Extent of laminin-5 assembly and secretion effect junctional epidermolysis bullosa phenotype.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin blistering disease with both lethal and nonlethal forms, with most patients shown to have defects in laminin-5. We analyzed the location of mutations, gene expression levels, and protein chain assembly of the laminin-5 heterotrimer in six JEB patients to determine how the type of genetic lesion influences the pathophysiology of JEB. Mutations within laminin-5 genes were diversely located, with the most severe forms of JEB correlating best with premature termination codons, rather than mapping to any particular protein domain. In all six JEB patients, the laminin-5 assembly intermediates we observed were as predicted by our previous work indicating that the alpha3beta3gamma2 heterotrimer assembles intracellularly via a beta3gamma2 heterodimer intermediate. Since assembly precedes secretion, mutations that disrupt protein-protein interactions needed for assembly are predicted to limit the secretion of laminin-5, and likely to interfere with function. However, our data indicate that typically the most severe mutations diminish mRNA stability, and serve as functional null alleles that block chain assembly by resulting in either a deficiency (in the nonlethal mitis variety) or a complete absence (in lethal Herlitz-JEB) of one of the chains needed for laminin-5 heterotrimer assembly.

Estimating transboundary transported anthropogenic sulfate deposition in Japan using the sulfur isotopic ratio.

High emissions of air pollutants from Northeast Asia are strongly influenced by air quality as well as by ecosystems. This study investigated the spatiotemporal variations in the sulfur isotopic ratio (δ34S) in atmospheric deposition at eleven monitoring stations in Japan from 2011 to 2016 and estimated the amount of transboundary transported anthropogenic sulfate (TRB) deposition using mass balance calculations. The δ34S of sulfate in precipitation ranged from -0.42 to +22.7‰. Sea salt (SS), TRB, and domestic anthropogenic sources (DOM) were the dominant sources of sulfate deposition in Japan. TRB sulfate deposition was largest on the Sea of Japan side, with an annual average value of 1.5 ±â€¯0.3-6.9 ±â€¯0.5 mg m-2 d-1 (36-44%), followed by Mt. Happo (4.5 ±â€¯0.1 mg m-2 d-1; 88%), the Pacific Ocean side (1.5 ±â€¯0.8, 4.3 ±â€¯0.9 mg m-2 d-1; 24-50%), and the remote islands in the North Pacific Ocean (1.1 ±â€¯0.2, 2.0 ±â€¯0.8 mg m-2 d-1; 19-32%). TRB sulfate deposition on the Sea of Japan side was 2-12 times higher in winter and 1-2 times higher in summer than that of DOM. In contrast, TRB sulfate deposition on the Pacific Ocean side was 1.5-3 times higher in summer than in winter due to high precipitation levels. In Tokyo, the annual contribution from DOM sulfate deposition is approximately three times higher than that from TRB. Annual TRB sulfate deposition is lowest at Ogasawara at 1.1 ±â€¯0.2 mg m-2 d-1, and the annual oceanic DMS contribution to sulfate deposition is high, accounting for 1.3 mg m-2 d-1 (20 ±â€¯6%). The contribution of Asian dust was estimated to be 1-5.2 mg m-2 d-1(3-6%), which occurred in a single Asian dust event on the Sea of Japan side.

Protective effect of Na+ and K+ against inactivation of (Na+ + K+)-ATPase by high concentrations of 2-mercaptoethanol at high temperatures.

Purified dog kidney (Na+ + K+)-ATPase (EC 3.6.1.3) was inactivated with high concentrations of 2-mercaptoethanol at 50-55 degrees C. The inactivation was prevented by NaCl or KCl, with KCl being more effective than NaCl (the former ion being about one order more efficient under a typical set of experimental conditions). A disulfide bond in the beta-subunit of the enzyme protein was prevented from reductive cleavage by NaCl or KCl in accordance with protection of the enzyme activity. Choline chloride did not exert a significant protective effect over a similar concentration range. (Na+ + K+)-ATPase was also inactivated with high concentrations of 2-mercaptoethanol in the presence of low concentrations of dodecyl sulfate. This inactivation was also prevented by NaCl or KCl, with the latter being again more efficient than the former. These results indicate that Na+ and K+ bound to their respective ion-binding sites on the alpha-subunit exert a protective effect on a disulfide bond on the beta-subunit. This suggests some sort of interaction between the alpha- and the beta-subunits.

Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase.

