Absence of Spatial Tuning in the Orbitofrontal Cortex

There is limited data in the literature to explicitly support the notion that neurons in OFC are truly action-independent in their coding. We set out to specifically test the hypothesis that OFC value-related neurons in area 13 m of the monkey do not carry information about the action required to obtain that reward – that activity in this area represents reward values in an abstract and action-independent manner. To accomplish that goal we had two monkeys select and execute saccadic eye movements to 81 locations in the visual field for three different kinds of juice rewards. Our detailed analysis of the response fields indicates that these neurons are insensitive to the amplitude or direction of the saccade required to obtain these rewards. Our data thus validate earlier proposals that neurons of 13 m in the OFC encode subjective value independent of the saccadic action required to obtain that reward.     

Modelling Thrombin Generation in Human Ovarian Follicular Fluid

A mathematical model is constructed to study thrombin production in human ovarian follicular fluid. The model results show that the amount of thrombin that can be produced in ovarian follicular fluid is much lower than that in blood plasma, failing to reach the level required for fibrin formation, and thereby supporting the hypothesis that in follicular fluid thrombin functions to initiate cellular activities via intracellular signalling receptors. It is also concluded that the absence of the amplification pathway to thrombin production in follicular fluid is a major factor in restricting the amount of thrombin that can be produced. Titration of the initial concentrations of the various reactants in the model lead to predictions for the amount of tissue factor and phospholipid that is required to maintain thrombin production in the follicle, as well as to the conclusion that tissue factor pathway inhibitor has little effect on the time that thrombin generation is sustained. Numerical experiments to determine the effect of factor V, which is at a much reduced level in follicular fluid compared to plasma, and thrombomodulin, illustrate the importance for further experimental work to determine values for several parameters that have yet to be reported in the literature.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

On the Specificity of Adaptive Mutations

Adaptive mutations are mutations that occur in nondividing or slowly dividing cells during prolonged nonlethal selection, and that appear to be specific to the challenge of the selection in the sense that the only mutations that arise are those that provide a growth advantage to the cell. The issue of the specificity has been controversial because it violates our most basic assumptions about the randomness of mutations with respect to their effect on the cell. Although a variety of experiments in several systems in both bacteria and yeast have claimed to demonstrate that specificity, those experiments have been subjected to a variety of technical criticisms suggesting that the specificity may not be real. Here I use the ebg system to provide evidence that when selection is applied to one specific nucleotide site within a gene, mutation occurs at that site but not at an alternative and equally mutable site within the same gene.     

Analysis of cellular factors that mediate nuclear export of RNAs bearing the Mason-Pfizer monkey virus constitutive transport element

There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)(+) RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin beta-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.     

ENHANCING EVOLUTION AND ENHANCING EVOLUTION

It has been claimed in several places that the new genetic technologies allow humanity to achieve in a generation or two what might take natural selection hundreds of millennia in respect of the elimination of certain diseases and an increase in traits such as intelligence. More radically, it has been suggested that those same technologies could be used to instil characteristics that we might reasonably expect never to appear due to natural selection alone. John Harris, a proponent of this genomic optimism, claims in his book Enhancing Evolution that we not only have it in our power to enhance evolution, but that we also have a duty to do so. In this paper, I claim that Harris' hand is strong but that he overplays it nevertheless. He is correct to dismiss the arguments of the anti‐enhancement lobby and correct to say that enhancement is permissible; but ‘good’ is different from ‘permissible’ and his argument for the goodness of enhancement is less convincing. Moreover, he is simply wrong to claim that it generates a duty to enhance.     

