Emergent decarboxylase activity and attenuation of α/β-hydrolase activity during the evolution of methylketone biosynthesis in tomato

Specialized methylketone-containing metabolites accumulate in certain plants, in particular wild tomatoes in which they serve as toxic compounds against chewing insects. In Solanum habrochaites f. glabratum, methylketone biosynthesis occurs in the plastids of glandular trichomes and begins with intermediates of de novo fatty acid synthesis. These fatty-acyl intermediates are converted via sequential reactions catalyzed by Methylketone Synthase2 (MKS2) and MKS1 to produce the n-1 methylketone. We report crystal structures of S. habrochaites MKS1, an atypical member of the α/β-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional α/β-hydrolase triad, Ser-His-Asp. Determination of the MKS1 structure points to a novel enzymatic mechanism dependent upon residues Thr-18 and His-243, confirmed by biochemical assays. Structural analysis further reveals a tunnel leading from the active site consisting mostly of hydrophobic residues, an environment well suited for fatty-acyl chain binding. We confirmed the importance of this substrate binding mode by substituting several amino acids leading to an alteration in the acyl-chain length preference of MKS1. Furthermore, we employ structure-guided mutagenesis and functional assays to demonstrate that MKS1, unlike enzymes from this hydrolase superfamily, is not an efficient hydrolase but instead catalyzes the decarboxylation of 3-keto acids.     

Heterozygosity–behavior and heterozygosity–fitness correlations in a salamander with limited dispersal

Inbreeding and genetic drift can decrease genetic heterozygosity, and this low heterozygosity can depress fitness, resulting in heterozygosity–fitness correlations (HFCs). HFCs are typically small in magnitude, a result often attributed to power of the analyses. Animal behaviors often affect fitness and are often heritable to some degree. We hypothesized that heterozygosity influences behavior, which, in turn, potentially influences fitness. Specifically, in red-backed salamanders, Plethodon cinereus, which have limited dispersal and the potential for inbreeding, we tested whether heterozygosity, as estimated from six microsatellite loci, affected home range size, juvenile growth, and survival. We found that salamanders with higher heterozygosity had larger home ranges and grew faster, which is indicative of reproductive success. However, we found no effects of heterozygosity on survival. We conclude that, because activity in P. cinereus is tightly linked to food uptake and mass gain, heterozygosity influences growth via effects on foraging behavior. Future research should investigate how the relationship between heterozygosity, behavior, and fitness may be affected or mediated by endocrine or immune systems.     

Light enough to travel or wise enough to stay? Brain size evolution and migratory behavior in birds

Brain size relative to body size is smaller in migratory than in nonmigratory birds. Two mutually nonexclusive hypotheses had been proposed to explain this association. On the one hand, the “energetic trade‐off hypothesis” claims that migratory species were selected to have smaller brains because of the interplay between neural tissue volume and migratory flight. On the other hand, the “behavioral flexibility hypothesis” argues that resident species are selected to have higher cognitive capacities, and therefore larger brains, to enable survival in harsh winters, or to deal with environmental seasonality. Here, I test the validity and setting of these two hypotheses using 1466 globally distributed bird species. First, I show that the negative association between migration distance and relative brain size is very robust across species and phylogeny. Second, I provide strong support for the energetic trade‐off hypothesis, by showing the validity of the trade‐off among long‐distance migratory species alone. Third, using resident and short‐distance migratory species, I demonstrate that environmental harshness is associated with enlarged relative brain size, therefore arguably better cognition. My study provides the strongest comparative support to date for both the energetic trade‐off and the behavioral flexibility hypotheses, and highlights that both mechanisms contribute to brain size evolution, but on different ends of the migratory spectrum.     

Is individual consistency in body mass and reproductive decisions linked to individual specialization in foraging behavior in a long‐lived seabird?

Individual specialization in diet or foraging behavior within apparently generalist populations has been described for many species, especially in polar and temperate marine environments, where resource distribution is relatively predictable. It is unclear, however, whether and how increased environmental variability – and thus reduced predictability of resources – due to global climate change will affect individual specialization. We determined the within‐ and among‐individual components of the trophic niche and the within‐individual repeatability of δ¹³C and δ¹⁵N in feathers and red blood cells of individual female southern rockhopper penguins (Eudyptes chrysocome) across 7 years. We also investigated the effect of environmental variables (Southern Annular Mode, Southern Oscillation Index, and local sea surface temperature anomaly) on the isotopic values, as well as the link between stable isotopes and female body mass, clutch initiation dates, and total clutch mass. We observed consistent red blood cell δ¹³C and δ¹⁵N values within individuals among years, suggesting a moderate degree of within‐individual specialization in C and N during the prebreeding period. However, the total niche width was reduced and individual specialization not present during the premolt period. Despite significant interannual differences in isotope values of C and N and environmental conditions, none of the environmental variables were linked to stable isotope values and thus able to explain phenotypic plasticity. Furthermore, neither the within‐individual nor among‐individual effects of stable isotopes were found to be related to female body mass, clutch initiation date, or total clutch mass. In conclusion, our results emphasize that the degree of specialization within generalist populations can vary over the course of 1 year, even when being consistent within the same season across years. We were unable to confirm that environmental variability counteracts individual specialization in foraging behavior, as phenotypic plasticity in δ¹³C and δ¹⁵N was not linked to any of the environmental variables studied.     

Test–retest stability of the oral niacin test and electrodermal activity in patients with schizophrenia

In schizophrenia, well-replicated findings support an attenuated niacin skin-flush response. We have previously reported a delayed skin-flush after niacin ingestion and also an association between niacin non-responding and electrodermal non-responding in schizophrenia. The stability of the niacin and electrodermal tests was now studied in a test–retest design. An additional aim was to assess the association previously found.Twenty-three patients with schizophrenia underwent two sessions 3 months apart during which an oral niacin test was conducted and electrodermal activity was measured. Despite similar values for niacin outcome variables at the group level, there was high intraindividual variation. Test–retest stability for the oral niacin test was thus low, although a trend toward correlation for the dichotomous response criterion was found. Most electrodermal measures correlated between baseline and retest. A significant association between the tests was again found; niacin non-responding implied electrodermal non-responding, providing further support for a common underlying aberration in schizophrenia.     

Internal consistency and test–retest reliability of an instrumented functional reaching task using wireless electromyographic sensors

The purpose of this study was to establish the internal consistency and test–retest reliability of the electromyographic and accelerometric data sampled from the prime movers of the dominant arm during an antigravity, within-arm’s length stand-reaching task without trunk restraint. Ten healthy young adults participated in two experimental sessions, approximately 7–10 days apart. During each session, subjects performed 15 trials of both a flexion- and an abduction-reaching task. Surface EMG and acceleration using wireless sensors were sampled from the anterior and middle deltoid. Reliability was established using Cronbach’s alpha, intraclass correlation coefficients (ICC 2, k) and standard error of measurements (SEM) for electromyographic reaction time, burst duration and normalized amplitude along with peak acceleration. Results indicated high degrees of inter-trial and test–retest reliability for flexion (Cronbach’s α range = 0.92–0.99; ICC range = 0.82–0.92) as well as abduction (Cronbach’s α range = 0.94–0.99; ICC range = 0.81–0.94) reaching. The SEM associated with response variables for flexion and abduction ranged from 1.55–3.26% and 3.33–3.95% of means, respectively. Findings from this study revealed that electromyographic and accelerometric data collected from prime movers of the arm during the relatively functional stand-reaching task were highly reproducible. Given its high reliability and portability, the proposed test could have applications in clinical and laboratory settings to quantify upper limb function.     

Preparation,characterization and in vitro activity of a docetaxel–albumin conjugate

Docetaxel is one of the most effective anticancer drugs. However, the current formulation of docetaxel contains Tween 80 and ethanol as the solvent, which can cause severe side effects. Consequently, the development of new type of formulation of docetaxel with high efficiency and low side effects is a very important issue. In this study, we explored the covalent linking of docetaxel and albumin via one organic linker. 6-Maleimidocaproic acid was applied to link the C2′ hydroxyl group of docetaxel with the cysteine-34 of albumin to obtain 1:1 docetaxel–albumin conjugate. The synthesized conjugate can control the release of docetaxel in the bovine serum. Furthermore, in vitro cell cytotoxicity experiments indicated that the docetaxel–albumin conjugate have high activities for human prostate cancer cell line PC3 and human breast cancer cell line MCF-7. The present study provides a valuable strategy for further development of a new type of docetaxel–albumin prodrug.     

Bacterial complementation as a means to test enzyme–ligand interactions

A bacterial complementation assay has been developed for the rapid screening of a large number of compounds to identify those that inhibit an enzyme target for structure-based inhibitor design. The target enzyme is the hypoxanthine phosphoribosyltransferase (HPRT). This enzyme has been proposed as a potential target for inhibitors that may be developed into drugs for the treatment of diseases caused by several parasites. The screening assay utilizes genetically deficient bacteria complemented by active, recombinant enzyme grown in selective medium in microtiter plates. By comparing absorbance measurements of bacteria grown in the presence and absence of test compounds, the effect of the compounds on bacterial growth can be rapidly assayed. IC₅₀ values for inhibition of bacterial growth are a reflection of the ability of the compounds to bind and/or inhibit the recombinant enzyme. We have tested this bacterial complementation screening assay using recombinant HPRT from the parasites Plasmodium falciparum and Trypanosoma cruzi, as well as the human enzyme. The results of these studies demonstrate that a screening assay using bacterial complement selection can be used to identify compounds that target enzymes and can become an important part of structure-based drug design efforts. Received: 4 December 1997 / Received revision: 17 March 1998 / Accepted: 26 March 1998     

Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity

The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E approximately 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E approximately 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E approximately 86).     

Defying the activity–stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability

The biotechnological applications of enzymes are limited due to the activity–stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity–stability trade-off can be overcome. In this review, the origin of the activity–stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity–stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel–Danson equilibrium model, catalytic antibodies, and life on cold planets.     

Changes in the protein and activity levels of the plastid NADH–plastoquinone–oxidoreductase complex during fruit development

Plastids contain an NADH dehydrogenase complex (Ndh complex) homologous to the mitochondrial complex I (EC 1.6.5.3). In this work, we have analysed the changes in the Ndh complex during ripening of pepper (Capsicum annum L., cv. Maor) and tomato (Lycopersicon esculentum Mill., cv. Marglobe) fruits. The Ndh complex was mainly present in the outer pericarp of tomato fruits, whereas it was evenly distributed in the pericarp of pepper. In both kinds of fruit we observed a decrease in the total amount of Ndh complex from the green to the red stage of development. This decrease corresponds to parallel decreases in the content and activity of the complex in plastids during the transition from chloroplasts to chromoplasts. Levels of plastidial quinol peroxidase activity were also higher during the first stages of tomato fruit development than during the latter stages of ripening. However, when referred to total plastid protein, the amount and activity of the Ndh complex in chloroplasts isolated from green fruits was higher than in chloroplasts isolated from leaves. These results strongly suggest that function of the Ndh complex, probably related to a plastidial electron transport chain, can be important during the first stages of fruit development.     

Spring and autumn territoriality in song sparrows: same behavior, different mechanisms?

Vertebrates show a diverse array of social behaviors associatedwith territoriality. Field and laboratory experiments indicatethat underlying themes—including mechanisms—mayexist. For example in birds, extensive evidence over many decadeshas implicated a role for testosterone in the activation ofterritorial aggression in reproductive contexts. Territorialityat other times of the year appeared to be independent of gonadalhormone control. One obvious question is—why this diversityof control mechanisms for an apparently similar behavior? Controlof testosterone secretion during the breeding season must balancethe need to compete with other males (that tends to increasetestosterone secretion), and the need to provide parental care(that requires lower testosterone concentrations). Regulationof aggressive behaviors by testosterone in the non-breedingseason may incur substantial costs. A series of experimentson the male song sparrow, Melospiza melodia morphna, of westernWashington State have revealed possible mechanisms to avoidthese costs. Song sparrows are sedentary and defend territoriesin both breeding and non-breeding seasons. Dominance interactions,territorial aggression and song during the non-breeding seasonare essentially identical to those during the breeding season.Although in the non-breeding season plasma testosterone andestradiol levels are very low, treatment with an aromatase inhibitordecreases aggression and simultaneous implantation of estradiolrestores territorial behavior. These data suggest that the mechanismby which testosterone regulates territorial behavior at theneural level remains intact throughout the year. How the hormonalmessage to activate such behavior gets to the brain in differentseason does, however, appear to be different.     

Duration of activity and period of circadian activity–rest rhythm in a photoperiod-dependent primate,Microcebus murinus

Under free-running conditions, the grey lesser mouse lemur expresses a circadian activity–rest rhythm with a period particularly short (22.50 ± 0.6 h) for a mammal. Light exerts a strong suppressive effect upon activity. After transfer from nycthemeral to free-running conditions the duration of activity was systematically increased. This extension took place in the first cycle and was characterized by both a phase advance in activity onset and an even larger phase delay in activity offset. This phenomenon was more pronounced after long day entrainment. Any shift of the light–dark cycle was followed the next day by a corresponding shift in activity onset. The phase response curve pattern was similar to that already described for nocturnal mammals. Due to the strength of light as a zeitgeber and the plasticity of the response to photic conditions, the mouse lemur appears as a convenient species for chronobiological studies on non-human primates.     

Prothoracic DUM neurons of the cricket Gryllus bimaculatus — responses to natural stimuli and activity in walking behavior

Summary Responses to sensory stimuli and spike activity uring walking were investigated in bilaterally symmetrical dorsal unpaired median (DUM) neurons of the cricket. Intracellular recordings within the prothoracic ganglion were made either in restrained animals or in stationary walking specimens whilst parameters of their intended locomotion were measured. Three types of DUM cells were distinguished morphologically and physiologically. DUMa neurons send axons through segmental nerves. They often generated spontaneously large action potentials with low frequencies. Most DUMa neurons showed multimodal sensitivity, preferentially to cereal wind puffs and 15 kHz sound. Mean latencies ranged from 25 to 349 ms. Their large intraindividual variability could be correlated with behavioral modes during walking. Generally, the spike frequency increased with increased forward speed, while it was not related to turning. DUMb neurons projected either through the anterior or posterior connectives, but seemed physiologically similar to DUMa neurons. DUMc neurons were H-shaped with axons in both pairs of connectives. No external stimulus led to discrete spikes, but the regular spontaneous activity was modulated following cereal wind puffs to a restrained animal. During wind evoked escape the spike activity of another DUMc cell was modulated in phase with the rhythmic running behavior. The possibly different functions of DUMa and DUMc neurons during walking are discussed.     

Liquid chromatography–mass spectrometry in forensic and clinical toxicology

This paper reviews liquid chromatographic–mass spectrometric (LC–MS) procedures for the identification and/or quantification of drugs of abuse, therapeutic drugs, poisons and/or their metabolites in biosamples (whole blood, plasma, serum, urine, cerebrospinal fluid, vitreous humor, liver or hair) of humans or animals (cattle, dog, horse, mouse, pig or rat). Papers published from 1995 to early 1997, which are relevant to clinical toxicology, forensic toxicology, doping control or drug metabolism and pharmacokinetics, were taken into consideration. They cover the following analytes: amphetamines, cocaine, lysergide (LSD), opiates, anabolics, antihypertensives, benzodiazepines, cardiac glycosides, corticosteroids, immunosuppressants, neuroleptics, non-steroidal anti-inflammatory drugs (NSAID), opioids, quaternary amines, xanthins, biogenic poisons such as aconitines, aflatoxins, amanitins and nicotine, and pesticides. LC–MS interface types, mass spectral detection modes, sample preparation procedures and chromatographic systems applied in the reviewed papers are discussed. Basic information about the biosample assayed, work-up, LC column, mobile phase, interface type, mass spectral detection mode, and validation data of each procedure is summarized in tables. Examples of typical LC–MS applications are presented.     

Plant–microbe interactions and nitrogen dynamics during wetland establishment in a desert stream

In late-successional steady state ecosystems, plants and microbes compete for nutrients and nutrient retention efficiency is expected to decline when inputs exceed biotic demand. In carbon (C)-poor environments typical of early primary succession, nitrogen (N) uptake by C-limited microbes may be limited by inputs of detritus and exudates derived from contemporaneous plant production. If plants are N-limited in these environments, then this differential limitation may lead to positive relationships between N inputs and N retention efficiency. Further, the mechanisms of N removal may vary as a function of inputs if plant-derived C promotes denitrification. These hypotheses were tested using field surveys and greenhouse microcosms simulating the colonization of desert stream channel sediments by herbaceous vegetation. In field surveys of wetland (ciénega) and gravelbed habitat, plant biomass was positively correlated with nitrate (NO₃ ^?) concentration. Manipulation of NO₃ ^? in flow-through microcosms produced positive relationships among NO₃ ^? supply, plant production, and tissue N content, and a negative relationship with root:shoot ratio. These results are consistent with N limitation of herbaceous vegetation in Sycamore Creek and suggest that N availability may influence transitions between and resilience of wetland and gravelbed stable states in desert streams. Increased biomass in high N treatments resulted in elevated rates of denitrification and shifts from co-limitation by C and NO₃ ^? to limitation by NO₃ ^? alone. Overall NO₃ ^? retention efficiency and the relative importance of denitrification increased with increasing N inputs. Thus the coupling of plant growth and microbial processes in low C environments alters the relationship between N inputs and exports due to increased N removal under high input regimes that exceed assimilative demand.     

Identification of a locality in snake venomα-neurotoxins with a singificant compositional similarity to marine snailα-conotoxins: Implications for evolution and structure/activity

Summary -neurotoxins from elapid snake venoms and-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an-neurotoxin from the false sea snakeLaticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller-conotoxins (from the marine snailsConus magus andConus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake-neurotoxins and their homologues. Six Proteins unrelated to erabutoxin b were compared to the-conotoxins to show that the extent of the erabutoxin b/-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely-cobratoxin fromNaja naja siamensis and cytotoxin V^II4 fromNaja mossambica mossambica, were also analyzed. The extent of the matching with the-conotoxins decreased in the series erabutoxin b>-cobratoxin>cytotoxin V^II4, and this also relates the order of similarity to the pharmacological properties of the-conotoxins.The-conotoxin-like area of the snake-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.     

β-Lactamase activity and resistance to penicillins in Myxococcus xanthus

Several mutants and other variants of Myxococcus xanthus HP100 were obtained with differences in their sensitivity to carbenicillin and other penicillin derivatives. The specific activities of -lactamase in different resistant organisms varied from strain to strain but were consistently higher than in HP100. The relative molecular mass (M _r ) of the enzyme in M. xanthus HP100 was found to be 22,300. In certain carbenicillin resistant strains a second fraction of -lactamase activity of molecular weight 186,000 presumed to be an octamer of the other form was present. The enzyme was found in cell free extracts and also in culture supernatants of all carbenicillin resistant mutants but not in culture supernatants of strain HP100. In all the carbenicillin resistant mutants a part of the intracellular enzyme activity was released by osmotic shock and this activity may be periplasmic. The forms of the enzyme present in the culture supernatants and released by osmotic shock were monomeric. Carbenicillin resistance was not transferable between strains by conjugation. One resistance allele inhibited the transfer of the R factor Sa between myxococci.Non-standard abbreviations C^S C^R sensitivity and resistance to carbenicillin - C _u ^R C _S ^R unstable and stable resistance to carbenicillin