Bio-derived hydroxystearic acid ameliorates skin age spots and conspicuous pores.

INTRODUCTION: We report on the preparation and efficacy of 10-hydroxystearic acid (HSA) that improves facial age spots and conspicuous pores. METHODS: The hydration of oleic acid into HSA was catalyzed by the oleate hydratase from Escherichia coli. Following treatment with HSA, collagen type I and type III was assessed in primary human dermal fibroblasts together with collagen type III, p53 protein levels and sunburn cells (SBC) after UVB irradiation (1 J cm-2 ) by immunohistochemistry on human ex vivo skin. UVB-induced expression of matrix metalloprotease-1 (MMP-1) was determined from full thickness skin by RT-qPCR. Modification of the fibroblast secretome by HSA was studied by mass-spectrometry-based proteomics. In a full-face, double blind, vehicle-controlled trial HSA was assessed for its effects on conspicuous facial pore size and degree of pigmentation of age spots in Caucasian women over an 8-week period. RESULTS: HSA was obtained in enantiomeric pure, high yield (≥80%). Collagen type I and type III levels were dose-dependently increased (96% and 244%; P < 0.01) in vitro and collagen type III in ex vivo skin by +57% (P < 0.01) by HSA. HSA also inhibited UVB-induced MMP-1 gene expression (83%; P < 0.01) and mitigated SBC induction (-34% vs. vehicle control) and reduced significantly UV-induced p53 up-regulation (-46% vs. vehicle control; P < 0.01) in irradiated skin. HSA modified the fibroblast secretome with significant increases in proteins associated with the WNT pathway that could reduce melanogenesis and proteins that could modify dermal fibroblast activity and keratinocyte differentiation to account for the alleviation of conspicuous pores. Docking studies in silico and EC50 determination in reporter gene assays (EC50 5.5 × 10-6  M) identified HSA as a peroxisomal proliferator activated receptor-α (PPARα) agonist. Clinically, HSA showed a statistically significant decrease of surface and volume of skin pores (P < 0.05) after 8 weeks of application and age spots became significantly less pigmented than the surrounding skin (contrast, P < 0.05) after 4 weeks. CONCLUSION: HSA acts as a PPARα agonist to reduce the signs of age spots and conspicuous pores by significantly modulating the expression of p53, SBC, MMP-1 and collagen together with major changes in secreted proteins that modify keratinocyte, melanocyte and fibroblast cell behavior.

INTRODUCTION: voici notre rapport sur la préparation et l'efficacité de l'acide 10-hydroxystéarique (AHS) qui atténue les taches de vieillesse faciale et améliore l'apparence des pores. MÉTHODES: l'hydratation de l'acide oléique en AHS a été catalysée par l'hydratase d'oléate à partir de l'Escherichia coli. Après un traitement par AHS, les collagènes de type I et de type III ont été analysés dans des fibroblastes dermiques humains primaires, ainsi que le taux de collagène de type III et de protéine p53, et les cellules provenant de coups de soleil (sunburn cells, SBC) après irradiation par UVB (1 J cm−2 ) par immunohistochimie sur de la peau humaine ex vivo. L'expression de la matrice métalloprotéase-1 (MMP-1) induite par les UVB a été déterminée à partir d'un échantillon de pleine épaisseur de peau par RT-qPCR. La modification du sécrétome des fibroblastes par l'AHS a été étudiée par analyse protéomique basée sur une spectrométrie de masse. Dans une étude du visage entier, en double aveugle, contrôlée par excipient, l'AHS a été évaluée pour ses effets sur la taille des pores apparents du visage et sur le degré de pigmentation de taches de vieillesse chez des femmes de race blanche sur une période de 8 semaines. RÉSULTATS: l'AHS a été obtenu à un haut rendement, énantiomérique pur (≥80 %). Les taux de collagènes de type I et de type III ont augmenté in vitro en fonction de la dose (96 % et 244 %; P < 0.01) et le collagène de type III dans de la peau ex vivo de +57 % (P < 0.01) lors d'un traitement par AHS. L'AHS a également inhibé l'expression génique MMP-1 induite par les UVB (83%; P < 0.01) et a atténuée l'induction des SBC (−34 % par rapport à l'excipient), et a réduit significativement la régulation à la hausse du p53 induite par les UV (−46% par rapport à l'excipient; P < 0.01) sur de la peau irradiée. L'AHS a modifié le sécrétome des fibroblastes avec des augmentations significatives des protéines associées à la voie WNT qui pouvaient réduire la mélanogenèse et des protéines qui pouvaient modifier l'activité des fibroblastes dermiques et la différenciation des kératinocytes pour une atténuation des pores apparents. Des études de docking in silico et la détermination de l'EC50 dans les dosages des gènes rapporteurs (EC50 5.5 × 9 10−6 M) ont identifié l'AHS comme un agoniste du récepteur-α activé par les proliférateurs de peroxysomes (peroxisomal proliferator activated receptor-α, PPARα). Cliniquement, l'AHS a permis une diminution statistiquement significative de la surface et du volume des pores de la peau (P < 0.05) après 8 semaines d'application, et les taches de vieillesse sont devenues significativement moins pigmentées par rapport à la peau environnante (contraste, P < 0,05) après 4 semaines. CONCLUSION: l'AHS agit comme un agoniste du PPARα pour réduire les signes de taches de vieillesse et l'apparence des pores par une modulation significative de l'expression de la protéine p53, des SBC, de la MMP-1 et du collagène avec des changements majeurs dans les protéines sécrétées qui modifient le comportement cellulaire des kératinocytes, des mélanocytes et des fibroblastes.

