Activation of cycasin to a mutagen for Saccharomyces cerevisiae by rat intestinal flora

Genetic test systems involving microorganisms and liver enzyme preparations may be insufficient to detect compounds that require breakdown by enzymes provided by the microbial flora of the intestinal tract. A method is described for providing such activation and for simultaneously testing the potential genetic activity of breakdown products in an indicator organism. Parabiotic chambers containing Saccharomyces cerevisiae genetic test organisms in one chamber were separated by a membrane filter from rat cecal organisms and test chemical contained in the other chamber. The genetic activities of cycasin breakdown products for mutation, gene conversion, and mitotic crossing-over in samples incubated aerobically are reported. Samples containing cycasin alone had a small but clearly increased frequency of genetic damage. Samples containing rat cecal organisms without cycasin showed no increase in genetic activity. Anaerobic incubation resulted in no increase in genetic activity in any of the samples.     

Genotoxic activity in vivo of the naturally occurring glucoside,cycasin, in the Drosophila wing spot test

Cycasin, methylazoxymethanol-β-glucoside, is a naturally occurring carcinogenic compound. The genotoxicity of cycasin was assayed in the Drosophila wing spot test. Cycasin induced small single and large single spots on feeding at 10 μmol/g medium. The presence of these spots indicates that cycasin is genotoxic in Drosophila melanogaster. Microorganisms which showed β-glucosidase activity for cleaving cycasin to toxic aglycon were isolated from gut flora of the Drosophila larvae. Consequently, the Drosophila wing spot test would be useful for mutagenicity screening of other naturally occurring glucosides.     

Studies on Cycasin,a New Toxic Glycoside,of Cycas revoluta Thunb

The enzymatic hydrolysis of cycasin with cycad-emulsin, prepared from the seeds of Cycas revoluta, is described. As the complete degradation products we obtained about one mole each of formaldehyde, nitrogen gas, and methanol per mole of cycasin, in addition to glucose, the sugar component. These products are the same as those found in the acid hydrolysis of cycasin which has been reported previously. Therefore, it is concluded that the aglycone of cycasin can not be liberated intact as a single component, but decomposes into those smaller molecules as above even by means of the enzymatic hydrolysis.     

Studies on the function of intracellular ribonucleases. V. Ribonuclease activity in ribosomes and polysomes prepared from rat liver and hepatomas

The RNase activity and properties of ribosome and polysome preparations from normal rat liver and some hepatomas have been examined. Polysome and ribosome preparations from the Novikoff, McCoy MDAB, and Dunning hepatomas had considerably higher specific RNase activity than corresponding preparations from normal rat liver, Novikoff ascites, or Morris 5123 hepatomas. The optimum pH of the RNase was approximately 8.5 for all samples tested, and the samples showed no evidence of latent RNase activity when treated with 3 M sodium chloride, EDTA, urea, or p-chloromercuribenzenesulfonic acid. The RNase activity appeared to be associated principally with breakdown products and/or subunits smaller than 80S. In the presence of Mg⁺⁺ ions, subunits could reaggregate to form monomer ribosomes indistinguishable from the natural products, but some of the reassociated ribosomes could contain RNase activity which had been bound to the smaller particles. Similar results were obtained with spermine. In the hepatomas, evidence was obtained for the preexistence of considerable amounts of the smaller, RNase-containing subunits in the cell. When a small amount of crystalline bovine pancreatic RNase was added to partly dissociated ribosomes, the RNase was found only in association with the smaller subunits, and little or no enzyme was taken up by ribosomes or polysomes. The results have led to the conclusion that RNase is not a normal constituent of the ribosome or polysome, but that RNase may become associated with these particulates if dissociation and reassociation take place. Some implications of these findings for the stability of messenger RNA and for the mechanism of its breakdown are discussed.     

基因组编辑产品的安全管理

基因组编辑技术是指利用特异性核酸酶的定点剪切活性与细胞内源DNA损伤修复活性,在基因组水平上对目的核酸序列或单个核苷酸进行定点修饰的基因工程技术,该技术可以对生物体基因组进行精确敲除、插入、单碱基突变或置换等编辑。目前,基因组编辑技术具有精确性、高效性及易操作性,应用范围日益扩大。文中简要概述了3种主要的基因组编辑技术工具及基因组编辑类型,介绍了美国、欧盟等国家和地区对基因组编辑产品的监管体制。同时,基于我国对转基因产品的安全管理原则与体系,初步提出了基因组编辑产品的安全管理思路。根据中间材料或产品中是否含有外源编辑酶蛋白基因成分对基因组编辑产品进行分类管理,含有外源编辑酶的材料应按现有转基因安全管理办法进行管理;中间材料或产品中不含有Cas9等编辑酶的材料应根据被编辑位点的特征进行具体分类管理。     

On the distribution of genotoxic factors in various organs of mice treated with cycasin

The distribution of genotoxic factors in various organs of mice treated orally with methylazoxymethanol-beta-D-glycoside (cycasin) was investigated using the DNA-repair host mediated assay. Indicator of genotoxic activity was a pair of streptomycin dependent Escherichia coli strains differing vastly in DNA repair capacity; uvrB/recA vs. uvr+/rec+. The animal-mediated assays were performed by injecting mixtures of the two strains i.v. and orally into mice, which were subsequently treated with the test chemical and from which the differential survival of the indicator bacteria present in several organs was determined. The same strains and selection procedures were also used for assessing the DNA-damaging activity in vitro. In the animal-mediated assays in which cycasin was applied orally, significant effects were observed at doses of 100 and 500 mg/kg body weight. The organ distribution of genotoxic factors in the host animal was as follows: the highest genotoxic activity was observed in the liver, followed by intestine and stomach; a clear effect was also observed in the kidneys and, to a lower extent, in the blood stream and in the lungs at the highest dose administered (500 mg/kg body weight). Under in vitro conditions a marginal genotoxic effect was observed even in the absence of liver homogenate, indicating that the test compound is possible activated (hydrolysed) by the E. coli cells. Therefore the genotoxic activity of cycasin observed in the gastrointestinal tract was not unexpected, since the substance was applied orally, thereby exposing the indicator bacteria in these organs to high levels of unmetabolised compound, especially in the stomach. In the intestine members of the microbial flora probably contribute to the metabolic activation of the test compound. The occurrence of genotoxic factors remote from the gastrointestinal tract shows that the present compound or active metabolites thereof penetrate through the intestinal barrier. The extraordinarily high genotoxic activity observed in the liver suggests that the compound is additionally activated in this organ. In compliance with previous in vitro findings this second activation step might lead to the formation of the highly reactive aldehydic form of methylazoxymethanol (MAMAL) mediated by dehydrogenases. Comparison with carcinogenicity studies indicates a good correlation between the distribution of genotoxic effects as determined in the present studies and the localisation of tumors in various organs of rodents treated with cycasin.     

Azoxyglycoside content and beta-glycosidase activities in leaves of various cycads

Azoxyglycoside contents in leaves of 32 cycad species belonging to 10 cycad genera and the seeds of 4 Encephalartos species were analyzed by HPLC with a YMC-PA03 amide column. Azoxyglycosides were detected in mature leaves of 14 cycad species including 2 Bowenia, 2 Lepidozamia, 1 Microcycas, and 1 Stangeria species, but not in mature leaves of 18 other cycad species; 2 of 3 Ceratozamia, 1 of 3 Cycas, 3 of 3 Dioon, 10 of 11 Encephalartos, 1 of 3 Macrozamia and 1 of 3 Zamia species analyzed. The ratios of beta-glycosidase activity toward cycasin and macrozamin in extracts from the leaves of 9 species belonging to 9 genera were measured. The hydrolysis of cycasin was higher in the leaf extracts of Cycas revoluta, Bowenia spectabilis, Stangeria eriopus and Ceratozamia mexicana, whereas in Lepidozamia hopei, the hydrolysis levels of cycasin and macrozamin were similar. On the other hand, activity toward macrozamin was higher in Dioon edule, Encephalartos villosus, Macrozamia miquelii and Zamia fischeri. The hydrolytic activities in most species were estimated to be sufficient for the release of methylazoxymethanol in leaves analogous to the cyanogenesis of cyanogenic plants. Therefore, hydrolysis of azoxyglycosides by endogenous glycosidase in leaves seems to occur by accidental injury of leaves. However, in M. miquelii leaf extract, hydrolytic activity toward macrozamin was high and the activity toward cycasin was very low, though only cycasin was found in the leaves of this species.     

