The polarography of cephalosporin C derivatives

WE report some preliminary measurements on the polarographic reduction of cephalosporin C, cephalothin [7-(2-thienylacetamido)-cephalosporanic acid] and cephaloridine [7-(2-thienylacetamido)-3-(1-pyridylmethyl)-3-cephem-4-carboxylic acid betaine]. Green, Page & Staniforth (1965) have shown that cephalosporin C derivatives give well-defined infrared and nuclear magnetic resonance spectra that can be used for their qualitative identification. Martin & Shaw (1965) have reviewed the application of methods such as ultraviolet spectrophotometry, paper chromatography and paper electrophoresis for cephaloridine, while Chapman, Page & others (1968) have described infrared and X-ray powder measurements for this compound. The application of mass spectroscopy to the structural elucidation of cephalosporin derivatives has been reported by Richter & Biemann (1965).     

Carbon catabolite regulation of the conversion of penicillin N into cephalosporin C

Cephalosporin C biosynthesis by Cephalosporium acremonium was delayed until most glucose in the medium was used. Addition of increasing concentrations of glucose up to 55 g/liter decreased cephalosporin C biosynthesis but stimulated growth. Sequential formation of penicillin N (an intermediate in the cephalosporin C biosynthetic pathway) and cephalosporin C was found when the culture was developed synchronously. Little cephalosporin C formation was observed until most penicillin N had already been formed. The sequential formation of penicillin N and cephalosporin C was due to the sequential formation of the "penicillin N synthetase system" and the "cephalosporin C synthetase system". Cells grown in the presence of glucose showed an increased accumulation of penicillin N and clear reduction of the conversion of penicillin N to cephalosporin C. Resting cell studies indicated that the glucose effect was due to the repression of one or more of the enzymes converting penicillin N into cephalosporin C. Little inhibition by glucose of the activity of these enzymes, once formed, was observed. Glucose did not effect significantly the pool sizes of either precursor amino acids of cephalosporin (alpha-aminoadipic acid and valine) or methionine (an inducer of penicillin N and cephalosporin C biosynthesis). On the basis of these data it is suggested that glucose catabolism specifically represses the enzyme system converting penicillin N into cephalosporin C.     

Effect of methionine on cephalosporin C and penicillin N production by a mutant of Cephalosporium acremonium.

A mutant with enhanced potential to utilize sulfate for cephalosporin C production was isolated from a strain of Cephalosporium acremonium. The mutant displayed potency levels more than twofold that of the parent in the presence of sulfate but its productivity was severely inhibited by more than 0.5% of methionine which gave high cephalosporin C production with the parent. In a complex medium norleucine stimulated cephalosporin C production by the mutant in the presence of sulfate, whereas it showed no effect on the parent. In an incubation system with sulfur-starved cells of the mutant, L-methionine, but not the D-isomer, gave lower cephalosporin C production and a delayed production of penicillin N. However, it exhibited a stimulatory effect in the presence of valine or alpha-aminoadipic acid, the constituent amino acids of the antibiotic. Norleucine showed a similar effect to that of L-methionine in the presence of sulfate. On the basis of these results, characteristics of the mutant are discussed in connection with the effect of methionine.     

Custom made C18 columns for the hydrophobic chromatography of cephalosporin C derivatives

"Hydrophobic chromatography", which is a variation of reverse phase chromatography, is applicable to the analysis of cephalosporin C derivatives, especially in fermentation broths. Unfortunately, there are no commercial C18 columns which are entirely suitable for this class of compounds. For this reason C18 columns were prepared by an in-situ bonding technique and were optimally designed for cephalosporin C derivatives. Mono-, di- and trifunctional octadecyl bonding agents were used with 10 microns silica of both 60 A and 100 A pore diameter. The best results were obtained with the difunctional agent, methyloctadecyldichlorosilane, and 100 A silica. "Endcapping" of residual silanol groups with a trimethylsilylation agent was optional, since good results were obtained with both a plain C18 column and one that was "endcapped".     

Studies on the cephalosporin C biosynthesis by fermentation

UsingCephalosporium acremonium ATCC 14553, we studied on the important factors in fermentation. Those factors were: carbon and nitrogen sources, methionine effect, adsorbent effect, fermentation variables in a batch fermentation, effect of reuse of its cellular biomass as a reusable nutrient, and 2,4-dinitrophenol effect on cephalosporin C production.     

解吸方法对头孢菌素C提取质量的影响

目的研究不同的解吸方法对头孢菌素C提取质量的影响。方法将头孢菌素C发酵上清液通过XAD16大孔吸附树脂进行吸附,然后分别采用单一浓度解吸(均一解吸)及梯度浓度解吸的方法进行解吸。结果梯度浓度解吸与单一浓度解吸相比,解吸液的纯度提高3.23%,解吸收率提高2.3%。结论梯度浓度解吸法比单一浓度解吸法具有更多的优势,值得推广和应用。     

Production and optimization studies of cephalosporin C by solid state fermentation

Production of cephalosporin C employing a strain, Cephalosporium sp. NCIM 1039 under solid state fermentation was optimized. Different substrates like wheat bran, wheat grains, rice grains, barley and rice bran were studied to optimize the best substrate. Wheat bran showed the highest antibiotic yield. The physical and chemical parameters were optimized. The maximum productivity of cephalosporin C (750 U/g) was achieved by employing wheat bran and with optimized nutritional and process parameters such as potato starch as additive 1% w/w, urea as additive 1% w/w, incubation period of 7 days, incubation temperature at 30 degrees C, inoculum level 10% w/v, moisture content of solid substrate 80% and pH 7.0.     

Immunomodulatory effects of two extracts of Panax ginseng C.A. Meyer

The effect of Panax ginseng extracts on cell-mediated immune functions in man has been investigated. Three groups, each consisting of twenty healthy volunteers, were treated under conditions of double blindness with capsules containing lactose (Control Group B), with capsules containing 100 mg of aqueous extract of the drug (Group A), and with capsules containing 100 mg of standardized extract of the drug (Group C). All the patients took one capsule every 12 h for 8 weeks. Blood samples were withdrawn before beginning the treatment, at the fourth week and at the eighth week. The immune parameters examined were the following: chemotaxis of PMNs, phagocytosis index (PHI), phagocytosis fraction (PHF), intracellular killing, total lymphocytes (T3), T helper (T4) subset, suppressor cells (T8) subset, blastogenesis of circulating lymphocytes, natural killer-cell activity (NK). Chemotaxis proved to be enhanced (p less than 0.05) already at the fourth week in Group A as well as in Group C; the increase became even more marked (p less than 0.001) at the eight week in subjects belonging to Group C. PHI and PHF proved to be enhanced (p less than 0.05) at the eighth week in subjects of Group A; these increases were found to be higher in subjects of Group C (p less than 0.001) already starting at the fourth week. Intracellular killing was shown to be significantly increased (p less than 0.05) already at the fourth week in Groups A and C; the increase becomes highly significant in both groups (p less than 0.001) at the eighth week; however, a significant increase (p less than 0.05) at the eighth week was also noticed in the placebo group (Group B).(ABSTRACT TRUNCATED AT 250 WORDS)     

响应面法优化头孢菌素C发酵条件

目的 研究顶头孢霉发酵制备头孢菌素C的过程，确定关键影响因素，优化发酵条件，提高发酵水平。 方法 采用Plackett-Burman方法对影响头孢菌素C发酵水平的11个因素进行考察，评估各因素的影响程度，筛查影响发酵水平的关键因素，再采用响应面方法建立这些因素与发酵水平间的数学模型，为发酵条件的优化提供指导。 结果 Plackett-Burman实验发现，金枪鱼浸膏浓度和蛋氨酸浓度是对头孢菌素C发酵产量影响最显著的2个因素，经响应面方法优化后头孢菌素C的产量从32.59g/L提高到了35.99g/L，提高了10.4%。 结论 合适浓度的金枪鱼浸膏和蛋氨酸可提高头孢菌素C发酵产量，通过统计优化方法，可有效提高头孢菌素C发酵水平。     

Strain improvement studies for cephalosporin C production by Cephalosporium acremonium

Treatment of spore suspension of Cephalosporium acremonium ATCC 48272 with UV rays and N-methyl-N-nitro-N-nitrosoguanidine (NTG) induced mutants capable of producing higher yields of cephalosporin C. Antibiotic yield was improved from 630 micrograms/ml to 1995 micrograms/ml of the broth resulting in a high yielding mutant.     

头孢菌素C发酵液流变特性的研究

通过测定头孢菌素C发酵液的粘度，研究非牛顿体系的流变特性，根据体系的凯松流体模型确定本构方程。     

Quinoline antifolate thymidylate synthase inhibitors: variation of the C2- and C4-substituents.

Modifications to the bicyclic ring system of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (1, CB3717) have led to the synthesis of a series of quinoline antifolates bearing a variety of substituents at the C2 and C4 positions. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with a disubstituted 6-(bromomethyl)quinoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 TS. As a measure of cytotoxicity, the compounds were tested for their inhibition of the growth of L1210 cells in culture. Good enzyme inhibition and cytotoxicity were found for compounds containing chloro, amino, or methyl substituents at the C2 position with chloro or bromo substituents at C4. The effect on enzyme inhibition of varying the N10 substituent of 2h was similar to that observed in the quinazolinone-containing antifolates, indicating that the quinoline compounds may be interacting with the enzyme in a similar way to the quinazolinones. Also, the introduction of a 2'-fluoro substituent into the benzoyl ring of several of the quinoline antifolates led to an increase in both TS inhibition and the inhibition of L1210 cell growth. These data demonstrate that the N3-H of the pyrimidine ring of the quinazolinone antifolates is not required for binding to TS if appropriate substituents are placed at the C2 and C4 positions of the bicyclic ring system.     

Stability of three cephalosporin antibiotics in AutoDose Infusion System bags

OBJECTIVE: To evaluate the physical and chemical stability of three commonly used cephalosporin antibiotic solutions packaged in AutoDose Infusion System bags stored and evaluated at appropriate intervals for up to 7 days at 23 degrees C and up to 30 days at 4 degrees C. SETTING: Laboratory. INTERVENTIONS: The test samples were prepared by adding the required amount of the cephalosporin antibiotic to the AutoDose Infusion System bags and diluting to the target concentration with 0.9% sodium chloride injection. MAIN OUTCOME MEASURES: Physical stability and chemical stability based on drug concentrations initially and at appropriate intervals over periods of up to 7 days at 23 degrees C and up to 30 days at 4 degrees C. RESULTS: All of the cephalosporin admixtures were clear when viewed in normal fluorescent room light and with a Tyndall beam. Measured turbidity and particulate content were low and exhibited little change. The cefazolin sodium-containing samples were colorless throughout the study. The admixtures with ceftazidime and ceftriaxone sodium had a slight yellow tinge initially, and the room temperature samples turned a frank yellow color after 5 days. The refrigerated samples did not change color. High-performance liquid chromatography analysis showed that cefazolin sodium and ceftriaxone sodium remained stable for 30 days and ceftazidime remained stable for 7 days at 4 degrees C. At room temperature, losses were much more rapid. Cefazolin sodium and ceftriaxone sodium retained at least 90% of their initial concentrations through 7 days and 5 days, respectively, when stored at 23 degrees C. Ceftazidime remained stable for only 1 day at 23 degrees C. CONCLUSION: Cefazolin sodium, ceftazidime, and ceftriaxone sodium exhibited physical and chemical stabilities consistent with those found in previous studies of these drugs. The AutoDose Infusion System bags did not adversely affect the physical and chemical stabilities of these three cephalosporin antibiotics.     

Compatibility and stability of linezolid injection admixed with three cephalosporin antibiotics

OBJECTIVE: To evaluate the physical compatibility and chemical stability of linezolid (Zyvox-Pharmacia) 200 mg/100 mL admixed with cefazolin sodium 1 gram, ceftazidime 2 grams, and ceftriaxone sodium 1 gram for 7 days at 4 degrees C and 23 degrees C. DESIGN: Controlled experimental trial. SETTING: Laboratory. INTERVENTIONS: The test samples were prepared by adding the required amount of the cephalosporin antibiotic to bags of linezolid injection 200 mg/100 mL. MAIN OUTCOME MEASURES: Physical stability and chemical stability based on drug concentrations initially and after 1, 3, 5, and 7 days of storage at 4 degrees C and 23 degrees C protected from light. RESULTS: All of the linezolid admixtures with cephalosporins were clear when viewed in normal fluorescent room light and with a Tyndall beam. Measured turbidity and particulate content were low and exhibited little change. The cefazolin sodium-containing samples were colorless throughout the study. The admixtures with ceftazidime and ceftriaxone sodium had a slight yellow tinge initially, and the room temperature samples became a frank yellow color after 5 days. The refrigerated samples did not change color. High-performance liquid chromatography analysis found little or no loss of linezolid in any sample stored at either temperature throughout the study. Cefazolin sodium and ceftazidime in the linezolid admixtures at 4 degrees C remained stable for 7 days, but at 23 degrees C cefazolin sodium was stable for 3 days and ceftazidime for only 24 hours before cephalosporin decomposition exceeded 10%. Ceftriaxone sodium was less stable in the admixtures; 10% loss occurred in 3 days at 4 degrees C and more than 20% loss occurred in 24 hours at 23 degrees C. CONCLUSION: Admixtures of linezolid 200 mg/100 mL with cefazolin sodium 1 gram and ceftazidime 2 grams were physically compatible and chemically stable for at least 7 days stored at 4 degrees C protected from light and for 3 days and 1 day, respectively, at 23 degrees C protected from light. Admixtures of linezolid with ceftriaxone sodium 1 gram exhibited a rapid rate of cephalosporin loss at 23 degrees C, which precludes admixture of the two drugs.     

Cefodizime, an aminothiazolyl cephalosporin. IV. Influence on the immune system

Studies concerning the activity of cefodizime (HR 221), on certain aspects of the immune response, were conducted. It was found that lymphocytes from Balb/c mice treated with 3 and 30 mg/kg/day of cefodizime display increased responsiveness to B-cell mitogens and specific antigens. Also, the amount of antigen specific antibody producing plaque forming cells was increased in these mice and was accompanied by a rise in the specific IgG haemagglutinin titer. These effects were not observed in lymphocytes obtained from NMRI mice that had been treated with cefodizime. Peritoneal macrophages from NMRI mice, treated with cefodizime prior to harvesting of the cells, contained increased levels of lysosomal enzymes, developed enhanced chemiluminescent reaction to stimuli and showed elevated pinocytosis rates. Furthermore, NMRI mice treated with cefodizime during the immunization, developed enhanced DTH-reaction, when challenged with the antigen (SRBC). The prophylactic treatment of Balb/c mice with cefodizime (2 X 30 mg/kg/day ip for 4 days) significantly prolonged the mean survival time of the animals after intravenous infection with Candida albicans 200/175 (16.7 days as against 3.5 days in the case of the controls). This stimulatory effect of cefodizime on the host defence system was not observed for NMRI mice. Treatment with latamoxef or cefoperazone under the same experimental conditions did not reduce the susceptibility of mice to C. albicans. The protective activity of cefodizime against C. albicans in Balb/c mice, may be due to the immuno-stimulatory activity of this agent.     

Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9

1. Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared. 2. Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes. Two N-terminal modifications (Delta3-20 and 17alpha-leader) of the individual P450s were additionally investigated. 3. Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity. CYP expression was higher with the Delta3-20 construct for CYP2C9 and with the 17alpha-presequence construct for CYP2C8. 4. Using torsemide as substrate, all methods gave catalytically active systems with K(m) values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the Delta3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17alpha- presequence constructs. OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes. 5. Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.     

利用神经网络预测头孢菌素C的生物合成

利用头孢菌素C发酵过程积累的数据,建立BP神经网络预估模型,实现以发酵前期的菌浓和pH对效价的预测,将此模型应用于生产实际,分别通过倒种和改变培养基中碳源组成的方法,使头孢菌素C的合成水平分别提高了11.8%和15.7%,表明模型具有较好的预测功能。     

紫外诱变和终产物抗性筛选头孢菌素C高产菌株

目的建立筛选头孢菌素C高产菌株的有效方法。方法出发菌株Cephalosporium acrem onium NS-1,经过紫外线诱变处理,筛选头孢菌素C高产菌株;然后通过头孢菌素C梯度平板筛选终产物抗性突变菌株。结果得到比出发菌株头孢菌素C产量提高45%的突变菌株RM-8。结论紫外线诱变结合终产物抗性筛选的方法是一种高效的头孢菌素C高产菌株选育方法 。     

Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9

Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared.

Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes. Two N-terminal modifications (A3-20 and 17a-leader) of the individual P450s were additionally investigated.

Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity. CYP expression was higher with the A3-20 construct for CYP2C9 and with the 17a-presequence construct for CYP2C8.

Using torsemide as substrate, all methods gave catalytically active systems with K_m values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the A3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17a- presequence constructs. OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes.

Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.     

Derivation of RNA aptamer inhibitors of human complement C5.

Specific aptamer inhibitors of the human complement C5 component were produced by the SELEX methodology of directed evolution of nucleic acid ligands. The SELEX procedure started with a pool of random-sequence, 2'F-pyrimidine-modified nuclease-stabilized RNA, and after twelve rounds of iterative C5 binding and nucleic acid amplification an evolved RNA pool was obtained which contained the highest affinity binders to the C5 protein. The evolved RNA pool was then cloned and sequenced, and individual clones were analyzed for binding and function. Twenty-eight clones (out of sixty) were identified which bound C5 (termed aptamers). Seven of these aptamers formed a closely related sequence homology family; these aptamers bound C5 with a Kd 20-40 nM and also inhibited human serum hemolytic activity. In addition, these aptamers inhibited zymosan-induced generation of C5a. Aptamer inhibition of both C5b and C5a suggests that aptamer binding inhibits cleavage of C5 by the C5 convertase of both pathways. One of the inhibitory aptamer sequences was truncated to yield a 38-mer 2'F RNA aptamer which retained C5 binding and inhibitory activity. The structure of this aptamer is predicted to be a stem-loop containing thirteen base pairs, and also containing two bulges. The affinity of this aptamer was improved by performing a second biased SELEX experiment, where the randomized starting RNA pool uses a template where the individual base compositions are biased toward a specific sequence. This second SELEX experiment produced an aptamer with a Kd of 2-5 nM which retained functional activity. Another SELEX to rat C5 produced an aptamer with binding and inhibitory properties virtually identical with the human aptamer. The human and rat aptamers are being evaluated for complement inhibition in vitro and in vivo as potential therapeutics for treatment of human disease.