Whole-mount in situ detection of microRNAs on Arabidopsis tissues using Zip Nucleic Acid probes.

MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ development, transition to flowering, and responses to abiotic/biotic stresses. To understand the biological role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the spatiotemporal regulation of their expression. Many methods have been used to define the set of organ-specific miRNAs by tissue dissection and miRNA profiling but none of them can describe their tissue and cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Arabidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the main steps of the procedure by robotized liquid handling has also been implemented in the protocol for best reproducibility of results, enabling running of ISH experiments at high throughput.

Transcriptome analysis reveals coordinated spatiotemporal regulation of hemoglobin and nitrate reductase in response to nitrate in maize roots.

Given the importance of nitrogen for plant growth and the environmental costs of intense fertilization, an understanding of the molecular mechanisms underlying the root adaptation to nitrogen fluctuations is a primary goal for the development of biotechnological tools for sustainable agriculture. This research aimed to identify the molecular factors involved in the response of maize roots to nitrate. cDNA-amplified fragment length polymorphism was exploited for comprehensive transcript profiling of maize (Zea mays) seedling roots grown with varied nitrate availabilities; 336 primer combinations were tested and 661 differentially regulated transcripts were identified. The expression of selected genes was studied in depth through quantitative real-time polymerase chain reaction and in situ hybridization. Over 50% of the genes identified responded to prolonged nitrate starvation and a few were identified as putatively involved in the early nitrate signaling mechanisms. Real-time results and in situ localization analyses demonstrated co-regulated transcriptional patterns in root epidermal cells for genes putatively involved in nitric oxide synthesis/scavenging. Our findings, in addition to strengthening already known mechanisms, revealed the existence of a new complex signaling framework in which brassinosteroids (BRI1), the module MKK2-MAPK6 and the fine regulation of nitric oxide homeostasis via the co-expression of synthetic (nitrate reductase) and scavenging (hemoglobin) components may play key functions in maize responses to nitrate.

Humic substances induce lateral root formation and expression of the early auxin-responsive IAA19 gene and DR5 synthetic element in Arabidopsis.

Humic substances (HS) have positive effects on plant physiology, but the molecular mechanisms underlying these events are only partially understood. HS exert auxin-like activity, but data supporting this hypothesis are under debate. To investigate the auxin-like activity of HS, we studied their biological effect on lateral root initiation in Arabidopsis thaliana. To this aim we characterised HS by means of DRIFT and (13)C CP/MAS NMR spectroscopy, and measured their endogenous content of IAA. We then utilised a combination of genetic and molecular approaches to unravel HS auxin activity in the initiation of lateral roots. The data obtained using specific inhibitors of auxin transport or action showed that HS induce lateral root formation mostly through their 'auxin activity'. These findings were further supported by the fact that HS used in this study activated the auxin synthetic reporter DR5::GUS and enhanced transcription of the early auxin responsive gene IAA19.

Expression of two maize putative nitrate transporters in response to nitrate and sugar availability.

A full-length cDNA encoding a putative high-affinity nitrate transporter (ZmNrt2.2) from maize was isolated and characterised, together with another previously identified transporter (ZmNrt2.1), in terms of phylogenesis, protein structure prediction and regulation of transcript accumulation in response to nitrate and sugar availability. The expression of both genes was evaluated by quantitative and semi-quantitative RT-PCR in response to nitrate and sugar supply and the in planta localisation of mRNA was studied by in situ hybridisation. Data obtained suggested similar genetic evolution and identical transmembrane structure prediction between the two deduced proteins, and differences in both regulation of their expression and mRNA localisation in response to nitrate, leading us to hypothesise a principal role for ZmNRT2.1 in the influx activity and the major involvement of ZmNRT2.2 in the xylem loading process. Our data suggest opposing sugar regulation by ZmNrt2.1 and ZmNrt2.2 transcription in the presence or absence of nitrate and the existence of both hexokinase-dependent and hexokinase-independent transduction mechanisms for the regulation of ZmNrt2.1 and ZmNrt2.2 expression by sugars.

Haemoglobin E in pregnancy. A case report. Diagnosis, familiar study and counselling, follow up until delivery and new-born observation.

Haemoglobin E is a beta chain variant quite common in Southeastern Asia. The case of a gravid Thai woman with a microcytic anaemia is reported. The diagnosis of homozygous haemoglobin E was suspected on the basis of ethnic considerations when analysis of her haemoglobin showed the absence of normal HbA1 and about 100% of a variant Hb with electrophoretic mobility with HbC and HbA2. Identification of the haemoglobin variant was performed by using an association of alkaline electrophoresis on agarose gel, acid electrophoresis on agarose gel, haemoglobin isoelectrofocusing, high performance liquid chromatography. A study of haemoglobin pattern in the partner, parents and siblings was also performed. Pregnancy continued without any problems until the 40th week when a caesarean section was performed due to a difficult labour with foetal distress. The haemoglobin pattern of the new-born was studied at birth and after 1 year; as expected, it was quite normal at birth and a heterozygous condition for HbE was observed after 1 year. HbE, in even heterozygous and homozygous states, gives a mild clinical picture but its association with other haemoglobinopathies, such as a double heterozygous state (i.e. HbE/beta Thalassaemia) gives rise to a severe transfusion dependent thalassaemia syndrome. It is the authors' opinion that only a strict interaction between obstetricians and pathologists is the possible correct answer to the new diagnostic question proposed by a rapidly evolving inter-ethnic society.

FATZ, a filamin-, actinin-, and telethonin-binding protein of the Z-disc of skeletal muscle.

We report the identification and characterization of a novel 32-kDa protein expressed in skeletal muscle and located in the Z-disc of the sarcomere. We found that this protein binds to three other Z-disc proteins; therefore, we have named it FATZ, gamma-filamin/ABP-L, alpha-actinin and telethonin binding protein of the Z-disc. From yeast two-hybrid experiments we are able to show that the SR3-SR4 domains of alpha-actinin 2 are required to bind the COOH-terminal region of the FATZ as does gamma-filamin/ABP-L. Furthermore, by using a glutathione S-transferase overlay assay we find that FATZ also binds telethonin. The level of FATZ protein in muscle cells increases during differentiation, being clearly detectable before the onset of myosin. Although FATZ has no known interaction domains, it would appear to be involved in a complex network of interactions with other Z-band components. On the basis of the information known about its binding partners, we could envisage a central role for FATZ in the myofibrillogenesis. After screening our muscle expressed sequence tag data base and the public expressed sequence tag data bases, we were able to assemble two other muscle transcripts that show a high level of identity with FATZ in two different domains. Therefore, FATZ may be the first member of a small family of novel muscle proteins.

ZASP: a new Z-band alternatively spliced PDZ-motif protein.

PDZ motifs are modular protein-protein interaction domains, consisting of 80-120 amino acid residues, whose function appears to be the direction of intracellular proteins to multiprotein complexes. In skeletal muscle, there are a few known PDZ-domain proteins, which include neuronal nitric oxide synthase and syntrophin, both of which are components of the dystrophin complex, and actinin-associated LIM protein, which binds to the spectrin-like repeats of alpha-actinin-2. Here, we report the identification and characterization of a new skeletal muscle protein containing a PDZ domain that binds to the COOH-terminal region of alpha-actinin-2. This novel 31-kD protein is specifically expressed in heart and skeletal muscle. Using antibodies produced to a fragment of the protein, we can show its location in the sarcomere at the level of the Z-band by immunoelectron microscopy. At least two proteins, 32 kD and 78 kD, can be detected by Western blot analysis of both heart and skeletal muscle, suggesting the existence of alternative forms of the protein. In fact, several forms were found that appear to be the result of alternative splicing. The transcript coding for this Z-band alternatively spliced PDZ motif (ZASP) protein maps on chromosome 10q22.3-10q23.2, near the locus for infantile-onset spinocerebellar ataxia.

Telethonin, a novel sarcomeric protein of heart and skeletal muscle.

In this paper we describe a novel 19 kDa sarcomeric protein named telethonin. The cDNA sequence discloses an open reading frame of 167 amino acids that does not resemble any known protein. Antibodies against a recombinant telethonin fragment were used for Western blot analysis, confirming the presence of this 19 kDa protein in heart and skeletal muscle and revealing an immunofluorescence pattern typical of sarcomeric proteins, overlapping myosin. The frequency of specific cDNA clones in different libraries indicates that the telethonin transcript is amongst the most abundant in skeletal muscle. In human, telethonin maps at 17q12, adjacent to the phenylethanolamine N-methyltransferase gene.

Semi-multiplex PCR technique for screening of abundant transcripts during systematic sequencing of cDNA libraries.

The systematic sequencing of cDNA libraries is an efficient approach for the identification of new genes, but the presence of abundant mRNAs is often a major problem. This paper describes a very simple method of "semi-multiplex PCR" that allows specific identification of such abundant transcripts before DNA sequencing without using nonrepresentative subtracted libraries. The PCR utilizes a series of forward primers specific for abundant transcripts with a pair of universal primers used for template generation. cDNA clones corresponding to abundant mRNAs are then revealed by double bands in agarose gel.

Identification of 4370 expressed sequence tags from a 3'-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization.

A systematic study on the mRNA species expressed in the human skeletal muscle is presented in this paper. To carry on this study, a new method has been developed for the construction of unbiased cDNA libraries specially designed for the production of ESTs corresponding to the 3'-end portion of the mRNAs. The method has been applied to human skeletal muscle, where the analysis of the transcription profile is particularly difficult for the presence of several very abundant transcripts. To detect and quantify high-level mRNAs, the first 1054 ESTs were obtained from randomly selected clones. The 10 most abundant transcripts accounted for > 45% of the clones. Subsequently, these transcripts were identified by filter hybridization, thus making DNA sequencing more productive. Overall, 4370 clones were identified: 3372 by DNA sequencing and 998 by filter hybridization. The number of groups of sequences identifying individual transcripts was relatively low compared with other tissues, resulting in a total of 934 groups out of 4370 ESTs. Of these, 719 groups were represented by only one sequence.

[Chronic digestive loss of plasma proteins evaluated with 51Cr-albumin in Billroth I and II gastrectomized patients]. / Perdas digestivas crônicas de proteínas plasmáticas avaliadas com albumina 51Cr em gastrectomizados à Billroth I e II.

The digestive clearance of albumin 51Cr was determined in patients submitted to gastrectomy due to peptic ulcer, 6 to 18 months after surgery. Patients were of both sexes, with ages varying from 24 to 70 years. Reconstruction after resection was according to Billroth I and II techniques (groups B and C). The group of control (group A) presented no digestive illness. Each group was represented by 10 individuals. The mean value and respective standard derivation for each group was respectively: Group A: 16,4 +/- 4,6; Group B: 22,0 +/- 8,1; Group C: 35,8 +/- 14,4. Comparing the mean values according to the Tukey test, significance was observed at the alpha = 0.05 level between A and C groups and B and C ones.