Atomic-Level Dynamical, Structural and Functional Investigation of a Membrane Protein Complex through Nuclear Magnetic Resonance Spectroscopy.

Biological cell membranes are vital boundaries that separate the intracellular elements from the extracellular environments, and they are fundamental regulators of a number of cellular and physiological phenomena. In fact, these biological processes are essentially based on biomolecular interactions within and between cells. A significant number of biologically important protein-protein and protein-lipid interactions in life science, for instance, signal transductions (an essential molecular machinery for sensory systems), electron transport chains (an essential scheme for respiration systems) and photosynthesis (one of our primary sources of energy), all take place at the membrane interface of cells. Furthermore, more than 30% of the human genome and 50% of known drug targets are membrane-associated proteins. Therefore, it is critical to establish techniques that will allow us to investigate these kinds of membrane complex systems and gain insights into biological phenomena for scientific and biomedical purposes. Despite their importance there are very few reports on the atomic-level structure and dynamics investigations of the combinatorial complexes composed of protein-protein interactions and protein-lipid interactions within bilayers. The lack of success in this area of research is largely attributed to the challenges imposed by membrane proteins when examined with the most commonly used biophysical techniques such as X-ray crystallography, electron microscopy and Nuclear Magnetic Resonance (NMR) spectroscopy. My dissertation concentrates on three novel components that will accelerate the understanding of these important and challenging systems. The first element is to gain atomic-level structure and dynamics determination of biologically important systems by developing new solid-state and solution NMR approaches that enhance resolution and the sensitivity of spectra, allowing for the robust NMR experiments for samples that have higher molecular weight. The second component pertains to the preparation of stable and well-behaved biologically relevant samples. The third is to retain the biological functions of membrane complex systems in order to investigate dynamical structure of membrane complexes. The combination of these three approaches has already led, for the first time, to the determination of structural and dynamical interactions in an intact mammalian membrane protein complex, rabbit Cytochrome-b5, and Cytochrome-P450, which metabolizes more than 50% of the pharmaceuticals in clinical use today.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86462/1/kyama_1.pd

Impaired Development of Somatotropes, Lactotropes and Thyrotropes in Growth-Retarded (grt) Mice

Congenitally primary hypothyroid growth-retarded (grt) mice exhibit a characteristic growth pause followed by delayed onset of pubertal growth. We characterized the developmental pattern of somatotropes, lactotropes and thyrotropes in the anterior pituitary, as well as plasma levels of their secretory hormones, in grt mice. Compared with normal mice, the weight of grt pituitary gland was similar at 8 weeks of age but significantly heavier after 12 weeks of age. Compared with normal mice, there were significantly fewer somatotropes in the grt pituitary until 8 weeks of age, but the number gradually increased up to 48 weeks. The number of lactotropes in grt mice was consistently lower than that in normal mice from 2 through 48 weeks, whereas the number of thyrotropes in the grt pituitary was consistently higher than in the normal pituitary. Thyrotropes in the grt pituitary exhibited hypertrophy and hyperplasia with less intensive thyroid-stimulating hormone (TSH) immunoreactivity than normal thyrotropes. In normal mice, the sum of the relative proportions of these cells plateaued at 8 weeks, where it remained up to 48 weeks of age. In grt mice, these proportions almost reached normal levels at 12 weeks of age but gradually declined after 24 weeks. Plasma growth hormone concentrations did not differ between grt and normal mice until 24 weeks of age. Compared with normal mice, grt mice exhibited significantly lower plasma prolactin and thyroxine levels but higher TSH levels. These findings indicate that development of somatotropes, lactotropes and thyrotropes in grt mice is impaired, being followed by altered hormone secretion

Two-Dimensional Band Dispersion of Ultra-Flat Hexagonal Bismuthene Grown on Ag(111) Bulk and Quantum-Well Films

Two-dimensional band dispersion of (2$\times$2) superstructure with Bi grown on Ag(111), which has been urged as an ultraflat hexagonal bismuthene, is investigated using angle-resolved photoemission spectroscopy (ARPES). The (2$\times$2)-Bi superstructure can be grown on the Ag(111) surface at low temperatures; it transforms into a surface alloy with a ($\sqrt{3}\times\sqrt{3}$) superstructure at 300 K. ARPES measurements reveal the consistency with the band structure of ultraflat bismuthene in previous reports. The band structure of (2$\times$2)-Bi surface remains unchanged with decreasing Ag layer thickness, indicating the limited penetration of Bi p-orbitals into the Ag layer.Comment: 6 pages, 4 figure

N,N’-Bis(2-chloroethyl)-N-nitrosourea (BCNU)-induced Apoptosis of Neural Progenitor Cells in the Developing Fetal Rat Brain

N,N’-bis(2-chloroethyl)-N-nitrosourea (BCNU) is one of the major drugs used in chemotherapy against malignant gliomas due to its effects, such as induction of bifunctional alkylation of DNA and formation of interstrand DNA cross-linkages, and induces cortical malformations in the fetal and neonatal rat brain. In this study, pregnant rats were treated with 7.5 mg/kg of BCNU on gestational day 13 (GD 13), and their fetuses were collected from 12 to 72 hours after BCNU treatment in order to examine the timecourses of morphological and immunohistochemical changes in neural progenitor cells in the developing brain. The number of pyknotic cells in the telencephalon peaked at 24 h and then gradually decreased until 72 h. The majority of these pyknotic cells were positive for cleaved caspase-3, a key executioner of apoptosis. The pyknotic cells showed the ultrastructural characteristics of apoptosis. The number of p53-positive cells began to increase prior to the appearance of apoptotic cells and p21-positive cells. The number of phosphorylated-histone H3-positive cells (mitotic cells) decreased from 24 to 36 h. The number of Iba1-positive cells (microglial cells) in the telencephalon increased from 12 to 48 h. These results suggest that BCNU induces p53-dependent apoptosis and reduces proliferative activity, resulting in reduction of the weight of the telencephalon and the thickness of the telencephalic wall in the fetal brain. This study will help to clarify the mechanisms of BCNU-induced fetal brain toxicity

