Some properties of hyalin: the calcium-insoluble protein of the hyaline layer of the sea urchin egg.

The principal protein component of the hyaline layer of sea urchin eggs is the calcium-insoluble protein first described by Kane and Hersh. The protein hyalin is abnormally high in acidic amino acids, almost devoid of basic amino acids, and characteristically rich in valine and proline. Essentially all of the cysteine present is found in the disulfide form; no evidence points to intermolecular disulfide linkages. Hyalin from several species has a minimal subunit weight of about 100,000, though evidence exists for a particle three times this weight in urea or guanidine hydrochloride from one species. Optical rotatory dispersion measurements indicate no alpha-helix content, though the dispersion has unique characteristic features. Addition of small quantities of calcium causes hyalin to gel to a birefringent fibrous form. The fibrous, birefringent form of hyalin is rendered isotropic upon addition of EDTA, but the birefringence is restored with re-addition of divalent cation.

The isolation of a major structural element of the sea urchin fertilization membrane.

Procedures for isolating the contents of the cortical granules from the ova of the sea urchin, Strongylocentrotus purpuratus, are reported. Dithiothreitol is used to remove the vitelline coat; the "demembranated" eggs are then subsequently activated with butyric acid. By means of these procedures, the hyaline protein and crystalline or paracrystalline material have been isolated from the cortical granules. The crystalline material consists of sheets of cylinders or tubules 150-200 A in diameter. This material is believed to be a major structural element of the fertilization membrane which, in the absence of the vitelline coat, does not form.

Immunochemical comparison of histidine-rich protein in keratohyalin granules and cornified cells.

(1) Combination of techniques for extraction and purification of histidine rich protein established by several investigators were employed for comparison of histidine-rich protein in granular cells and cornified cells of newborn rats. (2) Histidine-rich protein extracted from the same cell fraction by two different techniques either in 1 M potassium phosphate buffer (Ugel) or in 4 M urea (Dale) showed identical elution profiles on CM 52 cellulose ion exchange chromatography and the same SDS polyacrylamide gel electrophoretic patterns. (3) Histidine-rich protein from granular cells contained polypeptides of larger molecular sizes than those in histidine-rich protein from cornfield cells, although amino acid composition of the two histidine-rich protein was non-distinguishable (histidine residue was more than 7%). (4) Antibodies raised in rabbits by injection of histidine rich protein from granular cells and that from cornfield cells immunologically cross-reacted. Furthermore, the antisera were found to be reactive over both keratohyalin granules and cornified cells, but not epidermal cells of the lower strata.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins of newbonr rat skin. II. Keratohyalin and stratum corneum proteins.

Keratohyalin extracts from newborn rat epidermis were prepared by potassium phosphate and citric acid-detergent extraction procedures. These preparations were compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and amino acid analysis. The major band of the potassium phosphate extract has a molecular weight of 48,000. The major bands of the citric acid-detergent preparation have molecular weights of 64,000, 61,500, 57,000 and 54,000. Electrophoresis of S-carboxylmethylated (SCM)-fibrous protein results in two major bands of approximately 57,000 and 64,000. SDS gels of the two preparations of keratohyalin and the SCM-fibrous protein were compared with gels of the insoluble proteins of granular and eluted cornified cells. All of the major bands in the preparations of keratohyalin can be seen in gels of the granular preparation. The two SCM-fibrous protein bands correspond with two prominent bands in gels of the cornified cell preparation. Two bands of the citric acid-extracted keratohyalin sample also have the same mobility. The major band of the potassium phosphate-extracted preparation of keratohyalin corresponds with a third prominent band of the cornified cell preparation. These results suggest that biochemical components of the preparations of keratohyalin are present in both the granular and the cornified layers of newborn rat epidermis.

Bovine keratohyalin: anatomical, histochemical, ultrastructural, immunologic, and biochemical studies.

Salt extraction studies showed that keratohyalin (KH) could be solubilized and extracted from fresh bovine hoof epidermis. The solubility of KH varied in relation to the molarity of the salt solution used for extraction. Using this information, the extracted KH was aggregated in vitro by dialyzing the high salt extract against distilled water. Histochemical, ultrastructural, and immunologic studies of the resultant particles or macroaggregates showed that the latter had the same properties and immunogenicity as the KH granule in situ and produced antibodies against it. Fractionation of the macroaggregates by polyacrylamide gel electrophoresis demonstrated that the macroaggregates were compsed of sets of 20 polymers whose subunits or monomers had a molecular weight of 16,900. Amino acid analyses showed that the macroaggregates and the various fractionated polymers were similar and that the protein ahd 116 amino acid residues. Serine, arginine, glycine, glutamic acid, and histidine constituted 78% of all residues, and serine alone represented 27%. The molecular weight by amino acid analyses was 16,150 after correction for the 8% ribonucleic acid which appears to be complexed to the protein.

