Ginsenoside Rg1 exerts anti­apoptotic effects on non­alcoholic fatty liver cells by downregulating the expression of SGPL1.

Non­alcoholic fatty liver disease (NAFLD) has a high incidence, and can lead to liver cirrhosis and even hepatocellular carcinoma in severe cases. To the best of our knowledge, there is currently no safe and effective treatment for the management of this disease. Ginsenoside Rg1 (Rg1) is an active monomer derived from ginseng and notoginseng. In the present study, HHL­5 hepatocytes were used to establish an in vitro cell model of NAFLD by medium­ and long­chain fat emulsion treatment, and the effects of Rg1 on adipose accumulation, apoptosis and the expression levels of apoptosis­related proteins in HHL­5 hepatocytes were examined. The results demonstrated that Rg1 inhibited the accumulation of fat in HHL­5 cells, while inhibiting apoptosis, and Rg1 downregulated the expression levels of the pro­apoptotic protein Bax and upregulated the expression levels of the anti­apoptotic protein Bcl­2, indicating that Rg1 could promote the stability or integrity of mitochondria and exert an anti­apoptotic effect by regulating Bcl­2 family proteins. In addition, Rg1 markedly downregulated the expression levels of sphingosine­1­phosphate lyase 1 (SGPL1), a key enzyme in the sphingosine signaling pathway, in HHL­5 cells with steatosis, and increased the expression levels of the downstream pro­survival signals phosphorylated (p­)Akt and p­Erk1/2. Furthermore, overexpression of SGPL1 abolished the anti­apoptotic effect of Rg1 on SGPL1­overexpressing HHL­5 cells with steatosis, and downregulated the expression levels of pro­survival proteins, such as Bcl­2, p­Akt and p­Erk1/2, whereas the expression levels of pro­apoptotic Bax were markedly increased. In conclusion, although there are some reports regarding the protective effect of Rg1 on fatty liver cells, to the best of our knowledge, the present study is the first to report that Rg1 may exert an anti­apoptotic effect on fatty liver cells by regulating SGPL1 in the sphingosine signaling pathway. Rg1 is the main component of the prescription drug Xuesaitong in China; therefore, the findings of the present study may provide a theoretical molecular basis for the use of Rg1 or Xuesaitong in the treatment of patients with NAFLD.

Regulation of MCP-1 production in brain by stress and noradrenaline-modulating drugs.

While it is accepted that noradrenaline (NA) reduction in brain contributes to the progression of certain neurodegenerative diseases, the mechanisms through which NA exerts its protective actions are not well known. We previously reported that NA induced production of monocyte chemoattractant protein (MCP-1/CCL2) in cultured astrocytes mediated some of the neuroprotective actions of NA. We have now examined the regulation of MCP-1 production in vivo. Treatment of mice with the NA precursor l-threo-3,4-dihydroxyphenylserine induced the production of MCP-1 in astrocytes. In contrast, exposure to stress (a process known to elevate brain NA levels) produced only a moderate increase of MCP-1 because of the inhibitory activity of glucocorticoids released during the stress response. Similarly, corticosterone treatment of astrocytes caused a reduction of constitutive as well as the NA-induced MCP-1 production. When stressed rats had the production of glucocorticoids blocked by the selective inhibitor metyrapone, a large increase of MCP-1 concentration was observed in cortex, whereas propranolol (a beta adrenergic receptor blocker) avoided modifications of MCP-1 after stress. Desipramine (an inhibitor of NA reuptake) also caused an increase of MCP-1 in cortex. These data suggest that some phenomena caused by the alteration of NA or glucocorticoids could be mediated by MCP-1.

Purification capability of tobacco transformed with enzymes from a methylotrophic bacterium for formaldehyde.

Plants have the ability to remediate environmental pollution. Especially, they have a high purification capability for airpollution. We have measured the purification characteristics of foliage plants for indoor airpollutants--for example, formaldehyde (HCHO), toluene, and xylene--using a tin oxide gas sensor. HCHO is an important intermediate for biological fixation of C1 compounds in methylotrophs. The ribulose monophosphate pathway of HCHO fixation is inherent in many methylotrophic bacteria, which can grow on Cl compounds. Two genes for the key enzymes, HPS and PHI, from the methylotrophic bacterium Mycobacterium gastri MB19 were introduced into tobacco. In this article, the HCHO-removal characteristic of the transformant was examined by using the gas sensor in order to evaluate quantitatively. The purification characteristics of the transformant for toluene, xylene, and styrene were also measured. The results confirmed an increase of 20% in the HCHO-removal capability. The differences of the purification capabilities for toluene, xylene, and styrene were not recognized.

Products by the sphingosine kinase/sphingosine 1-phosphate (S1P) lyase pathway but not S1P stimulate mitogenesis.

Sphingosine 1-phosphate (S1P) functions as a ligand for the S1P/EDG family receptors. For years, intracellular signaling roles for S1P have also been suggested, especially in cell proliferation. Now, we have generated several mouse F9 embryonic carcinoma cell lines varying in expression of the S1P-degrading enzyme, S1P lyase (SPL) and/or sphingosine kinase (SPHK1). All these cell lines accumulated S1P compared to the wild-type F9 cells, but the amounts varied. We investigated the ability of these cells to proliferate under low serum conditions, as measured by a thymidine uptake assay. Although F9 cells over-expressing SPHK1 did exhibit enhanced DNA synthesis, other S1P-accumulating cells (SPL-null cells and SPL-null cells over-expressing SPHK1) did not. The overproduction of both SPL and SPHK1 resulted in the most striking mitogenic effect. Moreover, nM concentrations of sphingosine (or dihydrosphingosine) stimulated DNA synthesis in an SPL-dependent manner. These results indicate that products by the SPL pathway, not S1P itself, function in mitogenesis.

