Effect of the thermostat in the molecular dynamics simulation on the folding of the model protein chignolin

Molecular dynamics simulations of the model protein chignolin with explicit solvent were carried out, in order to analyze the influence of the Berendsen thermostat on the evolution and folding of the peptide. The dependence of the peptide behavior on temperature was tested with the commonly employed thermostat scheme consisting of one thermostat for the protein and another for the solvent. The thermostat coupling time of the protein was increased to infinity, when the protein is not in direct contact with the thermal bath, a situation known as minimally invasive thermostat. In agreement with other works, it was observed that only in the last situation the instantaneous temperature of the model protein obeys a canonical distribution. As for the folding studies, it was shown that, in the applications of the commonly utilized thermostat schemes, the systems are trapped in local minima regions from which it has difficulty escaping. With the minimally invasive thermostat the time that the protein needs to fold was reduced by two to three times. These results show that the obstacles to the evolution of the extended peptide to the folded structure can be overcome when the temperature of the peptide is not directly controlled.     

Delineation of the HLA-DR region and the residues involved in the association with the cytoskeleton

Whereas the association of major histocompatibility complex (MHC) class II molecules with the cytoskeleton and their recruitment into lipid rafts play a critical role during cognate T/antigen-presenting cell interactions, MHC class II-induced signals, regions, and residues involved in their association and recruitment have not yet been fully deciphered. In this study, we show that oligomerization of HLA-DR molecules induces their association with the cytoskeleton and their recruitment into lipid rafts. The association of oligomerized HLA-DR molecules with the cytoskeleton and their recruitment into lipid rafts occur independently. Furthermore, the association with the cytoskeleton is HLA-DR-specific, since oligomerization of HLA-DP triggers its recruitment only into lipid rafts. HLA-DR molecules devoid of both alpha and beta cytoplasmic tails did not associate with the cytoskeleton, but their recruitment into lipid rafts was unimpeded. Deletion of either the alpha or beta cytoplasmic tail did not affect the association of HLA-DR with the cytoskeleton and/or recruitment into lipid rafts. HLA-DR molecules that were devoid of the alpha cytoplasmic chain and that had their beta cytoplasmic chain replaced with the HLA-DP beta chain or with a beta chain in which the residues at positions Gly(226)-His(227)-Ser(228) were substituted by alanine no longer associated with the cytoskeleton. They were, however, still recruited into lipid rafts. Together, these results support the involvement of different regions of the cytoplasmic tails in the association and the recruitment of HLA-DR into different compartments. The differential behavior of HLA-DP and -DR with respect to their association with the cytoskeleton may explain the previously described difference in their transduced signals.     

A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope

The nuclear pore complex (NPC) is a multicomponent structure containing a subset of proteins that bind nuclear transport factors or karyopherins and mediate their movement across the nuclear envelope. By altering the expression of a single nucleoporin gene, NUP53, we showed that the overproduction of Nup53p altered nuclear transport and had a profound effect on the structure of the nuclear membrane. Strikingly, conventional and immunoelectron microscopy analysis revealed that excess Nup53p entered the nucleus and associated with the nuclear membrane. Here, Nup53p induced the formation of intranuclear, tubular membranes that later formed flattened, double membrane lamellae structurally similar to the nuclear envelope. Like the nuclear envelope, the intranuclear double membrane lamellae enclosed a defined cisterna that was interrupted by pores but, unlike the nuclear envelope pores, they lacked NPCs. Consistent with this observation, we detected only two NPC proteins, the pore membrane proteins Pom152p and Ndc1p, in association with these membrane structures. Thus, these pores likely represent an intermediate in NPC assembly. We also demonstrated that the targeting of excess Nup53p to the NPC and its specific association with intranuclear membranes were dependent on the karyopherin Kap121p and the nucleoporin Nup170p. At the nuclear envelope, the abilities of Nup53p to associate with the membrane and drive membrane proliferation were dependent on a COOH-terminal segment containing a potential amphipathic alpha-helix. The implications of these results with regards to the biogenesis of the nuclear envelope are discussed.     

