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1.
Skin Pharmacol Physiol ; 17(4): 200-6, 2004.
Article in English | MEDLINE | ID: mdl-15258452

ABSTRACT

We present here a new cosmetic formula system containing 3% ascorbic acid based on an optimized oil-in-water (O/W) emulsion. This formulation demonstrated a good long-term stability of the active ingredient and also of the emulsion itself. It could be deduced from in vitro release studies that this O/W emulsion enabled a better release of the hydrophilic active agent than an alternative W/O emulsion. By measuring the ultraweak photon emission, which is a well-established parameter for the oxidative stress in the skin, the high in vivo antioxidant capacity of 3% ascorbic acid was demonstrated after 1 week of product application. This placebo-controlled study also proved that ascorbic acid in an O/W cream reduced oxidative stress in human skin significantly better than the derivative sodium ascorbyl-2-phosphate, a more stable vitamin C replacement commonly used in cosmetic formulations. With increasing age, the number of papillae in the epidermal-dermal junction zone in human skin are reduced. This implies a possible consequence of reduced mechanical resistance of the skin and impaired supply of the epidermis with nutrients. In a 1-month placebo-controlled study on 25 human volunteers, a significant increase in the number of dermal papillae after application of the 3% ascorbic acid cream was demonstrated, using a confocal laser scanning microscope. Fine lines and wrinkles are a characteristic sign of aged and especially photo-aged skin. Application of 3% ascorbic acid in a 12-week placebo-controlled usage study indicated a significant reduction of facial wrinkles. Altogether, 3% ascorbic acid in a cosmetic O/W emulsion has been shown to be appropriately stable and to enable a good release of the active agent in vitro as a precondition for a high efficacy in vivo. Application in vivo resulted in a significant reduction of oxidative stress in the skin, an improvement of the epidermal-dermal microstructure and a reduction of fine lines and wrinkles in aged skin. These results were received within a relatively short period of time of product application.


Subject(s)
Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Cosmetics/pharmacology , Skin Aging/drug effects , Administration, Cutaneous , Antioxidants/administration & dosage , Ascorbic Acid/administration & dosage , Ascorbic Acid/chemistry , Controlled Clinical Trials as Topic , Cosmetics/chemistry , Diffusion , Drug Compounding/methods , Drug Stability , Emulsions , Excipients/administration & dosage , Female , Humans , Microscopy, Confocal/instrumentation , Middle Aged , Oxidative Stress/drug effects , Reproducibility of Results , Skin Aging/pathology , Treatment Outcome , Ultraviolet Rays/adverse effects
2.
J Mol Biol ; 314(4): 823-37, 2001 Dec 07.
Article in English | MEDLINE | ID: mdl-11734000

ABSTRACT

Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication.


Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Exotoxins/chemistry , Exotoxins/toxicity , Pseudomonas aeruginosa/chemistry , Virulence Factors , Amino Acid Sequence , Binding Sites , Computer Simulation , Crystallization , Crystallography, X-Ray , Exotoxins/genetics , Exotoxins/metabolism , Furin , Hydrogen Bonding , Hydrogen-Ion Concentration , Ligands , Models, Molecular , Mutation/genetics , Pliability , Protein Structure, Secondary , Protein Structure, Tertiary , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Sequence Alignment , Static Electricity , Structure-Activity Relationship , Subtilisins/metabolism , Pseudomonas aeruginosa Exotoxin A
3.
Proc Natl Acad Sci U S A ; 98(11): 5965-9, 2001 May 22.
Article in English | MEDLINE | ID: mdl-11353861

ABSTRACT

The hydrophobic interaction, the tendency for nonpolar molecules to aggregate in solution, is a major driving force in biology. In a direct approach to the physical basis of the hydrophobic effect, nanosecond molecular dynamics simulations were performed on increasing numbers of hydrocarbon solute molecules in water-filled boxes of different sizes. The intermittent formation of solute clusters gives a free energy that is proportional to the loss in exposed molecular surface area with a constant of proportionality of 45 +/- 6 cal/mol A(2). The molecular surface area is the envelope of the solute cluster that is impenetrable by solvent and is somewhat smaller than the more traditional solvent-accessible surface area, which is the area transcribed by the radius of a solvent molecule rolled over the surface of the cluster. When we apply a factor relating molecular surface area to solvent-accessible surface area, we obtain 24 cal/mol A(2). Ours is the first direct calculation, to our knowledge, of the hydrophobic interaction from molecular dynamics simulations; the excellent qualitative and quantitative agreement with experiment proves that simple van der Waals interactions and atomic point-charge electrostatics account for the most important driving force in biology.


