Metachromatic Material Research Articles

Sulfatides and demyelination. Subcellular localization in metachromatic leukodystrophy.

METACHROMATIC leukodystrophy (MLD) is a progressive, demyelinating disorder which is characterized chemically by the accumulation of a class of acidic glycolipids: the sulfatides. Pathologically, demyelination involves both the central nervous system (CNS) and peripheral nervous system, and granular, metachromatic deposits are spread throughout the neuraxis. Similar metachromatic granular material is found both intracellularly and as deposits in the tubules of the kidneys, adrenal glands, gallbladder, and liver.1 Austin et al2first suggested the presence of an enzyme defect in MLD, and Mehl and Jatzkewitz,3after demonstrating desulfation of sulfatides to cerebrosides in vitro, have shown a deficiency in a degradative enzyme system, cerebroside sulfatase, in kidney tissue from a patient with MLD.4,5Moser et al6studied the turnover of injected sodium sulfate S-35 in patients with MLD. In comparison with control groups, a greatly prolonged isotope turnover was found in MLD. They concluded that

The effect of ascorbic acid on monolayer cultures of three types of chondrocytes

A comparative study was made of chondrocytes from three types of embryonic chick cartilage when grown as monolayer cultures. Each type displayed a characteristic cellular pattern. Epiphyseal and articular chondrocytes (tibia) formed nodules of rounded cells in an abundant intercellular matrix. Hypertrophic chondrocytes (tibial diaphysis) produced a monolayer of polygonal cells which later became encapsulated with minimal intercellular matrix. Chonrocytes from Meckel's cartilage formed nodules of encapsulated cells, variously shaped, imbedded in a thick matrix. Ascorbic acid had a profound effect on cultures of all three types. Addition of 50 μg/ml to the medium resulted in an increase of metachromatic and fibrillar intercellular material and was essential for maintenance of typically chrondrified masses of cells.

Endocytosis of sugars in embryonic skeletal tissues in organ culture. IV. Lysosomal and other biochemical effects. General discussion.

ABSTRACT Cultivation of limb-bone rudiments in medium containing various non-metabolizable or poorly metabolizable sugars caused vacuolation of the perichondrial and articular cells and was accompanied by an increased synthesis and profuse secretion of lysosomal enzymes. Experiments with [14C]dextran indicated that the cytoplasmic vacuolation was due not to a higher rate of endocytosis, but rather to an abnormally long persistence of the pinocytotic vacuoles. Meta-chromatic material was lost from the cartilage only in the immediate vicinity of the vacuolated articular chondrocytes and the amount of hexosamine and hydroxyproline released into the medium was not much increased. However, the hexosamine and hydroxyproline of the sucrosetreated rudiments was much more susceptible to extraction with neutral salt than those of paired controls. Sucrose taken up by the cells was liberated when the rudiments were returned to normal medium; this release, unlike the secretion of lysosomal enzymes, was unaffected by the presence of hydrocortisone. It is probable that the primary lysosomes, formed in the enlarged Golgi region, are the vehicles for the secretion of lysosomal enzymes. No appreciable amount of enzymic activity was released into the medium from rudiments grown in the presence of 0·08 M sucrose, before 36 h; at, or just before this time, however, a net increase in synthesis of enzyme was observed. The role of endocytosis in the resorption of skeletal matrix is discussed.

Gaucher's disease: a genetic disease detected in skin fibroblast cultures.

Skin fibroblasts from three adult patients with chronic noncerebral Gaucher's disease, three children of one of the patients, three parents, and six normal individuals were grown in cell culture. Giant fibroblasts containing metachromatic material were seen in all cultures derived from affected individuals and heterozygous carriers but not in those derived from normal individuals.

Effect of cyanide intoxication on the metachromatic material found in the central nervous system.

