Oral glutamine inhibits tumor growth of gastric cancer bearing mice by improving immune function and activating apoptosis pathway.

Gastric cancer is one of the most common cancers in the world. It has been shown that exogenous glutamine (GLN) can inhibit the growth of tumor in vivo, but the relationship between GLN and gastric cancer has not been studied. The gastric cancer bearing mouse model was constructed and taken GLN orally at the same time, and the results found that oral GLN (1 or 2 g/kg/d) significantly inhibited the growth rate of tumor and reduce the weight of tumor tissues. Immunohistochemistry showed that oral GLN significantly reduced the PCNA index, which further proved that GLN could inhibit the growth of tumor cells. At the same time, TUNEL assay showed that oral GLN significantly enhanced the apoptosis levels of tumor cells. In addition, GLN reduced GSH levels in tumor tissues, but increased the levels of GSH in plasma, improved the T-lymphocyte transformation rate and NK cell activity, significantly inhibited the secretion of TNF-α and promoted the secretion of IL-2, thus regulating the immune function in vivo. Further detection of apoptosis pathway showed that oral GLN significantly enhanced the expression of pro-apoptotic factor Bad and inhibited the expression of Bcl-2. Meanwhile, GLN significantly increased the activities of Caspase-3, Caspase-8, caspase-9 and PARP. GSH activator NAC had a similar effect to GLN, which could improve the immune function and activate apoptosis pathway, while GSH inhibitor BSO significantly blocked the regulation of GLN, destroyed the immune balance and inhibited apoptosis, but IL-2 significantly blocked the anti-apoptotic effect of BSO. Therefore, oral GLN can improve immune function and activate apoptosis pathway through GSH, and then inhibit the growth of tumor in vivo.

In vitro dissolution absorption system (IDAS2): Use for the prediction of food viscosity effects on drug dissolution and absorption from oral solid dosage forms.

Existing in vitro dissolution or permeation models to predict food effect are mainly based on Pharmacopeias' compendial media, which specify such variables as pH, bile salts, lipolytic enzymes, and phospholipids content. However, the viscosity of food in the gastrointestinal (GI) tract is not taken into account, although it can affect both the dissolution of the oral solid dosage form and absorption of the released drug. Here, a new in vitro dissolution absorption system (IDAS2) is utilized, which comprises a dissolution apparatus USP2 (DISTEK) equipped with specially constructed permeability chambers containing Caco-2 monolayers, thereby allowing dissolution and transepithelial absorption to be ascertained simultaneously. The IDAS2 was used to evaluate the effect of medium viscosity on both the dissolution of oral solid dosage forms and absorption of released drugs. Such information, which is not ordinarily determined in dissolution and permeation studies, will be helpful to the formulators developing robust oral dosage forms. Commercially available solid dosage forms of ten model drugs from across all BCS classifications were used in this evaluation: metoprolol, minoxidil, and propranolol from BCS class 1; carbamazepine, ketoprofen, and simvastatin from BCS class 2; atenolol and ranitidine from BCS class 3; and acetazolamide and saquinavir from BCS class 4. The study revealed the applicability of IDAS2 as a tool for in vitro screening of dissolution and absorption of intact oral solid products to predict food viscosity effect. The most profound viscosity effect on dissolution and absorption was observed of solid dosage forms for the BCS class 2 compounds carbamazepine and simvastatin. A higher medium viscosity significantly slowed down the dissolution rate of tested BSC class 4 compounds acetazolamide and saquinavir, without significant effect on their absorption. The solid dosage forms least affected by the viscosity of the medium tested were the BCS class 1 compounds minoxidil and propranolol.

Simultaneous Analysis of Dissolution and Permeation Profiles of Nanosized and Microsized Formulations of Indomethacin Using the In Vitro Dissolution Absorption System 2.

The in vitro dissolution absorption system 2 (IDAS2), a recent invention comprised a conventional dissolution vessel containing 2 permeation chambers with Caco-2 cell monolayers mounted with their apical side facing the dissolution media, permits simultaneous measurement of dissolution and permeation of drugs from intact clinical dosage forms. The objectives of this study were (1) to assess the utility of IDAS2 in the determination of the effect of particle size on in vitro performance of indomethacin and (2) to find out whether the behavior in IDAS2 of 2 indomethacin products differing in particle size is correlated with their in vivo behavior. Indomethacin dissolution and permeation across Caco-2 cell monolayers were simultaneously measured in IDAS2; the dissolution and permeation profiles were simultaneously modeled using a simple two-compartment model. Compared to microsized indomethacin, the nanosized formulation increased the dissolution rate constant by fivefold, whereas moderately increasing the permeation rate constant and the kinetic solubility. As a result, the drug amount permeated across the Caco-2 cell monolayers doubled in the nanosized versus microsized formulation. The in vitro results showed a good correlation with in vivo human oral pharmacokinetic parameters, thus emphasizing the physiological relevance of IDAS2 data in predicting in vivo absorption.

