bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450cam)
Authors: Jones Jonathan P. , O'Hare Ellen J. , Wong Luet‐Lok 
Published: March 2003

Polychlorinated benzenes are recalcitrant environmental pollutants primarily because they are resistant to attack by dioxygenases commonly used by micro‐organisms for the biodegradation of aromatic compounds. We have investigated the oxidation of polychlorinated benzenes by mutants of the haem mono‐oxygenase CYP101 (cytochrome P450cam) from Pseudomonas putida with the aim of generating novel systems for their biodegradation. Wild‐type CYP101 had low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorinated benzenes. Increasing the active‐site hydrophobicity with the Y96F mutation increased the activity up to 100‐fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol. Decreasing the space available at the top of the active site with the F87W mutation to force the substrate to be bound closer to the haem resulted in a further 10‐fold increase in activity with most substrates. Introducing the F98W mutation, also at the top of the active site, decreased the NADH‐turnover rates but increased the coupling efficiencies, and > 90% coupling was observed for 1,3‐dichlorobenzene and 1,3,5‐trichlorobenzene with the F87W–Y96F–F98W mutant. The V247L mutation generally increased the NADH‐turnover rates, and the F87W–Y96F–V247L mutant showed reasonably fast NADH turnover (229 min−1) with the highly insoluble pentachlorobenzene without the need for surfactants or organic cosolvents. As all chlorophenols are degraded by micro‐organisms, novel biodegradation systems could be constructed in which CYP101 mutants convert the inert polychlorinated benzenes to the phenols, which are then readily degraded by natural pathways.
Characterization of carbonic anhydrase from Neisseria gonorrhoeae
Authors: Elleby Björn , Chirica Laura C. , Tu Chingkuang , Zeppezauer Michael , Lindskog Sven 
Published: March 2003

We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s−1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping‐pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O‐exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66→Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2‐dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO−, SCN− and N3− as HCA II, while CN− and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)‐substituted NGCA with acetazolamide, NCO−, SCN−, CN− and N3− resemble the corresponding spectra obtained with human Co(II)‐isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)‐induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2‐carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8–2.9 m GdnHCl was observed.
Processing of tumor necrosis factor by the membrane‐bound TNF‐α‐converting enzyme, but not its truncated soluble form
Authors: Itai Toshimitsu , Tanaka Masato , Nagata Shigekazu 
Published: March 2003

Tumour necrosis factor (TNF)‐α‐converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the proform of TNF‐α. In this study, we constructed expression vectors for the membrane‐bound full‐length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF‐α expression vector was introduced into human 293 cells, processing of TNF‐α to its mature form was enhanced by coexpressing mTACE, and this processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF‐α, although the culture medium of sTACE‐transfected cells could cleave a peptide containing the TNF‐α cleavage site. Fas ligand (FasL)‐transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF‐α indicated that both mTACE and sTACE could interact with the proform of TNF‐α. In the same assay, neither mTACE nor sTACE interacted with FasL. The catalytic domain‐lacking TACE mutant, which could also interact TNF‐α, showed a dominant negative effect on not only TNF‐α secretion but also FasL secretion. These results suggest that binding of the membrane‐anchored but not the soluble form of TACE to TNF‐α results in efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.
Transport of L‐carnitine in isolated cerebral cortex neurons
Authors: Wawrzeńczyk Agnieszka , Sacher Ayelet , Mac Magdalena , Nałecz Maciej J. , Nałecz Katarzyna A. 
Published: March 2003

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH‐group reagents (N‐ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4‐aminobutyrate (GABA) in a competitive way (Ki = 20.9 ± 2.4 mm). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1‐[2‐([(diphenylmethylene)amino]oxy)ethyl]‐1,2,5,6‐tetrahydro‐3‐pyridinecarboxylic acid hydrochloride (NO‐711) acid decreased carnitine accumulation while betaine, taurine and β‐alanine had no effect. The GABA transporters expressed inXenopus laevisoocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nałecz, D. Dymna, J. E. Mroczkowska, A. Broër, S. Broër, M. J. Nałecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.
The chondroitin sulfate chain of bikunin‐containing proteins in the inter‐α‐inhibitor family increases in size in inflammatory diseases
Authors: Mizon Charlotte , Mairie Christophe , Balduyck Malika , Hachulla Eric , Mizon Jacques 
Published: March 2003

