Distribution of Cytoskeletal Components in Endothelial Cells in the Guinea Pig Renal Artery

The cytoskeletal components of endothelial cells in the renal artery were examined by analysis of en face preparations under confocal laser scanning microscopy. Renal arterial endothelial cells were shown to be elongated along the direction of blood flow, while stress fibers ran perpendicular to the flow in the basal portion. Focal adhesions were observed along the stress fibers in dot-like configurations. On the other hand, stress fibers in the apical portion of cells ran along the direction of flow. The localizations of stress fibers and focal adhesions in endothelial cells in the renal artery differed from those of unperturbed aortic and venous endothelial cells. Tyrosine-phosphorylated proteins were mainly detected at the sites of cell-to-cell apposition, but not in focal adhesions. Pulsatile pressure and fluid shear stress applied over endothelial cells in the renal artery induce stress fiber organization and localization of focal adhesions. These observations suggest that the morphological alignment of endothelial cells along the direction of blood flow and the organization of cytoskeletal components are independently regulated

Hypoglycemic activity of Momordica charantia (bitter melon)

Diabetes mellitus (DM) represents a global health and economical problem. Many patients with DM in Asia, South America, India and East Africa have traditionally used the water extract of unripe fruits of Momordica charantia (bitter melon) as some form of complementary and alternative medicine. Studies of laboratory animals have shown the beneficial blood-glucose lowering and anti-diabetic effects of this remedy. Some oral components that bring lower blood glucose level have been isolated : charantin (sterol glycosides), charantin (polypeptide) and cucurbine-type triterpenes. Part of their actions are related to AMP-activated kinase and repression of the oxidative stress that is increased in DM. Most clinical reports are not fully convincing due to the lack of randomized control studies. The present article reviews the pharmacological and clinical effects of bitter melon with special emphasis on the anti-diabetic effects, and some effects that would require caution in the context of human trials

Inhibitory Effect of Fermented Papaya Preparation on Hydroxyl Radical Generation from Methylguanidine

We have previously shown that extremely high level of guanidino compounds such as methylguanidine (MG), known as a neurotoxin and also a nephrotoxin, generate reactive oxygen species (ROS) using an electron spin resonance (ESR) technique with spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In this in vitro study, the inhibitory effect of fermented papaya preparation (SAIDO-PS501:PS-501) on hydroxyl radical (·OH) generation from MG was examined using an ESR spectrometry, and it was found that PS-501 suppressed ·OH generation from MG in a dose-dependent manner. The ID50 value of PS-501 was 8 mg/ml. On the contrary, glucose itself did not suppress ·OH generation from MG up to100 mg/ml, whereas PS-501 almost completely suppressed ·OH generation from MG at a dose of 100 mg/ml. These results imply that PS-501 itself may have a beneficial effect of preventing ROS- and MG-related diseases

Natural antioxidants may prevent posttraumatic epilepsy: a proposal based on experimental animal studies.

Head injury or hemorrhagic cortical infarction results in extravasation of blood and breakdown of red blood cells and hemoglobin. Iron liberated from hemoglobin, and hemoglobin itself, are associated with the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS have been demonstrated to be involved in the mechanism of seizures induced by iron ions in the rat brain, an experimental animal model for posttraumatic epilepsy (PTE). ROS are responsible for the induction for peroxidation of neural lipids, i.e., an injury of neuronal membranes, and also could induce disorders in the excitatory and inhibitory neurotransmitters. Antioxidants, such as a phosphate diester of vitamin E and C (EPC-K1) and antiepileptic zonisamide, have been known to prevent the epileptogenic focus formation, or to attenuate seizure activities in the iron-injected rat brain. Natural antioxidants, such as alpha-tocopherol, and condensed tannins, including (-)-epigallocatechin and (-)-epigallocatechin-3-O-gallate, adenosine and its derivative, melatonin, uyaku (Lindera Strychnifolia), fermented papaya preparations, Gastrodia elata BI., and Guilingji, have been demonstrated to scavenge ROS and/or RNS and to be prophylactic for the occurrence of epileptic discharge in the iron-injected rat brain.</p

The Anticonvulsant Zonisamide Inhibits Hydroxyl Radical Generated from Methylguanidine

Methylguanidine (MG) is a known nephrotoxin and neurotoxin, and an intracisternal injection of MG can induce convulsions in experimental animals. In this in vitro study, we examined the inhibitory effects of the antiepileptic agent zonisamide (ZNS) on hydroxyl radicals (•OH) generated from MG by using an electron spin resonance (ESR) technique. ZNS scavenged •OH generated from MG in a dose-dependent manner through direct scavenging during the auto-oxidation of MG. The rate constant of ZNS reacting with the •OH was at a near diffusion-controlled rate. These findings indicate that ZNS might detoxify MG and could thus protect against convulsive disorders

Examining the effects of additives and precursors on the reactivity of rhodium alkyl nitrenes generated from substituted hydroxylamines

In this study, the reactivity of the alkyl nitrenes, generated from the substituted hydroxylamine precursors, was determined using the same rhodium catalyst. The results revealed that in competitive C–H insertion experiments, the regioselectivity between benzylic and tertiary C–H bonds could be modulated by adding Brønsted acids or changing the substituents on oxygen. This study enhances our understanding of the metallonitrene structures and provides valuable insights for further development of selective N-heterocycle syntheses

Role of KIFC3 motor protein in Golgi positioning and integration

KIFC3, a microtubule (MT) minus end–directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3−/− cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3−/− cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3−/− cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3−/− cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end–directed motors other than KIFC3, such as dynein, in kifC3−/− cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3−/− cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein

KIFC3, a microtubule minus end–directed motor for the apical transport of annexin XIIIb–associated Triton-insoluble membranes

We have identified and characterized a COOH-terminal motor domain–type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end–directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro. Flotation assay, coupled with detergent extraction, demonstrated that KIFC3 is associated with Triton X-100–insoluble membrane organelles, and that it overlaps with apically transported TGN-derived vesicles. This was confirmed by immunoprecipitation and by GST pulldown experiments showing the specific colocalization of KIFC3 and annexin XIIIb, a previously characterized membrane protein for apically transported vesicles (Lafont, F., S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413–1427). Furthermore, we proved that the apical transport of both influenza hemagglutinin and annexin XIIIb was partially inhibited or accelerated by overexpression of motor-domainless (dominant negative) or full-length KIFC3, respectively. Absence of cytoplasmic dynein on these annexin XIIIb–associated vesicles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-driven transport in epithelial cells

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond