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Connectin, also called titin, is the largest protein with a critical function as a molecular spring during contraction and relaxation of striated muscle; its mutation leads to severe myopathy and cardiomyopathy. To uncover the cause of this pathogenesis, zebrafish have recently been used as disease models because they are easier to genetically modify than mice. Although the gene structures and putative primary structures of zebrafish connectin have been determined, the actual primary structures of zebrafish connectin in heart and skeletal muscles remain unclear because of its large size and the PCR amplification-associated difficulties. In this research, using RT-PCR amplification from zebrafish heart and skeletal muscles, we determined the complete primary structures of zebrafish connectin in the I-band region at which mechanical property is modulated by alternative splicing. Our results showed that the domain structures of zebrafish connectins were largely similar to those of human connectins; however, the splicing pathways in the middle-Ig segment and the PEVK segment were highly diverse in every isoform. We also found that a set of 10 Ig domains in the middle-Ig segment of zebrafish connectin had been triplicated in human connectin. Because these triplicate regions are expressed in human leg and diaphragm, our findings may provide insight into the establishment of walking with limbs and lung respiration during tetrapod evolution.

Royal jelly (RJ), the exclusive food for queen bees, is taken as a dietary supplement because it is highly rich in nutrients. However, RJ is known to induce an anaphylactic response in some individuals. We evaluated in the present study the hypoallergenicity of alkaline protease-treated RJ in vitro and in vivo. We first confirmed that this treated RJ contained the same levels of vitamins, minerals and specific fatty acid as in untreated RJ. We then showed that the IgE-binding capacity of the treated RJ was very significantly reduced by conducting in vitro assays of the blood from RJ-sensitive patients. An in vivo skin-prick test on the RJ-sensitive patients also showed that, in the majority of the patients (3 out of 4 tested), the treated RJ did not evoke any allergenic response. It is thus advantageous to prepare hypoallergenic RJ by a protease enzyme treatment for its safe consumption.

The effective uptake of antigens (Ags) by specialized M cells of gut‐associated lymphoid tissues is an important step in inducing an efficient intestinal mucosal immune response. In this study, royal jelly (RJ) was found to stimulate the differentiation of M‐like cells from human Caco‐2 cells in an in vitro M cell model. Furthermore, RJ and protease‐treated royal jelly (pRJ) efficiently enhanced transcytosis of FluoSpheres® carboxylate‐modified microspheres from the apical side to the basolateral side in the model. Therefore, we evaluated the ability of pRJ to induce efficient mucosal immune responses in an in vivo nonhuman primate. Continuous oral administration of commercially available pRJ resulted in a significant enhacement of the antigen‐specific IgA response in stool sample. Interestingly, Caco‐2 monolayer assay demonstrated that ether extracts from pRJ efficiently increased the expression level of a universal M cell marker, glycoprotein 2 (gp2). These findings suggest that pRJ exhibits mucosal immunomodulatory properties via stimulation of effective uptake of Ags through M cells.

In a previous study (Y. Kimura et al., Biosci. Biotechnol Biochem., 70, 2583-2587, 2006), we found that new complex type N-glycans harboring Thomsen-Friedenreich antigen (Ga1 beta 1-3GalNAc) unit occur on royal jelly glycoproteins, suggesting the involvement of a new beta 1-3galactosyltransferase in the synthesis of the unusual complex type N-glycans. So far, such beta 1-3galactosyltransferase activity, which can transfer galactosyl residues with the beta 1-3 linkage to beta 1-4 GalNAc residues in N-glycan, has not been found among any eucaryotic cells. But using GalNAc(2)GlcNAc(2)Man(3)-GlcNAc(2)-PA as acceptor N-glycan, we detected the beta 1-3 galactosyltransferase activity in membrane fraction prepared from honeybee cephalic portions. This result indicates that honeybee expresses a unique beta 1-3 galactosyltransferase involved in biosynthesis of the unusual N-glycan containing a tumor related antigen in the hypopharyngeal gland.

Melinjo (Gnetum gnemon L.) seed extracts (MSEs) are rich in resveratrol dimers (gnemonoside A, C, D, gnetin C), trans-resveratrol, and other resveratrol derivatives. trans-Resveratrol is a widely studied caloric restriction mimetic. In mice fed a high-fat diet (HFD), trans-resveratrol protects against obesity, type 2 diabetes, and premature death. Here, treatment of HFD-fed mice with 2.0% MSE significantly reduced body weight gain (p

