Hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures

Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6β-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6β-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe²⁺ ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-¹⁴C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO₂ from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6β-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures.     

Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus: n-Butylamine as a Potent Inhibitor of the Transferase both in Vitro and in Vivo

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine, followed by its methylation by putrescine N-methyltransferase (PMT; EC 2.1.1.53). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus that contain hyoscyamine as the main alkaloid. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi-bi mechanism, in which the K_m values for putrescine and S-adenosyl-l-methionine are 277 and 203 μm, respectively, and the K_i value for S-adenosyl-l-homocysteine is 110 μm. PMT efficiently N-methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT, among which n-butylamine, cyclohexylamine, and exo-2-aminonorbornane were most inhibitory, with respective K_i values of 11.0, 9.1, and 10.0 μm. When n-butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine, and pseudotropine) drastically decreased at 1 mm of the exogenous monoamine, and the hyoscyamine content decreased by 52% at 6 mm, whereas the contents of 6β-hydroxyhyoscyamine and scopolamine did not change. Free and conjugated forms of polyamines were also measured. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool), and the spermine content also increased slightly, whereas the spermidine content decreased slightly. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. Similar results were also obtained in root cultures of Datura stramonium. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.     

Metabolism of Radiolabeled β-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-¹⁴C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether (VI) to ¹⁴CO₂ in stationary and in shaking cultures. ¹⁴CO₂ evolution was greater in stationary culture. ¹⁴CO₂ evolution from [γ-¹⁴C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O₂ rather than air (21% O₂) was the gas phase above the cultures. ¹⁴CO₂ evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed ¹⁴CO₂ evolution from both substrates in stationary cultures. [¹⁴C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.     

Biochemical and structural characterization of recombinant hyoscyamine 6β-hydroxylase from Datura metel L.

Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K_m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 μM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern–Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M^?1 and 0.56 M^?1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.     

Degradation of the γ-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4′-Ethoxy-3′-Methoxyphenyl)-2-(4″-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4′-ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [¹⁴C]lignin to ¹⁴CO₂. 1-(4′-Ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by α, β cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under ¹⁸O₂, ¹⁸O was incorporated into the β position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

New Insights into the Ligninolytic Capability of a Wood Decay Ascomycete

Wood-grown cultures of Daldinia concentrica oxidized a permethylated β-¹⁴C-labeled synthetic lignin to ¹⁴CO₂ and also cleaved a permethylated α-¹³C-labeled synthetic lignin to give C_α-C_β cleavage products that were detected by ¹³C nuclear magnetic resonance spectrometry. Therefore, this ascomycete resembles white-rot basidiomycetes in attacking the recalcitrant nonphenolic structures that predominate in lignin.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

The plasmid pGKE75-cat_A138T, which comprises pUC18 and the cat_A138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CAT_A138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-cat_A138T in an error-prone thermophile, generating the mutant plasmid pGKE75^αβ-cat_A138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75^αβ-cat_A138T contained no mutation in the cat_A138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75^αβ-cat_A138T is attributable to increases in intracellular CAT_A138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75^αβ-cat_A138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75^αβ-cat_A138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli.     

Pronghorn (Antilocapra americana) enamel phosphate δ18O values reflect climate seasonality: Implications for paleoclimate reconstruction

