THE CHEMICAL NATURE OF KERATOHYALIN GRANULES OF THE EPIDERMIS

Keratohyalin granules were isolated in the native form from the epidermis of newborn rats by the use of citric acid and a detergent. The isolated granules revealed a fine granular substructure in the electron microscope similar to that seen in situ. Analyses of amino acids by automated column-chromatography showed that proline and cystine are present in large proportions whereas histidine is present in a small amount. Accordingly, it was concluded that keratohyalin represents a sulfur-rich amorphous precursor of the horny cell content, rather than a sulfur-poor side product of the keratinization process, or a unique histidine-rich protein as proposed by in situ histochemical and radioautographic studies.     

Distribution of keratin and associated proteins in the epidermis of monotreme,marsupial, and placental mammals

The expression of acidic and basic keratins, and of some keratinization marker proteins such as filaggrin, loricrin, involucrin, and trichohyalin, is known for the epidermis of only a few eutherian species. Using light and high-resolution immunocytochemistry, the presence of these proteins has been studied in two monotreme and five marsupial species and compared to that in eutherians. In both monotreme and marsupial epidermis lamellar bodies occur in the upper spinosus and granular layers. Development of the granular layer varies between species and regionally within species. There is great interspecific variation in the size (0.1-3.0 microm) of keratohyalin granules (KHGs) associated with production of orthokeratotic corneous tissues. Those skin regions lacking hairs (platypus web), or showing reduced pelage density (wombat) have, respectively, minute or indiscernible KHGs, associated with patchy, or total, parakeratosis. Ultrastructural analysis shows that monotreme and marsupial KHGs comprise irregular coarse filaments of 25-40 nm that contact keratin filaments. Except for parakeratotic tissues of platypus web, distribution of acidic and basic proteins in monotreme and marsupial epidermis as revealed by anti-keratin antibodies AE1, AE2, and AE3 resembles that of eutherian epidermis. Antibodies against human or rat filaggrins have little or no cross-reactivity with epidermal proteins of other mammals: only sparse areas of wombat and rabbit epidermis show a weak immunofluorescence in transitional cells and in the deepest corneous tissues. Of the available, eutherian-derived antibodies, that against involucrin shows no cross-reactivity with any monotreme and marsupial epidermal tissues and that against trichohyalin cross-reacts only with cells in the inner root sheath and medulla of hairs. These results suggest that if involucrin and trichohyalin are present throughout noneutherian epidermis, they may have species-specific molecular structures. By contrast, eutherian-derived anti-loricrin antibodies show a weak to intense cross-reactivity to KHGs and corneous tissues of both orthokeratotic and parakeratotic epidermis in monotremes and marsupials. High-resolution immunogold analysis of loricrin distribution in immature keratinocytes of platypus parakeratotic web epidermis identifies labeled areas of round or irregular, electron-pale granules within the denser keratohyalin component and keratin network. In the deepest mature tissues, loricrin-like labeling is diffuse throughout the cytoplasm, so that cells lack the preferential distribution of loricrin along the corneous envelope that characterizes mature eutherian keratinocytes. Thus, the irregular distribution of loricrin in platypus parakeratotic tissues more resembles that which has been described for reptilian and avian keratinocytes. These observations on the noneutherian epidermis show that a stratum granulosum is present to different degrees, even discontinuous within one tissue, so that parakeratotic and orthokeratotic areas may alternate: this might imply that parakeratotic monotreme epidermis reflects the primitive pattern of amniote alpha-keratogenesis. Absent from anamniote epidermis and all sauropsid beta-keratogenic tissues, the ubiquitous presence of a loricrin-like protein as a major component of other amniote corneous tissues suggests that this is a primitive feature of amniote alpha-keratogenesis. The apparent lack of specific regionalization of loricin near the plasma membranes of monotreme keratinocytes could be an artifactual result of the immunofluorescence technique employed, or there may be masking of the antigenicity of loricrin-like proteins once they are incorporated into the corneous envelope. Nevertheless, the mechanism of redistribution of such proteins during maturation of monotreme keratinocytes is different from, perhaps more primitive, or less specialized, than that in the epidermis of eutherian mammals.     

