Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.
Amino Acids ; Bacillus subtilis/metabolism* ; Culture Media ; Fermentation ; Lipopeptides ; Peptides, Cyclic

BACKGROUND: The biosynthesis of melanin is initiated by the enzymatic oxidation of L-tyrosine to L-dopa by tyrosinase. Some precursors of melanin are cytotoxic, and melanoma cells are killed as a risk of exposare to excess tyrosine or dopa in the culture medium. However, there have been few observations of the effects of L-tyrosine on cultured normal human melanocyte. OBJECTIVE: In order to investigate whether exogenous tyrosine induces cytotoxicity in cultured normal human melanocytes as in melanoma cells, we examined the effects of L-tyrosine on proliferation and melanization in normal human melanocytes. METHODS: A melanocyte culture was produced with a modified TIC medium. L-tyrosine was added to the culture medium, 100, 200, 400, and 800uM. After 2 days of incubation, the proliferation was measured by methylthiazol tetrazolium(MTT) assay and sulforhodamine B(SRB) assay. The melanin contenis were also measured by the modified Whittaker's method. RESULTS: On MTT assay, the proliferation of melanocytes had been stirnulated significantly (p< 0.05) in all L-tyrosine added groups. On SRB assay, the proliferation of melanocytes had heen stimulated significantly (p<005) in 200, 400, 800uM of L-tyrosine added groups. The melanin contents had increased in all L-tyrosine added groups, and had increased significantly (p<0.05) in 400uM of L-tyrosine added group. CONCLUSION: L-tyrosine is not toxic to normal melanocytes, It stimulates the proliferation and melnization of cultured normal human melanocytes.
Dihydroxyphenylalanine ; Humans* ; Levodopa ; Melanins ; Melanocytes* ; Melanoma ; Monophenol Monooxygenase ; Tics ; Tyrosine*

BACKGROUND: The biosynthesis of melanin is initiated by the enzymatic oxidation of L-tyrosine to L-dopa by tyrosinase. Some precursors of melanin are cytotoxic, and melanoma cells are killed as a risk of exposare to excess tyrosine or dopa in the culture medium. However, there have been few observations of the effects of L-tyrosine on cultured normal human melanocyte. OBJECTIVE: In order to investigate whether exogenous tyrosine induces cytotoxicity in cultured normal human melanocytes as in melanoma cells, we examined the effects of L-tyrosine on proliferation and melanization in normal human melanocytes. METHODS: A melanocyte culture was produced with a modified TIC medium. L-tyrosine was added to the culture medium, 100, 200, 400, and 800uM. After 2 days of incubation, the proliferation was measured by methylthiazol tetrazolium(MTT) assay and sulforhodamine B(SRB) assay. The melanin contenis were also measured by the modified Whittaker's method. RESULTS: On MTT assay, the proliferation of melanocytes had been stirnulated significantly (p< 0.05) in all L-tyrosine added groups. On SRB assay, the proliferation of melanocytes had heen stimulated significantly (p<005) in 200, 400, 800uM of L-tyrosine added groups. The melanin contents had increased in all L-tyrosine added groups, and had increased significantly (p<0.05) in 400uM of L-tyrosine added group. CONCLUSION: L-tyrosine is not toxic to normal melanocytes, It stimulates the proliferation and melnization of cultured normal human melanocytes.
Dihydroxyphenylalanine ; Humans* ; Levodopa ; Melanins ; Melanocytes* ; Melanoma ; Monophenol Monooxygenase ; Tics ; Tyrosine*

We study the techniques of synthesis of disulfide bond-bearing cyclopeptides FIK. This experimentation with the material of Fmoc-aa use Solid-Phase synthesis after condensation by HBTU/HOBt/DIEA to synthesize linear peptide, then cyclopeptide was synthesized by creation of intramolecular disulfide bond by means of 12 oxidation of bis cysteine sulfhydryl of the linear peptide. The crude production was cleaved from the resin together with all protecting group and identified and separated by MALDI-MS and RP-HPLC. The peptide yield was 18%, after purification the purity was more than 97%. It was identified on MALDI-MS and Ellman reagent detection. This method is effective, simple, rapid and obtained good yield, and it's fit for the large-scale production.
Amino Acids ; chemistry ; Combinatorial Chemistry Techniques ; methods ; Disulfides ; chemistry ; Fluorenes ; chemistry ; Molecular Structure ; Peptides, Cyclic ; chemical synthesis ; chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

