Cytochrome c maturation: a complex pathway for a simple task?

Post-translational maturation of c-type cytochromes involves covalent attachment of haem to the apocytochrome polypeptide by two thioether bonds. In bacteria, haem attachment occurs in the periplasm, after the separate translocation of haem and the polypeptide across the cytoplasmic membrane. In Escherichia coli, delivery and attachment of the cofactor requires eight or nine specific proteins, which are believed to be organized in a membrane protein complex. After transport across the membrane, haem is attached covalently to the haem chaperone CcmE in an unusual way at a single histidine residue. However, haem binding to CcmE is transient and is succeeded by a further transfer to apocytochrome c. Both haem binding to and release from CcmE involve integral membrane proteins, CcmC and CcmF respectively, which carry a conserved tryptophan-rich motif in a periplasmic domain. Apocytochrome c polypeptides are synthesized as precursors and reach the periplasm by sec-dependent translocation. There they are prepared for haem binding by reduction of the cysteine residues in the motif Cys-Xaa-Xaa-Cys-His, which is characteristic of such proteins. This reduction is achieved in a thio-reduction pathway, whereby electrons are passed from cytoplasmic thioredoxin to the transmembrane protein DsbD, across the membrane, and on to the specific reductases CcmG/CcmH. The merging of the haem delivery and the thio-reduction pathways leads to the stereospecific insertion of haem into various type c cytochromes.     

Are we ready for a natural history of motor learning?

Here we argue that general principles with regard to the contributions of the cerebellum, basal ganglia, and primary motor cortex to motor learning can begin to be inferred from explicit comparison across model systems and consideration of phylogeny. Both the cerebellum and the basal ganglia have highly conserved circuit architecture in vertebrates. The cerebellum has consistently been shown to be necessary for adaptation of eye and limb movements. The precise contribution of the basal ganglia to motor learning remains unclear but one consistent finding is that they are necessary for early acquisition of novel sequential actions. The primary motor cortex allows independent control of joints and construction of new movement synergies. We suggest that this capacity of the motor cortex implies that it is a necessary locus for motor skill learning, which we argue is the ability to execute selected actions with increasing speed and precision.     

Phase-contrast versus off-axis illumination: is a more complex microscope always more powerful?

In this article, a practical demonstration suitable for any biology college classroom is presented. With the examples of a complex biological specimen (slug's radula) and a simple reference specimen (electron microscopical grid imprint in gelatin), both of which can be easily prepared, the capabilities of two imaging modes commonly used in optical microscopy are demonstrated. The results obtained under phase contrast (a rather sophisticated method, 1953 Nobel Prize to Zernike) and off-axis illumination (a very simple method) are compared. The off-axis illumination setup is capable of delivering noticeably better microscopic images of these two particular specimens, yet it can be easily assembled in a laboratory classroom. The outcome of such a demonstration is expected to be the realization on the part of the students that one needs to carefully choose the apparatus to address a given biological problem, with the "bottom line" being that a more complex one may not necessarily yield better results. An attempt to explain this "paradox" is presented, in the particular case presented here, partly from the physiology of vision perspective (the shape-from-shading problem). The overall aim of the present article is to induce in students critical thinking about the capabilities of a laboratory equipment in general and about data interpretation.     

Role of motor cortex in coordinating multi-joint movements: is it time for a new paradigm?

Reaching movements to spatial targets require motor patterns at the shoulder to be coordinated carefully with those at the elbow to smoothly move the hand through space. While the motor cortex is involved in this volitional task, considerable debate remains about how this cortical region participates in planning and controlling movement. This article reviews two opposing interpretations of motor cortical function during multi-joint movements. On the one hand, studies performed predominantly on single-joint movement generally support the notion that motor cortical activity is intimately involved in generating motor patterns at a given joint. In contrast, studies on reaching demonstrate correlations between motor cortical activity and features of movement related to the hand, suggesting that the motor cortex may be involved in more global features of the task. Although this latter paradigm involves a multi-joint motor task in which neural activity is correlated with features of movement related to the hand, this neural activity is also correlated to other movement variables. Therefore it is difficult to assess if and how the motor cortex contributes to the coordination of motor patterns at different joints. In particular, present paradigms cannot assess whether motor cortical activity contributes to the control of one joint or multiple joints during whole-arm tasks. The final point discussed in this article is the development of a new experimental device (KINARM) that can both monitor and manipulate the mechanics of the shoulder and elbow independently during multi-joint motor tasks. It is hoped that this new device will provide a new approach for examining how the motor cortex is involved in motor coordination.     

Time versus size: which characteristic of a neural response carries more information?

 Considering a variety of quite different candidate neural codes, Cheng and Wasserman [(1996) Biol Cybern 75, 93–103, 105–115] reported data which suggested that amplitude codes transmitted information more faithfully than temporal codes. Reanalyzing their data, the present study measured how well size and time represented information resident within the same neural response feature, namely the response peak. Responses from photoreceptor cells and optic nerve fibers in the peripheral visual system of Limulus polyphemus were therefore re-examined using signal detectability values provided by receiver-operating-characteristic analyses to compare how well the timing and the amplitude of the response peak represent information within a single cell and how well they transmit information between cells. Data were available for several cells in several different light-adaptation states onto which several different test-flash intensities had been superimposed. The present reanalysis of these data replicated the results yielded earlier by the peak candidate code, and compared them with the data produced by two different measurements of the timing of the same response peak. A relative-timing code was derived from measurements of the time that elapsed between the moment when the response exceeded a criterion potential and when it reached its peak. An absolute-timing code was derived from the time that elapsed between test flash onset and the peak. The results clearly indicated that the peak code represented information within cells better than either of the two timing codes. However, both peak and absolute-timing codes clearly transmitted the available information between cells more faithfully than the relative-timing code. These data lead to two conclusions. First, that the same response feature, when measured in different ways, can produce remarkably different information processing outcomes. In this particular case, time represented information resident within a cell less well than did size. Second, that the fidelity of information transmission between nerve cells may be relatively independent of the quantity of information resident within any particular cell. Received: 5 November 2001 / Accepted in revised form: 8 July 2002 Acknowledgements. We are deeply indebted to Huiqi Yin for her technical assistance with all the calculations and the graphics. We also sincerely thank Tony Hosking, who wrote the computer programs that extracted the code data for this project, and Brendan Marr, who assisted with data analysis. Correspondence to: G.S. Wasserman (e-mail: codelab@purdue.edu, Fax: +1-765-4961264)     

Enhancement of performances in a learning task in suckler calves after weaning and relocation: Motivational versus cognitive control?: A pilot study

Weaning in suckler calves influences performance in a learning task. The aim of the present study was to investigate whether the improved performance after weaning, including relocation, is due to differences in motivation for the reward or in learning abilities. Forty Aubrac calves were used; half of them were weaned from their dams at around eight months, the other half were weaned one month later. After weaning, calves were housed in groups of four in a new setting. From the day after weaning of the last group of calves, the animals were subjected to two tests: (1) an arena test, (2) a T-maze test where one arm led to either a social or a food reward. The T-maze test consisted of three sessions: in Session 1, trials were conducted until the animal acquired the task (i.e. did not take the unrewarded arm on three consecutive trials); in Session 2, the motivation for the reward was assessed via the walking time of the animal to reach the reward; in Session 3, the place of reward was reversed and the animals were trained until they acquired the new task.

Calves weaned for one day explored more (P < 0.05) and had lower heart rates during the arena test (P < 0.05) compared to the ones weaned for one month. During the T-maze test, calves weaned for one month versus one day did not differ in their capacities to learn the initial route (Session 1) or in their motivation for either the social or food reward (Session 2). Calves weaned for one day learned significantly faster (P < 0.05) the reversed route (Session 3) than calves weaned for one month. Hence, the better performances at reversal in the T-maze by calves that have just been weaned cannot be accounted for by a higher motivation for the reward. A better cognitive control of their behaviour due to a lower stress state is suggested by our results.     

Plant preference for ammonium versus nitrate: a neglected determinant of ecosystem functioning?

Although nitrogen (N) availability is a major determinant of ecosystem properties, little is known about the ecological importance of plants' preference for ammonium versus nitrate (β) for ecosystem functioning and the structure of communities. We modeled this preference for two contrasting ecosystems and showed that β significantly affects ecosystem properties such as biomass, productivity, and N losses. A particular intermediate value of β maximizes the primary productivity and minimizes mineral N losses. In addition, contrasting β values between two plant types allow their coexistence, and the ability of one type to control nitrification modifies the patterns of coexistence with the other. We also show that species replacement dynamics do not lead to the minimization of the total mineral N pool nor the maximization of plant productivity, and consequently do not respect Tilman's R* rule. Our results strongly suggest in the two contrasted ecosystems that β has important consequences for ecosystem functioning and plant community structure.     

Active formation of mixed-species grouse leks: a role for predation in lek evolution?

Behavioural ecologists have interpreted avian leks as products of sexual selection, in which males display socially to increase their opportunities to mate. However, without invoking reproductive queuing or kin selection, this paradigm does not necessarily explain why many males that fail to mate participate in leks. An alternative solution, that males also aggregate to reduce predation, has previously lacked compelling support. We show that mixed-species leks, comprising two congeneric grouse, form when single males or small groups of one species, the greater prairie chicken Tympanuchus cupido, join leks of another, the sharp-tailed grouse T. phasianellus. We documented the process by observing lek dynamics and comparing group sizes between mixed- and single-species leks. Joining implies that prairie chickens benefit from displaying with sharp-tailed grouse. The numbers of females of each species attending a lek increased with the number of conspecific, but not heterospecific, males. This suggests that the joining of heterospecifics is unlikely to increase mating opportunities, and leaves lowered predation risk as the most likely benefit of associating with heterospecifics. Active formation of mixed-species leks therefore suggests that predation may be sufficient to drive lek formation. The benefits of participation in mixed leks may be asymmetrical because prairie chickens display more and are less vigilant than sharp-tailed grouse.     

The chemistry of copper binding to PrP: is there sufficient evidence to elucidate a role for copper in protein function?

There has been an enormous body of literature published in the last 10 years concerning copper and PrP (prion protein). Despite this, there is still no generally accepted role for copper in the function of PrP or any real consensus as to how and to what affinity copper associates with the protein. The present review attempts to look at all the evidence for the chemistry, co-ordination and affinity of copper binding to PrP, and then looks at what effect this has on the protein. We then connect this evidence with possible roles for PrP when bound to copper. No clear conclusions can be made from the available data, but it is clear from the present review what aspects of copper association with PrP need to be re-investigated.     

Effects of temperature on maximum clinging ability in a diurnal gecko: evidence for a passive clinging mechanism?

The thermal dependence of performance of ectotherms, and particularly locomotor performance in lizards, has received much attention. However, only a single study has examined the effects of temperature on adhesive clinging ability in geckos, despite the importance of adhesion for many pad-bearing lizards and invertebrates. We set out to characterize the thermal response of clinging ability in the diurnal gecko, Phelsuma dubia in the temperature range of 15-35 degrees C. Our findings indicate that there is no significant trend in clinging ability for P. dubia with temperature and that there is high variation about the mean at all temperatures. These findings differ from other whole-organism studies of clinging performance and are suggestive of a passive clinging mechanism that is dominated by intermolecular van der Waals forces. These findings also suggest that clinging ability in this species is not under selective pressures resulting from thermal variation, and that P. dubia does not need to regulate body temperature closely to maximize clinging ability.     

Sequence-specific binding of single-stranded RNA: is there a code for recognition?

A code predicting the RNA sequence that will be bound by a certain protein based on its amino acid sequence or its structure would provide a useful tool for the design of RNA binders with desired sequence-specificity. Such de novo designed RNA binders could be of extraordinary use in both medical and basic research applications. Furthermore, a code could help to predict the cellular functions of RNA-binding proteins that have not yet been extensively studied. A comparative analysis of Pumilio homology domains, zinc-containing RNA binders, hnRNP K homology domains and RNA recognition motifs is performed in this review. Based on this, a set of binding rules is proposed that hints towards a code for RNA recognition by these domains. Furthermore, we discuss the intermolecular interactions that are important for RNA binding and summarize their importance in providing affinity and specificity.     

Utilization of hybrid oak hosts by a monophagous gall wasp: How little host character is sufficient?

We examined the utilization by the cynipid gall former, Andricus californicus, of oak trees ranging across a hybrid continuum from pure Quercus dumosa to pure Q. engelmannii. Two components of utilization were considered, the proportion of trees parasitized and the density of galls on parasitized trees. Although the wasp normally only parasitizes Q. dumosa, galls were found on trees with up to 85% of the genetic character of the non-host, as determined by a hybrid index. The proportion of hybrids utilized did not differ statistically from the proportion of pure Q. dumosa utilized. We consider competing hypotheses about the genetic mechanism behind host choice in the light of our results. The model that is most consistent with the observed incidence pattern is based on a cluster concept: A number of independent genetic factors in the host determine its acceptability, however, these factors are substitutable, such that no single factor is critical, but some threshold number of factors must be exceeded.     

The Q-cycle reviewed: How well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc₁ complex is reviewed. The data strongly support a mechanism in which the Q_o-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q_o-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c₁ and liberate the H⁺. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O₂ is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b_L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b_L to enhance the rate constant. The acceptor reactions at the Q_i-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b₁₅₀ phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc₁ complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b_L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.     

