OBJECTIVETo study the conditions on separation and regeneration of protoplast from Phellinus igniarius.

METHODThe effects of enzymolysis conditions of P. igniarius mycelia on yield of protoplast and culturing conditons on regeneration ratio of protoplast were investigated.

RESULTWhen the 8 days-old mycelia was hydrolysed by 1.5% of lywallzyme adding to driselase of 0. 5% and at 30 degrees C for 3 h and enzymolysis was stablized by sucrose as a stablisher of osmotic pressure, higher yield of P. igniarius protoplast was obtained. If 10 days-old mycelia was used as raw material of enzymolysis and manntol was selected as stablisher of osmotic pressure of enzymolysis, higher regeneration ratio of P. igniarius protoplast also would be obtained in following regeneration step at same time keeping higher yield. For the regeneration processing, it was beneficial for the regeneration of P. igniarius protoplast that PDA plusing mulberry ramulus was used as the culture medium of regeneration and manntol was selected as the osmotic pressure establisher of regeneration culture medium.

CONCLUSIONThe method and conditions to keep both higher yield and regeneration ratio of P. igniarius protoplast were obtained.

Culture Media ; pharmacology ; Fungal Proteins ; pharmacology ; Glucan Endo-1,3-beta-D-Glucosidase ; pharmacology ; Glycoside Hydrolases ; pharmacology ; Mannitol ; pharmacology ; Multienzyme Complexes ; pharmacology ; Osmotic Pressure ; Peptide Hydrolases ; pharmacology ; Polyporaceae ; drug effects ; physiology ; Protoplasts ; drug effects ; physiology ; Regeneration ; drug effects ; Sucrose ; pharmacology ; Temperature

Transcranial Doppler (TCD) was carried out to determine the resistive index (RI) values of normal canine cerebral arteries and its reproducibility and to evaluate the change of cerebral vascular resistance following diuretics administration. RI values of rostral cerebral artery (RCA) were compared between fontanelle window and temporal window. Normal ranges and reproducibility of the RI values were examined in the rostal cerebral artery (RCA) and caudal cerebral artery (CCA). And after administration of diuretics, TCD-derived RI values were measured at RCA and CCA. Cerebral vascular RI values of RCA and CCA were 0.55 +/- 0.05 and 0.55 +/- 0.03 in the normal dogs, respectively. There was no significant difference of RI between male and female; between fontanelle window and temporal window. Reproducibility of RI measurements between intraobserver and interobserver were relatively high. The RI of RCA and CCA were significantly increased 15 minutes after mannitol administration (p<0.01) and returned to baseline values by 30 minutes, but it did not significantly change after furosemide and saline administration. The results suggest that TCD is a useful test which can obtain reproducible results from any window and has the advantage of detecting subtle changes in cerebral vascular resistance.
Animals ; Cerebral Arteries/drug effects/*ultrasonography ; Diuretics/pharmacology ; Dogs/*physiology ; Feasibility Studies ; Furosemide/pharmacology ; Mannitol/pharmacology ; Reference Values ; Reproducibility of Results ; Ultrasonography, Doppler/*veterinary ; Vascular Resistance/drug effects/*physiology

A new method that uses the reduced scattering coefficients (micro'(s)) measured in rat's brain tissue in vivo after administration of hyperosmotic solution by bifurcated fiber optic probe is proposed in this paper. 60 SD rats were divided into three groups by randomization method, and then were treated by 0.9% NaCl, 20% Mannitol and 7.5% NaCl through vena caudalis, respectively. The changes of micro'(s) in every rat's local cortex were observed continuously by a bifurcated fiber optic probe in vivo in a mini-invasive way. No changes of micro'(s) were observed in the control group which was given by 0.9% NaCl, while the micro'(s), relative changes of the 20% mannitol group and 7.5% NaCl group increased by 7.3% +/- 1.7% and 12.8% +/- 2.9%, respectively. The results showed that there were significant differences among the three groups (P < 0.05). The micro'(s) of rat's local cortex observed by bifurcated fiber optic probe can be used for shedding light on the anhydration induced by hyperosmotic solution.
Animals ; Cerebral Cortex ; drug effects ; Female ; Male ; Mannitol ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Saline Solution, Hypertonic ; pharmacology ; Scattering, Radiation

OBJECTIVETo study the protective effect of cold autologous blood cardioplegic solution on the heart of infants with cyanotic congenital heart disease (CCHD).