Point mutations of Asp-376 of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase (the site of phosphorylation during the catalytic cycle) to Asn, Glu or Thr led to virtual abolishment of Na+/K(+)-ATPase activity and ouabain-binding capacity. Replacement of Lys-507 of the same subunit (the putative ATP-binding site) by Met resulted in decreases in Na+/K(+)-ATPase activity and ouabain-binding capacity. These results are in agreement with those reported for rabbit sarcoplasmic reticulum Ca2(+)-ATPase (Maruyama, K. and MacLennan, D.H. (1988) Proc. Natl. Acad. Sci. USA 85, 3314-3318).

Differential expression of the chemokine receptors by the Th1- and Th2-type effector populations within circulating CD4+ T cells.

The in vitro studies have proposed that human Th1 cells favor expression of CXCR3 or CCR5, whereas Th2 cells favor CCR3 and CCR4. In this study, the in vivo relevance of expression of these chemokine receptors on Th cells was investigated in patients with atopic dermatitis (AD) as the Th2-dominated disorder and nonatopic normal individuals. Flow-cytometric analysis using monoclonal antibodies against CXCR3, CCR5, CCR3, and CCR4 disclosed that a substantial proportion of memory (CD45RO+) CD4+ T cells in the blood of AD and normal patients expressed CXCR3, CCR5, or CCR4, but expression of CCR3 on these cells was negligible. Stimulation studies combined with intracellular cytokine staining revealed that the cells capable of producing Th2 cytokines, such as interleukin-4 (IL-4), IL-5, and IL-13, were restricted to the CCR4-expressing population within memory CD4+ T cells. Concerning Th1 cytokine production, interferon-gamma (IFN-gamma)-producing cells resided exclusively in CXCR3-expressing memory CD4+ T cells, although IFN-gamma production was found in both memory CD4+ T cells with and without CCR5 expression. We observed that CCR4-expressing memory CD4+ T cells in the blood were more increased in AD patients as compared with normal patients, whereas CXCR3-expressing memory CD4+ T cells were present in a lower frequency in AD than seen in normal patients. These results suggest that CXCR3 and CCR4, but not CCR5 or CCR3, appear to serve as the useful markers for identification of circulating Th1 and Th2 effector populations.

Emergence of resistance to acyclovir and penciclovir in varicella-zoster virus and genetic analysis of acyclovir-resistant variants.

We have characterized the differential actions of acyclovir and penciclovir against varicella-zoster virus (VZV) in cell culture by comparing the frequency of appearance of resistant viruses followed by their characterization. Cells were infected with cell-free virus and the cultures were successively treated with increasing concentrations of acyclovir or penciclovir. Drug-resistant viruses were selected in the presence of 6 microg/ml of acyclovir or penciclovir. The emergence frequency of resistant viruses was significantly higher following acyclovir exposure than following penciclovir exposure (Fisher's exact test, P<0.0001), possibly reflecting virus growth differences under these experimental conditions. Based on antiviral drug susceptibility and thymidine kinase (TK) activity assays, 11 acyclovir-resistant variants from seven experiments using three virus strains (Kawaguchi strain, Oka varicella vaccine strain and a clinical isolate from a zoster patient) were found to be TK-deficient. Sequence analysis of TK-deficient variants of the Kawaguchi strain revealed deletions that caused frameshifts, resulting in premature termination in the TK gene.

Suppression of recurrent genital herpes simplex virus type 2 infection by Rhus javanica in guinea pigs.

Rhus javanica has been shown to exhibit anti-herpes simplex virus (HSV) activity and potentiate the anti-HSV activity of acyclovir in vitro and in vivo. This extract was examined for its suppressive efficacy on recurrent genital infection in guinea pigs. Guinea pigs were primarily infected intravaginally with HSV type 2 (HSV-2). Prophylactic oral administration, at the dose corresponding to human use, of R. javanica significantly reduced the incidence, severity and/or frequency of spontaneous and severe skin lesions as compared with latently infected guinea pigs administered with water. This prophylactic efficacy was confirmed by the crossover administration, for more than 2 months, of R. javanica and water to the infected guinea pigs. Toxicity, such as weight loss, from R. javanica administration was not observed in the guinea pigs. When recurrent HSV-2 disease was induced by ultraviolet irradiation 3 months after primary infection, the prophylaxis with R. javanica was also significantly effective in reducing the severity of ultraviolet-induced skin lesions. Thus, prophylaxis of recurrent genital HSV-2 infection with R. javanica may preserve the efficacy of acyclovir by reducing both the use of acyclovir and the appearance of acyclovir-resistant viruses.

