An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum. In the analytical process applied with coupled-enzyme, the quality control of sarcosine oxidase (SOX) as a key enzyme is the first problem to be solved. In order to establish an efficient and laboratory-scale production of SOX, the recombinant sarcosine oxidase (r-SOX) gene was a high-level expression in E. coli induced with lactose on a large-scale fermentation in 300 L fermenter. The results suggested that the biomass concentration reached OD600 of 22 and the expression of recombinant sarcosine oxidase in E. coli accounted for about 25% of total soluble protein in culture after fermentation. The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography. The sarcosine oxidase with 97% purity, 25 U/mg specific activity and 92.4% activity recovery was obtained. The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography. This sarcosine oxidase was found to be a conjugated protein, yellow enzyme, which combined with FAD as prosthetic group by covalent linkage. The contaminant of catalase was not detected in the sample pool of this enzyme. In addition, a further test to the thermal stability of sarcosine oxidase was done. According to the above results, the development and utilization of this enzyme has been set up on a reliable foundation.
Escherichia coli ; Fermentation ; Recombinant Proteins ; biosynthesis ; Sarcosine Oxidase ; biosynthesis

Amino Acid Metabolism, Inborn Errors ; Humans ; Mitochondrial Diseases ; Sarcosine Dehydrogenase ; deficiency

BACKGROUND AND PURPOSE: Primary involvement of the peripheral nerves in myotonic dystrophy type I (MyD1) is controversial. We investigated whether the involvement of peripheral nerves is a primary event of MyD1 or secondary to another complication such as diabetes mellitus (DM). METHODS: The subjects comprised 12 patients with MyD1, 12 with DM and no peripheral nerve involvement, and 25 healthy volunteers. We measured multiple excitability indices in the median motor axons. The strength-duration time constant was calculated from the duration-charge curve, the threshold electrotonus and current-threshold relationships were calculated from the sequential subthreshold current, and the recovery cycle was derived from double suprathreshold stimulation. RESULTS: The depolarizing and hyperpolarizing threshold electrotonus were significantly reduced and exhibited increased refractoriness in the MyD1 group compared with the DM and control groups. The SDTC, superexcitability, and subexcitability were not significantly altered in the MyD1 group. CONCLUSIONS: The MyD1 group exhibited a depolarized axonal membrane potential. The significant differences in peripheral nerve excitability between the MyD1 group and the DM and normal control groups suggest that peripheral neuropathy is a primary event in MyD1 rather than a secondary complication of DM.
Axons ; Diabetes Mellitus ; Humans ; Membrane Potentials ; Myotonic Dystrophy ; Peripheral Nerves ; Peripheral Nervous System Diseases ; Sarcosine ; Thiocarbamates

INTRODUCTIONSarcosinaemia is a rare metabolic disorder which has not been reported in Asia.

CLINICAL PICTUREThe urine samples of 2 patients were screened as a routine metabolic screening offered for patients with mental retardation in our hospital. We used gas chromatography-mass spectrometry (GC-MS) which is capable of detecting abnormal pattern in amino acids and organic acids. Plasma sarcosine level was further quantified by GC-MS. The same methods were used in the investigations of asymptomatic family members. Urine examination by GC-MS revealed excessive amount of sarcosine in urine (normally undetectable) and their plasma sarcosine levels were raised. The 2 differential diagnoses of presence of sarcosine in urine--glutaric aciduria type II and folate deficiency--were ruled out by the absence of abnormal organic acids in the initial urine screen and by normal serum folate level respectively. Screening of the 2 families identified excessive sarcosine in urine in 2 siblings, one from each family. However, these 2 siblings of indexed patients thus identified have no neurological or developmental problem.

CONCLUSIONOur finding was consistent with the notion that sarcosinaemia is a benign condition picked up coincidentally during screening for mental retardation.

Amino Acid Metabolism, Inborn Errors ; complications ; diagnosis ; Child ; Child, Preschool ; China ; ethnology ; Family Health ; Female ; Gas Chromatography-Mass Spectrometry ; Hong Kong ; Humans ; India ; ethnology ; Intellectual Disability ; complications ; Sarcosine ; blood ; urine ; Sarcosine Dehydrogenase ; deficiency

