Expression, characterization, and gene knockdown of zebrafish doublecortin-like protein kinase

Doublecortin-like protein kinase (DCLK) is a protein Ser/Thr kinase expressed in brain and believed to play crucial roles in neuronal development. To investigate the biological significance of DCLK, we isolated cDNA clones for zebrafish DCLK (zDCLK) and found that there were five splice variants of the kinase. In this study, the catalytic properties of a major isoform of zDCLK, which we designated as zDCLK1, and of an N-terminal truncated mutant retaining the kinase domain were examined by expressing them in Escherichia coli. Mutational analysis of recombinant zDCLK suggested that the kinase was activated not only by phosphorylation at Thr-576 in the activation loop but also by autophosphorylation at the other site(s) in the catalytic domain. zDCLK significantly phosphorylated protein substrates such as myelin basic protein, histones, and synapsin I. Subcellular localization of zDCLK and its N-terminal deletion mutant implicated that microtubule-association of zDCLK is mediated through N-terminal doublecortin like domain of this enzyme. Western blotting analysis and whole mount in situ hybridization revealed that zDCLK was highly expressed in brain and eyes after 24-h post fertilization. Gene knockdown of zDCLK using morpholino-based antisense oligonucleotides induced significant increase of apoptotic cells in the central nervous systems and resulted in the increase of the morphologically abnormal embryos in a dose-dependent manner. These results suggest that zDCLK may play crucial roles in the central nervous systems during the early stage of embryogenesis.     

Inhibition of gut pacemaker cell formation from mouse ES cells by the c-kit inhibitor

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a "gut", from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified. By adding Glivec 10(-5)M, a tyrosine kinase receptor c-kit inhibitor, only during EB formation, we for the first time succeeded in suppressing in vitro formation of ICC in the ES gut. The ES gut without ICC did not exhibit any movements. However, it appeared that Glivec 10(-6)-10(-7)M rather increased number of ES guts with spontaneous movements associated with increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). These results suggest ICC is critical for in vitro formation of ES guts with spontaneous movements.     

Heart slice NMR

Nuclear magnetic resonance (NMR) spectroscopy of the heart is normally carried out using whole heart preparations under coronary perfusion. In such preparations, either radical changes in ionic composition of the perfusate or applications of numerous drugs would affect coronary microcirculation. This report communicates the first (31)P NMR spectroscopy study using a heart slice preparation (left ventricular slices) superfused with extracellular medium. The ratio of phosphocreatine concentration to ATP concentration was approximately 2.1. Also, intracellular pH and Mg(2+) concentration ([Mg(2+)](i)), estimated from the chemical shifts of inorganic phosphate and ATP, were comparable with those under retrograde perfusion. [Mg(2+)](i) was significantly increased by the removal of extracellular Na(+), supporting the essential role of Na(+)-coupled Mg(2+) transport in Mg(2+) homeostasis of the heart. Heart slice preparation could also be used to evaluate the potency of cardiac drugs, regardless of their possible effects on coronary microcirculation.     

Zebrafish relatively relaxed mutants have a ryanodine receptor defect, show slow swimming and provide a model of multi-minicore disease

Wild-type zebrafish embryos swim away in response to tactile stimulation. By contrast, relatively relaxed mutants swim slowly due to weak contractions of trunk muscles. Electrophysiological recordings from muscle showed that output from the CNS was normal in mutants, suggesting a defect in the muscle. Calcium imaging revealed that Ca(2+) transients were reduced in mutant fast muscle. Immunostaining demonstrated that ryanodine and dihydropyridine receptors, which are responsible for Ca(2+) release following membrane depolarization, were severely reduced at transverse-tubule/sarcoplasmic reticulum junctions in mutant fast muscle. Thus, slow swimming is caused by weak muscle contractions due to impaired excitation-contraction coupling. Indeed, most of the ryanodine receptor 1b (ryr1b) mRNA in mutants carried a nonsense mutation that was generated by aberrant splicing due to a DNA insertion in an intron of the ryr1b gene, leading to a hypomorphic condition in relatively relaxed mutants. RYR1 mutations in humans lead to a congenital myopathy, multi-minicore disease (MmD), which is defined by amorphous cores in muscle. Electron micrographs showed minicore structures in mutant fast muscles. Furthermore, following the introduction of antisense morpholino oligonucleotides that restored the normal splicing of ryr1b, swimming was recovered in mutants. These findings suggest that zebrafish relatively relaxed mutants may be useful for understanding the development and physiology of MmD.     