Determinants of the quantity of the stable SecY complex in the Escherichia coli cell.

While SecY in wild-type Escherichia coli cells is stable and is complexed with other proteins within the membrane, moderately overexpressed and presumably uncomplexed SecY was degraded with a half-life of 2 min. The fact that the amount of stable SecY is strictly regulated by the degradation of excess SecY was demonstrated by competitive entry of the SecY+ protein and a SecY-LacZ alpha fusion protein into the stable pool. Simultaneous overexpression of SecE led to complete stabilization of excess SecY. Overproduced SecD and SecF did not affect the stability of SecY, but plasmids carrying ORF12 located within the secD-secF operon partially stabilized this protein. In contrast, mutational reduction of the SecE content (but not the ORF12 content) led to the appearance of two populations of newly synthesized SecY molecules, one that was immediately degraded and one that was completely stable. Thus, the E. coli cell is equipped with a system that eliminates SecY unless it is complexed with SecE, a limiting partner of SecY. Our observations implied that in wild-type cells, SecY and SecE rapidly associate with each other and remain complexed.     

Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane

The exocyst is an octameric protein complex implicated in the tethering of post-Golgi secretory vesicles to the plasma membrane before fusion. The function of individual exocyst components and the mechanism by which this tethering complex is targeted to sites of secretion are not clear. In this study, we report that the exocyst subunit Exo70 functions in concert with Sec3 to anchor the exocyst to the plasma membrane. We found that the C-terminal Domain D of Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues on Exo70 that are critical for its interaction with phospholipids and the small GTPase Rho3. Further genetic and cell biological analyses suggest that the interaction of Exo70 with phospholipids, but not Rho3, is essential for the membrane association of the exocyst complex. We propose that Exo70 mediates the assembly of the exocyst complex at the plasma membrane, which is a crucial step in the tethering of post-Golgi secretory vesicles for exocytosis.     

Biochemical studies on the origin of the ATPase of the avian myeloblastosis virus

The adenosine triphosphatase activity of the avian myeloblastosis virus obtained from the blood of the virus-infected chicken was compared with that of the host cell myeloblasts. The specific activity of the viral enzyme is unusually higher than that of the myeloblasts. A significant difference in inhibitor sensitivity was observed with quercetin. When the virus was grown in chicken embryonic fibroblasts in culture, the resulting virus showed very little adenosine triphosphatase activity, comparable to that of the fibroblasts and similar sensitivity to inhibitors. Antibody raised against the purified enzyme of avian myeloblastosis virus inhibits the enzyme activity of the myeloblasts while the activity of the fibroblasts enzyme as well as that of fibroblast-grown virus remains unaffected.     

Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase

The kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase has been investigated employing the heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as substrate. Initial velocity measurements performed over a wide range of ATP and Kemptide concentrations indicated that the reaction follows a sequential mechanistic pathway. In line with this, the results of product and substrate inhibition studies, the patterns of dead end inhibition obtained employing the nonhydrolyzable ATP analogue, AMP X PNP (5'-adenylylimidodiphosphate), and equilibrium binding determinations, taken in conjunction with the patterns of inhibition observed with the inhibitor protein of the cAMP-dependent protein kinase that are reported in the accompanying paper (Whitehouse, S., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3682-3692), are best fit by a steady state Ordered Bi-Bi kinetic mechanism. Although the inhibition patterns obtained employing the synthetic peptide analogue in which the phosphorylatable serine was replaced by alanine were apparently incompatible with this mechanism, these inconsistencies appear to be due to some element of the structure of this latter peptide such that it is not an ideal dead end inhibitor substrate analogue. The data presented both here and in the accompanying paper suggest that both this substrate, analogue and the ATP analogue, AMP X PNP, do not fully mimic the binding of Kemptide and ATP, respectively, in their mechanism of interaction with the protein kinase. It is proposed that, as with some other kinase reactions, the configuration of the terminal anhydride bond of ATP assumes a conformation once the nucleotide is bound to the protein kinase that assists in the binding of either Kemptide or the inhibitor protein but not the alanine-substituted peptide and that AMP X PNP, because of its terminal phosphorylimido bond, cannot assume this conformation which favors protein (or peptide) binding.     

An Analysis of the Metabolic Theory of the Origin of the Genetic Code

A computer program was used to test Wong's coevolution theory of the genetic code. The codon correlations between the codons of biosynthetically related amino acids in the universal genetic code and in randomly generated genetic codes were compared. It was determined that many codon correlations are also present within random genetic codes and that among the random codes there are always several which have many more correlations than that found in the universal code. Although the number of correlations depends on the choice of biosynthetically related amino acids, the probability of choosing a random genetic code with the same or greater number of codon correlations as the universal genetic code was found to vary from 0.1% to 34% (with respect to a fairly complete listing of related amino acids). Thus, Wong's theory that the genetic code arose by coevolution with the biosynthetic pathways of amino acids, based on codon correlations between biosynthetically related amino acids, is statistical in nature. Received: 8 August 1996 / Accepted: 26 December 1996     

Characterization of the structures involved in localization of the SUN proteins to the nuclear envelope and the centrosome

The nuclear envelope forms a selective barrier that separates the cytoplasm from the nucleus. During mitosis the nuclear envelope breaks down so that the microtubule network can form contacts with the kinetochore and guide chromosome segregation. Previous studies have suggested a model in which the centrosome and the microtubule network may play a role in nuclear envelope breakdown through as yet unidentified interactions with proteins localized to the nuclear envelope. In the current study we characterized a nuclear envelope protein SUN2 and identified a substructure involved in its localization to the nuclear envelope. We found that a structurally related protein, SUN1, may be localized to the nuclear envelope through a different mechanism. Furthermore, the SUN2 protein can form different assemblies, including homodimers and heterodimers with SUN1. Finally, we provide evidence indicating that SUN1 and SUN2 may form a physical interaction between the nuclear envelope and the centrosome.     

On the use of the Price equation

This paper distinguishes two categories of questions that the Price equation can help us answer. The two different types of questions require two different disciplines that are related, but nonetheless move in opposite directions. These disciplines are probability theory on the one hand and statistical inference on the other. In the literature on the Price equation this distinction is not made. As a result of this, questions that require a probability model are regularly approached with statistical tools. In this paper, we examine the possibilities of the Price equation for answering questions of either type. By spending extra attention on mathematical formalities, we avoid the two disciplines to get mixed up. After that, we look at some examples, both from kin selection and from group selection, that show how the inappropriate use of statistical terminology can put us on the wrong track. Statements that are 'derived' with the help of the Price equation are, therefore, in many cases not the answers they seem to be. Going through the derivations in reverse can, however, be helpful as a guide how to build proper (probabilistic) models that do give answers.     

Specific binding of lectins with the nucleus of the sea urchin embryo and changes in the lectin affinity of the embryonic chromatin during the course of development

(1) Embryonic cells of sea urchins were made permeable by treating them with glycerol solution for the purpose of allowing penetration of macromolecules into the cell. With the use of such permeabilized cells, several kinds of fluorescent dye-labeled lectins were introduced into the cell, and it was found that some lectins showed notable affinity with the nucleus as compared with cytoplasmic structures. (2) Isolated chromatin was incubated with several kinds of fluorescent dye-labeled lectins in vitro, and the amount of lectins bound with the chromatin was measured by fluorometry. By means of this method, the lectin-binding capacity of chromatin was estimated and compared at various stages of development. It was found that lectins could be classified into three groups according to the mode of binding with the chromatin: (a) Extent of binding increased notably at the gastrula stage (Con A and RCA-120); (b) extent of binding showed a temporary decrease at the gastrula stage (TTA); and (c) very low level of binding was maintained throughout all stages, and no particular change was observed at any stage of development (WGA, SBA, and UEA-I). (3) These facts seem to suggest that lectin-binding components are contained in sea urchin chromatin, and that drastic changes occur in these components of chromatin at the stage of gastrulation. It was proposed that the lectin-binding components such as proteoglycans and glycoproteins may play regulatory roles in embryonic chromatin at early stages of development.     

