Diagnostic histochemistry of amylopectinosis (glycogenosis type IV).
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Glycogen Storage Disease↗
Biomedical subjects
F Wohlrab
Publications and source records attributed to F Wohlrab.
Quantitative properties of Müller cells in rabbit retina as revealed by histochemical demonstration of NADH-diaphorase activity.
Müller cells in rabbit retina were selectively stained by demonstrating NADH-diaphorase activity. After the sections were measured by light microscopy, the following properties of Müller cells were found: (a) independent of the retinal localization, one Müller cell corresponds with a retinal volume of 15,000 microns 3, i.e., with 11 photoreceptor cells, 2 neurons of the inner nuclear layer, and 0.3-1 ganglion cells; (b) the Müller cell population represents about 6.8% of the total retinal volume; (c) the sorbitol accumulation of Müller cells in diabetic retinopathy is said to be unable to cause osmotic damage.
Animals↗
Induction of nuclear deoxyribopyrimidine triphosphatase and sensitivity of clinical isolates of herpes simplex virus to (E)-5-(2-bromovinyl)-2'-deoxyuridine.
In a blind study, 21 clinical isolates of herpes simplex virus which had been typed using differential growth on guinea pig embryo versus chicken embryo cells were tested for the presence of the viral deoxyribopyrimidine triphosphatase. In all isolates of type 1, the triphosphatase was present in the nuclei of the infected cells, while none of the HSV-2 isolates induced a nuclear enzyme. In all isolates there was a complete correlation between the presence of nuclear deoxyribopyrimidine triphosphatase and sensitivity to 0.7 microgram/ml of E-5-(2-bromovinyl)-2'-deoxyuridine. The study suggests that the type-specific distribution of the triphosphatase is of general validity, including clinical isolates of herpes simplex virus, and could be used as a type-specific enzyme marker.
Animals↗
Horizontal cells of the rabbit retina: some quantitative properties revealed by selective staining.
Horizontal cells of the rabbit retina were selectively stained by demonstration of beta-hydroxybutyrate dehydrogenase activity. Thereafter, the size of the cell bodies, the distance between neighbouring cells, and the number of cells per mm2 were measured. In the area centralis, the horizontal cell bodies occupy only 4.2% of the total retinal area; in the far periphery, however, 12.8% of the retinal area consist of horizontal cell bodies. Furthermore, the horizontal cells of the retinal periphery have much larger cell bodies as compared with those of the retinal center. The far periphery, for these reasons, is concluded to be the optimal region for intracellular microelectrode recordings from rabbit retinal horizontal cells.
Animals↗
[Specificity of the histochemical determination of sorbitol dehydrogenase--comparative indicator and immunohistochemical studies on the rat kidney].
We have investigated the distribution of sorbitol dehydrogenase by comparative indicator- and immunohistochemical technique in rat kidney. In this connection we have tested some parameters which influence the histochemical demonstration of SoDH-activity by tetrazolium salt indicator technique (e.g. prefixation of tissue sections, elution behaviour of SoDH, influence of different dialysis membranes, composition of incubating medium). According these studies, the enzyme was demonstrated in native cryostate sections by membrane incubating method. For the immunohistochemical demonstration of SoDH specific antibodies were prepared against this enzyme in rabbits, and were used for indirect immunofluorescence method and for the unlabelled antibody enzyme (PAP) technique. A good agreement of sorbitol dehydrogenase localization was obtained with all the tested methods, but also some discrepancies was observed in enzyme topic (e.g. glomeruli). The results are discussed under methodical aspects in connection with the specifity of the used methods to localize the enzyme.
Animals↗
[Prostaglandin catabolism in the mammalian kidney--histochemical and electrophoretic studies].
