Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator.

1. Supernatant fluids from rat cerebral cortex, cerebellum, kidney, heart and liver contained more phosphodiesterase activity hydrolysing cyclic GMP than that hydrolysing cyclic AMP when assayed with sub-saturating concentrations of substrate. 2. These activities were resolved into several fractions by Sephadex G-200 gel filtration; no two tissues had similar activity profiles. 3. With every tissue examined, a fraction (fraction II) with a molecular weight of about 150,000 was obtained which hydrolysed cyclic GMP preferentially at sub-saturating substrate concentrations in the presence of micromolar concentration of Ca2+, millimolar concentration of Mg2+ and a protein activator. 4. The activity of fraction II accounted for about 60 percent in liver, more than 80 percent in heart and cerebellum, and almost 100 percent in cerebral cortex of the total activity for cyclic GMP hydrolysis, calculated from the activity profiles. 5. Km values of fraction II samples from kidney, heart and liver for cyclic GMP were 1.3, 1.7 and 5 muM respectively. 6. 3-Isobutyl-1-methylxanthine inhibited hydrolysis of cyclic GMP by fraction II with an I50 value of 3muM for heart and liver and 50 muM for cerebrum. 7. The activator protein, with an estimated molecular weight of about 30,000 was isolated from all the tissues listed in 1.8. The concentrations of activator protein and of the isolated enzyme, fraction II, did not correspond exactly.     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.     

Regulation of breathing during exercise

This paper reviewed in short neural and humoral factors which might be responsible for inducing exercise hyperpnea. As one of the neural factors afferent signals which arise in the exercising limbs and are transmitted via group III or IV high threshold sensory fibres were involved. The other neural factor is command signals originating in the central nervous system and being fed onto the respiratory center. Hypothalamic locomotor region is assumed to be a possible locus to integrate these peripheral and central neural signals. There are enough evidences to believe that humoral factors mediated via cardiac output is also essential for the hyperpnea. Changes in VCO2 is well correlated with those of VE in dynamic as well as in steady-state response. Oscillations in PaCO2 can be assumed to play a role to link metabolic CO2 changes to those in ventilation. Thus, no single factor can explain the whole process of exercise hyperpnea. Poon's optimization model may give a key to integrate complicated and coflicting experimental results in a unique concept.     

gamma-Ray-induced mutations in male germ cells of a recombination-defective strain (c3G) of Drosophila melanogaster

The gamma-ray (60Co) induction of the dumpy and Minute mutations, and the hyperploid exceptions during spermatogenesis was investigated by using a recombination-defective strain of Drosophila melanogaster, c3G and its wild-type strain, Oregon-R (c3G+). The results show that: (1) no essential difference exists in the response patterns for the dumpy and Minute mutations between these two strains; (2) however, a striking difference exists in the response pattern for the hyperploid exceptions. This is mainly due to an extraordinarily high sensitivity of the early spermatocytes of the c3G males to gamma-ray induction of these mutations. These findings possibly suggest that c3G gene may have some kind of role in the production of large structural changes by ionizing radiation, but not in the production of gene mutations.     

miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210–3p, and miR-224–5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210–3p or miR-224–5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210–3p, and miR-224–5p in sEVs was compared. The amount of miR-33a-5p and miR-210–3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224–5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210–3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.     

Preparation of optically active 4-chlorophenylalanine from its racemate by deracemization technique using transformant Escherichia coli cells

We have developed a method for the preparation of l-4-chlorophenylalanine from its racemate with Escherichia coli cells expressing a single foreign gene. l-4-Chlorophenylalanine was obtained in a high optical yield by the inversion of configuration of its d-form via the tandem reactions catalyzed by d-amino acid dehydrogenase (DadA) and branched-chain amino acid aminotransferase (BCAAT). While we constructed a plasmid for BCAAT utilizing the gene from Sinorhizobium meliloti ATCC 51124, the first enzyme DadA was the dadA-gene product from E. coli host cell itself, which was activated by the addition of l-alanine in the growth medium.     

Functional Analysis of Deep Intronic SNP rs13438494 in Intron 24 of PCLO Gene

The single nucleotide polymorphism (SNP) rs13438494 in intron 24 of PCLO was significantly associated with bipolar disorder in a meta-analysis of genome-wide association studies. In this study, we performed functional minigene analysis and bioinformatics prediction of splicing regulatory sequences to characterize the deep intronic SNP rs13438494. We constructed minigenes with A and C alleles containing exon 24, intron 24, and exon 25 of PCLO to assess the genetic effect of rs13438494 on splicing. We found that the C allele of rs13438494 reduces the splicing efficiency of the PCLO minigene. In addition, prediction analysis of enhancer/silencer motifs using the Human Splice Finder web tool indicated that rs13438494 induces the abrogation or creation of such binding sites. Our results indicate that rs13438494 alters splicing efficiency by creating or disrupting a splicing motif, which functions by binding of splicing regulatory proteins, and may ultimately result in bipolar disorder in affected people.