The Tropic Effect of Intrarectal Deoxycholate On Rat Colorectum Is Unaffected By Oral Metronidazole

Abstract. Intrarectal administration of sodium deoxycholate (SDC) enhances experimental colorectal carcinogenesis, an effect that is partly vitiated by oral metronidazole. the effect of topical SDC with or without concurrent metronidazole on colorectal cell proliferation was explored in male Sprague-Dawley rats ( n = 30) allocated to five groups. Two groups received thrice weekly intrarectal instillations of 1 ml N saline or 1 ml 0.12 m SDC. A third group received SDC plus metronidazole 22.5 mg/kg/day in the drinking water. Controls had no instillations or metronidazole alone. At time of killing (10 weeks), crypt cell production rate (CCPR) was determined by the stathmokinetic technique for four large-bowel segments. Saline had no significant effect on colorectal CCPR but SDC produced increases throughout, varying from 53% in the proximal colon to 222% in the rectum ( P < 0.01). Metronidazole did not reduce this effect, although given alone it reduced colonic CCPR by 40 to 50%. the direct tropic effect of bile acids could largely explain their cocarcinogenic properties. Since metronidazole does not prevent this increase in cell proliferation, its mildly protective role against cancer may reflect the presence of fewer anaerobes capable of degrading bile acids to carcinogenic metabolites.     

Isolation and characterization of a human interleukin 2 gene

An interleukin 2 (IL-2) gene was isolated from a Charon 4A human gene library. Electron microscopic examination of 15 heteroduplexes formed between the genomic DNAs and the IL-2 cDNAs demonstrated that the size of the IL-2 gene is about 5.1 +/- 0.5 kb and that there are at least two introns in this gene. Nucleotide sequence of the 5' flanking region of the IL-2 gene showed a homology with that of the corresponding region of the human immune interferon gene.     

Isolation and characterization of nucleases from a clinical isolate of Serratia marcescens kums 3958

Two new extracellular nucleases, nucleases SM1 and SM2, were purified from the culture fluid of S. marcescens kums 3958, a fresh clinical isolate. The purification was carried out by the following steps; ammonium sulfate precipitation, and DEAE-cellulose and Sephadex G-100 column chromatography. At the final step, nucleases SM1 and SM2 were purified about 3,700- and 1,000-fold, respectively. They were free from phosphomonoesterase and phosphodiesterase activities. The pIs were 8.1 and 7.5 for nucleases SM1 and SM2, respectively. The molecular weight was estimated to be 35,000 for both enzymes by SDS-polyacrylamide disc gel electrophoresis. The results of amino acid analyses showed that both the threonine and serine contents were higher in nuclease SM2 than in SM1. Furthermore, nuclease SM1 was more stable than nuclease SM2 at 4 degrees C. The other properties of the two enzymes were similar; pH optimum (8.0), Mg2+ or Mn2+ for activation, and inhibition by chemical reagents such as EDTA and pyrophosphate. No significant difference was found in base specificity between nucleases SM1 and SM2. Both enzymes specifically degraded double-stranded homopolymers, especially poly(I). poly(C), as well as yeast RNA and calf thymus DNA. They hardly degraded, however, single-stranded homopolymers such as poly(dA), poly(G), and poly(U).     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

Chemiluminescent assay of various enzyme activites and its application to enzyme immunoassays

A highly sensitive chemiluminescent assay for NAD(P)H have been developed. The principle of the method is as follows; NAD(P)H reduces molecular oxygen to superoxide anion (O) and hydrogen peroxide (H₂O₂) in the presence of 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS) as electron mediator. The produced O and H₂O₂ can be measured by chemiluminescent reaction using isoluminol (IL) and microperoxidase (m-POD). A linear relationship between chemiluminescence intensity and NAD(P)H concentration (log/log) was obtained ranged from 10^?9 mol/I to 10^?5 mol/I. This chemiluminescent reaction has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), β-D -galactosidase (β-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, β-Gal and ALP were 10^?18 mol, 10^?20 mol and 10^?18 mol per assay, respectively. The chemiluminescent assay of these enzymes applied to chemiluminescent enzyme immunoassay for 17α-hydroxy-progesterone and DNA hybridization assay using these enzymes as label.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.