Broad spectrum repairing properties of an extract of Aquaphilus dolomiae on in vitro and ex vivo models of injured skin.

BACKGROUND: A biological concentrate was produced from cultures of an Avène aquatic microflora isolate, namely Aquaphilus dolomiae. Some of the beneficial effects on diseased and damaged skin are thought to be due to the presence of this microorganism. AIMS: An extract of A. dolomiae (A. dolomiae extract-G2, ADE-G2) was evaluated for its wound-healing effects using in vitro and ex vivo models of injured skin. METHODS: The effect of ADE-G2 on the proliferation of fibroblasts, migration of keratinocytes and re-epithelialization of ex vivo wounded skin explants was measured. Antimicrobial protection by ADE-G2 was measured by analysing the gene expression of a panel of antimicrobial proteins (AMPs) in keratinocytes (RNASE7, S100A7, DEFB4A/B and DEFb103B), as well as the protein encoded by DEFB4A-B (hBD2) in the medium. RESULTS: ADE-G2 increased fibroblast proliferation and keratinocyte migration, as well as re-epithelialization of wounded ex vivo skin. ADE-G2 induced the expression of all AMP genes analysed in keratinocytes, as well as stimulated the release in to the medium of hBD2 peptide, encoded by DEFB4A/B. CONCLUSIONS: We have shown the broad spectrum of the repairing properties of the A. dolomiae extract, ADE-G2. These results support the use of ADE-G2 as a promising component for use in formulations aimed at repairing skin, limiting wound superinfection and preventing complicated wounds.

Protective effect of Aquaphilus dolomiae extract-G1, ADE-G1, on tight junction barrier function in a Staphylococcus aureus-infected atopic dermatitis model.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions and defects of the skin barrier. Bacterial infection with Staphylococcus aureus contributes to increased severity of AD by compromising the barrier further. A microorganism component of Avène Thermal Spring Water, Aquaphilus dolomiae, is thought to contribute to some of its beneficial effects to skin, eg AD alleviation. AIMS: Here, we have investigated the effects of an extract of A. dolomiae, A. dolomiae extract-G1 (ADE-G1), on the structural barrier function of keratinocytes, tight junction (TJ) protein expression and the expression of several genes altered in AD patients. METHODS: An epidermal cell culture model mimicking the AD environment and phenotype was used, in which S. aureus-infected cell cultures of normal human epidermal keratinocytes were exposed to a proinflammatory environment. Endpoints measured included the transepithelial electrical resistance (TER) and immunohistological staining of the epidermal TJ proteins, claudin and occludin. Additional analysis was made of several genes known to be differentially regulated in skin from AD patients (C-C motif chemokine ligand 20 (CCL20), interleukin-8 (IL-8), S100 calcium binding protein A7 (S100A7), defensin beta 4 (DEFB4) and filaggrin). RESULTS: Aquaphilus dolomiae extract-G1 strongly increased TER in non-infected cells and provided protection against infection by overcoming the decrease in TER induced by the infection with S. aureus. In infected cells exposed to a pro-inflammatory environment - depicting AD-like conditions - TER protection by ADE-G1 was still observed. Gene expression analysis of infected and pro-inflammatory stimulated cells indicated that ADE-G1 modulated the inflammatory response (induced IL-8 and attenuated CCL20 expression), increased antimicrobial activities (induced DEFB4 and A100A7) and strengthened barrier function (restored filaggrin expression). CONCLUSIONS: ADE-G1 reinforces barrier function and strongly protects TJ barrier disruption induced by bacterial infection and inflammation.

Fragility of epidermis in newborns, children and adolescents.

Within their first days of life, newborns' skin undergoes various adaptation processes needed to accommodate the transition from the wet uterine environment to the dry atmosphere. The skin of newborns and infants is considered as a physiological fragile skin, a skin with lower resistance to aggressions. Fragile skin is divided into four categories up to its origin: physiological fragile skin (age, location), pathological fragile skin (acute and chronic), circumstantial fragile skin (due to environmental extrinsic factors or intrinsic factors such as stress) and iatrogenic fragile skin. Extensive research of the past 10 years have proven evidence that at birth albeit showing a nearly perfect appearance, newborn skin is structurally and functionally immature compared to adult skin undergoing a physiological maturation process after birth at least throughout the first year of life. This article is an overview of all known data about fragility of epidermis in 'fragile populations': newborns, children and adolescents. It includes the recent pathological, pathophysiological and clinical data about fragility of epidermis in various dermatological diseases, such as atopic dermatitis, acne, rosacea, contact dermatitis, irritative dermatitis and focus on UV protection.

In vitro approaches to pharmacological screening in the field of atopic dermatitis.

In vitro models are valuable for evaluating potential active ingredients and other molecules used in medications for atopic dermatitis (AD). However, finding appropriate in vitro models can be problematic. Our strategy was to set up different in vitro models that would mimic the pathomechanisms of AD. We describe five such models - the AD keratinocyte model, the AD reconstructed human epidermis model, the adaptive immunity model, the innate immunity model and the pruritus model - which we have used to evaluate a new ingredient for emollients derived from a biological extract. The models chosen provide useful data for the pharmacological characterization of active ingredients in adjunctive treatments for AD.

