Male hybrid sterility of mice with the genomic region of the KitW mutation and the KitS allele from Mus spretus

Two congenic strains, C57BL-Kit^W and C57BL-Kit^S, were generated. The Kit^W allele originated from strain WB-Kit^W and the Kit^S allele from Mus spretus. The Kit^W/Kit^S males showed hybrid sterility with small testes, but the females were fertile. The development of the seminiferous tubules of Kit^W/Kit^S males stopped before the spermatocyte stage and they were almost free of sperm. The Kit gene is located at position 42 on chromosome 5. We investigated in the C57BL-Kit^S congenic strain which part of the chromosomal region adjacent to the Kit^S allele is introduced from SPR into a C57BL background. The region between positions 42 and 44 was derived from SPR. Eleven amino acid substitutions of the Kit^S cDNA were detected by comparison with the sequence data of the +^Kit cDNA from C57BL; seven were in the extracellular domain, one in the transmembrane domain, two in the kinase I domain, and one in the carboxy-terminal tail. The Kit mRNA derived from both Kit^W and Kit^S alleles was expressed in the sterile testes of Kit^W/Kit^S males.     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Effect of the chelation of metal cation on the antioxidant activity of chondroitin sulfates

The antioxidant potencies of chondroitin sulfates (CSs) from shark cartilage, salmon cartilage, bovine trachea, and porcine intestinal mucosa were compared by three representative methods for the measurement of the antioxidant activity; DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity. CSs from salmon cartilage and bovine trachea showed higher potency in comparison with CSs from shark cartilage and porcine intestinal mucosa. Next, CS from salmon cartilage chelating with Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺ were prepared, and their antioxidant potencies were compared. CS chelating with Ca²⁺ or Mg²⁺ ions showed rather decreased DPPH radical scavenging activity in comparison with CS of H⁺ form. In contrast, CS chelating with Ca²⁺ or Mg²⁺ ion showed remarkably enhanced superoxide radical scavenging activity than CS of H⁺ or Na⁺ form. Moreover, CS chelating with divalent metal ions, Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, showed noticeably higher hydroxyl radical scavenging activity than CS of H⁺ or Na⁺ form. The present results revealed that the scavenging activities of, at least, superoxide radical and hydroxyl radical were enhanced by the chelation with divalent metal ions.     

A comparison of the effects of subsoiling on plant uptake and leaching losses of sulphur and nitrogen from a simulated urine patch

Synthetic cow urine labelled with ³⁵S and ¹⁵N was applied to large, undisturbed, monolith lysimeters sampled from subsoiled and non-subsoiled areas of a grass/clover pasture. For one year following the urine application, the lysimeters were subjected to a combination of natural rainfall, simulated rainfall and simulated flood irrigations. Drainage from the lysimeters was sampled regularly and monthly (approx.) pasture cuts taken. At the end of the year, the lysimeters were destructively sampled in 50 mm depth increments for soil analysis. Leachates, plant samples and soil samples were analysed for ³⁵S and ¹⁵N.There were no significant differences in plant uptake of ³⁵S and ¹⁵N between the subsoiled and nonsubsoiled lysimeters. Initially grass showed a higher degree of labelling than clover. Total amounts of ³⁵S and ¹⁵N leached from the subsoiled lysimeters were approximately twice that leached from the nonsubsoiled ones. Leaching patterns differed substantially between the two nutrients.Total recoveries of ³⁵S (in plants, leachates and soil extracts) accounted for 82% of the applied ³⁵S for the subsoiled lysimeters and 72% for non-subsoiled ones. The unrecovered ³⁵S is considered to have been incorporated into soil organic matter. Total recoveries of ¹⁵N (in plants, soil and leachates) were similar to those for ³⁵S, but unrecovered ¹⁵N is attributed to loss by denitrification.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.     

