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G Imokawa

Publications and source records attributed to G Imokawa.

At least 55 records · Page 3Linked to original sources
PubMed1996

Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency?

Previously, we demonstrated that there is a marked reduction in the amount of ceramide in the stratum corneum of both lesional and nonlesional forearms in atopic dermatitis (AD), suggesting that an insufficiency of ceramides in the stratum corneum is an etiologic factor in atopic dry and barrier-disrupted skin. In this study, we investigated, as a possible mechanism involved in the ceramide deficiency, whether sphingomyelin (SM) metabolism is altered in AD as compared to normal controls. In stripped stratum corneum and biopsied whole epidermis of patients with AD, SM hydrolysis as measured at pH 4.7 using [choline-methyl-14C]sphingomyelin as a substrate were markedly increased by 27- and 7-fold, respectively. Radio-thin-layer chromatography of the reaction products revealed that, whereas the SM hydrolysis in age-matched normal controls were associated with sphingomyelinase (SMase) that degrades SM to yield ceramides and phosphorylcholine (PC), most of the SM hydrolysis detected in AD were attributable not to the SMase but to a hitherto undiscovered epidermal enzyme, SM acylase, which releases free fatty acid and sphingosyl-PC (Sph-PC) instead of ceramides. The potential of this acylase-like enzyme to generate Sph-PC through SM hydrolysis was corroborated by thin-layer chromatographic analysis of the reaction products obtained using porcine kidney acylase, followed by high-performance liquid chromatography-mass spectrometry. Furthermore, Sph-PC was also detected by high-performance liquid chromatography-mass spectrometry after incubation of SM with atopic stratum corneum samples. On the other hand, the stratum corneum of patients with contact dermatitis or chronic eczema exhibited neither increased SM hydrolysis nor the generation of Sph-PC upon radio-thin-layer chromatographic analysis. These findings suggest that SM metabolism is altered in AD, resulting in a decrease in levels of ceramides, which could be an etiologic factor in the continuous generation of atopic dry and barrier disrupted skin observed in AD.

Amidohydrolases↗
PubMed1996

Unusual wrinkle formation after temporary skin fixation followed by UVB irradiation in hairless mouse skin.

Generally, many wrinkles form on the human face, and temporary wrinkles eventually become permanent. We evaluated the effects of temporary skin fixation on wrinkle formation after UVB irradiation using the back skin of hairless mice. In the group treated with UVB irradiation immediately after production using cyanoacrylate resin of an artificial groove parallel to the midline, wrinkles formed parallel to the midline, an uncommon direction for wrinkle formation in this mouse model. These wrinkles did not disappear even when the skin was stretched. No such changes were observed in the group in which only the temporary groove alone was produced without UVB irradiation. In 3-D surface parameter analysis, all roughness parameters in the group treated with UVB irradiation immediately after production of an artificial groove were significantly increased relative to the age-matched control group. In contrast, no differences were observed between the group in which only the temporary groove alone was produced without UVB irradiation and age-matched controls. The results of this study suggest that both a temporary groove in the skin and UVB irradiation are necessary for wrinkle formation in the back skin of hairless mice.

Animals↗
PubMed1996

Characterization of melanogenesis in normal human epidermal melanocytes by chemical and ultrastructural analysis.

Chemical and ultrastructural studies were conducted to define the relationship between type of melanogenesis and fine structures of melanosomes in normal human epidermal melanocytes. Chemical analysis of epidermal melanin demonstrated that the ratio of eumelanin/pheomelanin varied individually, ranging from 1.31 to exclusively eumelanic. Ultrastructural analysis of fine structures of melanosomes revealed that spheroid melanosomes were frequently observed in melanocytes of the epidermis whose eumelanin/ pheomelanin ratio was less than 5. Conversely, ellipsoid melanosomes predominated in melanocytes of the epidermis whose ratio was more than 10. On the basis of these findings, it seems reasonable to conclude that 1) normal human epidermal melanocytes synthesize both eumelanin and pheomelanin and 2) pheomelanin synthesis may be characterized by the presence of spheroid melanosomes whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.

Adult↗
PubMed1995

Purification and biochemical characterization of membrane-bound epidermal ceramidases from guinea pig skin.

Ceramidase (CDase) catalyzes the hydrolysis of ceramides to yield sphingosine and fatty acid. In this paper, two forms of membrane-bound alkaline ceramidase, have been, for the first time, purified from guinea pig epidermis by chromatography on DEAE-cellulose, phenyl-Superose, HCA-hyroxyapatite, isoelectric focusing, Mono Q, and TSK-3000SW column. One species (CDase-I) migrated upon SDS-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 60 kDa; the other (CDase-II) was only partially purified with apparent M(r) of about 148,000 estimated by gel filtration. The specific activities of the two species increased by 1.130- (for CDase-I) and 400-fold (for CDase-II) over the original tissue extract. The activity of both enzymes for ceramide species decreased in the order of linoleoyl > oleoyl > palmitoylsphingosine. The optimal pH for enzyme activity was approximately 7.0-9.0 for CDase-I and 7.5-8.5 for CDase-II. Interestingly, both enzymes were inhibited by the reaction product sphingosine with a concentration for half-maximal inhibition (ID50) of 100-130 microM, compared to the apparent kinetic parameters with CDase-I (Km = 90 microM, Vmax = 0.62 unit) and CDase-II (Km = 140 microM, Vmax = 0.50 units). Some lipids, such as phosphatidylcholine and sphingomyelin, are also inhibitory with IC50 values of 50-250 microM, suggesting well controlled CDase activity by sphingolipid metabolites. These studies begin to elucidate a regulatory mechanism for the balance of the ratio of ceramide/sphingosine which can serve as an intracellular effector molecule in epidermis.

Amidohydrolases↗
PubMed1995

Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis.

We previously demonstrated that human keratinocytes produce and secrete endothelins (ET), which can be strong mitogens for human melanocytes. Ultraviolet B (UVB) exposure highly stimulated the paracrine linkage of endothelins between keratinocytes and melanocytes, indicating that they are keratinocyte-derived intrinsic mitogens in UVB-induced pigmentation. In this study, the role of ET-1 as a melanogen in UVB melanogenesis was investigated in vitro and in vivo. In the conditioned medium of keratinocytes exposed to UVB, melanin synthesis by human melanocytes, as measured by 14C-thiouracil incorporation, was significantly accentuated. This stimulatory effect was reduced by anti-ET-1 to the level of that in the non-UVB-exposed control, suggesting an essential role of ET-1 as an intrinsic melanogen in UVB-induced melanogenesis. In a parallel study, the addition of 10 nM ET-1 induced an increase in tyrosinase activity in cultured human melanocytes and was accompanied by elevated levels of tyrosinase and tyrosinase-related protein-1 mRNA expression as shown by Northern blotting. Reverse transcription-polymerase chain reaction of RNA isolated from the epidermis of human skin exposed to UVB revealed that, whereas in non-exposed sites ET-1, IL-1 alpha, and tyrosinase mRNA signals were scarcely detected, UVB-irradiation, with a dose of twice the minimal erythema dose, caused a significant increase in the expressions of the three genes 5 d after irradiation. These findings suggest that ET-1 is an important mediator for UVB-induced pigmentation in the epidermis in vivo.

Base Sequence↗
PubMed1995

Degree of ultraviolet-induced tortuosity of elastic fibers in rat skin is age dependent.

To elucidate differential effects of ultraviolet (UV) exposure on three-dimensional networks of elastic fibers during maturation of rat skin, Sprague-Dawley rat hind limbs were irradiated with suberythemal doses of UV light (UVB, 130 mJ/cm2, or UVA, 27 J/cm2) in three different time courses of exposure: 3-9 weeks old, 9-15 weeks old, and 3-15 weeks old. Three-dimensional arrangement with special reference to linearity of elastic fibers was quantified by image analysis using a scanning electron microscope after a combination of intravascular resin injection and selective digestion technique using formic acid. Among the three irradiation groups, the group irradiated with UVB or UVA between 3 and 15 weeks old (UVB, three times per week; UVA, five times per week) elicited the most marked decrease in the linearity of elastic fibers. Despite the same irradiation period, there was a significant difference in the decreased linearity between the two irradiation groups of 3-9 and 9-15 weeks old, with the former irradiation group exhibiting greater loss of linearity than the latter irradiation group. The magnitude of the decreased linearity was greater in the UVB-exposed groups than in the UVA-exposed group. These findings indicate that the three-dimensional linearity of elastic fibers is more susceptible to disruption by UV exposures during the growth period than that after the growth period.

Aging↗
PubMed1995

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.

Amino Acids↗
PubMed1995

Modulation of melanogenic protein expression during the switch from eu- to pheomelanogenesis.

Mammalian melanocytes can produce two basic types of melanin, eumelanin and pheomelanin, within discrete organelles termed melanosomes. The physiological signals that regulate this switch are extrinsic to the melanocyte, and include alpha-melanocyte stimulating hormone and the agouti protein. Tyrosinase, encoded at the albino locus, is the enzyme essential for the synthesis of both types of melanin, but other tyrosinase-related proteins (e.g. TRP1 encoded at the brown locus and TRP2 encoded at the slaty locus) regulate eumelanogenesis catalytically at steps distal to tyrosinase (as 5,6-dihydroxyindole-2-carboxylic acid oxidase and DOPAchrome tautomerase, respectively). The silver protein is another melanosomal protein, and although it has some limited homology to the tyrosinase-related proteins, it does not have any known enzymatic function and probably serves as a structural matrix protein. The role of each of those melanosomal proteins in pheomelanogenesis, however, is still unclear. In this study, we have compared the expression and catalytic functions of those proteins in pheomelanic and eumelanic hair bulb melanocytes. There was no detectable expression of TRP1 or TRP2, or either of their enzymatic activities, in hair bulbs of lethal yellow (Ay/a) newborn mice, and tyrosinase activity was present at a reduced level compared to that found in hair bulbs of black (a/a) newborn mice. Similar results were observed in regenerating hair bulbs of adult lethal yellow mice and in hair bulbs of 5- to 7-day-old agouti mice (A/A), an age where pheomelanin is produced predominantly. Expression of the silver protein was similarly not observed in hair bulbs of the pheomelanic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

[Vasodilating effects of NY-008 in ring preparation of rat aorta].

Vasodilating effects of NY-008 were compared to those of verapamil in isolated rat aorta. NY-008 at the dose of 3 x 10(-5) M relaxed 10-70 mM potassium chloride (KCl)-induced contraction. NY-008 relaxed preparations precontracted with 60 mM KCl concentration-dependently with an IC50 of (6.17 +/- 1.75) x 10(-6) M, and those precontracted with norepinephrine (NE) (10(-6) M) concentration-dependently, maximally by 90.0 +/- 2.86%, with an IC50 of (2.06 +/- 0.38) x 10(-5) M. At 10(-8)-3 x 10(-6) M, verapamil relaxed preparations precontracted with NE (10(-6) M) concentration-dependently, maximally by 55.9 +/- 5.56%. At 3 x 10(-5) M and 10(-4) M, NY-008 relaxed (10(-6) M)-induced phasic contractions in a Ca(2+)-free 1 mM EGTA-containing buffer, by 22.4 +/- 3.69% and 35.4 +/- 5.74%, respectively. In contrast, 3 x 10(-7) M verapamil did not. NY-008 concentration-dependently decreased the maximal responses to calcium chloride (CaCl2) in 60 mM KCl-depolarized preparations, and it shifted the ED50 values of CaCl2 to the right, whereas verapamil shifted the ED50 values of CaCl2 to the right without decreasing the maximal responses to CaCl2. At 10(-5)-3 x 10(-4) M, NY-008 concentration-dependently relaxed preparations precontracted with prostaglandin F2 alpha (10(-5) M) in a Ca(2+)-free, 0.5 mM EGTA-containing buffer, whereas 10(-7)-10(-5) M verapamil did not. These results suggest that NY-008 antagonized Ca2+ and decreased the Ca(2+)-sensitivity of contractile elements or inhibited contractile proteins in rat aorta.

Animals↗
PubMed1994

Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis.

Several genes critical to the enzymatic regulation of melanin production in mammals have recently been cloned and mapped to the albino, brown and slaty loci in mice. All three genes encode proteins with similar structures and features, but with distinct catalytic capacities; the functions of two of those gene products have previously been identified. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, while the slaty locus encodes tyrosinase-related protein 2 (TRP2), an enzyme with a single specific, but distinct, function as DOPAchrome tautomerase. Although the brown locus, encoding TRP1, was actually the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) has been in general dispute. In this study we have used two different techniques (expression of TRP1 in transfected fibroblasts and immunoaffinity purification of TRP1 from melanocytes) to examine the enzymatic function(s) of TRP1. The data demonstrate that the specific melanogenic function of TRP1 is the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to a carboxylated indole-quinone at a down-stream point in the melanin biosynthetic pathway. This enzyme activity appears to be essential to the further metabolism of DHICA to a high molecular weight pigmented biopolymer.