Subject(s)
Computer Simulation , Models, Molecular , Solutions/chemistry , Solvents/chemistry , Energy Transfer , Methane/chemistry , Water/chemistry
4.
Nat Struct Biol ; 6(9): 825-31, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10467093

ABSTRACT

The kinetic intermediate of RNase H is structured in a core region of the protein. To probe the role of this intermediate in the folding of RNase H, the folding kinetics of mutant proteins with altered native state stabilities were investigated. Mutations within the folding core destabilize the kinetic intermediate and slow refolding in a manner consistent with an obligatory intermediate model. Mutations outside of the folding core, however, do not affect the stability of the kinetic intermediate but do perturb the native state and transition state. These results indicate that interactions formed in the intermediate persist in the transition and native states and that RNase H folds through a hierarchical mechanism.


Subject(s)
Protein Folding , Ribonuclease H/chemistry , Ribonuclease H/metabolism , Amino Acid Substitution , Circular Dichroism , Enzyme Stability , Kinetics , Models, Molecular , Mutation , Protein Denaturation , Protein Engineering , Protein Structure, Secondary , Ribonuclease H/genetics , Thermodynamics , Urea
5.
Curr Opin Biotechnol ; 9(1): 80-6, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9503592

ABSTRACT

Hydrogen exchange techniques, with their residue-level specificity, exquisite sensitivity, and adaptability to many solution conditions, are becoming essential to the study of protein stability, folding and dynamics. Recent studies have elucidated the structures of intermediates formed transiently during protein folding and rare partially folded ensembles present at equilibrium. Analysis of hydrogen exchange mechanisms has revealed protein stability and kinetics at the level of individual residues.


Subject(s)
Hydrogen , Models, Molecular , Proteins/chemistry , Hydrogen Bonding , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Folding
6.
Biochemistry ; 36(23): 7256-63, 1997 Jun 10.
Article in English | MEDLINE | ID: mdl-9188727

ABSTRACT

The ribonuclease H (RNase H) family of enzymes selectively degrades the RNA strand of RNA-DNA hybrids. This activity is essential for retroviruses such as HIV and resides in a domain of the larger reverse transcriptase molecule. RNase H from Escherichia coli is the best-characterized member of the family and serves as a model for structure/function studies. Despite having almost identical alpha + beta folds, the isolated domain from HIV is inactive and much less stable than the E. coli homolog. The HIV domain also shows increased disorder in its C-terminal regions (E-helix and His-containing loop). We investigated the importance of this region by studying a variant of E. coli RNase H lacking these elements (RNHdeltaE). Despite the elimination of 33 of 155 residues (including a complete helix), this C-terminal deletion mutant folds cooperatively as a subdomain. Surprisingly, this protein lacking residues near the active site retains weak Mn2+-dependent activity. A peptide corresponding to the deleted E-helix is helical in isolation and stimulates the activity of the deletion mutant in vitro. These results have implications for the catalytic mechanism of RNase H and drug design targeted to HIV reverse transcriptase.


Subject(s)
Escherichia coli/enzymology , Ribonuclease H/chemistry , Amino Acid Sequence , Circular Dichroism , Crystallography, X-Ray , Enzyme Stability , Manganese/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Folding , Protein Structure, Secondary , Ribonuclease H/genetics , Ribonuclease H/metabolism , Sequence Deletion
7.
Nat Struct Biol ; 4(4): 298-304, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9095198

ABSTRACT

Folding of ribonuclease HI from Escherichia coli populates a kinetic intermediate detectable by stopped-flow circular dichroism. Pulse labelling hydrogen exchange reveals that this intermediate consists of a structured core region of the protein, namely helices A and D and beta-strand 4. This kinetic intermediate resembles both the acid molten globule of ribonuclease HI and rarely populated, partially unfolded forms detected under native conditions. These results indicate that the first portion of ribonuclease HI to fold is the most thermodynamically stable region of the native state, and that folding of this protein follows a hierarchical process.


Subject(s)
Protein Folding , Ribonuclease H , Circular Dichroism , Dose-Response Relationship, Drug , Escherichia coli/enzymology , Flow Injection Analysis , Kinetics , Models, Chemical , Models, Molecular , Urea/pharmacology
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