PURPOSE: The purpose of this study was to determine, using in vivo and in vitro 1H MRS (MR spectroscopy), the characteristic biochemical metabolites related with breast cancer, and to assess the clinical usefulness and limitations of this modality. MATERIALS AND METHODS: For in vivo 1H MRS, nine patients with breast cancer and two normal volunteers were examined on a 1.5 T MR imager equipped with facilities for spectroscopy. In order to localize the breast lesion, axial and sagittal T1-weighted images and fat-suppressed T2-weighted images were obtained just prior to MRS; MR spectra were acquired at TR=3000 msec and TE=144 msec. For in vitro 1H MRS, breast tumor and adja-cent normal tissue were extracted from 13 patients with breast cancer, and in two of these, both in vivo and in vitro 1H MRS were performed. All in vitro 1H MRS specimens were immediately immersed in liquid nitrogen, and then in a preparation of perchloric acid. For quantitative analysis of the MR spectra of cancerous and normal breast tissue, the paired t-test was used (p < 0.05). RESULTS: At1H MRS in vivo, choline and two lipids were identified at 3.21 ppm, and 1.33 ppm and 0.9 ppm, re-spectively. The distinction between cancerous and normal breast tissue was based on the higher level of choline (3.21 ppm) present in the former. At 1H MRS in vitro, on the other hand, mean and standard deviation (% standard deviation) for the various metabolites in cancerous and normal breast tissue were as follows: choline, 30.195 +/- 2.448(8.108) and 22.648 +/- 1.938(8.556); trimethylamine, 3.425 +/- 0.335(9.769) and 0.640 +/- 0.066(10.325); sarcosine, 3.425 +/- 0.335(9.769) and 0.640 +/- 0.099(15.394); lactate, 16.388 +/- 1.134(6.922) and 9.715 +/- 0.385(3.965); inositol, 1.970 +/- 0.282(14.334) and 3.859 +/- 0.502(13.020); and taurine, 6.614 +/- 0.556(8.412) and 10.748 +/- 1.206(11.222). High levels of choline (p=0.026), trimethylamine (p=0.001), sarco-sine (p=0.009), and lactate (p=0.009), and lower levels of inositol (p=0.006) and taurine (p=0.008) were char-acteristic findings in cancerous as compared with normal breast tissue, with significantly different results. CONCLUSION: 1H MRS both in vitro and in vivo showed that increased choline levels were present in cancerous breast tissue, but that normal tissue does not contain choline. The presence of choline could therefore be used as a marker for malignancy in breast lesions. Information provided by in vitro 1H MRS, together with the development of in vivo 1H MRS with high field strength and high resolution, may be very useful for the diagnosis of breast cancer.
Breast Neoplasms* ; Breast* ; Choline ; Diagnosis ; Hand ; Healthy Volunteers ; Humans* ; Inositol ; Lactic Acid ; Magnetic Resonance Spectroscopy* ; Nitrogen ; Sarcosine ; Spectrum Analysis ; Taurine

As part of an initial inquiry into the function of the outer membrane proteins (OMPs) of Helicobacter pylori Korean strain 51, we have conducted an extensive proteome analysis via quadrupole time of flight (Q-TOF) mass spectrometry (MS). Fifty one OMPs of H. pylori were purified using sarcosine and resolved via two-dimensional electrophoresis with immobilized pH gradient strips. The most abundant proteins were observed in the alkaline pI regions (6.0~11.0) at molecular masses between 10~100 KDa. Here, 15 spots were identified, representing 9 types of genes (KHP0852, KHP0853, KHP1353, KHP1017, KHP0172, KHP0076, KHP0617, KHP1069, KHP0614) from the sarcosin-insoluble fraction of H. pylori 51. These may be employed in the characterization of the OMPs of H. pylori 51, which will help to identify new potential target proteins for vaccine development and drug therapy.
Electrophoresis ; Helicobacter ; Helicobacter pylori ; Mass Spectrometry ; Membrane Proteins ; Membranes ; Proteins ; Proteome ; Proton-Motive Force ; Sarcosine ; Sprains and Strains

We purified a sarcosine oxidase from Bacillus sp. strain BSD-8 isolated from soil. We purified the enzyme by ammonium sulfate precipitation, DEAE-cellulose, Toyopearl hydrophobic and Sephadex G-75 molecular sieve chromatography and characterized the purified sarcosine oxidase. This sarcosine oxidase was a flavin enzyme containing a noncovalently bound flavin with the subunit molecular mass of 51 kDa. The optimal temperature for this enzyme was 60 degrees C and it showed its highest activity at pH 8.5. It was stable in the pH range of 8.0-10.0 and at the temperature of 60 degrees C. Estimated by Lineveaver-Burk plots, the K(m) of the enzyme was 3.1 mmol/L. Ag+, Hg2+, SDS and Tween 80 dramatically inhibted the enzyme activity, whereas Tween 20 and Triton X-100 had no effect on enzyme activity. The thermostability of this enzyme was better than reported sarcosine oxidases, and it could be applied in enzymatic measuring of creatinine.
Bacillus ; enzymology ; isolation & purification ; Bacterial Proteins ; chemistry ; isolation & purification ; metabolism ; Chemical Precipitation ; Enzyme Stability ; Sarcosine Oxidase ; chemistry ; isolation & purification ; metabolism ; Soil Microbiology