Animals↗
PubMed1994

The Pmel 17/silver locus protein. Characterization and investigation of its melanogenic function.

The silver mutation in mice causes progressive graying of hair due to the loss of functional follicular melanocytes. Recently the silver locus gene (called Pmel 17) has been cloned; its encoded product shares homology with a chick melanosomal matrix protein and a bovine retinal pigment epithelial protein. Although the sequence of the silver gene and the correlation of its expression with pigment production have been reported, its function in melanogenesis is still unknown. In an effort to characterize that function, we have synthesized the predicted carboxyl-terminal peptide of the mouse Pmel 17 protein and generated a rabbit polyclonal antibody (alpha PEP13) to it; that antibody recognized the silver protein specifically. The immunoaffinity-purified silver protein lacked all of the known melanogenic catalytic activities which other tyrosinase-related proteins (TRP) have, nor did it appear to modulate any of those TRP activities. Metabolic labeling experiments demonstrated that the silver protein disappears in vivo within a few hours, indicating that it is rapidly degraded, or quickly processed to lose its carboxyl terminus. Cross-reactivity experiments showed that a recently reported anti-melanosomal matrix protein antibody (alpha MX) also recognizes the silver protein, although at a different epitope from that of alpha PEP13. Using Western immunoblotting, we analyzed subcellular fractions isolated from B16 F10 melanoma cells and found that the silver protein was rich in the melanosome fraction but was absent from coated vesicles which deliver TRPs to melanosomes. These results suggest that the silver locus product is a melanosomal matrix protein which may contribute to melanogenesis as a structural protein, although the possibility remains that it also has a novel catalytic function in melanogenesis.

Amino Acid Sequence↗
PubMed1994

Ultraviolet-B irradiation deforms the configuration of elastic fibers during the induction of actinic elastosis in rats.

We used scanning electron microscopy combined with perfusion fixation, resin injection and a selective digestion procedure to determine the effects of ultraviolet-B (UVB) radiation on the three-dimensional architecture of elastic fibers of rat skin. Chronic irradiation with a suberythematous dose of UVB (3 times/week for 12 weeks) produced a tortuous deformation of the superficial elastic fibers in the skin of the rat sole which normally are linearly arranged. Using computer analysis, we evaluated 40 individuals elastic fibers every 3 weeks for 12 weeks following the irradiation. This procedure confirmed the increasing tortuosity of the fibers, which was related to a decline in the elastic property of the skin in situ. Fine elastic branches developed among the deformed fibers and eventually anastomosed to produce an irregular network in the superficial dermal connective tissue, which may correspond to the development of actinic elastosis, that is, the UV-related accumulation of elastic fiber material.

Animals↗
PubMed1994

Age-related changes in the elastic properties and thickness of human facial skin.

Using recently designed, commercially available, non-invasive instruments, we measured the thickness and elasticity of the skin of the face and ventral forearm in 170 women, and evaluated the effects of age and exposure to sunlight. Skin thickness decreased with age in ventral forearm skin, which has limited exposure to sunlight, but increased significantly in the skin of the forehead, corners of the eyes, and cheeks, which are markedly exposed to sunlight. Skin elasticity (Ur/Uf) decreased with age on both the face and forearm. The ratio of viscosity element to elasticity element (Uv/Ue) increased with age at all sites. However, delayed distension (Uv), immediate retraction (Ur), final distension (Uf), and immediate distention (Ue), as individual elements, decreased on the face and increased on the forearm with age. This tendency was more marked after correction for skin thickness. These results suggested the specificity of skin thickness and elasticity in the facial skin. Analysis using a four-element model showed no changes in the elasticity coefficient of Maxwell element on the forearm, but an increase on the face. This indicates quantitative or qualitative changes in elastic fibres in facial skin. Thus, sunlight appears to have a considerable effect on the thickness and physical properties of facial skin.

Adolescent↗
PubMed1994

DHICA oxidase activity of TRP1 and interactions with other melanogenic enzymes.

