Preparation and process optimization of microbial organic copper as a feed additive / Preparação e otimização do processo de cobre orgânico microbiano como aditivo alimentar

As an essential trace element for animals, copper significantly contributes to the growth and health of animals. Compared to inorganic trace elements, organic trace elements are better supplements; notably, they are acquired through microbial transformation. Therefore, we screened for copper-enriched microorganisms from high copper content soil to obtain organic copper. Sodium diethyldithio carbamate trihydrate was applied as a chromogenic agent for determining micro amounts of intracellular copper through spectrophotometry. In total, 50 fungi were isolated after the successful application of the screening platform for copper-rich microbes. Following morphological and molecular biology analyses, the N-2 strain, identified as Aspergillus niger sp. demonstrated showed better copper enrichment potential than others. Notably, the strain tolerance to copper was nearly thrice that of Saccharomyces cerevisiae, up to 1600mg/L. The content of the organic bound copper was 22.84mg Cu/g dry cell. Using the Central Composite Design (CCD) response surface method, we optimized the fermentation condition (inoculation amount, 13%; temperature, 28(C; pH, 5.0). Compared to the original strain results under the single factor fermentation condition, we reported an increase by 24.18% under the optimized conditions. Collectively, these findings provide a reference for uncovering new and low-cost organic copper additives.(AU)

Como elemento traço essencial para os animais, o cobre contribui significativamente para o crescimento e saúde dos animais. Comparado aos oligoelementos inorgânicos, os oligoelementos orgânicos são melhores suplementos; notavelmente, eles são adquiridos através de transformação microbiana. Portanto, nós selecionamos microorganismos enriquecidos com cobre de solos com alto teor de cobre para obter cobre orgânico. O carbamato de sódio diethyldithio trihidratado foi aplicado como agente cromogênico para a determinação de micro quantidades de cobre intracelular através da espectrofotometria. No total, 50 fungos foram isolados após a aplicação bem sucedida da plataforma de triagem para micróbios ricos em cobre. Após análises morfológicas e de biologia molecular, a cepa N-2, identificada como Aspergillus niger sp. demonstrou um melhor potencial de enriquecimento de cobre do que outras. Notavelmente, a tolerância da estirpe ao cobre foi quase três vezes maior que a da Saccharomyces cerevisiae, até 1600mg/L. O conteúdo de cobre ligado orgânico era de 22,84mg Cu/g de célula seca. Usando o método de superfície de resposta Central Composite Design (CCD), nós otimizamos a condição de fermentação (quantidade de inoculação, 13%; temperatura, 28C; pH, 5,0). Em comparação com os resultados da deformação original sob a condição de fermentação de fator único, relatamos um aumento de 24,18% sob as condições otimizadas. Coletivamente, estas descobertas fornecem uma referência para descobrir novos aditivos de cobre orgânico de baixo custo.(AU)

In search for better pharmacological prophylaxis for acute mountain sickness: looking in other directions.

Despite decades of research, the exact pathogenic mechanisms underlying acute mountain sickness (AMS) are still poorly understood. This fact frustrates the search for novel pharmacological prophylaxis for AMS. The prevailing view is that AMS results from an insufficient physiological response to hypoxia and that prophylaxis should aim at stimulating the response. Starting off from the opposite hypothesis that AMS may be caused by an initial excessive response to hypoxia, we suggest that directly or indirectly blunting-specific parts of the response might provide promising research alternatives. This reasoning is based on the observations that (i) humans, once acclimatized, can climb Mt Everest experiencing arterial partial oxygen pressures (PaO2) as low as 25 mmHg without AMS symptoms; (ii) paradoxically, AMS usually develops at much higher PaO2 levels; and (iii) several biomarkers, suggesting initial activation of specific pathways at such PaO2, are correlated with AMS. Apart from looking for substances that stimulate certain hypoxia triggered effects, such as the ventilatory response to hypoxia, we suggest to also investigate pharmacological means aiming at blunting certain other specific hypoxia-activated pathways, or stimulating their agonists, in the quest for better pharmacological prophylaxis for AMS.

Stachyose extract from Rehmannia glutinosa Libosch. to lower plasma glucose in normal and diabetic rats by oral administration.