Proliferation, differentiation and maturation of a mouse epidermal keratinocyte cell line

The spontaneous development of a cell line of neonatal mouse C3H/He epidermal cells is described. The culture has been serially passaged at 29 °C over 18 months in the absence of any dermal support. The cell morphology of the 18th passage is reported. During early growth phase, the morphology of the cell layers was similar to that observed in the basal and differentiating strata of the epidermis: numerous tonofilament bundles and desmosome-filament complexes were observed. During late growth phase, maturation and vertical stratification occurred: demonstrated by the tonofilament accumulation, cell organelle degradation, nuclear pyknosis, presence of keratohyalin granules and horny cell layers with thickened membranes. Hemidesmosome-like structures were shown. No basal lamina or membrane coating granules were detectable. The 18th passage cultured cells did not induce tumors in nude mice. This keratinocyte cell line is not permanent, however: a malignant transformation occurred after 25 subcultures which resulted in an undifferentiated cell population.     

Electron microscopic study of the cortical reaction of an ophiuroid echinoderm.

The egg coats of an ophiuroid echinoderm (Ophiopholis aculeata) are described by electron microscopy before and after fertilization. The unfertilized egg is closely invested by a vitelline coat about 40 A thick, and the peripheral cytoplasm is crowded with cortical granules five or six deep. During the cortical reaction, which rapidly follows insemination, exocytosis of cortical granules takes place. Some of the cortical granule material is evidently added to the vitelline coat to form a composite structure, the fertilization envelope, which is made up of a 400 A thick middle layer separating inner and outer dense layers, each about 50 A thick. The elevation of the fertilization envelope from the egg surface creates a perivitelline space in which the hyaline layer soon forms. The hyaline layer is about 2 micron thick, finely granular, and apparently derived from cortical granule material. The extracellular layers of the early developmental stages of ophiuroids and echinoids are quite similar in comparison to those of asteroids; this finding helps support Hyman's argument that the ophiuroids are more closely related to the echinoids than to the asteroids.     

Formation of horny cells: the fate of cell organelles and differentiation products in ruminal epithelium

Epithelial cells changing from the granular stage of differentiation to the horny stage are more numerous, and reveal sequential events of transformation in finer detail in the rumen epithelium than in other keratinizing epithelia thus far studied in the electron microscope. Studies of such cells indicate that transformation is initiated by the release of hydrolytic enzymes, as evidenced by the appearance of lysosomes. As lysosomes increase in number, the nucleus, ribosomes, mitochondria, Golgi apparatus, and mucous granules are gradually degraded. Furthermore, marked changes occur in permeability of the plasma membrane as voluminous amounts of the lysed cell components pass through and accumulate in the intercellular space in the form of an amorphous mass. Filaments, keratohyalin granules, and the content of the ER (ER-protein) are not lysed, revealing the action of released enzymes to be specific. During transformation, filaments become displaced toward the cell periphery and keratohyalin granules disperse and mix with the ER-protein in the cell center. Subsequently, the keratohyalin-ER-protein complex infiltrates the filament network whereby a fibrous-amorphous cell content is formed. Loss of fluids through the plasma membrane leads to reduction of cell volume and consolidation of the remaining cell content. The deep interdigitations formed between the cells ultimately interlock the outer part of the epithelium into a cohesive and protective stratum corneum.     

Plantar epidermis of the guinea pig and characteristics of the stratum corneum.

The guinea pig plantar epidermis was examined by light-microscopical histochemical methods and by transmission electron microscopy. Autolysis of cell structure was much less complete in guinea pig plantar horny layer than in the back, and stainable cytoplasm was retained in keratinized cells but organelles were lost except for some degraded ultrastructural remnants. By light microscopy the whole thickness of the horny layer showed bound phospholipid and bound cysteine, and there was a weak cystine reaction at the peripheries of the keratinized cells. In ultrastructure the keratohyalin contained slightly larger subparticles than in the back skin. The horny layer was not divisible into basal, intermediate and superficial regions as in hairy skin. The stratum lucidum of light microscopy was not defined in electron micrographs. Osmium-stained cytoplasmic material was retained in horny cells about to be desquamated, in contrast to the empty appearance of these cells in hairy skin. Epidermal cells in plantar skin have ultrastructural cytoplasmic processes which are longer than they are broad. In the horny layer these interdigitate with those of neighbouring cells and are held together by lateral demonsomal junctions. Probably this gives mechanical strength against shearing forces experienced by the plantar horny layer.     