Tyrosinase-related protein 1 (TRP1) maps to the brown locus in mice. Although the specific function of TRP1 has been in dispute, mutations in its structural gene result in the formation of brown rather than black melanin. We have investigated the melanogenic function of TRP1 by using immune-affinity purification of the protein and also by using transfection of its gene into fibroblasts to study its characteristics. We show that TRP1 has the ability to oxidize DHICA, a melanogenic intermediate derived from DOPAchrome. In addition, TRP1 has the ability to interact with tyrosinase and significantly stabilize the latter's catalytic function.

Animals↗
PubMed1994

Regional analysis of ceramides within the stratum corneum in relation to seasonal changes.

We studied the regional variations and seasonal changes of ceramide quantities in the stratum corneum of human skin. In summer, the total lipid amounts extracted from the stripped stratum corneum were highest in the forehead, followed by the chest and upper back, with the lowest level in the sole of the foot. In winter, while the total lipid distribution followed the same trend as that in summer, the absolute amounts slightly decreased, especially in the forehead and chest, as compared with those found in summer. Ceramide analysis showed that while a higher level was seen in the cubital fossa in summer, the level was highest in the forehead during winter. A seasonal comparison of ceramide mass revealed a slightly increased level in winter compared with summer, at almost all sites tested, except in the cubital fossa and dorsum pedis, reflecting seemingly enhanced keratinization in winter. These findings indicate that in young adults, the mass of ceramide remains steady at similar levels at various skin sites, providing maintenance of a pertinent water reservoir and the barrier function of the stratum corneum, even under different seasonal conditions.

Adult↗
PubMed1994

Pseudo-acylceramide with linoleic acid produces selective recovery of diminished cutaneous barrier function in essential fatty acid-deficient rats and has an inhibitory effect on epidermal hyperplasia.

Pseudo-acylceramides with different acyl properties were investigated for their capacity to restore diminished barrier function in essential fatty acid-deficient rats. Daily topical applications of synthetic pseudo-acylceramides containing ester-linked linoleic acid caused a dose-dependent, significant reduction of transepidermal water loss (TEWL). Both other pseudo-acylceramides with ester-linked oleic acid or saturated alkyl chains and ordinary ceramides exhibited a poor effect on recovery of TEWL. Furthermore, pseudoceramide containing ether-linked linoleic acid, which is biologically inactive in terms of degradation by hydrolytic enzymes, also induced a significant and similar increase in the barrier function. This restoration of barrier function by pseudo-acylceramides with linoleic acid was accompanied by suppressed DNA synthesis in the EFAD rat epidermis. In UVB-irradiated guinea pig skin, topical applications of the pseudo-acylceramides with linoleic acid immediately after the exposure significantly reduced epidermal hyperplasia, secondary to markedly diminished barrier disruption, whereas linoleic acid itself did not. A comparison of both the anti-hyperplasia and the barrier recovery effects in the series of pseudo-ceramide derivatives examined revealed that the suppressive effect on the induced epidermal hyperplasia was paralleled by the recovery of the barrier defect in EFAD rats. These findings directly suggest that acylceramide with an ester-linked linoleic acid has an essential role in the epidermal permeability barrier.

Animals↗
PubMed1994

Analysis of beta-glucocerebrosidase and ceramidase activities in atopic and aged dry skin.

To elucidate the mechanisms that are involved in the decrease of ceramide levels in atopic dry skin and in aged skin, we examined both the activities of beta-glucocerebrosidase, which is a major enzyme in ceramide production, and of ceramidase, which is an essential enzyme in ceramide degradation, in the stratum corneum of atopic dry skin and aged skin. The specimens of the stratum corneum of forearm skin were obtained by tape-stripping from 61 healthy volunteers and 23 patients with atopic uninvolved skin. The beta-glucocerebrosidase activity in the stratum corneum extracts was estimated using fluorescent 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. Ceramidase activity was determined using 14C-palmitoylsphingosine as the substrate. Among the atopic skin samples, neither beta-glucocerebrosidase nor ceramidase activities were different from those of age-matched healthy controls. Nor was the beta-glucocerebrosidase activity deficient in the aged skin samples as compared to that seen in samples from the young, healthy group. In contrast, there was an age-related upregulation in ceramidase activity. The results indicate that the decrease of ceramides in atopic dry skin may not be accompanied by reduced synthesis or by enhanced degradation, each of which is primarily attributable to the above two enzymes, respectively. The pathogenesis of aged dry skin can be explained, at least partially, in terms of elevated ceramidase activity, which results in a disturbance of the lamellar structure of the stratum corneum lipids.

Adolescent↗