Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana

Background

The ubiquitin ligase COP1, COnstitutively Photomorphogenic 1, functions in many biological responses in mammalian cells, but its downstream pathway remains unclear.

Results

Here, we identified FIP200, a key regulator of mammalian autophagy, as a novel COP1-interacting protein by yeast two-hybrid screening. The interaction was confirmed by a GST-pulldown assay. Split-GFP analysis revealed that interaction between COP1 and FIP200 predominantly occurred in the cytoplasm and was enhanced in cells treated with UV irradiation. Different forms of FIP200 protein were expressed in cultured mammalian cells, and ectopic expression of COP1 reduced one of such forms.

Conclusions

These data suggest that COP1 modulates FIP200-associated activities, which may contribute to a variety of cellular functions that COP1 is involved in.     

Zur maassanalytischen Bestimmung des Wismuths

Ohne Zusammenfassung     

A GC/MS-based metabolomic approach for diagnosing citrin deficiency

Citrin is the hepatic mitochondrial aspartate–glutamate carrier that is encoded by the gene SLC25A13. Citrin deficiency often leads to hyperammonemia, for which the current treatment concept is different from that for primary hyperammonemias. Metabolite level diagnosis, often referred to as chemical diagnosis, is not always successful in identifying citrin deficiency immediately or in a timely fashion. We previously made the chemical diagnosis of citrin deficiency in ten patients from nine families. In order to devise a more rapid and more accurate chemical diagnosis of this disorder than is currently available, we reinvestigated the gas chromatography/mass spectrometry-based urine metabolome in these patients. In patients aged 2 to 5 months, prominent biomarkers detected included one or more of the following metabolites: tyrosine, p-hydroxyphenyllactate, p-hydroxyphenylpyruvate, and N-acetyltyrosine, galactose, galactitol and galactonate, glucose, glucitol, and cystathionine. These biomarkers are less prominent in older patients, but are not increased in argininosuccinate synthetase deficiency or other hyperammonemias. α-Ketoglutaramate (KGM), a recently recognized urinary biomarker of primary hyperammonemias associated with defects of the urea cycle, was increased in most patients with citrin deficiency studied here in spite of normal urinary levels of glutamine (the immediate precursor of KGM), 5-oxoproline, glutamate, aspartate, and asparagine. Other important urinary biomarkers that should be measured for differential diagnosis of hyperammonemias, including orotate, uracil, and β-ureidopropionate, were not increased. The presence of citrulline and citrulline-derived metabolites was noted in all cases. The present study shows that noninvasive urine metabolomics, together with an analysis of selected metabolites or groups of metabolites, provides a more reliable and rapid chemical diagnosis of citrin deficiency than was previously available and more readily differentiates this disorder from other hyperammonemic syndromes.     

Five cases of beta-ureidopropionase deficiency detected by GC/MS analysis of urine metabolome

The clinical presentation of inborn errors of pyrimidine degradation varies considerably from asymptomatic to severe neurological illness. We have reported a method to screen for and make a chemical diagnosis of beta-ureidopropionase deficiency, leading to the discovery of the first asymptomatic case of this disease. In this method, the recovery of beta-ureidopropionate and beta-ureidoisobutyrate, the key biomarkers, was very high,and the adoption of GC/MS and targeted analysis enabled us to simultaneously obtain information related and unrelated to pyrimidine metabolism. The present study reports the results of a large-scale screening of 24,000 newborns using dried urine on filter paper. Identification of a total of four asymptomatic patients among newborns suggests the high incidence (1/6000) of this disease in Japan. While these newborns were asymptomatic, two additional cases detected at the age of 5 years as well as 3 months with this method for high-risk screening had autism and West syndrome, respectively.The key biomarkers and alpha-ureidobutyrate used as an internal standard were found to give not only their di-trimethylsilyl derivatives but also tri-trimethylsilyl derivatives, upon derivatization. The mass spectra and retention times of their tri-trimethylsilyl derivatives and data handling for quantification of the markers are presented.Identification of individuals with defects in pyrimidine metabolism would realize personalized medication in cancer chemotherapy with pyrimidine analogs such as 5-fluorouracil.     

