Application of surface response analysis to the optimization of penicillin acylase purification in aqueous two-phase systems

Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350–sodium citrate aqueous two-phase systems (ATPS) was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, sodium citrate and NaCl concentrations of 15.1, 11.0 and 8.52% respectively. However, under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%.     

Effect of trisodium citrate treatment on hybridoma cell viability

Trisodium citrate has been widely used to dissolve calcium alginate beads in order to estimate cell concentration in the beads. To obtain an accurate measurement of viable cell concentration in calcium alginate beads, the effect of trisodium citrate solutions on hybridoma cell viability was studied with regard to stage of growth and trisodium citrate concentration. The cells in the decline phase of growth were more sensitive to 30 minutes of trisodium citrate treatment than the cells in the exponential phase of growth. The cell viability did not decrease rapidly during citrate treatment regardless of cell growth phase and trisodium citrate concentration in the range of 1–1.5%. By using the commercially available sodium alginate, Keltone LV, dissolution time can be kept short enough (below 5 minutes) to keep the effect of trisodium citrate negligible to cell viability.     

Slide Processing for the Examination of Male Mammalian Meiotic Chromosomes

A rapid method was devised specifically for the cytological identification of translocations in the male mouse at late prophase to metaphase of meiotic division I, but the method should be useful for less specific objectives requiring examination of mammalian testicular material. For the adult mouse, masses of tubules from a single testis, freed of the testicular tunic, are placed in 3 ml of 0.7% sodium citrate for 15-20 min, and subsequently fixed in 50% acetic acid by the addition of 3 ml of glacial acetic acid to the hypotonic citrate. To facilitate handling of individual tubules by preserving their visible structure, the addition of fixative is at a rate which is grossly adjusted so that 2 ml will have been added at the end of 30 sec and the remaining 1 ml by the end of a minute. A single fixed tubule 1-2 cm long is placed lengthwise on a slide and covered with a drop of lactic-acetic orcein made as follows: Add 2 gm of synthetic orcein (G. T. Gurr) to a mixture of 50.0 ml of glacial acetic acid, 42.5 ml of 85% lactic acid, and 7.5 ml of distilled water. After staining for 10 min, a 22 × 50 mm cover slip is placed over the tubule, and it is allowed to stain for an additional 10 min. The majority of germinal cells will not be in late prophase or metaphase of the first meiotic division, therefore many preparations will be useless; however, slides with division figures are radidly selected as follows: Before squashing, examine under a microscope at a magnification of 150, and upon recognition of a single meiotic division, remove the slide and squash the preparation for subsequent detailed examination. As a consequence of the spermatogenic wave that progresses along the length of a tubule, a given slide will usually have many division figures or none at all, hence the limitation of 1 tubule per slide facilitates efficient discarding. Preliminary work with the Chinese hamster suggests that good preparations might be obtained from testes of various mammals when the volume of hypotonic solution is adjusted so as to compensate for varying testicular sizes by maintaining a 15:1 ratio of fluid to estimated volume of tissue.     

The differential requirement of albumin and sodium citrate on the development of in vitro produced bovine embryos

In vitro culture for bovine embryos is largely not optimal. Our study was to determine the components necessary for early embryo development. In experiment 1, IVF embryos were cultured for two days in CR1aa medium containing sodium citrate and BSA from two sources (Sigma vs. ICPbio), subsequently for additional five days with cumulus monolayer in 10% FBS CR1aa. We found that supplementation with both Sigma-BSA and sodium citrate significantly increased total blastocyst (BL) development compared with the ICPbio-BSA groups (37% vs. 19-21%), and enhanced the total number of high quality (C1 BL, IETS standard) blastocysts (26% vs. 11-17%) (P < 0.05). In experiment 2 with serum free and/or somatic free culture, we found that CR1aa culture can support a comparable embryo development with a supplement of Sigma BSA. The addition of sodium citrate did not increase blastocyst development in either the Sigma-BSA or the ICPbio-BSA groups. An inferior blastocyst development occurring in ICPbio-BSA culture (1-3%) could be rescued by culture in CRlaa supplemented with 10% FBS (29%), more importantly, by culture in CR1aa with a replacement of Sigma BSA (24%) (P <0.05). C1 blastocysts rescued by FBS and Sigma BSA in ICPbio-BSA culture possessed indistinguishable morphology to embryos developed in a Sigma-BSA, FBS and somatic co-culture system, showing similar cell number/blastocyst (129-180, P > 0.05). Our study found a beneficial effect of sodium citrate and BSA on the in vitro development of bovine IVF embryos during co-culture. We also determined that differential embryotrophic factor(s) contained in BSA and serum, probably not sodium citrate, is necessary for promoting competent morula and blastocyst development in cattle.     

The chemical state of the calcium reacting in the coagulation of blood

1. The widely accepted theory that calcium participates in the coagulation mechanism in the form of Ca(++) and acts as a catalyst is not in accord with several important experimental findings: (a) The anticoagulant action of sodium oxalate is much slower than the precipitation of ionized calcium as the oxalate salt. (b) Sodium citrate begins to depress prothrombin activity at a concentration at which ionized calcium is still present. The inability of tricalcium phosphate to adsorb prothrombin from citrated plasma indicates that citrate forms a complex with prothrombin and it is postulated that prothrombin is thereby inactivated. (c) In plasma which is decalcified, i.e. in which the Ca(++) is markedly reduced, the labile factor of prothrombin rapidly decreases. A concentration of 0.01 M sodium citrate sufficient to inhibit coagulation does not depress Ca(++) enough to cause diminution of the labile factor, whereas when the concentration is increased to 0.02 M the labile factor decreases as rapidly as in oxalated plasma. 2. It is postulated that calcium functions in coagulation not as Ca(++) but as combined with a component which is part of the prothrombin complex that is not adsorbed by tricalcium phosphate. A concentration of sodium citrate just sufficient to inhibit coagulation is not enough to remove calcium from the essential prothrombin component. The primary anticoagulant action of sodium citrate is therefore not decalcification but antiprothrombic. 3. It has been shown that citrated plasma is basically different from oxalated plasma in several important aspects. Unless cognizance is taken of these differences, serious errors and misinterpretations of experimental findings may be made.     

Effect of homogenization procedures on Escherichia coli counts in naturally contaminated cheese

A comparison was made between blending in 2% sodium citrate and stomaching in 0.1% peptone or 0.1% peptone - 1% Tween 80 for the enumeration of Escherichia coli in naturally contaminated cheeses. Statistical analysis of the results from 25 samples of cheese showed that there were no significant differences in recovery by the three methods at the 95% confidence level.     

Genotoxicity of five food preservatives tested on root tips of Allium cepa L

The effects of the food preservatives sodium benzoate (SB), boric acid (BA), citric acid (CA), potassium citrate (PC) and sodium citrate (SC) have been studied on root tips of Allium cepa L. Roots of A. cepa were treated with a series of concentrations, ranging from 20 to 100 ppm for 5, 10 and 20 h. The results indicate that these food preservatives reduced mitotic division in A. cepa compared with the respective control. Mitotic index values were generally decreased with increasing concentrations and longer treatment times. Additionally, variations in the percentage of mitotic stages were observed. The total percentage of aberrations generally increased with increasing concentrations of these chemicals and the longer period of treatment. Different abnormal mitotic figures were observed in all mitotic phases. Among these abnormalities were anaphase bridges, C-mitosis, micronuclei, lagging, stickiness, breaks and unequal distribution.     

Proceedings: Influence of tryptophan on the hypotensive effect of arachidonic acid

When arachidonic acid (AA) was given to a rabbit which was previously heparinized, its arterial pressure began to fall. Indomethacin erased this vasodepressive power without modifying the sensitivity of the cardiovascular apparatus to prostaglandins. Thus, it seems to depend on the biosynthesis of the prostaglandins. This hypotensive action is used as an indication of natural factors which modify the biosynthesis of prostaglandins in living subjects. Tryptophan was used on 8 male rabbits who had been fed normally, weighing 2.3-2.8 kg. The standard dose of .12 mg/kg of AA was injected in 80 seconds. 3 experiments were done without heparin, using a manometer in which the liquid had been made incoaguable by citrate. With 5 others, after injection of AA, the isotonic liquid with citrate was replaced by a manometer with heparin (20 mg/kg of heparin to avoid reabsorption of citrate during hypotension). Several injections of AA were given until a constant hypotensive action resulted; then 4 mg/kg of tryptophan were given and 10 minutes later, a new injection of AA. The results were: 1) With .12 mg/kg there was no hypotensive effect in the rabbit without heparin. 2) A previous treatment with 4 mg/kg of tryptophan achieved a fall of 5-15%. 3) In heparinized rabbits, after AA injections, there were falls of pressure which intensified and attained 25% 40 minutes after the heparin injection. 4) The injection of tryptophan obtained hypotensions of 45-50% in 5 rabbits. It is concluded that in rabbits tryptophan is an activator of the biosynthesis of prostaglandins.     

