Horse chestnut extract induces contraction force generation in fibroblasts through activation of Rho/Rho kinase

Contraction forces generated by non-muscle cells such as fibroblasts play important roles in determining cell morphology, vasoconstriction, and/or wound healing. However, few factors that induce cell contraction forces are known, such as lysophosphatidic acid and thrombin. Our study analyzed various plant extracts for ingredients that induce generation of cell contraction forces in fibroblasts populating collagen gels. We found that an extract of Horse chestnut (Aesculus hippocastanum) is able to induce such contraction forces in fibroblasts. The involvement of actin polymerization and stress fiber formation in the force generation was suggested by inhibition of this effect by cytochalasin D and by Rhodamine phalloidin. Rho kinase inhibitors (Y27632 and HA1077) and a Rho inhibitor (exoenzyme C3) significantly inhibited the force generation induced by the Horse chestnut extract. H7, which inhibits Rho kinase as well as other protein kinases, also significantly inhibited induction of force generation. However, inhibitors of other protein kinases such as myosin light chain kinase (ML-9), protein kinase C (Calphostin), protein kinase A (KT5720), and tyrosine kinase (Genistein, Herbimycin A) had no effect on force generation induced by Horse chestnut extract. These results suggest that the Horse chestnut extract induces generation of contraction forces in fibroblasts through stress fiber formation followed by activation of Rho protein and Rho kinase but not myosin light chain kinase or other protein kinases.     

Imaging of melanin distribution using multiphoton autofluorescence decay curves

Background/purpose: Multiphoton fluorescence lifetime imaging (FLIM) is a technique that produces an image based on differences in the decay rate of fluorescence from a sample. Based on this method, the DermaInspect was developed to observe human skin components non-invasively. In this study, we used the DermaInspect to study melanin in skin.
Methods: A human three-dimensional skin model containing melanocytes was embedded in an OCT compound, frozen and sectioned at 10 μm. The melanin distribution in each section was visualized by the DermaInspect using time-resolved single-photon counting and near-infrared femtosecond laser pulse excitation. The melanin distribution of the same sections was then visualized using the Fontana-Masson staining method.
Results: High-resolution images were generated from the ratio of a ₁/ a ₂ ( a ₁e^{− t /120}+ a ₂e^{− t /1100} was chosen to express the exponential fluorescent decay curve) obtained using the DermaInspect. Granules with a high a ₁/ a ₂ ratio, approximately 1 μm in diameter, were observed. Fontana-Masson staining identified these granules as melanin. This new technique was then applied for in vivo observation of melanin in human skin. 'Melanin caps' were visualized in the basal cell layer around the nuclei in images derived from the a ₁/ a ₂ ratio.
Conclusion: Our study confirms that FLIM can non-invasively provide data of the melanin distribution with almost the same quality as the conventional Fontana-Masson staining method, and demonstrates that FLIM is useful for in vivo observation of melanin granules in human skin.     

FOXA2 gene mutation in a patient with congenital complex pituitary hormone deficiency

We report a patient with congenital complex pituitary hormone deficiency (CPHD) with intestinal malrotation and anal atresia. We identified a de novo heterozygous mutation, c.664T > G (p.Cys222Gly), in the FOXA2 gene in this individual. This missense mutation had the potential to affect the DNA binding properties of the FOXA2 protein based on a protein structure prediction. Since a CPHD patient with another missense mutation and one other case with an entire gene deletion have also been reported, we speculated that a haploinsufficiency of the FOXA2 gene might be a genetic etiology for this disorder. Phenotypic similarities and differences among these three cases are also discussed.     

Stem cell factor–KIT signalling plays a pivotal role in regulating pigmentation in mammalian hair

Hair greying is one of the most distinct but least comprehended features of senescence. The signalling of stem cell factor (SCF) and its receptor KIT has been documented to regulate essential roles in the maintenance of embryonic melanocyte lineages and postnatal cutaneous melanogenesis, although little is known about its detailed mechanisms in postnatal hair pigmentation. To address this, anagen human hair follicles and C57BL/6 murine pelage were analysed in this study. Molecular biological analyses of murine follicular skin indicated a significant increase of membrane‐bound SCF expression, reaching its peak 8–16 days after anagen induction in concert with the escalation of cutaneous tyrosinase activity and corresponding pigmentation. Administration of KIT–neutralizing antibody abolished MITF and tyrosinase expressions, resulting in a reversible hair depigmentation in murine regenerated hair and human hair organ culture. Quantitative RT‐PCR of human hair follicles indicated that KIT expression as well as the expression of several melanogenic factors, including MITF, was significantly lower in unpigmented than in pigmented follicles. Taken together, these data revealed a pivotal role of SCF–KIT signalling in the maintenance of human hair follicle melanogenesis during the anagen cycle and its involvement in physiological ageing of the hair follicle pigmentary unit. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The formation of wrinkles caused by transition of keratin intermediate filaments after repetitive UVB exposure