The role of cattle in the volatile loss of nitrogen from a shortgrass steppe

The cycling and volatile loss of N derived from cattle urine at upland and lowland sites within the shortgrass steppe of eastern Colorado was studied, using¹⁵N-labelled urea as an N source. Losses of NH ₀ ³ were determined by direct measurement and by difference. Losses were higher from coarse (27% summer, 12% winter) than from fine textured (0–2%) soils. Immobilization and plant uptake of N accounted for significant amounts of added N. Extrapolating our plot measurements to a typical pasture, using spatially and temporally stratified urine deposition data, losses from upland sites were calculated to be 0.016 g N · m^-2 · y^-1, while losses from lowland sites were negligible. This resulted in an average loss of 0.011 g N · m^-2 · y^-1 for a pasture divided 70:30 between uplands and lowlands. The loss of urine N calculated assuming no spatial stratification would be sevenfold higher (0.076 g N · m^-2 · y^-1). Losses of NH ₀ ³ from urine, animal biomass removal, and NH₂O loss totaled only 0.07 g N · m^-2 · y^-1 , or about 25% of wet deposition input. We calculated a potential loss of NH ₀ ³ from senescing vegetation of 0.26 g N · m^-2 · y^-1, an order of magnitude larger than all other losses combined.     

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus–oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL⁺) and without (CCL^-) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL⁺), contralateral (CL⁻), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL⁻ cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL⁺ group; however, a higher proportion of embryos were obtained from the CCL⁻ group. Besides, COCs from the CL⁺ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL⁻ group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.     

Structure and Colonization Dynamics of Epiphytic Bacterial Communities and of Selected Component Strains on Tomato (Lycopersicon esculentum) Leaves

The sizes and compositions of bacterial populations found on leaves of greenhouse and field grown tomato plants were studied by dilution plating, fatty acid methyl ester analysis (FAME), and BIOLOG plates of isolates in pure cultures. In the greenhouse, overhead-irrigated plants sustained higher microbial populations (up to 10⁵ cfu g⁻¹) than soil-irrigated plants (10³ cfu g⁻¹). Strains isolated from overhead-irrigated plants grown in a vegetable garden (n=216) and from greenhouse-grown plants (n=114) were subjected to FAME analysis. Similarly, strains from soil-irrigated field-grown plants (n=83) were identified using BIOLOG plates. In each case, populations were dominated by a few genera. When concentrated phyllosphere washes (CPW) were sprayed on greenhouse-grown, soil-irrigated plants, leaf bacterial populations of more than 10⁵ CFU g⁻¹ were sustained for 4 days; sterile buffer-sprayed leaves sustained less than 10⁴ CFU g⁻¹. No significant enrichment of any strain isolated from the sprayed leaves could be detected by FAME identification of randomly selected colonies. However, when recurring leaf saprophytic species (both Gram-positive and Gram-negative) isolated from these experiments and from plants grown outdoors were tested for epiphytic colonization under stressful conditions, all could still be detected at various levels up to 4 days after inoculation, indicating differential epiphytic fitness. The non-epiphytic bacteriaEscherichia coli andAzospirillum brasilense disappeared from the leaf surface within the same experimental period.     

Propagation of lettuce (Lactuca sativa) breeding material by tissue culture

A tissue culture method using Murashige and Skoog's (MS) medium was devised to propagate healthy plants from field grown lettuce plants selected for seed production. Explants (2–3 mm long) from axillary buds were successfully grown on MS + 1.0 or 2.0 mg litre^-1 kinetin and 6.4 mg litre^-1 IAA to promote shoot growth. Concentrations of 0.5 and 4.0 mg litre^-1 kinetin gave poor shoot growth. The cultures were successfully rooted after 3–4 wk on MS + 6.4 mg litres^-1 IAA after transfer from MS + 1.0 mg litre^-1 kinetin and on MS + 4.8 mg litre^-1 IAA after transfer from MS + 2.0 mg litre^-1 kinetin. Concentrations of 3.2 and 8.0 mg litre^-1 IAA gave poor root initiation. Root initiation was more successful when cultures were grown at 40 Wm^-2 than in cultures grown at 5 Wm^-2. Rooted cultures were established in compost with a 90–95% success rate and the regenerated plants flowered c. 18 wk after the cultures were initiated.     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Determinants of the disposition of 14C-delta 9-tetrahydrocannabinol and 3H-delta 9-tetrahydrocannabinol.

Intragastric (i.g.) administration of ¹⁴C-THC plus ³H-THC to male Wistar and Fischer rats resulted in a more rapid disappearance of ¹⁴C than ³H from fresh blood or plasma. The concentrations of the two isotopes were equivalent from 1–4 hours when blood was analyzed after being dried. For each rat strain, apparent absorption of both isotopes was more rapid in fed than fasted rats and in young (130–150g) compared to older (250–260g) animals. The concentrations of ³H were significantly higher than ¹⁴C in the major organs which were analyzed fresh at 4 and 24 hours after drug administration, but the isotope levels were not different when the tissues were analyzed after lyophilization. Experiments indicate that tritiated water was produced metabolically from ³H-THC and was lost from blood and organs upon drying.The fresh blood levels of total ¹⁴C and unchanged ¹⁴C-THC were higher than total ³H and ³H-THC, respectively, from 40 min to 4 hrs following i.v. injection of ¹⁴C-THC plus ³H-THC in bile duct-cannulated rats. Similarly, the amount of ¹⁴C was higher than the amount of ³H in the urine. However, the concentration of ³H was higher than ¹⁴C in the bile after 20 min. The ³H level was higher than ¹⁴C at 4 hrs in the brain but lower than ¹⁴C in the liver, heart and spleen. Drying of body fluids and tissues before isotope analysis did not alter these results. It is concluded that the formation of tritiated water occurs in the gut after i.g. ³H-THC administration, and that the dispositions of ¹⁴C-THC and ³H-THC are not entirely equivalent following i.v. injection.     

Gamma densitometry for the measurement of skeletal density

A method is described for the measurement of the density of calcium carbonate materials from the attenuation of a narrow, collimated beam of gamma photons. For the measurement of density for slices, approximately 0.5 to 1.0 cm thick, from the skeletons of reef building corals, the optimum beam energy is 30–34 keV; and measurement is practical from approximately 22 to 100 keV. The potential utilities of five commercially available isotopic sources (¹⁰⁹Cd,¹²⁵I,²⁵³Gd,²¹⁰Pb and²⁴¹Am) are evaluated. Methods and results are presented for gamma densitometry using²¹⁰Pb and²⁴¹Am. The²¹⁰Pb point source had its principal gamma emission at 46.5 keV. Bremsstrahlung and high energy (800 keV) gamma emissions associated with the²¹⁰Pb decay grand-daughter were detected, and procedures were developed to accommodate the contribution of these emissions to the overall count rate. The attenuation of count rate by aluminium and aragonite absorbers closely followed simple theoretical considerations provided that narrow energy window settings were used at the radiation monitor. These theoretical considerations take account of the density of the material absorbing the radiation, and hence the density could be determined from the attenuation of the gamma beam. Increased accuracy was achieved by the use of²⁴¹Am and high speed counting equipment.²⁴¹Am has its principal gamma emission at 59.6 keV. The attenuation of this gamma beam follows simple theoretical considerations for targets with mass thicknesses from 0 to 6 g cm^-2. Aragonite from the shell of a giant clam was found to have slightly different properties in the absorption of gamma photons to aragonite from a coral skeleton. The differences were small but statistically significant.     

Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.     

An enriched preparation of basal-lateral plasma membranes from gastric glandular cells

Summary A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from nonglandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na⁺+K⁺)-ATPase, ouabain-sensitive K⁺-stimulatedp-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na⁺+K⁺)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg²⁺-ATPase, cytochromec oxidase, NADPH-cytochromec reductase, glucose-6-phosphatase, (K⁺+H⁺)-ATPase, DNA and RNA.     

Water relations of coastal plant communities near the ocean/freshwater boundary

Summary Salinity and isotope ratios were determined in water from several wells in the Florida Keys, and tidal inlets. Both D/H and ¹⁸O/¹⁶O ratios of water from wells and tidal inlets were highly correlated to their salinity. Water from standing pools was enriched in deuterium and oxygen-18 relative to their salinity because of evaporation processes. ¹⁸O/¹⁶O and D/H ratios of stem water from plants of several different communities at Sugar Loaf Key, ranging from hardwood hammocks to mangroves, were highly correlated to their predawn water potential. The correlation was consistent with the presence of high salinity in waters with high ¹⁸O and D content. Most individuals from each community were either utilizing water with isotopic characteristics typical of freshwater or of ocean water, while only a few individuals had stem water with isotopic ratios intermediate to these two water sources.     

Isolation and Molecular Characterization of Heavy Metal-Resistant Azotobacter chroococcum from Agricultural Soil and Their Potential Application in Bioremediation

Pollution of soil with heavy metals, herbicides, antibiotics and other chemicals is known to have a negative effect on microbial activities. Therefore, the aim of this study was to isolate cultures of Azotobacter sp. from polluted and unpolluted soils and to study the effect of these pollutants on their growth. A total of 120 Azotobacter sp. were isolated from soils irrigated with wastewater (contaminated soils) and groundwater (uncontaminated soils). These isolates were screened for resistance to heavy metals, herbicide and antibiotics. Also, the soils from which the cultures were isolated were analyzed for the concentrations of Zn²⁺, Cd²⁺, Cu²⁺, Pb²⁺ and Mn²⁺ they contained. Contaminated soil showed high levels of heavy metals as compared to uncontaminated soil. The size of the Azotobacter population in contaminated soil was lower than that in uncontaminated soil. Of the Azotobacter isolates, 64 that were recovered from contaminated soil exhibited high resistance to heavy metals (Hg²⁺, Cd²⁺, Cu²⁺, Cr³⁺, Co²⁺, Ni²⁺, Zn²⁺ and Pb²⁺) and herbicide 2,4-D compared to 56 isolates from uncontaminated soil. Also, isolates from contaminated soil showed high resistance to chloramphenicol, nitrofurantoin and co-trimoxazole compared to those isolated from uncontaminated soil. The majority of Azotobacter isolates from contaminated soil showed multiple-resistance to different metal ions and antibiotics. All isolates failed to grow at pH less than 6. Salt concentration (5%) was found to be inhibitory to all isolates. The most potent isolates from contaminated soil that showed multiresistance to all substances tested were identified on the basis of morphological and biochemical characteristics, and 16S rRNA as A. chroococcum. These resistant isolates could be employed in contaminated soils and/or bioremediation.     

Sources and fluxes of submarine groundwater discharge delineated by radium isotopes

This paper reports the results derived fromradium isotopes of a submarine groundwaterdischarge (SGD) intercomparison in thenortheast Gulf of Mexico. Radium isotopesamples were collected from seepage meters,piezometers, and surface and deep ocean waters.Samples collected within the near-shore SGDexperimental area were highly enriched in allfour radium isotopes; offshore samples wereselectively enriched. Samples collected fromseepage meters were about a factor of 2–3higher in radium activity compared to theoverlying waters. Samples from piezometers,which sampled 1–4 meters below the sea bed were1–2 orders of magnitude higher in radiumisotopes than surface waters. The twolong-lived Ra isotopes, ²²⁸Ra and²²⁶Ra, provide convincing evidence thatthere are two sources of SGD to the study area:shallow seepage from the surficial aquifer andinput from a deeper aquifer. A three end-membermixing model can describe the Ra distributionin these samples. The short-lived radium isotopes, ²²³Ra and²²⁴Ra, were used to establish mixing ratesfor the near-shore study area. Mixing wasretarded within 3 km of shore due to a strongsalinity gradient. The product of the mixingrate and the offshore ²²⁶Ra gradientestablished the ²²⁶Ra flux. This flux mustbe balanced by Ra input from SGD. The flux ofSGD within 200 m of shore based on the²²⁶Ra budget was 1.5 m³ min^?1.This flux agreed well with other estimatesbased on seepage meters and ²²²Rn.     

Anaerobic Biodegradation of the Lignin and Polysaccharide Components of Lignocellulose and Synthetic Lignin by Sediment Microflora

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses and [¹⁴C-polysaccharide]lignocelluloses were prepared from a variety of marine and freshwater wetland plants including a grass, a sedge, a rush, and a hardwood. These [¹⁴C]lignocellulose preparations and synthetic [¹⁴C]lignin were incubated anaerobically with anoxic sediments collected from a salt marsh, a freshwater marsh, and a mangrove swamp. During long-term incubations lasting up to 300 days, the lignin and polysaccharide components of the lignocelluloses were slowly degraded anaerobically to ¹⁴CO₂ and ¹⁴CH₄. Lignocelluloses derived from herbaceous plants were degraded more rapidly than lignocellulose derived from the hardwood. After 294 days, 16.9% of the lignin component and 30.0% of the polysaccharide component of lignocellulose derived from the grass used (Spartina alterniflora) were degraded to gaseous end products. In contrast, after 246 days, only 1.5% of the lignin component and 4.1% of the polysaccharide component of lignocellulose derived from the hardwood used (Rhizophora mangle) were degraded to gaseous end products. Synthetic [¹⁴C]lignin was degraded anaerobically faster than the lignin component of the hardwood lignocellulose; after 276 days, 3.7% of the synthetic lignin was degraded to gaseous end products. Contrary to previous reports, these results demonstrate that lignin and lignified plant tissues are biodegradable in the absence of oxygen. Although lignocelluloses are recalcitrant to anaerobic biodegradation, rates of degradation measured in aquatic sediments are significant and have important implications for the biospheric cycling of carbon from these abundant biopolymers.     

The biosynthetic origin of oxygen functions in phenylphenalenones of Anigozanthos preissii inferred from NMR- and HRMS-based isotopologue analysis

The biosynthetic origin of 9-phenylphenalenones and the sequence according to which their oxygen functionalities are introduced were studied using nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). ¹³C-labelled precursors were administered to root cultures of Anigozanthos preissii, which were simultaneously incubated in an atmosphere of ¹⁸O₂. Two major phenylphenalenones, anigorufone and hydroxyanigorufone, were isolated and analyzed by spectroscopic methods. Incorporation of ¹³C-labelled precursors from the culture medium and ¹⁸O from the atmosphere was detected. O-Methylation with ¹³C-diazomethane was used to attach ¹³C-labels to each hydroxyl and thereby dramatically enhance the sensitivity with which NMR spectroscopy can detect ¹⁸O by means of isotope-induced shifts of ¹³C signals. The isotopologue patterns inferred from NMR and HRESIMS analyses indicated that the hydroxyl group at C-2 of 9-phenylphenalenones had been introduced on the stage of a linear diarylheptanoid. The oxygen atoms of the carbonyl and lateral aryl ring originated from the hydroxyl group of the 4-coumaroyl moiety, which was incorporated as a unit.     

Investigation of Microorganisms in Bentonite Deposits

Microbiological characteristics of bentonite deposits were investigated as a natural analogue of microbial behavior in the buffer material for geological disposal of radioactive waste. Distributions of microorganisms in bentonite were examined at four sites in two different bentonite deposits in Japan. The sites included pond bottom, wetland, and wet mine gallery environments where bentonite layers have been left undisturbed for 2 to 30 years. Excavation was performed without using drilling water and the center parts of the cores were used for microbial examination. Plate counts with R2A medium of aerobic and anaerobic bacteria at the drilling mouth ranged from 10⁵ to 10⁷/g DW (dry weight) and from 10³ to 10⁶/g DW, respectively. The CFDA-AM (Carboxyfluorescein diacetate acetoxymethyl ester) cell counts ranged from 10⁶ to 10⁹/g DW. Bacterial numbers in the bentonite layers declined with distance from the drilling mouth; both aerobes and anaerobes were less than 10²/g DW and CFDA-AM cell counts less than 10⁶/g DW for core samples taken from approximately 1 m depth, except at the pond bottom. These results suggest that microbial activity in natural bentonite is lower than in typical soils and aquatic sediments and does not spread easily.