We have studied the effects of excitatory amino acids on the expression of the c-fos and c-jun mRNA in rat C6 glioma cells. The glutamate, N-methyl-D-aspartate (NMDA), and kainic acid (KA) increased c-fos mRNA level in a concentration-dependent manner. However, they did not affect c-jun mRNA level. In addition, forskolin and phorbol 12-myristate 13-acetate (PMA) increased c-fos mRNA level. Furthermore, PMA increased c-jun mRNA level whereas forskolin downregulated c-jun mRNA level. The glutamate, NMDA and KA, at a concentration of 0.25 mM, did not affect the basal c-fos and c-jun mRNA levels, and also did not affect forskolin- and PMA-induced responses. Furthermore, both forskolin and PMA itself increased the phosphorylation of ERK (extracellular signal regulated kinase) and CREB (cyclicAMP responsible element binding protein) proteins. The KA, NMDA, and glutamate did not affect forskolin-induced increase of ERK and CREB phosphorylation. The KA decreased PMA-induced increase of phosphorylation of ERK and CREB proteins, whereas glutamate and NMDA did not affect the phosphorylation of ERK and CREB proteins induced by PMA. These findings suggest that, in C6 glioma cells, c-fos mRNA induction induced by EAAs is not mediated by phosphorylation of ERK and CREB proteins.
Animals ; Colforsin ; Cyclic AMP Response Element-Binding Protein ; Excitatory Amino Acids ; Gene Expression* ; Glioma* ; Glutamic Acid* ; Kainic Acid ; N-Methylaspartate ; Phosphorylation ; Rats* ; Receptors, Glutamate* ; RNA, Messenger

Tetrahydrobiopterin (BH4) deficiency is caused by mutations in genes encoding enzymes involved in the synthesis and regeneration of BH4. The condition is usually accompanied by hyperphenylalaninemia (HPA) and deficiency of neurotransmitter precursors L-dopa and 5-hydroxytryptophan. BH4 deficiency is much rarer than classical phenylketonuria. Dihydropteridine reductase (DHPR) deficiency, an autosomal recessive genetic disorder, is a cause of malignant hyperphenylalaninemia due to BH4 deficiency. When left untreated, DHPR deficiency leads to neurologic deterioration at the age of 4 or 5 months, including psychomotor retardation, tonicity disorders, drowsiness, irritability, abnormal movements, hyperthermia, hypersalivation, and difficulty swallowing. Treatment of DHPR deficiency should be initiated as early as possible with BH4 supplementation and replacement of the neurotransmitter precursors L-dopa and 5-hydroxytryptophan. We report the first case of DHPR deficiency in Korea, a child diagnosed at 9 years of age by genetic testing.
5-Hydroxytryptophan ; Biopterin ; Child ; Deglutition ; Dihydropteridine Reductase ; Dyskinesias ; Fever ; Genetic Testing ; Humans ; Korea ; Levodopa ; Neurotransmitter Agents ; Phenylketonurias ; Regeneration ; Sialorrhea ; Sleep Stages

Hyperphenylalaninemia can result from defects in either the phenylalanine hydroxylase (PAH) enzyme or in the synthesis or recycling of the active pterin, tetrahydrobiopterin (BH4), which is an obligate co-factor for the PAH enzyme, as well as tyrosine hydroxylase and tryptophan hydroxylase. One of the most common causes of BH4 deficiency is a defect in the synthesis of 6-pyruvoyltetrahydropterin synthase (PTPS) enzyme. Patients present with progressive neurological disease such as mental retardation, convulsions and disturbance of tone and posture despite strict adherence to diet and good metabolic control. The authors report the first two cases of PTPS deficiency in the Philippines. Both are females with initial phenylalanine levels of more than 1300 umol/L who continued to develop neurologic deterioration despite good metabolic control and strict adherence to diet. Further investigation showed that they both had PTPS deficiency. Treatment was started with BH4, L-dopa/carbidopa, and 5-hydroxytryptophan (5HT) with concomitant significant improvements in their neurologic and developmental outcomes.

Human ; Female ; Child Preschool ; Infant ; Phenylalanine Hydroxylase ; Carbidopa ; Tyrosine 3-monooxygenase ; 5-hydroxytryptophan ; Tryptophan Hydroxylase ; Levodopa ; Sapropterin ; Intellectual Disability ; Philippines ; Phenylketonurias ; Pterins ; Seizures ; Diet ; Posture

OBJECTIVETo emphasize early differential diagnosis from patients with hyperphenylalaninemia (HPA) and to evaluate the treatment and long-term outcome of patients with tetrahydrobiopterin synthase (BH4) deficiency in Northern Chinese population.