It has been reported that the formation of wrinkles involves changes in the elastic properties of the dermis due to the denaturation of elastic fibers. Several studies have shown that the hydration condition of the stratum corneum is also important in wrinkle formation. It is, however, still unclear how the stratum corneum contributes to wrinkle formation. Here we investigated the relationship between the formation of wrinkles and changes in the physical properties and condition of the skin after repetitive ultraviolet B (UVB) irradiation of hairless mice (HR/ICR). Repetitive UVB irradiation caused wrinkles on the dorsal skin of the mice. The elasticity (E) of the stratum corneum of UVB-irradiated mice was significantly lower than that of age-matched control (unirradiated) mice. UVB exposure also caused a deterioration of the fibrous ultrastructure of keratin intermediate filaments (KIFs) in the skin. We conclude that the deterioration of KIFs in the stratum corneum caused by repetitive UVB irradiation decreases the elastic properties of the stratum corneum, resulting in the formation of wrinkles.     

An accumulation of glucosylceramide in the stratum corneum due to attenuated activity of beta-glucocerebrosidase is associated with the early phase of UVB-induced alteration in cutaneous barrier function

Epidermal glucosylceramide (GlcCer) metabolism is essential to the maintenance of skin homeostasis. Although exposure of the skin to ultraviolet (UV) radiation elicits dramatic physiological and biological changes in the epidermis attributable to barrier perturbation, wrinkle formation and inflammation, little is known about UV-induced changes in GlcCer metabolism. In this study, we have assessed β-glucocerebrosidase (GlcCer’ase) activity in murine epidermis before and after a single UVB irradiation and have compared it with GlcCer and ceramide (Cer) levels. GlcCer’ase enzymatic activity was significantly suppressed in a dose-dependent manner one day after UVB (70 mJ/cm²) irradiation despite a significant increase in GlcCer’ase mRNA. The marked decrease in enzyme activity was followed by an accumulation of GlcCer in the stratum corneum, which peaked at day 2. This decreased level of GlcCer’ase activity returned to 80% of the control level by day 3 followed by a return of GlcCer level to the control level by day 4. In the whole epidermis, significant increases in Cer and GlcCer levels occurred on day 3 and on day 2, respectively. These results suggest that UVB irradiation dramatically affects the metabolism of GlcCer to Cer in the epidermis (including the stratum corneum) and that this may be closely associated with the early and minor phase of UVB-induced alteration in cutaneous barrier function.     

Epidemiological survey of allergic diseases in first-year junior high school students in Wakayama Prefecture in 2003

Allergic diseases have been increasing in prevalence in developed countries, including Japan. The aim of the present report was to determine the prevalence of allergies in Wakayama prefecture using an epidemiological study. In total, 759 first-year students attending junior high school in Hidaka country, Wakayama prefecture, were surveyed. The results for 699 cases were then analyzed. A questionnaire regarding allergic diseases, specific IgE measurements performed using a MAST26 system (Hitachi Co., Ltd.), and total serum IgE levels measured using RIST (Pharmacia Co., Ltd.) were performed. The prevalence (present + past) of various allergic diseases was 37.9%. The prevalence of rhinitis, including pollinosis, was 31.0%, while that of atopic dermatitis was 26.2% and bronchial asthma was 11.3%. The positive rates for specific IgE antibodies against Japanese cedar pollen was 48.6%. The positive rate for Dermatophagoides farinae, timothy and housedust II were 44.2%, 29.6%, and 28.9%, respectively. Statistically significant differences were recognized between the students with allergic rhinitis, atopic dermatitis, allergic conjunctivitis or bronchial asthma and positive results for D. farinae, housedust II, cedar pollen, Penicillium, Cladosporium, or Aspergillus-specific antibodies. Regarding family history (two generations), a statistical significant difference between family history and positivity for specific IgE antibodies like D. farinae, housedust II, ragweed, cedar pollen or Cladosporium was observed. The total IgE titer was correlated with the number of positive allergen items. The increasing prevalence of various allergic diseases in developed countries remains a mystery, but the hygienehypothesis has attracted some attention. The findings of this epidemiologic study will contribute to basic data on the increasing prevalence of various allergic diseases.     

Preimplantation genetic diagnosis/screening by comprehensive molecular testing

Although embryo screening by preimplantation genetic diagnosis (PGD) has become the standard technique for the treatment of recurrent pregnancy loss in couples with a balanced gross chromosomal rearrangement, the implantation and pregnancy rates of PGD using conventional fluorescence in situ hybridization (FISH) remain suboptimal. Comprehensive molecular testing, such as array comparative genomic hybridization and next‐generation sequencing, can improve these rates, but amplification bias in the whole genome amplification method remains an obstacle to accurate diagnosis. Recent advances in amplification procedures combined with improvements in the microarray platform and analytical method have overcome the amplification bias, and the data accuracy of the comprehensive PGD method has reached the level of clinical laboratory testing. Currently, comprehensive PGD is also applied to recurrent pregnancy loss due to recurrent fetal aneuploidy or infertility with recurrent implantation failure, known as preimplantation genetic screening. However, there are still numerous problems to be solved, including misdiagnosis due to somatic mosaicism, cell cycle‐related background noise, and difficulty in diagnosis of polyploidy. The technology for comprehensive PGD also requires further improvement.