Withdrawal: Specific nephrotoxicity and cardiotoxicity of BT-CAL®, Sigma Anti-bonding Molecule Calcium Carbonate, in mice.

[This retracts the article on p. 7 in vol. 29, PMID: 23573102.].

Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury.

Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level.

Comparative effects of plant oils on the cerebral hemorrhage in stroke-prone spontaneously hypertensive rats.

OBJECTIVES: Since oils and fats can induce metabolic syndrome, leading to cardiovascular and cerebrovascular diseases, the present study was performed to find out whether the plant oils affect the cerebral hemorrhage in stroke-prone spontaneously hypertensive (SHR-SP) rats. METHODS: From 47 days of age, male SHR-SP rats were given drinking water containing 1% NaCl to induce hypertension, and simultaneously fed semi-purified diets containing 10% perilla oil, canola oil, or shortening. The onset time of convulsion following cerebral hemorrhage was recorded, and the areas of hemorrhage and infarction were analyzed in the stroke brains. RESULTS: In comparison with 58-day survival of SHR-SP rats during feeding NaCl alone, perilla oil extended the survival time to 68.5 days, whereas canola oil shortened it to 45.7 days. Feeding perilla oil greatly reduced the total volume of cerebral hemorrhage from 17.27% in the control group to 4.53%, while shortening increased the lesions to 21.23%. In a microscopic analysis, perilla oil also markedly decreased the hemorrhagic and infarction lesions to 1/10 of those in control rats, in contrast to an exacerbating effect of shortening. In blood analyses, perilla oil reduced blood total cholesterol and low-density lipoproteins which were increased in SHR-SP, but canola oil further increased them and markedly lowered platelet counts. DISCUSSION: Perilla oil delayed and attenuated cerebral hemorrhage by improving hyperlipidemia in hypertensive stroke animals, in contrast to the aggravating potential of canola oil and shortening. It is suggested that perilla oil should be the first choice oil for improving metabolic syndrome in hypertensive persons at risk of hemorrhagic stroke.

Health Span-Extending Activity of Human Amniotic Membrane- and Adipose Tissue-Derived Stem Cells in F344 Rats.

UNLABELLED: Aging brings about the progressive decline in cognitive function and physical activity, along with losses of stem cell population and function. Although transplantation of muscle-derived stem/progenitor cells extended the health span and life span of progeria mice, such effects in normal animals were not confirmed. Human amniotic membrane-derived mesenchymal stem cells (AMMSCs) or adipose tissue-derived mesenchymal stem cells (ADMSCs) (1×10(6) cells per rat) were intravenously transplanted to 10-month-old male F344 rats once a month throughout their lives. Transplantation of AMMSCs and ADMSCs improved cognitive and physical functions of naturally aging rats, extending life span by 23.4% and 31.3%, respectively. The stem cell therapy increased the concentration of acetylcholine and recovered neurotrophic factors in the brain and muscles, leading to restoration of microtubule-associated protein 2, cholinergic and dopaminergic nervous systems, microvessels, muscle mass, and antioxidative capacity. The results indicate that repeated transplantation of AMMSCs and ADMSCs elongate both health span and life span, which could be a starting point for antiaging or rejuvenation effects of allogeneic or autologous stem cells with minimum immune rejection. SIGNIFICANCE: This study demonstrates that repeated treatment with stem cells in normal animals has antiaging potential, extending health span and life span. Because antiaging and prolonged life span are issues currently of interest, these results are significant for readers and investigators.

Perilla oil improves blood flow through inhibition of platelet aggregation and thrombus formation.

The inhibitory effects of perilla oil on the platelet aggregation in vitro and thrombosis in vivo were investigated in comparison with aspirin, a well-known blood flow enhancer. Rabbit platelet-rich plasma was incubated with perilla oil and aggregation inducers collagen or thrombin, and the platelet aggregation rate was analyzed. Perilla oil significantly inhibited both the collagen- and thrombin-induced platelet aggregations, in which the thromboxane B2 formation from collagen-activated platelets were reduced in a concentration-dependent manner. Rats were administered once daily by gavage with perilla oil for 1 week, carotid arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Perilla oil delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 0.5 mL/kg. In addition, a high dose (2 mL/kg) of perilla oil greatly prevented the occlusion, comparable to the effect of aspirin (30 mg/kg). The results indicate that perilla oil inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is proposed that perilla oil could be a good candidate without adverse effects for the improvement of blood flow.

Secretome analysis of human oligodendrocytes derived from neural stem cells.

In this study, we investigated the secretome of human oligodendrocytes (F3.Olig2 cells) generated from human neural stem cells by transduction with the gene encoding the Olig2 transcription factor. Using mRNA sequencing and protein cytokine arrays, we identified a number of biologically important secretory proteins whose expression has not been previously reported in oligodendrocytes. We found that F3.Olig2 cells secrete IL-6, PDGF-AA, GRO, GM-CSF, and M-CSF, and showed prominent expression of their corresponding receptors. Co-expression of ligands and receptors suggests that autocrine signaling loops may play important roles in both differentiation and maintenance of oligodendrocytes. We also found that F3.Olig2 cells secrete matrix metalloproteinases and matrix metalloproteinase-associated proteins associated with functional competence of oligodendrocytes. The results of our secretome analysis provide insights into the functional and molecular details of human oligodendrocytes. To the best of our knowledge, this is the first systematic analysis of the secretome of oligodendrocytes.

Specific nephrotoxicity and cardiotoxicity of BT-CAL®, Sigma Anti-bonding Molecule Calcium Carbonate, in mice.

According to a high anti-osteoporotic efficacy of Sigma Anti-bonding Molecule Calcium Carbonate (SAC), repeated-dose toxicities of SAC were investigated to assess its feasibility as drug or functional food ingredient. Male ICR mice were given drinking water containing 0.006, 0.02 or 0.06% SAC for 4 weeks. SAC feeding decreased the body weights and feed and water consumptions of mice in a dose-dependent manner, especially, leading to severe emaciation and 70% death in 3 weeks in the high-dose (0.06%) group. Not only kidney and heart weights, but also the levels of blood urea nitrogen, creatinine, aspartate transaminase, and creatine phospokinase significantly increased after SAC administration, indicative of nephrotoxicity and cardiotoxicity. Such renal and cardiac toxicities were also confirmed by microscopic findings, exhibiting renal crystals and cardiac fibrosis, which may be due to the insoluble crystal formation and calcium overload, respectively. In conclusion, it is suggested that no observed adverse effect level of SAC is lower than 0.006% in mice, and that a long-term intake may cause serious adverse effects on renal and cardiac functions.

Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation.

The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow.

Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells.

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.

Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain.

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50) = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.