Fragility of epidermis and its consequence in dermatology.

The skin is the largest organ of the body, providing a protective barrier against bacteria, chemicals and physical insults while maintaining homeostasis in the internal environment. Such a barrier function the skin ensures protection against excessive water loss. The skin's immune defence consists of several facets, including immediate, non-specific mechanisms (innate immunity) and delayed, stimulus-specific responses (adaptive immunity), which contribute to fending off a wide range of potentially invasive microorganisms. This article is an overview of all known data about 'fragile skin'. Fragile skin is defined as skin with lower resistance to aggressions. Fragile skin can be classified into four categories up to its origin: physiological fragile skin (age, location), pathological fragile skin (acute and chronic), circumstantial fragile skin (due to environmental extrinsic factors or intrinsic factors such as stress) and iatrogenic fragile skin. This article includes the epidemiologic data, pathologic description of fragile skin with pathophysiological bases (mechanical and immunological role of skin barrier) and clinical description of fragile skin in atopic dermatitis, in acne, in rosacea, in psoriasis, in contact dermatitis and other dermatologic pathologies. This article includes also clinical cases and differential diagnosis of fragile skin (reactive skin) in face in adult population. In conclusion, fragile skin is very frequent worldwide and its prevalence varies between 25% and 52% in Caucasian, African and Asian population.

A novel frame-shift deletion causing analbuminaemia in an Italian paediatric patient.

BACKGROUND: Analbuminaemia (OMIM #103600) is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous or compound heterozygous subjects. The trait is caused by a variety of mutations within the albumin gene. DESIGN: We report here the clinical and molecular characterization of a new case of congenital analbuminaemia in a 4-year-old Italian girl diagnosed on the basis of the low level of circulating albumin (= 10.0 g L(-1)). The albumin gene was screened by single-strand conformation polymorphism and heteroduplex analysis and the mutated region submitted to DNA sequencing. RESULTS: The proband was found to be homozygous, and both parents heterozygous, for a novel deletion in exon 8 (c.920delT). The subsequent frame-shift should have given rise to a putative polypeptide chain of 304 amino acid residues, which we could not identify in the proband's serum. CONCLUSIONS: A novel analbuminaemia causing mutation was identified and characterized at the clinical level in a child. The molecular diagnosis of the trait is based on the rapid localization of the mutation within the albumin gene by single-strand conformation polymorphism and heteroduplex analysis, followed by DNA sequencing of the mutated region.

Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins.

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.

Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator.

Cell growth and differentiation are controlled in many tissues by paracrine factors, which often require proteolytic processing for activation. Metalloproteases of the metzincin family, such as matrix metalloproteases and ADAMs, recently have been shown to be involved in the shedding of growth factors, cytokines, and receptors. In the present study, we show that hydroxamate-based inhibitors of metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion of C(2)C(12) myoblasts and provoke myotube hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that have previously been shown to regulate muscle growth in vitro, such as insulin-like growth factor-I, calcineurin, and tumor necrosis factor-alpha. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in C(2)C(12) cells treated with HIMPs, as suggested by the presence of nonprocessed myostatin precursor only in hypertrophic myotubes. Myostatin is a known negative regulator of skeletal muscle growth, belonging to the transforming growth factor-beta/bone morphogenetic protein superfamily. These results indicate that metalloproteases are involved in the regulation of skeletal muscle growth and differentiation, that the proteolytic maturation of myostatin in C(2)C(12) cells may be directly or indirectly linked to the activity of some unidentified HIMP-sensitive metalloproteases, and that the lack of myostatin processing on HIMP treatment may be a mediator of myotube hypertrophy in this in vitro model.

Enzymatic properties of human hemalbumin.

The binding of hemin to the primary site of human serum albumin (HSA) has been reinvestigated using UV-Vis, CD and NMR techniques. The major fraction of bound hemin contains a five-coordinated high-spin iron(III) center, but a minor fraction of the metal appears to be in a six-coordinated, low-spin state, where a 'distal' residue, possibly a second histidine residue, completes the coordination sphere. The reduced, iron(II) form of the adduct contains six-coordinated low-spin heme. The distal residue hinders the access to the iron(III) center of hemin-HSA to small anionic ligands like azide and cyanide and destabilizes the binding of neutral diatomics like dioxygen and carbon monoxide to the iron(II) form. In spite of these limitations, the hemin-HSA complex promotes hydrogen peroxide activation processes that bear the characteristics of enzymatic reactions and may have biological relevance. The complex is in fact capable of catalyzing peroxidative reactions on phenolic compounds related to tyrosine and hydrogen peroxide dismutation. Kinetic and mechanistic studies confirm that the low efficiency with which peroxidative processes occur depends on the limited rate of the reaction between hydrogen peroxide and the iron(III) center, to form the active species, and by the competitive peroxide degradation reaction.