Biosequestering Potential of Spirulina platensis for Uranium

The uranium sequestering potential of Spirulina platensis was studied in batch mode, and its equilibrium was established in approximately 60 minutes. It had maximum sorption at pH 4.0 to 4.5. Equilibrium data is well represented by the Langmuir isotherm model followed by the Freundlich and Redlich-Peterson models. The interference of other cations and anions in solution was found to decrease sorption of the uranium, suggesting a competition for sorption sites on S. platensis. The desorption results showed that sodium citrate solution is effective, with 83% of uranium being recovered through nondestructive means.     

Improved resolution of flow cytometric measurements of Hoechst- and chromomycin-A3-stained human chromosomes after addition of citrate and sulfite

The resolution of bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 and chromomycin A3 can be increased by adding sodium citrate and sodium sulfite to the chromosomes shortly before measurement. A flow karyotype of a patient with chronic myelocytic leukemia is shown to illustrate that the addition of these compounds allows high-resolution measurements to be made and evaluated reliably from clinical samples.     

The effect of citric acid, lactic acid, sodium citrate and sodium lactate, alone and in combination with nisin, on the growth of Arcobacter butzleri

The importance of Arcobacter spp. as a cause of human foodborne illness is unresolved. Organic acids and their sodium salts, and nisin are preservatives commonly used in the type of foods from which the organism is recovered. In this study their effect on the growth of A. butzleri in culture, alone and in combination, was investigated. At 0.5%, 1.0% and 2.0% lactic and citric acids inhibited A. butzleri growth; 2% sodium lactate was effective in inhibiting growth over 8 h incubation but not over longer periods. Sodium citrate was more effective than sodium lactate. Nisin alone inhibited A. butzleri growth at 500 IU ml-1 over 5 h. It did not enhance the effect of sodium citrate inhibition but it did augment the effect of sodium lactate alone over 8 h.     

柠檬酸钠对L-组氨酸发酵代谢流分布的影响

目的：建立谷氨酸棒杆菌TL1105生物合成L-组氨酸的代谢网络模型,并进行代谢网络计量分析。方法：通过所构建的L-组氨酸代谢网络模型,利用MATLAB软件计算出添加柠檬酸钠和不添加柠檬酸钠发酵中后期代谢网络的代谢流分布。结果：在L-组氨酸分批发酵过程中,在发酵初期未添加柠檬酸钠的条件下流向戊糖磷酸途径（HMP）的代谢流为9.59,合成组氨酸的代谢流为8.91;在发酵初期添加2g/L柠檬酸钠的条件下流向HMP的代谢流为12.74,合成组氨酸的代谢流为9.61。结论：在发酵初期添加柠檬酸钠能够改变L-组氨酸生物合成途径的关键节点6-磷酸葡萄糖、丙酮酸及乙酰辅酶A的代谢流分布,保持糖酵解途径、三羧酸循环与HMP之间代谢流量平衡,有利于提高L-组氨酸生物合成途径的代谢流量,最终使流向组氨酸的代谢流增加了7.86%。     

Isolation of high quality RNA and construction of a suppression subtractive hybridization library from ramie (Boehmeria nivea L. Gaud.)

Isolation of high quality RNA from ramie (Boehmeria nivea L. Gaud.) is difficult due to its high levels of polyphenols, polysaccharides, pectin, fat, wax and other secondary metabolites. A modified procedure based on guanidinium isothiocyanate for RNA preparation of ramie was developed in this study. High concentrations (5%, v/v) of guanidinium isothiocyanate, PVP-4000, sodium citrate and sodium lauryl sarcosinate and β-mercaptoethanol were used in the extraction buffer, together with a low pH sodium acetate (pH 4.0) added to improve the RNA quality. The average yield was about 400 μg RNAg^?1 fresh leaves. One SSH library which was induced by ramie anthracnose was constructed by utilizing the RNA extracted through the present method. These results showed that our protocol was applicable for RNA isolation from recalcitrant ramie tissues.     