Three-dimensional microvasculature of the hair follicle.

The three-dimensional microvasculature of the hair follicle of the adult Wistar rat was demonstrated by scanning electron microscopy of vascular corrosion casts. The anagen hair follicle was surrounded by the basket-like capillary network which was supplied by the branches of the subcutaneous artery and drained into the veins continuous with the subcutaneous vein. The capillary network surrounding the anagen hair follicle was most dense at its bottom, and became sparse at its upper part. The telogen hair follicle was surrounded by only a few capillaries. Transmission electron microscopy showed that the capillaries around the hair bulb possessed fenestrations. Our results indicate that the microvasculature of the anagen hair follicle is so organised as to supply the hair bulb with abundant blood, which is the most important area for hair growth.

Pathogenesis in pili torti: morphological study.

To clarify the mechanism of pili torti formation, hair materials obtained from a patient with the typical classical type of pili torti were examined. Light microscopy, scanning electron microscopy (SEM), transmission electron microscopy, (TEM) and 3-D analysis revealed that: (1) The hair shaft was partly flattened. The hairs twisted clockwise or counter-clockwise on their own axis and some hairs were knotted. (2) Short hairs showed disruptions at the flattened or unflattened regions, at the knot, or at the root of branched hairs caused by longitudinal ruptures. (3) Light microscopically, cell vacuolation and irregularity in thickness of the outer root sheath (ORS) at the suprabulbar level were seen. At the middle to upper levels of the follicles, some eosinophilic pyknotic ORS cells were observed scattered around. (4) At the upper levels, the ORS and inner root sheath (IRS) partly revealed irregularity of thickness and formed an irregular-shaped hair canal. (5) By DACM staining, cytoplasmic SH fluorescence showed an irregular arrangement of the ORS cells. Some abnormal SH- or SS-positive cells were observed scattered throughout the ORS. (6) Ultrastructurally, the irregularities in thickness and cell shape of the IRS and ORS became visible at the level, where the Henle's layer keratinized. In the hair cortex, there was no abnormality of nuclei and the keratin fibers were normally produced, although fibers were partially wavy. (7) By 3-D analysis, hair twist was already seen at the middle level of the hair follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural characterization of microvasculature in photoaging.

The cutaneous microvasculature was examined by electron microscopy in order to compare its characteristics in photodamaged preauricular skin and in sun-protected postauricular sites of 15 Japanese women aged 58-81 years. The characteristic ultrastructural features of the microvasculature in photodamaged skin compared with those in sun-protected skin included dilated vessels embedded in elastin which depressed endothelial cells, vessels surrounded by a thick amorphous material composed of multiple laminations of a basement membrane-like material, and activated endothelial cells which had increased numbers of cytoplasmic organelles and pinocytotic vesicles. A novel finding of this study in photodamaged vessels was an increased formation of new vessels (angiogenesis) via two distinct pathways. In severe elastosis, activated endothelial cells with densely packed intracytoplasmic microfilaments extended large pseudopods into the elastotic material. In contrast, isolated mesenchymal cells, which possessed immature Weibel-Palade bodies, were scattered around pre-existing vessels within the Grenz zone. In some cases, many mesenchymal cells with electron-lucent cytoplasms aggregated and interconnected by cytoplasmic processes, which was followed by the formation of vascular structures. These results suggest that there are significant ultrastructural differences in vessels between photoaged and intrinsically aged facial skin and that the photodamaged microvascular system is characterized by the co-existence of regressive changes and angiogenesis.

Characterization of acyclovir susceptibility and genetic stability of varicella-zoster viruses isolated during acyclovir therapy.

We have characterized the susceptibility and genetic stability of varicella-zoster viruses (VZV) isolated from skin lesions of three patients with herpes zoster and six patients with varicella treated with conventional short-term acyclovir (ACV) administration. The susceptibilities to ACV of the serial isolates from the patients were examined, and there was no significant difference in the susceptibility to ACV among the isolates before and during the ACV treatment, indicating that conventional short-term ACV treatment did not generate ACV-resistant VZV infections. Polymerase chain reaction (PCR) analyses of these as well as seven thymidine kinase-deficient VZV strains derived from in vitro ACV treatment were carried out to examine their genomic stability. Five regions containing tandem direct reiterations (R1-R5) were amplified by PCR and compared, and the region containing the Pst I-site was also examined. PCR analyses demonstrated that the R1, R5 and the Pst I-sites were stable and useful in epidemiological studies even after ACV treatment. The R2, R3 and R4 sites were far less stable in these experimental conditions. In this paper we discuss the results of the PCR analyses with regard to the dynamics of VZV population in patients with VZV infection treated with conventional short-term ACV administration.