Stable oxygen isotope (δ¹⁸O) compositions from vertebrate tooth enamel are widely used as biogeochemical proxies for paleoclimate. However, the utility of enamel oxygen isotope values for environmental reconstruction varies among species. Herein, we evaluate the use of stable oxygen isotope compositions from pronghorn (Antilocapra americana Gray, 1866) enamel for reconstructing paleoclimate seasonality, an elusive but important parameter for understanding past ecosystems. We serially sampled the lower third molars of recent adult pronghorn from Wyoming for δ¹⁸O in phosphate (δ¹⁸O_PO4) and compared patterns to interpolated and measured yearly variation in environmental waters as well as from sagebrush leaves, lakes, and rivers (δ¹⁸O_w). As expected, the oxygen isotope compositions of phosphate from pronghorn enamel are enriched in ¹⁸O relative to environmental waters. For a more direct comparison, we converted δ¹⁸O_w values into expected δ¹⁸O_PO4* values (δ¹⁸O_W‐_PO4*). Pronghorn δ¹⁸O_PO4 values from tooth enamel record nearly the full amplitude of seasonal variation from Wyoming δ¹⁸O_W‐PO4* values. Furthermore, pronghorn enamel δ¹⁸O_PO4 values are more similar to modeled δ¹⁸O_W‐PO4* values from plant leaf waters than meteoric waters, suggesting that they obtain much of their water from evaporated plant waters. Collectively, our findings establish that seasonality in source water is reliably reflected in pronghorn enamel, providing the basis for exploring changes in the amplitude of seasonality of ancient climates. As a preliminary test, we sampled historical pronghorn specimens (1720 ± 100 AD), which show a mean decrease (a shift to lower values) of 1–2‰ in δ¹⁸O_PO4 compared to the modern specimens. They also exhibit an increase in the δ¹⁸O amplitude, representing an increase in seasonality. We suggest that the cooler mean annual and summer temperatures typical of the 18th century, as well as enhanced periods of drought, drove differences among the modern and historical pronghorn, further establishing pronghorn enamel as excellent sources of paleoclimate proxy data.     

A fully reversible 25-hydroxy steroid kinase involved in oxygen-independent cholesterol side-chain oxidation

The degradation of cholesterol and related steroids by microbes follows fundamentally different strategies in aerobic and anaerobic environments. In anaerobic bacteria, the primary C26 of the isoprenoid side chain is hydroxylated without oxygen via a three-step cascade: (i) water-dependent hydroxylation at the tertiary C25, (ii) ATP-dependent dehydration to form a subterminal alkene, and (iii) water-dependent hydroxylation at the primary C26 to form an allylic alcohol. However, the enzymes involved in the ATP-dependent dehydration have remained unknown. Here, we isolated an ATP-dependent 25-hydroxy-steroid kinase (25-HSK) from the anaerobic bacterium Sterolibacterium denitrificans. This highly active enzyme preferentially phosphorylated the tertiary C25 of steroid alcohols, including metabolites of cholesterol and sitosterol degradation or 25-OH-vitamin D₃. Kinetic data were in agreement with a sequential mechanism via a ternary complex. Remarkably, 25-HSK readily catalyzed the formation of γ-(¹⁸O)₂-ATP from ADP and the C25-(¹⁸O)₂-phosphoester. The observed full reversibility of 25-HSK with an equilibrium constant below one can be rationalized by an unusual high phosphoryl transfer potential of tertiary steroid C25-phosphoesters, which is ≈20 kJ mol⁻¹ higher than that of standard sugar phosphoesters and even slightly greater than the β,γ-phosphoanhydride of ATP. In summary, 25-HSK plays an essential role in anaerobic bacterial degradation of zoo- and phytosterols and shows only little similarity to known phosphotransferases.     

Radioimmunoassay for the quantitative determination of scopolamine

A radioimmunoassay for the determination of pmol amounts of the tropane alkaloid scopolamine has been developed. The assay uses tritiated [N-C³H₃]scopolamine of high specific activity (0.67 Ci/mmol) as tracer. The measuring range of the assay extends from 0.5 to 50 ng of scopolamine, and as little as 200 pg may be detected. The antiserum raised against a conjugate of scopolamine-N-β-propionic acid-human serum albumin is highly specific, and neither hyoscyamine, 6-hydroxyhyoscyamine, scopine, tropic acid nor other related alkaloids interfere in the scopolamine determination in crude plant extracts. This assay allows for the first time the rapid, sensitive and precise (CV = 2.5 %) determination of this alkaloid in unpurified extracts of scopolamine-containing plants. The distribution of scopolamine in Datura plants, as well as its diurnal changes in leaf concentrations, has been investigated in detail and a preliminary survey on the variability of scopolamine leaf concentrations in a population of Datura sanguinea plants is given.     

Production of Anti-LPS IgM by B1a B Cells Depends on IL-1β and Is Protective against Lung Infection with Francisella tularensis LVS

The role of IL-1β and IL-18 during lung infection with the gram-negative bacterium Francisella tularensis LVS has not been characterized in detail. Here, using a mouse model of pneumonic tularemia, we show that both cytokines are protective, but through different mechanisms. Il-18^-/- mice quickly succumb to the infection and showed higher bacterial burden in organs and lower level of IFNγ in BALF and serum compared to wild type C57BL/6J mice. Administration of IFNγ rescued the survival of Il-18^-/- mice, suggesting that their decreased resistance to tularemia is due to inability to produce IFNγ. In contrast, mice lacking IL-1 receptor or IL-1β, but not IL-1α, appeared to control the infection in its early stages, but eventually succumbed. IFNγ administration had no effect on Il-1r1^-/- mice survival. Rather, Il-1r1^-/- mice were found to have significantly reduced titer of Ft LPS-specific IgM. The anti-Ft LPS IgM was generated in a IL-1β-, TLR2-, and ASC-dependent fashion, promoted bacteria agglutination and phagocytosis, and was protective in passive immunization experiments. B1a B cells produced the anti-Ft LPS IgM and these cells were significantly decreased in the spleen and peritoneal cavity of infected Il-1b^-/- mice, compared to C57BL/6J mice. Collectively, our results show that IL-1β and IL-18 activate non-redundant protective responses against tularemia and identify an essential role for IL-1β in the rapid generation of pathogen-specific IgM by B1a B cells.     

The conversion of cholest-7-en-3beta-ol into cholesterol. General comments on the mechanism of the introduction of double bonds in enzymic reactions

Convenient syntheses of 6β-tritiated Δ⁷-cholestenol and 3α-tritiated Δ⁷-cholestene-3β,5α-diol are described. It was shown that the conversion of 6β-tritiated Δ⁷-cholestenol into cholesterol is accompanied by the complete retention of label. It was unambiguously established that the overall reaction leading to the introduction of the double bond in the 5,6-position in cholesterol occurs via a cis-elimination involving the 5α- and 6α-hydrogen atoms and that during this process the 6β-hydrogen atom remains completely undisturbed. Metabolic studies with 3α-tritiated Δ⁷-cholestene-3β,5α-diol revealed that under anaerobic conditions the compound is not converted into cholesterol. This observation, coupled with the previous work of Slaytor & Bloch (1965), is interpreted to exclude a hydroxylation–dehydration mechanism for the origin of the 5,6-double bond in cholesterol. It was also shown that under aerobic conditions 3α-tritiated Δ⁷-cholestene-3β,5α-diol is efficiently converted into cholesterol and that this conversion occurs through the intermediacy of 7-dehydrocholesterol. Cumulative experimental evidence presented in this paper and elsewhere is used to suggest that the 5,6-double bond in cholesterol originates through an oxygen-dependent dehydrogenation process and a hypothetical mechanism for this and related reactions is outlined.     

Two β-Galactosidases from the Human Isolate Bifidobacterium breve DSM 20213: Molecular Cloning and Expression,Biochemical Characterization and Synthesis of Galacto-Oligosaccharides

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The k _cat/K _m values for oNPG and lactose hydrolysis are 722 and 7.4 mM⁻¹s⁻¹ for β-gal I, and 543 and 25 mM⁻¹s⁻¹ for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.     

Biosynthetic Origin of Gibberellins A(3) and A(7) in Cell-Free Preparations from Seeds of Marah macrocarpus and Malus domestica

Cell-free preparations from seeds of Marah macrocarpus L. and Malus domestica L. catalyzed the conversion of gibberellin A₉ (GA₉) and 2,3-dehydroGA₉ to GA₇; GA₉ was also metabolized to GA₄ in a branch pathway. The preparation from Marah seeds also metabolized GA₅ to GA₃ in high yield; GA₆ was a minor product and was not metabolized to GA₃. Using substrates stereospecifically labeled with deuterium, it was shown that the metabolism of GA₅ to GA₃ and of 2,3-dehydroGA₉ to GA₇ occurs with the loss of the 1β-hydrogen. In cultures of Gibberella fujikuroi, mutant B1-41a, [1β,2β-²H₂]GA₄, was metabolized to [1,2-²H₂]GA₃ with the loss of the 1α- and 2α-hydrogens. These results provide further evidence that the biosynthetic origin of GA₃ and GA₇ in higher plants is different from that in the fungus Gibberella fujikuroi.     

Spectrin Tetramer Formation Is Not Required for Viable Development in Drosophila

The dominant paradigm for spectrin function is that (αβ)₂-spectrin tetramers or higher order oligomers form membrane-associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. We characterize the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type α-spectrin binds to both β- and β_H-chains with high affinity, resembling other non-erythroid spectrins. However, α-spec^R22S, a tetramerization site mutant homologous to the pathological α-spec^R28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to β_H-spectrin ∼1000-fold. Even though spectrins are essential proteins, α-spectrin^R22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. Our data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm.     

The chemical synthesis and biological oxidation of 7α-hydroxy[26-14C]cholesterol, 7-dehydro[26-14C]cholesterol and 26-hydroxy[26-14C]cholesterol

1. 26-Hydroxycholesterol was obtained by reducing the methyl ester of (±)-3β-hydroxycholest-5-en-26-oic acid, which was synthesized from 25-oxonorcholesterol. 2. Methods for preparing 7α-hydroxycholesterol and 7-dehydrocholesterol were modified to allow the micro-scale preparation of these [¹⁴C]sterols from [26-¹⁴C]-cholesterol. 3. 26-Hydroxycholesterol was oxidized more readily than 7α-hydroxycholesterol, 7-dehydrocholesterol or cholesterol by mitochondrial preparations from livers of mice, rats, guinea pigs, common toads (Bufo vulgaris) and Caiman crocodylus. 4. (±)-3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was oxidized very rapidly to ¹⁴CO₂ by mouse and guinea-pig mitochondria without evident discrimination between the two optical isomers. 5. An enzyme system that oxidizes 26-hydroxycholesterol to 3β-hydroxycholest-5-en-26-oic acid was identified in the soluble extract of rat-liver mitochondria. This enzyme could use NADP in place of NAD but was not identical with liver alcohol dehydrogenase (EC 1.1.1.1). 6. [26-¹⁴C]Cholesteryl 3β-sulphate was not oxidized by fortified mouse-liver preparations that oxidized [26-¹⁴C]cholesterol to ¹⁴CO₂.     

4-Methylumbelliferyl-β-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity β-N-acetylglucosaminidase-like enzyme (K_m, 100 μmol liter^-1) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-N-acetylglucosaminidase-like enzyme (K_m, <0.5 μmol liter^-1). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r² = 0.96) and Cyclidium sp. (r² = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.     

Oxygen isotope values in bone carbonate and collagen are consistently offset for New World monkeys

Stable oxygen isotopes are increasingly used in ecological research. Here, I present oxygen isotope (δ¹⁸O) values for bone carbonate and collagen from howler monkeys (Alouatta palliata), spider monkeys (Ateles geoffroyi) and capuchins (Cebus capucinus) from three localities in Costa Rica. There are apparent differences in δ¹⁸O_carbonate and δ¹⁸O_collagen among species. Monkeys from moist forest have significantly lower isotope values than those from drier localities. Because patterns are similar for both substrates, discrimination (Δ) between δ¹⁸O_carbonate and δ¹⁸O_collagen is relatively consistent among species and localities (17.6 ± 0.9‰). Although this value is larger than that previously obtained for laboratory rats, consistency among species and localities suggests it can be used to compare δ¹⁸O_carbonate and δ¹⁸O_collagen for monkeys, and potentially other medium-bodied mammals. Establishing discrimination for oxygen between these substrates for wild monkeys provides a foundation for future environmental and ecological research on modern and ancient organisms.     

Metabolism of the alpha-Pyridone Ring of Ricinine in Ricinus communis L

Chemically synthesized ricinine-3,5-¹⁴C was used to study the metabolism of this alkaloid in the plant which produces it, Ricinus communis L. In a time course study, ricinine-3,5-¹⁴C was administered to a series of castor plants (Ricinus communis L.) and the radioactivity recovered in the ricinine samples showed a decrease with increase in time. It was also observed that the alkaloid was translocated to the seed. The in vivo conversion of ricinine-3,5-¹⁴C to respiratory ¹⁴CO₂ occurred in both light and dark and indicated that the α-pyridone ring of ricinine could be degraded.