bluuebunny/crossref_metadata_2025 huggingface dataset:
Observations on fresh forage intake, ingesta particle size and nutrient digestibility in four species of macropod
Authors: Lentle R. G. , Hume I. D. , Stafford K. J. , Kennedy M. , Springett B. P. , Haslett S.
Published: February 2004

The particle-size distributions of the ingesta of the sacciform forestomach in free-ranging animals of a grazing macropod species [Macropus eugenii (tammar wallaby)], a grazer/browser [Macropus parma (parma wallaby)], a browser/grazer [Petrogale penicillata (brush-tailed rock-wallaby)] and a browser [Wallabia bicolor (swamp wallaby)] from Kawau Island, New Zealand, were compared with those of captive animals maintained on a standing ryegrass (Lolium perenne) sward. Nutrient digestibility was also measured in tammar and parma wallabies fed ryegrass or browse, i.e. fresh mahoe (Melicytus ramiflora) and this was related to particle-size distributions of the ingesta.There were significant differences in the particle size distributions of digesta from tammar and parma wallabies in the wild but not in captivity. In free-ranging animals the ingesta from both browsing species, the brush-tailed rock-wallaby and the parma wallaby, contained consistently greater proportions of coarse particles and smaller proportions of fine particles than did those of the tammar wallaby. These differences may be correlated with reported differences in their tooth morphologies. However, the presence of significant differences in particle-size distributions of the digesta between brush-tailed rock-wallabies and parma wallabies when constrained to grass, despite reported similarities in their tooth morphology, suggests that factors other than tooth morphology contribute to differences in the oral processing of food by browsing and grazing macropods. There were greater proportions of grass fragments in the coarse than in the finer fractions of ingesta from free-ranging brush-tailed rock-wallabies, indicating that this species is less effective at chewing grass.There were no overall differences between tammar and parma wallabies in the digestibilities of organic matter, neutral-detergent fibre (NDF) or acid-detergent fibre (ADF) but the NDF and ADF digestibilites of both species increased significantly with increase in the proportion of fine ingesta particles and with increase in mass of fermentative digesta.These findings indicate the importance of oral processing to digestive efficiency in macropods and the relationship between oral processing and tooth morphology.
Characterisation and cross-species utility of microsatellite markers within kangaroos, wallabies and rat kangaroos (Macropodoidea : Marsupialia)
Authors: Zenger K. R. , Eldridge M. D. B. , Pope L. C. , Cooper D. W.
Published: February 2004

The analysis of DNA using molecular techniques is an important tool for studies of evolutionary relationships, population genetics and genome organisation. The use of microsatellite genetic markers in marsupial studies is primarily limited by their availability and the success of amplification. Within this study, 29 macropodoid (kangaroos, wallabies and rat kangaroos) microsatellite loci were characterised in the tammar wallaby (Macropus eugenii) to evaluate the level of polymorphism and effects of cross-species amplification. Results indicated that 25 primer pairs amplified a single locus, with 21 exhibiting high levels of variability. The success of cross-species amplification was inversely proportional to the evolutionary distance between species. Therefore most macropodoid species can be studied using many of the available microsatellites, since source species are regularly distributed among macropodoid lineages. It was shown that M. eugenii had significantly (P < 0.01) reduced genetic diversity compared with source species. In addition, many microsatellite loci had reduced repeat arrays within M. eugenii, and all monomorphic loci sequenced had interruptions within the repeat arrays. The diversity differences are most likely caused by ascertainment bias in microsatellite selection for both length and purity. The results from this study highlight the need for caution when using genetic distance measures between divergent taxa, as the assumption of a specific mutation rate and/or type may be violated.
Lateralisation of magnetic compass orientation in silvereyes, Zosterops lateralis
Authors: Wiltschko Wolfgang , Munro Ursula , Ford Hugh , Wiltschko Roswitha
Published: February 2004

The ability of migratory silvereyes to orient was tested in the geomagnetic field with one eye covered. Silvereyes using only their right eye were able to orient in migratory direction just as well as birds using both eyes. Using only their left eye, however, the birds did not show a significant directional preference. These data indicate that directional information from the magnetic field is mediated almost exclusively by the right eye and processed by the left hemisphere of the brain. Together with corresponding findings from European robins and indications for a similar phenomenon in homing pigeons, they suggest that a strong lateralisation of the magnetic compass is widespread among birds.
Breeding biology of the red-capped robin
Authors: Dowling Damian K.
Published: February 2004

The breeding biology of the red-capped robin (Petroica goodenovii) (Petroicidae) was studied over two breeding seasons (2000–02) in Terrick Terrick National Park, Victoria. Breeding commenced in August and the last offspring fledged in January. Only females developed brood patches, built nests and incubated. However, both sexes fed the young. Clutch size ranged from one to three eggs, with a mean of 2.1, with clutches of three occurring relatively early in the season. For clutches of two, the period from laying of the first egg to hatching was 14 or 15 days, with a mean of 14.2. The time from hatching to fledging was 13–15 days, with a mean of 14. In all, 34% of nesting attempts successfully fledged offspring. On average, 0.62 fledglings were produced per nesting attempt, whilst 0.57 offspring reached independence. Nesting success peaked in October. Predation appears to be responsible for almost all nest failure, and predation rate varied over the season. Breeding pairs produced 0–3 broods per season (0–5 independent fledglings) and pairs that began nesting early in the season produced a greater number of independent offspring. Nestling weight was affected by both laying date and brood size. A comparative analysis within the Petroicidae, controlled for body size, revealed that species endemic to Australia have shorter incubation periods, and species from semi-arid and dry woodlands have longer incubation periods than other species. Findings from this study are discussed in relation to the breeding ecology of other members of the Petroicidae.
Mechanisms of heterospecific recognition in avian mobbing calls
Authors: Johnson Frances R. , McNaughton Elisabeth J. , Shelley Courtney D. , Blumstein Daniel T.
Published: February 2004

Some birds emit special calls, referred to as mobbing calls, when they detect a predator in their area. These calls are easily localisable and function to rally other individuals to help chase out a threatening intruder. Interestingly, individuals may respond to the mobbing calls of other sympatric species. To understand the mechanism underlying interspecific recognition it is essential to determine whether birds also respond to the mobbing calls of allopatric species. If, as has been suggested, learning is important for call recognition, then calls from allopatric species should not evoke mobbing. If, however, there are intrinsic (and possibly convergent) characteristics of mobbing calls, then novel calls from allopatric species should evoke mobbing. We conducted two playback experiments with apostlebirds (Struthidea cinera, Family Corcoracidae), Australian mud-nesters, to understand mechanisms underlying mobbing-call recognition. The first demonstrated that allopatric mobbing calls could elicit a response significantly greater than control stimuli, but less than that elicited by playback of conspecific calls. The second demonstrated that the dominant frequency was critical for eliciting mobbing, rather than the commonly assumed broad bandwidth. Taken together, these results suggest that experience with a particular species' call is not essential to elicit mobbing; rather, intrinsic aspects of the calls themselves may explain heterospecific recognition.
Seasonality and colony-size effects on the life-history characteristics of Rhytidoponera metallica in temperate south-eastern Australia
Authors: Thomas Melissa L.
Published: February 2004

In social insects, colony growth is assumed to follow a logistic growth curve, with small, newly founded colonies experiencing exponential growth (ergonomic stage) that slows down with increasing colony size and approaches an asymptote when the colony produces sexuals (reproductive stage). Environmental factors may also influence colony growth, particularly in temperate climates where colder temperatures in winter slow development. However, growth patterns are rarely studied in detail in social insects. In this study, I investigated colony size and seasonality effects on life-history parameters of the ponerine ant Rhytidoponera metallica. I followed the growth of 10 laboratory colonies monthly over two years in conjunction with monthly excavations of 5 field colonies. Colony composition was highly seasonal in both laboratory and field colonies, with pupae and larvae produced only during the warmer months. Males, however, were present in colonies throughout most of the year. An expected logistic growth pattern was found in the majority (4 of 6) of laboratory colonies that had positive growth, one colony followed a Gompertz growth pattern and another a power curve. Two laboratory colonies decreased in size and two colonies didn't change in size. The slowing of growth observed with increasing colony size in the majority of laboratory colonies was related to a decrease in per capita brood production with increasing colony size. Colony size also related to the presence of males: field colonies containing males were significantly larger than field colonies where males were absent. By using a combination of laboratory and field colonies, I was able to obtain information on seasonality of brood and male production, in addition to important demographic data on mortality and natality rates that is difficult to obtain in social insects using only field excavations.
Annual variation in arrival and departure times of carrion insects at carcasses: implications for succession studies in forensic entomology
Authors: Archer M. S.
Published: February 2004

A succession of insect species associate with decaying bodies, and because of the relatively predictable arrival and departure times of many species, this process is routinely used to estimate minimum post-mortem interval. Corpse fauna are compared with baseline data on succession rates, which are usually taken from decomposing animal carcasses. Baseline data are traditionally collected over a single year only; however, annual differences in weather patterns and insect population parameters probably alter succession rates. Surprisingly, this potential source of error has not previously been examined. Therefore, a two-year succession experiment was performed to investigate annual variation in succession rates. Sets of five still-born piglet carcasses were placed in a forest each season of both years. Six insects that occur year-round were selected: adults of Aleochara brachialis Jekel, Anotylus spp., Creophilus erythrocephalus Fabricius (Staphylinidae: Coleoptera), Ptomaphila lacrymosa Schreibers (Silphidae: Coleoptera), Pseudonemadus spp. (Leiodidae: Coleoptera), and larvae of Calliphora spp. (Calliphoridae: Diptera). Arrival and departure weeks to and from carcasses were measured in each season of both years. Two-way ANOVAs revealed significant season and year interactions on the arrival weeks of Anotylus spp., C. erythrocephalus, and Pseudonemadus spp., and significant differences in seasonal and yearly arrival times for A. brachialis, and P. lacrymosa. For larvae of Calliphora spp., there was a significant seasonal effect on arrival times. A significant interaction between season and year affected the departure weeks of all taxa. These results show that error in the estimates of minimum post-mortem interval can result if baseline succession data are collected only over one year.
A life course approach to diet, nutrition and the prevention of chronic diseases
Authors: Darnton-Hill I , Nishida C , James WPT
Published: February 2004

Objective:

To briefly review the current understanding of the aetiology and prevention of chronic diseases using a life course approach, demonstrating the life-long influences on the development of disease.

Design:

A computer search of the relevant literature was done using Medline-‘life cycle’ and ‘nutrition’ and reviewing the articles for relevance in addressing the above objective. Articles from references dated before 1990 were followed up separately. A subsequent search using Clio updated the search and extended it by using ‘life cycle’, ‘nutrition’ and ‘noncommunicable disease’ (NCD), and ‘life course’. Several published and unpublished WHO reports were key in developing the background and arguments.

Setting:

International and national public health and nutrition policy development in light of the global epidemic in chronic diseases, and the continuing nutrition, demographic and epidemiological transitions happening in an increasingly globalized world.

Results of review:

There is a global epidemic of increasing obesity, diabetes and other chronic NCDs, especially in developing and transitional economies, and in the less affluent within these, and in the developed countries. At the same time, there has been an increase in communities and households that have coincident under- and over-nutrition.

Conclusions:

The epidemic will continue to increase and is due to a lifetime of exposures and influences. Genetic predisposition plays an unspecified role, and with programming during fetal life for adult disease contributing to an unknown degree. A global rise in obesity levels is contributing to a particular epidemic of type 2 diabetes as well as other NCDs. Prevention will be the most cost-effective and feasible approach for many countries and should involve three mutually reinforcing strategies throughout life, starting in the antenatal period.
Biopsychology research in China
Authors: Lin Wenjuan , Sui Nan
Published: February 2004

The early studies on biopsychology in China were classified under the name of physiological psychology and comparative psychology. In 1979 the Division of Physiological Psychology of the Chinese Psychological Society was founded. Fifteen years later, the Brain‐Behavior Research Center was founded at the Institute of Psychology, Chinese Academy of Sciences. The objective of the Center was to establish a multidisciplinary scientific environment for conducting experimental research on the relationship between brain and behaviour as well as the interactions of the mind and body. A wide range of studies in biopsychology has been conducted in China. The most major research areas are: (1) Behavioural and physiological studies of stress: Research work includes the effect of early environment on stressful responses, interactions of behavioural and endocrine responses under stress, effects of emotional stress on immune function, stress and hypertension and the related role of interleukin‐1. (2) Conditioning and immunity: Studies focus on the effects of conditioning in the modulation of bidirectional immune function. Data from different experiments demonstrate that psychological processes are capable of influencing immune function. The neural substrates are also explored. (3) Memory and learning: Studies mostly concentrate on types of memory formation and training stimulus, effect of light exposure and corticosterone on learning and memory, and the role of the hippocampus in learning and memory. (4) Drug addiction: Work mainly focuses on long‐term aspects of addiction, including memory, novelty seeking, motivation‐related models, and the brain mechanisms underlying morphine psychological dependence.
A century of Chinese developmental psychology
Authors: Miao Xiaochun , Wang Wei
Published: February 2004

At the turn of last century, psychology, including developmental psychology, was introduced into China. Since then, developmental psychology has always been one of the main branches of Chinese psychology. This paper reviews the progress of Chinese developmental psychology in the last century, with special emphasis on its development during the recent 20 years. The paper consists of three sections. The first is a historical review. The 100 years' history of Chinese developmental psychology has been divided into three phases: first half of the 20th century; the period between 1950s and 1970s after the foundation of the People's Republic of China; and the period from the end of the 1970s up to the present. The authors briefly describe the initiation of Chinese developmental psychology, highlighting the research and related work in the field during the three phases. The second section of the paper introduces the research conducted by Chinese developmental psychologists during the last two decades in the following areas: cognitive development, language development, social development and socialization, the study of gifted children, cross‐cultural studies of child development, the study of only children, and the psychology of ageing. This section is the focus of the paper, because in this period, developmental psychology in China has progressed steadily on a sustained course and entered an unprecedented thriving period along with the State opening‐up policy and structural reformation. The last section is an appraisal of the past and prospects for the future. Here, the authors comment on the characteristics and achievements of Chinese developmental psychology in the 20th century and discuss future directions for its further development in terms of research scope and depth, research methods, indigenization, and application.
Heroin users in Australia: population trends
Authors: KAYA C. YALÇIN , TUGAI YULIYA , FILAR JERZY A. , AGRAWAL MANJU R. , ALI ROBERT L. , GOWING LINDA R. , COOKE RICHARD
Published: February 2004

The aim of this paper is to identify certain important population trends among heroin users in Australia for the period 1971 – 97, such as: population growth, initiation, i.e. the number who were initiated to heroin in a given year, and quitting, i.e. the number that quit using heroin. For this purpose, we summarize and extract relevant characteristics from data from National Drug Strategy Household Survey (NDSHS 1998) conducted in Australia in 1998. We devise a systematic procedure to estimate historical trends from questions concerning past events. It is observed from our findings that the size of the heroin user population in Australia is in a sharp increase, especially from the early 1980s onwards. The general trend obtained for the period 1971 – 97 is strikingly similar to that obtained by Hall et al. (2000) for the dependent heroin user population in Australia, even though their study was based on different datasets and a different methodology. In our reconstruction of the time history we also detect a levelling‐off prior to 1990. Initiation is also observed to be on a sharp increase. The latter trend is accompanied by a similar trend of quitting, perhaps indicating a relatively short heroin use career. A sharp decrease in both initiation and quitting is observed after 1990. In conclusion, in the case of the trend in the population of heroin users a high rate of growth has been identified that is consistent with the existing literature. In the process, we demonstrated that even a static survey such as NDSHS 1998 can, sometimes, be used to extract historical (dynamic) trends of certain important variables.
Improved side‐chain prediction accuracy using an ab initio potential energy function and a very large rotamer library
Authors: Peterson Ronald W. , Dutton P. Leslie , Wand A. Joshua
Published: February 2004

Accurate prediction of the placement and comformations of protein side chains given only the backbone trace has a wide range of uses in protein design, structure prediction, and functional analysis. Prediction has most often relied on discrete rotamer libraries so that rapid fitness of side‐chain rotamers can be assessed against some scoring function. Scoring functions are generally based on experimental parameters from small‐molecule studies or empirical parameters based on determined protein structures. Here, we describe the NCN algorithm for predicting the placement of side chains. A predominantly first‐principles approach was taken to develop the potential energy function incorporating van der Waals and electrostatics based on the OPLS parameters, and a hydrogen bonding term. The only empirical knowledge used is the frequency of rotameric states from the PDB. The rotamer library includes nearly 50,000 rotamers, and is the most extensive discrete library used to date. Although the computational time tends to be longer than most other algorithms, the overall accuracy exceeds all algorithms in the literature when placing rotamers on an accurate backbone trace. Considering only the most buried residues, 80% of the total residues tested, the placement accuracy reaches 92% for χ1, and 83% for χ1 + 2, and an overall RMS deviation of 1 Å. Additionally, we show that if information is available to restrict χ1 to one rotamer well, then this algorithm can generate structures with an average RMS deviation of 1.0 Å for all heavy side‐chains atoms and a corresponding overall χ1 + 2 accuracy of 85.0%.
Rapid evolution in conformational space: A study of loop regions in a ubiquitous GTP binding domain
Authors: Blouin Christian , Butt Davin , Roger Andrew James
Published: February 2004

The rapidly evolving subsets of a protein are often evident in multiple sequence alignments as poorly defined, gap‐containing regions. We investigated the 3D context of these regions observed in 28 protein structures containing a GTP‐binding domain assumed to be homologous to the transforming factor p21‐RAS. The phylogenetic depth of this data set is such that it is possible to observe lineages sharing a common protein core that diverged early in the eukaryotic cell history. The sequence variability among these homolog proteins is directly linked to the structural variability of surface loops. We demonstrate that these regions are self‐contained and thus mostly free of the evolutionary constraints imposed by the conserved core of the domain. These intraloop interactions have the property to create stem‐like structures. Interestingly, these stem‐like structures can be observed in loops of varying size, up to the size of small protein domains. We propose a model under which the diversity of protein topologies observed in these loops can be the product of a stochastic sampling of sequence and conformational space in a near‐neutral fashion, while the proximity of the functional features of the domain core allows novel beneficial traits to be fixed. Our comparative observations, limited here to the proteins containing the RAS‐like GTP‐binding domain, suggest that a stochastic process of insertion/deletion analogous to “budding” of loops is a likely mechanism of structural innovation. Such a framework could be experimentally exploited to investigate the folding of increasingly complex model inserts.
Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase
Authors: Yang Zhengrong W. , Tendian Susan W. , Carson W. Michael , Brouillette Wayne J. , Delucas Lawrence J. , Brouillette Christie G.
Published: February 2004

Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD+ synthetase led to this investigation of its potential effects on the structure and stability of this 60‐kD homodimeric enzyme. Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two‐state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, Ka, equal to 5.6 × 106/M. DSC curve analysis is consistent with this finding. The data are best fit to a three‐state sequential unfolding mechanism, most likely representing folded dimer ⇆ folded monomer ⇆ unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large Ka down to ∼106/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.
Oligomerization of hydrophobin SC3 in solution: From soluble state to self‐assembly
Authors: Wang Xiaoqin , Graveland‐Bikker Johanna F. , De Kruif Cornelis G. , Robillard George T.
Published: February 2004

Hydrophobin SC3 is a protein with special self‐association properties that differ depending on whether it is in solution, on an air/water interface or on a solid surface. Its self‐association on an air/water interface and solid surface have been extensively characterized. The current study focuses on its self‐association in water because this is the starting point for the other two association processes. Size‐exclusion chromatography was used to fractionate soluble‐state SC3. Real‐time multiangular light scattering detection of the eluate indicated that SC3 mainly exists as a dimer in buffer, accompanied with a small amount of monomer, tetramer, and larger aggregates. Dimeric SC3 has very likely an elongated shape, as indicated by the hydrodynamic radius determined by using dynamic light scattering (DLS) and fluorescence anisotropy measurements on dansyl‐labeled SC3. Size‐exclusion chromatography experiments also indicated that the protein oligomerizes very slowly at low temperature (4°C) but rather rapidly at room temperature. Ionic strength plays an important role in the oligomerization; a short‐lived monomeric SC3 species could be observed in pure water. Oligomerization was not affected by low pH but was accelerated by high pH. Fluorescence resonance energy transfer showed that dissociation occurred when the protein concentration was lowered; a large population of oligomers, presumably dimers, dissociate when the protein concentration is <4.5 μg/mL. This value is similar to the critical concentration for SC3 self‐assembly. Therefore, dimeric SC3 is indicated to be the building block for both aggregation in solution and self‐assembly at hydrophobic/hydrophilic interfaces.
Analysis and prediction of affinity of TAP binding peptides using cascade SVM
Authors: Bhasin Manoj , Raghava G.P.S.
Published: February 2004

The generation of cytotoxic T lymphocyte (CTL) epitopes from an antigenic sequence involves number of intracellular processes, including production of peptide fragments by proteasome and transport of peptides to endoplasmic reticulum through transporter associated with antigen processing (TAP). In this study, 409 peptides that bind to human TAP transporter with varying affinity were analyzed to explore the selectivity and specificity of TAP transporter. The abundance of each amino acid from P1 to P9 positions in high‐, intermediate‐, and low‐affinity TAP binders were examined. The rules for predicting TAP binding regions in an antigenic sequence were derived from the above analysis. The quantitative matrix was generated on the basis of contribution of each position and residue in binding affinity. The correlation of r = 0.65 was obtained between experimentally determined and predicted binding affinity by using a quantitative matrix. Further a support vector machine (SVM)‐based method has been developed to model the TAP binding affinity of peptides. The correlation (r = 0.80) was obtained between the predicted and experimental measured values by using sequence‐based SVM. The reliability of prediction was further improved by cascade SVM that uses features of amino acids along with sequence. An extremely good correlation (r = 0.88) was obtained between measured and predicted values, when the cascade SVM‐based method was evaluated through jackknife testing. A Web service, TAPPred (http://www.imtech.res.in/raghava/tappred/ or http://bioinformatics.uams.edu/mirror/tappred/), has been developed based on this approach.
Cooperativity in two‐state protein folding kinetics
Authors: Weikl Thomas R. , Palassini Matteo , Dill Ken A.
Published: February 2004

We present a solvable model that predicts the folding kinetics of two‐state proteins from their native structures. The model is based on conditional chain entropies. It assumes that folding processes are dominated by small‐loop closure events that can be inferred from native structures. For CI2, the src SH3 domain, TNfn3, and protein L, the model reproduces two‐state kinetics, and it predicts well the average Φ‐values for secondary structures. The barrier to folding is the formation of predominantly local structures such as helices and hairpins, which are needed to bring nonlocal pairs of amino acids into contact.
Lead optimization of antifungal peptides with 3D NMR structures analysis
Authors: Landon Céline , Barbault Florent , Legrain Michèle , Menin Laure , Guenneugues Marc , Schott Valérie , Vovelle Françoise , Dimarcq Jean‐Luc
Published: February 2004

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti‐infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystein‐rich antimicrobial peptides with the CSαβ scaffold (an α‐helix linked to a β‐sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CSαβ peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD‐135, and ETD‐151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD‐135 and ETD‐151 were engineered by site‐directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD‐135 and ETD‐151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.
Laser light‐scattering evidence for an altered association of βB1‐crystallin deamidated in the connecting peptide
Authors: Harms Michael J. , Wilmarth Philip A. , Kapfer Deborah M. , Steel Eric A. , David Larry L. , Bächinger Hans Peter , Lampi Kirsten J.
Published: February 2004

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in βB1‐crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble βB1 contained 13%–17% deamidation at Gln146 and Asn157. Static and quasi‐elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild‐type (wt) βB1 and the deamidated βB1 mutants, Q146E and N157D. Dimer formation occurred for wt βB1, Q146E, and N157D in a concentration‐dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of βB1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.
The role of phosphagen specificity loops in arginine kinase
Authors: Azzi Arezki , Clark Shawn A. , Ellington W. Ross , Chapman Michael S.
Published: February 2004

Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock‐and‐key mechanism. The second loop contacts the substrate with a valine in the methyl‐substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.
The PRESAT‐vector: Asymmetric T‐vector for high‐throughput screening of soluble protein domains for structural proteomics
Authors: Goda Natsuko , Tenno Takeshi , Takasu Hirotoshi , Hiroaki Hidekazu , Shirakawa Masahiro
Published: February 2004

A rapid unidirectional method for cloning PCR‐amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT‐vector cloning, is based on a T‐vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5′‐sequence of the “rear” PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.
Defining the minimum size of a hydrophobic cluster in two‐stranded α‐helical coiled‐coils: Effects on protein stability
Authors: Lu Stephen M. , Hodges Robert S.
Published: February 2004

The α‐helical coiled‐coil motif is characterized by a heptad repeat pattern (abcdefg)n in which residues a and d form the hydrophobic core. Long coiled‐coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two‐stranded, disulfide‐bridged parallel α‐helical coiled‐coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as •) was investigated. Alanine residues (denoted as ○) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions (○○○) was among the least stable coiled‐coil (Tm = 39.3°C and Urea1/2 = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette (•○•), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette (••○)(○••) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes (•○•). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette (•••) resulted in a further synergistic gain in stability of 1.7 kcal/mole (Tm = 54.1°C and Urea1/2 = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled‐coils is extremely context dependent and clustering is an important aspect of protein folding and stability.
Comparison of BRCT domains of BRCA1 and 53BP1: A biophysical analysis
Authors: Ekblad Caroline M.S. , Friedler Assaf , Veprintsev Dmitry , Weinberg Richard L. , Itzhaki Laura S.
Published: February 2004

53BP1 interacts with the DNA‐binding core domain of the tumor suppressor p53 and enhances p53‐mediated transcriptional activation. The p53‐binding region of 53BP1 maps to the C‐terminal BRCT domains, which are homologous to those found in the breast cancer protein BRCA1 and in other proteins involved in DNA repair. Here we compare the thermodynamic behavior of the BRCT domains of 53BP1 and BRCA1 and examine their ability to interact with the p53 core domain. The free energies of unfolding are of similar magnitude, although slightly higher for 53BP1‐BRCT, and both populate an aggregation‐prone partly folded intermediate. Interaction studies performed in vitro by analytical size‐exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry reveal that 53BP1‐BRCT interacts with p53 with a Kd in the low micromolar range. Despite their homology with 53BP1‐BRCT domains, the BRCT domains of BRCA1 did not bind p53 with any detectable affinity. In summary, although other studies have indicated that the BRCT domains of both BRCA1 and 53BP1 interact with p53 core domain, the quantitative biophysical measurements performed here indicate that only 53BP1 can bind. Although both proteins may be involved in the same DNA repair pathways, our study indicates that a direct role in p53 function is unique to 53BP1.
Areas of Interictal Spiking Are Associated with Metabolic Dysfunction in MRI‐negative Temporal Lobe Epilepsy
Authors: Shih Jerry J. , Weisend Michael P. , Lewine Jeffrey , Sanders John , Dermon Jamie , Lee Roland
Published: February 2004

Summary:  Purpose: The objective of our study was to determine noninvasively whether metabolic dysfunction is present in focal areas of interictal electrophysiologic abnormality and whether metabolic dysfunction correlates with frequency of spiking.

Methods: We used a prospective, power analysis–driven, age‐matched design to study 20 subjects with nonlesional temporal lobe epilepsy by using magnetoencephalography (MEG) and proton magnetic resonance spectroscopy (1H‐MRS). MEG was used to localize the source area of interictal spikes. 1H‐MRS measured integrated peak areas for N‐acetyl compounds (NAA) and choline‐containing compounds (Cho) in both hippocampi, the MEG spike zone, and the region contralateral to the MEG spike zone in all subjects. 1H‐MRS was performed in seven controls.

Results: Fifteen of 20 subjects had a lower NAA/Cho ratio in the MEG spike zone compared with the contralateral homologous region. NAA/Cho was significantly decreased in the MEG spike zone (p < 0.01). NAA/Cho ratios were not significantly different between the hippocampus ipsilateral and contralateral to the spike activity, or from control hippocampi. NAA/Cho ratios did not correlate with spike frequency.

Conclusions: Metabolic dysfunction is present in focal areas of interictal spiking in nonlesional temporal lobe epilepsy. These findings confirm that functional abnormalities can be detected in vivo in radiographically normal‐appearing cortex exhibiting abnormal excitability.
The Cognitive Outcome of Hemispherectomy in 71 Children
Authors: Pulsifer Margaret B. , Brandt Jason , Salorio Cynthia F. , Vining Eileen P. G. , Carson Benjamin S. , Freeman John M.
Published: February 2004

Summary: Purpose:Long‐term neuropsychological outcome was studied in 71 patients who underwent hemispherectomy for severe and intractable seizures at The Johns Hopkins Hospital between 1968 and 1997 and who agreed to participate. Seizures were due to cortical dysplasias (n = 27), Rasmussen syndrome (n = 37), or vascular malformations or strokes (n = 7). Both presurgical and follow‐up results are available and reported for 53 patients.

Methods:Patients and caretakers were interviewed, and patients were administered standard measures of intelligence, receptive and expressive language, visual‐motor skills, adaptive/developmental functioning, and behavior.

Results:Mean age at surgery was 7.2 years. At follow‐up, on average 5.4 years after surgery, 65% are seizure free, 49% are medication free, and, of those responding, none rated quality of life as worse than before surgery. Mean IQ was in the 70s for Rasmussen and vascular patients and in the 30s for cortical dysplasia patients. Language and visual‐motor skills were consistent with IQ. For Rasmussen patients only, language was significantly more impaired for left than for right hemispherectomy, both before surgery and at follow‐up. Adaptive skills were mildly impaired, with greatest impairment in the physical domain. Cognitive measures typically changed little between surgery and follow‐up, with IQ change <15 points for 34 of 53 patients; of the remainder, 11 declined and eight improved. Behavior was free of major problems, but social interactions and activities were limited.

Conclusions:The most significant predictor of cognitive skills at follow‐up was etiology, with dysplasia patients scoring lowest in intelligence and language but not in visual‐motor skills. Regardless of etiology, most patients showed only moderate change in cognitive performance at follow‐up.
Thyroid Function in Girls with Epilepsy with Carbamazepine, Oxcarbazepine, or Valproate Monotherapy and after Withdrawal of Medication
Authors: Vainionpää Leena K. , Mikkonen Kirsi , Rättyä Johanna , Knip Mikael , Pakarinen Arto J. , Myllylä Vilho V. , Isojärvi Jouko I.T.
Published: February 2004

Summary:  Purpose: Antiepileptic drugs may affect the serum thyroid hormone concentrations. The aim of this study was to evaluate thyroid function in 78 girls taking carbamazepine (CBZ), oxcarbazepine (OXC), or valproate (VPA) monotherapy for epilepsy and after withdrawal of the treatment.

Methods: Forty‐one girls taking VPA, 19 taking CBZ, and 18 taking OXC for epilepsy, as well as 54 healthy age‐matched controls, aged 8 to 18 years, participated in the study. All the girls were examined clinically, and their pubertal stage was assessed. Blood samples were obtained for thyroid hormone and antibody assays. These examinations were repeated after a mean follow‐up of 5.8 years to assess thyroid function, and 64 (82%) of 78 patients and 42 (78%) of 54 controls agreed to participate in the second evaluation.

Results: In the first evaluation, the mean serum thyroid hormone concentrations were lower in the girls taking CBZ [thyroxine (T4), 70.2; SD, 10.9 nM; and free thyroxine (FT4), 11.5; SD, 1.8 pM] or OXC (T4, 74.9; SD, 16.4 nM; and FT4, 11.3; SD, 1.8 pM) than in the control girls (T4, 96.6; SD, 15.1 nM, and FT4, 14.4; SD, 1.5 pM; p < 0.001, all comparisons). However, thyrotropin (TSH) concentrations were normal in the girls taking CBZ or OXC. Sixty‐three% of the girls taking CBZ and 67% of the girls taking OXC had serum T4 and/or FT4 levels below the lower limit of the reference range. The VPA‐treated girls with epilepsy had normal serum T4 and FT4 concentrations, but slightly increased TSH levels (3.3; SD, 1.5 mU/L; p < 0.01) compared with the control girls (2.5; SD, 1.0 mU/L). Normal serum hormone concentrations were restored in the patients who discontinued the medication.

Conclusions: Both CBZ and OXC reduce serum thyroid hormone concentrations in girls with epilepsy. Conversely, VPA is associated with normal serum thyroid hormone and increased thyrotropin levels. However, our results suggest that the changes in serum thyroid hormone and thyrotropin levels are reversible after withdrawal of the medication.
Seizure Semiology in the Elderly: A Video Analysis
Authors: Kellinghaus Christoph , Loddenkemper Tobias , Dinner Dudley S. , Lachhwani Deepak , O. Lüders Hans
Published: February 2004

Summary:  Purpose: To describe the seizure semiology of patients older than 60 years and to compare it with that of a control group of younger adults matched according to the epilepsy diagnosis.

Methods: Available videotapes of all patients aged 60 years and older who underwent long‐term video‐EEG evaluation at the Cleveland Clinic Foundation (CCF) between January 1994 and March 2002 were analyzed by two observers blinded to the clinical data. A younger adult control group was matched according to the epilepsy diagnosis, and their seizures also were analyzed.

Results: Fifty‐four (3.3%) of the 1,633 patients were 60 years or older at the time of admission. For 21 of them, at least one epileptic seizure was recorded. Nineteen patients had focal epilepsy (nine temporal lobe, two frontal lobe, two parietal lobe, eight nonlocalized), and two patients had generalized epilepsy. Seventy‐three seizures of the elderly patients and 85 seizures of the 21 control patients were analyzed. In nine elderly patients and 14 control patients, at least one of their seizures started with an aura. Eleven elderly patients and 19 control patients lost responsiveness during their seizures. Approximately two thirds of the patients in both groups had automatisms during the seizures. Both focal and generalized motor seizures (e.g., clonic or tonic seizures) were seen less frequently in the elderly.

Conclusions: Only a small percentage of the patients admitted to a tertiary epilepsy referral center for long‐term video‐EEG monitoring are older than 60 years. All seizure types observed in the elderly also were seen in the younger control group, and vice versa. Simple motor seizures were seen less frequently in the elderly.