Vibrio mimicus, marine bacteria pathogenic for fish, can causes acute gastroenteritis in human. Iron limmited condition like in human body, may change the surface structure of V. mimicus. In this study we obse'rved the effect of iron limmited condition on outer membrane protein of V. mimicus. Ethylenediamine-di (O-hydroxy-phenylacetic) acid (EDDA), an iron chelator, delayed the time to reach expotential growth of V. mimicus in brain heart infusion medium from 3 hours to 20 hours. Outer membrane protein of V. mimicus-CON (cultured in BHI) and V. mimicus-EDDA (cultured in BHI contain EDDA) were seperated by 1% sarcosine from total cell envelop. SDS-PAGE of V. mimicus-EDDA and V. mimicus-CON showed similar protein profiles contain 37 kDa major protein but 86 and 90 kDa protein were induced differently. Immunological properties of above protein were determined by ELISA and western blotting. 86 kDa EDDA- specific OMP was induced in V. mimicus (isolate 96-1), V. parahaemolyticus (serotype 09), V. alginolyticus (isolate 95-1), E. coli (human isolate) and V. vulnificus ATCC 27562 in iron limmited condition.
Bacteria ; Blotting, Western ; Brain ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gastroenteritis ; Heart ; Human Body ; Humans ; Iron* ; Membrane Proteins ; Membranes* ; Sarcosine ; Vibrio mimicus* ; Vibrio*

Prostate cancer (PCa) is the most commonly diagnosed visceral cancer in men and is responsible for the second highest cancer-related male mortality rate in Western countries, with increasing rates being reported in Korea, Japan, and China. Considering the low sensitivity of prostate-specific antigen (PSA) testing, it is widely agreed that reliable, age-independent markers of the presence, nature, and progression of PCa are required to facilitate diagnosis and timely treatment. Metabolomics or metabonomics has recently emerged as a novel method of PCa detection owing to its ability to monitor changes in the metabolic signature, within biofluids or tissue, that reflect changes in phenotype and function. This review outlines the physiology of prostate tissue and prostatic fluid in health and in malignancy in relation to metabolomics as well as the principles underlying the methods of metabolomic quantification. Promising metabolites, metabolic profiles, and their correlation with the presence and stage of PCa are summarized. Application of metabolomics to biofluids and in vivo quantification as well as the direction of current research in supplementing and improving current methods of detection are discussed. The current debate in the urology literature on sarcosine as a potential biomarker for PCa is reviewed and discussed. Metabolomics promises to be a valuable tool in the early detection of PCa that may enable earlier treatment and improved clinical outcomes.
Biomarkers ; China ; Humans ; Japan ; Korea ; Male ; Metabolome ; Metabolomics ; Organothiophosphorus Compounds ; Passive Cutaneous Anaphylaxis ; Phenotype ; Prostate ; Prostate-Specific Antigen ; Prostatic Neoplasms ; Sarcosine ; Urology

BACKGROUND: Various decellularization methods have been studied in order to develop tissue graft which is less immunogenic and more durable. This study was performed to investigate the physico-dynamic and histological effect of trypsin pretreatment on decellularization protocols. MATERIAL AND METHOD: Two groups of bovine pericardium specimen each underwent decellularization process based on SDS and Triton X-100 or N-lauroylsarcosinate and Triton X-100. Two more groups additionally underwent pretreatment with 0.1% Trypsin/0.1% EDTA. After decellularization process, mechanical tensile strength was tested, then iomechanical test of permeability and compliance was tested before and after fatigue test. Light microscopy and electron microscopy was performed to observe histological findings. RESULT: There was no difference in mechanical tensile strength between groups, but permeability and compliance was decreased in trypsin pretreated groups. Light microscopic and electron microscopic findings revealed damage of the extracellular matrix in trypsin pretreated groups and in groups which underwent the fatigue test also. CONCLUSION: Trypsin pretreatment in decellularizing process of bovine pericardium damages extracellular matrix and increases permeability and compliance of the bovine pericardium, but did not decrease tensile strength. Further studies are needed to use enzymatic treatments in decellularization protocols.
Biomedical Engineering ; Bioprosthesis ; Compliance ; Edetic Acid ; Electrons ; Extracellular Matrix ; Fatigue ; Light ; Mechanical Processes ; Microscopy ; Microscopy, Electron ; Octoxynol ; Pericardium ; Permeability ; Sarcosine ; Tensile Strength ; Transplants ; Trypsin