Inter‐α‐inhibitor (IαI) and pre‐α‐inhibitor (PαI) are the main members of a set of multichain serine proteinase inhibitors. Present in human plasma, they may be involved in control of the inflammatory process. They are composed of homologous heavy chains (H1 and H2 for IαI; H3 for PαI) covalently linked by a protein–glycosaminoglycan–protein cross‐link to bikunin, which is a chondroitin 4‐sulfate proteoglycan. During the acute‐phase response, biosynthesis of IαI and PαI is downregulated and upregulated, respectively. In this work, we provide evidence that, in inflammatory diseases, the chondroitin sulfate chain of bikunin increases in size proportionally to the severity of the inflammatory response. As a consequence, all IαI‐related components that contain bikunin are structurally modified. Therefore, the changes in glycosylation of the acute‐phase proteins are not restricted to N‐linked glycans but also affect glycosaminoglycans. The implications of these findings are discussed with regard to biosynthesis and biological role, especially the anti‐inflammatory effects of IαI‐related proteinase inhibitors.
De novo identification of cell‐type specific antibody–antigen pairs by phage display subtraction
Authors: Stausbøl‐Grøn Brian , Jensen Kim Bak , Jensen Kristian Hobolt , Jensen Morten Østergaard , Clark Brian F. C. 
Published: March 2003

The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte‐specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly nondifferentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody–antigen pairs that characterize a specific cell type.
Monogamy as a Rule Rather than Exception in Nocturnal Lemurs: the Case of the Fat‐tailed Dwarf Lemur, Cheirogaleus medius
Authors: Fietz Joanna 
Published: March 2003

According to present hypotheses on the evolution of life history traits and social systems in Malagasy lemurs, nocturnality and infant parking are associated with a solitary lifestyle and a polygynous mating system. However, theoretically extreme seasonality of reproduction could limit the number of females that can be monopolized by a given male and thus hinder the evolution of polygyny. The aim of this ongoing study is to test these contrasting expectations by looking at the social and mating system of the fat‐tailed dwarf lemur Cheirogaleus medius. This species hibernates for up to 7 mo, so that time for breeding and raising offspring is extremely limited.A mark‐recapture study in western Madagascar was combined with observations and radio‐tracking of 36 individuals during the rainy seasons from 1995 to 1998. According to these data, fat‐tailed dwarf lemurs live in permanent sleeping groups consisting of a male and a female (n = 8) or one male and two females (n = 1). One or two offspring from the previous year were frequently observed to sleep together with an adult pair. Members of each sleeping group were the exclusive users of their nest holes and home ranges. During the birth season, males and females took turns at baby‐sitting their offspring. Females without paternal help were unable to raise their offspring successfully. Since females did not exhibit oestrus synchrony, the ultimate selective factor favouring pair‐living could be obligate paternal investment. The results, together with the lack of sexual size dimorphism and relatively small testis size, suggest that the fat‐tailed dwarf lemur lives in family groups with a monogamous mating system. A review of the mating systems of nocturnal lemurs shows that monogamy appears to be the rule rather the exception.
Feeding Tactics of Low‐ranking Red Deer Stags at Supplementary Feeding Sites
Authors: Schmidt Karoline T. , Hoi Herbert 
Published: March 2003

We tested the hypothesis that low‐ranking red deer Cervus elaphus stags adjust feeding tactics according to the spatio‐temporal availability of the food. We observed free‐ranging red deer at two supplementary feeding sites in the Scottish Highlands. It was predicted that at spatially clumped food, low‐ranking stags would avoid high‐ranking stags, 1. spatially when food is available only for a short period, but 2. temporally when the food is available continuously.We observed several feeding tactics in low‐ranking stags and the prevalence of a feeding tactic was related to the spatial distribution of the food. When food was highly clumped some low‐ranking stags rushed in between feeding high‐ranking stags, took a bite and retreated to process the bite (sneaking tactic). Bite rate/min did not differ between sneaking (0.29 ± 0.05) and non‐sneaking low‐ranking stags (0.26 ± 0.05). Compared with non‐sneaking low‐ranking herd members, sneaking stags took larger bites, as reflected in a significantly longer handling time (125 ± 12.4 s/bite) and thus achieved a higher intake, although still considerably less than high‐ranking stags.The temporal availability of the food did not influence feeding tactics in low‐ranking stags. Low‐ranking stags always avoided higher‐ranking herd members spatially but did not shift their feeding bouts to times of the day when high‐ranking stags were not feeding.
Exploratory Activity of Rats in Three Different Environments
Authors: Genaro Gelson , Schmidek Werner Robert 
Published: March 2003

AbstractAdult male hooded (Long‐Evans) rats (Rattus norvergicus) were used to compare the efficiency of three types of tests (plain open field – OF, open field with a refuge – OD, and complex environment with a refuge – EX), for the evaluation of exploratory behaviour. The results confirmed that OF is a highly aversive situation with the animals showing elevated emotional response (defecation and urination) when compared to situations containing a refuge (OD and EX). Presence of a plain arena (OF and OD) does not elicit high exploratory performance, as shown by the delayed and reduced exit from the refuge in OD when compared to EX. Thus locomotor activity in OF (covered distance, number of rearings) probably reflects more of an escape reaction than a genuine exploratory behaviour. Conversely the presence of a complex environment seems to elicit a high exploratory performance expressed as a quick exit from the refuge, an increased time in the environment (more than 75% of the observation time) and intense locomotor patterns (large covered distances, great number of rearings) but with little emotional display (reduced defecation and urination). The results thus show the importance of considering ethological factors in the choice or development of laboratory tests.
Estimating Behavioral Transition Rates: Problems and Solutions
Authors: Losey George , Ross David , Higa Jonathan 
Published: March 2003

Continuous time Markov chain (CTMC) models offer ethologists a powerful tool. The methods are based on well‐established procedures for estimating the rates at which one state (e.g. resting) changes to some other set of states (e.g. feeding, fighting, etc.). Unfortunately, ethological data typically differ in a very critical manner from the type of data to which these methods are usually applied: ethological data are usually heavily censored in the sense that each behavioral state shows frequent transitions to several other possible states. This occurs when several competing processes can each end a bout.We used computer simulation of various behavioral models with known transition rates to investigate the unknown performance of four of the most popular statistical tests for screening data prior to application of CTMC models; this included a modification of one of these tests derived under the assumption of random censoring. Two of the four tests failed completely and would result in rejection of nearly all data even if the model did fit the assumptions of the CTMC methods. Only Barlow's total‐time‐on test performed with an acceptable α error rate under all conditions. None of the tests were particularly effective at detecting certain types of departures from the CTMC assumptions.Guidelines are given as to how much confidence should be attached to apparent changes in transition rates.
Different Reproductive Tactics in House Sparrows Signalled by Badge Size: Is There a Benefit to Being Average?
Authors: Václav Radovan , Hoi Herbert 
Published: March 2003

AbstractSexual selection models usually predict directional selection for ornamental traits because of intra‐ as well as inter‐sexual selection. Animals frequently face reproductive trade‐offs, such as between mating and parental effort. Provided that both are essential and have opposite effects on ornament expression, we may however not necessarily expect directional selection for ornament size. The house sparrow is an ideal species to study such a trade‐off, as the size of the male ornament, the black throat badge, seems to be inversely related to mating and parental effort. It has been suggested that large‐badged males invest more in female attraction and territory defence, while small‐badged males may invest more in parental care. In a nest‐box study, we show that females started to breed earliest and produced the largest clutches when mated to males with average‐sized badges that invested in paternal care more than other males. These results are discussed in view of inter‐ as well intra‐sexual selection. Overall, average‐badged males experienced the highest hatching failures, their chicks were in the poorest physical condition and they did not fledge more chicks than other males. It is therefore unlikely that the mating advantages that we observed could by themselves lead to stabilizing selection for badge size. Our results rather suggest that badge size in male house sparrows signals different reproductive tactics, which are adapted flexibly according to their physical condition and socio‐ecological situations.
Vocalizations of Amazon River Dolphins, Inia geoffrensis: Insights into the Evolutionary Origins of Delphinid Whistles
Authors: Podos Jeffrey , Da Silva Vera M. F. , Rossi‐Santos Marcos R. 
Published: March 2003

AbstractOceanic dolphins (Odontoceti: Delphinidae) produce tonal whistles, the structure and function of which have been fairly well characterized. Less is known about the evolutionary origins of delphinid whistles, including basic information about vocal structure in sister taxa such as the Platanistidae river dolphins. Here we characterize vocalizations of the Amazon River dolphin (Inia geoffrensis), for which whistles have been reported but not well documented. We studied Inia at the Mamirauá Sustainable Development Reserve in central Brazilian Amazônia. During 480 5‐min blocks (over 5 weeks) we monitored and recorded vocalizations, noted group size and activity, and tallied frequencies of breathing and pre‐diving surfaces. Overall, Inia vocal output correlated positively with pre‐diving surfaces, suggesting that vocalizations are associated with feeding. Acoustic analyses revealed Inia vocalizations to be structurally distinct from typical delphinid whistles, including those of the delphinid Sotalia fluviatilis recorded at our field site. These data support the hypothesis that whistles are a recently derived vocalization unique to the Delphinidae.
Sexually and Developmentally Dimorphic Grooming: A Comparative Survey of the Ungulata
Authors: Mooring Michael S. , Reisig Dominic D. , Niemeyer Jason M. , Osborne Eric R. 
Published: March 2003

AbstractGrooming is effective in removing fitness‐compromising ectoparasites, such as ticks. Tick‐removal grooming is regulated both by a central control mechanism (programmed grooming model) and by cutaneous stimulation from tick bites (stimulus‐driven model). The programmed grooming model predicts that organismic factors that impact the cost‐benefit ratio of grooming (e.g., sex and body size) will influence the rate of grooming. The ‘vigilance principle’ predicts that breeding males of sexually dimorphic species will groom less than females to maintain high levels of vigilance for rival males and oestrus females. The intraspecific body size principle predicts that juveniles will engage in more frequent grooming than larger‐bodied adults to compensate for the higher cost of tick infestation for smaller animals. To examine the generalizability of these predictions, we surveyed the grooming rate of 53 species/subspecies of ungulates (primarily Bovidae and Cervidae) in tick‐free zoological parks in which stimulus‐driven grooming was controlled for. Matched‐pair analysis supported both predictions. Males delivered fewer oral grooming episodes per hour than females in 85% of sexually dimorphic species (n = 40), but only 56% of monomorphic species (n = 11) exhibited sexually dimorphic grooming, which was not different from random. Juvenile oral episodes per hour exceeded that of adult females in 74% of surveyed species (n = 36). As predicted by the body size principle, the grooming rate of juveniles declined as juveniles grew larger and less vulnerable to tick infestation. In separate analyses of Bovidae and Cervidae to control for common ancestry, both families supported sexually dimorphic grooming, but developmentally dimorphic grooming was supported only for Bovidae. Our results indicate that sexually dimorphic grooming is widespread in the ungulate species surveyed, suggesting that programmed grooming evolved at least as early as the common ancestor to the Artiodactyla.
Pup Shoving by Adult Naked Mole‐Rats
Authors: Stankowich Theodore , Sherman Paul W. 
Published: March 2003

AbstractAdult naked mole‐rats (Heterocephalus glaber) characteristically perform an unusual behavior toward young: they shove small pups frequently and vigorously around the nest. We studied 15 litters in five captive colonies to quantify which adults shove pups, changes in shoving frequencies as pups develop, how external disturbances affect pup‐shoving frequencies, and behavior of juveniles that were not shoved as pups. In all litters and colonies the breeding female shoved pups significantly more often than any other individual. Breeding females also shoved adult colony mates, but at far lower rates than they shoved pups. Breeding males shoved pups about half as often as did breeding females. Together, the parents shoved pups ten times more often than did nonbreeders. Frequencies of pup shoving peaked when pups were 3–4 wk old, roughly coincident with weaning. When colonies were disturbed experimentally, frequencies of pup shoving increased dramatically, whereas rates at which nonbreeding adults were shoved decreased sharply. We separated four newly‐weaned litters and raised half the pups apart from their colony. When these litters were reunited 4–9 wk later, the unshoved (experimental) pups were the same size as the frequently shoved (control) pups, but the unshoved pups were significantly less likely to flee from a disturbance. Shoving of small pups encourages them to flee from danger, and also may enforce weaning.