Simplified procedure for the analysis of 3- and 4-hydroxyproline

A column chromatographic analysis of 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and γ-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before asparti acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collager, metabolism disorders.     

The mechanisms of citrate on regulating the distribution of carbon flux in the biosynthesis of uridine 5′-monophosphate by Saccharomyces cerevisiae

A whole cell biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l⁻¹ sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle.     

Genotoxicity of five food preservatives tested on root tips of Allium cepa L.

The effects of the food preservatives sodium benzoate (SB), boric acid (BA), citric acid (CA), potassium citrate (PC) and sodium citrate (SC) have been studied on root tips of Allium cepa L. Roots of A. cepa were treated with a series of concentrations, ranging from 20 to 100 ppm for 5, 10 and 20 h. The results indicate that these food preservatives reduced mitotic division in A. cepa compared with the respective control. Mitotic index values were generally decreased with increasing concentrations and longer treatment times. Additionally, variations in the percentage of mitotic stages were observed. The total percentage of aberrations generally increased with increasing concentrations of these chemicals and the longer period of treatment. Different abnormal mitotic figures were observed in all mitotic phases. Among these abnormalities were anaphase bridges, C-mitosis, micronuclei, lagging, stickiness, breaks and unequal distiribution.     

A Colchicine, Hypotonic Citrate, Air Drying Sequence for Foetal Mammalian Chromosomes

Mice 14 days pregnant were given 0.3 ml of 0.025% Colcemid (Demecolcine, Ciba) and killed after 1 hr. The livers of the foetuses were removed and broken up in 0.1% Colcemid in phosphate-buffered 0.85% NaCl and left 1.5 hr. The cell suspension was then centrifuged, resuspended in 1% sodium citrate for 20 min, centrifuged and the cells fixed in acetic-alcohol (1:3) for 30 min at 4°C. The cells were then resuspended (twice) in 45% acetic acid and dropped onto warm slides. After drying, the cells were stained for 30 min with lactic-acetic-orcein and examined under oil-immersion without a cover slip. Good numbers of well-spread mitotic figures were obtained.     

Histochemical Demonstration of Acid Phosphatase in Hard Tissues

The action of the following decalcifying solutions for the demonstration of acid phosphatase has been studied: buffer solution acetic-acetate 0.05 M, pH 5; 2, 5, 10, 20, and 50% formic acid and 20% sodium citrate in equal parts (pH 2.6, 3, 3.8, 4.2, and 5); 0.5 M citric acid-NaOH, pH 4.2; Versene solution, 5%, pH 7. A comparative study of fixatives has been made also (neutral formalin, 10-20%; formalin-chloral hydrate (Fishman), acetone and 80% alcohol). The best results were obtained with fixation at 4°C in 10-20% neutral formalin or formalin-chloral hydrate, for a period of 24 hr, and decalcification with 20% sodium citrate, 5% formic acid, in equal parts, pH 4.2, which can act on both specimens or sections for a period up to 2 wk with very little loss of enzyme. It is not necessary to reactivate the enzyme after decalcification; frozen sections should be used and should be washed in distilled water before proceeding with the demonstration of the enzyme (Gomori's method or azo dye coupling). Other fixatives (acetone and alcohol) and paraffin embedding produce a greater loss in enzymes and very irregular results.     

Chromosome Preparations from the Avian Allantoic Sac

An abundance of mitotic cells, a rapid and uniform response of cells to mitotic inhibition, ease in obtaining a monolayer of cells, clear and well-spread chromosomes make the allantois an ideal tissue for squash preparations. After a 45 min incubation of each embryo, still in the shell, with 0.02 ml of 0.05% Colcemid, 4-day avian embryos were treated in distilled water for 15 min or in 0.9% sodium citrate for 30-60 min and then fixed for at least 1 hr in 1:3 acetic-alcohol. Squash preparations were made after immersion of the allantois in 45% acetic acid for 5-10 mm. Phase contrast microscopy could be used, or permanent preparations made by freezing, air-drying and staining. Staining with Gram's iodine for 4 min followed by 1% crystal violet in 95% ethanol for 3 min is recommended. The allantois is well suited for use in biology laboratories to demonstrate avian chromosomes in different stages of mitosis.