A simple screening method using ion chromatography for the diagnosis of cerebral creatine deficiency syndromes

Cerebral creatine deficiency syndromes (CCDS) are caused by genetic defects in L-arginine:glycine amidinotransferase, guanidinoacetate methyltransferase or creatine transporter 1. CCDS are characterized by abnormal concentrations of urinary creatine (CR), guanidinoacetic acid (GA), or creatinine (CN). In this study, we describe a simple HPLC method to determine the concentrations of CR, GA, and CN using a weak-acid ion chromatography column with a UV detector without any derivatization. CR, GA, and CN were separated clearly with the retention times (mean ± SD, n = 3) of 5.54 ± 0.0035 min for CR, 6.41 ± 0.0079 min for GA, and 13.53 ± 0.046 min for CN. This new method should provide a simple screening test for the diagnosis of CCDS.     

Role of cell contact in the specification process of pigment founder cells in the sea urchin embryo

Effects of LiCl on the specification process of pigment founder cells were examined in the sea urchin Hemicentrotus pulcherrimus. If embryos were treated with 30 mM LiCl during 4-7 or 7-10 hours postfertilization, pigment cells increased significantly. Aphidicolin treatment indicated that this increase was due to the increase in the pigment founder cells. Interestingly, if the embryos were treated sequentially with LiCl and Ca2+-free seawater during 4-7 and 7-10 hr, respectively, they differentiated only about the same number of pigment cells as control embryos. Further, the increase was scarcely discerned when the embryos were treated with LiCl in the absence of Ca2+ during 7-10 hr. These results suggested that effect of LiCl would be ascribed to the increase in cell adhesiveness. In fact, LiCl-treated embryos were more difficult to be dissociated into single cells. Cell electrophoresis showed that the amount of the negative cell surface charges decreased considerably in LiCl-treated embryos. It was also found that the number of pigment cells seldom exceeded 100, even if embryos were exposed to a higher concentration of LiCl. This suggested that only a subpopulation of the descendants of veg2 blastomeres received the inductive signal emanated from the micromere progeny.     

pGp as the main product of bovine tRNA kinase

One of the Ser-tRNAs, Ser-tRNA^Sec, is converted to Sec-tRNA^Sec by Sec synthase. This Ser-tRNA^Sec is also converted to phosphoser-tRNA^Sec by tRNA kinase. In this study, we analyzed of the products of phosphorylation with tRNA kinase. [³H]Ser-tRNA^Sec purified on Sephacryl S-200 was phosphorylated with [-³²P]ATP by tRNA kinase. The product [³²P][³H]phosphoser-tRNA was purified on Sephacryl S-200 and hydrolyzed with ribonuclease T2. The chromatogram of this hydrolyzate on DEAE-cellulose in 7M urea buffer showed four peaks. The first peak of the pass-through fraction was seryl-adenosine liberated from the 3-terminal of the tRNA. The second peak, eluted before the third peak containing inorganic phosphate, was phosphoseryl-adenosine. The major compound in the fourth peak was pGp. As a control experiment, non-acylated tRNA^Sec was used as a substrate of phosphorylation and the product was analyzed. The chromatogram of the digest with ribonuclease T2 showed no peak of phosphoseryl-adenosine, but a peak of pGp was seen with the peak of inorganic phosphate. Thus, the major product in the presence of tRNA kinase was pGp, and a small but significant proportion of the radioactivity was found as phosphoserine in the presence of seryl residue on the 3-CCA terminal of tRNA^Sec. These results indicated that tRNA kinase phosphorylates not only Ser-tRNA to phosphoser-tRNA but also Gp of the 5-termini of tRNA to pGp. This study gives a new role to mammalian tRNA kinase.     

Molecular cloning of the complement C1r/C1s/MASP2-like serine proteases from the common carp (Cyprinus carpio)

The classical pathway of complement composed of C1, C4, and C2 is an antibody-dependent activation cascade that is present in jawed vertebrates. C1 is a Ca2+-dependent complex of C1q, C1r, and C1s, and analogous to an initiation complex of the lectin pathway of complement, which consists of the mannose-binding lectin (MBL) homologous to C1q and the MBL-associated serine proteases (MASPs) homologous to C1r and C1s. Thus divergence of Clq and MBL and that of C1r, C1s and the MASPs are considered to be crucial events in the establishment and evolution of the classical complement pathway. However, molecular information on the C1 subcomponents is very limited in lower vertebrates. Here we describe two distinct C1r/C1s/MASP2-like cDNA clones (C1r/s-A, C1r/s-B) isolated from the common carp (Cyprinus carpio). They share 83% identity at the amino acid level and have a domain structure similar to that of C1r/C1s/MASPs from other species. The serine protease domain of the carp homologues lacks the histidine loop and is encoded by a single exon containing an AGY codon for the active serine residue, as in mammalian C1r, C1s, and MASP2. Southern blot and PCR analyses indicated that the carp has at least three copies of the C1r/s-A gene and a single C1r/s-B gene. Although phylogenetic tree analysis does not definitively assign carp C1r/s-A and C1r/s-B, they might represent ancestral molecules which later diverged into C1r, C1s, and MASP2 of higher vertebrates.     

