Characterisation of cryptic plasmids in clinical isolates of Bacteroides fragilis.

A screening method for plasmids in the fragilis group of Bacteroides spp. was developed, taking account of the lysozyme resistance of these species; 26 strains, 24 of them B. fragilis, were investigated by this method. Eleven strains contained plasmids and up to three different plasmids were found in individual strains. The plasmids belonged to five different size classes of mol. wt (10(6] 2.8, 3.5, 3.6, 4.2 and 19. Plasmids of equal size showed homology; no homology was found between plasmids of different sizes. Plasmids of equal size showed identical restriction patterns with 17 restriction endonucleases. Restriction maps were constructed for the five classes of plasmid.

Isolation and purification of plasmids from Bacteroides fragilis using rubidium trichloroacetate density gradient centrifugation.

A rapid and easy final purification method is described for the isolation of plasmids from B. fragilis. Using RbTCA density gradient centrifugation in an airfuge ultracentrifuge ccc plasmid DNA can be separated from RNA, residual chromosomal DNA, linear and oc plasmid DNA. Pure ccc plasmid DNA is obtained from cultures of between 1 ml and 2 l in less than one day.

Microdisc-electrophoretic study of deoxyribonucleases in cow snout epidermis.

Acid and neutral deoxyribonucleases (DNases) of the cow snout epidermis were investigated by the microdisc-electrophoresis of polyacrylamide gels containing highly polymerized DNA and by isoelectric focusing techniques. The nucleases were characterized with respect to their pH optimum. An acid DNase at pH 5.0 was detected as a single distinct band after the electrophoretic separation. After isoelectric focussing also, only one acid DNase activity with an isoelectric point (IP) of 6.2 was detectable. Neutral DNases at pH 7.4 were demonstrated as major and minor bands by their different electrophoretic mobilities. In the isoelectric focusing system also, two neutral DNases, a major one (IP, 4.6) and a minor one (IP, 6.4), were found. Characterization with respect to their histologic location showed acid and neutral DNases across the epidermal layers with the highest activities in the upper layers, where DNA concentration had been shown to be lowest. In correlation with their subcellular distribution, the highest activities of both acid and neutral DNase were found in the 105,00 X g supernatant of the subcellular fractions.

Purification of a nuclease from human serum.

The purification procedure for a nuclease from human serum is described. It includes ammonium sulfate precipitation, chromatography on DEAE-Sephadex and on Sephacryl-S 200, and preparative electrophoresis. The enzyme purified about 2000-fold, is homogeneous in a sodium dodecyl sulfate electrophoretic system, where it has a mol. wt of 78,000. The pH optimum lies around pH 6.5; it is a sugar-nonspecific endonuclease.

Serum DNase activity after experimental, acute hemorrhagic-necrotizing pancreatitis in dogs: detection of a serum DNase isoenzyme.

The activity of a neutral serum DNase has been determined in dogs before and after experimental, acute hemorrhagic-necrotizing pancreatitis. Dog serum DNase corresponds in its conditions of activity and in its isoelectric point with the DNase I. Electrophoretically three DNase isoenzymes can be distinguished. 30 min after induction, an increase of DNase activity was observed. This activity increase is related to only one of the three DNase isoenzymes. After induction a DNase with an isoelectric point of 5.6 occurs. This enzyme is not found in healthy animals.

Deoxyribonucleases in herpes simplex virus type 1 and 2 infected primary rabbit kidney cells.

In primary rabbit kidney cells infected with herpes simplex virus four different neutral deoxyribonuclease activities can be detected by means of the deoxyribonuclease assay in DNA-containing polyacrylamide gels following their separation by disc-electrophoresis. The method is suitable to follow independently the change in each activity of the different enzymes using only about 5 X 10(5) cells for each assay during the time-course of infection. Under these conditions one enzyme activity is constant, two disappear while the activity of a fourth one present only in infected cells, increases.

Comparison of DNase, DNA-polymerase and RNA-polymerase activities present in the DNA-binding proteins of normal human dermis, epidermis, horny layer and psoriatic scales.

DNA-binding proteins (DBP) of normal human dermis, epidermis, horny layer and psoriatic scales represent a tissue-specific group of mostly nuclear nonhistone proteins. To analyse their function, the different DBP fractions were examined concerning the presence of DNase, DNA-polymerase and RNA-polymerase activities. DBP of normal epidermis and horny layer contain four different DNases. One DNase of both DBP fractions is active only at pH 5.0. Three DNases of epidermal DBP are active at a pH-range from 5.0--8.5, while the corresponding DNases of horny layer-DBP are most active at pH 7.4. Probably these DNases have changed their pH-optimum during keratinisation. DBP of psoriatic scales include no activity of these three DNases and the pH 5.0-DNases seem to have reduced DNA-affinity. Human dermis DBP contain quite another set of four DNases which hardly can be correlated to the DNases of epidermal DBP. DNA-polymerase activities are present in each fraction and derive from different DNA-polymerases. Two DNA-polymerases with pI-values of 4.5 and 9.3 may correspond to beta- and alpha-DNA-polymerase of eukaryotes, respectively. Further activity of proteins which are focussed at pH 6.5--7.2 and 8.2 could be detected. The proteins represent either tissue-specific DNA-polymerases or further thymidine monophosphate incorporating enzymes. Contrary, RNA-polymerase activity could not be enriched from correlating extracts by DNA-cellulose chromatography.

