Nitrogen inputs best predict farm field nitrate leaching in the Willamette Valley, Oregon.

Nitrate leaching is an important yet difficult to manage contribution to groundwater and surface water contamination in agricultural areas. We examine 14 farm fields over a four year period (2014-2017) in the southern Willamette Valley, providing 53 sets of annual, field-level agricultural performance metrics related to nitrogen (N), including fertilizer inputs, crop harvest outputs, N use efficiency (NUE), nitrate-N leaching and surplus N. Crop-specific nitrate-N leaching varied widely from 10 kg N ha-1yr-1 in hazelnuts to >200 kg N ha-1yr-1 in peppermint. Averaging across all sites and years, most leaching occurred during fall (60%) and winter (32%). Overall NUE was 57%. We used a graphical approach to explore the relationships between N inputs, surplus, crop N harvest removal and NUE by crop type. The blueberry site had high inputs and surplus, peppermint had high inputs but also high crop N removal and NUE and thus lower surplus, and most wheat crops had high NUE and evidence of using soil N. Annual N surplus was not well correlated with leaching, and leaching varied more by crop type and inputs. Grass seed and hazelnuts, which are dominant crop types in the southern Willamette Valley, were intermediate in terms of NUE, leaching and surplus. Of all performance metrics, N input was most closely aligned with field-level crop N harvest and nitrate leaching, therefore optimizing N inputs may well inform local efforts to reduce groundwater nitrate contamination.

Identification and characterization of a novel HIV-1 nucleotide-competing reverse transcriptase inhibitor series.

Several groups have recently reported on the identification of nucleotide-competing reverse transcriptase inhibitors (NcRTIs), a new class of RT inhibitors. NcRTIs reversibly inhibit binding of the incoming nucleotide to the RT active site but do not act as chain terminators, unlike the nucleos(t)ide reverse transcriptase inhibitor (NRTI) class. We identified a novel benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI chemical series. Structure-activity relationship evaluation of this series with both RT and viral replication assays led to the identification of compound A, a new NcRTI. Compound A inhibited HIV-1 RT in a primer extension assay (50% inhibitory concentration, 2.6 nM) but had no measurable activity against human DNA polymerase γ at 10 µM. It potently inhibited HIV-1 replication in vitro (50% effective concentration, 1.5 nM). The antiviral potency of compound A was unaffected by the presence of nonnucleotide RT inhibitor (NNRTI) mutations tested (L100I, K103N/Y181C, V106A, or Y188L). Notably, viruses encoding K65R were hypersusceptible to inhibition by compound A. Compound A also retained full activity against viruses encoding M184V. In vitro selection for resistant virus to compound A led to the selection of a single substitution within RT: W153L. A recombinant virus encoding the RT W153L was highly resistant to compound A (fold change, 160). W153 is a highly conserved residue in HIV RT and has not been previously associated with drug resistance. In summary, a novel NcRTI series with optimized antiviral activity, minimal cross-resistance to existing RT inhibitor classes, and a distinct resistance profile has been discovered. These results further establish NcRTIs as an emerging class of antiretroviral agents.

Structure, genetic mapping, and function of the cytochrome P450 3A37 gene in the turkey (Meleagris gallopavo).

Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B1 (AFB1). Previous studies in our laboratory have shown that the extreme sensitivity of turkeys to AFB1 is in part explained by efficient hepatic P450-mediated epoxidation to the toxic and reactive metabolite the exo-AFB1-8,9-epoxide (AFBO). Using 3'-5'-rapid amplification of cDNA ends (RACE), we amplified CYP3A37 from turkey liver RNA, the E. coli-expressed protein which efficiently epoxidates AFB(1). Turkey CYP3A37 has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. The turkey gene is organized into 13 exons and 12 introns. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10 (corresponding to chicken chromosome 14, GGA14). Because of the important role of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB(1), this study will contribute to the identifying allelic variants of this important gene in poultry.

Butylated hydroxytoluene chemoprevention of aflatoxicosis - effects on aflatoxin B(1) bioavailability, hepatic DNA adduct formation, and biliary excretion.

The extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB(1) toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB(1) bioactivation. To test whether dietary BHT alters hepatic AFB(1)-DNA adduct formation, excretion, and bioavailability of AFB(1)in vivo, turkeys were given diets with BHT (4000ppm) for 10 days, given a single oral dose of [(3)H]-AFB(1) (0.05microg/g; 0.02microCi/g), then sampled at intervals up to 24h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB(1)-DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M(1) (AFM(1)) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB(1), aflatoxin Q(1) or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB(1) bioavailability in addition to inhibition of bioactivation.

Structure and genetic mapping of the Cytochrome P450 gene (CYP1A5) in the turkey (Meleagris gallopavo).

Cytochromes P450 (P450) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. The recently cloned P450 gene (CYP1A5) encodes the primary protein responsible for epoxidation of aflatoxin B(1) (AFB(1)) in the turkey, an animal extremely sensitive to this mycotoxin. Hypersensitivity of turkeys to AFB(1) was first demonstrated by association with 'Turkey X Disease' which caused widespread deaths of turkeys and other poultry throughout Europe in the 1960s, later shown to be caused by AFB(1)-contaminated feed. In this study, comparative genomic approaches were used to selectively amplify and sequence the introns and 3' flanking region of CYP1A5. The structure of the CYP1A5 gene in the turkey is shown to be equivalent to that of the human CYP1A genes with seven exons of 38, 858, 127, 90, 124, 87 and 307 bp, respectively, and six introns. A single nucleotide polymorphism (SNP) in the 3' UTR was used to assign CYP1A5 to turkey linkage group M16 (equivalent to chicken chromosome 10). The results of this study provide the framework for identifying allelic variants of this biochemically important P450 gene in poultry.

Native root xylem embolism and stomatal closure in stands of Douglas-fir and ponderosa pine: mitigation by hydraulic redistribution.

Hydraulic redistribution (HR), the passive movement of water via roots from moist to drier portions of the soil, occurs in many ecosystems, influencing both plant and ecosystem-water use. We examined the effects of HR on root hydraulic functioning during drought in young and old-growth Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco] and ponderosa pine ( Pinus ponderosa Dougl. Ex Laws) trees growing in four sites. During the 2002 growing season, in situ xylem embolism, water deficit and xylem vulnerability to embolism were measured on medium roots (2-4-mm diameter) collected at 20-30 cm depth. Soil water content and water potentials were monitored concurrently to determine the extent of HR. Additionally, the water potential and stomatal conductance ( g(s)) of upper canopy leaves were measured throughout the growing season. In the site with young Douglas-fir trees, root embolism increased from 20 to 55 percent loss of conductivity (PLC) as the dry season progressed. In young ponderosa pine, root embolism increased from 45 to 75 PLC. In contrast, roots of old-growth Douglas-fir and ponderosa pine trees never experienced more than 30 and 40 PLC, respectively. HR kept soil water potential at 20-30 cm depth above -0.5 MPa in the old-growth Douglas-fir site and -1.8 MPa in the old-growth ponderosa pine site, which significantly reduced loss of shallow root function. In the young ponderosa pine stand, where little HR occurred, the water potential in the upper soil layers fell to about -2.8 MPa, which severely impaired root functioning and limited recovery when the fall rains returned. In both species, daily maximum g(s) decreased linearly with increasing root PLC, suggesting that root xylem embolism acted in concert with stomata to limit water loss, thereby maintaining minimum leaf water potential above critical values. HR appears to be an important mechanism for maintaining shallow root function during drought and preventing total stomatal closure.

Effects of dietary butylated hydroxytoluene on aflatoxin B1-relevant metabolic enzymes in turkeys.