The effects of bee pollen extract on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats in vitro and in vivo were investigated. Bone tissues were cultured for 48 hr in serum-free Dulbecco's modified Eagle's medium containing either vehicle or water- or ethanol-solubilized extracts (10, 100, or 1000 μg/ml of medium) obtained from the bee pollen of Cistus ladaniferus. Calcium content in the femoral-diaphyseal or -metaphyseal tissues was significantly increased in the presence of water-solubilized extract (100 or 1000 μg/ml) and ethanol-solubilized extract (1000 μg/ml). An increase was also observed in the presence of water-solubilized extract (100 μg/ml) obtained from Fagopyrum esculentum, Camellia sinesis, or Brassica napus L. Alkaline phosphatase activity and DNA content in the femoral-diaphyseal or -metaphyseal tissues in vitro were significantly increased in the presence of water-solubilized extract (100 or 1000 μg/ml) obtained from the bee pollen. The effects of the bee pollen extract (100 μg/ml) in increasing bone components were completely inhibited in the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis, in vitro. Moreover, the calcium content and alkaline phosphatase activity in the femoral-diaphyseal or -metaphyseal tissues were significantly increased by the oral administration of water-solubilized extracts (5 or 10 mg/100 g body weight) obtained from the bee pollen of Cistus ladaniferus once daily for 7 days. The DNA content in the diaphyseal or metaphyseal tissues was significantly increased by the oral administration of water-solubilized extract (10 mg/100 g) of bee pollen cistus. The dose of 1.0 mg/100 g caused a significant increase in the diaphyseal and metaphyseal alkaline phosphatase activity or the metaphyseal DNA content in vivo. This study demonstrates that the extract of bee pollen has an anabolic effect on bone components in rats in vitro and in vivo.

The bee pollen Cistus ladaniferus (C. ladaniferus) extract has an anabolic effect on bone metabolism. The effects of the fractionated extracts obtained from bee pollen on bone calcium content and osteoclast-like cell formation in vitro were investigated. Rat femoral-diaphyseal and -metaphyseal tissues were cultured for 48 hr in a medium containing either vehicle or a water-solubilized extract with the membrane fractions obtained from bee pollen. The active component of bee pollen in increasing calcium content in diaphyseal tissues was seen in the fraction of molecular weight (MW) of less than 1000, and it was not observed in fractions of greater than MW 1000. Culture with the fractionated bee pollen extract (25 or 50 µg/ml of medium) of less than MW 1000 caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. The parathyroid hormone (PTH; 10 ‐6 M)induced decrease in diaphyseal calcium content was significantly prevented in the presence of the fractionated bee pollen extracts (10 µg/ml) of less than MW 1000 or greater than MW 1000. Mouse marrow cells were cultured for 7 days in a medium containing PTH (10 ‐6 M) in the presence or absence of the fractionated bee pollen extract (10 or 50 µg/ml). The PTH-induced increase in osteoclast-like cell formation was markedly suppressed in the presence of extracts of less than MW 1000 as compared with that in the presence of fractions of greater than MW 1000. The effects of the fractionated bee pollen extracts of less than MW 1000 in increasing diaphyseal calcium content and in inhibiting PTH-induced osteoclastic cell formation were significantly decreased upon heat treatment for 20 and 60 min at 80°C. This study demonstrates that the active component of bee pollen C. ladaniferus extract, which stimulates bone formation and inhibits osteoclastic bone resorption, is the fraction with MW less than 1000.

The effect of the combination of beta-cryptoxanthin and zinc sulfate (zinc) on bone components in the femoral-diaphyseal and -metaphyseal tissues of young rats in vitro was investigated. Bone tissues were cultured for 48 h in a serum-free Dulbecco's modified Eagle's medium containing either vehicle, beta-cryptoxanthin (10(-9)-10(-7) M) or zinc sulfate (10(-6)-10(-4) M). The presence of beta-cryptoxanthin (10(-9) M) or zinc (10(-6) M) did not have a significant effect on calcium content in the femoral-diaphyseal or -metaphyseal tissues. However, culture which combined beta-cryptoxanthin (10(-9) M) and zinc (10(-6) M) caused a significant increase in calcium content in the femoral-diaphyseal and -metaphyseal tissues. Such an effect was not observed by the combination of beta-cryptoxanthin (10(-9) M) plus genistein (10(-6) M) or menaquinone-7 (10(-6) M), or zinc (10(-6) M) plus genistein (10(-6) M) or menaquinone-7 (10(-6) M). Also, the combination of beta-cryptoxanthin (10(-9) M) plus zinc (10(-6) M) caused a remarkable increase in alkaline phosphatase activity and deoxyribonucleic acid (DNA) in the femoral-diaphyseal and -metaphyseal tissues, while their application alone did not have an effect on the enzyme activity or DNA content in the femoral tissues. The effect of the combination of beta-cryptoxanthin (10(-9) M) plus zinc (10(-6) M) in increasing calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues was completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DBR), an inhibitor of transcriptional activity. This study demonstrates that the combination of beta-cryptoxanthin and zinc at a lower concentration has a synergistic effect on bone components in vitro.

Mutations in the EDA gene cause anhidrotic ectodermal dysplasia (EDA), with lesions in skin appendage formation. To begin to analyze EDA pathways, we have used expression profiling on 15,000-gene mouse cDNA microarrays, comparing adult mouse skin from wild-type, EDA-defective (Tabby) mice, and Tabby mice supplemented with the EDA-A1 isoform, which is sufficient to rescue multiple Tabby phenotypes. Given the sensitivity of the current microarray system, 8500 genes (60%) were estimated to be expressed, including transcription factors and growth-regulatory genes that had not previously been identified in skin; but only 24 (0.16%), one-third of them novel, showed significant differences between wild type and Tabby. An additional eight genes not included in the 15,000 gene set were shown to have expression differences by real-time RT-PCR. Sixteen of 32 affected genes were restored significantly toward wild-type levels in EDA-A1 transgenic Tabby mice. Significant up-regulation in Tabby skin was observed for several dermal matrix genes, including Col1a1, Col1a2, Col3a1 and SPARC: In contrast, down-regulation occurred for the NEMO/NF-kappa B pathway, already implicated in skin appendage formation, and even more markedly for a second pathway, JNK/c-jun/c-fos and their target genes, that has not previously been clearly associated with skin development. These data are consistent with the regulation of the NF-kappa B pathway by EDA, and support its involvement in the regulation of the JNK pathway as well.