METHODSFrom 1992 to 2005, a total of 618 patients with HPA were diagnosed and/or cared for in our outpatient clinic. Urinary pterin analysis, detection of dihydropteridine reductase (DHPR) activity in blood, and then BH4 loading tests were carried out to differentiate BH4 deficiency in these patients from classical phenylketonuria. BH4 deficient patients were treated with BH4, levodopa and 5-hydroxytryptophane (5-HTP) immediately while the diagnosis was done to disease. Patientso blood phenylalanine levels, psychomotor and intelligence development were followed up.

RESULTSA total of 38 cases were diagnosed as BH4 deficiency, all of them were revealed as 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency from the extremely decreased urine biopterin, normal DHPR activities and drop down of blood phenylalanine level to normal range within 4 to 8 hours after BH4 loading. The most common manifestations were progressively psychomotor and mental retardation to patients even after taking early dietary treatment. The patients were diagnosed and treated with drugs at the ages of 2.1 months to 13 years. With 4 patients died of pneumonia, 7 patients refused to treatment, only 27 patients were under treatment and followed up. The average full scale development or intelligence quotient (DQ/IQ) of patients who were treated within and after 6 months were 86+/- 10 or 66+/- 7 respectively. Development was not even in different aspects. A significant negative correlation was observed between the level of the DQ and the age of treatment commenced (r was -0.714, P< 0.01). Eleven patients experienced the extrapyramidal movement disorders, 3 of them combined with epilepsy. The extrapyramidal disorders were controlled by administration of levodopa.

CONCLUSIONThe differential diagnosis for BH4 deficiency should be carried out in all patients with HPA. PTPS deficiency is the most common form of BH4 deficiency in Northern Chinese population. The long-term outcome of these patients benefits from diagnosis and treatment with BH4, levodopa and 5-HTP as early as possible.

5-Hydroxytryptophan ; therapeutic use ; Asian Continental Ancestry Group ; genetics ; Biopterin ; analogs & derivatives ; deficiency ; therapeutic use ; Child, Preschool ; China ; Dihydropteridine Reductase ; blood ; Humans ; Infant ; Levodopa ; therapeutic use ; Phenylalanine ; blood ; Phenylketonurias ; drug therapy ; genetics ; metabolism ; Phosphorus-Oxygen Lyases ; deficiency ; genetics

This study is to investigate the effects of fluvastatin on the activation of p38 mitogen-activated protein kinase (p38 MAPK) and cAMP response element-binding protein (CREB1) in glomerular mesangial cells under high concentration of glucose. High concentration glucose and fluvastatin were used to stimulate the cultured rat glomerular mesangial cells (GMCs) in vitro. The protein expressions of p38 MAPK, CREB1, p-p38 MAPK and p-CREB1 were observed with Western blotting. TGF-beta1 and fibronectin (FN) mRNA were measured with reverse transcription and polymerase chain reaction (RT-PCR). The protein synthesis of laminine (LN) and type IV collagen in the supernatants of the GMCs were detected with radioimmunoassay. Compared with low glucose control group, the expressions of p-p38 MAPK, p-CREB1 were increased obviously in high glucose group, TGF-beta1 mRNA and FN mRNA, LN and type IV collagen in the supernatants were increased significantly in GMCs under high concentration glucose medium. The expression levels of p-p38 MAPK, p-CREB1, TGF-beta1 mRNA, and FN mRNA, LN and type IV collagen in the supernatants were significantly lower in the fluvastatin group than those in the high concentration glucose group. It is concluded that fluvastatin can inhibit over production of TGF-beta1 and ECM proteins in GMCs under high concentration of glucose, partly by regulating the phosphorylation of p38 MAPK and CREB1.
Amino Acids, Diamino ; metabolism ; Animals ; Cell Proliferation ; Cells, Cultured ; Collagen Type IV ; metabolism ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Fatty Acids, Monounsaturated ; pharmacology ; Fibronectins ; genetics ; metabolism ; Glucose ; administration & dosage ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Indoles ; pharmacology ; Male ; Mesangial Cells ; cytology ; metabolism ; Phosphorylation ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Transforming Growth Factor beta1 ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

No Abstract Available.
Histidine*