Detection of biomolecular interaction between biotin and streptavidin on a self-assembled monolayer using magnetic nanoparticles

For developing a magnetic bioassay system, an investigation to determine the presence of a specific biomolecular interaction between biotin and streptavidin was done using magnetic nanoparticles and a silicon substrate with a self-assembled monolayer. Streptavidin was immobilized on the magnetic particles, and biotin was attached to the monolayer-modified substrate. The reaction of streptavidin-modified magnetic particles on the biotin-modified substrate was clearly observed under an optical microscope. The magnetic signals from the particles were detected using a magnetic force microscope. The results of this study demonstrate that the combination of a monolayer-modified substrate with biomolecule-modified magnetic particles is useful for detecting biomolecular interactions in medical and diagnostic analyses.     

Cancer prevention by antioxidants

Various antioxidants in foods, such as phenolic compounds and carotenoids, were proven to have anticarcinogenic activity. In the case of carotenoids, the mixture of them was found to be very effective. In fact, the development of hepatoma in the high risk group of liver cancer, was significantly suppressed by the treatment with natural carotenoids mixture. The role of nitric oxide (NO) in carcinogenesis has been pointed out, since large quantity of NO has been detected in cancer tissues, and the expression of inducible NO synthase (iNOS) was found to correlate with tumor growth and metastasis. Recently, we found that NO possessed tumor initiating activity in mouse skin carcinogenesis. It has been suggested that some parts of pathological effects induced by NO may depend on peroxynitrite, an active metabolite of NO. Thus, we accessed the tumor initiating activity of peroxynitrite, and found that treatment with peroxynitrite (initiator) plus TPA (promoter) resulted in the formation of skin tumors. Under this experimental condition, it has been proven that natural antioxidants, such as curcumin and nobiletin, showed anti-tumor initiating effect. In the case of nobiletin, suppressive effect on iNOS induction has also been demonstrated. It is of interest that suppression of iNOS induction was also observed in phytoene synthase transgenic mouse. After administration of glycerol (a lung tumor promoter), lower induction of iNOS gene was observed in lung of the phytoene producing mice, comparing with that of control mice. Combinational use of various kinds of antioxidants distributed in foods, e.g., mixture of carotenoids and flavonoids, seems to be effective methods for cancer prevention.     

High efficiency electrochemical immuno sensors using 3D comb electrodes

To realize highly sensitive electrochemical immunoassays, a micro-fabricated three-dimensional (3D) electrode was fabricated and applied to enzyme immuno assay based on production of a redox species. The dimensions of the electrodes are 10 microm in width and 30 microm in height, with 20 microm spacing in between, and the 30 pairs of anode and cathode electrodes made up a single sensor. This structure lead to enhancement of the electrochemical reaction, nearly 100% of trap ratio of redox species. It can be applied to highly sensitive enzyme immuno sensing based on p-aminophenylphosphate (PAPP). Applicability of this technique to the immuno assay for one of the clinical diagnostic marker proteins (alpha-fetoprotein; AFP) from 6 to 500 ng/mL was demonstrated.     

MASTDISCS combi Carba plus,a simple method for discriminating carbapenemase‐producing Enterobacteriaceae,including OXA‐48‐type producers

Accurate and rapid detection of carbapenemases and identification of their types in Enterobacteriaceae are both still major challenges for clinical laboratories in attempting to prevent the intrusion and transmission of carbapenemase‐producing Enterobacteriaceae. This study aimed to evaluate the performance of the MASTDISCS combi Carba plus disc system in identification of different carbapenemase types, including OXA‐48‐type carbapenemase, for which no specific enzyme inhibitors have so far been available. The simple disc system discriminates carbapenemases, including OXA‐48‐types exhibiting low carbapenem minimum inhibitory concentrations, by targeting Enterobacteriaceae isolates with a EUCAST meropenem screening cut‐off of ≥0.25 mg/L.     

A cyto-embryological study of gastrulation in the sand dollar, Scaphechinus mirabilis

Processes of gastrulation in the sand dollar Scaphechinus mirabilis were compared with those in the sea urchin Hemicentrotus pulcherrimus , which seemed to show a typical pattern of gastrulation. Measurement of the archenteron length clearly demonstrated that invagination processes in H. pulcherrimus are divided into two phases, the primary and secondary invagination. On the other hand, invagination in S. mirabilis was revealed to continue at a constant rate. To see the movement of cells during gastrulation, embryos were labeled with Nile blue. In H. pulcherrimus embryos, labeled cells were observed along the full length of the archenteron, if the embryos had been labeled before and during the primary invagination. Labeled cells were never observed in the embryos stained after the primary invagination. In contrast, labeled cells were always discerned at the basal part of the archenteron in S. mirabilis , even if the embryos were stained after invagination had undergone considerable progress. The number of cells in the archenteron of S. mirabilis embryos increased with the advancement of gastrulation, while the numbers were almost constant in H. pulcherrimus . These results suggest that the cellular basis of gastrulation in S. mirabilis is quite different from that in well-known species of sea urchins.