubMed1987

Light microscopic immunocytochemical localization of pyruvate dehydrogenase complex in rat brain: topographical distribution and relation to cholinergic and catecholaminergic nuclei.

Pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) includes 3 catalytically active mitochondrial enzymes involved in the formation of cellular energy through the tricarboxylic acid cycle and in the synthesis of ACh. We sought to determine whether immunocytochemically detected PDHC was enriched in neurons of the rat CNS, and, if so, whether the perikarya containing higher levels of PDHC immunoreactivity were differentially distributed with respect to their size or location within nuclear groups containing ACh, catecholamines or other unidentified transmitters. Under the labeling conditions used in this study, the peroxidase-antiperoxidase immunoreaction product for PDHC was detectable principally in neuronal perikarya. The intensity of immunoreactivity within perikarya was variable as judged visually and by cellular, computer-assisted densitometry. In the forebrain, the most intensely labeled perikarya were seen in the medial septal nuclei, the nuclei of the diagonal band, the nuclei basalis, the dorsal and ventral striatum, and the entorhinal cortex. More caudally, intense immunoreactivity was detected in perikarya in the supraoptic hypothalamic nuclei, reticular thalamic nuclei, lateral substantia nigra, most of the tegmental nuclei, lateral nuclei of the trapezoid body, raphe pontis and obscuris, and the caudal part of the lateral reticular nuclei. In addition, many of the motor nuclei of the cranial nerves, including the dorsal motor nuclei of the vagus and the hypoglossal nuclei, and the nucleus ambiguus contained perikarya with intense PDHC labeling. Densitometry revealed no differences in intensity of immunoreactivity in soma of varying sizes. However, the intensity of neuronal labeling for PDHC was significantly greater in several nuclear groups that were shown in adjacent sections to contain cholinergic, but not catecholaminergic, enzymes. In contrast, the primary olfactory cortex, pyramidal cell layer of the regio inferior of hippocampus, and the Purkinje cell layer of the cerebellum were regions having perikarya with intense PDHC immunoreactivity but lacking both the synthetic and the degradative enzymes for ACh. These results provide the first morphological evidence that PDHC, a general metabolic enzyme complex, is enriched in selective perikarya that are heterogeneously distributed in brain and are especially abundant in many of the regions containing cholinergic neurons. The heterogeneity of PDHC immunoreactivity suggests that certain cholinergic as well as noncholinergic nuclei may be selectively vulnerable to mitochondrial diseases involving pyruvate utilization.

Animals↗