Quantitative cytochemical aspects of a combined Feulgen-Naphthol Yellow S staining procedure for the simultaneous determination of nuclear and cytoplasmic proteins and DNA in mammalian cells

The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained:

1. 1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis.

2. 2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95).

3. 3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity.

4. 4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining.

From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.     

Isolation of galactinol from leaves of Cucumis sativus

A simple procedure for the isolation of galactinol from leaves of Cucumimis sativus L. has been developed. The procedure yielded approx. 60% of the galactinol originally present in leaves with an apparent purity of 97%. Gas chromatographic and mass spectral analysis indicated that the compound was identical to galactinol isolated from sugar beet. The cucumber leaf galactinol was found to be suitable as a substrate for a galactosyl transferase enzyme which catalyses the formation of stachyose.     

Effect of medium composition on L-Lysine production by a variant strain ofCorynebacterium clutamicum ATCC 21513

Summary The strainCorunebacterium glutamicum ATCC 21513 grown in a medium containing 17.5% glucose, 5.5% ammonium sulphate and 2% yeast extract was found to produce (under laboratory conditions) high amounts of L-Lysine in this optimized medium. Lysine production, residual sugar and dry cell mass were measured as a function of fermentation time. It was observed that l g of cell mass produced 2.96 g of Lysine. The conversion efficiency was found to be 44%.     

Gas chromatographic–mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples

A gas chromatographic–mass spectrometric (GC–MS) procedure for the determination of stable isotope labelled glutathione has been applied to animal and human samples. The method, based on preparation of the N,S-ethoxycarbonyl methyl ester derivative of the intact peptide, is rapid and requires little or minor tissue treatment. The same method was applied to cysteine. The method was found to be reliable in terms of within-day and between-day precision, accuracy and linearity. The procedure was applied in humans and animals to determine in vivo the glutathione fractional synthesis rate using labelled cysteine infusion. The glutathione fractional synthesis rate was found to be 22.5%/day in blood from a healthy volunteer and 337±29%/day in rat liver.     

Purification of the bovine rod outer segment Na+/Ca2+ exchanger

Optimal conditions for solubilization and stabilization of the Na+/Ca2+ exchanger from rod outer segments were examined. The exchanger was found to be most stable at low detergent concentrations (7.5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), greater than or equal to 100 mM NaCl, pH 7.0-7.5, and with 0.1% added soybean asolectin. The sulfhydryl-modifying reagent, dithiothreitol, caused a loss of exchanger activity and was omitted throughout the purification procedure. These conditions were used to purify the Na+/Ca2+ exchanger from rod outer segments by a combination of selective solubilization, ion exchange, and wheat germ agglutinin chromatography. The procedure achieves a 336-fold increase in exchanger specific activity. The presence of exchanger activity most closely correlates with a polypeptide of molecular mass 215-kDa. Exchanger activity in both the crude rod outer segments and the purified exchanger is specifically dependent upon the presence of K+ in the assay medium; neither choline nor Li+ can substitute for K+.     

Determination of plasma propofol levels using gas chromatography—mass spectrometry with selected-ion monitoring

Propofol (2,6-diisopropylphenol, I.C.I. 35 868) is a rapid-acting, intravenous anesthetic agent recently introduced for the induction and maintenance of general anesthesia. This paper describes a gas chromatographic—mass spectrometric procedure using selected-ion monitoring for the determination of plasma propofol levels. The drug and the internal standard (thymol) were extracted from plasma into diethyl ether—pentane, and derivatized to their trimethylsilyl derivatives before analysis. The reproducibility of the daily standard curves had coefficients of variation ranging from 2.7% to 10.2%. The precision of the assay yielded a coefficient of variation ranging from 4.5% to 5.6%, and the concentration means for the seeded control samples were found to be within −1.6% to +0.6% of the theoretical values for propofol. No interfering peaks have been observed in application of this procedure to either normal volunteer or patient samples. The minimum detectable level under the conditions described was 0.20 ng propofol/ml plasma. This assay and a high-performance liquid chromatographic assay with fluorescence detection were both used to measure plasma propofol concentrations in 89 human plasma samples, and the correlation between the two methods was excellent.     

Hydrocarbons in green and blue-green algae

Liquid column chromatography and thin-layer chromatography were used to determine the total content of hydrocarbons and gas chromatography was used to evaluate composition of hydrocarbons in green algae (Chlorella kessleri, C. vulgaris, Chlorella sp.,Scenedesmus acutus, S. acuminatus, S. obliquus) and the blue-green alga (Spirulina platensis) cultivated under autotrophic or heterotrophic conditions. InC. kessleri cultivated under heterotrophic conditions the content of hydrocarbons was found to be about 10^-2 % (per dry mass), whereas under autotrophic conditions it was about 10^-3 % (per dry mass). The highest content of hydrocarbons was detected in species of the genusScenedesmus cultivated autotrophically (10^-1 %). Heptadecane and hexacosane were found as major alkanes, 1-heptadecene was detected among alkenes.     

Extraction and separation of urinary catecholamines as their diphenyl boronate complexes using C18 solid-phase extraction sorbent and high-performance liquid chromatography

The clinical utility of a one-step extraction procedure based on the retention of a diphenyl boronate-catecholamine complex on a C18 solid-phase extraction sorbent was investigated for the measurement of urinary catecholamines. Although recoveries with the extraction procedure were optimal over a relatively broad pH range (7.5-9.5), analytical factors such as sample loading and elution flow-rates, wash step and elution conditions, the concentration of catecholamines in urine to be extracted and the type of C18 sorbent used for extraction were found to influence the efficiency of this procedure and would therefore need to be controlled for optimal recoveries. Under optimal conditions the recovery of noradrenaline, adrenaline and dopamine from spiked urine was high and reproducible (mean recoveries were >85% for all catecholamines). The effectiveness of sample clean-up step was demonstrated by reverse phase, ion pair high-performance liquid chromatography with electrochemical detection. The method described was found to be suitable for the routine measurement of catecholamines in urine in clinical biochemistry laboratories. It has a high sample extraction throughput (40/h) and has adequate precision (between batch CV<8%) and sensitivity (LOD<30 nmol/l; LOQ<65 nmol/l) for all the catecholamines measured. The method has acceptable accuracy, showing a mean bias of 6.6% for noradrenaline, 7.3% for adrenaline and 6.8% for dopamine from the mean value of laboratories (N=69) participating in an External Quality Assurance scheme for greater than 12 months.     

Study of gaseous substrate fermentations: carbon monoxide conversion to acetate. 2. Continuous culture

The fermentation of gaseous substrates such as CO, H(2), and CO(2) may be performed in a continuous stirred tank reactor, as well as the traditional batch reactor. In this article, the conversion of carbon monoxide by Peptostreptococcus productus is demonstrated in a stirred tank reactor under both mass transfer-controlled and nonmass transfer-controlled conditions. Utilizing a non-steady-state procedure, intrinsic rates are evaluated under non-mass transfer-controlled conditions in a time period of only 5-6 hours. A steady-state procedure was used to evaluate CSTR performance under mass transfer-controlled conditions. The mass transfer coefficient was calculated, followed by the development of a model to predict CSTR behavior for this gas phase substrate.     

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.     

The preparation of tritiated tunicamycin

A relatively simple and inexpensive procedure was devised for the radiolabeling of the glycoprotein biosynthesis inhibitor, tunicamycin. The procedure is based on hydrogen exchange in alkaline solutions of tritiated water. It was noted that the antibiotic was much more alkali labile than model compounds such as uridine. The alkali stability of the inhibitor was studied to determine conditions for optimum labeling and yield. The effects of alkaline incubation on the inhibitory properties of the antibiotic were also investigated and it was found that the breakdown products are not effective inhibitors of the reaction that transfers N-acetylglucosamine-1-phosphate to dolichyl phosphate. The isolated radioactive tunicamycin homologs, however, retained all their inhibitory action. Incubation of tunicamycin in the presence of deuterated water and mass spectral analysis showed that under the conditions used for the tritiation of tunicamycin the major product exchanged six hydrogen atoms. The position of the tritium atoms in labeled tunicamycin was not determined. The radioactive label in these compounds was shown to be stable under physiological conditions and should be useful for investigations involving the action of these antibiotics.     

Electrophoretic separation of high-molecular hydrophobic proteins: apolipoprotein B and other protein constituents of low-density lipoproteins

Apolipoprotein B (apoB) and 40 different protein standards were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under various conditions. The system selected is capable of fractionating well the apoB and its high-molecular-weight species formed after treatment of serum lipoproteins by malonic dialdehyde. The basis for good resolution of protein bands is the use of complex gels combining in one block the areas with constant (T = 3 and 10%) or continuously changing (gradient gel with T = 3-6%) concentrations of acrylamide. The procedure allows one to create the optimal conditions for simultaneous separation of both high- and low-molecular-weight polypeptides. The method was used to determine the relative molecular mass (Mr) of apoB. When apoB was isolated in the presence of proteolytic inhibitors, its Mr was found to be equal to 540,000 +/- 9500 or 500,000 +/- 5700, depending on the linear regression equation applied. The results are in good accordance with values calculated from the amino acid sequence of mature apoB.     

Identification of bis-quaternary ammonium compounds using mild ionization mass spectrometry

Ethonium, an antimicrobial chemotherapeutic agent, was investigated by mass spectrometry (MS) under various ionization conditions: electron impact, field ionization, field desorption (FD) and fast atom ionization. FDMS was found to be the most suitable procedure for ethonium identification. Relation of the ED mass spectra to the distance between the nitrogen atoms in bis-quaternary ammonium compounds is discussed. It was shown that the most intensive ions with m/z 499, 315 in the FD mass spectra corresponded to the ethonium specific fragmentation and their occurrence in the spectra could serve as a sufficient criterion useful in qualitative and quantitative assay of the drug in the sample.     