Redesigning the Industrial Ecology of the Automobile

This article explores the potential of industrial ecology to inform the redesign of an existing industry: that which is concerned with the production, sale, and support of automobiles. In so doing, it brings together the concepts embedded in industrial ecology with issues of economic scale, product design or technology, process technology, and the way in which new combinations of these features can result in an alternative structure for the automotive industry that has the potential to enhance sustainability performance. In so doing, the article advances the general argument that the economic, technical, and spatial organization of production and consumption are co-determined in a manner that collectively shapes the industrial ecology of an industry. In contradistinction to the prevailing industry, the article then advances the concept of micro factory retailing as an alternative framework for the industry that would result in significantly different performance in terms of industrial ecology.     

Sequence and developmental expression of the mRNA encoding the seleno-protein of the sperm mitochondrial capsule in the mouse

We have characterized cDNA clones encoding the selenium-containing polypeptide of the keratinous mitochondrial capsule in mouse sperm. The longest open reading frame encodes a polypeptide 143 amino acids long which contains 21% cysteine and 27% proline and closely resembles the size and amino acid composition of bull mitochondrial capsule seleno-protein (V. Pallini, B. Baccetti, and A. G. Burrini, 1979, in "The Spermatozoon," D. W. Fawcett and J. M. Bedford, Eds., pp. 141-151, Urban & Schwartzenberg, Baltimore/Munich). The reading frame encoding the mitochondrial capsule seleno-protein ends with an amber stop codon suggesting that selenium is not incorporated cotranslationally into the protein by an opal suppressor selenocysteyl-tRNA as has been found for several eukaryotic and bacterial proteins. Northern blots using RNA extracted from purified spermatogenic cells and staged prepuberal mice suggest that the mitochondrial capsule seleno-protein mRNA is first transcribed in late meiotic cells and that the levels of the mRNA increase after meiosis in early haploid cells. Southern blots demonstrate that there is one copy of the gene in the mouse genome. The identification of this cDNA clone, in combination with previous work (K. C. Kleene, 1989, Development 106, 367-373) demonstrates that the mRNA for the mitochondrial capsule seleno-protein is translationally repressed with long homogenous poly(A) tracts in round spermatids and translationally active with shortened heterogenous poly(A) tracts in elongating spermatids.     

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo

The present study examines the indispensability of a nucleus or nucleus-deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)-damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV-damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV-damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV-damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos.     

Correlates of the duration of the egg collecting phase in the three-spined stickleback

Individual male three-spined sticklebacks in the field, often collected eggs for longer (up to 10 days) than had been reported for sticklebacks in captivity (3–6 days). The probability that a male stopped collecting eggs increased with the number of eggs already present, and possibly with the age of the eggs. Males with nests hidden under a plant were more likely to continue collecting than males with exposed nests. These results are discussed in the light of theoretical considerations that predict when males should stop collecting further eggs.     

Rethinking the Role of the Nervous System: Lessons From the Hydra Holobiont

Here we evaluate our current understanding of the function of the nervous system in Hydra, a non‐bilaterian animal which is among the first metazoans that contain neurons. We highlight growing evidence that the nervous system, with its rich repertoire of neuropeptides, is involved in controlling resident beneficial microbes. We also review observations that indicate that microbes affect the animal's behavior by directly interfering with neuronal receptors. These findings provide new insight into the original role of the nervous system, and suggest that it emerged to orchestrate multiple functions including host‐microbiome interactions. The excitement of future research in the Hydra model now relies on uncovering the common rules and principles that govern the interaction between neurons and microbes and the extent to which such laws might apply to other and more complex organisms.