Dynamin Isoforms Decode Action Potential Firing for Synaptic Vesicle Recycling

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.     

A novel,potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.     

Molecular Orbital Study of the Magnetic Properties of Pyrazine- and Pyrimidine-Bridged Copper(II) Complexes

Magnetic interactions in the three copper(II)-complex polymers, [Cu(PZ)(NO₃)₂]_n, [Cu(PM)(NO₃)₂(H₂O)₂]_n, and [Cu(PM)₂(NO₃)₂]_n are discussed on the basis of extended Hückel calculations inthe formulas PZ and PM stand for pyrazine and pyrimidine, respectively. Interactions between the Cu-3d orbitals and the lone-pair orbitals of pyrazine and pyrimidine are analyzed from the viewpoint of `through-space' and `through-bond' interactions using binuclear complexes to model the three copper(II) polymers. Three conclusions can be drawn from the orbital interaction analysis: (1) in the first polymer, a superexchange pathway is formed with the bond of Cu–-N and the through-bond interaction between the lone pairs of the nitrogen atoms of pyrazine will lead to an antiferromagnet state; (2) in the second polymer a superexchange pathway is formed with the bond of Cu–-N and the through-space interaction between the lone pairs of the nitrogen atoms of pyrimidine, and as a result an antiferromagnetic state will be preferred; and (3) in the third polymer., there is no effective pathway in respect of overlap interaction and the HOMO and the LUMO are actually degenerate, and thus a ferromagnetic state will arise. The band structures are analyzed to characterize the magnetic properties of the antiferromagnetic polymers, [Cu(PZ)(NO₃)₂]_n and [Cu(PM)(NO₃)₂(H₂O)₂], and the ferromagnetic polymer, [Cu(PM)₂(NO₃)₂]_n.     

Over-expression of the testis-specific gene TSGA10 in cancers and its immunogenicity

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real-time RT-PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over-expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.     

Relative biological effectiveness and tolerance dose of fission neutrons in canine skin for a potential combination of neutron capture therapy and fast-neutron therapy

To investigate the potential efficacy of fission neutrons from a fast-neutron reactor for the treatment of radioresistant tumors, the relative biological effectiveness (RBE) and tolerance dose of fission neutrons in canine skin were determined. The forelimbs of 34 healthy mongrel dogs received a single dose of fission neutrons (5.6, 6.8, 8.2, 9.6 or 11 Gy) or 137Cs gamma rays (10, 15, 20, 25 or 30 Gy). Based on observations of radiodermatitis for each radiation, the single-fraction RBE of fission neutrons in the sixth month was calculated as approximately 3. The tolerance doses of fission neutrons and gamma rays, defined as the highest doses giving no moist desquamation on the irradiated skin in the recovery phase, were estimated as 7.6 Gy and 20 Gy, respectively. The tolerance dose of 7.6 Gy of fission neutrons included 5.0 Gy of fast neutrons possessing high anti-tumor effects and 1.4 x 10(12) n/cm2 of thermal neutrons, which could be applicable to neutron capture therapy (NCT). The combination of fast-neutron therapy and NCT using a fast-neutron reactor might be useful for the treatment of radioresistant tumors.     

Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro

The developmental capacity of reconstructed bovine oocytes that contained nuclei from growing stage oocytes, 70-119 microm in diameter, was assessed after fertilization in vitro. Nuclei from growing stage oocytes of adult ovaries were transferred to enucleated, fully grown germinal vesicle (GV) stage oocytes. After culture in vitro, the reconstructed oocytes matured, forming the first polar body and MII plate. To supply the ability to form pronuclei, the resultant MII plate was transferred to enucleated MII oocytes, which were obtained by in vitro culture of cumulus-oocyte complexes. After fertilization in vitro, 11-15% of the reconstructed oocytes developed to morulae and blastocysts. To assess the ability to develop to term, a total of 27 late morulae and blastocysts were transferred to 19 recipient cows. Of the three cows that subsequently became pregnant, one recipient, who received two embryos derived from reconstructed oocytes with a nucleus from oocytes 100 to 109 microm in diameter, continued the pregnancy to Day 278 of gestation. This pregnancy, however, was unexpectedly a triplet pregnancy that included a set of identical twins and resulted in the premature birth of the calves, followed by death from lack of post-parturient treatment. These results show that bovine oocyte genomes are capable of supporting term development before the oocytes grow to their full size, which suggests that growing stage oocytes can be directly used as a source of maternal genomes.     

Evolutionary aspects of gobioid fishes based upon a phylogenetic analysis of mitochondrial cytochrome B genes

The Gobioidei is a large suborder in the order Perciformes and consists of more than 2000 species belonging to about 270 genera. The vast number of species and their morphological specialization adapted to diverse habits and habitats makes the classification of the gobioid fishes very difficult.A comprehensive estimation of the evolutionary scenario of all gobioid fishes using only morphological information is difficult for two major reasons: first, in addition to wide ecological diversification, there is a trend towards specialization and degeneration of morphological characters among these species; second, an appropriate outgroup of gobioid fishes has not been recognized.Based upon nucleotide sequence comparisons of gobioid mitochondrial cytochrome b genes, we established the phylogenetic relationships of their differentiation into many groups of morphological and ecological diversity. The phylogenetic trees obtained show that most species examined have diverged from each other almost simultaneously or during an extremely short period of time.     

Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

Photoautotrophic bacteria have developed mechanisms to maintain K⁺ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K⁺ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K⁺ (Kdp). The contributions of each of these K⁺ transport systems to cellular K⁺ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K⁺ uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K⁺ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K⁺ depletion. Correspondingly, Kdp-mediated K⁺ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K⁺ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K⁺ concentration under conditions of limited K⁺ in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K⁺ availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K⁺ levels under fluctuating environmental conditions.     

Editor's choice: Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging

The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics.