Molecular analysis of elastic properties of the stratum corneum by solid-state 13C-nuclear magnetic resonance spectroscopy.

To elucidate the precise molecular mechanisms underlying stratum corneum (SC) elasticity, we investigated the molecular dynamics of chemical residues within keratin fibers of human plantar SC under various conditions by cross polarization/magic angle spinning 13C-nuclear magnetic resonance. The intensities of nuclear magnetic resonance spectra responsible for amide carbonyl, C alpha methine, and side-chain aliphatic carbons in the intact SC decreased markedly with increasing water content of up to 30% in dry SC, and then remained constant at greater than 30%. Lipid extraction of intact SC with acetone/ether (1:1) did not induce any significant change in the nuclear magnetic resonance spectrum, whereas additional treatment with water, which released natural moisturizing factors (mainly amino acids), caused the SC to lose elasticity. The observed decrease in elasticity of the SC recovered after treatment with basic and neutral amino acids, but not after treatment with acidic amino acid. With the latter treatment, movement of amino acid molecules was significantly disturbed, suggesting a strong interaction with keratin fibers. Parallel studies of the complex elastic modulus of a pig SC sheet with a rheovibron also demonstrated that removal of natural moisturizing factor reduced the elasticity of the SC; this effect was also reversed by the application of basic and neutral amino acids, but not by the application of acidic amino acid. These findings suggest that structural keratin proteins, mainly consisting of 10-nm filaments, acquire their elasticity with the help of hydrated natural moisturizing factor via the reduction of intermolecular forces, probably through nonhelical regions between keratin fibers.

Epidermolysis bullosa simplex, Dowling-Meara type. A report of two cases with different types of tonofilament clumping.

Two cases of the Dowling-Meara type of epidermolysis bullosa simplex (EBS) are described. Both had severe blistering at birth, which improved gradually with age. Vesicles and small bullae clustering in a herpetiform fashion were seen in both cases. One showed mild pincer deformity of the nails, and in the other the nail plates were shed after subungual blistering, but regrew without deformity. Histopathology and ultrastructural study showed cytolysis of the basal cells in both cases, but ultrastructurally different forms of tonofilament clumps were present in epidermal keratinocytes. In one case there was typical round clumping of tonofilaments, and in the other a whisk-type clumping of tonofilaments. Cultured keratinocytes from the former produced round clumps of keratin filaments, but those from the latter did not. Review of previous reports of Dowling-Meara EBS revealed that cases could also be divided into two groups in terms of the type of tonofilament clumping at an ultrastructural level. The possibility of subtyping of Dowling-Meara EBS, and possible mechanisms of the blistering in this disease are discussed.

Phorbol ester- and calcium-induced reorganization of 180-kDa bullous pemphigoid antigen on the ventral surface of cultured human keratinocytes as studied by immunofluorescence and immunoelectron microscopy.

The hemidesmosome is an adhesion structure of the epidermal-dermal junction in keratinocytes. When keratinocytes migrate laterally or upward to differentiate, they must control the formation and disintegration of the hemidesmosomes. When keratinocytes are cultured in low-calcium (below 0.1 mM) medium, all cells behave like basal cells, adhere to the culture dish, and proliferate without differentiation. The calcium addition induces the differentiation. A bullous pemphigoid antigen, 180-kDa BPA, has been shown to be a component of the hemidesmosome. Using a monoclonal antibody to the 180-kDa BPA and a human squamous cell carcinoma cell line (DJM-1 cells), the fate of hemidesmosomes was studied after the addition of calcium to low-calcium-grown cells and 12-tetradecanoylphorbol-13-acetate (TPA) to high-calcium (1.87 mM) grown cells by immunofluorescence and immunoelectron microscopy. The antigen was distributed evenly as fine dots on the entire ventral surface of low-calcium cells, whereas they formed a peculiar, concentric ring or arch arrangement on the ventral surface of high-calcium cells. Immunoelectron microscopy revealed the deposits of gold particles at sites on the membrane surface, where some filamentous or electron-dense materials were associated, although the complete structure of hemidesmosomes was not formed. They deposited directly onto the membrane surface in low-calcium cells and with a distance of 20-50 nm from the membrane surface in high-calcium cells. The calcium addition caused a profound reduction of the 180-kDa BPA-positive area for 30 to 120 min and then formed the high-calcium-ring pattern after 4 to 6 h. A similar calcium response was seen in normal human keratinocytes. TPA (16 nM) treatment caused disintegration of the ring pattern in high-calcium DJM-1 cells. This was inhibited with a protein kinase C (PKC) inhibitor. H7 (20 microM). These results suggest that the hemidesmosome is a dynamic structure and PKC can be one of the major factors in controlling the hemidesmosome, since it is known that the low-high calcium shift induces a calcium influx and a PKC activation, and TPA activates PKC in keratinocytes.

[Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex].

The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti-keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of castration on volumes of the preoptic nucleus and the amygdala and on immunoreactivity of LH-RH fibers in the brain of the toad, Bufo japonicus.

We examined the influence of castration on the volumes of sexually dimorphic nuclei, the amygdala pars medialis (Am) and lateralis (Al), and the anterior part of the preoptic nucleus, and on the immunoreactivity of luteinizing hormone-releasing hormone (LH-RH) in brains of Japanese toads captured in spring and autumn. Animals were castrated (GnX) and half were implanted with testosterone (T) (GnX + T) and then killed and dissected after 1 month. Compared with sham-operated (Sham) toads, plasma androgen levels in autumn toads were significantly decreased by the castration, and those in both spring and autumn GnX toads were significantly elevated by the T implantation. The volume of Am in autumn toads was significantly reduced by GnX. Although not significant, changes in the volumes of the other nuclei, except for Al in spring toads, showed the following tendency 30 days after the operation: GnX + T greater than Sham greater than GnX. GnX did not alter LH-RH immunoreactivity in the median eminence. However, dense immunoreactive LH-RH fibers were found in the mesencephalic tegmental region in spring GnX toads but not in the other operation groups in both spring and autumn. LH-RH immunoreactivity was not altered in autumn toads. In spring GnX toads, thumb pads degenerated and evoked release calling was infrequent. These results suggest that (i) the volumes of sexually dimorphic nuclei, especially Am in the autumn toad, are androgen-dependent, and (ii) castration can modulate activity of the extrahypothalamic LH-RH-ergic projection in toad brain.

Extrahypothalamic projection of luteinizing hormone-releasing hormone fibers in the brain of the toad, Bufo japonicus.

Extrahypothalamic projection of luteinizing hormone-releasing hormone (LHRH) fibers in the brain of the toad (Bufo japonicus) was examined immunohistochemically by the avidin-biotin-peroxidase complex (ABC) method. Immunoreactive LHRH perikarya are localized in the nuclei medialis septi and of the diagonal band of Broca. A part of the LHRH fibers are sent anteriad to the medial and dorsal pallia. Some fibers reach the olfactory bulb. Dorsocaudally, LHRH neurons in the medial septum project their fibers to the deep layers of the optic tectum and the posterior mesencephalon including the nucleus pretrigeminalis, which is considered to be a generator of mate calling behavior, via the habenular and posterior thalamic regions. In addition, LHRH fibers which run caudad through the dorsal infundibular region and then the mesencephalic reticular formation were widely distributed in both the gray and the white matter of the medulla oblongata. These findings suggest that LHRH acts as a neurotransmitter or a neuromodulator in the various neuronal circuitries for reproductive behavior in the central nervous system, because LHRH has been considered to be related to amphibian seasonal breeding, and many regions where the immunoreactive LHRH fibers were observed are the loci concerned with mating behavior.

Projections of luteinizing hormone-releasing hormone and vasotocin fibers to the anterior part of the preoptic nucleus in the toad, Bufo japonicus.

The anterior part of the preoptic nucleus (APON) is a crucial locus for triggering male mate calling behavior in anuran amphibia. The projections to this locus of luteinizing hormone-releasing hormone and vasotocin fibers were immunohistochemically demonstrated in the brain of the toad (Bufo japonicus) using the avidin-biotin-peroxidase complex method. Immunoreactive (ir)-LH-RH perikarya are localized in the nucleus medialis septi and the nucleus of the diagonal band of Broca. A part of ir-LH-RH fibers arising from these nuclei project to the lateral part of the APON, where the APON neurons form their dendritic field, and often protrude into its medial neuronal cell mass. Meanwhile, a considerable number of vasotocin fibers arising from the ventral magnocellular part of the preoptic nucleus project anterior to these loci. These observations indicate that the LH-RH and vasotocin fibers may form ordinary and/or en passant synapses with the APON neurons to transmit peptidergic neuronal signals which are concerned with initiation of seasonal reproductive behavior.

An immunohistochemical study of seasonal changes in luteinizing hormone-releasing hormone and vasotocin in the forebrain and the neurohypophysis of the toad, Bufo japonicus.

Seasonal changes in luteinizing hormone-releasing hormone (LH-RH) and arginine vasotocin (AVT) were examined immunohistochemically in the toad forebrains and neurohypophyses. Strongly immunoreactive (ir-) LH-RH perikarya, from which dense ir-LH-RH fibers project to the median eminence, were localized in the medial septal nucleus and the nucleus of the diagonal band of Broca in the animals captured in the spring and the autumn. While, in the animals collected in the summer, ir-LH-RH perikarya and fibers were sparse, and immunoreactivity in the median eminences was weak. Artificially induced hibernation decreased the density of ir-LH-RH in the median eminence, in contrast with strong immunoreactivity in the control animals kept at room temperature. The amounts of ir-LH-RH in the median eminences of hibernating toads which were captured shortly before the breeding period varied conspicuously among individuals. The median eminences in migrating toads showed relatively weak LH-RH immunoreactivity. After the breeding, the immunoreactivity returned to the strong level that was observed in the spring and the autumn. These seasonal changes in ir-LH-RH seem to correspond to seasonal reproductive activity in this species. However, significant seasonal variations were not found in ir-AVT.