The histochemical distribution of protein bound sulfhydryl groups in human epidermis by the new staining method.

Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) which is nonfluorescent by itself but will react readily with -SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of -SH groups and S--S linkages in the human epidermis. The distribution of -SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S--S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared gradually. The distribution of S--S linkages appeared to be very low in the cytoplasm of keratinized cells. No substantial fluorescence was localized on keratohyalin granules even after reduction.     

Ultrastructural study of the neural fat-body system in the cockroach Periplaneta americana

The neural fat-body system of the ventral nerve cord in the cockroach Periplaneta americana was studied with the light and electron microscopes. This adipose tissue surrounds the connectives and extends over the ganglia. The adipose cells typically contain numerous extremely large lipid inclusions, pleomorphic lysosomes, and tightly packed glycogen granules. The neural lamella consists of a thick inner layer rich in collagen fibers and a thin outer layer of granular material. At points where the fat body is attenuated, this granular layer is split and the outer lamina is reflected superficially to ensheath and apparently to anchor the fat body.     

Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes.

In situ hybridization with fluorescent oligonucleotides was used to detect and localize microorganisms in the granules of two lab-scale upflow anaerobic sludge blanket reactors that had been fed for several months with either sucrose or a mixture of volatile fatty acids. Sections of the granules were hybridized with 16S rRNA-targeted oligonucleotide probes for Bacteria, Archaea, specific phylogenetic groups of methanogens, and two syntrophic propionate-oxidizing strains, MPOB and KOPROP1. Cells of the syntrophic strain KOPROP1 were not detected in either type of sludge granules. Hybridizations of the sucrose-fed granules showed an outer layer of mainly bacterial microcolonies with different morphologies. More inwards of these granules, a layer of different methanogenic microcolonies mixed with large colonies of the syntrophic strain MPOB could be detected. The MPOB colonies were intertwined with hydrogen- or formate-consuming methanogens, indicating their syntrophic growth. The granules fed with volatile fatty acids showed an outer layer of mainly bacteria and then a thick layer of Methanosaeta-like methanogens mixed with a few bacteria and a layer of methanogens mixed with syntrophic MPOB microcolonies. The centers of both sludge types consisted of large cavities and methanogenic microcolonies. These results indicate a juxtapositioning of syntrophic bacteria and methanogens and provide additional evidence for a layered microbial architecture of anaerobic granular sludge.     

Clinical response of captive common wombats (Vombatus ursinus) infected with Sarcoptes scabiei var. wombati

Seven common wombats (Vombatus ursinus) were exposed and two of these were re-exposed to Sarcoptes scabiei var. wombati (Acari: Sarcoptidae). For wombats exposed for the first time, five exposed to 5,000 mites on their shoulder developed moderate to severe parakeratotic mange after 11 wk compared with two given 1,000 mites that developed mild clinical signs of mange after 11 wk. For re-exposed wombats, one of two given 5,000 mites developed mild parakeratotic mange and the other developed severe parakeratotic mange. Initial signs of mange were erythema followed by parakeratosis, alopecia, excoriation and fissuring of parakeratotic crust and skin. Erythema usually became apparent within 14 days after exposure (DAE) or within 24 hrs of re-exposure. Parakeratosis was visible 14-21 DAE and alopecia first occurred 35-77 DAE. Clinical signs increased in severity over time and lesions spread slowly from the site of exposure. Mangy wombats scratched excessively, lost weight, and exhibited a significant neutrophilia compared with control wombats. Treatment of mange with three injections of ivermectin, 300 micrograms/kg, 10 days apart led to complete resolution of clinical signs. However mites were not entirely eliminated until wombats received a second regime of treatment.     