On the evolutionary origin of aging

It is generally believed that the first organisms did not age, and that aging thus evolved at some point in the history of life. When and why this transition occurred is a fundamental question in evolutionary biology. Recent reports of aging in bacteria suggest that aging predates the emergence of eukaryotes and originated in simple unicellular organisms. Here we use simple models to study why such organisms would evolve aging. These models show that the differentiation between an aging parent and a rejuvenated offspring readily evolves as a strategy to cope with damage that accumulates due to vital activities. We use measurements of the age-specific performance of individual bacteria to test the assumptions of the model, and find evidence that they are fulfilled. The mechanism that leads to aging is expected to operate in a wide range of organisms, suggesting that aging evolved early and repeatedly in the history of life. Aging might thus be a more fundamental aspect of cellular organisms than assumed so far.     

Towards a molecular explanation of the high performance of the tuna heart

The tuna heart is capable of sustaining cardiac outputs that are high relative to other active teleost species. The question as to whether there are known molecular mechanisms to explain this phenomenon is examined. Unfortunately, the evidence at present is scant but the paper attempts to put existing knowledge into a contextual framework. It is known that the high cardiac outputs in tuna are due to the maximum heart rates that are high relative to other active teleost species. While the normalized stroke volume (ml/kg body weight) in tuna is in the same range as that of trout several important points are worth noting. The stroke volume in the tuna heart is generated in the face of an afterload that is two-fold higher (due to high ventral aortic pressure) than that observed in the trout or other teleosts. Since the stroke volume is predicated on the strength of ventricular contraction, the question then arises as to whether this is a consequence of factors intrinsic or extrinsic to the design of the cardiomyocytes that make up the myocardium. Two important extrinsic factors must be noted: the substantially higher normal operating temperatures and the apparent cardiac hyperplasia in the tuna. While the merit of these factors is discussed, the paper focuses on intrinsic factors. There is very little that has been determined to date that would indicate substantive changes in the design of the myocyte in the tuna heart compared to that of trout. Are these extrinsic factors alone then sufficient to account for the impressive cardiac output capabilities in the tuna?     

Extracellular superoxide dismutase inhibits inflammation by preventing oxidative fragmentation of hyaluronan

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.     

Interaction of salt with ether- and ester-linked phospholipid bilayers

A distinguishing feature of Archaeal plasma membranes is that their phospholipids contain ether-links, as opposed to bacterial and eukaryotic plasma membranes where phospholipids primarily contain ester-links. Experiments show that this chemical difference in headgroup-tail linkage does produce distinct differences in model bilayer properties. Here we examine the effects of salt on bilayer structure in the case of an ether-linked lipid bilayer. We use molecular dynamics simulations and compare equilibrium properties of two model lipid bilayers in NaCl salt solution – POPC and its ether-linked analog that we refer to as HOPC. We make the following key observations. The headgroup region of HOPC “adsorbs” fewer ions compared to the headgroup region of POPC. Consistent with this, we note that the Debye screening length in the HOPC system is ∼ 10% shorter than that in the POPC system. Herein, we introduce a protocol to identify the lipid-water interfacial boundary that reproduces the bulk salt distribution consistent with Gouy-Chapman theory. We also note that the HOPC bilayer has excess solvent in the headgroup region when compared to POPC, coinciding with a trough in the electrostatic potential. Waters in this region have longer autocorrelation times and smaller lateral diffusion rates compared to the corresponding region in the POPC bilayer, suggesting that the waters in HOPC are more strongly coordinated to the lipid headgroups. Furthermore, we note that it is this region of tightly coordinated waters in the HOPC system that has a lower density of Na⁺ ions. Based on these observations we conclude that an ether-linked lipid bilayer has a lower binding affinity for Na⁺ compared to an ester-linked lipid bilayer.     

Endosomal microdomains: Formation and function

It is widely recognized that after endocytosis, internalized cargo is delivered to endosomes that act as sorting stations. The limiting membrane of endosomes contain specialized subregions, or microdomains, that represent distinct functions of the endosome, including regions competing for cargo capture leading to degradation or recycling. Great progress has been made in defining the endosomal protein coats that sort cargo in these domains, including Retromer that recycles transmembrane cargo, and ESCRT (endosomal sorting complex required for transport) that degrades transmembrane cargo. In this review, we discuss recent work that is beginning to unravel how such coat complexes contribute to the creation and maintenance of endosomal microdomains. We highlight data that indicates that adjacent microdomains do not act independently but rather interact to cross-regulate. We posit that these interactions provide an agile means for the cell to adjust sorting in response to extracellular signals and intracellular metabolic cues.     

