Mn(II) oxidation is catalyzed by heme peroxidases in "Aurantimonas manganoxydans" strain SI85-9A1 and Erythrobacter sp. strain SD-21.

A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, "Aurantimonas manganoxydans" strain SI85-9A1 and Erythrobacter sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of A. manganoxydans containing stainable heme. The addition of both Ca(2+) ions and H(2)O(2) to the enriched protein from Aurantimonas increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 microM min(-1) mg(-1). Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in Erythrobacter sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from A. manganoxydans. The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes.

Use of computer simulation to teach a systems approach to metabolism.

Use of a systems approach, as embodied in the computer simulation model of metabolism of a dairy cow, Molly (Baldwin, 2005), is ideal for teaching nutrition. This approach allows the overall complexity of the comprehensive system to be broken down into smaller manageable subunits that are easier to visualize. Quantitative interactions among nutrients supplied and metabolic production processes can be observed over extended time periods. Using Molly, undergraduate animal science students are able to observe detailed effects of changing dietary inputs, altering genetic milk production potential, and exogenously manipulating metabolism on metabolism of the whole cow. This paper demonstrates how Molly is used in the classroom to teach a systems approach to nutrition using example simulations. Three simulation examples demonstrate exercises examining effects of recombinant bovine somatotropin administration, dietary protein, and amino acid supplementation and nitrogen efficiency on milk production and cow metabolism. These and similar examples have been used to teach nutrition, metabolism, and lactation to undergraduate students for the past 20 yr.

Valuation of milk composition and genotype in cheddar cheese production using an optimization model of cheese and whey production.

A mass balance optimization model was developed to determine the value of the kappa-casein genotype and milk composition in Cheddar cheese and whey production. Inputs were milk, nonfat dry milk, cream, condensed skim milk, and starter and salt. The products produced were Cheddar cheese, fat-reduced whey, cream, whey cream, casein fines, demineralized whey, 34% dried whey protein, 80% dried whey protein, lactose powder, and cow feed. The costs and prices used were based on market data from March 2004 and affected the results. Inputs were separated into components consisting of whey protein, ash, casein, fat, water, and lactose and were then distributed to products through specific constraints and retention equations. A unique 2-step optimization procedure was developed to ensure that the final composition of fat-reduced whey was correct. The model was evaluated for milk compositions ranging from 1.62 to 3.59% casein, 0.41 to 1.14% whey protein, 1.89 to 5.97% fat, and 4.06 to 5.64% lactose. The kappa casein genotype was represented by different retentions of milk components in Cheddar cheese and ranged from 0.715 to 0.7411 kg of casein in cheese/kg of casein in milk and from 0.7795 to 0.9210 kg of fat in cheese/kg of fat in milk. Milk composition had a greater effect on Cheddar cheese production and profit than did genotype. Cheese production was significantly different and ranged from 9,846 kg with a high-casein milk composition to 6,834 kg with a high-fat milk composition per 100,000 kg of milk. Profit (per 100,000 kg of milk) was significantly different, ranging from $70,586 for a high-fat milk composition to $16,490 for a low-fat milk composition. However, cheese production was not significantly different, and profit was significant only for the lowest profit ($40,602) with the kappa-casein genotype. Results from this model analysis showed that the optimization model is useful for determining costs and prices for cheese plant inputs and products, and that it can be used to evaluate the economic value of milk components to optimize cheese plant profits.

NFL1, a Nicotiana tabacum LEAFY-like gene, controls meristem initiation and floral structure.

The Arabidopsis LEAFY (LFY) gene product induces cells of the shoot apical meristem to differentiate into floral primordia by acting as a master regulator of downstream floral homeotic genes. Tobacco, an allotetraploid, possesses two homologous genes, NFL1 and NFL2, which are 97% identical in amino acid sequence and share 73% amino acid sequence identity with LFY. In order to test whether the highly conserved tobacco orthologue, NFL1, shares functional identity with LFY, we created transgenic tobacco and Arabidopsis plants that constitutively express the NFL1 cDNA. Our results indicate that NFL1 plays a critical role in the allocation of meristematic cells that differentiate lateral structures such as leaves and branches, thereby determining the architecture of the wild-type tobacco shoot. NFL1 also regulates floral meristem development and does so through the control of cell proliferation as well as cell identity. Surprisingly, unlike ectopic LFY expression, which can act as a floral trigger, ectopic NFL1 expression does not promote severe precocious flowering in Nicotiana tabacum suggesting that variations in amino acid sequence among members of the LFY-like gene family have led to divergence in the functional roles of these genes.

