Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haematological values during normal reproduction of the maternal and the fetal rabbit

1. Haematological values of non-pregnant/non-lactating, pregnant as well as lactating rabbits and 28-day-old fetuses were measured. 2. The haemoglobin content in does decreased during the observed periods from 122 +/- 8 g/l to 100 +/- 11 g/l. In 28-day-old fetuses it was 85 +/- 0 g/l. 3. The erythrocyte count in 28-day-old fetuses was 2.4 X 10(12)/l. In the does, the erythrocyte count was 5.2 X 10(12)/l in week 4 of gestation. The erythrocyte volume in fetuses was about 45% higher than that of the doe. 4. In fetuses the leucocyte count was approximately one ninth that of the mother in week 4 of gestation (0.41 +/- 0.08 X 10(9)/l vs 3.8 +/- 0.4 X 10(9)/l).     

High Yield Isolation of Nuclei from Plant Protoplasts

Nuclei were isolated from protoplasts obtained from Parthenocissus tricuspidata Crown Gall callus tissues. The effect of various isolation procedures, detergent or ultrasonication, on yield and quality of nuclei was studied. A standard procedure, based on the use of 5 × 10^?3% Triton × 100 — 6% PVP — 20% glycerol, may be carried out in 30 min and gives 80 to 90% yield of nuclei in which RNA polymerase activity is retained.     

Cellulolytic and non-cellulolytic bacteria in rat gastrointestinal tracts.

Lactobacillus and Bifidobacterium species were the predominant organisms isolated from small intestinal (jejunal) contents of rats, and lactic acid was the only organic acid detected. The numbers of cellulolytic bacteria in small intestines were low (approximately 10(3)/g). The fermentation in ceca was different from that in intestines, as, in addition to small amounts of lactic acid, high concentrations of volatile fatty acids were detected. The mixed cecal microflora was able to digest cellulose (pebble-milled Whatman no. 1) and cabbage. High numbers of cellulolytic bacteria were found (0.5 X 10(8) to 12.2 X 10(8)/g; 6% of total viable bacteria). The predominant celluloytic organism isolated was Bacteroides succinogenes. Ruminococcus flavifaciens was isolated from a few animals. The kinds and numbers of the predominant non-cellulolytic organisms isolated from rat ceca were similar to those described by previous workers.     

Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models

We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQ's therapeutic potential in two different mice colon cancer models [1,2-dimethyl hydrazine (DMH) and xenografts]. We also examined TQ effects on the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion in vitro. Mice were treated with saline, TQ, DMH, or combinations once per week for 30 weeks and the multiplicity, size and distribution of aberrant crypt foci (ACF) and tumors were determined at weeks 10, 20 and 30. TQ injected intraperitoneally (i.p.) significantly reduced the numbers and sizes of ACF at week 10; ACF numbers were reduced by 86%. Tumor multiplicity was reduced at week 20 from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long-term; tumors did not re-grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly (P < 0.05) delayed the growth of the tumor cells. Using a matrigel artificial basement membrane invasion assay, we demonstrated that sub-cyto-toxic doses of TQ (40 microM) decreased C26 cell invasion by 50% and suppressed growth in three-dimensional spheroids. Apoptotic signs seen morphologically were increased significantly in TQ-treated spheroids. TUNEL staining of xenografts and immunostaining for caspase 3 cleavage in DMH tumors confirmed increased apoptosis in mouse tumors in response to TQ. These data should encourage further in vivo testing and support the potential use of TQ as a therapeutic agent in human colorectal cancer.     

An efficient procedure for isolation of nuclei from plant protoplasts

Summary The present communication describes an easy, efficient and rapid method for isolation of nuclei from plant protoplasts. Release of nuclei is accomplished by disruption of protoplasts in an appropriate buffer containing a very low concentration (0.01%) of the detergent Triton X-100. The pH of the nuclei isolation buffer (5.3) played a critical role in the recovery of stable nuclei in large numbers. Supplementation of buffer (10 mM MES) with spermine (0.1 mM), dithiothreitol (2.5 mM), ethylenediaminetetraacetic acid (2.5 mM) and Nad and KCl (10 mM each) improved nuclear yield and quality. With the method developed it is possible to routinely recover 95% nuclei from the protoplasts within 30 minutes. The nuclear preparations are of high purity with little detectable cytoplasmic contamination and no clumping of the nuclei. The structural integrity of the nuclei has been assessed and confirmed by Nomarski differential interference contrast optics and ultrastructural observations.     