The cDNA which encodes the mouse counterpart of human plasma hyaluronan-binding protein (PHBP) was isolated and characterized. The clone contained an insert of 2153 bp, which contained the 1674-bp open reading frame coding for a polypeptide of 558 amino acid residues. The amino acid sequence of mouse PHBP predicted from the nucleotide sequence of cDNA shows reasonable homology to that of human PHBP. Like human PHBP, the amino acid sequence predicted from the nucleotide sequence of mouse PHBP cDNA exhibited significant homology to that of human hepatocyte growth factor activator (HGFA).

Human scalp skins were treated with 20 mM ethylenediaminetetraacetic acid and terminal hair follicles were extracted with the epidermis. Some terminal hair follicles were morphologically preserved well and provided opportunity to examine three-dimensional distribution of CAM5.2 (K8, 52.5 kD) reactive Merkel cells. In anagen terminal hair of the scalp numerous immunoreactive Merkel cells were distributed in the presumptive bulge area. Distinct swelling as in the bulge of human vellus hair was usually absent; however, in rare instances anagen terminal hair demonstrated unilateral prominent swelling with dense aggregation of Merkel cells. In telogen hair the bulge becomes indistinguishable from the regressed end of the club hair follicle but Merkel cells continued to be abundant. We found morphologic variation of the bulge such as formation of knoblike swellings and villous projections. Interestingly, Merkel cells were also located in these structures. Palisading stockade-like nerve endings were observed surrounding the follicular epithelium at the sebaceous gland level. Merkel cells were sparse in this follicular segment. Variable number of Merkel cells were also scattered in the infundibulum of terminal hair in no association with peripheral nerves.

The concept of sequential cytotoxicity, which states that successive chemical attacks on cellular constituents can be more deleterious to neoplasms than normal cells, was evaluated using a series of 3,5-bis(benzylidene)-1-diethylphosphono-4-oxopiperidines 1 and related phosphonic acids 2, which were screened against a panel of malignant and normal cell lines. The compounds proved to be not only potent cytotoxins (71% of the CC(50) figures are submicromolar) but to display greater cytotoxicity to the neoplastic cells. QSAR revealed that both cytotoxic potencies and selective toxicity were increased by a rise in the electron-withdrawing properties and a decrease in the hydrophobicity of the aryl substituents. Utilisation of the PL10 concept and evaluation of druglike properties revealed 1c as the lead tumour-specific cytotoxin. This molecule activated caspase-3 in HL-60 cells but not in the HSC-2 cell line. While 1c caused internucleosomal DNA fragmentation in HL-60 cells, it did not elicit this effect in either HSC-2 and HSC-4 cells. Clearly 1c exerts its cytotoxic potencies by different mechanisms and such pleiotropy is likely the principal reason for the remarkable display of preferential toxicity towards malignant cells of the compounds in series 1 and 2.

The origin of Merkel cells is uncertain, although current evidence by immunohistochemical keratin marker studies favors an epidermal derivation. We studied the expression of keratin species in Merkel cells of human fetus and adult using 19 anti-keratin antibodies. Epidermal and dermal Merkel cells contained not only simple epithelium-type but also some stratified epithelium-type keratins. Interestingly, expression of some keratins was different between epidermal and dermal Merkel cells, for example, AN3 (50, 58 kD) and CKB1 (50 kD) recognized epidermal Merkel cells, but not dermal Merkel cells. These results suggest that surrounding keratinocytes influence the expression of cytokeratins in Merkel cells or that dermal Merkel cells undergo a modification from keratin-producing epidermal Merkel cells to a more neural cell type by the association with nerve endings in the upper dermis. On the other hand, certain cytokeratin polypeptides recognizable with Ks19.1 (40 kD), CK5 (45 kD), and CAM5.2 (52.5 kD) were expressed in both epidermal and dermal Merkel cells. The expression of simple epithelium-type keratins in Merkel cells remained even after the epidermal basal cells gradually lost their expression.

Tetradecane (TD), testosterone (TS), and dimethyl sulfoxide (DMSO) were separately inuncted on rabbit pinnas once a day; the pinnas were biopsied on days 1, 3, 7, and 28. Untreated pinnas and squalane-treated pinnas were used as controls. Three-dimensional images of sebaceous glands were reconstructed from their serial histologic sections using a computer-image analysis system. By stereometry, the sebaceous gland volumes gradually increased during the course of treatments with TS and DMSO, whereas the volumes of TD-treated sebaceous glands increased, reached the maximum on day 7, and then decreased. The acinus number was increased by all the substances, the DMSO-treated sebaceous glands on day 28 showing the largest number of acini. The volumes of individual acini increased until day 28 with TS treatment, whereas with TD and DMSO treatments they increased, reached the maximum on day 7, and then decreased. Stereographically, each untreated control sebaceous gland was composed of 20-30 pear-shaped acini. On day 28, acini of TS-treated sebaceous glands were swollen and ovoid in shape, DMSO-treated sebaceous glands resembled a bunch of grapes, and acini of TD-treated sebaceous glands were widely scattered and showed globular swelling arising from an elongated tube. Hyperplastic responses of sebaceous glands are quite different according to substances applied topically.

