Understanding skin barrier differences: a demographic, cultural, and medical diversity viewpoint.

Important differences exist in the physiology of the stratum corneum barrier according to demographic, cultural, and medical factors. Understanding these differences is crucial to choosing strategies for optimum clinical management.

Update on the structure and function of the skin barrier: atopic dermatitis as an exemplar of clinical implications.

The healthy stratum corneum allows optimum permeability of water and provides the first line of defense against pathogenic and environmental assaults. The barrier functions of the stratum corneum are interrelated, coregulated, and interdependent. Research has demonstrated that three lipid species, which usually comprise 10% of the stratum corneum, are crucial to both its structure and its function; these must be present in sufficient quantities and in the correct proportions to provide optimum barrier function. The clinical implications of how the skin barrier works--and is supported and restored--can be seen in the current and emerging understanding of atopic dermatitis management.

The chemistry of skin cleansers: an overview for clinicians.

Cleansers and other skin care products can be agents either of stratum corneum damage and skin barrier dysfunction or of maintaining or restoring healthy stratum corneum barrier structure and function. To guide patients toward beneficial choices most suitable for their individual skin conditions and needs, clinicians must be aware of and understand the ingredients in such skin care products and their potential effects on the stratum corneum barrier. In cleansers specifically, clinicians should be aware particularly of the benefits and potential problems associated with chemical components of surfactants, preservatives, and fragrances.

A lifetime of well skin care: practical recommendations for clinicians and patients.

The skin is an indicator of overall health throughout life, and the skin's lifelong care and environment are reflected with aging. The goal of skin care education by clinicians is to teach and reinforce habits that will support and maintain optimum stratum corneum barrier function throughout life and, when dermatologic conditions or injuries arise, that will aid in recovery of barrier function.

Skin-cleansing and care principles for special pediatric populations.

Good skin care has two overall goals: to support and maintain healthy stratum corneum function and to help restore barrier function perturbed by disease processes or injuries. In this article, we discuss the special attention that is required in the initial skin care of newborns, and we address what measures, beyond the basic skin care principles, are required for patients with conditions such as atopic dermatitis, acne, contact and allergic dermatitis, and diaper rash.

Tolerance of skin care regimen in healthy, full-term neonates.

PURPOSE: To assess the tolerance of a baby cleanser and lotion (both lightly fragranced) on healthy, full-term neonates. MATERIALS AND METHODS: Twenty-six infant-mother pairs were enrolled in a 6-week, nonrandomized, controlled-use study that took place in the routine setting of a pediatric clinic and mothers' homes. During study weeks 1 to 6, neonates were bathed by their mother with water and a test cleanser (JOHNSON'S® HEAD-TO-TOE® Baby Wash). During study weeks 1 to 3, mothers also applied test lotion (JOHNSON'S® Baby Lotion) to the babies' skin immediately after bathing and one to three times/day on bathing and non-bathing days. During study weeks 4 to 6, no lotion was applied. At baseline and weeks 3 and 6, the infants' pediatrician or mother or both performed visual skin assessments. RESULTS: Twenty-three infant-mother pairs completed the study. The mean age of neonates at enrolment was 17.4 days (range, 13-28 days). Pediatrician observations found no clinical signs of irritation, erythema, or dryness with any significant difference in scores of these parameters compared with baseline throughout the study. Assessment of skin softness, smoothness, dryness, and overall skin condition was very good at baseline and remained so with minimal changes throughout the study. Mothers reported improvements versus baseline (P ≤ 0.05) in overall skin appearance, moisturization, softness, and smoothness on the arms and legs at weeks 3 and 6. A total of four (15.4%) subjects experienced adverse events. For three of the subjects, the investigator suspected that the adverse events were unrelated to either of the test products. In one participant, the cause of the adverse event could not be determined. CONCLUSION: The use of a lightly fragranced nonstinging baby cleanser, with or without a lightly fragranced baby lotion, was well tolerated by newborns and resulted in observable skin benefits per the pediatricians' and mothers' assessment.

Active rhodanese lacking nonessential sulfhydryl groups has increased hydrophobic exposure not observed in wild-type enzyme.

Mutation of all nonessential cysteine residues to serines in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). bis-ANS binding studies have shown that C3S has more hydrophobic exposure than WT, although both have similar secondary structures suggesting the flexibility of its structure. Activity of C3S falls once it binds bis-ANS, and covalent binding of bis-ANS to C3S is induced by light. bis-ANS binds to C3S in its C-terminal domain as is shown by gel electophoresis and proteolysis. bis-ANS binding makes the C-terminal domain more susceptible to trypsin cleavage.

