Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

Design, synthesis, and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors

Aiming to develop selective anticancer drugs, we designed and synthesized three disulfides bearing a folic acid moiety as candidate folate receptor (FR)-targeted prodrugs of thiolate histone deacetylase inhibitors. Among them, compound 1 displayed growth-inhibitory activity toward folate receptor-positive MCF-7 breast cancer cells. The activity of 1 was significantly reduced by free folic acid, suggesting that cellular uptake of 1 is mediated by FR.     

Direct evidence that an increase in aortic norepinephrine level in response to insulin-induced hypoglycemia is due to increased adrenal norepinephrine output

This study reports on the major source of circulating norepinephrine that is known to increase, progressively, during sustained hypoglycemia induced by intravenous insulin administration. Plasma concentrations of epinephrine, norepinephrine, and dopamine were simultaneously determined for adrenal venous and aortic blood in dogs anesthetized with sodium pentobarbital. The model used allowed us to perform a functional adrenalectomy (ADRX), while continuously monitoring the adrenal medullary secretory function. Under basal conditions, the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine were 0.169 +/- 0.074, 0.067 +/- 0.023, and 0.011 +/- 0.003, respectively. Plasma concentrations (ng/mL) of aortic epinephrine, norepinephrine, and dopamine were 0.132 +/- 0.047, 0.268 +/- 0.034, and 0.034 +/- 0.009. Following insulin injection (0.15 IU/kg, i.v.), the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine increased gradually (p less than 0.05), reaching the values of 0.918 +/- 0.200, 0.365 +/- 0.058, and 0.034 +/- 0.007 30 min after insulin administration. Similarly, aortic epinephrine, norepinephrine, and dopamine concentrations (ng/mL) increased significantly (p less than 0.05) to 0.702 +/- 0.144, 0.526 +/- 0.093, and 0.066 +/- 0.024. The aortic glucose concentration (mg/dL) was diminished from 81.8 +/- 4.1 to 36.9 +/- 3.4 (p less than 0.01). After taking the blood sample at 30 min following insulin administration, ADRX was immediately performed. Five minutes after the onset of ADRX, the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine increased further to 1.707 +/- 0.374 (p less than 0.05), 0.668 +/- 0.139 (p less than 0.05), and 0.052 +/- 0.017.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence and release of calcitonin gene-related peptide in rat stomach

Immunoreactive (IR)-calcitonin gene-related peptide (CGRP) was identified throughout the entire stomach of rats, being most highly concentrated in the pyloric region, and the concentrations in muscular layers being higher than those in mucosal layers. In addition, IR-CGRP was also present in the venous effluent from isolated perfused rat stomach, and its release was stimulated by dibutyryl cyclic AMP or theophylline but not by glucagon. Gel chromatography as well as HPLC of both tissue extracts and gastric perfusate showed three identical major peaks of IR-CGRP, one of which coeluted with synthetic CGRP. These results suggest that CGRP in the stomach plays a role in the regulation of gastric function.     

Changes in important apoptosis-related molecules in the endothelin-1-induced hypertrophied cardiomyocytes: effect of the pretreatment with eicosapentaenoic Acid

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1-induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1-administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)-treated group, and the ET-1 group pretreated with EPA (10 microM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1-induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1-induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1-administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.     

The effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of Suaeda salsa under saline conditions

Suaeda salsa, a leaf succulent shrub in the family Chenopodiaceae, is one of the most important halophytes in China. Suaeda salsa produces dimorphic seeds (soft brown seeds and hard black seeds). Seeds of S. salsa were collected from the coastal salt flats near Huanghua City, China. Experiments were conducted to determine the salinity-alleviating effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of S. salsa. Brown seeds had a higher germination rate than black seeds in all experiments. Black seeds were more sensitive to salt in the absence of light in comparison to brown seeds. Brown seeds absorbed water more quickly in comparison to black seeds and were found to be more tolerant of salt stress. Our results showed that 1-aminocyclopropane-1-carboxylate (ACC, the immediate precursor of ethylene), nitrite, GA₄ and BA improved seed germination in the presence of salt. However, nitrate, GA₁, GA₃ failed to alleviate salt stress. ABA inhibited seed germination and seedling growth. Possible mechanisms involved in the alleviation of salt stress in S. salsa seeds and the ecological adaptation of the seeds to the environment are discussed.     

MTH1, an oxidized purine nucleoside triphosphatase, protects the dopamine neurons from oxidative damage in nucleic acids caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

We previously reported that 8-oxoguanine (8-oxoG) accumulates in the cytoplasm of dopamine neurons in the substantia nigra of patients with Parkinson's disease and the expression of MTH1 carrying an oxidized purine nucleoside triphosphatase activity increases in these neurons, thus suggesting that oxidative damage in nucleic acids is involved in dopamine neuron loss. In the present study, we found that levels of 8-oxoG in cellular DNA and RNA increased in the mouse nigrostriatal system during the tyrosine hydroxylase (TH)-positive dopamine neuron loss induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MTH1-null mice exhibited a greater accumulation of 8-oxoG in mitochondrial DNA accompanied by a more significant decrease in TH and dopamine transporter immunoreactivities in the striatum after MPTP administration, than in wild-type mice. We thus demonstrated that MTH1 protects the dopamine neurons from oxidative damage in the nucleic acids, especially in the mitochondrial DNA of striatal nerve terminals of dopamine neurons.