Urinary metabolic profile of phenylketonuria in patients receiving total parenteral nutrition and medication

Nutrition and drugs are main environmental factors that affect metabolism. We performed metabolomics of urine from an 8‐year‐old patient (case 1) with epilepsy and an 11‐year‐old patient (case 2) with malignant lymphoma who was being treated with methotrexate. Both patients were receiving total parenteral nutrition (TPN). We used our diagnostic procedure consisting of urease pretreatment, partial adoption of stable isotope dilution, gas chromatography/mass spectrometry (GC/MS) measurement and target analysis for 200 analytes including organic acids and amino acids. Surprisingly, their metabolic profiles were identical to that of phenylketonuria. The neopterin level was markedly above normal in case 1, and both neopterin and biopterin were significantly above normal in case 2. Mutation analysis of genomic DNA from case 1 showed neither homozygosity nor heterozygosity for phenylalanine hydroxylase deficiency. The metabolic profiles of both cases were normal when they were not receiving TPN. TPN is presently prohibited for individuals who have inherited disorders that affect amino acid metabolism. Although the Phe content of the TPN was not the sole cause of the PKU profile, its effect, combined with other factors, e.g. specific medication or possibly underlying diseases, led to this metabolic abnormality. The present study suggests that GC/MS‐based metabolomics by target analysis could be important for assuring the safety of the treatments for patients receiving both TPN and methotrexate. Metabolomic profiling, both before and during TPN, is useful for determining the optimal nutritional formula not only for neonates, but also for young children who are known heterozygotes for metabolic disorders or whose status is unknown. Copyright © 2009 John Wiley & Sons, Ltd.     

Optical fiber sensor for the measurement of electric field intensity and voltage (OPSEF)

An optical fiber sensor for measuring high electric field intensity and voltage, which utilizes the electro-optic effect of bismuth silicone oxide single crystal, has been developed successfully, and the fundamental characteristics have been evaluated. The electric field intensity distribution inside the coaxial electrodes has been measured with this sensor. The measured results are in agreement with the theoretical values.     

Synthesis and characterization of β-O-tosyldehydroserine as a precursor of dehydroamino acids

β-O-Tosyldehydroserine 3 is prepared directly by the oxidation of the corresponding serine derivative 1 with dimethyl sulfoxide which is activated by p-toluenesulfonyl chloride. The enol tosylate 3 retains reactivities characteristic of aldehydes like those expected for the corresponding dehydroserine derivative 2. A typical reaction of 3 includes substitution of the tosyl group with nucleophiles such as primary and secondary amines, leading to other dehydroamino acids.     

Die Methode zur Bestimmung des Schmelzpunktes

Ohne Zusammenfassung     

Urinary 2-hydroxy-5-oxoproline,the lactam form of α-ketoglutaramate,is markedly increased in urea cycle disorders

α-Ketoglutaramate (KGM) is the α-keto acid analogue of glutamine, which exists mostly in equilibrium with a lactam form (2-hydroxy-5-oxoproline) under physiological conditions. KGM was identified in human urine and its concentration quantified by gas chromatography/mass spectrometry (GC/MS). The keto acid was shown to be markedly elevated in urine obtained from patients with primary hyperammonemia due to an inherited metabolic defect in any one of the five enzymes of the urea cycle. Increased urinary KGM was also noted in other patients with primary hyperammonemia, including three patients with a defect resulting in lysinuric protein intolerance and one of two patients with a defect in the ornithine transporter I. These findings indicate disturbances in nitrogen metabolism, most probably at the level of glutamine metabolism in primary hyperammonemia diseases. Urinary KGM levels, however, were not well correlated with secondary hyperammonemia in patients with propionic acidemia or methylmalonic acidemia, possibly as a result, in part, of decreased glutamine levels. In conclusion, the GC/MS procedure has the required lower limit of quantification for analysis of urinary KGM, which is markedly increased in urea cycle disorders and other primary hyperammonemic diseases.     

Selective CO2 conversion to formate conjugated with H2O oxidation utilizing semiconductor/complex hybrid photocatalysts

Photoelectrochemical reduction of CO(2) to HCOO(-) (formate) over p-type InP/Ru complex polymer hybrid photocatalyst was highly enhanced by introducing an anchoring complex into the polymer. By functionally combining the hybrid photocatalyst with TiO(2) for water oxidation, selective photoreduction of CO(2) to HCOO(-) was achieved in aqueous media, in which H(2)O was used as both an electron donor and a proton source. The so-called Z-scheme (or two-step photoexcitation) system operated with no external electrical bias. The selectivity for HCOO(-) production was >70%, and the conversion efficiency of solar energy to chemical energy was 0.03-0.04%.