Activation parameters for the spontaneous and pressure-induced phases of the dissociation of single-ring GroEL (SR1) chaperonin.

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range 0.5-3.0 kbar. The kinetics were studied as a function of temperature in the range 15-35 degrees C. The dissociation processes at each pressure and temperature showed biphasic behavior. The slower rate (k1,obs) was confirmed to be the self-dissociation of SR1 at any specific temperature at atmospheric pressure. This dissociation was pressure independent and followed concentration-dependent first-order kinetics. The self-dissociation rates followed normal Eyring plots (In k1,obs/T vs. 1/T) from which the free energy of activation (deltaG++ = 22 +/- 0.3 kcal mol(-1)), enthalpy of activation (deltaH++ = 18 +/- 0.5 kcal mol(-1)), and entropy of activation (deltaS++ = -15 +/- 1 kcal mol(-1)) were evaluated. The effect of pressure on the dissociation rates resulted in nonlinear behavior (ln k2,obs vs. pressure) at all the temperatures studied indicating that the activation volumes were pressure dependent. Activation volumes at zero pressure (V++o) and compressibility factors (beta++) for the dissociation rates at the specific temperatures were calculated. This is the first systematic study where the self-dissociation of an oligomeric chaperonin as well as its activation parameters are reported.

Prodan fluorescence mimics the GroEL folding cycle.

The non-covalent fluorescent probe 6-propionyl-2-(dimethylamino) naphthalene sulfonate (prodan) binds to hydrophobic surfaces exposed on the surface of GroEL. Under identical experimental conditions free prodan exhibits a green emission peak of intensity 390,000 cps at 520 nm. However prodan bound to GroEL, GroEL-ATP, and GroEL-ATP-GroES shows emission peaks of intensities 500,000, 540,000, and 480,000 cps at 515, 512 and 515 nm, respectively, thus mimicing the way hydrophobic surfaces on GroEL become exposed during the folding cycle. Other hydrophobic probes like bis-ANS and dansyl lysine were unable to detect the minor changes in hydrophobic exposure of GroEL after it binds to ATP, although they were able to detect hydrophobic exposure in GroEL itself.

Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound, inactivated, and reactivated by GroEL.

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.

Dissociation of the single-ring chaperonin GroEL by high hydrostatic pressure.

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range of 1-2.5 kbar. The kinetics of the dissociation of SR1 in the absence and presence of Mg2+, KCl, and nucleotides were monitored using light scattering. The major aim of this investigation was to understand the role of the double-ring structure of GroEL by comparing its dissociation with the dissociation of the single ring. At all the pressures that were studied, SR1 dissociates much faster than the GroEL 14mer. As observed with the GroEL 14mer, SR1 also showed biphasic kinetics and the dissociated monomers do not reassociate readily back to the oligomer. Unlike the GroEL 14mer, the observed rates for SR1 dissociation are independent of the concentrations of Mg2+ and KCl in the studied range. The effects of nucleotides on the observed rates, in the absence or presence of Mg2+ and KCl, are not very significant. The heterogeneity induced in the GroEL molecule with the double-ring structure by ligands such as Mg2+, KCl, and nucleotides is not observed in the case of SR1. This indicates that the inter-ring negative cooperativity in the double-ring GroEL has a major role in this regard. The results presented in this investigation demonstrate that the presence of a second ring in the GroEL 14mer is important for its stability in an environment where the functional ligands of the chaperonin are available.

The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor.

The molecular chaperone DnaK and trigger factor (TF), a ribosome-associated protein with folding activity, have been implicated in assisting nascent polypeptides to acquire a three-dimensional structure on Escherichia coli ribosomes. We asked whether ribosomes that lack trigger factor would recruit DnaK for synthesis and folding of nascent peptides. For these analyses, translating ribosomes with a homogeneous population of nascent peptides were isolated. Truncated forms of rhodanese and E. coli translation initiation factor 3 (IF3) were generated with tandem rare arginine codons in the coding sequence. These codons cause strong translational pausing during coupled transcription/translation in E. coli extracts, generating nascent polypeptides on ribosomes. Protein synthesis in the TF(-) extract was initiated with biotin-Met-tRNA(f). Ribosomes with nascent polypeptides were isolated by interaction of the N-terminal biotin with streptavidin on magnetobeads. These translating ribosomes that lack TF contain the molecular chaperone DnaK in considerably less than stoichiometric amounts.

Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro.

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNA(f). All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E. coli. Addition of large amounts of E. coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause-site peptides seen in the expression of wild-type CAT. Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing.

Rhodanese can partially refold in its GroEL-GroES-ADP complex and can be released to give a homogeneous product.

Molecular chaperones GroEL and GroES facilitate reactivation of denatured rhodanese which folds poorly unless the process is assisted. The present work tests the hypothesis that more extensively unfolded forms of rhodanese bind tighter than those forms that appear later in the folding pathway. The study of the interaction of different urea-induced forms of rhodanese with GroEL suggests that species preceding the domain folded form bind directly and productively to GroEL. Rhodanese partially folds while in the GroEL-GroES-ADP complex, but it does not significantly reach an active state. Partially folded rhodanese can be released from the GroEL-GroES-ADP complex by subdenaturing concentrations of urea as a homogeneous species that is committed to fold to the native conformation with little or no partitioning to the aggregated state. Dilution of denatured rhodanese to the same final concentration gives less active enzyme and significant aggregation. Urea denaturation studies show that active rhodanese released from complexes behaves identically to native enzyme, while spontaneously folded rhodanese has a different stability. These results are interpreted using a previously proposed model based on studies of unassisted rhodanese folding [Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120-128. Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63-70].

Conformational heterogeneity is revealed in the dissociation of the oligomeric chaperonin GroEL by high hydrostatic pressure.

We investigated the dissociation of tetradecameric GroEL by high hydrostatic pressure in the range of 1-2.5 kbar. Kinetics of the dissociation of GroEL in the absence and presence of Mg(2+) and/or KCl were monitored using light scattering. All of the kinetics were biphasic in nature. At any given pressure, only monomers and 14mers were produced, and below 2.5 kbar, the 14mers only partially dissociated to monomers, which did not significantly reassemble on depressurization. Under identical reaction conditions, the observed dissociation rates decreased by only 2-fold when the concentration of GroEL was increased by 20-fold. At 2.5 kbar the observed rates decreased exponentially with the increase in [KCl] and reached a minimum at approximately 75mM. Similarly, the rates decreased with the increase in [Mg(2+)] and reached a minimum at approximately 3 mM Mg(2+). In the presence of saturating amounts of Mg(2+) (10 mM) and KCl (100 mM), the rates were much faster than with 10 mM Mg(2+) alone. The results could be rationalized in terms of the presence of GroEL heterogeneity, which could not be assessed easily by common techniques such as sedimentation velocity, HPLC, gel electrophoresis, and dissociation by chaotropes. This heterogeneity is evidence of subpopulations of GroEL that dissociate at different pressures. At low pressures, the oligomer without added Mg(2+) only partially dissociates to monomers, leading to an apparent plateau in the kinetics, whereas in the presence of Mg(2+) the species are converted to a tighter Mg(2+)-bound species, leading to a much slower dissociation process. The presence of KCl in the sample also leads to similar heterogeneity.

Isolation and characterization of rhodanese intermediates during thermal inactivation and their implications for the mechanism of protein aggregation.

The initial steps of heat-induced inactivation and aggregation of the enzyme rhodanese have been studied and found to involve the early formation of modified but catalytically active conformations. These intermediates readily form active dimers or small oligomers, as evident from there being only a small increase in light scattering and an increase in fluorescence energy homotransfer from rhodanese labeled with fluorescein. These species are probably not the domain-unfolded form, as they show activity and increased protection of hydrophobic surfaces. Cross-linking with glutaraldehyde and fractionation by gel filtration show the predominant formation of dimer during heat incubation. Comparison between the rates of aggregate formation at 50 degrees C after preincubation at 25 or 40 degrees C gives evidence of product-precursor relationships, and it shows that these dimeric or small oligomeric species are the basis of the irreversible aggregation. The thermally induced species is recognized by and binds to the chaperonin GroEL. The unfoldase activity of GroEL subsequently unfolds rhodanese to produce an inactive conformation and forms a stable, reactivable complex. The release of 80% active rhodanese upon addition of GroES and ATP indicates that the thermal incubation induces an alteration in conformation, rather than any covalent modification, which would lead to formation of irreversibly inactive species. Once oligomeric species are formed from the intermediates, GroEL cannot recognize them. Based on these observations, a model is proposed for rhodanese aggregation that can explain the paradoxical effect in which rhodanese aggregation is reduced at higher protein concentration.