The goal of this review is to present a comprehensive survey of the many intriguing facets of creatine (Cr) and creatinine metabolism, encompassing the pathways and regulation of Cr biosynthesis an...

http://www.afboard.com/library/creatinemetabolism.pdf

Creatine and Creatinine Metabolism

https://publications.mpi-cbg.de/Matyash_2008_1086.pdf

Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.

Improved DNA: liposome complexes for increased systemic delivery and gene expression.

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Multicenter Evaluation of a Sequence-Based Protocol for Subtyping Shiga Toxins and Standardizing Stx Nomenclature

The present invention provides an apparatus for purifying nucleic acids such as DNA and RNA or proteins such as enzymes and antibodies from microorganisms such as viruses and bacteria or animal and plant tissues, the apparatus being capable of fast processing of a plurality of samples. More specifically, the present invention provides an apparatus for purifying nucleic acids or proteins using magnetically attractable particles, the apparatus comprising a plurality of piston pumps; a plurality of nozzles capable of having a plurality of disposable tips which are automatically attachable/detachable; and a mechanism which is capable of dispensing a desired amount of a reagent to be used subsequently in the next step into the same number of sections as that of the samples with high accuracy and at a high rate, while a mixture of a sample and a reagent are being mixed (stirred) by the pumps and nozzles, the apparatus being capable of rapidly processing a plurality of samples, wherein a series of steps from mixing (stirring) of the magnetically attractable particles and concentration of the sample to purification is automated with reduced wastes.

Apparatus for purifying nucleic acids and proteins

PROBLEM TO BE SOLVED: To purify the subject DNA in a high purity for a short time by adding a cytolytic liquid containing a chaotropic substance, an organic solvent and a nucleic acid-bonding solid-phase carrier to a microorganism or a cell containing a plasmid DNA, washing the resultant solid-phase carrier adsorbing the plasmid DNA thereon and then treating the washed solid-phase carrier with an eluent. SOLUTION: A cytolytic liquid at pH 3-6 containing a chaotropic substance comprising a guanidine thiocyanate and sodium acetate-hydrochloric acid, etc., an extracting liquid comprising an organic solvent, etc., such as a buffer solution-saturated phenol and a nucleic acid-bonding solid-phase carrier comprising particles, etc., containing a superparamagnetic metallic oxide are added, mixed or brought into contact with a microorganism or a cell holding a plasmid DNA to adsorb the plasmid DNA on the solid-phase carrier. The solid-phase carrier adsorbing the plasmid DNA thereon is then washed with a wash liquid and the plasmid DNA is eluted from the washed solid-phase carrier with an eluent to thereby extract and purify the plasmid DNA from the microorganism or cell in high purity for a short time without requiring complicated operations.

Extraction and purification of plasmid dna

Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification

PROBLEM TO BE SOLVED: To purify an RNA in a high purity for a short time without requiring complicated operations by adding a lytic liquid containing a chaotropic substance, an organic solvent and a nucleic acid-bonding solid-phase carrier to a biological material such as a cell under acidic conditions, washing the resultant solid-phase carrier adsorbing the RNA thereon and treating the washed solid-phase carrier with an eluent. SOLUTION: A lytic liquid at pH 3-6 containing a chaotropic substance such as a guanidine thiocyanate, an extracting liquid comprising an organic solvent such as a water-saturated or a buffer solution-saturated phenol and a nucleic acid-bonding solid-phase carrier comprising particles, etc., containing a superparamagnetic metallic oxide are added, mixed or brought into contact with a biological material such as a cell under acidic conditions to adsorb an RNA contained in the biological material on the solid-phase carrier. The solid-phase carrier adsorbing the RNA thereon is then washed with a wash liquid and the RNA is eluted from the washed solid-phase carrier with an eluent to thereby extract and purity the RNA from the biological material such as the cell in a high purity for a short time without requiring complicated operations.

Extraction and purification of rna

Background: Many different methods have been used to assay amylase activity, using nitrophenylated oligosaccharides as substrate; however, the hydrolysis steps in these methods are complex.

Methods: We developed a new continuously monitoring assay for amylase activity in biological fluids, using 2-chloro-4-nitrophenyl-4- O -β-d-galactopyranosylmaltoside (GalG2CNP) as the substrate; this assay was used with anti-human salivary amylase monoclonal antibodies for specific determination of the pancreatic isoenzyme. Amylase converted GalG2CNP into β-d-galactopyranosylmaltose and 2-chloro-4-nitrophenol, which was measured at 405 nm.

Results: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and β-d-galactopyranosylmaltose and did not undergo transfer reactions. The within-assay CVs (n = 20) for total amylase (T-AMY) and pancreatic amylase (P-AMY) were 0.6–1.6% and 0.5–2.5%, respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were 0.8–3.7% and 0.6–4.1%, respectively. T-AMY and P-AMY activities in serum or urine obtained by the proposed method correlated well with those determined by the 2-chloro-4-nitrophenyl 4- O -β-d-galactopyranosyl-β-maltotetraoside method or the modified IFCC method.

Conclusions: This novel assay for T-AMY and P-AMY measures both activities stoichiometrically, directly, and easily, and may be suitable for routine procedures.

/pdf/total-and-pancreatic-amylase-measured-with-2-chloro-4-4ix3k0to18.pdf

Yoshihisa Kawamura

Papers

Apparatus for purifying nucleic acids and proteins

Extraction and purification of plasmid dna

Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification

Extraction and purification of rna

Total and Pancreatic Amylase Measured with 2-Chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside