Structural basis of aggregate binding by the AAA+ disaggregase ClpG.

Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a ß-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.

Temperature Matters: Bacterial Response to Temperature Change.

Temperature is one of the most important factors in all living organisms for survival. Being a unicellular organism, bacterium requires sensitive sensing and defense mechanisms to tolerate changes in temperature. During a temperature shift, the structure and composition of various cellular molecules including nucleic acids, proteins, and membranes are affected. In addition, numerous genes are induced during heat or cold shocks to overcome the cellular stresses, which are known as heat- and cold-shock proteins. In this review, we describe the cellular phenomena that occur with temperature change and bacterial responses from a molecular perspective, mainly in Escherichia coli.

Cytoplasmic molecular chaperones in Pseudomonas species.

Pseudomonas is widespread in various environmental and host niches. To promote rejuvenation, cellular protein homeostasis must be finely tuned in response to diverse stresses, such as extremely high and low temperatures, oxidative stress, and desiccation, which can result in protein homeostasis imbalance. Molecular chaperones function as key components that aid protein folding and prevent protein denaturation. Pseudomonas, an ecologically important bacterial genus, includes human and plant pathogens as well as growth-promoting symbionts and species useful for bioremediation. In this review, we focus on protein quality control systems, particularly molecular chaperones, in ecologically diverse species of Pseudomonas, including the opportunistic human pathogen Pseudomonas aeruginosa, the plant pathogen Pseudomonas syringae, the soil species Pseudomonas putida, and the psychrophilic Pseudomonas antarctica.

Silymarin inhibits melanin synthesis in melanocyte cells.

OBJECTIVES: The aim was to search for inhibitors of melanogenesis from natural resources. METHODS: The inhibitory effect of silymarin on melanogenesis in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab, was studied. KEY FINDINGS: Silymarin significantly prevented melanin production in a dose-dependent manner with an IC50 value (concentration producing 50% maximal inhibition) of 28.2 microg/ml, without effects on cell viability. Also, silymarin inhibited L-DOPA oxidation activity of tyrosinase, the rate-limiting melanogenic enzyme, in cell based-systems but it did not directly affect cell-free tyrosinase activity. Furthermore, Western blot analysis indicated that silymarin decreased the expression of tyrosinase protein. CONCLUSIONS: This study suggests that the depigmenting effect of silymarin might be attributable to inhibition of tyrosinase expression and that silymarin may be useful as a natural skin-lightening agent.

Alternative evaluation method in vitro for the water-resistant effect of sunscreen products.

BACKGROUND/PURPOSE: Sunscreen products today represent a trend of providing not only simple sun protection factor (SPF)/protection of UVA (PFA) but also other additional benefits. For example, as popularized by seasonless use of sunscreens, the special function of water resistance or sand proof is added to sunscreens as well as for leisure. Because a human in vivo test is time consuming and expensive, a screening process has been tried using an accurate in vitro system. In this study, we suggest the development of an in vitro test that can predict the result of in vivo water resistance of sunscreens. METHODS: Water resistance is presented as a comparison of initial SPF and water-exposed SPF by immersion and washing. In order to be comparable with the in vivo test, water immersion and flow were defined as the basic statements. Also, substrate, revolutions per minute (r.p.m.)--rotative velocity--of propeller inducing water flow, and time of immersion were defined as controlled factors. Considering the strength, separation of test material and adhesive texture, a PMMA plate was selected as suitable among commercial substrates: Transpore tape, VITRO SKIN, and PMMA plate. Also, when the PMMA plate was adhered on the wall of a water bath, the water turbulence of the rotational propeller alone was not strong enough to wash off the test material from the substrate. Therefore, PMMA plates were fixed on the axis. In this experiment, the most important thing is whether this in vitro system can predict correctly. Hence, we tried to match the in vitro water resistance following from our control factors and water resistance value of the in vivo test. RESULTS: We found the immersion time and r.p.m. of controlled factors to obtain the target water resistance using design of experiment, MiniTab statistical package. Response optimization yielded the optimal in vitro conditions of 150 r.p.m./60 min. The repeatability and reproducibility of this in vitro system were also good in validation studies. CONCLUSIONS: This study enables to modify an in vivo water resistance test and predict the result of in vivo water resistance by the manufacture of effective equipment and choosing a suitable substrate. Compared with in vivo results, our in vitro system is more time and cost effective, and provides reliable results.

Suitability of macrophage inflammatory protein-1beta production by THP-1 cells in differentiating skin sensitizers from irritant chemicals.

BACKGROUND: Worldwide restrictions in animal use for research have driven efforts to develop alternative methods. OBJECTIVE: The study aimed to test the efficacy of the macrophage inflammatory protein-1beta (MIP-1beta) assay for testing chemicals' skin-sensitizing capacity. METHODS: The assay was performed using 9 chemicals judged to be sensitizing and 7 non-sensitizing by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of 4 doses, 0.01x, 0.1x, 0.5x, or 1x IC(50) (50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and the MIP-1beta level in the supernatants was determined. Skin sensitization by the test chemicals was determined by MIP-1beta production rates. The MIP-1beta production rate was expressed as the relative increase in MIP-1beta production in response to chemical treatment compared with vehicle treatment. RESULTS AND CONCLUSION: When the threshold MIP-1beta production rate used was 100% or 105% of dimethyl sulfoxide, all the sensitizing chemicals tested (dinitrochlorobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone, dinitrofluorobenzene, benzocaine, nickel, chromium, and 5-chloro-2-methyl-4-isothiazolin-3-one) were positive, and all the non-sensitizing chemicals (methyl salicylate, benzalkonium chloride, lactic acid, isopropanol, and salicylic acid), with the exception of sodium lauryl sulfate, were negative for MIP-1beta production. These results indicate that MIP-1beta could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.

Influence of polyol and oil concentration in cosmetic products on skin moisturization and skin surface roughness.

BACKGROUND/PURPOSE: The purpose of this study is to investigate the optimum combination of polyols and oils in moisturizing cosmetic products to improve the human skin moisturization and skin surface roughness. Polyols and oils are essential ingredients in skin care products, but it is still not understood how their concentrations affect their efficacy and sensory properties on human skin. We investigated the effect of polyol and oil concentration on skin properties by noninvasive methods. METHODS: The polyols consisted of glycerin and butylenes glycol in a ratio of 1:1 and the oils consisted of equal parts of hydrogenated polydecene, cethyl ethylhexanoate and pentaerythrityl tetraethylhexanoate. All cosmetic products were made in O/W emulsions in concentrations ranging from 0% to 30% for polyols and from 0% to 35% for oils. We investigated the effect on water content and skin surface roughness on the forearm after application of the cosmetic products. The skin water contents were measured by a Corneometer CM825 and the skin surface roughness by visual coring of skin surface biopsies in the scanning electron micrographs. RESULTS: In the first study, we found that the water content of the skin correlated highly with the polyol (up to 30%) and oil (up to 12%) concentrations, respectively. At two hours after application, the correlation coefficients were 0.971 and 0.985, respectively (P<0.01). Skin surface roughness not only showed a strong concentration dependence on polyols and oils (up to 6%). In the second study, we investigated the optimum combination of polyols and oils to improve the skin moisturization and skin surface roughness by the Response surface methodology. The water content of the skin surface was high in the ratio of polyol to oil (30:12 and 25:30). The skin surface roughness was improved considerably in the ratio of polyols to oil (30:6 and 30:35). CONCLUSIONS: Our results indicated that the skin surface properties were improved in the different ratios of their concentrations because they are influenced by not one ingredient but the interaction between polyols and oils. In this study, we could recommend the optimum concentration of polyols and oils to improve the skin surface properties. Further studies will be performed with other ingredients such as surfactants, lipids and so on.

Comparison and correlation between stinging responses to lactic acid and bioengineering parameters.

In evaluating the safety of a novel cosmetic product or a new chemical, it is important to assess susceptible population. One group of subjects is known to stingers who are more likely to experience sensory effects such as stinging and burning after contacting with cosmetics. The purpose of the study is to measure skin biophysical parameters noninvasively in stingers and non-stingers and to see their correlations with stinging responses. 298 women were evaluated by modified lactic acid stinging test with 5% lactic acid solution rather than classic 10% solution because of strong reaction in Asian populations. Transepidermal water loss (TEWL), skin hydration, sebum content, and pH were measured using the bioengineering instruments in an environment-controlled room. Correlations between stinging responses and skin biophysical parameters were statistically analysed. There was a positive correlation between stinging responses and TEWL evaluation. However, no correlations was observed between stinging responses and other parameters such as skin hydration, sebum content, and pH. Our data indicate that there is a relationship between the degree of stinging and the skin barrier function. However, we believe that various additional studies are necessary to characterize skin of stingers and the pathogenesis.

Correlation between a Cutometer and quantitative evaluation using Moire topography in age-related skin elasticity.