METHODSNinety-six infants with CCHD who underwent cardiopulmonary bypass (CPB) were randomly and equally divided into three groups: histidine-tryptophan-ketoglutarate (HTK) solution, cold non-autologous blood cardioplegic solution, and cold autologous blood cardioplegic solution. The right auricular tissues were taken before aortic cross-clamping and at 30 minutes after aortic declamping, and ATP level and energy charge (EC) in the myocardium were measured. Venous blood was collected before and immediately after CPB, and the serum levels of creatine kinase (CK)-MB and cardiac troponin I (cTnI) were measured. The clinical parameters, such as the re-beat time and re-beat rate during CPB, cardiac index, dependence on positive inotropic agents, and left ventricular ejection fraction (LVEF) at 2 hours after CPB, the incidence rate of arrhythmia within 24 hours after CPB, and postoperative complications and mortality, were recorded.

RESULTSAt 30 minutes after aortic declamping, the three groups showed significantly decreased ATP and EC levels (P<0.05), and the cold autologous blood group had significantly higher ATP and EC levels than the other two groups (P<0.05). Immediately after CPB, the three groups showed significantly increased serum levels of CK-MB and cTnI (P<0.05), and the cold autologous blood group had significantly lower serum levels of CK-MB and cTnI than the other two groups (P<0.05). The cold autologous blood group had significantly better outcomes than the other two groups in terms of the re-beat time during CPB and the dependence on positive inotropic agents and LVEF at 2 hours after CPB (P<0.05).

CONCLUSIONSCold autologous blood cardioplegic solution is superior to HTK and cold non-autologous blood cardioplegic solutions in preserving myocardial energy and reducing myocardial injury in infants with CCHD who undergo CPB, thus providing a better protective effect on the heart.

Cardioplegic Solutions ; pharmacology ; Cardiopulmonary Bypass ; Energy Metabolism ; Female ; Glucose ; pharmacology ; Heart Defects, Congenital ; metabolism ; surgery ; Humans ; Infant ; Infant, Newborn ; Male ; Mannitol ; pharmacology ; Myocardium ; metabolism ; Potassium Chloride ; pharmacology ; Procaine ; pharmacology ; Ventricular Function, Left

This study is to investigate the effects of concentration, intestinal section, pH, paracellular route, substrate/inhibitor of enzyme (CYP3A) and proteins (P-gp, MRP2, SGL1) on the absorption of forsythoside A. The absorption of three concentrations (2.6, 5.2, and 10.4 microg x mL(-1)) of forsythoside A in different intestinal segments was studied with phenol red as the marker by rat circulation in situ. The results showed that the residue of forsythoside A with different concentrations had little significant difference from that obtained after perfusing via duodenum, jejunum, ileum and colon, which indicated that the absorption of forsythoside A was passive diffusion and had no difference in different segments of rat intestine. The residue of forsythoside A increased to 466.160 and 463.429 microg respectively when cyclosporine (4 microg x mL(-1)) or midazolam (50 micromol x L(-1)) was added to the circulation fluid, which showed significant difference compared to the control group (P < 0.05). Moreover, the residue of forsythoside A showed a tendency of increase with the increase of cyclosporine or midazolam. When digoxin (50 micromol x L(-1)) or EDTA (10 microg x mL(-1)) was added to the circulation fluid, the residue of forsythoside A decreased to 325.110 and 369.888 microg respectively, which showed significant difference as compared to the control group (P < 0.05). Besides, the residue of forsythoside A showed a tendency of reduction with the increase of digoxin or EDTA. However, there is no significant change in the absorption of forsythoside A when the different concentrations of mannitol were added to the circulation fluid. The results above indicated that the absorption of forsythoside A was mainly passive diffusion and involved paracellular route at the same time. In addition, the substrates of P-gp or CYP3A had dose-dependent effect on the absorption of forsythoside A.
Animals ; Colon ; metabolism ; Cyclosporine ; pharmacology ; Digoxin ; pharmacology ; Dose-Response Relationship, Drug ; Duodenum ; metabolism ; Edetic Acid ; pharmacology ; Glycosides ; administration & dosage ; pharmacokinetics ; Hydrogen-Ion Concentration ; Ileum ; metabolism ; Intestinal Absorption ; Jejunum ; metabolism ; Male ; Mannitol ; pharmacology ; Midazolam ; pharmacology ; Rats ; Rats, Sprague-Dawley