Simvastatin, but not pravastatin, inhibits the proliferation of esophageal adenocarcinoma and squamous cell carcinoma cells: a cell-molecular study.

BACKGROUND AND OBJECTIVE: Long-term statin therapy has been shown to protect against several cancers, including esophageal cancer (EC). While the mechanisms underlying this effect are not clear. We investigated the effect of hydrophobic simvastatin and hydrophilic pravastatin on the proliferation of EC cells and sought to explore the underlying mechanisms. METHODS: Esophageal adenocarcinoma OE-19 cells and esophageal squamous cell carcinoma Eca-109 cells were treated with different concentrations of simvastatin or pravastatin for 24 h and 48 h. Cell proliferation was assessed by Cell Counting Kit-8 assay. Malondialdehyde (MDA) levels were measured by thiobarbituric acid (TBA) assay. mRNA and protein expression of COX-2 were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively; The expression of prostaglandin E2 (PGE2) was measured by ELISA. RESULTS: Simvastatin, but not pravastatin, significantly inhibited the proliferation of OE-19 and Eca-109 cells in a dose- and time-dependent manner, accompanying with the increasing of the MDA level. Moreover, simvastatin suppressed the expression of COX-2 and PGE2 in both OE-19 and Eca-109 cells in a dose-dependent manner. CONCLUSIONS: Lipophilic simvastatin, but not hydrophilic pravastatin, had significant inhibitory effects on the proliferation of Eca-109 and OE-19 cells. The reduction of COX-2 and PGE2 by simvastatin suggested that the inhibitory effect of simvastatin on the proliferation of EC cells may be independent of its lipid-lowering effect. Simvastatin may be a promising agent for the prevention and treatment of EC.

Melatonin Attenuates Pain Hypersensitivity and Decreases Astrocyte-Mediated Spinal Neuroinflammation in a Rat Model of Oxaliplatin-Induced Pain.

Neuroinflammatory response in spinal dorsal horn has been demonstrated to be a critical factor in oxaliplatin-induced pain. Melatonin has been shown to have anti-inflammatory and anti-allodynia effects in both preclinical and clinical studies. In the present study, we investigated the role of systemic administration of melatonin on oxaliplatin-induced pain. Intraperitoneal (i.p.) injection with oxaliplatin induced significantly mechanical allodynia and thermal hyperalgesia. Melatonin (i.p.) significantly alleviated mechanical allodynia and thermal hyperalgesia in the oxaliplatin but not sham-treated rats. The attenuation of nociceptive response persisted at least to 3 days after melatonin injection, throughout the entire observing window. Immunohistochemistry showed that oxaliplatin induced a significant increase of glial fibrillary acidic protein (GFAP) immunodensities, which could be suppressed by melatonin. Western blotting showed that GFAP protein levels were significantly elevated in the oxaliplatin-vehicle group. Melatonin significantly decreased oxaliplatin-induced upregulation of GFAP expressions. Oxaliplatin injection also enhanced the messenger RNA (mRNA) expressions of cytokines including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) and chemokines including monocyte chemoattractant protein-1 (MCP-1) and monocyte inflammatory protein-1 (MIP-1α) in the spinal dorsal horn, which could be significantly repressed by melatonin. In vitro study showed that mRNA levels of TNF-α, IL-1ß, MCP-1, and MIP-1α in primarily astrocytes were significantly increased after lipopolysaccharide (LPS, 1 µg/ml) stimulation. Melatonin (10 and 100 µM) greatly inhibited synthesis of these inflammatory mediators, in a dose-related manner. Conclusively, our data provide a novel implication of anti-nociceptive mechanism of melatonin in chemotherapy-related pain.

[Physiological responses of different cucumber cultivars seedlings to iso-osmotic Mg (NO3) 2 and NaCl stress].

