Effective encapsulation of proteins into size-controlled phospholipid vesicles using freeze-thawing and extrusion

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-l-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 microm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-)(1) and cooling rate of -140 degrees C min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 +/- 20 nm.     

A new alpha,beta,gamma,delta-unsaturated carboxylic acid and three new cyclic peroxides from the marine sponge,Monotria japonica,which selectively lyse starfish oocytes without affecting nuclear morphology

The marine sponge Monotria japonica contains cytolytic constituents, which have been fractionated to a new alpha,beta,gamma,delta-unsaturated carboxylic acid designated monotriajaponide A (1) and three new cyclic peroxides designated monotriajaponides B (2), C (3), and D (4), in addition to a known peroxide (5) and a known alpha,beta-unsaturated ester (6). The structures were determined on the basis of spectroscopic data. Compounds 1-5 lysed immature starfish (Asterina pectinifera) oocytes without affecting nuclear morphology at the minimum effective concentrations of 50, 6.3, 6.3, 6.3, and 13 microg/mL, respectively. On the other hand, compound 6, at the minimum effective concentration of 25 microg/mL, lysed both oocyte's plasma membrane and the nuclear envelope.     

Salusins: newly identified bioactive peptides with hemodynamic and mitogenic activities

The discovery of endogenous bioactive peptides has typically required a lengthy identification process. Computer-assisted analysis of cDNA and genomic DNA sequence information can markedly shorten the process. A bioinformatic analysis of full-length, enriched human cDNA libraries searching for previously unidentified bioactive peptides resulted in the identification and characterization of two related peptides of 28 and 20 amino acids, which we designated salusin-alpha and salusin-beta. Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. Intravenous administration of salusin-alpha or salusin-beta to rats causes rapid, profound hypotension and bradycardia. Salusins increase intracellular Ca2+, upregulate a variety of genes and induce cell mitogenesis. Salusin-beta stimulates the release of arginine-vasopressin from rat pituitary. Expression of TOR2A mRNA and its splicing into preprosalusin are ubiquitous, and immunoreactive salusin-alpha and salusin-beta are detected in many human tissues, plasma and urine, suggesting that salusins are endocrine and/or paracrine factors.     

Molecular mechanism of adaptive cytoprotection induced by ethanol in human gastric cells

Adaptive cytoprotection is the process by which the pretreatment of cells with low concentrations of a noxious agent prevents the damage caused by a subsequent exposure of those cells to higher concentrations of that same agent. In this study, a human gastric carcinoma cell line was used to examine the molecular mechanism of adaptive cytoprotection induced by ethanol. Pretreatment of cells with 1%-4% ethanol made cells resistant to a subsequent exposure to 8% ethanol. This adaptive cytoprotection was accompanied by an increase in prostaglandin E2 synthesis and was partially inhibited by inhibitors of cyclooxygenase-2, but not by an inhibitor of cyclooxygenase-1. Furthermore, the adaptive cytoprotection was not dependent on newly synthesized proteins and was inhibited by a protein tyrosine kinase inhibitor. Based on these results, it is proposed that the stimulation of cyclooxygenase-2-dependent prostaglandin E2 synthesis, which is regulated post-translationally by protein tyrosine phosphorylation, plays an important role in adaptive cytoprotection induced by ethanol in gastric cells.     

Molecular evaluation of phylogenetic significances in the highly divergent karyotypes of the genus Gonocephalus (Reptilia: Agamidae) from tropical Asia

The Oriental large-bodied crested dragons of the genus Gonocephalus are known to include two distinct karyomorphs. To evaluate their phylogenetic significances, we conducted phylogenetic analyses of the genus together with other agamid genera on the basis of 862 base positions of mitochondrial 12S and 16S rRNA genes. Results suggested the presence of two distinct lineages within Gonocephalus, of which one, represented by G. robinsonii that has a 2n = 32 karyotype, was closer to other Oriental agamid genera than to the other congeneric lineage. Monophyly of the latter, characterized by unique chromosomal arrangement among agamid genera (2n = 42 karyotype), was confirmed. It is thus likely that states of morphological characters shared between the two lineages are derived through convergence, or represent symplesiomorphy. Our results also suggest that the karyological similarity between G. robinsonii and several Australian agamids, pointed out in a previous study, is actually attributable to homoplasy rather than their recent common ancestry.     

Systemic and microvascular responses to hemorrhagic shock and resuscitation with Hb vesicles

A phospholipid vesicle encapsulating hemoglobin (Hb vesicle, HbV) has been developed to provide O(2)-carrying capacity to plasma expanders. Its ability to restore systemic and microcirculatory conditions after hemorrhagic shock was evaluated in the dorsal skinfold window preparation of conscious hamsters. The HbV was suspended in 8% human serum albumin (HSA) at Hb concentrations of 3.8 g/dl [HbV(3.8)/HSA] and 7.6 g/dl [HbV(7.6)/HSA]. Shock was induced by 50% blood withdrawal, and mean arterial pressure (MAP) at 40 mmHg was maintained for 1 h by the additional blood withdrawal. The hamsters receiving either HbV(3.8)/HSA or HbV(7.6)/HSA suspensions restored MAP to 93 +/- 14 and 93 +/- 10 mmHg, respectively, similar with those receiving the shed blood (98 +/- 13 mmHg), which were significantly higher by comparison with resuscitation with HSA alone (62 +/- 12 mmHg). Only the HSA group tended to maintain hyperventilation and negative base excess after the resuscitation. Subcutaneous microvascular blood flow reduced to approximately 10-20% of baseline during shock, and reinfusion of shed blood restored blood flow to approximately 60-80% of baseline, an effect primarily due to the sustained constriction of small arteries A(0) (diameter 143 +/- 29 microm). The HbV(3.8)/HSA group had significantly better microvascular blood flow recovery and nonsignificantly better tissue oxygenation than of the HSA group. The recovery of base excess and improved tissue oxygenation appears to be primarily due to the increased oxygen-carrying capacity of HbV fluid resuscitation.     

A newly established neuronal rho-0 cell line highly susceptible to oxidative stress accumulates iron and other metals. Relevance to the origin of metal ion deposits in brains with neurodegenerative disorders

From human neuroblastoma-derived SILA cells we have established a rho-0 cell line that is deficient in both respiration and mitochondrial DNA. Lactate dehydrogenase activity, lactate production, and growth in the medium without glucose indicate that these cells shift from aerobic to anaerobic metabolism. Electron microscopic observations revealed abnormal mitochondria with unique cristae structures. Staining with MitoTracker dye showed that the mitochondrial transmembrane potential was reduced by 30-40% from the parent cell levels. These cells were markedly susceptible to H(2)O(2) and died apparently by a necrotic mechanism, a process blocked by deferoxamine in the parent cells but not rho-0 cells. Analysis by inductively coupled plasma-mass spectrometry revealed an approximately 3-fold accumulation of iron in the rho-0 cells at confluence (n = 4-6, three clones, *p < 0.05). Iron and four other metals were all elevated in the cells of one of the rho-0 clones and were similar to control levels in the control cybrid cells, which were replenished with normal mitochondrial DNA. Their sensitivity to H(2)O(2) was also similar to that of the parent cells. These results indicate that a newly established neuronal related rho-0 cell line is highly susceptible to active oxygen species and that these toxicity effects appear to be related to an accumulation of transition metals, which probably occurs through the respiratory impairment.     

Role of adiponectin in preventing vascular stenosis. The missing link of adipo-vascular axis

Obesity is more linked to vascular disease, including atherosclerosis and restenotic change, after balloon angioplasty. The precise mechanism linking obesity and vascular disease is still unclear. Previously we have demonstrated that the plasma levels of adiponectin, an adipose-derived hormone, decreases in obese subjects, and that hypoadiponectinemia is associated to ischemic heart disease. In current the study, we investigated the in vivo role of adiponectin on the neointimal thickening after artery injury using adiponectin-deficient mice and adiponectin-producing adenovirus. Adiponectin-deficient mice showed severe neointimal thickening and increased proliferation of vascular smooth muscle cells in mechanically injured arteries. Adenovirus-mediated supplement of adiponectin attenuated neointimal proliferation. In cultured smooth muscle cells, adiponectin attenuated DNA synthesis induced by growth factors including platelet-derived growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), basic fibroblast growth factor, and EGF and cell proliferation and migration induced by HB-EGF. In cultured endothelial cells, adiponectin attenuated HB-EGF expression stimulated by tumor necrosis factor alpha. The current study suggests an adipo-vascular axis, a direct link between fat and artery. A therapeutic strategy to increase plasma adiponectin should be useful in preventing vascular restenosis after angioplasty.     

2,4,6-trinitrotoluene-induced reproductive toxicity via oxidative DNA damage by its metabolite

Several epidemiological studies and animal experiments showed that 2,4,6-trinitrotoluene (TNT), a commonly used explosive, induced reproductive toxicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of TNT on the male reproductive system in Fischer 344 rats. TNT administration induced germ cell degeneration, the disappearance of spermatozoa in seminiferous tubules, and a dramatic decrease in the sperm number in both the testis and epididymis. TNT increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in sperm whereas plasma testosterone levels did not decrease. These results suggest that TNT-induced toxicity is derived from direct damage to spermatozoa rather than testosterone-dependent mechanisms. To determine the mechanism of 8-oxodG formation in vivo , we examined DNA damage induced by TNT and its metabolic products in vitro . 4-Hydroxylamino-2,6-dinitrotoluene, a TNT metabolite, induced Cu(II)-mediated damage to 32 P-labeled DNA fragments and increased 8-oxodG formation in calf thymus DNA, although TNT itself did not. DNA damage was enhanced by NADH, suggesting that NADH-mediated redox reactions involving TNT metabolites enhanced toxicity. Catalase and bathocuproine inhibited DNA damage, indicating the involvement of H 2 O 2 and Cu(I). These findings suggest that TNT induces reproductive toxicity through oxidative DNA damage mediated by its metabolite. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by TNT and other nitroaromatic compounds.     

A practical synthesis of 3,4-diethoxybenzthioamide based on Friedel-Crafts reaction with potassium thiocyanate in methanesulfonic acid

The synthesis of 3,4-diethoxybenzthioamide, the key intermediate for OPC-6535, is achieved by employing Friedel-Crafts reaction of 1,2-diethoxybenzene with potassium thiocyanate in methanesulfonic acid at ambient temperature.