Ultrastructure and composition of connective tissue in hyalinosis cutis et mucosae skin.

Skin biopsies from a patient with hyalinosis cutis et mucosae (HCM) were studied by routine histology, electron microscopy, biochemical extractions, and immunofluorescence for extracellular matrix proteins. The upper dermis consisted of large hyaline regions mainly composed of noncollagenous proteins. A portion of this material was solubilized by reduction in 8 M urea. Anti-sera against these proteins revealed multiple antigens most of which were also detectable in normal skin. The hyaline regions showed a reduced content of collagens, particularly of thick fibrils and of fibronectin. The basal lamina around capillaries and at the dermal-epidermal junction appeared as multiple, concentric layers of amorphous laminae intercalated with thin collagen fibrils. They consisted of collagens type III and IV and of laminin as shown by immunofluorescence. Antibodies could also be raised against laminin of HCM skin which showed strong cross-reactions with authentic mouse laminin. Cultured fibroblasts from the HCM lesion showed increased synthesis of noncollagenous proteins at the expense of newly synthesized collagens. Some but not all of these noncollagenous proteins were also produced by fibroblasts from normal skin. The above data indicate that the hyaline material in HCM originates from the overproduction of noncollagenous proteins, most of which are normal constituents of human skin.

Desmosomes, filaments, and keratohyaline granules: their role in the stabilization and keratinization of the epidermis.

Components of desmosomes, filaments, and keratohyaline granules were studied by electron microscope and biochemical methods to clarify their role in the stabilization and keratinization of the epidermis. Isolated desmosomes are composed of 76% protein, 17% carbohydrate, and 10% lipid. The bulk of protein consists of a "spectrin"-like fibrous protein, presumably present in the plaque, and of glycoproteins in the desmosomal interspace. The main component of filaments, prekeratin, is a low-sulfur alpha-protein composed of a pair of three-chain subunits with non-alpha-helical segments separated by 200 A-long alpha-helical regions. The major component of isolated keratohyaline granules, the amorphous particulate material, is formed by a high-sulfur protein with a single-type of polypeptide chain. Polypeptide chains comparable to those found in prekeratin and keratohyaline granules were recovered from extracts of horny cells. Within the living part of the epidermis, filaments hypothetically form a cytoskeletal system which is anchored to desmosomes by a filamentous plaque protein. Glycoproteins are involved in the formation of strong junctions between the cells which enable the living part of the epidermis to respond as a whole to mechanical stress. The stratum corneum is stabilized by a similar system in a consolidated state which is less extensible. Horny cells are enveloped by a thickened membrane and the interfilamentous spaces are filled with various proteins including the sulfur-rich amorphous protein found in keratohyaline granules.

An autopsy case of atypical infantile motor neuron disease with hyaline intraneuronal inclusions.

We describe a 5-year-old boy who had had a progressive motor weakness with bulbar palsy and spasticity of the lower extremities since age 3 years and who died of bronchopneumonia after about two and a half years of the illness. Neuropathologic examination revealed combined degenerative processes in the upper and lower motor neurons, the spinocerebellar and olivocerebellar systems, and the ventral thalamic nuclei. Lewy body-like intraneuronal hyaline inclusions, which ultrastructurally showed irregular accumulations of trilaminar membranous profiles, were detected in the spinal anterior horn, Clarke's dorsal nucleus, facial nerve nucleus, inferior olivary nucleus, and substantia nigra. This case could be considered as a unique form in the group of the infantile motor neuron diseases associated with multi-systemic degenerations in the central nervous system.

Hemorrhages with optic nerve drusen. A differentiation from early papilledema.

The distinction between early papilledema and drusen of the optic disk may be made more difficult by the presence of hemorrhages. We report five patients with drusen of the optic disk and associated hemorrhages. The distinguishing characteristics of both conditions are described.

Adenomas in glycogen storage disease type 1. Two cases with unusual histologic features.

Two cases of hepatic adenomas arising in patients with glycogen storage disease type I are presented. In both cases, the adenomas showed unusual histological features including marked fatty change, a polymorphonuclear infiltrate, Mallory hyalin, and lamellar fibrosis. In addition, one case was associated with amyloidosis. Such features have only recently been described in adenomas and have not been reported previously in association with glycogen storage disease type I.

Secretory meningioma. A distinct subtype of meningioma.