Using the prostaglandins PGE2, PGF2 alpha and PGB1 as substrates for demonstration of NAD-15-PGDH activity and of 13,14,15-Keto-PGF2 alpha for demonstration of NAD-9-PGDH activity these enzymes were localized in native sections of rat kidney by membrane incubating technique. Both enzymes are active in the same tubular structures of kidney cortex. Simultaneously homogenate supernatant of kidney cortex was separated by micro-electrophoretic technique. NAD-15-PGDH exist in rat kidney in multiple forms, which are varied in number (1 to 3) during postnatal development. One or 2 fractions of NAD-9-PGDH are detected in rat kidney cortex. In the kidney cortex of rabbit and guinea pig also multiple forms of NAD-15-PGDH exist, but not in the kidney cortex of pig. No multiple forms of NAD-9-PGDH are detectable in the kidney of pig, guinea pig, and rabbit. The histochemical and electrophoretical results suggest an important relationship between prostaglandin catabolizing activity and nephrogenesis in the early development of rat kidney. The functional significance of the detected multiple forms of NAD-15-PGDH and the variation in the number of fractions during ontogenesis in rat kidney are yet not clear.
Animals↗
Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.
Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.
Animals↗
[Histochemical determination of beta-hydroxybutyrate dehydrogenase in the brush border of the cortex epithelium of the rat kidney].
NAD-beta-hydroxybutyrate dehydrogenase activity was investigated in the kidneys of male and female adult Wistar rats using membrane incubating technique. In this connection the enzyme activity presents an other topochemical pattern as in comparison with conventional incubating technique. In the kidney cortex an additional reaction was demonstrated in the convoluted portion of proximal tubules. In connection with the results of biochemical investigations the soluble enzyme is characterized as a nonmitochondrial beta-hydroxybutyrate dehydrogenase of proximal kidney tubules (brush border membrane enzyme?).
Animals↗
[Enzyme histochemistry and diagnostic histopathology].
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Enzymes↗
Deoxyribopyrimidine triphosphatase activity specific for cells infected with herpes simplex virus type 1.
Nuclei from baby hamster kidney cells infected with herpes simplex virus type 1 contain a virus-specific deoxyribonucleoside triphosphate degrading activity. The reaction proceeds at 4 degrees C and can thus be distinguished from host enzymes. Under these conditions the enzyme is specific for deoxyribopyrimide triphosphates and catalyzes pyrophosphate cleavage to produce the monophosphates, dUTP being the best substrate followed by dCTP and dTTP. The appearance of the activity after infection parallels that of viral DNA-synthesis-related functions. Of a series of eight temperature-sensitive mutants tested, two (tsD and tsK) exhibit significantly decreased triphosphatase levels after infection at nonpermissive temperature, whereas a viral deoxypyrimidine kinase-deficient mutant induced wild-type levels.
Animals↗
[On the histotopochemistry of prostaglandin F2 alpha metabolizing enzymes in rat kidney (author's transl)].
Using PGF2 alpha as substrate we have investigated the demonstration and localization of NAD- and NADP-15-hydroxyprostaglandin dehydrogenase in the kidney of developing and adult rat kidney. Under histochemical conditions an adequate demonstration of the soluble PGDH in native sections is possibly by membrane incubating technic in presence of specific coenzymes. The effectors PMS and KCN showed a decreasing effect on the enzyme activity. In the developing and adult rat kidney the activity of NAD-PGDH was localized predominantly to medullary rays and inner cortex. We have found the reaction product in the following kidney substructures: Pars recta tubuli, distal convoluted tubule, ascending limb of Henle, collecting tubule and lower in proximal convoluted tubule and in the glomerular cells. The NADP-PGDH was localized only in the cortex structures, but the reaction was uneven and the localization of the reaction product was diffuse. The results are discussed in connection with the specificity of the reaction.
Aging↗
Thymidylate synthetase from Escherichia coli K12. Purification, and dependence of kinetic properties on sugar conformation and size of the 2' substituent.
Thymidylate synthetase from Escherichia coli K12 has been purified 3600-fold by a series of chromatographic procedures. The final preparation had a specific activity of 1.47 units/mg protein and was approximately 80% pure. The enzyme is a dimer of relative molecular mass, Mr, 64000 composed of two subunits of Mr 32,000 each. Its isoelectric point is 4.7 and it is stimulated by Mg2+. Michaelis constants for (+)5,10-methylene-5,6,7,8-tetrahydrofolate [(+)CH2H4folate] were 0.014 mM in the case of methylation of 2'-deoxyuridine-5'-phosphate (dUMP) and 0.55 mM when it served as methyl-group donor for 2'-fluoro-2'-deoxyuridine-5'-phosphate (dUflMP); the corresponding Km values for dUMP and dUflMP were 0.01 mM and 0.11 mM, respectively. The activation energies for the two reactions were found to be 72.8 kJ/mol (methylation of dUMP) and 66.1 kJ/mol (methylation of dUflMP). The data support a recognition mechanism between thymidylate synthetase and that fraction of the nucleotide the sugar moiety of which is in the 2'-endo-3'-exo conformation.