Cyanide effect on metachromatic bodies in rat brain white matter, noting possible relation to myelin

Enzymatic events during cartilage differentiation in the chick embryonic limb bud

Chondroitin sulfate and an unidentified sulfated mucopolysaccharide insensitive to hyaluronidase are synthesized by the prospective limb region of stage 15–16 chick embryos and stage 17–18 limb buds more than 2 days before the first appearance of metachromatic material. Sulfate-binding mucopolysaccharide material with the same electrophoretic mobility as chondroitin sulfate becomes entirely sensitive to hyaluronidase in stage 19 and older limb buds. The activity of four enzymes presumed to be involved in the pathway of chondroitin sulfate biosynthesis has been demonstrated before histological evidence of cartilage in both cell-free extracts of stage 19 chick limbs and in cultured aggregates of dissociated cells from the same source. The developmental kinetics of three of these enzymes, UDPG dehydrogenase, UDPGNAc-4-epimerase, and the sulfate-activating enzymes, have been examined; the first two enzymes are involved in the synthesis of the polysaccharide backbone of chondroitin sulfate, and the last system is responsible for sulfation of the polysaccharide chain. Although all three enzymes are present in low levels in limb bud tissue before chondrogenic differentiation, the rise in their specific activity is related to the development of cartilage both in vivo and in vitro. The fourth and most specific enzyme catalyzing the formation of a polysaccharide from UDPGNAc and UDPGA has merely been identified in both fresh stage 20 limb buds and aggregates cultured for more than 6 days. The products of the four enzymatic reactions have been identified. The relationship of these results to a theory of biochemical differentiation based upon restriction or enhancement of preexisting metabolic potentialities has been discussed.

Beta-aminopropionitrile and the mucopolysaccharides of embryonic chick aortae.

THERE is some evidence to suggest that mucopolysaccharides are involved in the connective tissue disorder of lathyrism, which is produced when certain experimental animals are exposed to the toxic effect of β-aminopropionitrile (BAPN) and related compounds. Thus, Menzies and Mills1 found that the metachromatic material of the ground substance was increased in lathyritic rat aortae, and Grant, Hathorn and Gillman2 found an increased content of hexosamine in the aortae of lathyritic rats. Levene and Gross3 showed that an abnormality of collagen can be demonstrated very soon after the injection of BAPN into chick embryos. We have investigated the aortic mucopolysaccharides in this experimental model, to see if changes in the mucopolysaccharides occurred in parallel with the changes in collagen.

Congenital metachromatic leukodystrophy. Report of a case.

The pathologic findings of a case of congenital metachromatic leukoencephalopathy are described in a newborn infant who died 20 hours after birth. Metachromatic material was found particularly in cerebral white matter. No metachromatic material was demonstrated, however, in central nervous system neurones or in cells of kidneys, lymph nodes and liver where large, vacuolated cells were found, probably because of the easy solubility in formalin of the deposited substance.

NUCLEOLONEMA AND PARS AMORPHA IN ROOT MERISTEMATIC CELLS OF VICIA FABA

In osmium-fixed, Epon-embedded root meristematic cells of Vicia faba stained according to the Feulgen – methylene blue procedure, the interphase nucleolus is seen to consist of a metachromatic cabbage-green colored material in which are embedded numerous unstained vacuole-like structures of varying size. By using an orange Wratten filter in the illumination system, it is observed that the metachromatically stained nucleolar material is, in fact, made up of two components, each segregated into zones distinguishable by differences in staining intensity. The first more lightly stained component is found in the central and bordering regions of the nucleolus as well as in a number of areas of varying width extending radially in between them. The second more intensely stained component is observed in areas of varying length and width usually occupying the intermediate regions of the nucleolar mass. Under electron microscopy, the first component is seen to consist of densely packed fibrils and granules some 150 Å in diameter; in places, those constituents are assembled into seemingly coarse, thread-like structures approximately 0.1 μ in diameter. The second component, on the other hand, is made up mainly, if not exclusively, of tightly packed fibrils 60 to 100 Å in diameter. Judging from their topographical distribution and ultrastructural characteristics, it would seem as if the two nucleolar components just described correspond respectively to what is usually referred to as nucleolonema and pars amorpha in animal nucleoli. In addition to nucleolonema and pars amorpha, two ultrastructurally and topographically distinct types of vacuole are recognizable within the interphase nucleolus of Vicia faba. The vacuoles of the first type are usually large or medium-sized, contain loosely and uniformly scattered fibrils and granules approximately 150 Å in diameter, and are observed exclusively within the nucleolonematic zones of the nucleolus. The vacuoles of the second type are small, contain only loosely dispersed fibrils, 60 to 100 Å in diameter, and are confined to the pars amorpha of the nucleolus.

