Practical application of cellular bioenergetics to the care of aged skin.

In human skin fibroblasts in vitro, procollagen-1 and NAD(+)/NADH were reduced in three strains of adult fibroblasts compared with neonatal fibroblasts. The levels of both procollagen-1 and NAD(+)/NADH were increased in the adult fibroblasts by treatment for 24 (NAD energy) or 48 h (procollagen-1) with a complex containing niacinamide, Pal-KTTKS peptide and an olive oil fatty acid derivative (Olivem(®)), especially in combination with a natural extract from dill (Lys'lastine V(®)). In one of the adult fibroblast strains evaluated, these changes in procollagen-1 and NAD(+)/NADH in response to the complex of bioactives were in parallel with increased expression of mRNA biomarkers related primarily to dermal matrix and basement membrane structure, including COL1A1, COL3A1, COL5A1, COL14A1, ELN and LOXL2, in addition to SOD2, NAMPT and TGFBR3; MMP1 was decreased in expression. In general, these mRNA biomarker effects were maintained or boosted by the addition of Lys'lastine V, particularly at 1%, and were similar to the fold changes in mRNA expression in neonatal compared with adult fibroblasts. These results indicate that the complex of niacinamide, Pal-KTTKS and Olivem, especially with addition of Lys'lastine V, increases the NAD(+)/NADH bioenergy level of adult skin fibroblasts in parallel with increased expression of skin structure biomarkers in vitro to levels similar to those in younger fibroblasts. Thus, niacinamide, Pal-KTTKS, Olivem and Lys'lastine V are promising bioactive candidates for inclusion in cosmetic formulations.

Skin stem cell hypotheses and long term clone survival--explored using agent-based modelling.

Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation.

Application of genomics to breakthroughs in the cosmetic treatment of skin ageing and discoloration.

The use of global gene expression profiling, also known as transcriptomics or genomics, provides a means to identify key pathways affected in ageing skin that can be improved with appropriate cosmetic compounds. Aspects of skin ageing that can be addressed include matrix production, barrier, lipid synthesis, antioxidant capacity and hyperpigmentation. Gene expression profiling together with in vitro human skin cell cultures for compound screening and verification have led to the identification of cosmetic compounds and an understanding of the biological effects of compounds such as niacinamide, Pal-KTTKS, hexamidine, retinyl propionate and sodium dehydroacetate. In addition, understanding of the decreased antioxidant capacity of aged skin has led to the identification of new antiageing ingredients, olive-derived fatty acid ethoxylates, which have been shown to restore antioxidant enzymes in skin keratinocytes and fibroblasts. Gene expression profiling of age spots has also provided an understanding of the role of undecylenoyl phenylalanine in reducing melanin production by an adrenergic receptor mechanism in melanocytes. The use of these compounds in cosmetic formulations for skin care can aid improvements in the appearance of aged skin, including the improved appearance of fine lines, wrinkles and age spots.

Dysregulation of transforming growth factor beta signaling in scleroderma: overexpression of endoglin in cutaneous scleroderma fibroblasts.

OBJECTIVE: As an initial approach to understanding the basis of the systemic sclerosis (SSc; scleroderma) phenotype, we sought to identify genes in the transforming growth factor beta (TGF beta) signaling pathway that are up-regulated in lesional SSc fibroblasts relative to their normal counterparts. METHODS: We used gene chip, differential display, fluorescence-activated cell sorter, and overexpression analyses to assess the potential role of TGF beta signaling components in fibrosis. Fibroblasts were obtained by punch biopsy from patients with diffuse cutaneous SSc of 2-14 months' duration (mean 8 months) and from age- and sex-matched healthy control subjects. RESULTS: Unexpectedly, we found that fibroblasts from SSc patients showed elevated expression of the endothelial cell-enriched TGF beta receptor endoglin. Endoglin is a member of the nonsignaling high-affinity TGF beta receptor type III family. The expression of endoglin increased with progression of disease. Transfection of endoglin in fibroblasts suppressed the TGF beta-mediated induction of connective tissue growth factor promoter activity. CONCLUSION: SSc is characterized by overproduction of matrix; that is, genes that are targets of TGF beta signaling in normal fibroblasts. Our findings suggest that lesional SSc fibroblasts may overexpress endoglin as a negative feedback mechanism in an attempt to block further induction of profibrotic genes by TGF beta.

A complex of nuclear pore proteins required for pore function.