A comparison between flow cytometric ploidy investigation and chromosome analysis of 32 human colorectal tumors

The correlation between flow cytometric ploidy investigation and classic chromosome analysis was studied in 32 human colorectal tumors. Flow cytometry was performed by nuclei isolation and DNA staining with ethidium bromide. Chromosome analysis was done after incubation with colcemid. In 12 cases, chromosome identification was possible by grouping according to the Denver system or by Q-banding. Generally, the measured DNA content corresponded well with the content expected from chromosome analysis, giving an average difference of 4%. In nine tumors, the measured DNA content was 4-18% higher than expected. Some of these discrepancies could be due to difficulties in identifying the corresponding cell populations in heterogeneous tumors. However, in general the number of cell populations and their quantitative representation by the two methods were statistically well correlated. The results indicate that flow cytometric ploidy investigation of colorectal tumors with the present technique is a reliable method, but also that a combination of both techniques may yield additional information about tumor cytogenetics.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Reciprocal translocations in germ cells of male mice receiving external gamma-irradiation

Data reported in the literature up to 1985 on reciprocal translocation induction in male mouse germ cells by external gamma-ray doses ranging from 0.5 to 6.0 Gy delivered at fixed dose rates were analyzed. On the assumption of a non-threshold linear dose response, zero effect at zero dose, and a center of distribution lying on an approximately straight line, calculations were made of linear regression coefficients. These coefficients (b), as a function of the dose rate (P), were well fitted by two straight lines: b = (3.15 +/- 0.59 log P) X 10(-6) for dose rates from 0.01 to 0.1 mGy/min; and b = (7.52 +/- 3.86 log P) X 10(-6) for dose rates ranging from 0.06 to 1.2 X 10(3) mGy/min. The intersection point of these two lines determined the so-called threshold level of the dose rate, namely, 4.6 X 10(-2) mGy/min, at which the effectiveness of external gamma-irradiation is not expected to exceed 2.36 X 10(-6)/mGy. In addition, experiments were undertaken in which yields were recorded of reciprocal translocations in germ cells of male mice exposed to 0.9 Gy of gamma-radiation at dose rates ranging from 6.14 X 10(-3) to 6.14 X 10(2) mGy/min (6 levels); comparisons were made with data published up to 1985 from similar studies using other fixed doses. To do this, translocation yields were expressed as relative yields (F) and their relationship to the dose rate (P) for the individual fixed doses was represented by an equation of the type: F = alpha + beta log P. For most of the equations, the regression coefficients were in good agreement and a single relationship was obtained to represent them. From the analysis performed it follows that, within the 0.6-6.0 Gy dose range, the pattern of the F vs. P relationship is unaffected by the dose. This supports the initial assumption that for the dose range up to 6.0 Gy the dose response for the reciprocal translocation yield is a non-threshold straight-line relationship.     

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs.

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.     

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.     

Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells

A rapid isolation procedure of nucleoli from detergent purified nuclei of some tumor and tissue culture cells is described. The procedure makes use of a non-ionic detergent, Nonidet P40 and sodium deoxycholate to purify nuclei followed by the addition of Ca²⁺ or Mg²⁺ to strengthen the nucleoli against sonication. Enzymatically active (with respect to nucleolar RNA polymerase) nucleoli containing undegraded nucleolar RNAs may be isolated from a mouse hepatoma MH134, Ehrlich ascites tumor, HeLa cells, L cells and C3H2K cells with this procedure.     

Removal of bacteria from water by adhesion to cross-linked poly(vinylpyridinium halide).

