Studies of novel bioactive substances in the spent media of cell lines using protein-free culture system]

Many human cell lines have been maintained in fetal bovine serum (FBS)-supplemented medium. These produce and secrete many substances such as transferrin, alpha 1-antitrypsin, alpha 2-macroglobulin, alkaline phosphatase, gamma-glutamyltranspeptidase, creatine kinase, carcino-embryonic antigen, alpha-fetoprotein, and cytokines including colony stimulating factors and transforming growth factors and further they may produce small amounts of unknown substances. Usually, small amounts of substances have to be concentrated as highly as possible for detection, but FBS interferes with procedures. A protein-free culture system in an ideal method for detecting small quantities of substances which originate from cell lines without interference by FBS. Our protein-free culture system can be available in every laboratory since this is not only an economical method, but also an effective method for the saving of purification procedures. Moreover, this is a most suitable method for surveying unknown substances derived from cell lines.     

The action of membranotropic compounds with various structures on the parameters of the phase transition of dimyristoylphosphatidylcholine

The effect of substances of different nature on the thermodynamic characteristics of dimyristoylphosphatidylcholine (DMPC) phase transition by the differential scanning microcalorimetry has been studied. The substances disposed in hydrophobic part of membrane--alpha-tocopherol, ubiquinone Q10, ionol and vitamin K3 cause the decrease of enthalpy and cooperativity of phase transition. The substances which have the side hydrocarbon chain (tocopherol and ubiquinone Q10) compared with ones without it (ionol and vitamin K3) and reduced quinones (Q10 and vitamin K3) compared with the oxidized ones have stronger influence on the enthalpy and cooperativity of transition. The inclusion of the local anesthetic dicaine disposed mainly in the zone of polar heads of phospholipids into DMPC membranes decreases the temperature of phase transition considerably and practically does not change the cooperativity. A possibility to use the method of differential scanning microcalorimetry to estimate the localization of membrane tropic substances within lipid bilayer is under discussion.     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

Indices of oxidative stress. 2. Lipid peroxides

Two methods of the determination of lipid peroxidation products have been compared which are based on Fe(II) oxidation by them at acid pH values in the presence of xylenol orange which binds Fe(III) have been compared. The first method uses cumene hydropeoxide as an internal standard. In the second one, lipid peroxides are previously reduced by triphenylphosphine and these substances content is measured as a difference of the production of complexes with xylenol orange and iron ions in the control (with reduction) and experimental sample (without reduction). The optimization of measurement conditions is described. The levels of lipid peroxides in goldfish tissues assayed simultaneously by two methods were similar. The method with cumene hydroperoxide needs less amounts of biological material; moreover, there is no necessity in a calibration curve. Effects of hyperoxia on lipid peroxide levels in goldfish tissues were studied with the cumene method. Within the first hours of hyperoxia this index increased 13-times in the liver and 2-times in the brain and muscle. The further exposure rebounded this parameter to the initial level. Levels of lipid peroxides positively correlated with levels of end products of lipid peroxidation (thiobarbiturate acid reactive substances) in the goldfish tissues. The method of quantification of lipid peroxides with cumene is recommended for wide using in biological investigations.     

Chemotaxis of cholera vibrios: a study method and the relation to different substances

The chemotaxis of V. cholerae in response to 56 different substances (amino acids, carbohydrates, salts, etc.) has been studied by the methods of visual observation and quantitative determination. Attractants, neutral substances and one repellent have been revealed. Adler's method (1973) has been modified with regard to the requirements for the working procedures in handling the causative agents of highly dangerous infections.     

High performance thin layer chromatography (HPTLC), an improved technique for screening lichen substances

Abstract: High performance thin layer chromatography (HPTLC) is a method that can be used for screening lichen substances. It is as simple to use as standard TLC, but has many advantages: It is more sensitive, it is possible to run more samples in a shorter period of time, and the amount of solvent used is much smaller. The material needed and the methods used are described in detail. Horizontal chromatogram development was used. Since two of the solvents used in system B have been substituted, and since the properties of the HPTLC plates are slightly different, our results are not entirely in accordance with the standardized TLC method. A revised table for the identification of 69 lichen substances (obtained from 62 taxa) is accordingly presented.     

Verification of a REACH Environmental Prioritization System Against Regulatory Risk Indices

A prioritization method that was developed to rank chemical substances on the basis of their environmental impact was applied to 230 new substance notifications from earlier European chemical legislation (67/548/EEC). The method encompasses three steps: (1) assigning an environmental hazard score, (2) assigning an environmental exposure score, and (3) combining these two scores into one priority score. In this study, the resulting scores ranged between 4 (highest priority) and 15 (lowest priority). The scores were compared with results from risk assessments available for 138 of the 230 substances. For most substances in these assessments, a priority score of 12 or higher was associated with no or limited risk, while a score of 11 or lower represented high risk or led to the conclusion that risk reduction measures were required. This categorization applied to all but 15 of the 138 substances. The method was also used for ranking the first 15 Substances of Very High Concern (SVHC) under REACH regulation, to compare the priority scores of new substances to those of the SVHC. In sum, the prioritization method seems to be valuable to identify substances of concern with respect to the environment.     

Improved extraction of PCR-quality community DNA from digesta and fecal samples

Several DNA extraction methods have been reported for use with digesta or fecal samples, but problems are often encountered in terms of relatively low DNA yields and/or recovering DNA free of inhibitory substances. Here we report a modified method to extract PCR-quality microbial community DNA from these types of samples, which employs bead beating in the presence of high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA, and with subsequent DNA purification by QIAamp columns [referred to as repeated bead beating plus column (RBB + C) method]. The RBB + C method resulted in a 1.5- to 6-fold increase in DNA yield when compared to three other widely used methods. The community DNA prepared with the RBB + C method was also free of inhibitory substances and resulted in improved denaturing gradient gel electrophoresis (DGGE) profiles, which is indicative of a more complete lysis and representation of microbial diversity present in such samples.     

Electron microscopic detection of PAS-positive substances with thiosemicarbazide

Summary The authors have elaborated a suitable method of detection of PAS-positive substances with chemicals in common use-thiosemicarbazide-both on semi-thick sections and tissue blocks for electron microscopic purposes. The method is sufficiently sensitive even in the presence of only a small amount of PAS-positive substances. They found that the specifity of this method is the same as that in the PAS method used for light microscopy. The structure of the cells remains surprisingly well preserved, especially in comparison with other methods in current use.     

A new theory of infarction-genesis in cats

It can be shown that a number of substances that are derived from incubation have a good blocking capacity in myocardial infarction. There exists also a natural substance (intrinsic factor, i.f.) which can also block infarction. The same substances also inhibit arrhythmias of ischaemic origin. Thus a common root for myocardial infarction and arrhythmias could be inferred. A new dynamic method (T/2 velocity) was elaborated for evaluating anti-infarction drugs instead of using static T/2 values. Oxidation of i.f. in vitro and in vivo enhances the blocking capacity of artificial drugs reagented previously each other. The natural substance (i.f.) lost its blocking capacity after oxygenation, thus may be unable to compete with the above mentioned artificial substances. Ligation of the coronaries i.e. its myocardial infarction producing effect can also be inhibited by the same substances. No matter how infarction is produced, the same substances can block it. Since after cutting the vagi KCl elicits the same phenomena their reflex origin can be excluded.     

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

On the establishing of permissible relations between MACC- and MACT-values of harmful substances

The establishment of MACC (Ceiling Values) and MACT (Time Weighted Average Values) for chemical substances permits a substantial more precise evaluation of the exposure to harmful substances at the work place as if only one limit value is given without reference to a Ceiling Value or a Time Weighted Average Value, respectively. The derivation of MACC- and MACT-Values is proposed from toxicometric data that are gained during animal experiments with chemical substances. For acutely toxic substances (e.g. irritants) the quotient F = MACC/MACT ought to be = 1. For cumulative acting substances the permissible relation of both values is determined from the magnitude of the zone of chronic effect (Zch) as F = 0.4. Zch. The condition MACC less than less than Limac takes into consideration at the same time. The frequency and the duration of MACT exceedings need not to be regulated with this procedure. A method is proposed for the derivation of the length of sampling periods necessary from the toxicological point of view starting from the Zch.     

Neurochemical applications of liquid chromatography with electrochemical detection

Liquid chromatography with electrochemical detection (LCEC) has been shown to have unique advantages for the determination of many substances of neurochemical interest. The technique is rapid, sensitive, and relatively inexpensive. In addition, it avoids the need for radiolabelled substances, the formation of volatile derivatives, or reactions which generate fluorescent products. LCEC is widely used for the measurement of the catecholamines and their metabolites and has recently gained acceptance for determination of the neurochemically important tryptophan metabolites. The method is also capable of assessing the activity of a number of neurologically important enzymes. The review which follows is intended to provide a brief overview of the LCEC technique and a guide to recent literature exemplifying its neurochemical applications.     

Chemoluminescence study of the influence of phagocytosis of peripheral blood leukocytes using a perfluorocarbon-containing blood substitute

In an animal experimental model (rats) the influence of which model substances for blood substitution of GDR origin may have on the phagocytosis behaviour of leukocytes of the peripheral blood was investigated by means of luminol-amplified chemoluminescence. After applying the model substance in an amount corresponding to 10% of the circulating blood volume a reversible increase of luminol-amplified chemoluminescence could be observed. The values referred to the portion of neutrophilic granulocytes, however, showed no significant differences compared to the control groups. The opsonizing capacity of the serum towards cymosan revealed a temporary deficit after applying blood substitution substances of GDR origin. The conclusion is drawn that the functions of leukocytes of the peripheral blood recorded by the applied method are not depressed by the model substances used.     

PCR inhibitors – occurrence,properties and removal

The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false‐negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix‐specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.     

The biolistic process

The biolistic process is a new process which employs high velocity microprojectiles to deliver substances into cells and tissues. It has been described in different ways and has been referred to as the particle gun method, the microprojectile method, the gene gun method, the particle acceleration method, the bio-blaster method. The inventors of the process have coined the term ‘biolistic’ (biological ballistics) to describe both the process and any associated apparatus used to shoot biological materials into living targets.     

The electronic structure and radiorprotective activity of a series of substituted iminodihydrofurans]

Compounds that increase the survival rate of lethally exposed hybrid (CBA x C57B1/6)F1 mice have been revealed within the series of iminodihydrofurans. The quantum-chemical estimates, made by the MNDO method, show that substances which are capable of donor-acceptance interaction with DNA nucleotides and have the energy of donor orbitals, comparable with the electron structure of nucleotides, possess radioprotective efficacy.     

Evaluation of natural substances from Evolvulus alsinoides L. with the purpose of determining their antioxidant potency

In recent years, great attention has been given to the search for natural compounds or extracts with the purpose of medical use. Evolvulus alsinoides L. (Convolvulaceae) is a plant used in traditional medicine of East Asia in many indications and has known nootropic and anti-inflammatory activity. However, the bioactive constituents have been described poorly in the literature. Four substances isolated from the ethanol extract of E. alsinoides by means of polyamide and Silica-gel chromatography are reported here. Their molecular structures were determined using NMR analyses. There were identified as scopoletin, umbelliferone, scopolin and 2-methyl-1,2,3,4-butanetetrol. The quantity of these substances was determined using HPLC-UV and GC-FID detection. Antioxidant activity of the isolated substances was measured by DPPH assay using the SIA method. Antioxidant activity and total phenolic content of the prepared fractions are also described. The prepared fractions and isolated substances did not exhibit any significant activity in DPPH test.     

Characterization of polypeptide antibiotics of the polymyxin series by liquid chromatography electrospray ionization ion trap tandem mass spectrometry.

A selective reversed phase liquid chromatography/mass spectrometry (LC/MSn) method is described for the identification of related compounds in commercial polymyxin B samples. Mass spectral data for these polypeptide antibiotics were acquired on a LCQ ion trap mass spectrometer equipped with an electrospray ionization probe operated in the positive ion mode. The LCQ ion trap is ideally suited for the identification of the related substances because it provides on-line LC/MSn capability. The main advantage of this hyphenated LC/MSn technique is the characterization of novel related substances without time-consuming isolation and purifications procedures. Using this method six novel related substances were partially identified in a polymyxin B bulk sample.