A family of proteins bearing novel N-acetylglucosamine residues has previously been found to be required to form functional nuclear pores. To begin to determine which of the proteins in this family are essential for pore function, antisera were raised to each of three members of the family, p62, p58, and p54. With these antisera, it was possible to deplete nuclear reconstitution extracts of the proteins and to test the depleted nuclei for nuclear transport. In the course of the experiments, it was found that the three proteins exist as a complex; antisera to any one, while specific on a protein blot, coimmunoprecipitated all three proteins. This complex of pore proteins is stable to 2 M salt, 2 M urea, and the detergent Mega 10, indicating the presence of specific and tight protein-protein interactions. By gel filtration, the complex has a molecular mass of 550-600 kD. Nuclei containing pores depleted of the complex are found to be defective for nuclear transport; moreover, we observe a strict linear correlation between the amount of complex present in nuclei and the amount of nuclear transport of which those nuclei are capable. Thus, the p62-p58-p54 complex defines a group of proteins with strong protein-protein interactions that form a unit of pore structure essential for pore function.

Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored.

Biochemically altered nuclear pores specifically lacking the N-acetylglucosamine-bearing pore proteins were constructed in a nuclear assembly extract in order to assign function to these proteins. The depleted pores do not bind nuclear signal sequences or actively import nuclear proteins, but they are functional for diffusion. These defects can be fully repaired by assembly with readded Xenopus pore glycoproteins. Strikingly, isolated rat pore glycoproteins also restore transport. Electron microscopy reveals that depleted pores have largely normal morphology. Thus, the pore glycoproteins are not required for assembly of the nuclear envelope, the major structures of the pore, or a pore diffusional channel. Instead, they are essential for active protein import and, unexpectedly, for construction of the part of the pore necessary for signal sequence recognition.

Nuclear transport in vitro.

In this paper, progress towards the goal of understanding communication between the nucleus and cytoplasm using an in vitro system is reviewed. To probe the mechanism of nuclear targeting, we developed an in vitro transport system and have begun to dissect the highly selective process of nuclear transport. The basic parameters of transport were defined using an easily isolated nuclear protein, nucleoplasmin. To study the interaction of nuclear targeting signals with the pore, an artificial nuclear transport substrate was constructed, which consists of human serum albumin coupled to the signal sequence of the SV40 T-antigen. A similar peptide-protein conjugate was made using a mutant signal sequence. These conjugates were fluorescently labeled and/or tagged with gold and tested for transport in the in vitro system. High levels of nuclear transport of the wild-type signal sequence-containing protein were observed, while no transport of the mutant signal sequence-containing protein was seen. Thus, the in vitro system correctly recognizes the single amino acid change between the wild-type and mutant signal sequences. We found that the observed nuclear transport was completely dependent on the presence of ATP. Using the in vitro system we identified a specific inhibitor of nuclear transport, the lectin wheat germ agglutinin (WGA), which we find binds directly to the nuclear pore. Probing blots of nuclear proteins with 125I-WGA identified a family of nuclear pore glycoproteins, including one major glycoprotein of 62K (K = 10(3)Mr) molecular weight. With the inhibitor and the in vitro assay, it has been possible to experimentally separate nuclear transport into two steps: (1) a step in which the signal sequence-bearing protein binds to the pore, followed by (2) a step in which the protein translocates through the pore. It is this second step which is the ATP-dependent step of transport, since pore binding but not translocation was seen to occur in the absence of ATP.

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy-dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Bürglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non-nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.

Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression.

Derivatives of plasmid pRK290 that are useful for cloning and for analyzing gene expression in a wide variety of Gram-negative bacteria are described. A smaller broad host range plasmid derived from RK2, with properties similar to that of pRK290, is also described.

The intramembrane topography of the mannitol-specific enzyme II of the Escherichia coli phosphotransferase system.

The D-mannitol-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is an integral cytoplasmic membrane protein responsible for concomitant transport and phosphorylation of this hexitol. We have investigated the intramembrane topography of this enzyme/permease using proteases, membrane-impermeable reagents, and antibodies against the purified protein. The results of these experiments suggest that this protein spans the membrane in a single orientation with a sizeable proportion of its mass extending into the cytoplasm, but with little of the polypeptide exposed at the outside surface of the membrane. Such an orientation is consistent with the reception and transport roles of the mannitol Enzyme II in E. coli.