Isolation of alpha-1-antitrypsin from human plasma by partitioning in aqueous biphasic systems of polyethyleneglycol-phosphate

The partitioning of alpha-1-antitrypsin was assayed in biphasic aqueous systems containing potassium phosphate and two polyethyleneglycols of molecular mass 600 and 1000, respectively. In order to isolate the alpha-1-antitrypsin from serum plasma, the partitioning behaviour of human serum albumin, its principal contaminant, was also studied. Several aqueous two-phase systems with different partitioning properties were obtained by varying the PEG1000/PEG600 mass proportion. In systems with PEG1000/PEG600 mass ratio of 8, the optimal difference between the partition coefficients of both proteins was found. Under such conditions, a satisfactory purification was carried out by a three-step extraction procedure. By applying this method the alpha-1-antitrypsin specific activity increased severalfold (nearly 10 times) with a yield of 43%.     

Preliminary application of liquid chromatography–electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C₁₈-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC–MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/ml.     

Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungus<Emphasis Type="Italic">Cladosporium phlei</Emphasis>

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 10³ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively. There was an equal contribution of the reported research by the first two authors.     

Characterization of Salmonella and possible interfering strains using GC profiling and factor analysis

Fatty acid methyl ester data of Salmonella and potentially interfering strains obtained by derivatization gas chromatography were investigated with principal components and factor-discriminant analysis. Salmonella strains could be distinguished using both data-handling techniques. The biochemical basis for this characterization was found to be in strong agreement with the higher mass range part of pyrolysis — direct chemical ionization mass spectrometric data representing diglyceride information. The quality of the analytical procedure as judged by longer term reproducibility and component assignment was found to be good. In combination with sophisticated data-handling methods this derivatization gas chromatographic approach can be a useful diagnostic tool for Salmonella characterization.     

Characterization and inhibition effects of some metal ions on carbonic anhydrase enzyme from Kangal Akkaraman sheep

In this work, the carbonic anhydrase (CA) enzyme was purified from Kangal Akkaraman sheep in Sivas, Turkey with specific activity value of 6681.57 EU/mg and yield of 14.90% with using affinity column chromatography. For designating the subunit molecular mass and enzyme purity, sodium dodecyl sulfate polyacrylamide gel electrophoresis method was used and single band for this procedure was obtained. The molecular mass of CA enzyme was found as 28.89 kDa. In this study, the optimum temperature and optimum pH were obtained from 30 and 7.5. V_max and K_m values for p‐nitrophenylacetate substrate of the CA were determined from Lineweaver–Burk graphs. Additionally, the inhibitory results of diverse heavy metal ions (Hg⁺, Fe²⁺, Pb²⁺, Co²⁺, Ag⁺, and Cu²⁺) on sheep were studied. Indeed, CA enzyme activities of Kangal sheep were investigated with using esterase procedure under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC₅₀) and K_i values were obtained.     

Poly-3-hydroxybutyrate production byAzotobacter chroococcum

Thirty-seven soil isolates and mutants ofAzotobacter chroococcum tested for poly-3-hydroxybutyrate (PHB) production using Sudan black B staining method were found to be positive. One mutant showed a higher number of PHB-producing cells and maximum number of granules per cell. Using 2% glucose and 15 mmol/L ammonium acetate, PHB production was found to be maximum at 36 and 48 h of growth under submerged cultivation and under stationary cultivation, respectively. PHB production was found to be higher on sucrose and commercial sugar (as carbon sources) as compared to glucose and mannitol. As commercial sugar is cheaper than sucrose it was selected as carbon source for PHB production, that being found to be maximum at 1% concentration. Inorganic nitrogen sources seemed to have no stimulatory effect on the production of PHB. However, ammonium acetate (15 mmol/L) was found to be best for PHB production. Peptone (0.2 %) gave a better yield of PHB under both growth conditions. Using all optimized conditions, PHB production was studied in ten selected strains. Two of them were found to be best PHB producers under both growth conditions, one producing 621 and 740 μg/g dry mass under submerged cultivation and under stationary cultivation, respectively, while the second one produced 589 and 733 μg/g.     

Occurrence of a thermoacidophilic cell-bound exo-pectinase inAlicyclobacillus acidocaldarius

Alicyclobacillus acidocaldarius was able to degrade pectin under thermoacidophilic conditions of high temperature and acidity. Both extracellular and cell-bound pectolytic activities were found (28 and 72% of total activity, respectively). WhenA. acidocaldarius was subjected to lysozyme or sonication, more than 50% of the activity was found to be bound with the cell debris. The cell-bound enzyme presented principally exopectolytic activity. SDS-PAGE and zymogram showed that the estimated molar mass of the crude enzyme was 52 kDa. pH optimum was between 1.5 and 2.0 and the enzyme was thermostable at 70°C for 1 h at pH 2.0.