The hypoglycemic effects of water extract and stachyose extract (Part III) from Rehmannia glutinosa Libosch. were investigated in this paper by oral administration to normal, glucose- and adrenaline-induced hyperglycemic and alloxan-induced diabetic rats. The results showed that Part III had the effect of lowering fasted plasma glucose level and partially preventing hyperglycemia induced by glucose (2.5 g x kg(-1), i.p.) and adrenaline (300 microg x kg(-1), i.p.), respectively, but no obvious dose-dependent effect was found when it was administered at the doses of 100, 200 and 400 mg x kg(-1) for 6 days, i.g. In alloxan-induced diabetic rats, Part III (200 mg x kg(-1) for 15 days, i.g.) gave a significant decrease in blood glucose level. The results suggested that Part III, which is mainly composed of stachyose from Rehmannia glutinosa Libosch., had a significant hypoglycemic effect in glucose- and adrenaline-induced hyperglycemic and alloxan-induced diabetic rats.

[Determination of thyroxine enantiomers in human plasma with normal high performance liquid chromatography-chiral complex exchange mobile phase].

An HPLC method was developed with normal HPLC-chiral complex mobile phase for the separation and determination of D- and L-thyroxine enantiomers (D- and L-T4) in human plasma. The method includes extraction of thyroxine from plasma and separation of thyroxine enantiomers on HPLC silica column with chiral eluent containing L-proline, cupric acetate and triethylamine (TEA). The sensitivity of the method was 0.1 mg/L. The precisions of inter-day and intra-day, linearity, extraction recovery, and stability of T4 enantiomers in plasma and in deproteinized plasma were determined for the validation of the method. Baseline enantioseparation of the compounds containing D- and L-T4 was achieved. Meantime we determined the concentrations of D-T4 and L-T4 in plasma of 15 volunteers with euthyroid, hypothyroid and hyperthyroid symptoms. Liquid chromatographic method based on the described procedure was useful for the determination of D- and L-thyroxine in patient plasma and for pharmacokinetics investigation.

Action of free radical in podophyllic acid piperindyl hydrazone nitroxide radical on its antitumor activity and toxicity.

AIM: To study the action of free radical in the spin-labeled podophyllotoxin derivative, podophyllic acid piperindyl hydrazone nitroxide radical (GP-1) on its antitumor activity and toxicity, by comparison with those of its free radical reduced product, podophyllic acid piperindyl hydrazone (GP-1-H). METHODS: After treatment with GP-1 and GP-1-H, the inhibitory effects on the growth of mouse transplantable tumors were determined; MTT formazan formation, [3H]deoxythymidine ([3H]TdR) incorporation, cell cycle progression, and mitotic index of SGC-7901 or L1210 cells were measured; the acute toxicity and immune function of mice were assayed. RESULTS: At doses of 1/6 and 1/12 LD50, the inhibitory rates against Lewis lung carcinoma were 60.3% and 42.1% (GP-1), 38.9% and 10.3% (GP-1-H), respectively; more effective antitumor activity of GP-1 against P388, HePS, and S-180 than that of GP-1-H were found. In vitro, GP-1 exhibited more powerful inhibitory effects on the proliferation and DNA synthesis of SGC-7901 and L1210 cells than GP-1-H. GP-1 and GP-1-H arrested the L1210 cells at G2/M phase with a corresponding decrease of the cells in G1 phase, and increased the mitotic index of the cells; but the effects of GP-1-H were weaker than those of GP-1. After treatment with doses of 1/4 and 1/8 LD50 for 5 d, no significant difference on immune function of mice between GP-1 and GP-1-H was found. CONCLUSION: GP-1 had more powerful antitumor activities than GP-1-H. The free radical in the spin-labeled podophyllotoxin derivative, GP-1, played an important role in its antitumor activity.

Pharmacokinetics of m-nifedipine in rabbits after intravenous injection.