The plucked hairs and biopsied hair follicles of the scalp were obtained from a female patient with monilethrix. By scanning electron microscopy, the plucked hairs showed a typical moniliform feature composed of alternated nodes and inter- nodes. By computer stereography, reconstructed three-dimensional models of in vivo hair structures showed that the diameter of hair shaft was partially reduced in the keratogenous zone and that the reduction was severe in the hair cortex and cuticle layers but mild in the inner root sheath. By transmission electron microscopy, a significant degeneration of hair matrix cells was found, and a zig-zag disarray of cortical tonofibrils and invaginations of the hair cuticle cells into the cortex were noticed in the suprabulbar portion. In some hairs, the hair bulbs showed no degeneration, but a degeneration of cortical cells and invaginations of hair cuticle were seen in some portions in the developing zone of the cortex. This suggests that not every hair matrix is damaged in a synchronized fashion, but individual hairs are affected independently. Furthermore, in all the hair and hair follicles examined, cytoplasmic vacuolations were always seen in the various layers, and abnormal formation of tonofibrils was often observed in the cortex. In monilethrix, a cell abnormality may inherently be present in the hair tissue and, when such abnormality occurs severely, the cortical cells are particularly affected in the hair matrix. This seems to result in. a decrease in number of cortical cells and thinning of the hair shaft.

Objective: To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM). Methods and results: We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30–50μm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 ± 5.4min (mean ± S.D., n = 61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 ± 3.2min (mean ± S.D., n = 53 monocytes). Conclusions: Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.

The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.

Multiple familial trichoepithelioma (MFT) is an autosomal dominant skin disease characterized by the presence of many small tumors predominantly on the face. To map the causative gene, we performed linkage analysis with microsatellite markers in three American families. We found a significant linkage of a gene for MFT to chromosome 9p2l. The maximum combined lod score was 3.31 at D9S171 at theta = 0. The disease locus was defined to a 4-cM region between IFNA and D9S126. Because several tumor suppressor genes including p16 and p15 have been mapped to this region, the gene for MFT may also be a tumor suppressor.

The cDNA encoding for human liver isozyme I of biliverdin-IX beta reductase was cloned from human liver cDNA libraries. The constructed cDNA of 853 bp in length contained an entire reading frame coding 206 amino acid residues. It was found that isozyme 1 of biliverdin-IX beta reductase is identical to human erythrocyte NADPH-flavin reductase recently reported in a communication by Chikuba et al., Biochem. Biophys. Res. Commun., 198, 1170-1176 (1994). We, further, characterized this mRNA by Northern blot analyses of poly(A) RNA from four different human fetal tissues and eight different human adult tissues showed hybridization mainly to liver RNA in fetal tissues and to skeletal muscle and liver RNA in adult tissues. Southern blot analysis indicated that isozyme I of biliverdin-IX beta reductase appeared to be a single copy gene. Insertion of the enzyme-coding sequence into an expression vector pET-3c yielded relatively high amounts of the active enzyme in E. coli. The amino terminal sequence of the recombinant protein was identical to that of native enzyme, indicating that E. coli also removed the N-terminal methionine to produce the mature form. The recombinant and native biliverdin-IX beta reductases were indistinguishable as far as their mobility on SDS-PAGE gel, immunoreactivity and specific activity were concerned.

The expression of a cell proliferation-associated human nuclear antigen was immunohistochemically studied in human anagen hair and hair follicles using the monoclonal antibody Ki-67. The reaction of Ki-67 in mature anagen hair follicles was observed in the hair matrix cells and outer root sheath (ORS) cells. Nuclear staining was seen in a small number of matrix cells and in some ORS cells; this finding corresponded to the thymidine or bromodeoxyuridine labeling studies previously reported. In addition, there were two different patterns of cytoplasmic staining in the ORS: strong staining of the innermost cells (IMC) and weaker staining of the other ORS cells in the isthmus. Ki-67 reactivity of the IMC layer was observed at each stage of anagen and was regularly seen from the upper bulb to the isthmus. Ki-67 is a commercially available antibody that detects cycling cells. However, the IMCs in anagen hair follicles showed cytoplasmic labeling by Ki-67 from the matrix cells in the upper bulb to the distal portion of the isthmus.

The mechanism of formation of trichorrhexis invaginata, which is one of the characteristic hair anomalies seen in Nether ton's syndrome, was studied using plucked hairs obtained from 3 patients with the typical clinical features. The biopsy specimens used to examine the hair root were obtained from the scalp of one of the patients and the eyebrow area of the other. The scanning electron microscopic observation of plucked hairs showed trichorrhexis invaginata, tortion nodule, pili torti, and trichorrhexis nodosa. The light microscopic sections of nodules of plucked hairs revealed invaginated hair cuticle into the cortex. Transmission electron microscopy revealed cleavages and electron-dense depositions in the cortex of plucked hair nodules; such changes were not seen in newly formed nodules in follicles in biopsy specimens. In the keratogenous zone, a zigzag pattern of cortical fibers and invaginations of hair cuticle cells into the cortex were observed. Histochemical staining for -SH groups and S-S linkages using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM) was performed on frozen sections of plucked hairs from patients and normal controls. Keratinized cortical cells of patients possessed more -SH groups than the controls and a strong fluorescence for -SH was sometimes seen in the cortex of nodules. It is concluded that the invagination of affected hairs is caused by softness of the cortex in the keratogenous zone, which may result from an incomplete conversion from -SH to S-S in proteins in cortical fibers.

The hair follicles and the eyes of pallid mice (C57/6J-Pa/Pa) and those of black mice (C57/6J−+/Pa) were examined ultrastructurally, histochemically, and biochemically to determine the cause of pigment dilution. The pigment cells in the hair follicles and the eyes of pallid mice have less mature melanosomes than those of black mice. In the hair follicles the pallid melanosomes were transferred into keratinocytes and became aggregated. In the eyes they were already aggregated within the pigment cells and were digested in acid phosphatase-positive lysosomes. The activity of acid phosphatase, a marker of lysosomal enzymes, was significantly higher in pallid hair follicles and eyes than in black hair follicles and eyes. Dopa reactions at light and electron microscopical levels indicated that the pigment cells in each tissue produced a large amount of Dopa oxidase when compared with those in each black ocunterpart. However, the rate of hydroxylation of L-tyrosine–3,5– 3 H was significantly lower in the pallid eyes than in black eyes, while this rate was significantly higher in pallid hair follicles than in black hair follicles. Immediate digestion of melanosomes within the pigment cells, i.e. autophagocytosis, seemed to explain the low activity in the pallid eyes. The diluted coat and eye colors of pallid mice are, therefore, not related to low Dopa oxidase activity but to immaturity of melanosomes and high activities of lysosomal enzymes; these enzymes seem to digest many of these immature melanosomes and contribute to the diluted coat and eye colors of pallid mice.

A new fluorogenic maleimide DACM, i.e., N(7-dimethyl amino-4-methyl coumarinyl) maleimide, does not fluoresce by itself. It is specifically combined with --SH groups and becomes fluorescent (lambda ex: 400 nm, lambda em: 485 nm). At this range of excitation the frozen skin section has no native fluorescence because the emission maximum of DACM does not overlap with any of aromatic residues of proteins such as tryptophane. S--S groups can be demonstrated with DACM by inhibiting native --SH and then reducing SS to --SH. --Sh was generally abundant at the bulb region. --SH of hair cortex was more concentrated at keratogenous zone. Further up in the follicle, --SH of hair cortex was gradually decreased, although at the region of isthmus, --SH reaction of the hair cortex was still moderately strong. Outer root sheath had --SH from the upper bulb to the surface of the epidermis. One the other hand, no S--S was demonstrable at the bulb region. At the level of keratogenous zone, however, S--S linkages of hair cortex and inner root sheath began to appear and further up in the follicle S--S linkages were increased gradually. Outer root sheath had no S--S linkages by DACM staining up until it is keratinized along the hair canal in the upper follicle. The concentration of --SH and S--S thus seemed to be reciprocal; --SH is found in non-keratinized tissues, whereas S--S is abundant in keratinized areas. These findings are at some variance with conventional data, which suggest, for example, that --SH groups disappear suddenly above the keratogenous zone.

Immunoglobulin heavy and light chains and C3 were detected on amyloid deposits in cutaneous nodular amyloidosis (NA) by a direct immunofluorescence method. These immunoglobulin chains and C3 could be removed except lambda (lambda) light chain after treating sections with 0.05% Tween 20 solution, 0.1 M glycine buffer pH 7.4 or 0.5% Triton X solution. Lambda light chain was detected consistently after these treatments. Amyloid filaments were not altered with these treatments. Immunoelectron microscopy demonstrated persistent antigenicity of lambda light chain on amyloid filaments. It was suggested that lambda light chain is an inherent component of amyloid filaments in NA, whereas other chains and complement represent nonessential absorption on amyloid material.

Cross-banded filamentous aggregations (CBFA) with a periodic banding pattern (850–1250 A) have been found frequently in the stroma of squamous cell carcinomas and malignant melanomas of the skin. In these tumors, the aggregations were commonly associated with the basal lamina and abnormal fibrils. No CBFA was found where collagen fibrils did not show degradative changes. Less frequently, CBFAs were found in the skin lesions of primary and secondary amyloidoses, in inflammatory foci of a halo nevus, and in other skin lesions. CBFAs were similar to the Luse's bodies which have previously been found in normal and abnormal tissues of the central nervous system. Although some investigators have equated the aggregates with the fibrous-long-spacing (FLS) type of collagen, FLS collagen prepared in our laboratory showed a longer periodicity (2,200 A) than that of CBFA. Staining with ruthenium red significantly increased the electron density of CBFA, suggesting that CBFA contains acid mucopolysaccharides.