In this study, the effects of iso-osmotic solution of Mg (NO3) 2 and NaCl on seedling growth, leaf lipid peroxidation, antioxidant enzyme activities, and osmotic adjustment substance accumulation were investigated using three cucumber cultivars with different ecotypes. Then salt tolerance was evaluated by membership function method. The results revealed that under the stress of 60 and 80 mmol x L(-1) Mg(NO3) 2 solution and its isotonic 90 and 120 mmol x L(-1) NaCl solution, the seedling traits such as height, stem diameter, leaf area, fresh and dry mass of aerial part and underground parts, and antioxidant enzymes activity were obviously decreased with the increasing concentration of Mg( NO3)2 and its isotonic NaCl in the three cucumber cultivars. Moreover, the inhibitory effects became more obvious with the increasing concentration of either Mg(NO3)2 or NaCl solution. MDA content and membrane lipid peroxidation were enhanced in cucumber seedlings. Among the three cultivars, SJ31-1 changed less than the other two cultivars regarding the reduced amplitudes of biomass, and activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and the increased amplitude of MDA. In addition, Mg (NO3)2 solution inhibited seedling growth more strongly than isotonic NaCl solution did, such difference was relatively significant with increasing the concentration of solution. The contents of proline, soluble amino acids, and soluble sugars varied depending on the cucumber genotype and salt type. The increased amplitude of proline content was the largest in SJ31-1, and that of soluble sugars was the largest in Lubai 19 when growing under salt treatment. The change of these parameters in Xintaimici was in between. Soluble sugars and soluble proteins were predominant osmolytes unde NaCl stress, whereas proline and soluble proteins were main osmolytes under Mg (NO3) 2 stress. Comprehensive evaluation showed that salt tolerance of the three cucumber cultivars was in order of SJ31-1 > Xintaimici > Lubai 19.

Clinical review: Ethics and end-of-life care for critically ill patients in China.

Critical care medicine in China has made great advances in recent decades. This has led to an unavoidable issue: end-of-life ethics. With advances in medical technology and therapeutics allowing the seemingly limitless maintenance of life, the exact time of death of an individual patient is often determined by the decision to limit life support. How to care for patients at the end of life is not only a medical problem but also a social, ethical, and legal issue. A lot of factors, besides culture, come into play in determining a person's ethical attitudes or behaviors, such as experience, education, religion, individual attributes, and economic considerations. Chinese doctors face ethical problems similar to those of their Western counterparts; however, since Chinese society is different from that of Western countries in cultural traditions, customs, religious beliefs, and ethnic backgrounds, there is a great difference between China and the Western world in regard to ethics at the end of life, and there is also a huge controversy within China.

[Distribution and accumulation characteristics of nutrients in solar greenhouse soil in Ji'nan, Shandong Province of East China].

Taking the solar greenhouses with different cultivating years and vegetables in Ji'nan as test objects, this paper studied the amounts and frequency distribution of soil nutrients and the relationships between cultivating years and soil nutrients accumulation characteristics, and analyzed the factors causing soil salinization and acidification by fitting soil nutrients contents with cultivating years and vegetables. In the greenhouses, the contents of soil alkali-hydrolysable nitrogen, available phosphorus, available potassium, organic matter, and electrical conductivity were significantly higher than those in the open field, with an increment of 135.3%, 475.2%, 290.1%, 97.7%, and 188.7%, respectively, but the soil pH value was 0.31 lower than that of open field. The frequency distribution of soil nutrients presented a normal curve. Differences were observed in the soil nutrients contents in the greenhouses with different cultivating vegetables. The soil alkali-hydrolysable nitrogen content and electrical conductivity were in the order of tomato > cucumber > sweet pepper, soil organic matter content and pH value were cucumber > sweet pepper > tomato, soil available phosphorus content was cucumber > tomato > sweet pepper, and soil available potassium content was tomato > cucumber > sweet pepper. There was a mild tendency of soil acidification in soil alkali-hydrolysable nitrogen and available potassium. The decrease of soil pH was closely related to the accumulation of alkali-hydrolysable nitrogen. The soil nutrients accumulation in the greenhouses had the similar patterns, i. e. , rapid accumulation in the first two cultivating years, slowed down in the third and fourth year, and kept stable later, demonstrating a dynamic balance on the whole. All the nutrients contents were positively accumulated, while soil pH presented negatively. In the greenhouses with different cultivating vegetables, there was a significant correlation between soil nutrients and cultivating years, which could be fitted by conic curve or cubic curve.