Site of graviperception in roots: a re-examination

Two lines of evidence have been cited to support the assertion that the root cap is the sole site of graviperception in the root. The first evidence is based on surgical removal of the cap, which abolishes the response to gravity. This is sufficient to conclude that the cap is involved in gravitropism, but not to conclude that the cap is the site of graviperception. The second is based on the results of centrifugation experiments, in which different parts of the plant are subjected to different centrifugal forces. The data from such experiments have been cited to support the conclusion that the perception of gravity is limited to the rootcap. However, these data actually support the conclusion that gravity is perceived throughout the root tip, and not only in the root cap. We believe that the data support the conclusion that the root cap is involved in root gravitropism, but that there is inadequate evidence to conclude that the cap is the sole site of graviperception.     

GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli.

Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli.     

RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli

RuvABC is a complex that promotes branch migration and resolution of Holliday junctions. Although ruv mutants are hypersensitive to UV irradiation, the molecular event(s) that necessitate RuvABC processing in vivo are not known. Here, we used a combination of two-dimensional gel analysis and electron microscopy to reveal that although ruvAB and ruvC mutants are able to resume replication following arrest at UV-induced lesions, molecules that replicate in the presence of DNA damage accumulate unresolved Holliday junctions. The failure to resolve the Holliday junctions on the fully replicated molecules correlates with a delayed loss of genomic integrity that is likely to account for the loss of viability in these cells. The strand exchange intermediates that accumulate in ruv mutants are distinct from those observed at arrested replication forks and are not subject to resolution by RecG. These results indicate that the Holliday junctions observed in ruv mutants are intermediates of a repair pathway that is distinct from that of the recovery of arrested replication forks. A model is proposed in which RuvABC is required to resolve junctions that arise during the repair of a subset of nonarresting lesions after replication has passed through the template.     

Reasoning in bioethics

It is striking that some arguments in the bioethical literature seem implausible, counterintuitive, and even ridiculous when reported to competent moral agents. When examined, these arguments bear uncanny resemblances to the discourse of patients with debilitating mental disorders. I examine the kinds of irrationality involved, and discuss the fact that such irrationality is worrying in a discipline that purports to serve as a guide for real-life practical reasoning. I offer some thoughts about correctives that we might use to temper some of the odd opinions that bedevil our subject in the name of ethical analysis. It seems that one ought to be suspicious of neatly rational arguments that produce counterintuitive conclusions, but the alternative seems to be that we explore new constructions of old problems in Bioethics such that our discussion of them does justice to what we regard as of fundamental significance to our lives together as human beings.     

Long-range, pattern-dependent contextual effects in early human visual cortex

The standard view of neurons in early visual cortex is that they behave like localized feature detectors. Here we demonstrate that processing in early visual areas goes beyond feature detection by showing that neural responses are greater when a feature deviates from its context compared to when it does not deviate from its context. Using psychophysics, fMRI, and electroencephalography methodologies, we measured neural responses to an oriented Gabor ("target") embedded in various visual patterns as defined by the relative orientation of flanking stimuli. We first show using psychophysical contrast adaptation and fMRI that a target that differs from its context results in more neural activity compared to a target that is contained within an alternating sequence, suggesting that neurons in early visual cortex are sensitive to large-scale orientation patterns. Next, we use event-related potentials to show that orientation deviations affect the earliest sensory components of the target response. Finally, we use forced-choice classification of "noise" stimuli to show that we are more likely to "see" orientations that deviate from the context. Our results suggest that early visual cortex is sensitive to global patterns in images in a way that is markedly different from the predictions of standard models of cortical visual processing.