The effect of sodium ethylenediaminetetra-acetate (na2 edta) on the histochemical demonstration of some enzyme activities in neonatal mouse molar tissues

Summary Décalcification of neonatal mouse tissues, including the first molar germs, by 10 per cent ethylenediaminetetra-acetate (Na₂EDTA) containing 7.5 per cent polyvinylpyrrolidone (PVP) was found to increase the histochemical reactions of lactate and malate dehydrogenase activities, as compared with non-decalcified preparations. The activity of glucose-6-phosphate dehydrogenase was not significantly affected by the demineralization procedure. Adenosinetriphosphatases (ATPases) in the endothelial cells and in the nuclei of most of the cells in the cryostat sections (8 ) showed enhanced activities after the EDTA-treatment (48 hours at 4° C). ATPase in matrix-forming odontoblasts and ameloblasts, however, was consistently inhibited.The biochemical test indicated that the EDTA- and PVP-solutions, according to Fullmer (1966), allowed only a trace amount of protein leakage into the decalcification fluid.Preservation and retention of protein molecules and a simultaneous removal of contaminating ions and de-masking of active sites of the enzymes might be responsible for some of the effects observed in enzyme histochemical registrations.     

A modification of tooth germ cultivation in vitro and in ovo

Mandibular molar anlages excised from 17-day mouse foetuses were cultured in vitro or in ovo (on the chorioallantoic membrane). In both cases, the explants were underlain either with a Millipore filter or with a piece of fibrin foam. Tooth germs were harvested after 7 days of cultivation and processed histologically. Spatial arrangement was highly preserved in the tooth germs cultured in vitro on fibrin foam. In vitro cultures on Millipore filters revealed significant flattening of tooth germs, caused especially by the collapse of enamel organ and the pulp. The cytodifferentiation of tooth germs cultured in vitro on both substrates (Millipore filter, fibrin foam) was characterized by the presence of odontoblasts, polarizing ameloblasts and predentine. The cytodifferentiation of tooth germs cultured in ovo on Millipore filters placed on chorioallantoic membrane was characterized by the presence of odontoblasts, ameloblasts, predentine, dentine and enamel. However, the flattening of these explants was identical with the changes of the explants cultured on Millipore filters in vitro. In ovo cultivation on the fibrin foam failed to bring satisfactory results.     

The secretory ameloblast of the mini-pig foetus

Electron microscopic investigations of secretory ameloblasts from deciduous tooth germs of mini-pig foetuses and investigations of the ability of various fixatives to preserve these cells in tooth germs immersion-fixed in toto 5 min, 10 min, 15 min, 20 min and 40 min after death of the mother gave the following results:

Maturation ameloblasts of the porcine tooth germ do not express amelogenin

 Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. Accepted: 14 January 1999     

The development of activity of lactate dehydrogenase (EC 1.1.1.27) in the areas of nigro-striatal pathway during the ontogenesis of pig

The development of activity of lactate dehydrogenase was investigated by histochemical methods under the light and electron microscopes and also by biochemical methods. The studies were carried out in the areas of the nigro-striatal pathway of 7 foetal groups, two days-old piglets, three weeks old piglets and adult pigs. It was demonstrated that the activity of LDH appears early in the prenatal ontogenesis. High activity of the enzyme appears in the studied areas of the brain already in foetuses about 60 days old (100 mm) afterwards it rises and the highest activity is observed in about 86 days old foetuses. It then decreases just before birth. After birth in two days and three weeks old piglets the LDH activity decreases further. In adults a high activity of the examined enzyme was confirmed.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

The Effect of Red and Far-red Light on Subsequent Enzyme Activity in Arena Coleoptiles

The effect of red light (660 nm), far-red light (730 nm) and dark treatment on the subsequent enzyme activity in homogenates of Avena coleoptiles was investigated. The activities of succinic dehydrogenase (SDH), lactic dehydro-genase (LDH) and glucose-6-P dehydrogenase (GDH) were investigated. The activity of SDH was greatest in material receiving continuous darkness. LDH and GDH activity was stimulated by both light treatments compared with the dark values. Little or no difference in enzyme activity was found using either a single 15 min flash of light or continuous light for 24 h. Admixtures of extracts from dark treated and light treated material in a 1:1 ratio gave unexpected levels of enzyme activity. In all cases such admixtures gave much less than the anticipated enzyme activity.     