Ghrelin Receptor in Two Species of Anuran Amphibian, Bullfrog (Rana catesbeiana), and Japanese Tree Frog (Hyla japonica)

We have identified cDNA encoding a functional growth hormone secretagogue-receptor 1a (GHS-R1a, ghrelin receptor) in two species of anuran amphibian, the bullfrog (Rana catesbeiana), and the Japanese tree frog (Hyla japonica). Deduced receptor protein for bullfrog and Japanese tree frog (tree frog) was comprised of 374- and 371-amino acids, respectively. The two receptors shared 86% identity, and are grouped to the clade of the tetrapod homologs by phylogenetic analysis. In functional analyses, ghrelin and GHS-R1a agonists increased intracellular Ca2+ concentration in GHS-R1a-transfected-HEK293 cell, but ligand selectivity of ghrelin with Ser3 and Thr3 was not observed between the two receptors. Bullfrog GHS-R1a mRNA was mainly expressed in the brain, stomach, and testis. In the brain, the gene expression was detected in the diencephalon and mesencephalon, but not in the pituitary. Tree frog GHS-R1a mRNA was predominantly expressed in the gastrointestinal tract and ovary, but not detected in the pituitary. In bullfrog stomach but not the brain, GHS-R1a mRNA expression increased after 10 days of fasting. For tree frog, GHS-R1a mRNA expression was increased in the brain, stomach and ventral skin by 10 days of fasting, and in the stomach and ventral skin by a dehydration treatment. Intracerebroventricular injection of ghrelin in dehydrated tree frog did not affect water absorption from the ventral skin. These results suggest that ghrelin is involved in energy homeostasis and possibly in osmoregulation in frogs

Study of Beam Profile Measurement at Interaction Point in International Linear Collider

At the international linear collider, measurement of the beam profile at the interaction point is a key issue to achieve high luminosity. We report a simulation study on a new beam profile monitor, called the pair monitor, which uses the hit distribution of the electron-positron pairs generated at the interaction point. We obtained measurement accuracies of 5.1%, 10.0%, and 4.0% for the horizontal, vertical, and longitudinal beam size, respectively, for 50 bunch crossings.Comment: 13 pages, 8 figures, 2 table

Surgical outcomes in patients with small cell lung cancer: comparative analysis of computed tomograpy-detected patients with others

Background: It is shown that low-dose computed tomography (CT) screening is useful for a reduction in lung-cancer- specific mortality in heavy smokers. However, the information about effectiveness according to the histological types of lung cancer has not been adequately investigated especially small cell lung cancer (SCLC). The present study was performed to see the clinical benefit of CT screening in patients with SCLC following thoracotomy. Methods: We retrospectively reviewed the outcome in patients with early stage SCLC who initially underwent thoracotomy. The clinical stages and actuarial survival were estimated according to the three means of detection of SCLC: chest CT, radiographic screen, and symptomatically prompted cases. Results: Sixty-nine patients (men/women, 63/6; mean age, 70 years) with SCLC underwent thoracotomy between 1991 and 2010 including chest CT (n = 13), radiographic screening (n = 39), and symptomatically prompted cases (n = 17). Pathological staging information included stage IA (n = 25), IB (n = 8), IIA (n = 13), IIB (n = 5), IIIA (n = 11), and IIIB (n = 7). Median survival time was 30.0 (95% confidence interval (CI): 22.0 to 57.0) months, with overall survival at 5 years of 34.3% (95% CI, 23.47 to 47.3). Nine patients (69%) with stage I were detected by CT which was significantly higher than those in other detection arms. However, there were no significant differences in the survival between CT and other detection arms. Conclusions: CT examination may be useful for detection in early stage SCLC potentially suitable for surgery, but the contribution to better clinical outcome in patients with SCLC remains unclear.ArticleWORLD JOURNAL OF SURGICAL ONCOLOGY. 11:61 (2013)journal articl

Distribution and Sequence of Pyknotic Cells in Rat Fetuses Exposed to Busulfan

Busulfan, an antineoplastic bifunctional-alkylating agent, is known to induce developmental anomalies. In the present study, we examined the distribution and sequence of pyknotic cells in rat fetal tissues exposed to busulfan. Pregnant rats on gestation day 13 were administered intraperitoneally 30 mg/kg of busulfan, and fetal tissues were examined at 6, 12, 24, 36, 48, 72 and 96 hours after treatment (HAT). Pyknosis of component cells was observed markedly in the brain, moderately in the eyes and spinal cord and mildly in the craniofacial tissue, mandible, limb buds, tail bud, ganglions, alimentary tract, lungs, kidneys, pancreas and liver. In the brain, mitotic inhibition was also detected. Most of the pyknotic cells were considered to be apoptotic cells judging from the results of TUNEL staining and electron microscopic examination. Commonly in the above-mentioned tissues, pyknotic cells began to increase at 24 HAT, peaked at 36 or 48 HAT and disappeared at 96 HAT, which is when the histological picture returned to normal in most tissues except for the brain, spinal cord and eyes. The present study clarified the outline of busulfan-induced apoptosis in rat fetuses