Histochemically identifiable amyloid associated with actinic keratosis was studied with the electron microscope. Typical amyloid filaments forming islands identical to collagen strands were found in the upper dermis. Beyond the lesion of actinic keratosis, no amyloid was found. In the healing wound of a previously biopsied lesion, amyloid was regenerated only when the epidermis covered the wound. In such epidermis-covered granulation tissue, electron-dense amorphous substance was initially formed in the upper dermis and amyloid filaments subsequently emerged within it. In both original and regenerated lesions, perivascular spaces were free from amyloid, and actinic elastosis was absent. It was concluded that secondary amyloidosis associated with actinic keratosis is produced by local fibroblastic cells under an abnormal influence of the epidermis.

We studied the recovery phase of immune response-associated (Ia)-positive or ATPase-positive epidermal Langerhans cells (ELCs) after ultraviolet B (UVB)-induced depletion by using mouse ear epidermal sheets. An area 3 mm in diameter was irradiated with 300 nm UVB light (40 mJ/cm2). A time sequence study was carried out to 56 days. During this period the Ia-positive ELC population increased stepwise, i.e., first a rapid increase between day 7 and day 14, which we called the early recovery phase, and next a gradual increase between day 42 and day 56, which we called the late recovery phase. During the early recovery phase, we found polymorphous ELCs in the irradiated area which were giant or normal in size, dendritic or round in shape, and single or paired in distribution. Electron microscopy revealed some of round and some of paired ATPase-positive ELCs to be in metaphase or telophase of mitotic division. Within the entire observation period of our study, there was no evidence suggesting migration of ELCs from hair follicles or from the nonirradiated epidermis. These findings indicated that mitosis of ELCs contribute to their repopulation during the early recovery phase.

The mouse monoclonal antibody AFH1 was produced using formalin-fixed, sham paraffin-embedded human melanoma cell culture line A375 as immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against formalin- or acid alcohol-fixed paraffin-embedded tissue as well as formalin- or acid alcohol-fixed unembedded lesions. Ninety-seven nevomelanocytic lesions, neurofibromas, epithelial lesions, and a plasmacellular infiltrate were evaluated. AFH 1 was immunoreactive on 54 of 55 nevocytic lesions (98.2%), 15 of 16 primary melanomas (93.7%), a lentigo maligna, and nests in 21 of 21 dysplastic nevi (100%). Of 100 consecutive basal melanocytes of intraepidermal melanoma cells counted in each lesion, mean AFH1 immunoreactivity for nonnested basal melanocytes in nevocellular nevi was 3.8%; for dysplastic nevi, 13.8%; and for intraepidermal melanoma cells, 78.0%. When nonnested basal melanocytes were subdivided into cytologically normal and abnormal cell groups, AFH1 immunoreactivity was 9.4% and 72.6%, respectively. AFH1 recognition of the lentiginous portion of dysplastic nevi corresponds statistically to the appearance of abnormal melanocyte cytology, nest formation, or both. Using 50% immunoreactive nonnested melanocytes as the criterion, AFH1 seems to distinguish primary melanoma from dysplastic nevi with a sensitivity of 93.8% and a specificity of 95.8%.

A new monoclonal antibody NKH-1 was developed using human subepidermal basement membrane zone substances as immunogen. NKH-1, IgG1 kappa light chain, labeled proteins in the subbasement membrane zone in a linear fashion. It also labeled oxytalan fibers and elaunin fibers in the papillary dermis. Mature elastic fibers were labeled only in their peripheral microfibrils (elastofibrils) and the center core of elastin was nonreactive. Basal lamina itself was not decorated with NKH-1 even at the immunoelectron microscopic level. Skin appendages such as eccrine and apocrine glands, arrector pili muscle, hair follicle, and sebaceous gland were surrounded with NKH-1 -positive substances. This substance was in linear fashion closely associated with the basal lamina but deposited linearly outside of it. Species specificity tests were performed only in nonprimates: mouse and guinea pig skins were nonreactive with NKH-1. NKH-1 seems to recognize a new substance in the sub- basal lamina region (subbasal lamina proteins) which cross-react with elastic fiber microfibrils.

The apocrine duct extends from the apocrine secretory segment to the pilosebaceous follicle. Because apocrine glands are limited to a few areas, and even in these areas are few in number, the structure of their ducts has been largely ignored in descriptions of the ultra-structure of the epidermis and its appendages. Charles (1) and Zelickson (2) have briefly described the apocrine duct in their studies which were concerned primarily with the apocrine secretory segment.

This study was undertaken in order to describe the fundamental differences between normal skin and the oral mucosa mainly from the standpoint of keratinization.A brief survey of the literature dealing with the human oral mucosa reveals that Sognnaes and Albright (1,2), Fasske and Themann (3), and Zelickson and Hartmann (4) have done electron microscopic work on the human oral epithelium, Listgarten (5) and Kurahashi and Takuma (6) on the human gingival epithelium, Melcher on the oral connective tissue (7), and Frithiof and Wersall on some organelles (8). Until now, however, no electron microscopic work has been done on the posterior buccal mucosa of the human oral cavity, although the anterior buccal mucosa has been studied by Zelickson and Hartmann (4); and there has been no comparative study of the human mucous membrane with either adult or embryonic human skin.In this study it was found that the normal human posterior buccal mucosa is very similar to the immature embryonic epidermis in all aspects; and that despite a complete lack of keratinization, the plasma membranes of the posterior buccal epithelial cells in upper layers become considerably thicker than those of the lower layers, thus suggesting that this thickening process is a phenomenon independent of keratinization.MATERIALS AND METHODSBiopsy specimens were taken from the normal human buccal mucosa, posterior or lateral to thesecond molar tooth, of ten healthy white men and women between the ages of 17 and 50. Five specimens were obtained from the oral lesions of pemphigus vulgaris, and one from the lesion of stomatitis of the posterior buccal mucosa. All specimens were immediately sliced into small pieces, fixed in 1% osmic acid in veronal buffer (pH 7.2) containing 4.5% sucrose. After fixation for one hour, the tissues were dehydrated through a series of graded alcohols and propylene oxide, and were embedded in Araldite. Thin sections were then cut with an LKB Ultrotome at 400-600 A. The sections were stained first with 1% uranyl acetate in 50% etha-nol, and then, before completely dried, re-stained with lead citrate (9). Some specimens were stained for acid phosphatase before osmic acid fixation using the method of Barka and Anderson (10).RESULTSBasement membrane and basal layer. The basement membrane measured approximately 600 A in thickness. Occasionally, phagocytes, lymphocytes, and leukocytes were seen crossing the basement membrane (Fig. 1). In such areas the subepithelial reticular fibrils were thick, and re-duplication of the basement membrane, as reported by Caulfield and Wilgram in erythema multiforme (11), was observed (Fig. 1). The basal cells here contained fewer tonofilaments than the epidermal basal cells (Fig. 1). Intercellular spaces were rather wide, with numerous slender villi of the basal cells projecting into them (Fig. 1). Some basal melanocytes contained well-developed, rough-surfaced endoplasmic reticulum and were actively producing all gradients of maturation of melanin granules in their melanosomes (Fig. 2). The dendritic processes of these melanocytes, found between the basal cells and squamous cells, contained a greater number of more mature (larger and denser) melanin granules than did the body of the melanocytes. This phenomenon can be explained by assuming that the melanocytes inject their melanin granules into the surrounding cells as soon as the granules reach maturity. The basal cells and the squamous cells contained varying numbers of injected melanin granules (Fig. 2).As the basal cells moved up into the squamous512ULTRASTRUCTURES OF THE BUCCAL MUCOSA513*-i

With the advent of the electron microscope, investigators of elastic fiber have used two major approaches: first, the method of teasing out tissue fragments with or without additional shadow casting technics (replication method) (1-7), and second, that of using tissue sections (8-14). Several workers in each group have shown interest in the elastic tissue of the skin, either normal (2-4, 6, 10), or abnormal (9, 11-13).

Calcifying epithelioma is a rare, solitary tumor which often occurs in young individuals (1), most frequently on the face and the arms (1, 2). Since Malherbe and Chenantais (3) described “calcified epithelioma of sebaceous glands” in 1880, the histogenesis of this tumor has been attributed to various skin appendages such as sebaceous glands (3, 4) and hair (2, 5, 6, 7). Lever and Griesemer (7) regarded the basophilic cells in calcifying epithelioma as primary epithelial germ cells with a tendency to differentiate into keratotic hair cells. Forbis and Helwig (1), in their recent study on 240 specimens, agreed with the opinion of Lever (2) and Lever and Griesemer (7) that calcifying epithelioma arises from primitive cells of the hair matrix. They pointed out that the broad dense transitional zone from basophilic to shadow cells of this tumor, where sulfhydryl groups are abundant, is analogous to the kera-togenous zone of the hair. They also demonstrated that hyalinized shadow cells in some instances show a brilliant birefringence, as does the keratogenous zone of the hair.

As an extension of our histochemical studies of the skin (1, 2), the distribution and activity of cholinesterases were studied in the skin of the rat. The development of cholmesterase activity was followed during the histogenesis of the skin, with special reference to the nerve elements.

Although there have been several electron microscopic studies of human axillary apocrine glands (1, 2, 3, 4) the exact mechanism of apocrine secretion remains an enigma. Hibbs (1) denied true apocrine secretion in normal human axillary apocrine glands because he was unable to find the secretory cells in the process of extruding its contents. In addition, he could not visualize any secretion product within the lumen. He assumed, therefore, that the principal mode of secretion is by droplet formation at the apex of the cell and that secretion by disruption of the cell probably occurs only under extreme conditions, if at all. Montagna (2) suggested that the secretory granules are dissolved in the apical cytoplasm and excreted either by a fragmentation of the luminal microvilli or by oozing out through the plasma membrane. Charles (3) also failed to see cellular debris in the lumen, but did report disruption of the apical membrane through which he thought the contents of the secretory cell had been extruded. Observations similar to Mon-tagna's, e.g., that the secretory granules are dissolved in the apical cytoplasm, were made by Kurosumi et al. (4) ; they further described the formation of expanded apical secretory projections and were inclined to defend the classical decapitation secretion theory. They denied that secretion occurred through fragmentation of the luminal microvilli. The following report will be concerned primarily with the maturation of the apocrine secretory granules and their mechanism of excretion from the cell. A multiplicity of secretory mechanisms of the apocrine glands was found in this investigation, including decapitation secretion.