BACKGROUND/PURPOSE: As aging occurs, our skin gets more wrinkles, becomes drier and loses its elasticity. Validating the evaluation of skin elasticity is especially important, because it is not as visible as other signs of aging such as wrinkles. So it is needed that the method for measuring skin elasticity is able to reflect perception about the change of the skin state. METHODS: Here, the correlation between age and the parameters given by a Cutometer is identified and the main parameters that reflect the decreases in skin elasticity in terms of ages are presented. Also, Moire's system, an evaluation method to quantify the sensory value of viewing, is developed. A five-grade standard of Moire topographic photo scale on the face is used to evaluate the state of skin elasticity and lifting 20- to 61-year-old women. Based on this photo standard, scoring is performed using a five-grade system by three specialists to obtain the consensus score. The score is compared with the result of a Cutometer. RESULTS: Significant negative correlations between age and results of a Cutometer (r=-0.687-0.725), Moire's topography scores (r=-938), were found. Some Cutometer parameters and the decreases in skin elasticity in terms of ages were highly correlated (r=-0.687-0.725). The results from Moire system and flexibility as sensory evaluation also had a very high correlation with age (r=-0.765-0.932). Finally, we have shown the significance of the correlation between the result of a Cutometer and the score of Moire topography (r=0.711). CONCLUSIONS: It is considered that Cutometer parameters R7 and R2 are used as main parameters to assess skin elasticity and aging. And our studies using Moire topography on the face have confirmed that instrumental measurements reflect the decrease in skin elasticity, which is perceived visually.

Comparison of objective and sensory skin irritations of several cosmetic preservatives.

There are many cosmetic ingredients, such as preservatives and fragrances, known to elicit adverse effects. The aim of this study was to investigate the side-effects of cosmetic preservatives, by evaluating objective and subjective skin irritation. The method comprised of 2 parts. In part 1, we tried to compare 24-hr patch test results with the sensory irritation potential of several preservatives. In part 2, skin cumulative irritation test for 21 days and sensory irritation test were performed to compare various combinations of preservatives in 4 types of formulations. Our data showed that methylparaben, ethylparaben, propylparaben, butylparaben, phenoxyethanol (PE) and chlorphenesin (CPN) have similar objective skin irritation potential at the minimal inhibitory concentration of each preservative, but CPN has higher potential than other preservatives in subjective irritation. Sensory irritation of preservatives changed according to formulation type, and PE combined with CPN highly increased irritation. There was correlation between antimicrobial activity and skin objective irritation but not sensory irritation. Influence on skin sensory irritation varies with the combination of preservatives. Therefore, for the development of new preservatives and cosmetics, it is important to evaluate skin sensory irritation of preservatives used in cosmetic products according to the type of formulations.

Expression of vanilloid receptor 1 in cultured fibroblast.

Although the vanilloid receptor 1 (VR1) was originally discovered on primary sensory neurons, its broad tissue expression in non-neuronal cells has been reported on. Recently, VR1 expression was clearly demonstrated in a variety of cutaneous components, such as keratinocytes, glandular epithelium, mast cells and sebocytes, except for melanocytes and fibroblasts. However, we demonstrated the VR1 expression in the cultured human skin fibroblasts as follows. Previously cloned human VR1 primers that corresponded to the expected size of 680 bp by reverse transcriptase polymerase chain reaction were identified on the fibroblasts, the same as was noted for the positive control, the HaCaT cells. A positive immunoreactivity of the VR1 was observed both on fibroblasts and on HaCaT cells by Western blotting analysis. Fibroblasts treated with capsaicin, an agonist to the VR1, induced significant changes of the membrane current and the intracellular calcium level, and these changes were antagonized by capsazepin. Capsaicin treatment also showed a positive immunocytochemistry result. Our results suggest the existence of VR1 on fibroblasts; this receptor is likely to be influenced by ligand-dependent activation.

An in vitro phototoxicity assay battery (photohaemolysis and 3T3 NRU PT test) to assess phototoxic potential of fragrances.

The purpose of this study was to compare the in vivo and in vitro phototoxicity potentials of 13 fragrances. We used the 3T3 neutral red uptake phototoxicity (3T3 NRU PT) test and the photohaemolysis test as in vitro phototoxicity assays. In the 3T3 NRU PT test, all of the fragrances were non-phototoxic. Six fragrances were phototoxic in the photohaemolysis test. Three of the six photohaemolytic fragrances were phototoxic in the guinea-pig photoirritation test. These phototoxic fragrances did not cause cellular phototoxicity, but showed a photohaemolytic reaction. The photohaemolysis test was more sensitive than the 3T3 NRU PT test for screening for the phototoxicity of fragrances. The accuracy of this in vitro phototoxicity test battery was 82%. It is thought that the major phototoxic mechanism of fragrances is cell membrane damage. We suggest that a battery composed of the 3T3 NRU PT test and the photohaemolysis test is a simple and effective model for the in vitro phototoxicity assay of fragrances.