Isolation and characterization of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme.

The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alphabetagamma) with subunit masses of 100 kDa (alpha), 35 kDa (beta), and 25 kDa (gamma). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the alpha subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the beta subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the gamma subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents.

Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of 14C-U Leu given to a non-growing 20-g mouse and a flooding dose of 3H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d(-1) in liver, 14 and 16% d(-1) in brain and 15 and 14% d(-1) in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d(-1) in gastrocnemius, 58, 71 and 62% d(-1) in gut, 8.3, 8.4 and 7.9% d(-1) in heart, 32, 23 and 25% d(-1) in kidney, 160, 90 and 80% d(-1) in liver, 57, 5.5 and 57% d(-1) in soleus and 56, 3.4 and 57% d(-1) in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d(-1) for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d(-1) for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR.

A rodent model of protein turnover used to design an experiment for measuring the rates of channeling, recycling and protein synthesis.

We described previously a mechanistic model of whole-body protein turnover in rodents. Channeling was defined as the flow of amino acids from the extracellular compartment to aminoacyl tRNA and protein synthesis. Recycling was defined as the flow of amino acids from protein degradation to aminoacyl tRNA (protein synthesis) without mixing with the intracellular pool of amino acids. In this paper, the model is applied to tissues and whole body and is used to develop an experimental protocol for estimating protein fractional synthesis rate, recycling and channeling. Channeling, recycling and protein synthesis must be estimated simultaneously because changes in specific radioactivities over time are highly dependent on the rate of protein synthesis. Injection-specific radioactivities, body weights and experimental variation were used with the model to generate data at different rates of recycling and channeling. The data generated were then used to determine the best time points and experimental method to estimate percentages of recycling, channeling and protein synthesis rate by the iterative Method of Maximum Likelihood. Specific radioactivity at each time point was based on simulated data from three rodents at each of six time points. Predicted protein synthesis rates were within 5%/d of observed rates for all methods. Predicted rates of recycling and channeling were generally within 15% of observed rates except recycling in muscle at high channeling and high recycling. Standard deviations of the predictions of percentages of channeling and recycling were between 0.148 and 44.5% for the pulse dose method, 0.0655 and 197% for the continuous infusion method and 0.351 and 962% for the flooding dose method. The experimental design that yields the best estimates of channeling, recycling and protein synthesis is the pulse dose. Changes in amino acid specific radioactivities in the extracellular, aminoacyl tRNA and protein pools were greatest and should be measured at 2, 6, 10, 40, 70 and 100 min in the pulse method.

In vitro studies on the initial reactions of anaerobic ethylbenzene mineralization.

Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) [mg of protein](-1) and an apparent K(m) for ethylbenzene of approximately 60 microM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.

A model of whole-body protein turnover based on leucine kinetics in rodents.

The measurement of fractional synthesis rate is based on the following assumptions: amino acids for protein synthesis are supplied by an intracellular pool; amino acids from protein degradation are not recycled preferentially to protein synthesis; and proteins turn over at a homogeneous rate. To test these assumptions, a mechanistic, theoretical model of protein turnover for a nongrowing 26-g mouse was developed on the basis of data from the literature. The model consisted of three protein pools turning over at fast (102 micromol Leu, t1/2= 11.5 h), medium (212 micromol Leu, t1/2 = 16.6 h) or slow (536 micromol Leu, t1/2 = 71.5 h) rates and extracellular (1.69 micromol Leu), leucyl-tRNA (0.0226 micromol Leu) and intracellular (5.72 micromol Leu) amino acid pools that exchanged amino acids. The flow of amino acids from the protein pools to the leucyl-tRNA pool determined the amount of recycling. The flow of amino acids from the extracellular pool to aminoacyl tRNA determined the amount of channeling. Two flooding dose data sets were used to evaluate specific radioactivity changes predicted by the model. Predictions of specific radioactivities using flooding dose, pulse dose or continuous infusion methods indicated that the model can be a useful tool in estimating the rates of channeling and recycling. However, it was found that use of data from flooding dose experiments might cause inaccurate predictions of certain fluxes.

Recycling, channeling and heterogeneous protein turnover estimation using a model of whole-body protein turnover based on leucine kinetics in rodents.

In the companion paper, a whole-body, mechanistic model of protein turnover in a rodent was described and evaluated with independent data sets that used the flooding dose method. On the basis of fitted fluxes, the model was able to predict specific radioactivity changes in the protein and free leucine pools and whole-body protein fractional synthesis rate (FSR). In this paper, results of model simulations of specific radioactivity changes in the flooding dose, pulse dose and continuous infusion methods were compared and the influence of recycling, channeling and multiple protein pools on model behavior were analyzed. For all methods, the percentage of channeling must be estimated to determine whether the extracellular or intracellular pool specific radioactivities better approximate the aminoacyl tRNA pool specific radioactivity. Recycling also affects the specific radioactivity of the aminoacyl-tRNA pool and therefore must be estimated. An analysis of fits of the flooding dose data indicated that 100% channeling was occurring, but the percentage of recycling could not be determined. Multiple protein pools turning over at different rates overestimated FSR by 2-3% at early time points (5 min) and underestimated FSR by 3-6% at 60 min in the flooding dose method. For the pulse dose method, FSR was underestimated by 40-50% at 5 min and underestimated by 9-10% at 60 min. An increase in time to measure FSR caused a decrease in the estimate of FSR (18% over 3 h) for the flooding dose method and an increase in the estimate of FSR (144% over 3 h) for the pulse dose method.

Annojahnin from Annona jahnii: a possible precursor of mono-tetrahydrofuran acetogenins.

A new cytotoxic Annonaceous acetogenin, annojahnin (1), was isolated from the twigs of Annona jahnii (Annonaceae) by bioactivity-directed fractionation using lethality to brine shrimp. Compound 1 represents an unusual type of C-37 Annonaceous acetogenin, lacking either tetrahydrofuran (THF) or epoxide rings, bearing a keto group at C-10, and possessing a double bond located two methylenes away from a vicinal diol. The structure and absolute configuration of 1 were elucidated by 1H and 13C NMR, COSY, and single-relayed COSY and from chemical derivatives. 4-Deoxy-18/21-trans-annomontacin 10-one (4) and 4-deoxy-18/21-cis-annomontacin-10-one (5), two semisynthetic mono-THF acetogenins, were prepared from 1 by reactions that mimic the biogenetic pathways. These acetogenins showed selective cytotoxicities, comparable or superior to adriamycin, among six human solid tumor cell lines. Reduction of the 10-keto of 1, to the racemic 10-OH derivative (3), retained the bioactivities as did the conversion of 1 to 4 and 5.

New bioactive triterpenoids from Melia volkensii.

Bioactivity-directed fractionation of the root bark of Melia volkensii resulted in the isolation of two new natural products, meliavolkinin (1) and melianin C (3), together with two known compounds, 1,3-diacetylvilasinin (2) and melianin B (4). Jones oxidation of 4 gave compounds 3, 23,24-diketomelianin B (5), and 16,23,24-triketomelianin B (6). The structures of the new compounds were elucidated by spectral and chemical data. Compounds 1-6 all showed marginal cytotoxicities against certain human tumor cell lines, while 5 showed selective cytotoxicities for the human prostate (PC-3) and pancreatic (PACA-2) cell lines with potencies comparable to those of adriamycin.

Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1.

Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO2 under denitrifying conditions, as demonstrated by conversion of 69% of [14C]ethylbenzene to 14CO2. In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H2(18)O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.

SARs of annonaceous acetogenins in rat liver mitochondria.

The Annonaceous acetogenins represent a class of compounds with diverse bioactivities, including promising cytotoxicites. These are due, at least in part, to inhibition of complex I in the oxidative phosphorylation pathway in mitochondria. Fourteen Annonaceous acetogenins were tested in a rat liver mitochondrial oxygen uptake assay to probe additional structure-activity relationships. In this subcellular assay, the activity of non-adjacent bis-THF ring acetogenins depends on the distance between the two THF rings; the activity decreases to that of a mono-THF ring acetogenin if the distance is too long. When one THF ring is replaced with a tetrahydropyran ring, the activity remains comparable. The configuration of the THF ring, in mono ring compounds, seems to be more important than stereochemical differences in the rings of adjacent bis-THF ring compounds. Bullatacin, an adjacent bis-THF ring acetogenin, was used as a standard compound in every run to normalize the data.

Diagnostic information as a commodity.

In estimating the cost-effectiveness of diagnostic procedures, it is helpful to treat diagnostic information as a commodity with a unit price. The amount of useful information provided by a test result can be measured in binary units (bits), and the unit price of the information produced by the test result can be expressed in dollars per bit in much the same way that the price of gold is given in dollars per ounce. This allows comparison of the unit prices of various diagnostic tests, examination of the effect of multiple testing, and calculation of the most cost-effective conditions for screening tests.