TCA循环中间产物对酿酒酵母胞内代谢关键酶活性的影响

对酿酒酵母在添加苹果酸、柠檬酸和琥珀酸的混合培养基与其在YEPD培养基中胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活力差异进行了对比分析。结果表明:添加苹果酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.82%、57.23%、39.15%、12.10%;添加柠檬酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶的酶活分别下降50.17%、42.20%、48.40%;添加琥珀酸使胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.16%、34.16%、50.87%、50.87%、12.37%。丙酮酸激酶、异柠檬酸脱氢酶和苹果酸脱氢酶对3种有机酸的耐受性较差,葡萄糖-6-磷酸脱氢酶、乙醇脱氢酶对3种有机酸的耐受具有选择性。     

Effect of hypoxia on phosphoesterases,oxidative and glycolytic enzymes in the rabbit common carotid artery

Summary An enzyme-histochemical study was performed on the rabbit common carotid artery at periods ranging from one to seventeen days following double-ligation and injections of human , human pre- serum lipoproteins and a physiologic saline into the lumen. The alterations in enzyme activities compared to the contralateral carotid (control) were studied for DPN diaphorase, succinic acid dehydrogenase (SDH), lactic acid dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-pH DH), ATPase, AMPase, acid and alkaline phosphatase. At earlier time intervals there was a general reduction in oxidative enzyme and ATPase activities concomitant with a general increase in LDH and acid phosphatase activities. At later times oxidative enzyme and ATPase activities returned to their control levels and actually increased in the thickened intima. LDH and acid phosphatase activities remained above the control levels, especially in the thickened intima. Focal areas, presumably necrotic, demonstrated complete loss of activity for all enzymes studied. AMPase activity did not differ from the controls throughout this study, while G-6-pH DH and alkaline phosphatase activities were found only sparsely in the adventitia. The same general pattern of alteration in enzyme activity was found regardless of the substance injected into the ligated artery. Arguments for the use of this experimental model for studies on the pathogenesis of atherosclerosis are given.     

Demonstrability of some oxidative enzymes in early rodent embryos with and without fixation

Several oxidative enzymes [NADH-TR (reduced nicotinamide-adenine dinucleotide-tetrazolium reductase), NADPH-TR (reduced nicotinamide-adenine dinucleotide phosphate-tetrazolium reductase), SDH (succinic dehydrogenase) and LDH (lactate dehydrogenase)] were studied by histochemical means during early development of rat and mouse. All investigated enzymes could be easily demonstrated in zygote and also to some extent in somitic stages without any pretreatment. However, in cleavage and early postimplantation stages enzyme activity could be revealed only after the embryos were pretreated in some way. This pretreatment can be fixation with formalin or acetone, freezing and thawing, slight mechanical damage or very prolonged incubation time. The formazan granules as a sign of enzymatic activity were present in all stages of embryonic development and were more abundant in reactions for NADH-TR and LDH than in reactions for NADPH-TR and SDH. Our results suggest that the investigated enzymes are present in all embryonic cells during early development. It seems that the permeability of embryonic cells for histochemical media must be increased otherwise the histochemical reactions cannot be accomplished.     

Localization of lead and fluoride in cultured tooth germs by laser microprobe mass analysis

Trace elements can influence dental health, possibly by altering tooth resistance during preeruptive development. Therefore, it was investigated whether lead and fluoride would be incorporated into the calcifying matrices or the cellular parts of tooth germs in vitro. Using laser microprobe mass analysis, the localization of lead and fluoride was studied in the different layers or tooth germs that had been cultured in a medium to which PbCl₂ of NaF had been added in different concentrations. Both elements could only be detected in the dentine layer. Hence, the enamel organ in the secretory stage of tooth development excludes lead and fluoride from the enamel, even when enamel formation by the ameloblasts is visibly disturbed. Furthermore, there seemed to be a process of saturation in the accumulation of lead and fluoride in the dentine.     

Histochemical studies on the olfactory glomeruli of squirrel monkey

Summary Detailed histochemical studies on the distribution of various oxidative and dephosphorylating groups of enzymes have been made in the olfactory glomeruli of the squirrel monkey. The olfactory glomeruli showed strongly positive activity for succinic dehydrogenase, lactic dehydrogenase, monoamine oxidase, alkaline phosphatase, adenosinetriphosphatase and simple esterase. They showed moderately positive activity for cytochrome oxidase, specific cholinesterase, 5'nucleotidase; mildly positive activity for acid phosphatase; and negligible activity for nonspecific cholinesterase and glucose-6-phosphatase. The glomeruli did not show the presence of any thiamine pyrophosphatase-positive Golgi apparatus. The blood vessels surrounding the glomeruli were strongly positive for the nonspecific cholinesterase test. The significance of these results are discussed briefly.     

Histochemical correlates of changes in the primate brain associated with varying environmental light conditions

Summary Squirrel monkeys were kept in 3 groups of 2 each maintained on one of the following light schedules: 12-12 hour light/dark, continuous light, and continuous dark. The animals (one from each group) were sacrificed after a period of 81/2 and 111/2 months and histochemical techniques for some oxidative and hydrolytic enzymes were employed in cryostat section of the brains of these animals. Lactic dehydrogenase (LDH), an enzyme involved in glycolytic metabolism, showed enhanced enzyme activity compared to the other two groups in a number of areas (e.g., corpus geniculatum laterale, nucleus n. oculomotorii and trochlearis, nucleus interpeduncularis, locus coeruleus, reticular formation, cerebellar cortex, etc.). LDH showed more pronounced enzyme changes in the neuropil, whereas the neurons showed somewhat more enhanced activity in the succinic dehydrogenase preparations. Butyrylcholinesterase and simple esterase, however, did not show any difference in enzyme reaction in the three groups under study. Similarly, certain areas (e.g., colliculus superior, griseum pontis, central gray, vestibular and cochlear nuclei, choroid plexus and ependyma) did not show any enzyme difference in all the eight enzyme preparations. The epiphysis showed a decrease in monoamine oxidase activity in the constant light-exposed animals compared to the other two groups. The significance of these findings is discussed.     