Effects of Na+ and K+ on Artemia salina (Na,K)-ATPase solubilized with a zwitterionic detergent, CHAPS.

The membrane bound (Na,K)-ATPase prepared from Artemia salina nauplii was solubilized with a zwitterionic detergent, 3[3(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and then purified on a Bio-Gel A-1.5 m column in the presence of the detergent. 1) Upon solubilization, both NaCl and KCl protected the enzyme against loss of activity, KCl being more effective than NaCl. 2) Gel filtration of the solubilized enzyme on a Bio-Gel A-1.5 m column in the presence of 5 mM CHAPS resulted in loss of the enzyme activity even when one of the cations was added. Most of the phospholipids in the solubilized enzyme preparation were removed during the gel filtration (delipidation) and 10-25 phospholipids were left on a protomer (alpha beta) of the enzyme irrespective of the cation present during the gel filtration. With the addition of exogenous phospholipids, the activity was restored. The activity of the enzyme delipidated in the presence of KCl was restored to 3-4 times higher than in the case of that delipidated in the presence of NaCl. 3) Relipidation experiments with a fluorescent phospholipid, dansyl phosphatidylethanolamine (Dans-PE), suggested that the enzyme delipidated in the presence of KCl reassociated with phospholipids more firmly than the enzyme delipidated in the presence of NaCl. From these results we concluded that K+ stabilized the (Na,K)-ATPase more effectively than Na+, even when the enzyme was delipidated.

Ultrastructural studies of vitiligo, Vogt-Koyanagi syndrome, and incontinentia pigmenti achromians.

Studies of early progressive vitiligo, Vogt-Koyanagi syndrome, and incontinentia pigmenti achromians were made by electron microscopy. At the periphery of the depigmented lesion, the melanocytes had several subcellular abnormalities, ie, vacuolization of the cytoplasm, aggregation of melanosomes, autophagic vacuoles, fatty degeneration, pyknosis or homogeneous cytoplasmic degeneration, and others. Numerous nerve endings were seen in close contact with the basal lamina of the epidermis or even within the epidermis. Direct continuities between Schwann cell basal lamina of nerve endings and the basal lamina of the melanocytes were observed. Nerve endings could be associated with either normal or abnormal melanocytes.

Quantitative analysis of the proliferation of epidermal cells using a human skin organ culture system and the effect of DbcAMP using markers of proliferation (BrdU, Ki-67, PCNA).

The process of re-epithelialization of a wound in the epidermis comprises the following steps: proliferation of epidermal basal cells, migration of epidermal cells to the wound surface, and cell differentiation. In the present study, we evaluated the proliferation of epidermal basal cells, an important process in wound healing, in the wound margin using a human skin organ culture system and immunohistochemical labeling with bromodeoxyuridine (BrdU), Ki-67, and proliferating cell nuclear antigen (PCNA), as markers of cell proliferation. Dibutyryl cyclic adenosine monophosphate (DbcAMP) is a derivative of cAMP and has been shown to modulate human keratinocyte proliferation. The proliferation of keratinocytes was promoted by DbcAMP and particularly strong effects in terms of BrdU labeling index, Ki-67-positive ratio, and PCNA-positive ratio, were seen at 10(-5) M. The skin organ culture system presented here uses adult preputial skin and is a simple technique that uses easily available materials. In addition to identifying S phase cells using BrdU as an index of cell proliferation, the immunohistochemical method for evaluating the expression of Ki-67 and PCNA is very simple. Accordingly, the method described here seems to be useful for evaluating cell dynamics in wound healing.

Proboscis amputation facilitates the study of mosquito (Diptera: Culicidae) attractants, repellents, and host preference.

Proboscis amputation has facilitated the study of mosquito behavior. Using humans as a host is very important in the study of mosquito attractants, repellents, and host preference. However, mosquito bites cause potential medical problems because of hypersensitivity and perhaps secondary bacterial infection, even using laboratory mosquitoes. Moreover, once a normal female mosquito bites and feeds on human blood, it cannot be used in subsequent probing tests. These problems were resolved by proboscis amputation. Variation of attraction among humans was examined effectively without bites using proboscis-amputated Aedes albopictus Skuse. Proboscis-amputated and normal mosquitoes also showed equal repellency against 1% L-lactic acid. Although the mosquitoes lacked the tip of the labium and some sensilla, they alighted on human forearms in the same way as normal mosquitoes. Because proboscis-amputated mosquitoes continued to probe avidly, they could be used repeatedly, thereby reducing the number of mosquitoes required for experimentation. The use of proboscis-amputated mosquitoes would promote various studies of mosquito attraction or repellency with no risk of hypersensitivity and secondary bacterial infection by mosquito bites.