Carbohydrate Conformation↗
[On the localization of sorbitol dehydrogenase in the rat kidney (author's transl)].
We have investigated the localization and demonstration of sorbitol dehydrogenase activity in the rat kidney by comparative histochemical-electrophoretical technique. Under histochemical conditions an adequate demonstration of the activity of the sorbitol dehydrogenase in native sections is possible by membrane incubating technique in presence of PMS and KCN. In the glomerulum the strong and uniform reaction with regard to the specifity is not clear. In the glomerula of semithin sections from glutaraldehyde fixed, incubated and Epon-embedded cryostate sections the reaction product is localized predominantly in the maesangium cells. By micropolyacrylamidgel gradient electrophoresis of the supernatant from the kidney cortex and of isolated glomerula 2 sorbitol dehydrogenase fractions were found. The results are discussed in connection with the specifity of the reaction.
Animals↗
2'-Deoxy-2'-fluorouridine-5'-phosphate: an alternative substrate for thymidylate synthetase from Escherichia coli K12.
The substrate specificity of 2'-deoxy-2'-substituted uridines and their 5'-phosphates towards thymidylate synthetase from Escherichia coli K12 was investigated. Besides the natural substrate 2'-deoxyuridine-5'-phosphate (dUMP), only 2'-deoxy-2'-fluorouridine-5'-phosphate (dUflMP) was a substrate. The KM of dUflMP is 11 times higher than that of dUMP, while the Vmax values are virtually the same. It is concluded that the size of the 2'-substituent and not its polarity (and the concomitant conformational change) determines substrate specificity of thymidylate synthetase.
Deoxyuracil Nucleotides↗
On the histochemistry of prostaglandin-dehydrogenase.
Using PGF2alpha as substrate the PGDH-activity was localised in the male and in the female rat kidney by histochemical technique. The reaction is independent of the presence of NAD, but with NAD the reaction intensity is enhanced. The effectors phenazine methosulphate, Triton X 100 and Tween 80 showed no enhancing effect on the enzyme activity. In the rat kidney we have found the reaction product predominantly in the inner cortex (straight portion of the proximal tubules), however, sex dependent differences of PGDH-activity and localization are visible.
15-Oxoprostaglandin 13-Reductase↗
A model study for histochemical tetrazolium reduction by flavine enzymes.
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Dihydrolipoamide Dehydrogenase↗
[On the histochemistry of xanthine dehydrogenase (author's transl)].
We have investigated the enzymes of the XDH-XOD-system by histochemical technique based on a model study for histochemical tetrazolium reduction by flavine enzymes. We have not found a reaction in native sections from liver, kidney and duodenum of adult male Wistar rats. The effectors PMS, Triton X-100 and Tween 80 were inefficient. A strong reaction was observed after a short fixation (10 min) of the sections with glutaraldehyde predominantly in the duodenal epithel cells. The intensity of the reaction was much stronger in sections of liver, kidney and duodenum by influence of the effector PMS and the detergents Triton X-100 and Tween 80. The application of the membrane technique does not show advantages in the histochemical demonstration of the XDH-XOD-system. The specificity of the reaction is discussed under the aspect of the complexity of the XDH-XOD-system shown by biochemical investigations.
Animals↗
The problem of diabetic angiolopathy in sand rats (Psammomys obesus).
No diabetic angiolopathy was found in the retinas, kidneys and skeletal muscles of protodiabetic and overtly diabetic sand rats. The terminal blood vessels were investigated using histological, enzyme histochemical, immunofluorescence microscopic, autoradiographic and electron microscopic methods. There seems to be little or no connection in sand rats between the diabetic metabolism syndrome and the metabolic process leading to angiolopathy.
Animals↗