A glomus tumor of the multiple type was examined by electron microscopy. Except for a single layer of endothelial cells lining the vascular lumen, the remainder of the tumor cells are smooth muscle cells. What have been called “glomus” or “epithelioid” cells in the past are transversely sectioned smooth muscle cells. There is no ultrastructural similarity to the hemangiopericytoma.

Electron microscopic studies were performed on the hair cone and intraepidermal hair canal formation in the scalp and eyebrow skin using 11 Negro embryos of the menstrual ages of 9 to 18 weeks. While most of the previous observations of light microscopists were confirmed, information on several fine structural features was added. (1) The hair cone formation represented the very initial keratinization of the cells of future Henle and Huxley layers. These cells were connected by desmosomes, tight junctions, and intercellular cement. (2) The intraepidermal portion of the hair canal was formed by keratinization and vacuolization of the organized hair canal matrix cells. Lysosome-like dense bodies were present and thought to play a role in self-destruction of the matrix cells. (3) The cellular envelopes of the hair cone cells and of the wall cells of the hair canal became twice as thick as the plasma membranes of their matrix cells. An 80–100 A thick cytoplasmic band, i.e., the newly synthesized marginal band, was found to be responsible for this thickening.

Merkel cells identical to those described in the skin were found in the gingival mucosa and in the vermilion border of the lip. They were often clustered in rete ridges but also found individually in the basal layer. Unmyelinated axons (neurites) of presumably a sensory afferent nerve surrounded the basal half of these cells within the epithelium. Occasional desmosomes connected the Merkel cell to the neighboring keratinocytes, but neither desmosomes nor other junctional devices were found between the Merkel cell and the neurite. The Merkel cell is characterized by membrane-surrounded dense granules, measuring between 800 and 2000 A, which seemed to derive from Golgi saccules. Some Merkel cells contained lysosome-like dense bodies and transferred melanosomes.

Animal Material. Twenty-five rats of the Wistar strain were used. Twelve of these were embryos (3 each on the 12th, 14th, 16th and 18th days of gestation), 2 were new-born (the period of gestation varied from 19 to 21 days), 3 were ten-day-old, 3 were fifteen-day-old and 5 were adults. The adult rats weighed approximately 100 g. New-born and older rats were killed by a blow on the neck without anesthesia. Hair was shaved off the back, and skin specimens were taken from there and cut into small square pieces. These pieces were immediately immersed for about five minutes into ice-cold 0.1 M Sorensen's phosphate buffer and then used for frozen sections. (The buffer had a pH of 7.6 for the study of the succinic dehydrogenase system and a pH of 7.4 for the study of the malic and lactic dehydrogenase systems). In embryos, skin was removed from the back while they were still alive. Frozen sections, approximately 20 in thickness, were made from the fresh skin specimens. Histochem feat Demonstration of the Succinic Dehydrogenase System (SDS). Ogawa and Zimmerman's method (7) was employed to demonstrate the succinic dehydrogenase system. As a rule, nitroneotetrazolium chloride (nitro-NT) was used as an electron acceptor. Occasionally, however, nitro-blue tetrazolium (nitro-BT) was used, instead.

Perifollicular nerve endings of the human lanugo hairs have been studied with the electron microscope. The endorgans are arranged in palisade along the periphery of the follicle and are composed of an axon and an enveloping Schwann sheath. Spaces between the endorgan are largely free from collagen and cellular elements. This arrangement of endorgans perpendicular to the follicle may be most efficient to detect the movement of the follicle, and the empty spaces between the organs permit a maximum deformation of the terminal axon. The axon produces a subcytomembrane dense layer at the follicular end but is, with rare exceptions, separated from the external root sheath cells by the basal lamina. The endorgan can attach to any portion of the follicle including the cellular junctions, indicating that there is no specialization on the follicular side at the site of attachment. In fact, half-desmosomes and subbasal dense plaques are regularly seen on the basal surface of the follicular basal cells.

The formation of the intradermal eccrine sweat duct lumen began in the 15-week-old embryo with a dissolution of the desmosomal attachment plaques, followed by the separation of apposing inner cells and subsequent formation of microvilli at the luminal surfaces. The luminal cells possessed numerous microvilli and crypts and a periluminal band of tonofilaments. In the secretory portion of older embryos (22 weeks old), columnar secretory cells extended from the basement membrane to the luminal border. The presence in these secretory cells of microvilli, Golgi elements, mitochondria, small secretory vesicles, dense secretory granules and abundant endoplasmic reticulum, implies that preparation for secretory functioning begins in embryonic life. The pyramidal myoepithelial cells differentiated from the basal cells seen in younger embryo, and rested upon a basement membrane. In the oldest embryo examined (22 weeks) there was as yet neither intercellular canaliculi nor sufficient difference in the amount of glycogen to enable one to distinguish between dark and light secretory cells. Isolated cilia of 9 + 0 type were occasionally found in the luminal cells of the duct and the secretory segment.