A versatile Ca2+ ion-sensitive minielectrode with a microincubation chamber

A new, versatile Ca2+ ion-sensitive minielectrode with a microincubation chamber was designed for the direct, continuous monitoring of changes in Ca2+ ion activity in microgram tissue samples. The sample can be stirred in the microincubation chamber and kept at a constant temperature through thermostatisation. Samples with a protein content ranging from 10 to 40 micrograms are required for the measurement. This is two to three orders of magnitude less than necessary for measurement of Ca2+ ion activity with conventional, commercially available Ca2+ ion-sensitive electrodes. The device should be useful for a variety of applications in many research areas where sample volumes are small. Some examples are presented in this communication using mitochondria and microsomes from nine different rat tissues. In these experiments it is shown that with mitochondria from all tissues a steady-state ambient free Ca2+ concentration between 0.6 and 0.8 microM is reached, though the Na+ sensitivity of ruthenium red-induced Ca2+ efflux from these mitochondria varies considerably in dependence on the tissue. The additional presence of microsomes resulted in a steady-state Ca2+ concentration between 0.1 and 0.2 microM.     

An in vivo evaluation of induction of abnormal sperm morphology in mice by landfill leachates

Although several reports have demonstrated the acutely toxic and genotoxic effects of landfill leachates in microbial organisms, plants and aquatic animals, the effects of pollutants present in these leachates have not been clarified yet in terrestrial animals. This study mainly aimed to evaluate a potential genetic effect of raw and simulated leachates from Orita-Aperin and Oworonsoki landfills in south-west Nigeria by use of the murine sperm-head abnormality test. These landfills neither have a synthetic membrane liner at the bottom, nor a natural layer of compacted soil with the desired hydraulic conductivity, nor a run-off control system. As a result, the leachates produced are discharged into the environment. Samples designated as Orita-Aperin Raw Leachate (OARL), Orita-Aperin Simulated Leachate (OASL), Oworonsoki Raw Leachate (OWRL) and Oworonsoki Simulated Leachate (OWSL) were analyzed in the sperm-head abnormality test at concentrations (v/v) of 1%, 2.5%, 5%, 10% and 25%. Mice were given 0.5 ml sample per day for five consecutive days by intraperitoneal injection. Each dose group comprised seven mice, and a 5-week exposure period was utilized. The data show that the test mixtures induced a dose-dependent, statistically significant increase (P<0.05) in the number of sperm with abnormal morphology. Physico-chemical analysis of the test samples shows that they contained constituents that are capable of inducing mutation in biologic system. The interaction of some of these constituents with the genetic material in the differentiating cells during spermatogenesis may be responsible for these observations. This is relevant in environmental waste management, and for the assessment of the hazardous effects of the chemicals in landfill leachates.     

The pH dependence of breakdown of various purified brain proteins by cathepsin D preparations

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range. The formation of initial breakdown products and the further catabolism of the breakdown products was dependent on pH; hence the pattern of peptides formed from glial fibrillary acidic protein was different in incubations at different pH's. The relative activity of the enzyme preparations differed, depending on the substrate: with tubulin and S-100 as substrates, rat brain cathepsin D was the most active and the bovine spleen enzyme was the least active. With calmodulin and glial fibrillary acidic protein as substrates, rat brain and spleen cathepsin D activities were similar, and bovine brain cathepsin D showed the lowest activity. Actin breakdown fell between these two patterns.The rates of breakdown of the substrates were different; expressed as μg of substrate split per unit enzyme per h, with rat brain cathepsin D activity was 8–9 with calmodulin and S-100, 4 with glial fibrillary acidic protein, 1.8 with actin, and 0.9 with tubulin. The results show that there are differences in the properties of a protease like cathepsin D, depending on its source; furthermore, the rate of breakdown and the characteristics of breakdown are also dependent on the substrate.We recently measured the breakdown of brain tubulin by cerebral cathepsin D in a continuing study of the mechanisms and controls of cerebral protein catabolism (Bracco et al., 1982a). We found that tubulin breakdown is heterogeneous, that membrane-bound tubulin is resistant to cathepsin D but susceptible to thrombin (Bracco et al., 1982b), and that cytoplasmic tubulin was in at least two pools, one with a higher, another with a lower, rate of breakdown. The pH optimum of tubulin breakdown by cerebral cathepsin D differed significantly from the pH optimum of hemoglobin breakdown by the same enzyme.These findings showed that the properties of breakdown by a cerebral protease depend on the substrate. To further examine this dependence of properties of breakdown on the substrate, we now report measurements of pH dependence of breakdown of several purified proteins (tubulin, actin, calmodulin, S-100, glial fibrillary acidic protein [GFA]) from brain by cathepsin D preparations from three sources, rat brain, bovine brain, and bovine spleen. We also compare the rate of breakdown of the various proteins with the rate of hemoglobin breakdown.     

Rapid filter assay for the detection of DNA polymerase activity: direct identification of the gene for the DNA polymerase from Thermus aquaticus

A method for rapid identification of DNA polymerase activity employing an activated DNA substrate covalently bound to nitrocellulose membranes is described. Samples containing DNA polymerase are spotted and the membranes are incubated in an appropriate polymerization buffer containing radioactively labelled dNTPs. By autoradiography of the dried filters, DNA polymerase activity can be directly identified. The method can be used for fast and large-scale screening of chromosomal expression libraries for heterologous DNA polymerases characterized by activity optima different from those of the host organisms. We have identified the gene of the thermostable DNA polymerase from Thermus aquaticus in an expression library of Escherichia coli.     

Effects of sorghum grain tannins and dietary protein on the activity of liver UDP-glucuronyltransferase

The activity of liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17), an enzyme known to detoxify phenolic compounds, was measured in chicks and rats fed high- (HTS) and low-tannin sorghums (LTS). In an initial investigation, activity was significantly elevated in chicks fed HTS-soybean meal diets over those fed the LTS control diet. Other studies were designed to differentiate between the effects due to tannin and those resulting from a protein deficiency which had previously been reported to increase the activity of this enzyme. In general, only a relatively small part of the increased activity observed by feeding HTS to chicks could be attributed to a tannin-induced protein deficiency. The same phenomenon of elevated activity produced by feeding HTS to chicks was not observed in the rat. These results would suggest that sorghum tannins, or their breakdown products, are absorbed and activating UDP-glucuronyltransferase in the chick, but not the rat.     

Extraction of collagenase from the involuting rat uterus.

Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.     

The gravimetric method for the determination of residual moisture in freeze-dried biological products

The gravimetric test for the determination of residual moisture in freeze-dried biological products performed in a humidity- and temperature-controlled room with the use of scrupulous gravimetric analytical technique can be used to accurately determine residual moisture in freeze-dried biological products such as antihemophilic factor (human) or honey bee venom allergenic extract. This method determines the first water of hydration of sodium tartrate dihydrate (7.93%) to within 1.3% of the calculated value with a relative standard deviation of 0.3% for 10 replicates. For this gravimetric procedure, freeze-dried samples containing from 1.12 to 4.4% residual moisture had relative standard deviations ranging from 3.6 to 9.1%. Samples containing less than 1.0% residual moisture by the gravimetric method such as intravenous immune globulin and antihemophilic factor (human) had relative standard deviations ranging from 16.7 to 47.0%. Relative standard deviations for residual moisture tests performed on comparable samples by the Karl Fischer and thermogravimetric methods showed similar variability.     

