BACKGROUND: Gamma linoleic acid (GLA, Epogam) is considered a safe and effective modality in the treatment of atopic dermatitis (AD) in which impaired function of the enzyme, delta-6-desaturase, has been reported to result in reduced levels of GLA, desaturated fatty acids. OBJECTIVE: We performed this study to observe the changes of skin surface conditions measured objectively by bioengineering methods in relation to clinical improvement after treatment with GLA (Epogam®) in children with AD. METHODS: Thirty-four children with AD were treated with GLA (Epogam®) and evaluated with clinical parameters.The changes of skin surface conditions were monitored by non-invasive experimental instruments. RESULTS: There was a significant decrease of transepidermal water loss (TEWL) and gradual improvements in clinical severity after 12 weeks of GLA (Epogam®) treatment. The change of skin surface pH was statistically significant on the antecubital fossa and abdomen except the popliteal fossa. The other parameters including skin surface hydration and skin surface lipid did not show consistent changes. CONCLUSION: Clinical improvement of AD with GLA (Epogam) seemed to be achieved by the reduction of TEWL.
Abdomen ; Bioengineering ; Child ; Dermatitis, Atopic* ; Fatty Acids ; Humans ; Hydrogen-Ion Concentration ; Linoleic Acid* ; Linoleoyl-CoA Desaturase ; Skin* ; Water

Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly y-linolenic acid. In this process, delta6-Fatty acid desaturase (D6D) plays a key role due to its enzymatic properties that catalyze the delta6 site dehydrogenation of precursor linoleic acid (18:2delta(9, 12) n-6) and a-linolenic acid (18:3delta(9, 12, 15) n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acids (HUFA) synthesis pathways. After we have isolated and cloned the gene coding delta6-fatty acid desaturase from Thamnidium elegans As3.2806 (GenBank accession number DQ099380), our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR I and Kpn I , respectively. Then we ligated the digested DNA with T4 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of delta6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4 kb from the EcoR I digested sample, in which a 1.3kb 5' upstream sequence (GenBank accession number DQ309425) of delta6-fatty acid desaturase gene containing several putative regulatory elements including TATA. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans detla6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3 kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans delta6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.
Base Sequence ; Cloning, Molecular ; DNA Primers ; Linoleoyl-CoA Desaturase ; genetics ; Molecular Sequence Data ; Mucorales ; enzymology ; genetics ; Polymerase Chain Reaction ; methods

Delta6-fatty acid desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated fatty acids. A cDNA sequence putatively encoding a delta6-fatty acid desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel delta6-fatty acid desaturase gene which had an open reading frame of 1377bp coding 458 amino acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus delta6-fatty acid desaturase gene (RAD6) was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total fatty acids were extracted from the induced cells and methylated. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Fatty acids analysis showed that the coding product introduced a new double bond at delta6 position of appropriate fatty acid substrates including C16:1, C17:1, C18:1, linoleic acid and alpha-linolenic acid without chain length specificity of fatty acids. Furthermore, modification of sequence flanking AUG codon of this delta6-fatty acid desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of delta6-fatty acid desaturase gene.
Cloning, Molecular ; Electroporation ; Fungal Proteins ; biosynthesis ; genetics ; Genetic Vectors ; Linoleic Acid ; metabolism ; Linoleoyl-CoA Desaturase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Rhizopus ; enzymology ; genetics

Gamma-linolenic acid (GLA, C18:3delta6 ,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C18:2delta9,12) in the production of gamma-linolenic acid. A deficiency of GLA may have occurred when delta6-fatty acid desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-fatty acid desaturase was expressed intracellularly in P. pastoris and gamma-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.
Cloning, Molecular ; Fatty Acids ; analysis ; Gas Chromatography-Mass Spectrometry ; Linoleoyl-CoA Desaturase ; genetics ; Mortierella ; enzymology ; genetics ; Pichia ; genetics ; Plasmids ; gamma-Linolenic Acid ; analysis

BACKGROUND: Atopic dermatitis (AD) is related to a deficiency of delta-6-desaturase, an enzyme responsible for converting linoleic acid to gamma-linolenic acid (GLA). Evening primrose oil (EPO) as a source of GLA has been of interest in the management of AD. OBJECTIVE: The aim of this randomized, double-blinded, placebo-controlled clinical study is to evaluate the efficacy and safety of EPO in Korean patients with AD. METHODS: Fifty mild AD patients with an Eczema Area Severity Index (EASI) score of 10 or less were enrolled and randomly divided into two groups. The first group received an oval unmarked capsule containing 450 mg of EPO (40 mg of GLA) per capsule, while placebo capsules identical in appearance and containing 450 mg of soybean oil were given to the other group. Treatment continued for a period of four months. EASI scores, transepidermal water loss (TEWL), and skin hydration were evaluated in all the AD patients at the baseline, and in months 1, 2, 3, and 4 of the study. RESULTS: At the end of month 4, the patients of the EPO group showed a significant improvement in the EASI score (p=0.040), whereas the patients of the placebo group did not. There was a significant difference in the EASI score between the EPO and placebo groups (p=0.010). Although not statistically significant, the TEWL and skin hydration also slightly improved in the EPO patients group. CONCLUSION: We suggest that EPO is a safe and effective medicine for Korean patients with mild AD.
Capsules ; Clinical Study* ; Dermatitis, Atopic* ; Eczema ; gamma-Linolenic Acid ; Humans ; Linoleic Acid ; Linoleoyl-CoA Desaturase ; Oenothera biennis* ; Skin ; Soybean Oil ; Water