Tadao Takeuchi

HISTOCHEMICAL DETECTION OF PHOSPHORYLASE IN ANIMAL TISSUES

Amylophosphorylase in animal tissues has been demonstrated in frozen sections by use of activators and primer in a modified Yin and Sun method. A suitable technique for this purpose was newly devised. It was experimentally proved that the synthesis of glycogen from glucose-1-phosphate in tissues was specifically due to phosphorylase activity itself. Amylophosphorylase was histochemically demonstrated in muscle fibers of skeletal and heart muscles, smooth muscles in certain organs, epithelium of esophagus etc. The reactions were shown in cytoplasms and the nuclei were not stained. The phosphorylase reaction of muscle was very closely related to striated structure in muscle fibers.

A Pathological Study of Minamata Disease in Japan

SummaryThe pathological features and experimental results of Minamata disease are summarized. This disease is an unusual neurological disorder resulting from eating a large amount of fish and shellfish of Minamata bay in Japan. Minamata disease is a toxic encephalopathy characterized by cerebellar atrophy of granule-cell type, preferential injury of both calcarine regions and degenerative disturbances, to a lesser degree, of other cortical areas. Other parts of the nervous system may occasionally be disturbed. There are little or no remarkable changes in organs other than the nervous system, except for an occasional fatty degeneration of the liver and kidney, erosion in the digestive tract, and hypoplasia of the bone marrow.This disease affects not only human beings, but also various animals eating seafood from the bay, and it is pathologically demonstrated that even fish in the bay can be affected with the disease. Chemically, an appreciable quantity of mercury is demonstrated in organs of the human and animal autopsy cases. The ratio between the mercury content of the brain and that of the liver and kidney reveals a characteristic pattern of organomercury poisoning. Experimentally, Minamata disease is produced by feeding animals with fish and shellfish from the bay. The identical symptoms and pathological findings have been produced by administering to animals some organic mercury compounds, particularly alkyl-mercuric compounds such as alkylmercuric sulfide.At the present stage it is assumed that Minamata disease is caused by eating fish and shellfish containing an alkylmercuric compound with the R-Hg-S-group, which is produced under specific circumstances by yet unknown processes.ZusammenfassungDie pathologischen Merkmale der Minamata-Krankheit, einer durch den reichlichen Genuß von Fischen und Muscheln aus der Minamatabucht in Japan verursachten, ungewöhnlichen neurologischen Störung, und darauf bezügliche experimentelle Ergebnisse werden zusammengefaßt. Die Minamata-Krankheit ist eine toxische Encephalopathie, die durch Kleinhirnrindenatrophie vom Körnerzelltyp, den bevorzugten Befall der Calcarina und zu einem geringeren Grad durch degenerative Schädigungen anderer Rindengebiete gekennzeichnet ist. Manchmal sind Läsionen auch an anderen Stellen des Nervensystems zu finden. In anderen Organen treten keine oder nur geringfügige Veränderungen auf, außer gelegentlich Zellverfettung in Leber und Niere, Erosionen der Darmschleimhaut und Hypoplasie des Knochenmarkes.Nicht nur Menschen, sondern auch verschiedene Tiere, die sich von den Meerestieren in der Bucht ernähren, werden von der Krankheit befallen; es wird mit pathologischen Befunden belegt, daß sogar die in der Bucht lebenden Fische erkranken. Chemisch ist bei der Autopsie an Menschen und Tieren eine beträchtliche Menge von Quecksilber in den Organen nachzuweisen. Das Verhältnis des Quecksilbergehaltes im Gehirn zu dem in der Leber und Niere zeigt die Wesenszüge einer organischen Quecksilbervergiftung. Auf experimentellem Wege wird die Minamata-Krankheit durch die Fütterung von Tieren mit Fischen und Muscheln aus der Bucht hervorgerufen. Gleichartige Symptome und pathologische Befunde wurden erzielt, indem Tieren organische Quecksilberverbindungen, besonders Alkylquecksilberverbindungen wie Alkylquecksilbersulfid, verabreicht wurden.Gegenwärtig wird angenommen, daß die Minamata-Krankheit durch den Genuß von Fischen und Muscheln verursacht wird, welche eine unter besonderen Umständen auf eine bis jetzt noch unbekannte Art und Weise entstandene Alkylquecksilberverbindung mit der R-Hg-S-Gruppe enthalten.

Fetal Minamata disease. A neuropathological study of two cases of intrauterine intoxication by a methyl mercury compound.

Clinical and pathological features of the disease process in 2 children who were affected by a congenital form of cerebral palsy are reported. Both children were born near Minamata bay in families in which cases of Minamata disease had occurred. Pathological examination of the central nervous system showed lesions very similar in type and distribution to those found in adult patients suffering from Minamata disease. The disease is caused by intoxication with alkyl-mereury compounds and is contracted by rating fish from Minamata bay, the waters of which contain mercury originating from industrial wastes. Since the 2 children never ate fish or shellfish, it is concluded that cerebral palsy was caused by mercury intoxication acquired prenatally from the mother, although the mothers did not show manifestations of Minamata disease.

HISTOCHEMICAL DEMONSTRATION OF BRANCHING ENZYME (AMYLO-1,4 →1,6-TRANSGLUCOSIDASE) IN ANIMAL TISSUES

The procedure for the histochemical demonstration of branching enzyme (amylo-1,4 → 1,6-transglucosidase) activity is given. Procedures to determine the reliability of the results of localization of enzymatic activity and the histochemical relation between branching enzyme and amylophosphorylase are discussed. The results in normal tissues of the rabbit are presented. The distribution of amylo-1,4 → 1,6-transglucosidase in animal tissue cells is quite similar to that of amylophosphorylase. The quantitative balance of each enzyme in tissue cells is changed respectively according to various conditions. Greatest concentration of enzymatic activity is seen in liver, skeletal muscle, heart muscle, smooth muscle of various organs, nervous tissues and epithelium of certain organs.

