Basic fibroblast growth factor supports expansion of mouse compact bone-derived mesenchymal stem cells (MSCs) and regeneration of bone from MSC in vivo

Some progress has been made in development of methods to regenerate bone from cultured cells, however no method is put to practical use. Here, we developed methods to isolate, purify, and expand mesenchymal stem cells (MSCs) from mouse compact bone that may be used to regenerate bone in vivo. These cells were maintained in long-term culture and were capable of differentiating along multiple lineages, including chondrocyte, osteocyte, and adipocyte trajectories. We used standard cell isolation and culture methods to establish cell cultures from mouse compact bone and bone marrow. Cultures were grown in four distinct media to determine the optimal composition of culture medium for bone-derived MSCs. Putative MSCs were subjected to flow cytometry, alkaline phosphatase assays, immunohistochemical staining, and several differentiation assays to assess cell identity, protein expression, and developmental potential. Finally, we used an in vivo bone formation assay to determine whether putative MSCs were capable of regenerating bone. We found that compact bone of mice was a better source of MCSs than the bone marrow, that growth in plastic flasks served to purify MSCs from hematopoietic cells, and that MSCs grown in basic fibroblast growth factor (bFGF)-conditioned medium were, based on multiple criteria, superior to those grown in leukemia inhibitory factor-conditioned medium. Moreover, we found that the MSCs isolated from compact bone and grown in bFGF-conditioned medium were capable of supporting bone formation in vivo. The methods and results described here have implications for understanding MSC biology and for clinical purpose.     

Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.     

A Multicenter,Randomized, Double-Blind,Placebo-Controlled Trial of High-Dose Rebamipide Treatment for Low-Dose Aspirin-Induced Moderate-to-Severe Small Intestinal Damage

Background

Low-dose aspirin (LDA) frequently causes small bowel injury. While some drugs have been reported to be effective in treating LDA-induced small intestinal damage, most studies did not exclude patients with mild damage thought to be clinically insignificant.

Aim

We conducted a multicenter, randomized, double-blind, placebo-controlled trial to assess the efficacy of a high dose of rebamipide, a gastroprotective drug, for LDA-induced moderate-to-severe enteropathy.

Methods

We enrolled patients who received 100 mg of enteric-coated aspirin daily for more than 3 months and were found to have more than 3 mucosal breaks (i.e., erosions or ulcers) in the small intestine by capsule endoscopy. Eligible patients were assigned to receive either rebamipide 300 mg (triple dose) 3 times daily or placebo for 8 weeks in a 2:1 ratio. Capsule endoscopy was then repeated. The primary endpoint was the change in the number of mucosal breaks from baseline to 8 weeks. Secondary endpoints included the complete healing of mucosal breaks at 8 weeks and the change in Lewis score (an endoscopic score assessing damage severity) from baseline to 8 weeks.

Results

The study was completed by 38 patients (rebamipide group: n = 25, placebo group: n = 13). After 8 weeks of treatment, rebamipide, but not placebo, significantly decreased the number of mucosal breaks (p = 0.046). While the difference was not significant (p = 0.13), the rate of complete mucosal break healing in the rebamipide group (32%, 8 of 25) tended to be higher than that in the placebo group (7.7%, 1 of 13). Rebamipide treatment significantly improved intestinal damage severity as assessed by the Lewis score (p = 0.02), whereas placebo did not. The triple dose of rebamipide was well tolerated.

Conclusions

High-dose rebamipide is effective for the treatment of LDA-induced moderate-to-severe enteropathy.

Trial Registration

UMIN Clinical Trials Registry UMIN000003463     

Effect of taurine on growth hormone and prolactin secretion in rats: possible interaction with opioid peptidergic system

The effect of taurine on growth hormone (GH) and prolactin (PRL) secretion was investigated in the urethane-alpha-chloralose anesthetized rats, considering the interaction with endogenous opioid peptidergic system. Intraventricular injection of taurine (0.25 and 1.0 mumol) stimulated GH and PRL secretion in a dose-dependent manner. However, 4.0 mumol taurine failed to show these effect. The intravenous infusion of naloxone (4 mg/kg b.w.) completely inhibited both the GH and PRL secretion induced by taurine (1.0 mumol). The combined treatment of taurine (1.0 mumol) and FK33-824 (Met-enkephalin derivative, 100 micrograms/kg b.w., i.v.) significantly increased GH and PRL responses induced by taurine or FK33-824 alone. These results indicate that taurine is an effective stimulator of GH and PRL secretion in rats, and that the mechanism of this action involves the opioid peptidergic system in the hypothalamus.     