The effects of estradiol on the occurrence of mast cells in the rat uterus

Summary The number of mast cells found per transverse section of uteri taken from intact rats was lower in estrus (5.8 cells) than in diestrus (11.7 cells); cell counts in ovariectomized rats more closely approximated those in estrus animals. Treatment of ovariectomized rats with a single dose of estradiol (0.5 μg/100 g body weight) or cortisol did not significantly change the number of mast cells in uterus, mesometrium or skin within 24 hr. Unlike metachromatic material in mast cells elsewhere in the body, that in uterine and mesometrial cells occurs in a diffuse, hyalin form and rarely as discrete granules. These observations are discussed in relation to previously reported findings that estrogen stimulates release of histamine from the rat uterus. If it can be assumed that mast cells are the estrogen-sensitive repositories of histamine, failure to detect cytological changes in the present experiments after treatment with exogenous hormones suggests that release of the amine from mast cells of the rat uterus or mesometrium is merocrine or holocrine in nature depending on dosage and duration of hormone treatment. Alternatively, estrogen may activate uterine sites of histamine synthesis and storage other than mast cells.

THE EFFECT OF ANTISERUM, ALONE AND WITH HYDROCORTISONE, ON FOETAL MOUSE BONES IN CULTURE.

1. The effects of normal rabbit serum and of rabbit antiserum to whole foetal mouse tissues, on the isolated limb bones of late foetal mice were studied in organ culture, and the influence of hydrocortisone on these effects was investigated. 2. Unheated normal serum caused slight loss of metachromatic material from the cartilage matrix, and some resorption of both cartilage and bone. 3. In unheated antiserum to foetal mouse tissues, the terminal cartilage was smaller and less metachromatic than in paired controls in normal serum, while osteoclasis was so intense that in many explants the bone had almost disappeared. The amount of necrosis varied with different batches of antiserum. 4. The changes produced by normal serum and antiserum could be largely prevented by heating the sera to 57 degrees C for 45 minutes. 5. The effects could also be inhibited by the addition of hydrocortisone to the unheated sera; as little as 0.1 microg hydrocortisone per ml of medium had a well marked protective action. 6. It is suggested that (a) unheated antiserum causes a release of lysosomal enzymes with consequent breakdown of intercellular material, (b) this release is due to an indirect action on the lysosome via an increased permeability of the cell membrane, (c) hydrocortisone does not affect the antigen-antibody reaction, but inhibits the autolytic changes that normally follow this reaction, possibly by stabilising both the lysosomal and cell membranes.

HISTOCHEMICAL STUDIES ON THE ROLE OF THE MAST CELL IN CALCERGY.

Experiments were performed on rats, using special histochemical stains for the demonstration of metachromatic materials, lead, calcium, phosphate and carbonate, to study calcergy, the induction of soft-tissue calcification by topical treatment with certain compounds such as lead acetate. In simple calcergy, when lead acetate is directly injected into the subcutis, it is seen to impregnate the collagen fibers in the injection site without noteworthy participation of the mast cells. The lead-treated area secondarily attracts calcium, phosphate and possibly carbonate. In mastocalcergy, the lead acetate is injected intravenously and local calcification is produced by the simultaneous subcutaneous injection of a mast-cell discharger such as polymyxin. Here, the mast cells in the treated area first show degranulation without any mineralization, but this is soon followed by lead uptake on the mast-cell granules with secondary attraction of calcium, phosphate and possibly carbonate. Finally, the discharged and calcified mast-cell granules disintegrate into a dustlike, fine precipitate which is transferred to the circumjacent collagen fibers where it initiates an intense process of mineralization, eventually leading to the complete petrification of the polymyxin-injected area. Apparently, in addition to its already well established functions of histamine, heparin and serotonin production, the mast cell also plays a role in mineralization.