A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges.

Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.

Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion.

ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membrane-distal region. Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.

Structural characterization of the oligosaccharide chains of human alpha1-microglobulin from urine and amniotic fluid.

Human alpha1-microglobulin (alpha1-m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily, was isolated by sequential anion-exchange chromatography and gel filtration from the urine of hemodialized patients and from amniotic fluid collected in the week 16-18 of pregnancy. The carbohydrate chains of the protein purified from the two sources, which are organized in two Asn-linked and one Thr-linked oligosaccharides, were structurally characterized using matrix-assisted laser desorption ionization and electrospray mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and O-glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary structures attached in the case of urinary alpha1-m and only diantennary glycans in the amniotic fluid protein. The main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary glycans. The position of the oligosaccharide chains in a three-dimensional model of the protein, produced using the automated Swiss-Model service, is also discussed.

Genetic variants showing apparent hot-spots in the human serum albumin gene.

The molecular defects of three different slow-migrating genetic variants of human serum albumin, albumins Kamloops (formerly RIH), Stirling and Amsterdam, previously characterized only by electrophoretic and dye-binding studies, are now reported. Two of them are proalbumin variants: sequential analysis of the purified whole proteins has established the mutation responsible for albumin Kamloops as -1Arg-->Gln, and for albumin Stirling as -2Arg-->His. A Glu-->Lys substitution in position 570 of the mature albumin molecule was determined in albumin Amsterdam by sequential analysis of two abnormal tryptic fragments. The three alloalbumins are caused by single-base changes all of which seem to represent hot-spots in the albumin gene. The possible functional consequences of the presence of a circulating alloalbumin are discussed.

Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin.

Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp314-->Val (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg-2-->Cys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: GAT-->GTT (albumin Brest) and CGT-->TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg-6. In addition, part of normal albumin had lost Asp1. Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding.

Analysis of human serum albumin variants by mass spectrometric procedures.

A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed io isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.

Genetic variation in human serum albumin: a 313 Lys-->Asn mutation in albumin reading identified by PCR analysis.

An early case of bisalbuminaemia was reported in this journal in 1964, with the name Albumin Reading added later. Its use in electrophoretic comparisons led to some new variants being described as 'of the Reading type' on this basis alone. Protein sequencing and DNA studies have since found the single point mutation 313 Lys-->Asn common to this type, but the eponymous variant has not, until recently, been available for study. We now report on its characterisation using PCR analysis with allele-specific oligonucleotide primers, a method also applicable to studies of the population distribution of variants. We also draw attention to the need to link clinically-significant effects to individual variants of known structure.

Structural characterization of three genetic variants of human serum albumin modified in subdomains IIB and IIIA.

Three new genetic variants of human serum albumin have been detected in Italy by routine clinical electrophoresis. Albumin Milano Slow is common in Northern Italy, while albumins Liprizzi and Trieste, which are fast migrating, are rare and local variants. Isoelectric focusing analysis of the CNBr fragments obtained from the carboxymethylated alloalbumins in all cases localized the mutation to fragment CB5 (residues 330-446). The modified CNBr fragments were isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptides revealed that all the variants are caused by single point mutations: Trieste, Lys359-->Asn, Milano Slow, Asp375-->His, and Liprizzi, Arg410-->Cys. These results were confirmed by sequence determination of a variant V8 peptide in the case of Trieste, and by DNA sequence analysis for the other two variants. The DNA analysis showed a G-->C transversion at nucleotide position 11969 for albumin Milano Slow, and a C-->T transition at position 13251 for Liprizzi. The latter represents a mutation at a hypermutable CpG dinucleotide site. Albumins Trieste and Milano Slow, as most of the variants thus far described, have mutations involving residues on the surface of the molecule. In contrast, albumin Liprizzi represents the first example of a mutation in the most active binding pocket of the molecule, placed in subdomain IIIA.

Effect of genetic variation on the fatty acid-binding properties of human serum albumin and proalbumin.

In the circulation, non-esterified fatty acids are transported by albumin which also facilitates their removal from donor cells and uptake into receptor cells. We have studied whether genetic variations in the albumin molecule can affect its in vivo fatty acid-binding properties. The fatty acids bound to 25 structurally different variants and to their wildtype counterparts, isolated from heterozygous carriers, were determined gas chromatographically. The variants were proalbumins, albumins with single amino acid substitutions and glycosylated or truncated albumins. In eight cases the total amount bound to the variants was diminished (0.4-0.8-fold), and in seven cases the load was increased to 1.3 or more of normal. Twenty-one fatty acids were quantitated, and for 19 alloalbumins significant deviations from normal were found. Usually, changes in total and individual fatty acid binding were of the same type, but several exceptions to this rule was found. The glycosylated albumin Casebrook showed the largest changes, the total load and the amount of bound palmitate was 8.6 and 14 times, respectively, the normal. The most pronounced changes and the majority of cases of increased binding were caused by molecular changes in domain III. Mutations in domain I, II and the propeptide resulted in smaller effects, if any, and these were often reductions in binding.