AIM: To study the dose effects on pharmacokinetics of m-Nif. METHODS: Fifteen rabbits were divided into 3 groups receiving i.v. m-Nif 0.5, 1, and 2 mg.kg-1. Plasma levels of m-Nif were determined with HPLC method. RESULTS: The concentration-time data were fitted with 2-compartment model. After i.v. 1 mg.kg-1, the parameters were: Vd = 0.37 +/- 0.10 L.kg-1, T1/2 alpha = 6.4 +/- 2.9 min, T1/2 beta = 84 +/- 22 min, AUC = 94 +/- 16 mg.min.L-1, Cl = 0.65 +/- 0.13 L.kg-1.h-1. No statistically significant difference was found in Cl and T1/2 beta between 3 dose groups. AUC (standardized to body weight) was correlated with doses. CONCLUSIONS: m-Nif was distributed widely and eliminated at a fairly rapid rate in the rabbits. No dose-dependent pharmacokinetics was found after i.v. m-Nif 0.5-2 mg.kg-1. m-Nifedipine, 2, 6-dimethyl-3, 5-dicarbomethoxy-4-(3'-nitrophenyl)-1, 4-dihydropyridine (m-Nif) is a new calcium channel blocker. Dihydropyridine calcium channel antagonists are mainly used for the treatment of hypertension and angina[1]. Nifedipine is susceptible to photodegradation, but m-Nif is stable when exposed to light. The 2 drugs have the same antihypertensive effect[2]. So far, no report has been found on pharmacokinetics of m-Nif. Using a high performance liquid chromatographic (HPLC) method, we studied the dose effects on the pharmacokinetics of i.v. m-Nif 0.5, 1, and 2 mg.kg-1 in conscious rabbits.

Pharmacokinetics of 4-[4"-(2",2",6",6"-tetramethyl-1"-piperidinyloxy) amino]-4'-demethylepipodophyllotoxin in mice bearing sarcoma 180.

AIM: To study the pharmacokinetics of 4-[4"-(2",2",6",6"-tetramethyl- 1"-piperindyloxy) amino]-4'-demethylepipodophyllotoxin (GP-7) in mice bearing sarcoma 180. METHOD: Using HPLC with a uv detector at 285 nm. RESULTS: The plasma concentration-time course of GP-7 in mice was best fitted to a 2-compartment open modle after iv 20, 60 mg.kg-1. At both doses the plasma T1/2 beta was around 40 min. The highest concentration was found in liver and lung. The level of GP-7 was higher in tumor than in kidney, spleen, and bone marrow after ip 20 mg.kg-1 for 10 d. Urinary excretion of GP-7 as unchanged drug accounted for about 20% of the administered doses 72 h after injection. CONCLUSION: GP-7 disappeared more slowly from the plasma of mice bearing sarcoma 180, distributed extensively over the tissues and was partially excreted from urine. The concentrition of GP-7 in tumor was higher.

[HPLC determination of 4-[4"-(2",2",6",6"-tetramethyl-1"-piperidinyloxy) amino]-4'-demethylepipodophyllotoxin in rat plasma and studies of its pharmacokinetics].

4-[4"-(2",2",6",6"-tetramethyl-1"-piperidinyloxy) amino]-4'- demethylepipodophyllotoxin (GP-7) is a new podophyllotoxin spin-labeled derivative. Its primary effect is the antitumor activity on transplanted mouse tumors and cultured tumor cells. This paper describes a method for its determination using HPLC with UV detection and the determination of its pharmacokinetic parameters in rats. A Shimadzu LC-6A liquid chromatograph equipped with a Shimadzu SPD-6AV multiwavelength detector and a Chromatopac C-R3A data processor was used. The separation was performed on a Zorbax-ODS column (5 microns, 4.6 mm x 150 mm) with a mobile phase of methanol--water--glacial acetic acid (59:41:0.6). The flow-rate was 1.0 ml.min-1 and detection was made at 285 nm. A plasma specimen (0.2 ml) was spiked with 22.6 micrograms.ml-1 internal standard (podophyllic acid piperidinyl hydrazone nitroxide radical, GP-1) and extracted with ether--dichloromethane (3:1). The extract was evaporated at 45 degrees C. The residue was taken up with 0.1 ml of the mobile phase and 20 microliters aliquots were injected into the system. The calibration curve was linear in the range from 2 to 200 micrograms.ml-1 with r = 0.9997. The detection limit was 0.2 microgram.ml-1 and the recovery of GP-7 from rat plasma was 94.3%-100.9%. The relative standard deviations for within- day and between-day were 2.29%-4.64% and 5.55%-7.70%, respectively. After iv injection of GP-7 10, 20 and 30 mg.kg-1, the concentrations of the drug in rat plasma were determined. The pharmacokinetic parameters of GP-7 were obtained by using MCPKP program on a COMPAC-486 computer. The data obtained fitted a two-compartment open model, and the mean T1/2 beta value was 39.8 +/- 10.8 min.

The amiloride resistance gene, car1, of Schizosaccharomyces pombe.

Amiloride, an inhibitor of various sodium transporters, is toxic to Schizosaccharomyces pombe at low concentration in minimal but not in rich media. Amiloride-resistant mutants were isolated and shown to represent a new locus (car1 for changed amiloride resistance) on chromosome I. The car1 gene was cloned and sequenced. Sequence analysis revealed an open reading frame of 526 amino acids with a predicted molecular weight of 58,545 Da. It has 52% hydrophobic residues and belongs to the class of 12-transmembrane-domain transport proteins. Gene disruption of car1 results in increased amiloride resistance. car1 has sequence similarity to proteins from Candida associated with resistance to benomyl, methotrexate and cycloheximide. No single physiologically identifiable component of sodium transport appeared to be lost. We propose that car1 serves an uptake function, perhaps as a symport with an unknown substrate and this carrier may transport amiloride into the cell. Further, we suggest that amiloride toxicity at low concentrations is not due to its effect on sodium transport but, rather, depends on intracellular interference with an unknown biosynthetic pathway.

Effects of 4-[4''-(2'',2'',6'',6''-tetramethyl-1''-piperidinyloxy)amino]-4'-de methylepipodophyllotoxin on immune function in mice.

4-[4''-(2'',2'',6'',6''-Tetramethyl-1''-piperidinyloxy)amino]-4'- demethylepipodophyllotoxin (GP-7) 10-40 mg.kg-1 ip daily for 7 d reduced the specific antibody formation of splenocytes, serum agglutinin titer, and hemolysin HC50 in mice immunized with SRBC. GP-7 inhibited the footpad delayed hypersensitivity reaction and decreased the weights of spleen and thymus, but did not affect the phagocytic function of the peritoneal macrophages. In vitro the proliferation of mouse splenic lymphocytes activated by Con A was markedly inhibited by GP-7 in a concentration- dependent manner. At concentrations of 0.05-5 mg.L-1, the inhibition rates were 24-96%. These results suggested that GP-7 was an immunosuppressive agent.

Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance.

[Antitumor activity of 4-(4''-(2'',2'',6'',6''-tetramethyl-1''-piperidinyoxy)amino)-4'- demethyl epipodophyllotoxin in vitro].

The antitumor activity of a new podophyllotoxin spin-labeled derivative, 4-(4''-(2'',2'',6'',6''-tetramethyl-1''-+piperidinyoxy)amino)-4'- demethylepipodophyllotoxin (GP-7) First synthesized by us, was studied in vitro. It was found that the proliferation of SGC-7901 cells was markedly inhibited by GP-7 depending the concentration and exposure time. At concentration of 0.04-100 mg/L, the inhibition rates were 15.5-92.6% ID50 was 0.42 mg/L. After exposure to GP-7 greater than 0.5 mg/L for 24, 48, 72 and 96 h, the inhibition rates of cells were 25.1, 49.0, 71.4 and 84.9% respectively. The dose-response curve of GP-7 on SGC-7901 cell was similar to that of etoposide (VP-16). The colony formation of SGC-7901 cell was also inhibited by GP-7 in a concentration dependent fashion with ID50 1.63 mg/L. At concentrations of 0.1-0.5 mg/L, the inhibitory effects were stronger than that of VP-16. GP-7 decreased the mitotic index (MI) of SGC-7901 cell and had no effect on microtubule assembly and disassembly in vitro, which suggested that GP-7 did not act on M phase.

Anticancer drugs. II. Synthesis and biological evaluation of spin labeled derivatives of podophyllotoxin.

The spin labeled derivatives of podophyllotoxin, 4-[4''-(2'',2'',6'',6''-tetramethyl-l''-piperidinyloxy)amino] -4'-demethylepipodophyllotoxin(GP-7,3) and N-podophyllic acid-N''-[4-(2,2,6,6-tetramethyl-l-piperidinyloxy)] thiosemicarbazide(GP-4,5) were synthesized and tested for their anticancer activity against the mouse solid tumors S180 and HepA in vivo, and the mouse lymphocytic leukemia L1210 and human stomach carcinoma SGC-7901 cells in vitro. At equitoxic concentrations, the anticancer activity of GP-7(3) was found to be similar to that of the clinically used VP-16(2). The toxicity of GP-7(3) (LD50231.2 mg/Kg) is 3.3 times lower than that of VP-16 (LD50 69.5 mg/Kg). GP-7(3) exhibits low subchronic toxicity. The total chemical yield of GP-7 (26%) is 4 times higher than that of VP-16 (6%) (based on podophyllotoxin). Therefore, GP-7(3) seems to be a promising new entry into the podophyllotoxin class of anticancer drugs.