Down-regulation of rat brain adenosine A1 receptors at the end of pregnancy.

The status of the adenosine A1 receptor/adenylyl cyclase (A1R/AC) transduction pathway in rat brain was analysed at the end of pregnancy using different approaches. Pregnancy at term caused a significant decrease in the Bmax value obtained by saturation binding assays using [3H]DPCPX as radioligand, suggesting a down-regulation of adenosine A1 receptor. Moreover, A1 receptor immunodetection in pregnant rat membranes and the level of mRNA coding A1 receptor were significantly decreased. This loss of A1 receptor was associated with a significant increase in receptor affinity, since the KD value from the [3H]DPCPX saturation curve and Ki for N6-cyclohexyladenosine (CHA) were decreased in pregnant rats. Surprisingly, CHA-mediated inhibition of adenylyl cyclase was increased, reflecting enhanced receptor responsiveness. On the other hand, immunoblotting of different alphaGi-protein isoforms revealed a significant increase in alphaGi3 level in membranes from pregnant rats. Pre-incubation of membranes with anti-alphaGi3 antibody blocked the guanosine triphosphate (GTP) or CHA inhibitory effect on adenylyl cyclase in both pregnant and non-pregnant rats, pointing to alphaGi3 as the main isoform involved in the A1 receptor response. These results suggest that, at the end of pregnancy, there is a down-regulation of adenosine A1 receptors counterbalanced with a strengthened functionality, probably due to an increase in both alphaGi3 protein and receptor affinity.

A rat model of distractibility: effects of drugs modifying dopaminergic, noradrenergic and GABAergic neurotransmission.

A procedure for analyzing effects of drugs on distractibility is proposed. Rats are trained to traverse a straight runway with a sucrose solution as reinforcement. Once the response has been acquired, an additional runway ending in an empty box is connected. The time spent investigating this additional runway is the measure of distractibility. Amphetamine, 1 mg/kg i.p., increased distractibility. In rats that were never reinforced, amphetamine at a dose of 1 mg/kg reduced the time spent in the additional runway. This shows that the effects of amphetamine in the reinforced animals cannot be interpreted as enhanced exploration. Furthermore, the benzodiazepines diazepam (2 and 4 mg/kg, i.p.) and chlordiazepoxide (2.5, 5 and 10 mg/kg, i.p.), known to enhance exploration of novel environments, did not affect the time spent in the additional runway in sucrose-reinforced animals. It was concluded that the procedure indeed is a model of distractibility. The dopamine antagonist cis(Z)-flupenthixol, at a dose of 0.25 mg/kg, i.p., blocked the effects of amphetamine, 1 mg/kg. Flupenthixol itself, in doses of 0.25 and 0.5 mg/kg, did not affect the time spent in the additional runway. This suggests that enhanced dopaminergic activity indeed is responsible for the effects. This proposal is further supported by experiments showing that the noradrenaline precursor dihydroxyphenylserine (10 mg/kg + carbidopa, 50 mg/kg, both i.p.) and the noradrenergic neurotoxin DSP4 (50 mg/kg, i.p.) had no effect on distractibility. Moreover, amfonelic acid, a dopamine releaser with slight or no effect on noradrenergic neurotransmission, had effects very similar to those of amphetamine when given in doses of 0.25 and 0.5 mg/kg, i.p. A lower dose, 0.125 mg/ kg, was ineffective. Taken together, these data suggest that enhanced dopaminergic neurotransmission increases distractibility in the rat. However, both amphetamine and amfonelic acid may stimulate serotonin release. Until serotonergic drugs have been tested, a contribution of this transmitter cannot be ruled out. The distraction procedure may constitute an animal model of some kinds of disordered information processing.

Properties of fructose 1,6-diphosphate aldolases from spores and vegetative cells of Bacillus cereus.

Fructose 1,6-diphosphate aldolase from cells of Bacillus cereus appears to be typical Class II aldolase as judged by its functional and physical properties. Spore and vegetative cell aldolase had similar enzymatic, immunochemical, and heat resistance properties in the absence of calcium, but they differed in their thermal stabilities in the presence of calcium, their Stokes' radii, their mobility in acrylamide gel electrophoresis, and their molecular weights. The pH optimum for both enzymes was 8.5, and their K(m) with respect to substrate was 2 x 10(-3)m. Highly purified spore and vegetative cell aldolases were both heat labile with half-lives of 4 min at 53 C and pH 6.4. In the presence of 3 x 10(-2)m solution of calcium ions, the stability of the spore protein increased 12-fold but the vegetative form became more heat labile. The enhanced stability of the spore aldolase was not diminished by dialysis or gel filtration but was lost after chromatography on diethylaminoethyl cellulose at pH 7.4. Aldolase from vegetative cells exists in an equilibrium mixture of two molecular weights, 115,000 and 79,000 in the approximate ratio of 1:4, respectively. The molecular weight of spore aldolase is 44,000. Spore aldolase was more mobile during electrophoresis than its vegetative cell counterpart because of its smaller size.