Immunohistochemical Localization of Manserin,a Novel Neuropeptide Derived from Secretogranin II,in Rat Adrenal Gland,and its Upregulation by Physical Stress

We recently identified a novel 40-amino acid neuropeptide designated manserin from the rat brain (Yajima in NeuroReport 15: 1755–1759, 2004). Manserin is highly expressed in pituitary and hypothalamic nuclei, which suggests that it plays a role in the endocrine system. In this study, we employed immunohistochemical methods to investigate the presence of manserin in rat adrenal glands, as well as its regulation by physical stress. Immunohistochemical analysis using anti-manserin antibody showed that manserin is present in the rat adrenal medulla but not in the cortex. When the colocalization of manserin and phenylethanolamine N-methyltransferase (PNMT), an epinephrine-synthesizing enzyme, was examined, virtually all PNMT-positive cells expressed manserin. Interestingly, the immunoreactivity of manserin was significantly increased when the rats were exposed to water-immersion restraint stress. These results demonstrate for the first time that adrenal manserin, a novel neuropeptide, may have a potential physiological role under stress-inducing conditions.     

Tissue‐dependent induction of apoptosis by matrix metalloproteinase stromelysin‐3 during amphibian metamorphosis

Matrix metalloproteinases (MMPs) are a superfamily of Zn²⁺‐dependent proteases that are capable of cleaving the proteinaceous component of the extracellular matrix (ECM). The ECM is a critical medium for cell–cell interactions and can also directly signal cells through cell surface ECM receptors, such as integrins. In addition, many growth factors and signaling molecules are stored in the ECM. Thus, ECM remodeling and/or degradation by MMPs are expected to affect cell fate and behavior during many developmental and pathological processes. Numerous studies have shown that the expression of MMP mRNAs and proteins associates tightly with diverse developmental and pathological processes, such as tumor metastasis and mammary gland involution. In vivo evidence to support the roles of MMPs in these processes has been much harder to get. Here, we will review some of our studies on MMP11, or stromelysin‐3, during the thyroid hormone‐dependent amphibian metamorphosis, a process that resembles the so‐called postembryonic development in mammals (from a few months before to several months after birth in humans when organ growth and maturation take place). Our investigations demonstrate that stromelysin‐3 controls apoptosis in different tissues via at least two distinct mechanisms. Birth Defects Research (Part C) 90:55–66, 2010. © 2010 Wiley‐Liss, Inc.     

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.     

Increases of Calcium,Phosphorus, and Magnesium in Both the Right and Left Fibrous Trigones of Human Heart with Aging

We previously reported that reduced platelet endogenous antioxidant enzymes activities are related to the low plasma zinc level in patients with end-stage renal failure (ESRF). In this study, we attempt to evaluate whether dietary zinc deprivation reduces the activities of endogenous antioxidant and then enhances oxidative stress in the unstimulated platelet of normal and 5/6 nephrectomized (Nx) rats because increased platelet oxidative stress is suggested to involve in the incidence of thrombotic and atherosclerotic diseases. Male Sprague–Dawley rats (n = 48) were fed a zinc-deficient diet and deionized distilled water for 1 week to induce reduction of plasma zinc level. Half of the rats continued on this diet for 4 weeks as zinc-deplete group, and the other half were maintained on the same diet but with zinc-supplemented water (120 mg/L zinc sulfate solution) to correct the reduction of plasma zinc level as zinc-replete group. Half of each group underwent 5/6 Nx, while the other half underwent sham operation. Another 12 normal rats were fed standard rat chow (containing 23.4% protein and 50 ppm zinc) and drank deionized distilled water as normal control rats. In zinc-deplete rats including sham-operated and 5/6 Nx rats exhibited lower endogenous antioxidant enzymes activities such as reduced glutathione (GSH), superoxide dismutase (SOD), and glutathione peroxidase (GPX) and higher malondialdehyde (MDA) levels than normal control rats in the unstimulated platelets. However, in zinc-replete rats including sham-operated and 5/6 Nx rats have a normal endogenous antioxidant enzymes activity and normal MDA levels in the unstimulated platelets. We suggest that in uremia, the low plasma zinc level may be a risk factor for thrombotic and atherosclerotic diseases because it reduces the activities of endogenous antioxidant enzymes and increases oxidative stress in the unstimulated platelet. Supported by grant 92-117 from Taipei Veterans General Hospital     

Single molecule FRET for the study on structural dynamics of biomolecules

Single molecule fluorescence resonance energy transfer (FRET) is the technique that has been developed by combining FRET measurement and single molecule fluorescence imaging. This technique allows us to measure the dynamic changes of the interaction and structures of biomolecules. In this study, the validity of the method was tested using fluorescence dyes on double stranded DNA molecules as a rigid spacer. FRET signals from double stranded DNA molecules were stable and their average FRET values provided the distance between the donor and acceptor in agreement with B-DNA type helix model. Next, the single molecule FRET method was applied to the studies on the dynamic structure of Ras, a signaling protein. The data showed that Ras has multiple conformational states and undergoes transition between them. This study on the dynamic conformation of Ras provided a clue for understanding the molecular mechanism of cell signaling switches.     

Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone--a potent NR2B-selective N-methyl D-aspartate (NMDA) antagonist for the treatment of pain

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index=4200 vs NR2B binding IC(50)). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.     

Novel scaffold for cathepsin K inhibitors

Pyrrolopyrimidine, a novel scaffold, allows to adjust interactions within the S3 subsite of cathepsin K. The core intermediate 10 facilitated the P3 optimization and identified highly potent and selective cathepsin K inhibitors 11-20.     

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.