Six meningiomas with abundant hyaline inclusions (pseudopsammoma bodies) were studied. As seen by light and electron microscopy, hyaline inclusions are composed of material of varying structures located in intracellular lumina lined by microvilli. A remarkable pericytic proliferation within the vessel walls was found in five cases. In all six cases, immunohistochemical examination for multiple antigens showed positive staining for carcinoembryonic antigen and epithelial membrane antigen in inclusions and surrounding cells. Weak positivity was found for keratin and secretory component in five cases and for alpha-1-antitrypsin and IgM in four cases. It is concluded that secretory meningioma is a distinct type of meningioma, usually meningothelial in type. It shows characteristic light-microscopic, ultrastructural, and immunohistochemical features of epithelial and secretory differentiation with accumulation of secretory material in the form of hyaline inclusions; marked vascular pericytic proliferation is also frequently present.

Scanning electron microscopy of human drusen.

Drusen are small, yellowish deposits that form under the retinal pigment epithelium (RPE) with senescence or under certain pathological conditions. The present study examined these structures under the scanning electron microscope. Tissue came from four eyes of 66- and 75-year-old donors who demonstrated widespread drusen of the posterior fundus noted on postmortem examination. Specimens were prepared by either detaching the RPE from Bruch's membrane, or by cryofracturing the tissue for cross-sectional views. Drusen appeared to be composed of irregularly-shaped globular masses and of distinct spherical entities. These particles varied greatly in size, and were situated between the RPE's basement membrane and the outer collagenous zone of Bruch's membrane. Surface views showed drusen components to be embedded in the collagenous zone of Bruch's membrane. Pits corresponding to the sizes of the globular and spherical masses imply that some particles were lost during tissue processing. Fractured cross sections of the irregularly-shaped globular masses revealed a homogeneous, granular matrix with no distinct ultrastructural features, while some of the fractured spherical components demonstrated an internal core. Transmission electron microscopic analysis on the same specimens that were subjected to SEM corroborated these observations. Analytical x-ray microanalysis (Kevex, Foster City, CA) in the SEM revealed major peaks for calcium and phosphorous in the crystalline spherical components, and primarily potassium and chloride in the globular structures.

Severe antithrombin III deficiency in an infant associated with multiple arterial and venous thromboses.

Inherited antithrombin III (AT-III, heparin cofactor) deficiency is a rare condition, presenting with thrombotic disease in adult life. This paper reports an 8 months old South African Black male infant with multiple large vessel venous and arterial thromboses, and E. coli septicaemia. This was associated with an extremely low plasma AT-III level. Micronodular cirrhosis and intracytoplasmic hyaline globules in the liver cells were present. These globules were eosinophilic, and PAS-positive after diastase. They measured approximately 5 mu to 30 muin diameter, occurred singly in the liver cells and were located mainly in the periportal areas. The histological findings in the liver are similar to those observed in alpha 1-antitrypsin (AAT) deficiency in which the intracytoplasmic globules represent accumulation of altered AAT. Immunochemical studies carried out on formalin fixed tissue failed to detect cross reaction material with anti-alpha 1 antitrypsin or anti-AT III antiserum. This is the first case report of AT-III deficiency presenting in infancy. It is also the first case associated with distinctive liver pathology. The available data presented are insufficient to distinguish between an inborn defect and acquired caused of the severely depressed AT-III plasma level and the distinctive liver pathology.

Ovarian malignant mixed mesodermal tumor: the occurrence of hyaline droplets containing alpha 1-antitrypsin.

Twenty-one of 22 cases of malignant mixed mesodermal tumor (including carcinosarcoma) of the ovary were found to contain periodic acid-Schiff(PAS)-positive, diastase-resistant hyaline droplets ranging in diameter from less than 1 micrometer to approximately 50 micrometers. Droplets were located both inside and outside undifferentiated mesenchymal cells, predominantly within myxomatous areas. In 13 cases, droplets also occurred in epithelial structures, including areas mimicking ovarian carcinoma. Immunoperoxidase staining for alpha-fetoprotein was consistently negative, but droplets and cell cytoplasm reacted strongly with anti-alpha 1-antitrypsin. Analysis of the structure of droplet antitrypsin may be necessary to determine the origin of the droplets themselves and the possible usefulness of serum alpha 1- antitrypsin as a tumor marker in ovarian carcinoma and malignant mixed mesodermal tumor.

Mallory bodies in scar adenocarcinoma of the lung.

A 75 year old man presented with metastatic carcinoma of the brain. At autopsy a primary scar adenocarcinoma of the lung was found. By light microscopy the cytoplasm of the tumor cells was seen to contain hyalin masses with tinctorial properties identical to those of the Mallory bodies seen in human alcoholic liver disease and in hepatocellular carcinoma. The hyalin stained positively with anti-Mallory body antibody by the peroxidase antiperoxidase technique, and had the characteristic filamentous and granular amorphous appearance of alcoholic hyalin by electron microscopy. Our observation is of interest in light of recent information relating Mallory bodies to prekeratin, and vitamin A deficiency to lung cancer.

Pulmonary hyalinizing granuloma. Electron microscopic and immunologic studies.

We present a case of pulmonary hyalinizing granuloma (PHG). On light microscopy, the pulmonary nodular lesions consisted of extracellular, eosinophilic hyaline lamellae. Histochemical stains of the hyaline lamellae for amyloid were focally positive and the diagnosis of amyloidosis was seriously considered; however, on electron microscopic examination, the hyaline lamellae consisted of electrondense, compact, amorphous material quite unlike fibrillar amyloid. Although circulating immune complexes containing IgA were detected in our patient, immunoperoxidase stains did not reveal immunoglobulins in the hyaline lamellae. This case illustrates the value of electron microscopy in differentiating PHG from amyloidosis and supports the hypothesis that PHG represents an exaggerated immune response.

Peyer's patches in experimental Salmonella dublin infection in calves. Microvascular and epithelial changes contributing to atrophy of lymphoid follicles.

Six calves were infected per os with Salmonella dublin and killed nine hours to seven days later. Early changes included occlusion of capillaries with a hyaline material, particularly in the ileal Peyer's patch (PP). Central areas of the follicles contained hemorrhages and edema. In later stages the follicle-associated epithelium (FAE) of both the jejunal and ileal PP was fused with the adjacent epithelium and the follicles were collapsed. As judged from 5'nucleotidase histochemistry, follicles were depleted of lymphocytes whereas reticular cells were retained. Carbonic anhydrase (CA) histochemistry showed a decreased reaction in the ileal FAE and a reduced amount of CA reactive material in the follicles of the ileal PP, indicating loss of FAE differentiation and function. Hyaline material and fibrinous thrombi were seen occluding the blood capillaries and the lymphatics, respectively. The villi were atrophied and covered with thick fibrin deposits. Using antifibrinogen antibodies, immunoperoxidase stained fibrin in the lymphatics and the lumenal deposits but not the hyaline material in the capillaries. Reaction for CA indicated that this hyaline material originated from erythrocytes. Factors contributing to the follicle atrophy may include anoxia due to stasis in the microcirculation with the formation of erythrocyte thrombi, and reduced lymphopoiesis due to a decrease in the stimulating factors provided by the FAE.

Drusen of the optic nerve associated with retinitis pigmentosa.

Globular excrescences of the optic nerve associated with retinitis pigmentosa have been interpreted as drusen or as astrocytic hamartomas. The histopathologic and ultrastructural findings of globular excrescences of the peripapillary region of the optic nerve associated with retinitis pigmentosa were described in a 22-year-old patient who died in a car accident. From our findings we conclude that the globular excrescences of the optic nerve associated with retinitis pigmentosa are definitely drusen and not astrocytic hamartomas.

A histochemical approach to the differentiation of lichen planus from lupus erythematosus.

A histochemical technic utilizing a maleimide-derived fluorochrome for the detection of disulfide bonds in subepidermal hyaline bodies is described. Lichen planus and the skin lesions of lupus erythematosus, which may have similar clinical, histologic, and immunofluorescent presentations, tend to demonstrate different patterns of reactivity with this reagent. This technic, then, may be used to distinguish between these two cutaneous disorders.

Massive cutaneous hyalinosis. Identification of the hyalin material as monoclonal kappa light chains, adhesive 90 kD glycoprotein, and type I collagen.

The hyalin material in massive cutaneous hyalinosis, a disease characterized by extensive tumorous periodic acid-Schiff-(PAS) positive extracellular cutaneous deposits, has been elucidated by biochemical and immunologic methods. Three major components were found: kappa light chains, a mannose-rich glycoprotein, and type I collagen. Trace amounts of fibrinogen, fibronectin, laminin, IgG, pregnancy-specific glycoprotein, albumin, and keratan sulfate, but not keratin, were also present. The kappa light chains were monoclonal, cryoprecipiting, and more basic than the kappa chains from two myeloma patients. The glycoprotein, which could not be identified as any known glycoprotein, had an apparent molecular weight of 90,000 D. Amino acid analysis showed that glutamic acid, aspartic acid, leucine, and threonine were abundant, whereas hydroxyproline, hydroxylysine, and sulfhydryl amino acids were absent. The carbohydrate content of the protein was approximately 20%. The major monosaccharides were mannose and N-acetylglucosamine. Galactose, N-acetylneuraminic acid and fucose also were present. The third major component of the hyalin material was identified as type I collagen. A humoral immune response to the storage material was found: the patient's serum contained IgM and IgG class antibodies against the mannosylglycoprotein (90 kD glycoprotein) and against type I collagen.