Parathormone-Induced Osteolysis in Dogs

Fragments of the parietal bone and tibia of fourteen young dogs were studied by x-ray microradiography of undemineralized sections, alpharadiography, and acid mucopolysaccharide staining of demineralized sections. Eight of these dogs received parathormone in different doses and were killed after intervals ranging from three hours to four days. In the parathormone-treated animals, sites of resorption (osteolysis) were recognized in the middle of the trabeculae of cancellous bone and in the interstitial lamellae of compact bone. These sites were characterized by more numerous enlarged and confluent lacunae inhabited by hypertrophic osteocytes containing metachromatic material and surrounded by basophilic or metachromatic matrix of lower density. A dose of 300 units of parathyroid extract and a dose of 1000 units over three hours did not produce osteolysis. A dose of 600 units over three days and to a greater extent a dose of 1000 units over twenty-four hours produced the effects described. Osteocyte hypertrophy and hyperactivity caused by parathormone were often followed by degeneration and death of the cells.

A Chemocytological Investigation of the Avian Adrenal Medulla

The relative proportions of adrenaline (A) and noradrenaline (NA) containing celluler areas have been planimetrically estimated in the twelve avian species representing several orders and families. The study reveals a great variability in percentages of A and NA-containing cells in the suprarenal medulla of these species. Three possibilities have been forwarded to explain this hormonal diversity. These are. i) methylation of medullary hormones is dependent on the cortico-medullary ratio, ii) relative abundance of A and NA in a species is determined by the degree of aggression and predation behavior manifested by it and iii) differing association of A and NA in this class of vertebrates may bear some relation to the suggested avian phylogeny. Amongst these probabilities, the last one appears to fit in rather closely to our data. This assumes an efficacious accomplishment or hormonal methylation in the course of avian evolutionary ascent.The present report also indicates that the chromaffin tissue of birds under investigation is rich in acetal phosphatides, metachromatic substances and acid phosphatase. Two species (H. smyrnensis and A. phoenicurus), however, fail to reveal any trace of medullary metachromatic materials. This as well as the concept of ‘basic cytochemical unit’ in avian adrenal medulla have been pointed out and briefly discussed.

SPORULATION AND THE DEVELOPMENT OF RESISTANCE IN SPORING CULTURES OF CLOSTRIDIUM WELCHII

The growth of a strain of Clostridium welchii type A in Ellner's medium was studied and the development of spores was correlated with the emergence of forms resistant to deleterious influences including heat, desiccation and thiomersalate. A qualitative increase in heat resistance was demonstrated, coincident with spore maturation over the course of 12 hr. The granular appearance of the bacterial cytoplasm prior to sporulation, as revealed by Gram's stain, precedes the development of acid‐fast material and does not seem to be associated with the presence of sudanophilic or metachromatic material.

Peripheral nerve changes in the diagnosis of metachromatic leucodystrophy.

SummaryA histopathological study was made on peripheral nerves and spinal roots from biopsies and autopsies of 8 cases with metachromatic leucodystrophy (sulphatide lipidosis), 4 cases of globoid cell leucodystrophy (Krabbe's disease), 9 cases with other progressive neurological disorders of infancy and childhood and 4 controls. The sural nerve was found to give satisfactory biopsy specimens and a suitable biopsy technique is described. At histological examination, brown metachromatic deposits were revealed by the cresyl violet‐acetic acid method of v. Hirsch & Peiffer in all cases with metachromatic leucodystrophy, but were never observed in any of the other cases examined. The granular metachromatic material was localized mainly to Schwann cells and histiocytes which were accumulated in clusters around capillaries of the endo‐ and perineurium. For diagnostic purposes the most reliable and so far pathognomonic change in peripheral nerve biopsy specimens was considered to be the demonstration of brown metachromatic granular deposits in perivascular phagocytes. It is concluded that histological examination of peripheral nerves can serve as a useful supplementary tool in the diagnosis of this genetically transmitted degenerative disorder during life.