Cross-linked poly(vinylpyridinium halide) was found to have a novel and remarkable ability to remove bacteria from water. For example, when 10 g (wet weight) of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) was contacted with 20 ml of suspensions of Escherichia coli (9.7 X 10(4) to 9.7 X 10(7)/ml), Salmonella typhimurium (8.0 X 10(6) to 1.1 X 10(7)/ml), Streptococcus faecalis (5.0 X 10(7)/ml), Staphylococcus aureus (8.1 X 10(7)/ml), and Pseudomonas aeruginosa (3.2 X 10(5)/ml) under stirring in sterilized physiological saline at 37 degrees C, 99% of the viable cells of these bacteria were removed in 2 to 6 h. When suspensions of these bacteria (10(5) to 10(8) cells per ml) were passed through a column (20 mm by 100 cm) of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) at 37 degrees C with a flow rate of 0.8 to 1.4 bed volumes per h, 97 to 100% of the viable cells were eliminated from the suspensions during the treatment. Mechanistic studies demonstrated that cross-linked poly(vinylpyridinium halide) irreversibly captured these bacteria alive during the treatment. That is, total organic carbon was removed during the treatment, and the bacteria which adhered to the resin proliferated on the bacterial medium. The adhesion capacity was estimated to be 10(10) cells per g (dry weight). Total organic carbon was also removed even when the bacteria were killed by heat treatment before the column studies.     

Effect of prostaglandin F2 alpha on propulsive activity of the isolated segmental colon of the guinea-pig.

The effects of prostaglandin F2alpha (PGF 2alpha) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated. Using experimental conditions under which spontaneous propulsive activity was negligible, PGF2alpha (5X10(-8)X1X10(-6)M), added to the bathing medium increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1X10(-7)g/ml). The contractions produced by PGF2alpha (5X10(-7) -1X10(-5)M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1X10(-7) g/ml). From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF2alpha may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

The core proteins of 35S hnRNP complexes. Characterization of nine different species

Ribonucleoprotein complexes (hnRNP) containing fragments of heterogeneous nuclear (hn)RNA and sedimenting at 35-40 S were isolated from the nuclei of HeLa S3 cells using the pH 8.0/diffusion technique. These hnRNP complexes are thought to be part of the hnRNA processing apparatus. The major protein components (core proteins) were identified by their constant ratios in native particles and in 35S hnRNP particles reconstituted in vitro. All of the core proteins, with one exception, show an increase in Mr on sodium dodecylsulfate (NaDodSO4)/polyacrylamide gels containing 8 M urea, indicative of secondary structure elements resistant to denaturation by NaDodSO4. The nine core proteins found by us are: A1 [Mr(NaDodSO4) 31 X 10(3)/Mr (urea) 38 X 10(3), apparent isoelectric point, pIapp 9.3], A2 (32.5 X 10(3)/39 X 10(3), 8.4), B1a (35.5 X 10(3)/41 X 10(3), 8.8), B1b (35.5 X 10(3)/44 X 10(3), 8.3), B1c (35.5 X 10(3)/43 X 10(3), 5.7) B2 (37 X 10(3)/42 X 10(3), 9.15), C1 (39 X 10(3)/46 X 10(3), 9.2), C2 (40.5 X 10(3)/45 X 10(3), 5.55) and C3 (38.5 X 10(3)/37 X 10(3), 4.8). Individual proteins were electroeluted from two-dimensional gels and their amino acid composition determined. Difference indices were calculated and show a group of closely related basic proteins (A1, A2, B1a, B1b, B2, C1), two related slightly acidic proteins (B1c, C2) and a distinct acidic member (C3). Two-dimensional analysis of tryptic fragments and one-dimensional separation of peptides after V8 protease treatment support these data. Peptide mapping of the proteins A1 and A2 from bovine and human cells yields identical fragments indicating a high degree of cross-species conservation. An additional protein (D4: 44 X 10(3)/55 X 10(3), greater than 9.5) was found, which preferentially associates with heavier, oligomeric hnRNP structures. Only traces of actin are present in the 35S hnRNP fraction. All core proteins are modified by charge. A large part of the charge isomers arises by phosphorylation, which has been shown by labeling with 32PO4 in vivo and with [gamma-32P]ATP in vitro. In vitro the phosphate transfer is mediated by an endogenous protein kinase associated with the 35S hnRNP complexes. The major core protein A1 exists in two conformeric forms (A1 and A1x) of which only A1x serves as phosphate acceptor in vivo.     

Adaptation of the interspersed repetitive sequence polymerase chain reaction to the isolation of mouse DNA probes from somatic cell hybrids on a hamster background

A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, L1. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds.