Biotin-deficient rats were raised on a purified ration containing raw egg white plus avidin. Urea synthesis and excretion were compared between the biotin-deficient and the pair-fed control rats. 24hrurinary urea excretion and the specific activities of carbamylphosphate synthetase, ornithine transcarbamylase, and arginase in the liver mitochondria fraction were no different between these two groups. The net urea production in the liver slice and in the isolated perfused liver of the biotin-deficient rats was similar to that of the pair-fed control. Thus the conclusion must be that biotin is not in urea in mea biosynthesis in the rat.
Animal ; Avitaminosis/metabolism ; Biotin* ; Liver/metabolism* ; Rats ; Urea/biosynthesis* ; MH - ; Substances: ; Urea ; Biotin

The located culture of cells on patterned surfaces is useful for tissue engineering, biosensor development and fundamental research of cell biology. It is presented here a rapid fabrication method of Biotin-Avidin protein layers micropattern, which is based on soft-lithography technology. The bovine aortic endothelial cells are cultured on the micropatterned surface. It is found that cell location can be controlled on the scale of individual cell by this method.
Animals ; Avidin ; metabolism ; Biotin ; metabolism ; Cattle ; Cells, Cultured ; cytology ; Tissue Engineering

3-Methylcrotonyl-CoA carboxylase(MCC) is a biotin-dependent enzyme involved in the leucine metabolism. We describe a patient with MCC deficiency who manifested with Reye syndrome-like illness with status epilepticus, metabolic acidosis, hypoglycemia, hyperammonemia, elevated liver enzymes and neurologic impairments after a viral gastroenteritis and then suffered from Lennox-Gastaut syndrome. Urinary organic acid analysis revealed increased excretions of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. This patient was managed with a leucine restriction diet and supplementation of biotin and carnitine, which was not so effective. He suffered from neurologic sequelae such as Lennox-Gastaut syndrome, motor and cognitive impairements.
Acidosis ; Biotin ; Carnitine ; Diet ; Gastroenteritis ; Humans ; Hyperammonemia ; Hypoglycemia ; Leucine ; Liver ; Metabolism ; Status Epilepticus

The aim of this study is to determine the regulatory mechanism of PTEN-induced putative kinase protein 1 short isoform (PINK1S) in cytoplasm. By co-immunoprecipitation (Co-IP) assay, we identified that PINK1S interacted with the beta regulatory subunit of Casein Kinase 2 (CK2β), but not with the catalytic subunits CK2α1 and CK2α2. Furthermore, cells were transfected with PINK1S and CK2β, and then PINK1S was purified by immunoprecipitation. After detecting the phosphorylated proteins by Phos-tag Biotin, we found that CK2β overexpression increased auto-phosphorylation of PINK1S. Finally, we generated CK2β knockdown cell lines by RNA interference. Purified PINK1S from CK2β knockdown cells significantly reduced its auto-phosphorylation compared with control cells. These results suggested that CK2β functions as a regulatory subunit of PINK1S kinase complex promoted its activation by self-phosphorylation.
Biotin ; Casein Kinase II ; metabolism ; Cell Line ; Gene Knockdown Techniques ; Humans ; Phosphorylation ; Protein Kinases ; metabolism ; Pyridines ; RNA Interference ; Transfection

To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.
Bacillus subtilis ; Biotin ; chemistry ; Eukaryotic Cells ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; Promoter Regions, Genetic ; Trans-Activators

Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.
Aminoacyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Biotin ; Cysteine Endopeptidases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genes, Reporter ; Ligation ; Mutant Proteins ; genetics ; metabolism

OBJECTIVETo study the influence of heat-induced epitope retrieval (HIER) on endogenous avidin-binding activity (EABA) and to establish an effective way to block EABA in immunohistochemistry.

METHODSSystematically screening EABA in 164 (679 samples) formalin-fixed and paraffin-embedded human tissues including 76 (102 samples) normal tissues and 88 (577 samples) tumor tissues as well as 4 (80 samples) formalin-fixed and paraffin-embedded rat normal tissues using tissue array (tissue chip), HIER, immunohistochemistry and egg white solution blocking. In addition, EABA was also examined in 9 (15 samples) human frozen tissues.

RESULTS(1) EABA was detected in frozen tissues. (2) No staining for EABA was seen in formalin-fixed and paraffin-embedded tissues. (3) EABA was revealed after the tissues treated with microwave HIER. (4) The density of signal for EABA was variable from tissue to tissue and cell to cell. (5) The signals of EABA expressed in scatter or diffuse in tissues and in granular form in cytoplasm. (6) EABA was found in a wide range of epithelial tissues, especially in gland epithelia of normal and tumor tissues. These included kidney, adrenal cortex, liver, C cells of thyroid gland, oxyphil cells of parathyroid, fundal gland of stomach, sebaceous gland of skin, duct of salivary; oncocytoma and papillary adenocarcinoma of kidney and thyroid gland, adenolymphoma of parotid, carcinoma of liver cell, adenocarcinoma of stomach, colon, prostate, gall bladder and endometrium, and so on. (7) EABA was easier revealed by higher pH value buffer (EGTA pH 9.0) than that with lower pH value (EDTA pH 8.0 and citrate pH 6.0). (8) The revealed EABA could be effectively blocked using 20% egg white solution.

CONCLUSIONSHIER could unmask EABA in formalin-fixed and paraffin-embedded tissues. The unmasked EABA present in a wide range of human normal and tumor tissues as well as in rat normal tissues. The EABA could influence routine immunohistochemistry staining when using (strept)avidin-horseradish peroxidase detective system. The egg white solution could effectively block EABA and eliminate the influence of EABA on immunohistochemistry.

Animals ; Avidin ; antagonists & inhibitors ; metabolism ; Biotin ; metabolism ; Cells, Cultured ; Egg Proteins ; pharmacology ; Epithelial Cells ; metabolism ; Epitopes ; Female ; Hot Temperature ; Humans ; Immunohistochemistry ; Male ; Neoplasms ; metabolism ; pathology ; Rats

OBJECTIVETo report the clinical diagnosis, treatment and follow-up of children with holocarboxylase synthetas(HCS) deficiency and explore the gene mutation spectrum of the disease.

METHODSEleven children with HCS deficiency were enrolled. Mass spectrometry analysis and biotinidase activity determination were used for diagnosis of HCS deficiency. HCS gene mutations were analyzed by PCR directed sequencing methods. Ten patients received oral biotin treatment (10-40 mg/d). Clinical effects of biotin treatment were observed.

RESULTSAll 11 cases developed apathetic, lethargy and metabolic acidosis at different degrees, and 10 cases presented with skin lesions. The average blood 3-hydroxyisovaleryl-carnitine concentrations and urinary 3-methylcrontonylglycine and methylcitrate concentrations increased significantly. The biotinidase activity increased, being higher over 30% of the normal reference value. Four mutations in HCS gene were identified, and they were c.1522C>T (R508W), c.1088T>A (V363D), c.126G>T (E42D) and c.1994G>C (R665P) (a new variant) and the frequency was 50%, 29%, 7% and 14% respectively. The symptoms disappeared in 10 cases 1-2 weeks after biotin treatment, and blood and urinary abnormal metabolites were gradually reduced to normal 2-6 months after treatment.

CONCLUSIONSHCS deficiency is characterized by nervous system damage, skin lesions and metabolic acidosis. Mass spectrometry analysis, biotinidase activity determination and gene mutation analysis may be helpful in the definite diagnosis of this disorder. The effect of early biotin treatment is satisfactory. The mutations R508W and V363D might be hot-spots in Chinese children with HCS deficiency.

Biotin ; therapeutic use ; Biotinidase ; metabolism ; Carbon-Nitrogen Ligases ; genetics ; Child, Preschool ; Female ; Holocarboxylase Synthetase Deficiency ; diagnosis ; therapy ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation

Multiple carboxylase deficiency (MCD) includes autosomal recessive holocarboxylase synthetase (HLCS) deficiency and biotinidase (BTD) deficiency, which are caused by and gene mutations respectively. Neonatal screening for HLCS deficiency is based on 3-hydroxyisovaleryl carnitine in dry blood filter paper, and BTD deficiency is based on BTD activity determination. HLCS deficiency and BTD deficiency are characterized by neurocutaneous syndrome and organic aciduria, however, they are different in onset age, neurological symptoms and metabolic decompensation, which needed to be differentiated from acquired biotin deficiency or other genetic metabolic diseases. The diagnosis of the disease requires a combination of biochemical characteristics of hematuria, enzyme activity determination and genetic test. Routine biotin doses are effective for most MCD patients. This consensus is intended to benefit early screening and diagnosis of MCD.
Biotin/therapeutic use* ; Biotinidase Deficiency/therapy* ; Carbon-Nitrogen Ligases/metabolism* ; Consensus ; Holocarboxylase Synthetase Deficiency/genetics* ; Humans ; Infant, Newborn ; Multiple Carboxylase Deficiency/drug therapy* ; Neonatal Screening

Biotinylated chitosan nanoparticles (Bio-CS-NP) were prepared for the active delivery to cancer cells and its characterization was investigated in this study. The preparation process included two steps. First, biotinylated chitosan ( Bio-CS ) was obtained through a reaction between sulfosuccinimidobiotin and chitosan (CS). Second, Bio-CS-NP were prepared by the precipitation of Bio-CS with sodium chloride solution. With a biotin reagent box, the conjugation densities of biotin on the surface of Bio-CS-NP were determined. The morphology and diameter of the nanoparticles were assayed by transmission electron microscope (TEM) and laser light scattering particle analyzer, respectively. The uptake of nanoparticles by human hepotacarcinoma HepG2 cells, for example, Bio-CS-NP and chitosan nanoparticles (CS-NP) without any modification, was quantitatively examined. The results indicated that the conjugation densities of biotin on the surface of Bio-CS-NP were 2.2 biotin CS. Bio-CS-NP were spherical, smooth on the surface. The average diameter was 296.8 nm. The polydispersion index was 0.155. The uptake of Bio-CS-NP by HepG2 cells was much higher than that of CS-NP (P < 0.05). It demonstrated that Bio-CS-NP can be applied as a new vehicle to actively deliver anticancer drugs to tumor cells. The method for the determination of biotin was simple and practical.
Biotin ; analogs & derivatives ; chemistry ; metabolism ; Biotinylation ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Chitosan ; chemistry ; metabolism ; Drug Carriers ; Drug Compounding ; Drug Delivery Systems ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Nanoparticles ; Particle Size ; Succinimides ; chemistry ; metabolism