Evaluation of the impact of DNA extraction methods on BAC bacterial community composition measured by denaturing gradient gel electrophoresis

Aims: The impact of DNA extraction methods on biological activated carbon (BAC) DNA yield and bacterial community was evaluated. Methods and Results: Three different DNA extraction methods were compared: method a, method b and method c. Method c with ultrasonic pretreatment improved cell lysis efficiency (from 34% to 87%) and DNA yield [from 10·58 μg g^?1 (dry wt) of carbon to 21·42 μg g^?1 (dry wt) of carbon]. denaturing gradient gel electrophoresis profiles obtained by method c recovered the five seeded bacteria (Bacillus subtilis Strain WSO 6, Pseudomonas putida Strain WSO 7, Acinetobacter lwoffii WSO 10, Pseudomonas pertucinogena WSO 11 and Brevibacterium mcbrellneri WSO 13). Conclusions: The results showed method c with ultrasonic pretreatment was the most successful for the analysis of BAC bacterial community because it was effective in the detachment of bacteria and cell lysis, thereby resulting in good yields. Significance and Impact of Study: These results must be taken into consideration when extracting DNA for analysing BAC bacterial community.     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.     

Diversity of antigenic determinants of porcine zona pellucida revealed by monoclonal antibodies

Monoclonal antibodies derived from ten hybrid cell clones, generated against porcine zona pellucida gave strong immunofluorescence with zona but the pattern varied from patchy, thin rim to heavy precipitation type of rim. Five of the 6 monoclonals studied prevented the binding of the porcine epididymal sperm to homologous oocytesin vitro, whereas the sixth one was partially effective. All of the 6 monoclonale of this batch inhibited the lysis of zonae by proteolytic enzymes even at dilutions up to 1 × 10⁻³. Three of the four monoclonals prepared in a subsequent batch gave strong immunofluorescent reactions and had high titres as determined by enzyme immunoassay. These monoclonals did not, however, protect the zonae against lysis by proteolytic enzymes. These properties are suggestive of the heterogeneity of the antigenic determinants in zona and emphasize the employment of appropriate bioassays for screening and selection of bioeffective antibodies.     

Production and Purification of a New Chillproofing Enzyme and Its Properties

Chillproofing enzyme was obtained from broth cultures of Serratia marcescens B–103. This extracellular enzyme, tentatively, named the S-enzyme was highly purified from the culture supernatant by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G–200 and column chromatography on DEAE-Sephadex A–50.

The purified preparation appeared homogeneous on a ultracentrifugation with a sedimentation coefficient of 3.14 S and a molecular weight of 38,000~45,000 determined by the method of Whitaker.

The S-enzyme hydrolyzed various proteins at pH 4~6 and at low temperature hydrolyzed nitrogenous substances which may cause chill haze in beer. So the chillproofing activity of the S-enzyme may be due to its proteolytic activity.

The S-enzyme was stable at 4°C at pH 5~10.5. It was completely inactivated by heating at 60°C for 10 min, and was inactivated by Hg²⁺ and Pb²⁺ and activated by Mn²⁺, Ca²⁺. Mg²⁺ and Zn²⁺     

Auxin requirements of sycamore cells in suspension culture

Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this `auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.

There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10⁶ cells) on day 2 followed by a decline to 45 nanograms per 10⁶ cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10⁶ cells) rises slowly to a peak of 1.4 nanograms per 10⁶ cells by day 9 then decreases rapidly to 0.2 nanograms per 10⁶ cells by day 15 (early declining phase) and 0.08 nanograms per 10⁶ cells by day 23 (early stationary phase).

     

Partial inactivation of the mosquitocidal activity of Clostridium bifermentans serovar malaysia by extracellular proteinases

Summary Clostridium bifermentans serovar malaysia is toxic to mosquito larvae. During large-scale preparation in a fermentor, the bacteria enter the sporulation stage after 5 h culture, whereupon high larvicidal activity is obtained (LC₅₀ 48 h on Anopheles stephensi = 3.1 × 10^–5). The toxicity becomes maximal around 3–5 h later (LC₅₀ 48 h = 1.3 × 10^–5) and remains unchanged until sporangium lysis. An important loss of toxicity is then observed when the cells lyse. This loss appears to be due to the fact that C. bifermentans serovar malaysia synthesizes and excretes, mainly during vegetative growth, metallo- and/or cystein-proteinases, which are active between pH 6.0 and pH 8.0. Extracellular proteinases are most likely responsible in large part for the decrease in toxic activity concomitant with cell lysis. Lysis is however prevented by addition of 10 mM ethylenediaminetetraacetic acid to the culture medium before forespore formation, and under these conditions the larvicidal activity can be maximized. Offprint requests to: L. Nicolas     

Monilidiols,Characteristic and Bioactive Metabolites of Benomyl-resistant Strains of Monilinia fructicola

A new polygalacturonase was found in a culture filtrate of Aureobasidium pullulans. The enzyme was purified and obtained in crystalline form with 10% recovery. The crystalline enzyme was a homogeneous protein by analyses by sedimentation and electrophoresis. The enzyme was most active around pH 4.5, and stable in the pH range of 4 to 6. Its molecular weight was 42,000 and its isoelectric point was pH 6.0. The enzyme was an endo-polygalacturonase, catalyzing the cleavage of glycosidic bonds of polygalacturonic acid at random.

The enzyme had less protopectinase activity than those of the endo-polygalacturonases that were isolated as protopectin-solubilizing enzymes from Trichosporon penicillatum, Kluyveromyces fragilis, and Galactomyces reessii. Some characteristics were compared with the endo-polygalacturonases, which have potent protopectinase activity.     

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng, and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 × 10⁴ copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.     

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

Frequently measured mammalian cell culture process indicators include viability and total cell concentration (TCC). Cell lysis, an additional important process characteristic that substantially contributes to the overall product purity profiles, is often not addressed in detail. In the present study, an inexpensive and simple application of the Bradford assay is developed to determine the residual protein content (RPC) in cell culture supernatants. The reliability and reproducibility of the method are tested in a long‐term study and compared with lysis quantification via the DNA measurement. The results show that its performance is more robust and accurate over time and the respective concentration range. Additionally, both methods are used for cell lysis process monitoring in a recombinant Chinese hamster ovary fed‐batch process. In the presented process, by applying the established assay, the lysis rate k _DL is determined to be constant over time at 4.6 × 10 ^?4 lysed cell concentration (LCC) per TCC and time (LCC/TCC/h). In contrast, DNA data did not confirm the constant lysis rate due to variations of the content per cell during cultivation. Thus, information on the RPC can facilitate the determination of the optimal harvest time point with respect to purity and in improving process characterization.     

Structural homogeneity of mitochondrial DNA in the mitochondrial nucleoid of Physarum polycephalum

Mitochondrial DNA (mtDNA) of Physarum polycephalum was isolated gently by CsCl centrifugation. The mtDNA was linear with molecular weights ranging from 25·10⁶ to 45·10⁶ and heterogeneous in size. Nevertheless, thermal transition profiles of the mtDNA suggested that this DNA fraction was more homogeneous than nuclear DNA. Exhaustive digestions of this DNA with restriction endonucleases yielded unique fragments, and then the total of their molecular weights of each digest was around 45·10⁶. This value is equivalent to the maximum molecular weight estimated using electron microscopy and electrophoresis. Moreover, EcoRI digests of the mtDNA fractionated by the sucrose gradient showed unequimolar quantities of large fragments and a high background between bands. These results suggest that the mtDNA of Physarum has a homogeneous base sequence, and that the size heterogeneity of the mtDNA is attributable to degradation of the DNA under isolation procedures. The mtDNA was cleaved by EcoRI and XhoI to yield 16 and 7 fragments, respectively. A physical map of these fragments was constructed using the routine mapping procedures. The physical map showed that the mitochondrial genome of Physarum was linear with a molecular weight of 45·10⁶. We concluded therefore that the mitochondrial nucleoid is a structure in which the homogeneous mtDNA is highly amplified.     

Purification and Properties of API–2b→API–2c Converting Protease from Streptomyces griseoincarnatus Strain No. KTo–250, an API–2 Producer

Among three alkaline protease inhibitors (API-2a, -2b, -2c) produced by Streptomyces griseoincarnatus strain No. KTo-250, API-2b was converted to API-2c in the growing system.

The cultural conditions were examined exclusively for the production of API-2b→ API-2c converting protease in the culture filtrate. The protease was purified about 1080-folds by salting-out with ammonium sulfate, column chromatography on DEAE-cellulose and gel filtration on Sephadex G–100.

The optimal and maximal caseinolytic activities of the protease were around pH 9.0 and at 28°C, respectively. The protease activity was inhibited by EDTA and DFP, but not by PCMB, o-phenanthroline, TPCK, TLCK, AP-I and S-SI. The protease was a DFP and EDTA-sensitive alkaline protease, and it required Ca²⁺ ion for its activity and stability.     

Isolation and partial characterization of replication intermediates in Chironomus polytene chromosomes

DNA replication was investigated in cells with polytene chromosomes. The cells were obtained from the salivary glands of the dipteran Chironomus tentans. Polytene chromosomes are characterized by a specific and constant band — interband structure formed by the lateral association of homologous chromatids side by side. — The salivary gland DNA was labelled by injection of radioactive precursor into the living animal, extracted with a neutral nondenaturing buffer at 25° C and finally characterized by agarose gel electrophoresis. Radioactive DNA pulse-labelled for 30–60 min was released from the polytene chromosomes during cell lysis in the form of double-stranded fragments. The fragments, which show a heterogeneous appearance in gel electrophoresis, are probably produced in the living cell by the joining of several Okazaki fragments. The release of the fragments from the polytene chromosome is prevented by lysis at 0° C instead of 25° C. The size of the double-stranded fragments range between 3.75–6×10⁶ D. Moreover, after a time-lag the fragments are joined together to produce a high-molecular weight DNA. The existence of these nascent DNA fragments is discussed in relation to an earlier proposal that each band in the polytene chromosome may function as a separate replication unit.     

Speciation of copper(II) in natural waters in the presence of ligands of high and intermediate strength

Abstract

A new procedure is presented for the determination of the ligands of copper(II) in natural waters, based on titration with the metal ion, monitored by measuring the concentration of copper(II) sorbed on the carboxylic resin Amberlite CG 50. The data are treated by the Ruzic linearization method to obtain the concentration of the ligands and the conditional stability constant of the complexes. Ligands with reaction coefficient α_M higher than 0.1 K*w/V are detected, where K* is the ratio of the concentration of sorbed metal to the concentration of free metal in solution, which can be evaluated from the sorption equilibria of copper(II) on Amberlite CG 50, w is the amount of water in the resin phase, and V the volume of the solution phase. Some natural waters at high and low salinity were examined. The ligand concentration determined in these samples ranged from around 50 to 2000 nM, while the original copper concentrations from 11 to 130 nM. The ligand concentration was always much higher than that of copper(II). The conditional stability constants were very high, particularly in low salinity waters, where values as high as K’= 10^15.7 were obtained. In high salinity waters values around 10⁹ were found for the complex formation constant of the ligands titrated with copper(II). The investigation was also extended to a model solution, containing EDTA, obtaining K’ = 10^15.5, in acceptable agreement with that evaluated from the literature values.     

Production of protoplasts from Fusarium scirpi by lytic enzymes from Streptomyces tsusimaensis

Summary The ability of serveral strains of Streptomyces to degrade cell walls from Fusarium scirpi was tested by plating them on agar containing a cell wall preparation derived from the fungus. In this assay, S. tsusimaensis was most effective in producing a clear zone of lysis during growth on the opaque medium. This Streptomyce strain was subsequently grown in liquid culture containing cell walls as the sole carbon source and the exoenzymes were isolated from the culture broth. The enzyme preparation produces a clear zone of lysis when filled into wells in the cell wall agar and was used to prepare protoplasts from F. scirpi. The protoplast yield was 1x10⁹ protoplasts/ml of enzyme solution from 35 mg dry weight of Fusarium mycelium. Protoplasts could be regenerated at a frequency of up to 80%.     

Accumulation of 6-Azauridine by Mutants of Brevibacterium ammoniagenes

From the inosine producing mutants of Brevibacterium ammoniagenes, KY 13761 was selected as a strain which produced 6-azauridine from 6-azauracil.

The conditions for the conversion were examined and the intermitent feeding of 6-azauracil was found effective for the accumulation.

In order to increase the accumulation, prototrophic revertants were induced from KY 13761 and KY 13021 was selected. By intermitent feeding of 6-azauracil of a final concentration of 6 mg per ml, a maximal accumulation, 12.4 mg/ml, of 6-azauridine was obtained with KY 13021.     

Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.     

Investigation on silent bacterial infections in specimens from pregnant women affected by spontaneous miscarriage

Miscarriage is one of the main complications occurring in pregnancy. The association between adverse pregnancy outcomes and silent bacterial infections has been poorly investigated. Ureaplasma parvum and urealiticum, Mycoplasma genitalium and hominis and Chlamydia trachomatis DNA sequences have been investigated by polymerase chain reaction (PCR) methods in chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from females with spontaneous abortion (SA, n = 100) and females who underwent voluntary interruption of pregnancy (VI, n = 100). U. parvum DNA was detected in 14% and 15% of SA and VI, respectively, with a mean of bacterial DNA load of 1.3 × 10⁻¹ copy/cell in SA and 2.8 × 10 ⁻³ copy/cell in VI; U. urealiticum DNA was detected in 3% and 2% of SA and VI specimens, respectively, with a mean DNA load of 3.3 × 10⁻³ copy/cell in SA and 1.6 × 10⁻³ copy/cell in VI; M. hominis DNA was detected in 5% of SA specimens with a DNA load of 1.3 × 10⁻⁴ copy/cell and in 6% of VI specimens with a DNA load of 1.4 × 10⁻⁴ copy/cell; C. trachomatis DNA was detected in 3% of SA specimens with a DNA load of 1.5 × 10⁻⁴ copy/cell and in 4% of VI specimens with a mean DNA load of 1.4 × 10⁻⁴ copy/cell. In PBMCs from the SA and VI groups, Ureaplasma spp, Mycoplasma spp and C. trachomatis DNAs were detected with a prevalence of 1%–3%. Bacteria were investigated, for the first time, by quantitative real-time PCR (qPCR) in chorionic villi tissues and PBMCs from women affected by SA and VI. These data may help to understand the role and our knowledge of the silent infections in SA.     

The effect of substituted amides of pyrazine-2-carboxylic acids on flavonolignan production in <Emphasis Type="Italic">Silybum marianum</Emphasis> culture <Emphasis Type="Italic">in vitro</Emphasis>

In vitro plant tissue and cell cultures were used to study herbicide effects on growth, selected metabolic activities and other phenomena. The effect of abiotic elicitors, two newly synthesized substituted amides of pyrazine-2-carboxylic acids (synthesized at the Department of Pharmaceutical Chemistry and Drug Control, School of Pharmacy in Hradec Kralove), on the flavonolignan accumulation in callus and suspension culture of Silybum marianum (L.) Gaertn. was investigated. The compounds markedly influenced production of flavonolignans in an in vitro culture. Particularly after the elicitation with a solution of compound 3-methylamide 5-tert-butylpyrazine-2-carboxylic acid at a concentration of 3.71×10⁻⁷ mol 1⁻¹ and within 72 h of elicitation, an increase in flavonolignan production by 893 % in suspension culture versus control took place. The flavonolignan accumulation in callus culture after the elicitation with a solution of 5-brom-2-hydroxyphenylamide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid was also increased by about 1039% (24 hour elicitation and concentration of 2.59×10⁻⁴ mol 1⁻¹).