Unlike most secretory cells, high extra cellular calcium inhibits rather than stimulates hormonal secretion in several cells such as parathyroid cells, Juxtaglomerular cells and osteoclast. To gain further insight into the common but unique stimulus-secretion coupling mechanism in these cells, bovine parathyroid slices were incubated in various conditions of Krebs-Ringer (KR) solution containing essential amino acids. Parathyroid cells showed the inverse dependency of secretion on extra cellular calcium concentration as we expected. Ammonium acetate overcame the inhibitory effect of 2.5 mM of calcium and the maximum effect was as much as the five times of the basal value, while there was a little additive effect under 0 mM CaCl2. PTH secretion was biphasic according to the change of extra cellular osmolarity and the lowest response was observed at 300 mOsm/l. In Na-rich KR solution, high concentration of nigericin (> 10(-4)M) completely overcame the inhibitory effect of 2.5 mM CaCl2 and the maximum stimulatory effect was 8 times greater whereas it was only 2 times greater without CaCl2. In K-rich KR solution that abolished the K-gradient between the extra cellular solution and the cytoplasm, the rate of PTH secretion increased, and furthermore the addition of nigericin increased the rate of secretion significantly. The results above suggested that the osmotic swelling of the secretory vesicle in parathyroid cells might promote exocytosis as in Juxtaglomerular cells. We propose that the swelling of the vesicle is also prerequisite for secretion in several cells inhibited paradoxically by Ca++, whatever the signal transduction pathway for swelling of the secretory granules induced by the lowering of Ca++ in cytoplasm are.
Acetates/pharmacology ; Animal ; Body Fluids/*metabolism ; Cattle ; Cell Membrane Permeability ; Ionophores/pharmacology ; Mannitol/pharmacology ; Nigericin/pharmacology ; Osmosis ; Parathyroid Glands/drug effects/*secretion ; Parathyroid Hormones/*secretion ; Support, Non-U.S. Gov't

OBJECTIVETo detect the influence of mannitol on cerebral blood flow of post-resuscitation children by transcranial Doppler (TCD).

METHODSThe blood flow changes of left side middle cerebral artery (MCA) and extracranial internal carotid artery (EICA) were monitored by TCD in 21 post-resuscitation children. TCD waveforms, peak velocity (Vp), diastolic velocity (Vd), mean velocity (Vm) and pulsitility index (PI) of MCA and EICA were monitored daily and 30 minutes before and after the first dose of mannitol. Glasgow scores were estimated in the meantime. The patients were divided into 2 groups according to the patterns of diastolic flow. Patients in group I showed no diastolic flow or retrograde diastolic flow (n = 9), and patients in group II had positive diastolic flow (n = 12).

RESULTSVp, Vd, Vm, PI of MCA and EICA in group I patients had no significant change after the administration of mannitol and the Glasgow scores were much lower than that of group II patients (P < 0.05). TCD waveforms showed no improvement in group I patients and their outcomes were poor. Vd, Vm and Vp of MCA and Vd of EICA in group II patients increased, PI of MCA decreased (P < 0.05), while Vp, Vm, PI of EICA had no significant change. TCD waveforms recovered within one week. All the patients in this group survived.

CONCLUSIONSThe results suggested that mannitol could improve cerebral perfusion in patients with mild brain damage. These patients usually had increased diastolic blood flow in the early stage. Lack of or retrograde diastolic flow of TCD waveforms might be associated with severe brain damage with poor outcome. Mannitol had no effect on improving cerebral blood flow in these patients.

Adolescent ; Blood Flow Velocity ; Cardiopulmonary Resuscitation ; Cerebrovascular Circulation ; drug effects ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Mannitol ; pharmacology ; Middle Cerebral Artery ; physiopathology ; Ultrasonography, Doppler, Transcranial ; methods

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
Acrylamides/pharmacology* ; Animals ; Apoptosis/drug effects* ; Cryopreservation ; Disaccharides/pharmacology* ; Electrolytes/pharmacology* ; Glutamates/pharmacology* ; Glutathione/pharmacology* ; Heart/physiology* ; Heart Transplantation/methods* ; Hepatocyte Growth Factor/antagonists & inhibitors* ; Histidine/pharmacology* ; Histone Deacetylase Inhibitors/pharmacology* ; Intracellular Signaling Peptides and Proteins/antagonists & inhibitors* ; Male ; Mannitol/pharmacology* ; Oxidative Stress/drug effects* ; Phenylenediamines/pharmacology* ; Proto-Oncogene Proteins/antagonists & inhibitors* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; YAP-Signaling Proteins

OBJECTIVEStudy blood vessel injury and gene expression indicating vascular endothelial cell apoptosis induced by mannitol with and without administration of anti-oxidative vitamins.

METHODSHealthy rabbits were randomly divided into four groups. Mannitol was injected into the vein of the rabbit ear in each animal. Pre-treatment prior to mannitol injection was performed with normal saline (group B), vitamin C (group C) and vitamin E (group D). Blood vessel injury was assessed under electron and light microscopy. In a second experiment, cell culture specimen of human umbilical vein endothelial cells were treated with mannitol. Pre-treatment was done with normal saline (sample B), vitamin C (sample C) and vitamin E (sample D). Total RNA was extracted with the original single step procedure, followed by hybridisation and analysis of gene expression.

RESULTSIn the animal experiment, serious blood vessel injury was seen in group A and group B. Group D showed light injury only, and normal tissue without pathological changes was seen in group C. Of all 330 apoptosis-related genes analysed in human cell culture specimen, no significant difference was seen after pre-treatment with normal saline, compared with the gene chip without pre-treatment. On the gene chip pre-treated with vitamin C, 45 apoptosis genes were down-regulated and 34 anti-apoptosis genes were up-regulated. Pre-treatment with vitamin E resulted in the down-regulation of 3 apoptosis genes.

CONCLUSIONVitamin C can protect vascular endothelial cells from mannitol-induced injury.

Animals ; Antioxidants ; pharmacology ; Apoptosis ; Endothelial Cells ; cytology ; pathology ; Gene Expression Regulation ; Humans ; Mannitol ; chemistry ; pharmacology ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; chemistry ; Oxidation-Reduction ; Rabbits ; Vitamins ; metabolism

Chilling-sensitive rice varieties acquire chilling tolerance when their roots are exposed to water stress for short time. Caffeine-sensitive calcium signal was involved in this procedure. By using total RNA differential display, a chilling induced cDNA(ICT: induction of chilling treatment) was isolated from roots of chilling-sensitive rice variety. It was determined that it is a novel cDNA by homology searching. The transcript level of ict mRNA is up-regulated under chilling stress, it is decreased to low level when the samples were transferred to standard culture conditions. Pre-treated with mannitol for two hours is beneficial to inducing ICT level of expression. This chilling induction was inhibited by caffeine, suggesting that it may play a putative role in signal transduction of caffeine-sensitive calcium.
Cold Temperature ; DNA, Complementary ; genetics ; isolation & purification ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; drug effects ; Mannitol ; pharmacology ; Oryza ; drug effects ; genetics ; Plant Roots ; drug effects ; genetics ; RNA, Messenger ; drug effects ; genetics ; metabolism