Regulation of astrocytic glutamate transporter expression by Akt: evidence for a selective transcriptional effect on the GLT-1/EAAT2 subtype.

In the nervous system, astrocytes express different ratios of the two glial glutamate transporters, glutamate transporter subtype 1 (GLT-1) and glutamate/aspartate transporter (GLAST), but little is known about the signaling pathways that independently regulate their expression. Treatment with dibutyryl-cAMP, epidermal growth factor (EGF) or other growth factors both induces expression of GLT-1 and increases expression of GLAST in astrocyte cultures. The induction of GLT-1 is correlated with morphological and biochemical changes that are consistent with astrocyte maturation. Pharmacological studies suggest that phosphatidylinositol 3-kinase (PI-3K) and the nuclear transcription factor-kappaB (NF-kappaB) may be involved in the induction of GLT-1 expression. In several signaling systems Akt, also known as protein kinase B (PKB), functions downstream of PI-3K. In these present studies we used lentiviral vectors engineered to express dominant-negative (DN), constitutively active (CA), or null variants of Akt to study the possible involvement of Akt in the regulation of GLT-1. Expression of DN-Akt attenuated the EGF-dependent induction of GLT-1. Expression of CA-Akt caused a dose- and time-dependent increase in GLT-1 protein, increased GLT-1 mRNA levels, increased dihydrokainate-sensitive (presumably GLT-1 mediated) transport activity, and caused a change in astrocyte morphology to a more stellate shape, but had no effect on GLAST protein levels. Finally, the expression of CA-Akt increased the expression of a reporter construct containing a putative promoter fragment from the human homolog of GLT-1, called EAAT2. From these studies, we conclude that Akt induces the expression of GLT-1 through increased transcription and that Akt can regulate GLT-1 expression without increasing GLAST expression in astrocytes.

The role of N-glycosylation in function and surface trafficking of the human dopamine transporter.

The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.

Is Na(+) required for the binding of dopamine, amphetamine, tyramine, and octopamine to the human dopamine transporter?

The role of Na(+) in the recognition of blockers by the dopamine transporter is accomodated by a model with a cation site that overlaps with the blocker binding domain, and a distal Na(+) site that interacts with this cation site and perhaps with the blocker binding domain itself. The present study addresses the application of this model to the recognition of substrates by the dopamine transporter, focusing on conditions that should reveal a stimulatory effect, if present, of Na(+) on substrate binding. Recognition was studied via the inhibition of binding of [(3)H]WIN 35,428 (2beta-carbomethoxy-3beta-(4-fluorophenyl) [(3)H]tropane), a cocaine analog, to the human dopamine transporter in human embryonic kidney 293 cells. Little or no changes in binding were noted for dopamine, d-amphetamine, p-tyramine, or dl-octopamine by increasing [Na(+)] from 2 mM to 20 mM with co-varying Br(-), both at pH 7.4 and 7.0. In 74-mM Tris-HBr or -HCl, only dopamine and d-amphetamine showed binding increases upon raising Na(+), leveling off with NO(3)(-) or SO(4)(2-) but not Br(-) as anion at approximately 60 mM Na(+), consonant with a partly stimulatory action of Br(-). An Na(+) free, low 5-mM Tris-HEPES buffer was used for studying Na(+) curves truly starting at 0 mM, and, with SO(4)(2-) as the anion, no stimulation of binding by Na(+) was observed. This suggested that the stimulations observed in high (74 mM) Tris(+) buffer by Na(+) were not a direct effect of Na(+) but rather a disinhibitory effect of Na(+) in removing Tris(+) inhibition that depended upon substrate. Tris(+) IC(50) values in Na(+) free buffer were not lower for dopamine and d-amphetamine than p-tyramine and dl-octopamine. No evidence was found for a stronger inhibitory effect of Na(+) for dopamine and dl-octopamine potentially offsetting Tris(+) disinhibition. All results together support the existence of a substrate domain overlapping with a cation site that also binds Tris(+); a distal Na(+) site interacts with this cation site and with the substrate domain by negative allosterism and is additionally impacted by Cl(-). Importantly, interactions between sites vary with the type of substrate, and, in membrane preparations, Na(+) is not required for, or stimulatory to, the binding of any of the four substrates studied unlike the binding of the cocaine analog WIN 35,428.