The ZW sex chromosomes of <Emphasis Type="Italic">Gekko hokouensis</Emphasis> (Gekkonidae,Squamata) represent highly conserved homology with those of avian species

Populations of the gecko lizard Gekko hokouensis (Gekkonidae, Squamata) on Okinawajima Island and a few other islands of the Ryukyu Archipelago, Japan, have the morphologically differentiated sex chromosomes, the acrocentric Z chromosome and the subtelocentric W chromosome, although the continental representative of this species reportedly shows no sex chromosome heteromorphism. To investigate the origin of sex chromosomes and the process of sex chromosomal differentiation in this species, we molecularly cloned the homologues of six chicken Z-linked genes and mapped them to the metaphase chromosomes of the Okinawajima sample. They were all localized to the Z and W chromosomes in the order ACO1/IREBP–RPS6–DMRT1–CHD1–GHR–ATP5A1, indicating that the origin of ZW chromosomes in G. hokouensis is the same as that in the class Aves, but is different from that in the suborder Ophidia. These results suggest that in reptiles the origin of sex chromosomes varies even within such a small clade as the order Squamata, employing a variety of genetic sex determination. ACO1/IREBP, RPS6, and DMRT1 were located on the Z long arm and the W short arm in the same order, suggesting that multiple rearrangements have occurred in this region of the W chromosome, where genetic differentiation between the Z and W chromosomes has been probably caused by the cessation of meiotic recombination.     

Significance of light-induced hook exaggeration as reinforced by the concomitant anatomical change of germinating tomato seeds

Progression of the apical hook of tomato, Solanum lycopersicum, exaggerated by phytochrome mediation at the early germination stage, is followed in detail macroscopically and anatomically, and its proposed significance, i.e., survival by securing the seed coat release in the field, is reinforced by new findings. Furthermore, after self-release or artificial removal of the seed coat and the endosperm, no hook exaggeration occurs any more. Similar light-induced hook exaggeration (LIHE) is also found in carrot, parsley and Cryptotaenia japonica, which share some seed characteristics with tomato. These findings also support the above-stated significance.Key words: apical hook, carrot, cotyledons, Cryptotaenia japonica, endosperm, field germination, light action, parsley, phytochrome, seed coat, Solanum lycopersicumContrary to many other seed species,^1–3 the apical hook of germinating tomato seeds is exaggerated by red (R) and far-red light (FR) given in a pulse or continuously, mediated by the very low and low fluence responses of phytochromes.⁴ Also, an R high-irradiance response is probably involved.⁴ Based on some simulation experiments for germination in the field, we have proposed that LIHE may play a role, not in breaking through compacted soil surface, but in securing to release the seed coat at some depth of soil in response to light coming through soil gaps.⁴ Here we present additional observations and experimental data not published yet and reinforce the proposed significance of the recently discovered photo-response.     

Input-output analysis of in vivo photoassimilate translocation using Positron-Emitting Tracer Imaging System (PETIS) data

The Positron-Emitting Tracer Imaging System (PETIS) is introduced for monitoring the distribution of (11)C-labelled photoassimilates in Sorghum. The obtained two-dimensional image data were quantitatively analysed using a transfer function analysis approach. While one half of a Sorghum root in a split root system was treated with either 0, 100, or 500 mM NaCl dissolved in the nutrient solution, tracer images of the root halves and the lower stem section were recorded using PETIS. From the observed tracer levels, parameters were estimated, from which the mean speed of tracer transport and the proportion of tracer moved between specified image positions were deduced. Transport speed varied between 0.7 and 1.8 cm min(-1) with the difference depending on which part of the stem was involved. When data were collected in the lowest 0.5-1 cm of the stem, which included the point where the roots emerge, transport speed was less. Rapid changes in NaCl concentration, from 0 to 100 mM, resulted in short-term increases of assimilate import into the treated root. This response represented a transient osmotic effect, that was compensated for in the medium-term by osmotic adaptation. Higher concentrations of NaCl (500 mM) resulted in distinctly less photoassimilate transport into the treated root half. The present results agree with earlier observations, showing that transport of (11)C-labelled photoassimilates measured with the PETIS detector system can be quantified using the method of input-output analysis. It is worth noting that with the PETIS detector system, areas of interest do not need to be defined until after data collection. This means that unexpected behaviour of a plant organ will be seen, which is not necessarily the case with conventional detector systems looking at predefined areas of interest.     

Limited phylogenetic distribution of a long tandem-repeat cluster in the mitochondrial control region in Bubo (Aves,Strigidae) and cluster variation in Blakiston’s fish owl (Bubo blakistoni)

To investigate the phylogenetic position of Blakiston’s fish owl (Bubo blakistoni), we sequenced the mitochondrial (mt) DNA control region and cytochrome b (cyt b) for nine Bubo species. Maximum-likelihood analyses of combined control region and cyt b sequences, and cyt b sequences alone, showed that species formerly placed in genus Ketupa comprise a monophyletic group. Unexpectedly, we discovered a long cluster of 20–25 tandem repeat units 77 or 78 bp long in the third control region domain in four of the nine Bubo species for which the control region was sequenced (B. blakistoni, B. flavipes, and B. ketupu in the Ketupa clade; B. lacteus), leading to overall control region lengths of 3.0–3.8 kpb estimated from agarose gel electrophoresis. The control region in B. lacteus is the longest (3.8 kbp) reported to date in vertebrates. Sequencing of eight repeat units at each end of the cluster in 20 B. blakistoni individuals detected several types of repeat units 77 or 78 bp long, and six patterns in the order of unit types. The occurrence of a repeat cluster in all three species examined in the Ketupa clade suggests their common ancestor also had a cluster, whereas a maximum parsimony tree showed repeat-unit types grouping by species, rather than by paralog groups, suggesting independent origins of the clusters. We reconcile these results with a turnover model, in which the range in cluster-length variation and unit types at the 5′ end are hypothetically functionally constrained by the protein-binding function of the control region, but otherwise there is a continual turnover of units in evolutionary time, with new unit types arising through mutations, proliferating by duplication of single and double repeat blocks, and being lost through deletion. Estimated free energies for reconstructed secondary structures of single and especially pairs of repeat units were higher than for homologous single-unit blocks in species lacking a repeat cluster, supporting slipped-strand mispairing as the mechanism of cluster turnover.     

Purification and identification of activating enzymes of CS-0777, a selective sphingosine 1-phosphate receptor 1 modulator, in erythrocytes

CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ～10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug.     

Identification of an indispensable amino acid for ppGpp synthesis of Escherichia coli SpoT protein

Amino acid substitutions were introduced into a structurally flexible and highly conserved region of Escherichia coli SpoT protein. SpoT protein changed from Asp to Ala at the 293rd position did not restore cell growth of E. coli CF8295 (delta relA, delta spoT) and did not accumulate ppGpp in the cell, suggesting that the Asp293 is indispensable for ppGpp synthesis of the protein.     

Improved fish lymphocyte culture for chromosome preparation

Cytogenetic methodology is still underdeveloped in fishes compared with mammals. Culture condition for fish lymphocytes was optimized to improve chromosome preparation using the rainbow trout (Oncorhynchus mykiss) as a model after changing the combination of parameters such as mitogens, incubation periods, media, cell components, and freshness of blood. The optimized culture condition included isolation of lymphocytes from fresh blood by a stirring method, their culture in medium 199 supplemented with 10% FBS, 18g/ml of phytohemagglutinin (PHA-W) and 100g/ml of lipopolysaccharide (LPS) as mitogens, and harvested at 6 days after culture. This condition provided a notably increased mitotic index (MI) of 4.3–10.0% in rainbow trout lymphocytes. In addition, the condition was highly reproducible as shown by the similar level of MI in cultured lymphocytes from 181 individuals without failure. Applicability of this method in a wide range of fish groups was also proven with MI of 1.1–13.3% in cultured lymphocytes from other 16 freshwater species of Acipenseridae, Anguillidae, Salmonidae, Cyprinidae, and Centrarchidae, and five marine species of Sparidae, Kyphosidae, Paralichthyidae, and Scorpaenidae. Chromosome preparations of improved quality by the present method were successfully applied for the replication R-banding with incorporation of 5-bromo-2-deoxyuridine and direct R-banding fluorescence in situ hybridization.     

Possible involvement of dynorphin A release via mu1-opioid receptor on supraspinal antinociception of endomorphin-2

It has been demonstrated that the antinociception induced by i.t. or i.c.v. administration of endomorphins is mediated through mu-opioid receptors. Moreover, though endomorphins do not have appreciable affinity for kappa-opioid receptors, pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine markedly blocks the antinociception induced by i.c.v.- or i.t.-injected endomorphin-2, but not endomorphin-1. These evidences propose the hypothesis that endomorphin-2 may initially stimulate the mu-opioid receptors, which subsequently induces the release of dynorphins acting on kappa-opioid receptors to produce antinociception. The present study was performed to determine whether the release of dynorphins by i.c.v.-administered endomorphin-2 is mediated through mu-opioid receptors for producing antinociception. Intracerebroventricular pretreatment with an antiserum against dynorphin A, but not dynorphin B or alpha-neo-endorphin, and s.c. pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine dose-dependently attenuated the antinociception induced by i.c.v.-administered endomorphin-2, but not endomorphin-1 and DAMGO. The attenuation of endomorphin-2-induced antinociception by pretreatment with antiserum against dynorphin A or nor-binaltorphimine was dose-dependently eliminated by additional s.c. pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine or a selective mu(1)-opioid receptor antagonist naloxonazine at ultra low doses, which are inactive against mu-opioid receptor agonists in antinociception, suggesting that endomorphin-2 stimulates distinct subclass of mu(1)-opioid receptor that induces the release of dynorphin A acting on kappa-opioid receptors in the brain. It concludes that the antinociception induced by supraspinally administered endomorphin-2 is in part mediated through the release of endogenous kappa-opioid peptide dynorphin A, which is caused by the stimulation of distinct subclass of mu(1)-opioid receptor.