HISTOCHEMICAL DEMONSTRATION OF PHOSPHORYLASE

Phosphorylase is histochemically demonstrable in certain tissues of various organs. This enzyme is more labile than phosphatase, lipase etc. and it is less resistant to postmortem decomposition. Therefore, it is best demonstrated in fresh frozen sections. In Takeuchi and Kuriaki’s original method, the solubility of polysaccharide newly formed from glucose-iphosphate by the enzyme activity from the incubating mixture may cause difficulty in the procedure . Next we tried to utilize organic solvents (alcohol, methanol and acetone), for

Distribution of amylophosphorylase in various tissues of human and mammalian organs.

Phosphorylase forms from glucose-1-phosphate in tissue sections a polysaccharide which agrees best with amylose, amylopectin and a lightly branched glycogen, judging from the reactions with iodine, the periodic acid Schiff method and Bests carmine stain. Phosphorylase is demonstrated most intensely in striated skeletal, glossal and cardiac muscle, in tracheal and skeletal cartilage, in the smooth muscle of large and medium arteries, uterus, tubes, vagina, bladder etc., in liver cells and renal collecting tubules, sweat glands and their ducts, hair sheath cells, stratified squamous epithelia of epidermis, tongue, esophagus, cervix uteri and vagina, in some adrenal cortical cells and in nervous tissue. The iodine reaction technique with activator and primer, is the most suitable for the histochemical demonstration of phosphorylase in animal tissues.

An Autopsy Case of Minamata Disease (Methylmercury Poisoning)—Pathological Viewpoints of Peripheral Nerves

The outbreak of methylmercury poisoning in the geographic areas around Minamata Bay, Kumamoto, Japan in the 1950s has become known as Minamata disease. Based on earlier reports and extensive pathological studies on autopsied cases at the Kumamoto University School of Medicine, destructive lesions in the anterior portion of the calcarine cortex and depletion predominantly of granular cells in the cerebellar cortex came to be recognized as the hallmark and diagnostic yardstick of methylmercury poisoning in humans. As the number of autopsy cases of Minamata disease increased, it became apparent that the cerebral lesion was not restricted to the calcarine cortex but was relatively widespread. Less severe lesions, believed to be responsible for the motor symptoms of Minamata patients, were often found in the precentral, postcentral, and lateral temporal cortices. These patients also frequently presented with signs of sensory neuropathy affecting the distal extremities. Because of few sufficiently comprehensive studies, peripheral nerve degeneration has not been universally accepted as a cause of the sensory disturbances in Minamata patients. The present paper describes both biopsy and autopsy findings of the peripheral nerves in a male fisherman who died at the age of 64 years and showed the characteristic central nervous system lesions of Minamata disease at autopsy. A sural nerve biopsy with electron microscopy performed 1 month prior to his death showed endoneurial fibrosis and regenerated myelin sheaths. At autopsy the dorsal roots and sural nerve showed endoneurial fibrosis, loss of nerve fibers, and presence of Büngners bands. The spinal cord showed Wallerian degeneration of thefasciculus gracilis (Golls tract) with relative preservation of neurons in sensory ganglia. These findings support the contention that there is peripheral nerve degeneration in Minamata patients due to toxic injury from methylmercury.

HISTOCHEMICAL AND ELECTRON MICROSCOPIC OBSERVATIONS OF GLYCOGEN SYNTHESIZED FROM GLUCOSE-1-PHOSPHATE BY PHOSPHORYLASE AND BRANCHING ENZYME IN HUMAN MUSCLE

Polysaccharides synthesized histochemicaily from glucose-i -phosphate by amylophosphorylase alone or by both phosphorylase and branching enzyme in human skeletal muscle fibers were observed with the electron microscope. Histochemically the newly formed polysaccharides were stained blue in smaller muscle fibers and violet or red purple in larger ones with the iodine reaction. Low-density, amorphous areas contaming denser cloudy material with a few small particles were noted in the cytoplasmic matrix to the exclusion of all other components or organelles. It was considered that phos )horylase and branching enzyme might be localized in these.

HISTOCHEMICAL DEMONSTRATION OF A PATHWAY FOR POLYSACCHARIDE SYNTHESIS FROM URIDINEDIPHOSPHOGLUCOSE

ehunonides svnul hcshzcd hsisi ochensictolly frolns glum(use1 -pbuophnul e (( -1-F) ui-si mug pninser, nodemuylic F’ton. 1. Beta glucumronidasc activity in rat liver by 8-hydroxy-quinsolin-glutcutronuide tcchnsique. Tuventyfounr houmr fixation in chloral hydrate formahinu. Note diffuse cytopluosmic activity amiol marked locahizaItous uns bile cuopillary is-all-s nond penicanunohicoilnor cytoplasm.

Pathologic observations of the Minamata disease.

Minamata is a small industrial town with a total population of about 50,000. It is near the south west coast of Kyushu, the most southerly of the main Japanese islands. There is an old large artificial-fertilizer factory, the effluent from which empties into a small Minamata Bay. In this factory, a small amount of vinyl chloride compounds were experimentally manufactured during 1949-1951, and their production on a large scale has been opened since 1952 to 1953, separately from the fertilizer production. From the end of 1953 a mysterious nervous illness began sporadically to affect the villagers, especially the fishermen, and was called the “Minamata Disease ”. The outbreak was investigated by several departments of our University. The pathologic investigations and experiments were made by us. It was explained that the illness was caused from the eating of a large amount of fish and shellfish contaminated by the effluent of the new industrial department. The toxic agent seems to have some relation to the manufacturing of the vinyl chloride compounds.