Toward an understanding of structure and function of ion channels

The second half of the 1980s is certain to be considered a turning point in the study of ion channels. Within the last few years, monumental advances in the application of molecular biology, single-channel recording, and direct molecular characterization have been brought to bear on the problem of relating the molecular structure of the ion channel proteins to their function in the cell membrane. Structure-function relationships can now be studied at a level of detail that was unimagined a decade ago. Recently, advances made with the techniques of molecular biology appear to have dominated the literature in this field; however, innovative strategies of structural characterization and electrical measurements of functioning channels in native and artificial membranes continue to break new ground. This paper is a selective review of current progress in understanding structure-function relationships in ion channels. The relative usefulness of determining amino acid sequences of channel proteins together with the resulting deductions about 3-dimensional structure and function will be evaluated with respect to the potential importance of studying the channel molecules more directly by biochemical, immunological, and electrophysiological methods. A full understanding of the details of channel structure and its relationship to function may be realized in the near future as a result of the interdisciplinary application of biophysical, biochemical, and molecular biological techniques.     

Toward an understanding of biochemical equilibria within living cells

Four types of environmental effects that can affect macromolecular reactions in a living cell are defined: nonspecific intermolecular interactions, side reactions, partitioning between microenvironments, and surface interactions. Methods for investigating these interactions and their influence on target reactions in vitro are reviewed. Methods employed to characterize conformational and association equilibria in vivo are reviewed and difficulties in their interpretation cataloged. It is concluded that, in order to be amenable to unambiguous interpretation, in vivo studies must be complemented by in vitro studies carried out in well-characterized and controllable media designed to contain key elements of selected intracellular microenvironments.     

Toward understanding the different function of two types of parenchyma cells in bamboo culms

The bamboo, woody monocot, has two types of parenchyma cells in the ground tissues of its culm, in contrast to a single type of parenchyma cell in rice, maize and other major crop species. The distribution of cell wall components, including lignin, (1-->3), (1-->4)-beta-D-glucans (MGs), the highly-substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans (lbXs) in ground parenchyma tissue of Phyllostachys heterocycla var. pubescens culms was studied at various developmental stages using light microscopy (LM), UV-microscopy, transmission electron microscopy (TEM) and immunolabeling techniques. The short parenchyma cell walls were lignified in 2-month-old bamboo culms just as the long parenchyma cell walls were. The lignified regions were confined to the portions in contact with the long parenchyma cell walls, while the walls at the cell corner region never lignified, even in 7-year-old culms. Significant differences were also found in the hemicellulose distribution between the short and long parenchyma cell walls. In bamboo parenchyma tissue, MGs were localized in short parenchyma cell walls and few were found in long parenchyma cell walls in both young and 7-year-old culms. The distribution of hsGAXs was similar to that of MGs in young culms, but they only appeared in the cell corner region of short parenchyma cells in old culms. Low-branched xylans were distributed in the lignified, but not in unlignified parenchyma cell walls. Based on this evidence, the differences of function in both short and long parenchyma cells in a bamboo culm are discussed.     

Toward an understanding of the structural basis of translation

The recently solved X-ray crystal structures of the ribosome have provided opportunities for studying the molecular basis of translation with a variety of methods including cryo-electron microscopy - where maps give the first glimpses of ribosomal evolution - and fluorescence spectroscopy techniques.     

Toward an understanding of the brain substrates of reward in humans

A network of brain regions has been implicated in food-reward processing. now provide evidence that this network is differentially modulated by anticipation versus receipt of a food reward and suggest an additional effect of valence of the stimulus.     

Toward xeno-free culture of human embryonic stem cells

The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.     

Toward an individual-level understanding of vigilance: the role of social information

A gregarious lifestyle affords the benefit of collective detectionof predators through the many-eyes effect. Studies of vigilanceare generally concerned with exploring the relationship betweenvigilance rates and group size. However, a mechanistic understandingof the rules individual animals use to achieve this group-levelbehavior is lacking. Building on a previous modeling approach,we suggest that individuals reconcile their own private informationagainst the social information they receive from their groupmates in order to decide whether to feed or be vigilant at anyone time. We present a novel modeling approach utilizing a Markovchain Monte Carlo process to describe the transition betweenvigilant and nonvigilant states. Many of our assumptions arebased qualitatively on recently published experimental observations.We vary the amount of social information and the fidelity withwhich individuals process this information and show that thishas a profound effect on the individual vigilance rate, theindividual vigilant bout length, and the proportion of vigilantindividuals at any one time. A wide range of group-level vigilancepatterns can be obtained by varying simple behavioral characteristicsof individual animals. We find that generally, increasing theamount of, and sensitivity to, social information generatesa more cooperative vigilance behavior. This model potentiallyprovides a theoretical and conceptual framework for examiningspecific real-life systems. We propose analyzing individual-baseddata from real animals by considering their group to be a connectednetwork of individuals, with information transfer between them.     