We have shown previously that the extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is due to a combination of efficient AFB(1) activation by cytochrome P450s (CYPs) 1A and deficient detoxification by glutathione S-transferases (GSTs). Phenolic antioxidants such as butylated hydroxytoluene (BHT) have been shown to be chemoprotective in some animal models due, in part, to modulation of AFB(1)-relevant phase I and/or phase II activities, and we wished to determine whether BHT has a similar effect in turkeys. Ten-day-old male turkeys were maintained on diets amended with 1000 or 4000 ppm of BHT for 10 days, then sampled. Hepatic microsomal CYP 1A activity as well as conversion of AFB(1) to the putative toxic metabolite, the exo-AFB(1)-8,9-epoxide (AFBO), were significantly lower compared with control. Conversely, dietary BHT significantly increased activities of several isoforms of hepatic cytosolic GST, as well quinone oxidoreductase (QOR). Western immunoblotting confirmed that dietary BHT increased expression of homologues to rodent GST isoforms Yc1, Yc2 and Ya. There was, however, no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. In total, our data indicates that dietary BHT modulates a variety of AFB(1)-relevant phase I and phase II enzymes, while having no measurable effect towards specific AFB(1) detoxification by GST.

Inhibition of human cytochrome P450 2E1 by nicotine, cotinine, and aqueous cigarette tar extract in vitro.

Cigarette smoke is a complex mixture containing, among other chemicals, pyridine alkaloids and N-nitrosamines. Carcinogenic tobacco-specific N-nitrosamines, N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are both activated by cytochrome P450 (CYP) 2E1 in rats. Previous reports indicate that nicotine and the main nicotine metabolite, cotinine, reduce the mutagenicity of both NNK and NDMA in Salmonella typhimurium. To study the mechanism of this effect, we examined inhibition of CYP 2E1 activity, as assessed by p-nitrophenol (pNP) hydroxylation, by nicotine, cotinine, and an aqueous cigarette tar extract (ACTE) in human 2E1-expressing microsomes. At all substrate concentrations (0-1.25 mM) nicotine was a significantly more potent inhibitor of CYP 2E1 activity compared to cotinine. Estimated Ki values for nicotine and cotinine (both at 10 mM) were 13 mM (2 mg/ml) and 308 mM (54 mg/ml) respectively. The Ki for ACTE was 0.2 mg/ml at a concentration of 0.32 mg/ml. This rank order for inhibition was also seen when the data was expressed as IC(50). When compared on a mass/vol basis, ACTE was a significantly more potent CYP 2E1 inhibitor relative to nicotine and cotinine. Double-reciprocal plots indicated that nicotine and ACTE inhibited by a competitive, while cotinine inhibited CYP 2E1 by an uncompetitive mechanism. Although the contribution of nicotine to ACTE-mediated 2E1 inhibition is probably modest, pyridine alkaloid-mediated CYP 2E1 inhibition is a possible mechanism for the observed inhibition of NNK and NDMA mutagenicity by nicotine and cotinine in vitro.

Metabolism of aflatoxin B1 by normal human bronchial epithelial cells.

Aflatoxin B, (AFB1) is a potent hepatocarcinogen in animal models and a suspected carcinogen in humans. High concentrations of AFB, have been found in respirable grain dusts, and may therefore be a risk factor for human lung cancer in certain occupations. To study the potential for AFB, activation in human lung, cytochrome P-450 (CYP)-mediated activation and glutathione S-transferase (GST)-mediated detoxification of AFB1 were examined in cultured normal human bronchial epithelial (NHBE) cells. Cells were exposed to 0. 15 microM or 1.5 microM AFB, for 48 h and media was collected for metabolite analysis by high-performance liquid chromatography (HPLC). At 0. 15 microM, AFB1 was metabolized only to the detoxified metabolite aflatoxin Q1 (AFQ1). At 1.5 microM AFB1, both aflatoxin M1 (AFM1), and AFQ1 were produced. Cells pretreated with 50 degrees M 3-methylcholanthrene (3MC), a CYP 1A inducer, for 72 h prior to 0.15 microM AFB1, produced the activated AFB1 8,9-epoxide (AFBO). Similarly, microsomes prepared from 3MC-pretreated cells formed AFBO, but microsomes from noninduced cells did not. While AFB1-DNA adducts were not detected at low AFB1 concentrations in untreated NHBE, 3MC induction caused the production of AFB1-DNA adducts at 0.015 and 0.15 microM AFB1. Western immunoblots showed that the primary CYP isoforms responsible for AFB1 activation in the liver, 1A and 3A4, to be constitutively expressed in NHBE cells. Expression of CYP 1A was significantly increased in 3MC-pretreated cells, while CYP 3A4 expression increased slightly, but not to the extent of the 1A isoforms. The principal AFBO detoxifying enzyme, glutathione S-transferase (GST), was constitutively expressed in NHBE cells, and was increased approximately twofold by 3MC pretreatment. Cytosolic fractions from neither control nor 3MC-induced NHBE had measurable AFBO conjugating activity, indicating that these cells may lack AFB1-relevant GST activity. From these data, it appears that NHBE cells activate AFB1 inefficiently, but possess CYPs reportedly responsible for metabolism of AFB1. These data support earlier findings showing modest CYP-mediated AFB1 activation in human airways, but indicate that exposure to polycyclic aromatic hydrocarbons (PAHs), such as 3MC, which induce CYP(s) that specifically activate AFB1 may increase the harmful effects of AFB1 exposures in human airways.

Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair.

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-A resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu(475), located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.

The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site.

The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 A resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 A long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO-AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals significant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis.

Crystal structure of the NADP+-dependent aldehyde dehydrogenase from Vibrio harveyi: structural implications for cofactor specificity and affinity.

Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi, catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP(+). Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-A resolution as a partial complex with the cofactor NADP(+) and to 2. 1-A resolution as a fully bound 'holo' complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2'-hydroxy or the 2'-phosphate groups of the adenosine ribose moiety of NAD(+) or NADP(+), respectively. In the NADP(+)-dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack.

Biochemical basis for the extreme sensitivity of turkeys to aflatoxin B(1).

Poultry are the most susceptible food animal species to the toxic effects of the mycotoxin aflatoxin B(1) (AFB(1)). Feed contaminated with even small amounts of AFB(1) results in significant adverse health effects in poultry. The purpose of this study was to explain the biochemical mechanism(s) for this extreme sensitivity. We measured microsomal activation of AFB(1) to the AFB(1)-8,9-epoxide (AFBO), the putative toxic intermediate, as well as cytosolic glutathione S-transferase (GST)-mediated detoxification of AFBO, in addition to other hepatic phase I and phase II enzyme activities, in 3-week-old male Oorlop strain turkeys. Liver microsomes prepared from these turkeys activated AFB(1) in vitro with an apparent K(m) of 109 microM and a V(max) of 1.25 nmol/mg/min. Preliminary evidence for the involvement of cytochromes P450 (CYP) 1A2 and, to a lesser extent, 3A4 for AFB(1) activation was assessed by the use of specific mammalian CYP inhibitors. The possible presence of avian orthologues of these CYPs was supported by activity toward ethoxyresorufin and nifedipine, as well as by Western immunoblotting using antibodies to human CYPs. Cytosol prepared from turkey livers exhibited GST-mediated conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB), but at a much lower rate than that observed in other species. Western immunoblotting indicated the presence of alpha and sigma class GSTs and another AFB(1)-detoxifying enzyme, AFB(1)-aldehyde reductase (AFAR). Turkey liver cytosol also had quinone oxidoreductase (QOR) activity. Importantly, cytosol exhibited no measurable GST-mediated detoxification of microsomally activated AFB(1), indicating that turkeys are deficient in the most crucial AFB(1)-detoxification pathway. In total, our data indicate that the extreme sensitivity of turkeys to AFB(1) may be attributed to a combination of efficient AFB(1) activation and deficient detoxification by phase II enzymes, such as GSTs.

Comparative DNA cross-linking by activated pyrrolizidine alkaloids.

The toxicity and bioactivity of pyrrolizidine alkaloids (PAs), common constituents of hundreds of plant species, and in herbal remedies and folk medicines prepared thereof, are probably due to their ability to form DNA cross-linking. We investigated DNA cross-linking activity by chemically-activated PAs from four different structural classes in Madin-Darby bovine kidney (MDBK) cells and in pBR322 DNA. In cell culture, alpha,beta-unsaturated macrocyclic diester pyrroles dehydrosenecionine (DHSN), dehydroriddelliine (DHRD) and the saturated macrocyclic diester pyrrole dehydromonocrotaline (DHMO) were significantly more potent cross-linkers than the simple necine base (retronecine) and an N-oxide (indicine N-oxide; INO) as determined by alkaline elution. The proportion of total DNA cross-links that were proteinase K-resistant (DNA-DNA cross-links) induced by the various pyrroles ranged from 0.08 (DHRN) to 0.67 (DHSN). Those pyrroles that were potent cross-linkers of cellular DNA also cross-linked, in a dose-dependent manner, Bam HI-digested pBR322 DNA as assessed by a gel retardation assay. The possible functional relevance of pyrrole-DNA cross-links was determined by their ability to interrupt PCR amplification of a 1.129 kb segment of pBR322. Dehydrosenecionine completely inhibited amplification, while DHMO was of intermediate potency, while DHRN and INO had no effect. Taken together, these studies suggest that structural features, most notably the presence of a macrocyclic diester, confer potent cross-link activity to PAs. In any event, DNA-DNA cross-linking is probably biologically relevant as indicated by their interference with DNA replication.

Pyrrolizidine alkaloid plants, metabolism and toxicity.

More than 350 PAs have been identified in over 6,000 plants in the Boraginaceae, Compositae, and Leguminosae families (Table 1). About half of the identified PAs are toxic and several have been shown to be carcinogenic in rodents. PA-containing plants have worldwide distribution, and they probably are the most common poisonous plants affecting livestock, wildlife, and humans. In many locations, PA-containing plants are introduced species that are considered invasive, noxious weeds. Both native and introduced PA-containing plants often infest open ranges and fields, replacing nutritious plants. Many are not palatable and livestock avoid eating them if other forages are available. However, as they invade fields or crops, plant parts or seeds can contaminate prepared feeds and grains which are then readily eaten by many animals. Human poisonings most often are a result of food contamination or when PA-containing plants areused for medicinal purposes. This is a review of current information on the diagnosis, pathogenesis, and molecular mechanisms of PA toxicity. Additional discussion includes current and future research objectives with an emphasis on the development of better diagnostics, pyrrole kinetics, and the effects of low dose PA exposure.

Pyrrolizidine alkaloids crosslink DNA with actin.

Pyrrolizidine alkaloids (PAs) are toxic constituents of hundreds of plant species, some of which people are exposed to in herbal products and traditional remedies. The bioactivity of PAs are related, at least in part, to their ability to form DNA-protein complexes (DPC). Previous studies from our laboratory indicated a possible role for actin in PA-induced DPCs. Nuclei prepared from Madin-Darby bovine kidney (MDBK) and human breast carcinoma (MCF-7) cells were treated with the pyrrolic PAs dehydrosenecionine (DHSN) and dehydromonocrotaline (DHMO). DPCs were purified and then analyzed by Western immunoblotting. Actin was found in DPCs induced by both DHSN and DHMO, but not in those from control nuclei. Actin was also present in DPCs induced by cisplatinum and mitomycin C, two bifunctional cross-linkers. In separate experiments, DHSN and DHMO were crosslinked to a mixture of HindIII digested lambda phage with varying amounts of glutathione (GSH), cysteine, or methionine to identify the stoichiometry of competition between DNA and alternate nucleophiles for crosslink formation with pyrroles. GSH and cysteine, but not methionine, competed with lambda phage for DNA crosslinking, indicating that reduced thiols may have a role in nucleophilic reactions with pyrroles in the cell. While actin involvement in cisplatinum-induced DPCs is documented, the discovery of actin crosslinking in PA or mitomycin C-treated cells or nuclei is, to our knowledge, novel. Pyrrole-induced DPC formation with actin, a protein with structural and/or regulatory importance proteins, may be a significant mechanism for PA toxicity and bioactivity.

Structural basis for specificity of papain-like cysteine protease proregions toward their cognate enzymes.

Synthetic peptides corresponding to the proregions of papain-like cysteine proteases have been shown to be good and selective inhibitors of their parental enzymes. The molecular basis for their selectivity, quite remarkable in some cases, is not fully understood. The recent determination of the crystal structures of three distinct papain-like cysteine protease zymogens allows detailed structural comparisons to be made. The reasons for the specificity shown by each proregion toward its cognate enzyme are explained in terms of the three-dimensional structure of the proregion and the interface between the mature enzyme and the proregion. These comparisons reveal that insertion and substitution of amino acids within the proregion cause major rearrangement of sidechains on the enzyme/proregion interface, allowing detailed surface and charge recognition.

Procedure to calculate the molecular envelope from a partial model.

Density-modification algorithms have gained, in recent years, a widespread use in the early stages of protein structure determination, especially in combination with the single isomorphous replacement, the multiple isomorphous replacement and the multiple-wavelength anomalous dispersion methods, where density modification usually leads to a significant improvement in the quality and interpretability of the initial electron-density map. The current computer programs which are used to perform this task combine several approaches, an important component of which is the solvent-flattening procedure. The latter procedure depends crucially on the correct determination of the molecular envelope. The solvent-flattening procedure has also been applied to the electron-density maps calculated from partial mod- els obtained from the molecular replacement method. In such case the envelope calculated in the standard way does not always encompass entirely the missing part. It has been found that the standard application of the density-modification method (as implemented by programs SQUASH and DM) to a map calculated from a molecular replacement model containing approximately 60% of the molecule, led to little improvement in the map interpretability. Here, it is shown that a significant improvement of the map can be achieved when a better envelope is used in the procedure. Various methods of calculating the molecular envelope have been evaluated, the effect of the shape of the envelope on the modified electron-density map has been investigated and an improved procedure to calculate the envelope from a partial molecular replacement model is proposed.

Aflatoxin B1 activation in human lung.

Inhalation exposure to the carcinogen aflatoxin B1 (AFB1) in certain occupations is considerable. Because circumstantial epidemiological evidence suggests that AFB1 inhalation may cause primary lung cancer, we investigated AFB1 activation by human lung microsomes. Microsomes were incubated with [3H]AFB1 (124 microM), and activation to the AFB1-8,9-epoxide was measured as the AFB1-glutathione (AFB1-GSH) conjugate by HPLC. The formation of AFB1-GSH was in the range of 0.05-0.073 fmol/mg protein/min. The role of cytochrome P450 (CYP) 3A in this activation was investigated by oxidation of nifedipine (a prototype substrate for CYP 3A), by immunoinhibition, and by immunoblot analysis. Nifedipine oxidation varied from 0.2 to 19.2 pmol/mg protein/min in microsomes from different subjects, but did not correlate with AFB1 activation. Anti-human polyclonal CYP 3A4 IgG inhibited AFB1 activation. CYP 3A isoforms were immunoestimated to be in the range of 0.01-1.90 pmol/mg protein. Neither CYP 1A2 nor associated activity was detected in the lung microsomes. These data indicate that human lung microsomes activate AFB1 to form the exo-AFB1-8,9-epoxide and that CYP(s) of the 3A subfamily may be responsible for this activity. The relatively low amount of AFB1 activation in human lung compared to that in human liver can be explained by the scarcity of CYP-containing cells in the lung. In situ AFB1 activation and resultant carcinogenic risk are distinctly possible in occupational settings where inhalation of AFB1-contaminated dusts occurs.

Novel active site in Escherichia coli fructose 1,6-bisphosphate aldolase.

The molecular architecture of the Class II E. coli fructose 1,6-bisphosphate aldolase dimer was determined to 1.6 A resolution. The subunit fold corresponds to a singly wound alpha/beta-barrel with an active site located on the beta-barrel carboxyl side of each subunit. In each subunit there are two mutually exclusive zinc metal ion binding sites, 3.2 A apart; the exclusivity is mediated by a conformational transition involving side-chain rotations by chelating histidine residues. A binding site for K+ and NH4+ activators was found near the beta-barrel centre. Although Class I and Class II aldolases catalyse identical reactions, their active sites do not share common amino acid residues, are structurally dissimilar, and from sequence comparisons appear to be evolutionary distinct.