The biosynthesis of rat serum albumin. Metabolism of the NH2-terminal hexapeptide of proalbumin

Proalbumin differs from serum albumin in containing a leading hexapeptide segment, Arg-Gly-Val-Phe-Arg-Arg. This propeptide is removed in the Golgi complex immediately prior to secretion of the albumin, but its fate and possible functions are unknown. We have tested for the presence of the propeptide and its immediate catabolic products in rat liver and plasma and have studied both the disappearance of 3H-propeptide after intravenous injection and the breakdown of synthetic propeptide by rat liver cell components and plasma in vitro. We found no detectable propeptide or its two pentapeptide derivatives in rat liver or plasma at a sensitivity of less than 1 microM. Injected 3H-propeptide was completely cleared from blood within 2 min. No binding of free propeptide to serum albumin was observed. Liver cell fractions as well as blood plasma degraded added propeptide, with the highest activity being observed in smooth microsomes, the Golgi-enriched fraction, and plasma membrane. These preparations chiefly removed the terminal arginine residues, whereas enzymes in the cytosol degraded the peptide completely to amino acids. The activity in plasma resided largely in an alpha-globulin with molecular mass of about 280,000 Da which appears to be carboxypeptidase N. We conclude that the liberated propeptide is quickly broken down within the liver cell and does not accumulate in an amount sufficient to exert feedback or other effects on albumin synthesis.     

Occurrence of azoxyglycosides in Macrozamia riedlei and their effects on the free-living isolated Nostoc PCC 73102

The presence of azoxyglycosides in the Australian cycad Macrozamia reidlei was examined using high performance liquid chromatography. Cycasin and macrozamia were present in all tissues examined, cycasin being three to 17 times more abundant than macrozamin. The symbiotic organ, the coralloid root, contained 0.16% [g/g (fresh weight)] cycasin and 0.01 % (same unit) macrozamin. Addition of these azoxyglycoside concentrations to nitrogen-fixing Nostoc PCC 73102 cultures, a filamentous heterocystous cyanobacterium originally isolated from Macrozia, inhibited light and dark nitrogenase (acetylene reduction) activity. No effects were observed on in vitro glutamine synthetase activity or net in vivo CO₂ fixation. Cycasin (1.6%) and macrozamin (0.1%), i.e. 10 times the concentrations observed in the coralloid root, decreased phycobiliprotein content by 25 and 45%, 1 and 4 hr after the addition, respectively. The relative distribution of individual phycobiliproteins was not affected.     

Carbapenemase Producing Bacteria in the Food Supply Escaping Detection

Carbapenem antimicrobials are critically important to human health and they are often the only remaining effective antibiotics for treating serious infections. Resistance to these drugs mediated by acquired carbapenemase enzymes is increasingly encountered in gram-negative bacteria and is considered a public health emergency. Animal origin food products are recognized as a potential source of resistant organisms, although carbapenem resistance has only recently been reported. In western countries there are active resistance surveillance programs targeting food animals and retail meat products. These programs primarily target beef, pork and poultry and focus exclusively on E. coli, Salmonella, Campylobacter spp. and Enterococcus spp. This global surveillance strategy does not capture the diversity of foods available nor does it address the presence of resistance gene-bearing mobile genetic elements in non-pathogenic bacterial taxa. To address this gap, a total of 121 seafood products originating in Asia purchased from retail groceries in Canada were tested. Samples were processed using a taxa-independent method for the selective isolation of carbapenem resistant organisms. Isolates were characterized by phenotypic antimicrobial susceptibility testing, PCR and DNA sequencing. Carbapenemase producing bacteria, all bla_OXA-48, were isolated from 4 (3.3%) of the samples tested. Positive samples originated from China (n=2) and Korea (n=2) and included squid, sea squirt, clams and seafood medley. Carbapenemase producing organisms found include Pseudomonas, Stenotrophomonas and Myroides species. These findings suggest that non-pathogenic bacteria, excluded from resistance surveillance programs, in niche market meats may serve as a reservoir of carbapenemase genes in the food supply.     

Na+-Dependent and Phlorizin-Inhibitable Transport of Glucose and Cycasin in Brain Endothelial Cells

Abstract: Although cycasin (methylazoxymethanol β- d -glucoside) is proposed to be a significant etiological factor for the prototypical neurodegenerative disorder Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex, the mechanism underlying transport of cycasin across the blood-brain barrier (BBB) is unknown. We examined cycasin transport in cultured bovine brain endothelial cells, a major element of the BBB. Cycasin was taken up into endothelial cells in a dose-dependent manner with maximal uptake observed at a concentration of 10 µ M . Cycasin uptake was significantly inhibited by α-methyl- d -glucoside, a specific analogue for the Na⁺-dependent glucose transporter (SGLT), by the SGLT inhibitor phlorizin, by replacement of extracellular NaCl with LiCl, and by dinitrophenol (DNP), an inhibitor of energy metabolism. In addition, cycasin produced inward currents in a whole-cell voltage clamp configuration. Peak currents were observed at 10 µ M with a trend toward reduction at higher concentrations, and currents were clearly blocked by α-methyl- d -glucoside, phlorizin, and DNP. In addition, cycasin never evoked currents in Na⁺-free extracellular solution. These results suggest that cycasin is selectively transported across brain endothelial cells, possibly across the BBB by a Na⁺/energy-dependent glucose transporter.     

Immunization of guinea pigs with Neisseria gonorrhoeae: strain specificity and mechanisms of immunity.

Infection of subcutaneusly implanted chambers in guinea pigs conferred immunity against homologous infection of other chambers in the same animals. However, attempts to immunize guinea pigs by subcutaneous injection of filtered fluid from infected chambers, or with small doses of formalin-killed, chamber gonococci were not successful. Thus, neither organisms grown in vivo nor their extracellular products appeared to be exceptionally immunogenic. In immunizing tests with different isolates of gonococci adapted to growth in guinea-pig chambers, cross-immunity to chamber infection with low challenge doses was detected only between two of six isolates. The killing of gonococci in chambers of immunized animals, which occurred only after homologous challenge or with the heterologous strain showing cross-immunity, was not due primarily to humoral factors in the chamber fluid but probably to an enhanced effectiveness of phagocytosis. The serum of immunized animals was bactericidal for homologous strains and for the strain showing cross-immunity but not for strains showing no cross-immunity. Hence, serum bactericidal activity might be a useful indicator for investigating the specificity of immunity produced by different gonococcal strains.     

Polymerase chain reaction analysis of laboratory generated bioaerosols

The common methods for analyzing bioaerosols are based on maintaining organism viability and quantifying culturability which may result in the underestimation of microbial concentrations. The present study employed a well-developed technique that only requires cellular DNA to identify organisms. Polymerase chain reaction (PCR) was chosen to amplify specific DNA sequence from an organism, to detect and semi-quantify organisms. Suspensions ofFrancisella tularensis were aerosolized in a chamber, and air samples were collected using impingers. Samples were analyzed using limiting dilution PCR, and the results compared with those from a traditional plate counting. Results indicated that the limiting dilution PCR provides a new way to identify and quantify bioaerosols that does not rely on viability and culturability. Therefore, the method would provide a more reliable estimate of airborne bacterial concentrations compared to traditional plate counts.