CHANGES IN CONNECTIVE TISSUE AND INTESTINE CAUSED BY VITAMIN A IN AMPHIBIA, AND THEIR ACCELERATION BY HYDROCORTISONE

In view of the theory that an excess of vitamin A causes release of cathepsins from intracellular lysosomes, hypervitaminosis A was induced orally in the larvae of Xenopus laevis. It was predicted that the tails of these amphibia would undergo resorption prior to metamorphosis, since the presence of abundant lysosomes, associated with measurable increases of catheptic activity, had previously been demonstrated in the resorbing tails of amphibia during metamorphosis. This prediction was confirmed; after 3 to 4 weeks of hypervitaminosis A, the tails of treated animals underwent partial resorption. Other transitory appendages, the rostral tentacles, collapsed after 2 weeks of treatment with an excess of vitamin A, an effect analogous to the collapse of rabbits' ears after intravenous papain. These effects were related to the loss of metachromatic extracellular material in these appendages. Excess of vitamin A caused kyphoscoliosis and prognathos in the larvae. The hypervitaminotic larvae always developed a mucinous diarrhea, which was associated with a remarkable overgrowth of metachromatic goblet cells of the intestine. The entire intestine of the treated animals was more advanced in development than that of control larvae at equivalent stages. All the effects of hypervitaminosis A were accelerated by the simultaneous administration of hydrocortisone. This was held to be due to liberation of vitamin A from hepatic stores by the steroid, and is in contrast to the retardation of hypervitaminosis A by hydrocortisone in vitro.

The Development of Mast Cells in the Vascularized Cornea

The origin and physiological functions of the connective tissue mast cells have been the source of considerable speculation and experimentation since they were first described by Ehrlich. In recent years many workers have helped to clarify a few of the biochemical potentialities of these cells.3,14,17,19,20The role of mast cells in wound healing as well as the production of connective tissue ground substance via the manufacture of sulphated mucopolysaccharides still remains in doubt. Asboe-Hansen1has noted that the number of mast cells varies, in general, with the quantity of metachromatic ground substance present. Specific examples cited include myxedematous skin and the synovial membrane. However, if mast cells do contribute to the formation of ground substance, the property is not exclusively theirs. No mast cells appear in regenerating rat tendon25; they are similarly absent in the aortic media, which is rich in metachromatic material.5In studies of

Hyaluronic acid in the uterine wall of the guinea pig during the sexual cycle.

THE amount and function of acid mucopolysacchar-ides in the mammalian uterus are not yet known. A metachromatic staining material has been described in the endometrial stroma of the human uterus1–3 during the proliferative phase of the sexual cycle, and recently the presence of a periodic acid–Schiff positive material in the uterus of swine during the sexual cycle has been established4. Nothing definite has been found in the literature concerning the possible importance of hyaluronic acid in the physiology and pathology of the uterus. Investigations concerning the acid mucopolysaccharides of the uterus have been undertaken, since alteration in the quantity or quality of the ground substance may prove to be important in the pathogenesis of uterine infections and possibly in other disorders.

Late infantile metachromatic leukodystrophy. The nature of the chromotrope.

In 1910 Alzheimer and Baroncini1reported the first case of metachromatic leukodystrophy in an adult, and in 1933 metachromatic leukodystrophy occurring in late infancy was reported by Greenfield.2Since that time efforts have been made by many authors to elucidate the nature of a metachromatic material seen in the white matter of the morbid brain. Greenfield3considered the metachromatic material to be a galactolipid on the basis of a positive periodic acid-Schiff (PAS) reaction of material. However, as is well known, not only the glycolipids, but also polysaccharides, mucopolysaccharides, mucoproteins, glycoproteins, unsaturated fatty acids, and phospholipids are PAS-positive.4Greenfield mentioned in his paper that this method is not very specific in that it stains many of the more complex lipids such as myelin, and lipochrome, as well as many of the fat granule cells in areas of ischemic softening. In 1954 Feigin5reported a