Sugar preferences and xylose metabolism of a mammal pollinator, the Namaqua rock mouse (Aethomys namaquensis).

The sugar preferences of 10 Namaqua rock mice, Aethomys namaquensis, were assessed using pairwise combinations of 30% (w/w) solutions of sucrose, glucose, fructose, xylose, and a mixture of equal parts of glucose and fructose. The tests were designed to control for side biases that were apparent in preliminary experiments. The mice preferred sucrose to fructose and glucose. Xylose, although the least preferred sugar, was willingly consumed by the rodents (up to 5.8 mL in 24 h). This contrasts with the strong rejection of xylose by nectarivorous birds on which similar preference tests were performed. The efficiency of xylose absorption and metabolism by A. namaquensis was investigated by measuring dietary intake, blood xylose levels, and urinary and fecal xylose output. Again in contrast to the birds, the apparent absorption efficiency of xylose was found to be very high at 97%, but exactly how the xylose is metabolized requires further study. Xylose is thought to be only slowly metabolized by mammals, and it is possible that intestinal bacteria may serve this purpose, like the ruminal bacteria that break down xylans in plant tissue.     

Xylose utilization and short-chain fatty acid production by selected components of the intestinal microflora of a rodent pollinator (Aethomys namaquensis)

Namaqua rock mice (Aethomys namaquensis) consume nectar xylose when visiting Protea flowers. Whole-animal metabolism studies suggest that the gastrointestinal microflora plays an important role in xylose metabolism in A. namaquensis. We collected caecal contents under anaerobic conditions, cultured caecal microflora both aerobically and anaerobically, and assessed caecal microbial xylose utilization using a ¹⁴C-xylose incubation assay. All four mice sampled hosted culturable caecal micro-organisms that tested positive for xylose utilization. These were classified by 16S rRNA based taxonomy as: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Shigella boydii, Arthrobacter sp. and members of the fungal genera Aspergillus and Penicillium. Cultures of these isolates were then analyzed by gas chromatography to determine the types and quantities of short-chain fatty acids produced by xylose fermentation. These results are discussed in the context of other studies of gut microflora in vertebrates.     

The effect of different oral antibiotics on the gastrointestinal microflora of a wild rodent (Aethomys namaquensis)

Gut sterilization via the oral administration of antibiotics facilitates physiological studies of the nutritionally important relationship between intestinal microflora and the host. However, the composition of gut flora is extremely variable, and as a result, the efficacy of antibiotics in achieving gut sterilization varies considerably between species. We tested the effectiveness of three antibiotic cocktails in sterilizing the gut of a rodent pollinator, the Namaqua rock mouse (Aethomys namaquensis). The cocktails were (1) streptomycin sulfate and bacitracin (previously used with domestic mice and rats), (2) chloramphenicol and bacitracin (based on antibiotic screening tests performed on faecal flora) and (3) Baytril 10% oral solution (a veterinary antimicrobial agent containing enrofloxacin). We tested for antibiotic inactivation by determining bacterial viability through fluorescence staining of faecal samples. We also tested techniques to maintain sterility during antibiotic treatment without the benefit of a laminar flow cabinet. Antibiotics were administered orally in food and water consumed ad libitum over 4 consecutive days. Antibiotic effectiveness was assessed by culturing anaerobic bacteria from faecal samples collected before and after each antibiotic treatment. Treatment with Baytril 10% oral solution eliminated or significantly reduced faecal flora, whereas other antibiotics did not. This study clearly demonstrates the importance of testing the effectiveness of antibiotics before their use in studies that involve antibiotic-treated subjects, particularly if these are species previously untested.     

Intestine lengths of Southern African savanna rodents and insectivores: intra- and interspecific comparisons

Comparative intestine lengths of 15 Southern African savanna rodents and insectivores are presented. Differences in relative intestine length (ratio between hind gut length to head/body length) between seasons were significant in Aethomys chrysophilus . In various species, breeding females, with their increased energy requirement, had hind gut lengths that were well above average. These females also accounted for the differences between sexes which were significant in two species ( Aethomys chrysophilus and A. namaquensis ). Within species no correlation was found between relative intestine length and body size (length and weight) of the animals. Between species the correlation was significant and the larger species had relatively longer intestines. Three groups can be distinguished according to the different feeding types of the species. The omnivorous Praomys natalensis has by far the longest intestine. It is followed by a group of species that can be considered as granivore/herbivore. The species that are partly or predominantly insectivorous have the shortest intestine. The results of the field study are found to be in agreement with reports from laboratory experiments concerning adaptations of intestine length to different environmental situations.     

The metabolism of methoxyethylmercury salts

The metabolism of methoxy[(14)C]ethylmercury chloride in the rat has been investigated. After a single subcutaneous dose a small proportion is excreted unchanged in urine and a larger amount in bile with some resorption from the gut. The greater part of the dose is rapidly broken down in the tissues with a half-time of about 1 day to yield ethylene and inorganic mercury. Ethylene is exhaled in the breath and the mercury migrates to the kidney and is excreted in urine. A small proportion of the dose appears as carbon dioxide in the breath and about 12% in urine as a mercury-free metabolite. It is possible that the breakdown of methoxyethylmercurychloride to ethylene and inorganic mercury is not catalysed by an enzyme system.     

How important is the relationship between Protea humiflora (Proteaceae) and its non-flying mammal pollinators?

The interaction between Protea humiflora and its non-flying mammal pollinators was investigated with the aim of quantifying the relationship for both the plants and mammals involved. We manipulated flower density by inflorescence removal and determined the effect upon mammal captures, and excluded mammals to determine their effect upon seed set. Of three mammal species captured frequently, the smallest, Acomys subspinosus (18dž SD g, Rodentia: Muridae), demonstrated the strongest relationship with P. humiflora. Breeding coincided with flowering, and pollen constituted 33.5ᆯ.5% by volume (max 80%, n=19) of winter scats. Acomys captures on control grids were twice those on inflorescence-removal grids during flowering. Aethomys namaquensis (51ᆢ g, Rodentia: Muridae) included flower products in its diet (pollen comprising 3.2dž.9% of winter scats, n=73), but captures were not affected by inflorescence removal. Aethomys is a seasonal breeder and juveniles entered the population 2 months after flowering. Elephantulus edwardii, although an insectivore (48lj g, Macroscelidea: Macroscelididae), carried more pollen grains on its nose than the rodents, acquired while foraging in inflorescences for insects. Pollen comprised 3.1LJ.4% (n=23) of winter scats. From the plant perspective, seed set was significantly reduced in caged inflorescences and in those >20 cm above the ground, presumably due to limited accessibility by terrestrial animals. We conclude that small mammals are responsible for around half (56%) of the effective seed set in P. humiflora.     

Xylose as a nectar sugar: from biochemistry to ecology

Studies of nectar sugar composition in the Proteaceae, an ancient southern hemisphere plant family, have demonstrated that xylose comprises up to 39% of nectar sugar in two genera, Protea and Faurea, and may therefore represent a substantial fraction of the energy available to pollinators of these plants. Although insect and bird pollinators of Protea species are averse to xylose, mice (Aethomys namaquensis) will drink pure xylose, which is metabolized either by gut bacteria or by the mouse tissues. In the form of xylan polymers, the pentose sugar -xylose is a structural component of plant cell walls, and there is considerable biotechnological interest in xylose fermentation. Bacteria and yeasts convert -xylose to -xylulose and thence via the pentose phosphate pathway to fructose-6-phosphate, which is either oxidized or fermented to ethanol. Gut symbionts of rodent pollinators may be analogous to ruminal xylose-metabolizing bacteria. The presence of xylose in Protea and Faurea nectar remains puzzling in view of pollinator aversions: even for rodent pollinators, it is the least preferred nectar sugar. In the generalized pollination systems of the Proteaceae, a coevolutionary explanation for nectar xylose as an attractant for mammalian pollinators is probably less likely than one involving plant physiology, with xylose in phloem sap being secreted passively into the nectar.     

Metabolic distribution of radioactivity in Sprague-Dawley rats and B6C3F1 mice exposed to 1,3-[2,3-14C]-butadiene by whole body exposure

The uptake of 1,3-[2,3-(14)C]-butadiene and its disposition, measured as radioactivity in urine, faeces, exhaled volatiles and CO(2) during and following 6 h whole body exposure to 20 ppm butadiene has been investigated in male Sprague-Dawley rats and B6C3F1 mice. Whilst there were similarities between the two species, the uptake and metabolic distribution of butadiene were somewhat different for rats and mice. The major differences observed were in the urinary excretion of radioactivity and in the exhalation of 14C-CO(2). After 42 h from the start of exposure, 51.1% of radioactivity was eliminated in rat urine compared with 39.5% for mouse urine. 34.9% of the recovered radioactivity was exhaled by rats as 14C-CO(2), compared with 48.7% by mice. Excretion of radioactivity in faeces was similar for both species (3.8% for rats and 3.4% for mice). The tissue concentrations of 14C-butadiene equivalents measured in liver, testes, lung and blood of exposed mice were 0.493, 0460, 0.457, and 1.626 nmol/g tissue, respectively. The values for the corresponding rat tissues were 0.869, 0.329, 0.457, and 1.626 nmol butadiene equivalents/g tissue, respectively. For rats, 6.2% of recovered radioactivity (0.288 nmol butadiene equivalents/g tissue) was retained in carcasses whereas for mice the amount was 3.6% (0.334 nmol butadiene equivalents/g tissue). There were also some significant differences between the metabolic conversion of 1,3-[2,3-(14)C]-butadiene and excretion by mice following the 20 ppm whole body exposure compared to previously reported data for nose-only exposure to 200 ppm butadiene [Richardson et al., Toxicol. Sci. 49 (1999) 186]. The main difference between the high- and low-exposure studies was in the exhalation of 14C-CO(2). At the 200 ppm exposure, 40% of the radioactivity was exhaled as 14C-CO(2) by rats whereas 6% was measured by this route for mice. The proportional conversion of butadiene to CO(2) by mice was significantly greater at the low exposure concentration compared with that reported for the higher concentration. This shift was not observed for rats. The difference between species could be caused by a saturation of metabolism in mice between 20 and 200 ppm for the pathways leading to CO(2). Restraint or error in collection of CO(2) in the 200 ppm study could also be factors.     

Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae

Xylose transport, xylose reductase, and xylitol dehydrogenase activities are demonstrated in Saccharomyces cerevisiae. The enzymes in the xylose catabolic pathway necessary for the conversion of xylose to xylulose are present, although S. cerevisiae cannot grow on xylose as a sole carbon source. Xylose transport is less efficient than glucose transport, and its rate is dependent upon aeration. Xylose reductase appears to be a xylose inducible enzyme and xylitol dehydrogenase activity is constitutive, although both are repressed by glucose. Both xylose reductase and xylitol dehydrogenase activities are five- to tenfold lower in S. cerevisiae as compared to Candida utilis. In vivo conversion of (14)C-xylose in S. cerevisiae is demonstrated and xylitol is detected, although no significant levels of any other (14)C-labeled metabolites (e. g., ethanol) are observed.     

Catabolism and elimination of cholesterol in germfree rats

Three-month old germfree and conventional male rats were maintained on a complete steam-sterilized, semisynthetic diet. After intravenous injection of cholesterol-26-(14)C the animals were housed in a plastic metabolism chamber for 72 hr. Expired CO(2) was collected throughout the period. The conventional rats released 50% more (14)C as (14)CO(2) than the germfree animals. The total amount of the label recovered as (14)CO(2) during the 72 hr period amounted to 30% and 19% respectively, of the original dose. In both conventional and germfree rats the release of (14)CO(2) accounted for approximately 75% of the (14)C recovered in forms other than the original cholesterol-26-(14)C; 15-20% was found incorporated in water-soluble and fat-soluble fractions other than 3Beta-OH sterol of liver and carcass while the remainder was excreted with feces and urine. After the 72 hr period the specific activities of the cholesterol in plasma and liver were lower in conventional than in germfree animals. The data express the accelerating effect of the intestinal microflora on systemic cholesterol catabolism. They demonstrate that the release of (14)CO(2) from cholesterol-26-(14)C in the intact rat is a suitable and convenient indicator of the oxidative catabolism of cholesterol.     

A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [14C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.     

Alkylthioacetic acids (3-thia fatty acids) are metabolized and excreted as shortened dicarboxylic acids in vivo

The metabolism of 1-14C-labeled long-chain alkylthioacetic acids (3-thia fatty acids) which are blocked for normal beta-oxidation by a sulfur atom in the beta-position has been investigated in vivo. Most of the injected radioactivity (greater than 50%) was excreted in the urine within the first 48 h. The recovered and identified metabolites were all short sulfoxydicarboxylic acids. The main metabolite from dodecylthioacetic acid was carboxypropylsulfoxy acetic acid. Some bis(carboxymethyl)sulfoxide (dithioglycolic acid sulfoxide) was also found. The main metabolite from nonylthioacetic acid was carboxyethylsulfoxyacetic acid. No sulfones were found. Less than 1% of the 1-14C from the dodecylthioacetic acid was recovered in respiratory CO2 and about 3% of the 1-14C from nonylthioacetic acid. [1-14C]Dodecyl-sulfonylacetic acid was recovered almost quantitatively as carboxypropylsulfonylacetic acid in the urine after 3 h. A significant fraction (10% of the dodecylthioacetic acid was recovered in the phospholipids and triacylglycerols from liver and epidymal fat pad 4 h after injection. These experiments show that the alkylthioacetic acids undergo an initial omega-oxidation followed by beta-oxidation to short dicarboxylic acids.     

Absence of microbial mineralization of lignin in anaerobic enrichment cultures

The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin.     

Absence of microbial mineralization of lignin in anaerobic enrichment cultures.

The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin.     

Caffeine,quercetin and alizarin stimulate the exhalation of metabolic products of [14C]-N-nitrosodiethylamine in mice

Naturally occurring plant products belonging to different chemical classes namely alizarin, an anthraquinone, caffeine, a methylxanthine derivative and quercetin, a flavonol were studied for their effect on elimination of metabolites of [14C]-N-nitrosodiethylamine (14C-NDEA) through respiration in mice. Treatment with caffeine, quercetin and alizarin at doses of 200, 9 and 9 microg/ml respectively, in drinking water enhanced the exhalation of 14CO2, one of the major end products of NDEA metabolism. Radioactive CO2 exhaled in 60 min increased by 2, 1.61 and 1.4-folds in animals treated with caffeine, quercetin and alizarin for 8 weeks respectively. This increase in exhalation in caffeine-treated animals was achieved even in 2 weeks. These compounds had no adverse effects on the absorption of radioactive NDEA from the gut of the animals as shape and time of 14CO2 peak was similar in i.p. and orally administered [14C-NDEA]. Increased detoxification/elimination of the carcinogen could be one of the mechanisms for the anticarcinogenic properties of these phytochemicals in lung tumorigenesis induced by orally administered NDEA.     

Methanol metabolism in acatalasemic mice

The methanol metabolism in acatalasemic mice was studied by administering [14C]methanol and [14C]formic acid to acatalasemic and normal mice and determining the radioactivity of exhaled carbon dioxide. Methanol metabolism was also studied in acatalasemic and normal mice treated with 3-amino-1,2,4-triazole (AT), which is known to be an inhibitor of catalase (EC 1.11.1.6). The metabolism of methanol and formic acid was inhibited in acatalasemic mice as seen by reduced [14C]CO2 production. Similar results were obtained when AT was given prior to the methanol injection into the normal and acatalasemic mice. The results indicate the peroxidative activity of catalase plays the major role in the methanol metabolism in mice. On the other hand similar studies with [1-14C] ethanol showed that the metabolism of ethanol was not inhibited in acatalasemic mice.     

Metabolism of 5-thio-D-glucopyranose and 6-thio-D-fructopyranose in rats.

5-Thio-α-d-[U-¹⁴C]glucopyranose and 6-thio-β-d-[U-¹⁴C]fructopyranose were administered orally and intravenously to rats. On intravenous administration of 5-thio-d-[U-¹⁴C]glucopyranose, 1% was oxidized to [¹⁴C]carbon dioxide, 93% was excreted in the urine, and 1.6% was retained in the carcass. On oral administration of 5-thio-d-[U-¹⁴C]glucopyranose, 1% was exhaled as [¹⁴C]carbon dioxide, 90% was excreted in feces and urine, and 4% was retained in the carcass after 72 h. On intravenous administration of 6-thio-β-d-[U-¹⁴C]fructopyranose, 56% was exhaled as [¹⁴C]carbon dioxide, 23% was excreted in the urine, and 7.5% was retained in the carcass; after oral administration, 35% was oxidized to [¹⁴C]carbon dioxide, 50% was excreted in feces and urine, and 6% was retained in the animal after 72 h.On intravenous administration of 5-thio-d-glucose to fasted male rats, blood d-glucose levels increased at lower doses than on oral administration. A dose of 50 mg/kg raised blood d-glucose to 226 mg/100 ml within 2.5 h after intravenous but only to 173 mg/100 ml within 2 h after oral administration from basal level of 70–90 mg/100 ml. Blood d-glucose concentration returned to normal levels within 9 h in both cases. 6-Thio-d-fructopyranose showed no diabetogenic action. The LD₅₀ of 6-thio-d-fructopyranose was 11,200 mg/kg when tested in mice.     

Periodate-oxidized adenosine inhibits the formation of dimethylselenide and trimethylselenonium ion in mice treated with selenite

The metabolic detoxification of selenite and many other selenium compounds involves a series of S-adenosylmethionine-dependent methylations yielding dimethylselenide (DMSe), which is exhaled, and trimethylselenonium ion (TMSe), which is excreted in the urine. This paper shows that periodate-oxidized adenosine (Adox) inhibits these methylation reactions in vivo and increases the toxicity of selenite. When Adox was injected in mice at 100 mumol/kg 30 min before injection of [75Se]selenite at 0.4 mg Se/kg the appearances of [75Se]DMSe in the breath and [75Se]TMSe in the liver were completely inhibited for 90 min. This was mediated by accumulation of S-adenosylhomocysteine, the methyltransferase inhibitor, in the livers of Adox-treated mice due to inhibition of its hydrolase enzyme. During 24 h, Adox-treated mice excreted no detectable urinary [75Se]TMSe and exhaled only 20% as much [75Se]DMSe as controls. The urine of Adox-treated mice also contained S-adenosylhomocysteine at a level (ca. 4 mM), 200 times that of untreated mice, which provided a convenient index of methylation potential in the intact animal. When three groups of three mice each were injected with 100 mumol Adox/kg, selenite at 4 mg Se/kg, or a combination of the two, the mice receiving the combination were dead within 2 days, while the mice in the other two groups all survived at least 4 days. These results verify the enzymatic nature of selenium methylation in vivo, support its importance in detoxification, and indicate the value of Adox in further studies of selenium metabolism.     

Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2)

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (14)CO(2) assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO(2)-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO(2)-MAR results were supported by stable isotope analysis of (13)C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of (13)CO(2). In conclusion, the novel HetCO(2)-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Diauxic growth of Clostridium thermosaccharolyticum on glucose and xylose

Abstract Clostridium thermosaccharolyticum growing on medium containing glucose and xylose exhibits classical diauxic growth in which glucose is utilized during the first phase. The lag period between growth phases is associated with induction of synthesis of a xylose transport system together with the enzymes xylose isomerase and xylukokinase. Xylose metabolism by this organism is therefore shown to be inducible and subject to repression by glucose. Xylose utilization by cells adapted to this carbon source is also prevented immediately upon addition of glucose to the culture, suggesting a direct inhibitory effect of glucose on xylose uptake or metabolism.