L-lactic acid as a mosquito (Diptera: Culicidae) repellent on human and mouse skin.

The attraction of Aedes albopictus (Skuse) to hands and forearms of human subjects treated with several concentrations of L-LA solution were studied in a test chamber containing proboscis-amputated mosquitoes. Fewer mosquitoes alighted on L-LA treated human skin than on water-treated control skin. Similar results were found using normal mosquitoes following L-LA and water treatment of mouse skin. The relative repellent effects of L-LA varied with concentration. The minimum repellent concentration was lower than previously reported for human skin. The number of alightments decreased at increasing concentrations of L-LA, demonstrating the absolute repellency of L-LA. Unlike previous reports suggesting that L-LA attracted mosquitoes, our studies using human and mouse skin showed that L-LA exhibited both relative and absolute repellency.

Infundibulocystic basal cell carcinoma.

We report on a 72-year-old male patient with a solitary, dome-shaped tumor on the preauricular region. Histologically, it showed an intradermal basaloid cell tumor composed of many anastomosing epithelial cords. There were multiple infundibular cystic structures containing corneocytes lined by follicular infundibular epithelium and bud-like structures simulating the follicular germs throughout the neoplasm. Follicular bulbs and papillae were absent within the tumor. The stroma was scant and not highly fibroplastic. The tumor was diagnosed as infundibulocystic basal cell carcinoma, a recently described, uncommon variant of basal cell carcinoma.

Dyskeratosis congenita: a light microscopic and ultrastructural study.

We have examined, histologically and ultrastructurally, a case of dyskerato-sis congenita in a 46-year-old man. Clinically, a net-like pigmentation with partial poikiloderma atrophicans vasculare was observed. Dystrophic changes of the nails and whitish thickening of the oral mucosa were also present. Histological examination showed atrophy of the epidermis, disappearance of the rete ridges and cleft formation of dermo-epidermal junction. Vacuoles could be seen on electron microscopy in the cytoplasm of basal cells and above the basal lamina. Tonofibrils in the cytoplasm of basal cells were decreased in number and size. Duplication or multiplication of basal lamina was also seen. From these results, it appears that the vacuoles observed ultrastructurally correspond to the cleft of the dermo-epidermal junction seen by light microscopy and that atrophy of the epidermis and/or disappearance of rete ridges result from sporadic degeneration of basal cells.

A specific cutaneous lesion revealing myelodysplastic syndrome.

We report on an 86-year-old man with an ulcerated nodule on his left lower leg. Peripheral blood examination and bone marrow findings were compatible with the refractory anemia with an excess of blasts in transformation (RAEB-T) which is typical of the myelodysplastic syndrome (MDS). Because histological examination showed an infiltration of atypical cells of myeloid origin, this lesion was diagnosed as a specific lesion of MDS. Sometimes, only a subjective symptom, such as a skin lesion, precedes the diagnosis of MDS.

An ultrastructural and immunohistochemical study of pigmented dermatofibrosarcoma protuberans (Bednar tumor).

A case of Bednar tumor on the right shoulder of a 47-year-old Japanese woman is reported. Histological examination showed plump, spindle cells arranged in a storiform pattern in central areas of the tumor and a diffuse infiltration of the dermal stroma, which was frequently extended into the subcutis at the periphery of the tumor. The tumor contained a fairly identified population of dendritic pigmented cells. Ultrastructurally, most cells had folded nuclei, were spindle-shaped and had long, slender cytoplasmic projections. Dendritic pigmented cells, which were dispersed among neoplastic cells, contained premelanosomes and mature melanosomes. Immunohistochemically, tumor cells exhibited positive reactions for vimentin and CD 34 and failed to show a positive reaction for neuron specific enolase, HMB-45 or S-100 protein. Factor X IIIa was only expressed on tumor cells around melanin-containing cells, which reacted positively with antibodies to S-100 protein and vimentin. These results indicate that the phenotype of tumor cells around melanin-containing cells differs from other tumor cells and that this difference may be caused by the relationship of tumor cells and melanin-containing cells.