Expression of cytoplasmic antigens linked to orthokeratosis during the development of parakeratosis in newborn mouse tail epidermis

An unusual example of two different types of epidermal differentiation occurs side by side in adult mouse tail. The neonate shows only orthokeratotic differentiation, but develops parakeratotic scales in precise patterns over the first two weeks of life, retaining orthokeratotic differentiation at the necks of hair follicles. Certain human IgG antibodies from patients receiving bone marrow transplants bind only to cytoplasmic components of orthokeratotic epithelium. As markers of orthokeratotic differentiation in indirect immunofluorescent studies, these antibodies allowed the timing and location of the change in epidermal differentiation. A specific loss of 'orthokertotic' antigens in the natural switch from orthokeratosis to parakeratosis was demonstrated. The sequence of the orthokeratotic antigens disappearance suggested that the switch to parakeratotic differentiation occurred at a supra-basal level.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

Expression of Cytoplasmic Antigens Linked to Orthokeratosis During the Development of Parakeratosis in Newborn Mouse Tail Epidermis

Abstract. An unusual example of two different types of epidermal differentiation occurs side by side in adult mouse tail. The neonate shows only orthokeratotic differentiation, but develops parakeratotic scales in precise patterns over the first two weeks of life, retaining orthokeratotic differentiation at the necks of hair follicles. Certain human IgG antibodies from patients receiving bone marrow transplants bind only to cytoplasmic components of orthokeratotic epithelium. As markers of orthokeratotic differentiation in indirect immunofluorescent studies, these antibodies allowed the timing and location of the change in epidermal differentiation. A specific loss of 'orthokeratotic' antigens in the natural switch from orthokeratosis to parakeratosis was demonstrated. The sequence of the orthokeratotic antigens disappearance suggested that the switch to parakeratotic differentation occurred at a supra-basal level.     

Fine Structure of the Oocyst Wall of Eimeria nieschulzi

SYNOPSIS. Oocysts of Eimeria nieschulzi from the laboratory rat, Rattus, norvegicus , were studied by scanning and transmission electron microscopy. Oocysts had a rough outer wall with apparent random depressions. The oocyst wall is composed of 2 layers: an osmiophilic outer layer consisting of a rough external and smooth internal surface, and a relatively thick, electron-lucent inner layer. The outer layer is composed of a dense, coarsely granular matrix. The inner layer consists of homogeneous fine granular material interspersed with coarse osmiophilic granules and contains one closely applied membrane on the outermost surface. Several raised lenticular areas are seen on the coarse outer surface of the inner layer. These layers are 102 (75–128) and 176 (135–204) nm thick, respectively.
The sporocyst wall is thin, consisting of 3 to 4 unit membranes, and measures 27 (18–34) nm thick.     

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

Summary Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel patched appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.     

Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca: Pelecypoda)

Summary The fine structure of oyster leucocytes resembles to a great extent, that of typical eucaryotic cells. Organelles which have been described for the first time in this report are light granules, dense granules, protocentriole and X structure. Light microscopy reveals two morphological types of oyster leucocytes: agranular and granular. Based upon nuclear morphology and cytoplasmic compositions revealed in electron microscopy, at least three types of agranular and one type of granular cells are recognized.In the Giemsa-stained preparations, granular leucocytes exhibit three distinct types of cytoplasmic granules: refractile, dark blue, and pink, which presumably correspond to light granules Type A, B, and C seen in the electron micrographs. A granular leucocyte may contain one or more types of granules. Cytochemical investigations show that oyster leucocytes contain at least three hydrolytic enzymes: non-specific esterases, acid, and alkaline phosphatase. The latter two enzymes constitute 63% of the enzyme activity detected. These intracellular enzymes may be associated with the light granules and/or lysosome-like bodies.It is also demonstrated that the granular leucocyte population is significantly higher (P<0.001) in the oysters experimentally infected with Bacillus mycoides (72.19±4.71%) as contrasted with that of the controls (37.18±4.48%).Leucocytes in progressive stages of degeneration are also described.Contribution No. 71 from Marine Research Laboratory, University of Connecticut.The initial phase of this investigation was carried out at the Department of Zoology, Rutgers, The State University, New Brunswick, New Jersey, and supported by Public Health Service Research Grant AI-00781 from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, awarded to Dr. L. A. Stauber. Supported by a grant from the University of Connecticut Research Foundation and Faculty Summer Fellowship to S. Y. Feng.     