Review of the genotoxicity and carcinogenicity of antischistosomal drugs; is there a case for a study of mutation epidemiology? Report of a task group on mutagenic antischistosomals

One of the interests of ICPEMC is to identify situations in which the possible induction of inherited defects in man by mutagen exposure could actually be studied. The large-scale use of mutagenic drugs in field programmes against schistosomiasis, mainly during the 1970's, was considered a possible case. An ICPEMC task group approached the problem by (1) updating the genetic toxicology data base for antischistosomal drugs, and (2) reviewing possible study areas. Expertise was combined from genetic toxicology, mutation epidemiology and tropical medicine.It was considered that: (a) if any, hycanthone would be the most appropriate candidate drug for study; (b) it would be virtually impossible to meet the basic requirements of an appropriate mutation epidemiology study, in endemic countries; (c) as more defined genetic endpoints would be selected (e.g. sentinel phenotypes) the required large sample sizes would seem prohibitive, since documentation on past programmes is limited and local demography would render the reliable tracking of substantial numbers of offspring of treated persons an almost impossible task; (d) in most endemic countries proper diagnosis and registration of inherited defects is largely lacking; (e) the problems encountered in demonstrating inherited effects in humans after heavy or chronic exposure to established animal mutagens such as ionizing radiation and cancer chemotherapy, in combination with the ambiguous nature of the animal germ cell data with hycanthone, do not particularly warrant large expectations; (f) since non-mutagenic antischistosomal drugs are now in use, the problem is academic and of low priority in the endemic countries whose medical and research resources are often limited. Thus, studying offspring of hycanthone-treated people to demonstrate the mutagenic potential of the drug in man is not a viable enterprise. (See, however, note added in proof, p. 75.)     

Activation of development in mammals: is there a role for a sperm cytosolic factor?

In mammalian oocytes, fertilization-associated calcium [Ca2+]i oscillations are responsible for the activation of development. The mechanism(s) by which the sperm triggers the initial [Ca2+]i rise and supports long-lasting oscillations is not resolved. It has been proposed that the sperm may interact with receptors in the oocyte's plasma membrane and engage intracellular signaling pathways that result in Ca2+ release. A different line of investigation suggests that upon sperm-oocyte fusion, a sperm cytosolic factor is released into the oocyte which interacts with unknown cytosolic targets, and generates [Ca2+]i oscillations. We will discuss the most recent evidence for both lines of thought and demonstrate that injections of sperm crude extracts (SF) into mammalian oocytes trigger [Ca2+]i oscillations that support in vitro parthenogenetic development to the blastocyst stage.     

Abnormal motor patterns in the framework of the equilibrium-point hypothesis: a cause for dystonic movements?

Until now, the equilibrium-point hypothesis (λ model) of motor control has assumed nonintersecting force-length characteristics of the tonic stretch reflex for individual muscles. Limited data from animal experiments suggest, however, that such intersections may occur. We have assumed the possibility of intersection of the characteristics of the tonic stretch reflex and performed a computer simulation of movement trajectories and electromyographic patterns. The simulation has demonstrated, in particular, that a transient change in the slope of the characteristic of an agonist muscle may lead to temporary movement reversals, hesitations, oscillations, and multiple electromyographic bursts that are typical of movements of patients with dystonia. The movement patterns of three patients with idiopathic dystonia during attempts at fast single-joint movements (in the elbow, wrist, and ankle) were recorded and compared with the results of the computer simulation. This approach considers that motor disorders in dystonia result from faulty control patterns that may not correlate with any morphological or neurophysiological changes. It provides a basis for the high variability of dystonic movements. The uniqueness of abnormal motor patterns in dystonia, that precludes statistical analysis across patients, may result from subtle differences in the patterns of intersecting characteristics of the tonic stretch reflex. The applicability of our analysis to disordered multijoint movement patterns is discussed. Received: 26 July 1993/Accepted in revised form: 22 December 1993     

Molecular phylogenetics of Iberian wall lizards (Podarcis): is Podarcis hispanica a species complex?

Phylogenetic relationships between species and morphotypes of Podarcis wall lizards from the Iberian Peninsula and north Africa were estimated using partial 12S rRNA and cytochrome b mitochondrial DNA sequences. All species except Podarcis hispanica form monophyletic units. P. hispanica is paraphyletic, although all identified morphotypes are monophyletic. These morphotypes represent highly divergent lineages showing 10-15% pairwise sequence divergence with the cytochrome b gene. The data suggest that P. hispanica is a species complex. We recommend using P. hispanica* until additional sampling delimits the number and ranges of species currently referred to P. hispanica. P. carbonelli, which has recently been raised to species status, is confirmed as a genetically distinct form. P. atrata is genetically distinct, but much more closely related to some populations of P. hispanica than previously thought.     

The Q-cycle reviewed: How well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.