Air pollution-induced tanning of human skin.

BACKGROUND: Melanism is more frequent in animals living in polluted areas on urban-industrial sites. Given that an increasing number of people are exposed to elevated air pollution levels, it is possible that environmental pollutants affect melanogenesis in human skin. Epidemiological studies have shown that exposure to traffic-related air pollutants such as diesel exhaust particles (DEP) is associated with more clinical signs of hyperpigmentation. However, mechanistic evidence linking DEP exposure to pigmentation has been elusive. OBJECTIVES: To develop an ex␣vivo skin model to allow for repetitive topical application of relevant ambient DEP, and to provide proof of concept in humans. METHODS: We measured skin pigmentation, melanin and pigmentation-associated gene expression, and evaluated oxidative stress. RESULTS: Repetitive exposure of ex␣vivo skin to DEP at nontoxic concentrations increased skin pigmentation. This increase was visible to the naked eye, time dependent, and associated with an increase in melanin content and the transcription of genes involved in de novo melanin synthesis. Similarly, in healthy participants (n = 76), repetitive topical application of DEP at nontoxic concentrations increased skin pigmentation. DEP-induced pigmentation was mediated by an oxidative stress response. After the application of DEP, epidermal antioxidants were depleted, lipid peroxidation and oxidative DNA damage were enhanced, and in a vehicle-controlled, double-blind clinical study DEP-induced pigmentation was prevented by the topical application of an antioxidant mixture. CONCLUSIONS: Similar to solar radiation, air pollutants cause skin tanning. As eumelanin is an antioxidant, it is proposed that this response serves to protect human skin against air pollution-induced oxidative stress.

Molecular evidence that oral supplementation with lycopene or lutein protects human skin against ultraviolet radiation: results from a double-blinded, placebo-controlled, crossover study.

BACKGROUND: Increasing evidence suggests photoprotection by oral supplementation with ß-carotene and lycopene. OBJECTIVES: To examine the capacity of lycopene-rich tomato nutrient complex (TNC) and lutein, to protect against ultraviolet (UV)A/B and UVA1 radiation at a molecular level. METHODS: In a placebo-controlled, double-blinded, randomized, crossover study two active treatments containing either TNC or lutein were assessed for their capacity to decrease the expression of UVA1 the radiation-inducible genes HO1, ICAM1 and MMP1. Sixty-five healthy volunteers were allocated to four treatment groups and subjected to a 2-week washout phase, followed by two 12-week treatment phases separated by another 2 weeks of washout. Volunteers started either with active treatment and were then switched to placebo, or vice versa. At the beginning and at the end of each treatment phase skin was irradiated and 24 h later biopsies were taken from untreated, UVA/B- and UVA1-irradiated skin for subsequent reverse transcriptase polymerase chain reaction analysis of gene expression. Moreover, blood samples were taken after the washout and the treatment phases for assessment of carotenoids. RESULTS: TNC completely inhibited UVA1- and UVA/B-induced upregulation of heme-oxygenase 1, intercellular adhesion molecule 1 and matrix metallopeptidase 1 mRNA, no matter the sequence (anova, P < 0·05). In contrast, lutein provided complete protection if it was taken in the first period but showed significantly smaller effects in the second sequence compared with TNC. CONCLUSIONS: Assuming the role of these genes as indicators of oxidative stress, photodermatoses and photoageing, these results might indicate that TNC and lutein could protect against solar radiation-induced health damage.

Pycnogenol® effects on skin elasticity and hydration coincide with increased gene expressions of collagen type I and hyaluronic acid synthase in women.

INTRODUCTION AND OBJECTIVES: In recent years there has been an increasing interest in the use of nutritional supplements to benefit human skin. Molecular evidence substantiating such effects, however, is scarce. In the present study we investigated whether nutritional supplementation of women with the standardized pine bark extract Pycnogenol® will improve their cosmetic appearance and relate these effects to expression of corresponding molecular markers of their skin. MATERIALS AND METHODS: For this purpose 20 healthy postmenopausal women were supplemented with Pycnogenol for 12 weeks. Before, during and after supplementation, their skin condition was assessed (i) by employing non-invasive, biophysical methods including corneometry, cutometry, visioscan and ultrasound analyses and (ii) by taking biopsies and subsequent PCR for gene expression analyses related to extracellular matrix homeostasis. RESULTS: Pycnogenol supplementation was well tolerated in all volunteers. Pycnogenol significantly improved hydration and elasticity of skin. These effects were most pronounced in women presenting with dry skin conditions prior to the start of supplementation. The skin-physiological improvement was accompanied by a significant increase in the mRNA expression of hyaluronic acid synthase-1 (HAS-1), an enzyme critically involved in the synthesis of hyaluronic acid, and a noticeable increase in gene expression involved in collagen de novo synthesis. CONCLUSIONS: This study provides skin-physiological and for the first time molecular evidence that Pycnogenol supplementation benefits human skin by increasing skin hydration and skin elasticity. These effects are most likely due to an increased synthesis of extracellular matrix molecules such as hyaluronic acid and possibly collagen. Pycnogenol supplementation may thus be useful to counteract the clinical signs of skin aging.

Transformation of a novel direct-repeat repressor element into a promoter and enhancer by multimerisation.

Studies on the regulation of interferon (IFN) responsive genes have mainly been centred on the highly conserved IFN stimulated responsive elements (ISREs) which can mediate type I and II IFN inducibility. To date little is known about other functional cis-acting regulatory motifs in IFN responsive genes. We report here on the identification of a repressor element in the human MxA gene defined to a 19 base pair (bp) region which houses a 9 bp direct repeat. DNA-specific protein binding on this element is not affected by IFN treatment and is distinct from ISRE binding proteins. Remarkably, contrary to expectations, when the repressor element is multimerised and spliced, in either orientation, to a reporter gene it behaves like a functional, constitutive promoter. Positioning the multimerised element in front of the SV40 enhancerless promoter also led to enhanced expression. The same protein(s) seem to bind to both the single repressor element and its multimerised form. This discovery of phenotypic reversal on a repressor element via multimerisation may have important implications in vivo.

Gene expression in skeletal muscle in response to stretch and force generation.

Striated muscle is a tissue in which gene expression is influenced to a large extent by mechanical signals. This includes the regulation of gene expression-associated muscle fiber phenotype determination, which depends on which protein isoform genes are transcribed, as well as muscle fiber mass accretion, which appears to involve some translational regulation. Although muscle synthesizes a set of highly specialized proteins it has a remarkable ability to adapt by expressing different isoforms of the same protein so that it acquires the appropriate contractile characteristics. Our work has focused on the myosin heavy chain (HC) genes as these encode the myosin cross bridge, which is responsible for muscle intrinsic velocity of contraction and economy of force development. RNA analyses after cast immobilization of the limb with the muscle in the lengthened or shortened position and/or with electrical stimulation were used to determine the effects of altered mechanical signals on gene transcription. When the soleus muscle was immobilized in the shortened position in the young animal it did not fully differentiate into a slow postural-type muscle. Even in the adult, the soleus muscle if deprived of stretch and contractile activity switches back to transcribing the fast myosin HC gene. The converse was true when the fast rabbit tibialis anterior was subjected to immobilization in the lengthened position and/or electrical stimulation. Both stretch alone and stimulation alone caused repression of the fast type and activation of the slow myosin genes. The reprogramming of the fast muscle was more complete when the stretch was combined with stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stretch and force generation induce rapid hypertrophy and myosin isoform gene switching in adult skeletal muscle.

Using electrical stimulation to control force generation and limb immobilization to alter the degree of stretch, we have studied the role of mechanical activity in inducing hypertrophy and in determining fast and slow muscle fibre phenotype. Changes in gene expression were detected by analysing the RNA in hybridization studies employing cDNA probes specific for fast and slow myosin heavy chains and other genes. As a result of overload in the stretched position, the fast contracting tibialis anterior muscle in an adult rabbit is induced to synthesize much new protein and to grow by as much as 30% within a period as short as 4 days. This very rapid hypertrophy was found to be associated with an increase of up to 250% in the RNA content of the muscles and an abrupt change in the species of RNA produced. Both stretch alone and electrical stimulation alone caused repression of the fast-type genes and activation of the slow-type genes. it appears that the fast-type IIB genes are the default genes, but that the skeletal slow genes are expressed as a response to overload and stretch. These findings have implications as far as athletic training and rehabilitation are concerned.

The complete sequence of the human beta-myosin heavy chain gene and a comparative analysis of its product.

We have isolated and sequenced the gene and the cDNA coding for the human cardiac beta-myosin heavy chain (designated MYH7). The gene is 22,883 bp long. The 1935 amino acids of this protein (Mr223,111) are encoded by 38 exons. The 5' untranslated region (86 bp) is split by two introns. The 3' untranslated region is 114 bp long. Three Alu repeats were identified within the gene and a fourth one in the 3' flanking intergenic region. The molecular organization of this gene reflects the conservative pattern with respect to size, coding ratio, and number or position of introns characteristic of vertebrate sarcomeric myosin heavy chain genes. The protein sequence of the human beta-heavy chain was compared with corresponding (homologous) sequences of rabbit, rat, and hamster as well as with the (heterologous) embryonic heavy chain sequences of rat, chicken, and man. The results show that protein subregions responsible for basic functions of myosin heavy chains (nucleotide binding and actin binding) are very similar in homologous and heterologous heavy chains. Regions that differ in their primary sequences in heterologous heavy chains appear to be highly conserved within mammalian beta-myosin heavy chains. Constant and variable subregions of heavy chains are discussed in terms of functional significance and evolutionary relatedness.

Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin.

A cDNA clone coding for an internal fragment of slow-cardiac beta-myosin heavy chain was isolated from a lambda gt10 human skeletal muscle library. Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to a beta-galactosidase vector and expressed in Escherichia coli. Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites. Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region. This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule. Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured. They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution. Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains. We thus observed that one long heavy chain fragment synthesized by E. coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules. Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution. Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component. All interactions were non ATP-sensitive. We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiac beta-MHC. The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family.

Isolation and characterization of the complete human beta-myosin heavy chain gene.

The entire gene coding for the human beta-myosin heavy chain has been isolated from genomic EMBL3A phage libraries by chromosomal walking starting from clone gMHC-1, reported earlier (Appelhans and Vosberg 1983). gMHC-1 has been shown to carry coding information for the C-terminal two-thirds of beta-myosin heavy chain, which is expressed in cardiac muscle and in slow skeletal muscle fibers (Lichter et al. 1986). Three DNA clones were identified as overlapping with gMHC-1 by restriction mapping and DNA sequencing. They span a 30-kb region in the genome. About 22 kb extend from the initiation codon ATG to the poly(A) addition site. The clones include about 4 kb of 5' flanking sequences upstream of the promoter. Comparisons of beta- and alpha-myosin heavy chain sequences indicate that gene duplication of the cardiac myosin heavy chain isogenes preceded the mammalian species differentiation.