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

Histochemical demonstration of changes in liver cell enzymes in pregnant mice infected with coxsackie B3 virus

Summary Sequential changes in liver cell morphology in mice infected with Coxsackievirus B₃ have been studied using enzymic markers for early cell damage. Elevated alkaline phosphatase two days after infection was the only change identified consistent with the presence of virus in the tissue. Reduced glucose-6-phosphatase, and succinic dehydrogenase were associated with cytoplasmic vacuolation and fatty deposition which were clearly apparent 6 to 8 days after infection. Lysosomal acid phosphatase was increased at this stage in periportal areas and reduced in centrilobular regions. comparison of the Coxsackievirus induced changes with those induced by a direct liver pathogen—hepatitis virus, suggests that the histological and histochemical changes are probably of an indirect causation.     

Ultrastructure of secretory ameloblasts in the house musk shrew, Suncus murinus, Insectivora

Tooth germs from neonatal house musk shrews, Suncus murinus, were used for the study. The tooth morphogenesis was compared electron microscopically to that of Primates. In the tooth germ at the bell stage, the ameloblast was 3 x 50 microns in size, columnar in shape and had several tubular-type Golgi apparatus which were at the distal end of the cell. Most mitochondria were noted at the proximal end of ameloblasts. Tomes' processes were 1 micron in width, protruded 10 microns from the ameloblast and had many dense bodies and two kinds of vesicles. They were morphologically different from human ameloblasts and enamel rods.     

Histophysiological studies of prostaglandin F2a on isolated organs: I. Effect of prostaglandin F2a on the heart

The physiological and histochemical effects of PGF_2a on isolated rabbit hearts were examined. The results showed a positive inotropic effect. The coronary flow increased. From the histochemical studies, adenosine triphosphatase (ATP-ase) and succinic dehydrogenase activities were increased while that of alkaline phosphatase was decreased. Glycogen granules were depleted. These findings were discussed on a histophysiological basis.     

Studies on the pathophysiology of acute renal failure. II. A histochemical study of the proximal tubule of the rat following administration of mercuric chloride.

Acute renal failure was induced in male rats by the subcutaneous injectioon of 4 mg HgC12 per kg body weight. Enzyme activities of the proximal tubule were studied histochemically at six time intervals from 15 min to 24 h. The enzyme studied were alkaline phosphatase, 5'-nucleotidase, acid phosphatase, alpha-glycerophosphate dehydrogenase (NAD-independent), malic dehydrogenase, succinic dehydrogenase, latic dehydrogenase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase. Decreases in activity were observed for alkaline phosphatase and 5'-nucleotidase after 15 min. Acid phosphatase was decreased after 30 min. These three enzymes returned to control levels after 3 h, but malic dehydrogenase and alpha-glycerophosphate dehydrogenase were decreased at this time interval. Succinic dehydrogenase was first decreased after 6 h. The earliest morphological changes detectable by light microscopy were observed in pars recta tubules in the medullary rays after 6 h, a time when all enzymes studied showed widespread decreased activity throughout the proximal tubule. After 24 h, the pars convoluta appeared morphologically normal but the pars recta was necrotic and exhibited calcification, whereas enzyme activity was decreased (absent in some cases) in both pars convoluta and pars recta. These results support the hypothesis that Hg++, when given in a sublethal dose, is associated with early histochemical changes in the brush border of the proximal tubule, which may be related to early changes in sodium reabsorption and to the subsequent development of acute renal failure. The observation that changes in plasma membrane-associated enzymes occur early and prior to alterations in enzymes of mitochondria and the endoplasmic reticulum suggests that Hg++ interacts initially with the plasma membrane.     

Enhanced steroidogenesis in hen ovary after malonate administration.

The activities of glucose-6-phosphate dehydrogenase, delta 5-3 beta-hydroxysteroid dehydrogenase and succinic dehydrogenase have been studied histochemically in the ovary of normal and malonate treated hens. Following malonate treatment, glucose-6-phosphate dehydrogenase and delta 5-3 beta-hydroxysteroid dehydrogenase showed increased activities while succinic dehydrogenase activity was diminished significantly. The significance of the above changes has been discussed. The ascorbic acid and cholesterol contents of the ovary were determined to get additional information about steroidogenesis in the gland under such treatment.