Angiotensin II Reduces Lipoprotein Lipase Expression in Visceral Adipose Tissue via Phospholipase C β4 Depending on Feeding but Increases Lipoprotein Lipase Expression in Subcutaneous Adipose Tissue via c-Src

Metabolic syndrome is characterized by visceral adiposity, insulin resistance, high triglyceride (TG)- and low high-density lipoprotein cholesterol-levels, hypertension, and diabetes—all of which often cause cardiovascular and cerebrovascular diseases. It remains unclear, however, why visceral adiposity but not subcutaneous adiposity causes insulin resistance and other pathological situations. Lipoprotein lipase (LPL) catalyzes hydrolysis of TG in plasma lipoproteins. In the present study, we investigated whether the effects of angiotensin II (AngII) on TG metabolism are mediated through an effect on LPL expression. Adipose tissues were divided into visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) for comparison. AngII accelerated LPL expression in SAT but, on the contrary, suppressed its expression in VAT. In both SAT and VAT, AngII signaled through the same type 1 receptor. In SAT, AngII increased LPL expression via c-Src and p38 MAPK signaling. In VAT, however, AngII reduced LPL expression via the G_q class of G proteins and the subsequent phospholipase C β4 (PLCβ4), protein kinase C β1, nuclear factor κB, and inducible nitric oxide synthase signaling pathways. PLCβ4 small interfering RNA experiments showed that PLCβ4 expression is important for the AngII-induced LPL reduction in VAT, in which PLCβ4 expression increases in the evening and falls at night. Interestingly, PLCβ4 expression in VAT decreased with fasting, while AngII did not decrease LPL expression in VAT in a fasting state. In conclusion, AngII reduces LPL expression through PLCβ4, the expression of which is regulated by feeding in VAT, whereas AngII increases LPL expression in SAT. The different effects of AngII on LPL expression and, hence, TG metabolism in VAT and SAT may partly explain their different contributions to the development of metabolic syndrome.     

The ADP-ribosylation factor 1 gene is indispensable for mouse embryonic development after implantation

ADP-ribosylation factor (Arf) 1 is thought to affect the morphologies of organelles, such as the Golgi apparatus, and regulate protein trafficking pathways. Mice have six Arf isoforms. In knockdown experiments with HeLa cells, no single Arf isoform among Arf1–5 is required for organelle morphologies or any membrane trafficking step. This suggests that the cooperation of two or more Arfs is a general feature. Although many cell biological and biochemical analyses have proven the importance of Arf1, the physiological roles of Arf1 in mice remain unknown. To investigate the activity of Arf1 in vivo, we established Arf1-deficient mice. Arf^−/− blastocysts were identified at the expected Mendelian ratio. The appearance of these blastocysts was indistinguishable from that of wild-type and Arf^+/− blastocysts, and they grew normally in an in vitro culture system. However, Arf^−/− embryos were degenerated at E5.5, and none survived to E12.5, suggesting that they died soon after implantation. These data establish for the first time that the Arf1 gene is indispensable for mouse embryonic development after implantation.     

Soybean-derived phosphatidylinositol inhibits in vivo low concentrations of amyloid beta protein-induced degeneration of hippocampal neurons in V337M human tau-expressing mice

In our previous reports using primary cultured rat hippocampal neurons, pathophysiological concentrations (< or =10 nM) of amyloid beta proteins (Abetas) showed neurotoxicity via a phosphatidylinositol metabolism disorder, and soybean-derived phosphatidylinositol protected the neurons against the Abeta's neurotoxicity. In the present study, such a neurotoxic effect of Abeta and a neuroprotective effect of phosphatidylinositol were examined in vivo using transgenic mice expressing V337 M human tau. Intrahippocampal CA1 injection of 1.5 mul of 100 nM or 1 microM Abeta25-35 increased the number of degenerating neurons with an apoptotic feature in bilateral hippocampal CA1, CA2, CA3 and dentate gyrus regions in 1 month, demonstrating an in vivo neurotoxic effect of Abeta at lower concentrations after diffusion. Intrahippocampal co-injection or intracerebroventricular administration of 1.5 microl of 500 nM phosphatidylinositol prevented the Abeta25-35-induced neuronal degeneration in all the hippocampal regions, while co-injection of another acidic phospholipid, phosphatidylserine (1.5 microl, 500 nM) with Abeta25-35 showed no protective effects. Thus, exogenously applied phosphatidylinositol appeared to minimize the toxic effects of Abeta in vivo. These results suggest that soybean-derived phosphatidylinositol may be effective in the treatment of Alzheimer's disease.     

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.     

Larval development of the oriental lancelet, Branchiostoma belcheri, in laboratory mass culture

We are successfully maintaining a laboratory colony of the lancelet Branchiostoma belcheri bred in the laboratory. Based on living individuals in this mass culture, morphological characteristics from the seven-day larval to benthic juvenile stages have been studied. Most striking was that later larval development of B. belcheri showed great individual variation even in a rather stable culture environment. Metamorphosis first occurred on 60 days post fertilization (dpf) and was continuously observed throughout the present study up to 100 dpf. Morphological traits such as the number of primary gill slits and body size at the start of metamorphosis are apparently affected by culture condition. Body size measured in the largest individuals showed nearly linear growth at 0.087 mm/day. The variability found in larval development calls for caution when developmental stages and chronological ages are compared between populations. However, the developmental flexibility of this animal also raises the possibility that growth and sexual maturation could be controlled artificially in captivity.