Brain energy metabolism in global brain oedema.

Different degrees of severity in global brain oedema were induced by varying amounts of water intoxication (50, 100, 150, and 200 ml Aqua dest./kg b.wt. intravenously) in groups of six cats, which were functionally nephrectomized. Animals loaded with physiological saline and sham-operated served as controls. Two hours following the water load, the tissue concentrations of CrP, ATP, ADP, AMP, pyruvate, glucose, and lactate were determined by optical enzymatic analysis. The results show disturbances in brain energy metabolism dependent on the severity of the brain oedema. The high energy compounds and in consequence the ATP/ADP-ratio, and respectively the energy charge potential, fall in direct relationship to the severity of the brain oedema. Lactate and lactate-pyruvate ratio increase. The energy source of the cell, glucose as well as pyruvate, significantly falls in the group with severe brain oedema. The results of the brain energy metabolism were compared with our previous study concerning the brain water content, rCBF and CPP in global brain oedema (Meinig et al. 1973). The results show that the disturbances of energy metabolism are directly related to the rCBF and are not dependent on CPP over a wide range.

In situ assay of poxvirus-induced and virion-contained deoxyribonucleases in DNA polyacrylamide gels.

Cowpox virus and vaccinia virus WR specific DNases were identified by an in situ assay using DNA-containing acrylamide gels. A DNase present in the isolated virions of strain cowpox and vaccinia WR was identified. Its properties as determined by the in situ assay resembled those of the well-characterized DNase V1. The assay was also used to follow the time course of 'acid' DNase induction by cowpox virus in primary chick embryo fibroblasts.

[Different deoxyribonuclease-activities in bull seminal plasma (author's transl)]. / Unterschiedliche Desoxyribonuclease-Aktivitäten im Seminalplasma von Bullen

By means of the in situ assay of deoxyribonucleases in DNA-containing polyacrylamide gels after separation by micro-disc-electrophoresis different deoxyribonucleases are detectable in bull seminal plasma. There are two groups of acid deoxyribonuclease-activities with a pH optimum at pH 5.0, one with a pH optimum at pH 7.4 and an additional one with a pH optimum at pH 8.5.

Deoxyribonucleases activities in human leukemic cells and mouse lymphoblasts.

The deoxyribonuclease activities from human lymphocytes have been compared with the activities from acute lymphocytic leukemic cells and mouse leukemic cells L5178Y using the in situ detection of deoxyribonucleases in DNA-containing polyacrylamide gels following their separation by micro-disc-electrophoresis. A neutral deoxyribonuclease activity is completely missing in leukemic cells of untreated patients while a group of acid deoxyribonuclease activities is increased. A similar deoxyribonuclease pattern can be seen in L5178Y cells. Under medical treatment the increment of the acid deoxyribonuclease activities disappears and the neutral deoxyribonuclease activity reappears.

Characterization of different deoxyribonucleases in human lymphocytes.

Four groups of deoxyribonuclease activities from human lymphocytes have been characterized by deoxyribonuclease assay in DNA-containing polyacrylamide gels following their separation by disc-electrophoresis. All activities hydrolyse DNA endonucleolytically. One neutral deoxyribonuclease found in the cytoplasmic fraction prefers native or UV-irradiated DNA over denatured DNA as substrate and is a 5'-monoester former. Two groups of acid deoxyribonuclease activities are detectable in the nuclear fraction. Both are 3'-monoester formers. One is as well active with denatured DNA as with native DNA, the other one shows the same activity with native and UV-irradiated DNA but lower activity with denatured DNA. An alkaline deoxyribonuclease activity, also localized in the nucleus, is a 5'-monoester former, and prefers denatured or UV-irradiated DNA as substrate.

Separation of deoxyribonucleases (DNases) of normal human stratum corneum and psoriatic scales by micro-disc-electrophoresis.

Normal stratum corneum and psoriatic scales were homogenized and a differential centrifugation was performed. The DNase activity of the individual fractions was investigated by micro-dis-electrophoresis. At pH 5 only in the 600xg pellet and 105.000xg supernatant of normal keratin DNase activity could be observed. However, all psoriatic fractions showed distinct enzyme activities. At pH 7.4 little psoriatic DNase activity could only be demonstrated in the 105.000xg supernatant. Except from the 15.000xg pellet all fractions of normal stratum corneum displayed marked activities. In addition the 105.000xg supernatant showed two different DNase bands.

Deoxyribonuclease in human parotid saliva.

Deoxyribonuclease (EC 3.1.4.5, EC 3.1.4.6) activities in human parotid saliva were examined by microdisc electrophoresis. Three fractions, differing in their electrophoretic mobility and in their optimal incubation conditions, were characterized. The various enzyme activities were tested at pH 5.0 in 100 mmol with l-minus 1 Na-acetate buffer or at pH 7.04 in 100 mmol with l-minus 1 Tris-HCl-buffer in the presence of different combinations of MgCl(2), CaCl(2), EDTA, and Na(2)SO(4).