No abstract

Gonadotropin (GTH)-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that inhibits GTH secretion in mammals and birds by acting on gonadotropes and GnRH neurons within the hypothalamic-pituitary-gonadal axis. GnIH and its orthologs that have an LPXRFamide (X = L or Q) motif at the C terminus (LPXRFamide peptides) have been identified in representative species of gnathostomes. However, the identity of an LPXRFamide peptide had yet to be identified in agnathans, the most ancient lineage of vertebrates, leaving open the question of the evolutionary origin of GnIH and its ancestral function(s). In this study, we identified an LPXRFamide peptide gene encoding three peptides (LPXRFa-1a, LPXRFa-1b, and LPXRFa-2) from the brain of sea lamprey by synteny analysis and cDNA cloning, and the mature peptides by immunoaffinity purification and mass spectrometry. The expression of lamprey LPXRFamide peptide precursor mRNA was localized in the brain and gonad by RT-PCR and in the hypothalamus by in situ hybridization. Immunohistochemistry showed appositions of lamprey LPXRFamide peptide immunoreactive fibers in close proximity to GnRH-III neurons, suggesting that lamprey LPXRFamide peptides act on GnRH-III neurons. In addition, lamprey LPXRFa-2 stimulated the expression of lamprey GnRH-III protein in the hypothalamus and GTHβ mRNA expression in the pituitary. Synteny and phylogenetic analyses suggest that the LPXRFamide peptide gene diverged from a common ancestral gene likely through gene duplication in the basal vertebrates. These results suggest that one ancestral function of LPXRFamide peptides may be stimulatory compared with the inhibitory function seen in later-evolved vertebrates (birds and mammals).

We developed a quantum mechanical/molecular mechanical (QM/MM) free energy geometry optimization method by which the geometry of a quantum chemically treated (QM) molecule is optimized on a free energy surface defined with thermal distribution of the surrounding molecular environment obtained by molecular dynamics simulation with a molecular mechanics (MM) force field. The method called QM/MM reweighting free energy self-consistent field combines a mean field theory of QM/MM free energy geometry optimization developed by Yamamoto (Yamamoto, T. J. Chem. Phys.2008, 129, 244104) with a reweighting scheme for updating the MM distribution introduced by Hu et al. (Hu, H., et al. J. Chem. Phys.2008, 128, 034105) and features high computational efficiency suitable for exploring the reaction free energy surface of extensive protein conformational space. The computational efficiency with improved treatment of a long-range electrostatic (ES) interaction using the Ewald summation technique permits one to take into account global conformational relaxation of an entire protein of an enzyme in the free energy geometry optimization of its reaction center. We applied the method to an enzymatic reaction of a substrate complex of psychrophilic α-amylase from Antarctic bacterium Pseudoalteromonas haloplanktis and succeeded in geometry optimizations of the reactant and the product of the catalytic reaction that involve large conformational changes of protein loops adjacent to the reaction center on time scales reaching sub-microseconds. We found that the adjacent loops in the reactant and the product form in different conformations and produce catalytic ES potentials on the reaction center.

Conformational flexibility of proteins provides enzymes with high catalytic activity. Although the conformational flexibility is known to be pivotal for the ligand binding and release, its role in the chemical reaction process of the reactive substrate remains unclear. We determined a transition state of an enzymatic reaction in a psychrophilic α-amylase by a hybrid molecular simulation that allows one to identify the optimal chemical state in an extensive conformational ensemble of protein. The molecular simulation uncovered that formation of the reaction transition state accompanies a large and slow movement of a loop adjacent to the catalytic site. Free energy calculations revealed that, although catalytic electrostatic potentials on the reactive moiety are formed by local and fast reorganization around the catalytic site, reorganization of the large and slow movement of the loop significantly contributes to reduction of the free energy barrier by stabilizing the local reorganization.

The metastatic potency of NPC tissue and the prognosis of the patients with NPC can be estimated by measuring MVD and the expression of VEGF in NPC tissue. Drugs that have inhibitory actions on angiogenesis could be useful to prevent metastasis of NPC cells in the patients.

A new high resolution PET scanner dedicated to animal studies has been designed, built and tested. The system utilizes 240 block detectors, each of which consists of a new compact position-sensitive photomultiplier tube (PS-PMT) and an 8 x 4 BGO array. A total number of 7,680 crystals (480 per ring) are positioned to form a 508 mm diameter of 16 detector rings with 7.2 mm pitch and 114 mm axial field of view (FOV).The system is designed to perform activation studies using a monkey in a sitting position. The data can be acquired in either 2D or 3D mode, where the slice collimators are retracted in 3D mode. The transaxial resolution is 2.6 mm FWHM at the center of the FOV, and the average axial resolution on the axis of the ring is 3.3 mm FWHM in the direct slice and 3.2 mm FWHM in the cross slice. The scatter fraction, sensitivity and count rate performance were evaluated for a 10 cm diameter cylindrical phantom. The total system sensitivity is 2.3 kcps/kBq/ml in 2D mode and 22.8 kcps/kBq/ml in 3D mode. The noise equivalent count rate with 3D mode is equivalent to that with 2D mode at five times higher radioactivity level. The applicable imaging capabilities of the scanner was demonstrated by animal studies with a monkey.

No abstract

To clarify interactions between the cytoskeleton and activity of L-type Ca(2+) (Ca(L)) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca(2+) current [I(Ba(L))] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-alpha-actin and anti-alpha-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I(Ba(L)) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I(Ba(L)) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I(Ba(L)) due to another unknown mechanism, e.g., a direct block of Ca(L) channels. In summary, actin filament disruption of VSM cells inhibits Ca(L) channel activity, whereas disrupting the microtubules does not.