Expression of hornerin in stratified squamous epithelium in the mouse: a comparative analysis with profilaggrin.

We have recently identified a novel protein named hornerin, the structural features of which are most similar to those of profilaggrin, an essential protein for keratinization of epidermal tissues. In this study we examined the expression of hornerin compared with that of profilaggrin in various mouse tissues. Hornerin was expressed in the upper epidermis of newborn mouse skin, as was profilaggrin. In addition, both hornerin and profilaggrin were expressed in the tongue, esophagus, and forestomach. In all four tissues, immunostaining for hornerin and profilaggrin showed a granular pattern, and most of the signals for the two proteins were co-localized on keratohyalin granules. This was confirmed by double immunoelectron microscopy. Within keratohyalin granules, hornerin was detected more frequently in the periphery, whereas profilaggrin was equally distributed. A quantitative RT-PCR revealed that both genes were expressed at highest levels in the forestomach and at the next highest levels in skin. Profilaggrin mRNA was most abundant in the forestomach. In skin, the amount of hornerin mRNA was more than fourfold greater than the amount of profilaggrin mRNA. These results form the basis for a better understanding of possible overlapping and/or differential functions of hornerin and profilaggrin.     

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.     

Degenerative and regenerative changes in epidemrmal organ culture: A morphological study wity reference to membrane-coating granules

Summary Membrane-coating granules (MCG) are poorly understood lamellate organelles unique to keratinized epithelia. This study provides data on a skin model for future in vitro investigations of MCG. Porcine ear epidermal, organ cultures were used under standard cell culture conditions. This system was selected because it is easily established and, following a degenerative period in which MCG are lost, regenerates to form a highly differentiated epidermis. The epidermis appeared healthy during the first 2 din vitro and contained MCG but lost keratohyalin granules (KHG). Overt degenerative changes were evident in the upper epidermis on Day 3, and MCG were now bloated. By Day 4 only one to three layers of viable undifferentiated cells remained. In the overlying necrotic epidermis MCG were rare, presumably due to the bursting of bloated MCG. Epidermal regeneration began around Day 5 and by Day 7 there, were 8 to 13 layers, including a, rudimentary parakeratotic stratum corneum (up to 4 layers). The stratum granulosum (two to three layers) now containe immature KHG and poorly lamellate MCG, but only amorphous material extracellularly. By Day 11 there were three to four layers of granular cells as in vivo, and an orthokeratotic stratum corneum (two to four layers). Improved cornification coincided with an increased number of mature KHG and cross-banded MCG, and lamellate MCG contents extracellularly. This model of epidermal regeneration will factiliate studies into the role played by MCG in keratinization because the epithelium initially lacked MCG but later expressed all the major morphologic features of epidermis. Furthermore the mechanisms by which MCG translaction and extrusion are effected may be probed, by the inclusion of such agents as antimicrotubular drugs and calcium ionophores. This work was supported by a grant from Unilever Research, Port Sunlight, England.     

Age-related changes in the ultrastructural organization of the human gingival epithelium

By means of transmissive and scanning electron microscopy 103 gingival bioptates in practically healthy persons at the age of 18-80 years have been studied. At ageing essential changes take place in all structural elements of the epithelium. The basal membrane is intermittent and loose. In cytoplasm of the cells of the basal layer epithelium the amount of microfilaments increases essentially, and as a result it becomes electron opaque. Tonofibrillar fasciculi of the spinous layer cells are fragmented, their contours are indistinct. In cytoplasm of the granular layer cells amount of keratohyalin granules increases, their size becomes large and their typical form is lost. In cytoplasm of the basal, spinous and granular layer cells the amount of organells decreases. Mitochondria acquire the appearance of electron translucent cavities with discomplexic, and sometimes, destroyed cristae. Rather great changes occur in intercellular interrelations. In all the layers some intercellular spaces are widen, in the spaces formed isolated desmosomes and other debries of cellular structures are formed. Sharp changes of microrelief of the granular layer epitheliocytes are observed. The ultrastructural rearrangements of epitheliocytes, revealed in the human gingiva, demonstrate certain disturbances in keratinization processes, in mechanical firmness, as well as in barrier function of the epithelial layer.