Molecular and functional characterization of organic cation/carnitine transporter family in mice

Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of carnitine leads to fatal systemic carnitine deficiency. We have already shown that a defect of the organic cation/carnitine transporter OCTN2 is a primary cause of systemic carnitine deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for carnitine and significantly lower carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of carnitine transport activity and may have a different role from other members of the OCTN family.     

Potential of the mite-pathogenic fungus Neozygites floridana (Entomophthorales: Neozygitaceae) for control of the cassava green mite Mononychellus tanajoa (Acari: Tetranychidae)

The cassava green mite, Mononychellus tanajoa (Bondar), is an exotic pest in Africa and is the target of a classical biological control programme. Field data from the Neotropics, where it is indigenous, are presented for the first time, charting the variation in abundance of M. tanajoa over several seasons. This was highly variable, with a characteristic trough mid-year and a peak at the turn of the year. This pattern corresponded positively with rainfall levels, appearing to fit a phenology also characteristic of African studies, where rainfall at the start of the wet season promotes a leaf flush and so growth in M. tanajoa populations. Analyses implied some impact of leaf-inhabiting predatory mites (predominantly Neoseiulus idaeus Denmark & Muma) and a considerable impact of the fungal pathogen Neozygites floridana Fisher on M. tanajoa populations. This pathogen was not observed in the host population for several (generally dry) periods implying survival outside the host, perhaps as resting spores. This is a particularly desirable characteristic of a biological control agent. It is therefore proposed that N. floridana might be of particular use in drier cassava-growing areas where rainfall at the outset of the wet season is not sufficiently intense to cause heavy M. tanajoa mortality but may be sufficient to stimulate epizootics of the fungal pathogen, protecting the flush of new cassava growth.     

The synthesis and role of taurine in the Japanese eel testis

In teleost fish, the progestin 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is an essential component of the spermatogenesis pathway. In a series of investigations on the mechanisms underlying progestin-stimulated spermatogenesis, we have found that DHP up-regulates the expression of cysteine dioxygenase1 (CDO1) in the Japanese eel testis. CDO1 is one of the enzymes involved in the taurine biosynthesis pathway. To evaluate whether taurine is synthesized in the eel testis, cysteine sulfinate decarboxylase (CSD), another enzyme involved in taurine synthesis, was isolated from this species. RT-PCR and in vitro eel testicular culture revealed that although CSD was also expressed in eel testis, neither DHP nor other sex steroids affect CSD mRNA expression in a similar manner to CDO1. Using an in vitro eel testicular culture system, we further investigated the effects of DHP on taurine synthesis in the eel testis. HPLC analysis showed that DHP treatment significantly increases the taurine levels in the eel testis. These results suggest that DHP promotes taurine synthesis via the up-regulation of CDO1 mRNA expression during eel spermatogenesis. Furthermore, we observed from our analysis that although taurine does not induce complete spermatogenesis, it promotes spermatogonial DNA synthesis and the expression of Spo11, a meiosis-specific marker. These data thus suggest that taurine augments the effects of sex steroids in the promotion of spermatogonial proliferation and/or meiosis and hence that taurine plays important roles in spermatogenesis.     

Synthesis of [¹¹C]uric acid, using [¹¹C]phosgene, as a possible biomarker in PET imaging for diagnosis of gout

The synthesis and in vivo evaluation of (11)C -labeled uric acid ([(11)C]1), a potential imaging agent for the diagnosis of urate-related life-style diseases, was performed using positron emission tomography (PET) image analysis. First, the synthesis of [(11)C]1 was achieved by reacting 5,6-diaminouracil (2) with (11)C-labeled phosgene ([(11)C]COCl(2)). The radiochemical yield of [(11)C]1 was 37±7% (decay-corrected based on [(11)C]COCl(2)) with specific radioactivities of 96-152GBq/μmol at the end of synthesis (n=6). The average time of radiosynthesis from the end of bombardment, including formulation, was about 30min with >98% radiochemical purity. Second, the synthetic approach to [(11)C]1 was optimized using 5,6-diaminouracil sulfate (3) with [(11)C]COCl(2) in the presence of 1,8-bis(dimethylamino)naphthalene. [(11)C]1 was synthesized in 36±6% radiochemical yield, 89-142GBq/μmol of specific radioactivities, and 98% radiochemical purity by this method (n=5). This allowed the synthesis of [(11)C]1 to be carried out repeatedly and the radiochemical yield, specific radioactivities, average time of synthesis, and radiochemical purity of [(11)C]1 were similar to those obtained using 2. PET studies in rats showed large differences in the accumulation of radioligand in the limbs under normal and hyperuricemic conditions. Thus, an efficient and convenient automated synthesis of [(11)C]1 has been developed, and preliminary PET evaluation of [(11)C]1 confirmed the increased accumulation of radioactivity in the limbs of a rat model of hyperuricemia.     

Metabolic impact of overexpression of liver glycogen synthase with serine-to-alanine substitutions in rat primary hepatocytes

To investigate the effect of elevation of liver glycogen synthase (GYS2) activity on glucose and glycogen metabolism, we performed adenoviral overexpression of the mutant GYS2 with six serine-to-alanine substitutions in rat primary hepatocytes. Cell-free assays demonstrated that the serine-to-alanine substitutions caused constitutive activity and electrophoretic mobility shift. In rat primary hepatocytes, overexpression of the mutant GYS2 significantly reduced glucose production by 40% and dramatically induced glycogen synthesis via the indirect pathway rather than the direct pathway. Thus, we conclude that elevation of glycogen synthase activity has an inhibitory effect on glucose production in hepatocytes by shunting gluconeogenic precursors into glycogen. In addition, although intracellular compartmentation of glucose-6-phosphate (G6P) remains unclear in hepatocytes, our results imply that there are at least two G6P pools via gluconeogenesis and due to glucose phosphorylation, and that G6P via gluconeogenesis is preferentially used for glycogen synthesis in hepatocytes.