Regulation of prothoracic gland ecdysteroidogenic activity leading to pupal metamorphosis

The prothoracic glands of early last (fifth) instar larvae of the silkworm are inactive with regard to ecdysteroidogenesis and unresponsive to prothoracicotropic hormone (PTTH) [J. Insect Physiol. 31 (1985) 455]. In an attempt to elucidate the hormonal mechanisms that cause the inactivity, we compared the effects of PTTH, dibutyryl cyclic AMP (dbcAMP), a cAMP phosphodiesterase inhibitor (IBMX), juvenile hormone analogue (JHA) and 20-hydroxyecdysone (20E) on secretory activity of the third, fourth and fifth instar glands. Among the factors examined, feedback inhibition by 20E was indicated to be the most likely factor. Inhibition was moderate in the third and early fourth instars while 20E strongly inhibited the glands of middle fourth instar larvae. The inhibitory effect of 20E was reduced by removal of the brain and corpora allata. Once the glands were suppressed by 20E to the degree of exhibiting neither secretory activity nor responsiveness to PTTH, dbcAMP or IBMX did not elicit ecdysone secretion at all. Thus the feedback inhibition may shut down ecdysteroidogenesis although it is obscure whether it affects the intracellular transductory cascade from the PTTH receptor through cAMP. Taken together, this evidence suggests that inactivity of the gland in the early fifth instar is brought about by feedback inhibition of the glands by 20E occurring in the late fourth instar, and that this inactivity is maintained by the juvenile hormone found in the early fifth instar.     

Genomic heterogeneity in a natural archaeal population suggests a model of tRNA gene disruption

Understanding the mechanistic basis of the disruption of tRNA genes, as manifested in the intron-containing and split tRNAs found in Archaea, will provide considerable insight into the evolution of the tRNA molecule. However, the evolutionary processes underlying these disruptions have not yet been identified. Previously, a composite genome of the deep-branching archaeon Caldiarchaeum subterraneum was reconstructed from a community genomic library prepared from a C. subterraneum-dominated microbial mat. Here, exploration of tRNA genes from the library reveals that there are at least three types of heterogeneity at the tRNA(Thr)(GGU) gene locus in the Caldiarchaeum population. All three involve intronic gain and splitting of the tRNA gene. Of two fosmid clones found that encode tRNA(Thr)(GGU), one (tRNA(Thr-I)) contains a single intron, whereas another (tRNA(Thr-II)) contains two introns. Notably, in the clone possessing tRNA(Thr-II), a 5' fragment of the tRNA(Thr-I) (tRNA(Thr-F)) gene was observed 1.8-kb upstream of tRNA(Thr-II). The composite genome contains both tRNA(Thr-II) and tRNA(Thr-F), although the loci are >500 kb apart. Given that the 1.8-kb sequence flanked by tRNA(Thr-F) and tRNA(Thr-II) is predicted to encode a DNA recombinase and occurs in six regions of the composite genome, it may be a transposable element. Furthermore, its dinucleotide composition is most similar to that of the pNOB8-type plasmid, which is known to integrate into archaeal tRNA genes. Based on these results, we propose that the gain of the tRNA intron and the scattering of the tRNA fragment occurred within a short time frame via the integration and recombination of a mobile genetic element.     

Morphological and functional characterization of a novel Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase-immunoreactive,follicle-like structure on the gill septum of Japanese banded houndshark, <Emphasis Type="Italic">Triakis scyllium</Emphasis>

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranch fish, however, the ionoregulatory function of the gills is still poorly understood. Although mitochondria-rich (MR) cells have also been found in elasmobranch fish, these cells are considered to function primarily in acid-base regulation. In this study, we found a novel aggregate structure made up of cells with basolaterally-expressed Na⁺/K⁺-ATPase (NKA), in addition to NKA-immunoreactive MR cells that have already been described in the gill filament and lamella. The cell aggregates, named follicularly-arranged NKA-rich cells (follicular NRCs), were found exclusively in the epithelial lining of the venous web in the cavernous region of the filament and the inter-filamental space of the gill septum. The follicular NRCs form a single-layered follicular structure with a large lumen leading to the external environment. The follicular NRCs were characterized by: (i) well-developed microvilli on the apical membrane, (ii) less prominent infoldings of the basolateral membrane and (iii) typical junction structures including deep tight junction between cells. In addition, large numbers of vesicles were observed in the cytoplasm and some of them were fused to the lateral membrane. The follicular NRCs expressed Na⁺/H⁺ exchanger 3 and Ca²⁺ transporter 1. The follicular NRCs thus have the characteristics of absorptive ionoregulatory cells and this suggests that the elasmobranch gill probably contributes more importantly to body fluid homeostasis than previously thought.