Toward an understanding of microbial communities through analysis of communication networks

Bacteria receive signals from diverse members of their biotic environment. They sense their own species through the process of quorum sensing, which detects the density of bacterial cells and regulates functions such as bioluminescence, virulence, and competence. Bacteria also respond to the presence of other microorganisms and eukaryotic hosts. Most studies of microbial communication focus on signaling between the microbe and one other organism for empirical simplicity and because few experimental systems offer the opportunity to study communication among various types of organisms. But in the real biological world, microorganisms must carry on multiple molecular conversations simultaneously between diverse organisms, thereby constructing communication networks. We propose that biocontrol of plant disease, the process of suppressing disease through application of a microorganism, offers a model for the study of communication among multiple organisms. Successful biocontrol requires the sending and receiving of signals between the biocontrol agent and the pathogen, plant host, and microbial community surrounding the host. We are using Bacillus cereus, a biocontrol agent, and the organisms it must interact with, to dissect a communication network. This system offers an excellent starting point for study because its members are defined and well studied. An understanding of signaling in the B. cereus biocontrol system may provide a model for network communication among organisms that share a habitat and provide a new angle of analysis for understanding the interconnections that define communities. This revised version was published online in August 2006 with corrections to the Cover Date.     

Toward an understanding of the molecular basis of quantitative disease resistance in rice

Rice crops are severely damaged by diseases caused by bacterial, fungal, and viral pathogens. Application of host resistance to these pathogens is the most economical and environmentally friendly approach to solve this problem. Quantitative resistance conferred by quantitative trait loci (QTL) is a valuable resource for the improvement of rice disease resistance. Although numerous resistance QTL against rice diseases have been identified, these resources have not been used effectively in rice improvement because the genetic control of quantitative resistance is complex and the genes underlying most of the resistance QTL remain unknown. This review focuses on the latest molecular progress in quantitative disease resistance in rice. This knowledge will be helpful for characterizing more resistance QTL and turning the quantitative resistance into actual resources for rice protection.     

Toward an understanding of mechanisms regulating plant response to biochar application

Plant growth and development are affected by several environmental factors, among which soil nutrient availability. Biochar addition to soil is recognized to exert beneficial effects on soil fertility and thus plant growth; furthermore, it is a promising option for climate change mitigation. However, multi-species studies and meta-analyses have indicated considerable variations in biochar responses among plant species. To date, information on the biochar effect on plants, especially at molecular level, is still scarce. Using a multi-target approach with a model plant such as tomato, we demonstrate that biochar has a negligible effect on soil nutrient content and plant growth, even if it misbalances the plant photosynthetic machinery, as well as mechanisms recognizing pathogen-derived molecules. Ethylene could be one of the signal-molecule driving the alteration of tomato-pathogen recognition signaling by inactivation of vesicle trafficking. All these modifications could be at the basis of the increased susceptibility of biochar-treated plants to pathogen attack. Further organ-specific and tissue-specific multi-level studies, from high-resolution internal processes towards high-throughput external phenotyping, coupled with powerful biostatistic and informatic analysis, will help to decipher, in a network-type fashion, all the factors and signaling mechanisms related to the complex interaction between different plant, soil and biochar types.     

Toward a molecular understanding of pleiotropy

Pleiotropy refers to the observation of a single gene influencing multiple phenotypic traits. Although pleiotropy is a common phenomenon with broad implications, its molecular basis is unclear. Using functional genomic data of the yeast Saccharomyces cerevisiae, here we show that, compared with genes of low pleiotropy, highly pleiotropic genes participate in more biological processes through distribution of the protein products in more cellular components and involvement in more protein-protein interactions. However, the two groups of genes do not differ in the number of molecular functions or the number of protein domains per gene. Thus, pleiotropy is generally caused by a single molecular function involved in multiple biological processes. We also provide genomewide evidence that the evolutionary conservation of genes and gene sequences positively correlates with the level of gene pleiotropy.     

Toward understanding the dynamics of membrane-raft-based molecular interactions

The cell membrane is a 2-dimensional non-ideal liquid containing dynamic structures on various time-space scales, and the raft domain is one of them. Existing literature supports the concept that raft dynamics may be important for its formation and function: the raft function may be supported by stimulation-induced raft association/coalescence and recruitment of various raftophilic molecules to coalesced rafts, and, importantly, they both may happen transiently. Thus, one must always consider the limited association time of a raft or a raftophilic molecule with another raft, even when one interprets the results of static experiments, such as immunofluorescence and pull-down assays. Critical considerations on the chemical fixation mechanism and immunocolocalization data suggest that the temporary nature of raft-based molecular interactions may explain why colocalization results are sensitive to subtle variations in experimental conditions employed in different laboratories.     

Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase

The motions of key residues at the substrate binding site of lactate dehydrogenase (LDH) were probed on the 10 ns to 10 ms time scale using laser-induced temperature-jump relaxation spectroscopy employing both UV fluorescence and isotope-edited IR absorption spectroscopy as structural probes. The dynamics of the mobile loop, which closes over the active site and is important for catalysis and binding, were characterized by studies of the inhibitor oxamate binding to the LDH/NADH binary complex monitoring the changes in emission of bound NADH. The bound NAD-pyruvate adduct, whose pyruvate moiety likely interacts with the same residues that interact with pyruvate in its ternary complex with LDH, served as a probe for any relative motions of active site residues against the substrate. The frequencies of its C=O stretch and -COO(-) antisymmetric stretch shift substantially should any relative motion of the polar moieties at the active site (His-195, Asp-168, Arg-109, and Arg-171) occur. The dynamics associated with loop closure are observed to involve several steps with motions from 1 to 300 microms. Apart from the "melting" of a few residues on the protein's surface, no kinetics were observed on any time scale in experiments of the bound NAD-pyr adduct although the measurements were made with a high degree of accuracy, even for final temperatures close to the unfolding transition of the protein. This is contrary to simple physical considerations and models. These results show that, once a productive protein/substrate complex is formed, the binding pocket is very rigid with very little, if any, motion apart from the mobile loop. The results also show that loop opening involves concomitant movement of the substrate out of the binding pocket.