Environmental change and hedonic cost functions for automobiles

This paper focuses on how changes in the economic and regulatory environment have affected production costs and product characteristics in the automobile industry. We estimate “hedonic cost functions” that relate product-level costs to their characteristics. Then we examine how this cost surface has changed over time and how these changes relate to changes in gas prices and in emission standard regulations. We also briefly consider the related questions of how changes in automobile characteristics, and in the rate of patenting, are related to regulations and gas prices.

Functional domains for assembly of histones H3 and H4 into the chromatin of Xenopus embryos

Histones H3 and H4 have a well defined structural role in the nucleosome and an established role in the regulation of transcription. We have made use of a microinjection strategy using Xenopus embryos to define the minimal structural components of H3 and H4 necessary for nucleosome assembly into metazoan chromosomes in vivo. We find that both the N-terminal tail of H4, including all sites of acetylation, and the C-terminal α-helix of the H4 histone fold domain are dispensable for chromatin assembly. The N-terminal tail and an N-terminal α-helix of H3 are also dispensable for chromatin assembly. However, the remainder of the H3 and H4 histone folds are essential for incorporation of these proteins into chromatin. We suggest that elements of the histone fold domain maintain both nucleosomal integrity and have distinct functions essential for cell viability.

RacF1, a Novel Member of the Rho Protein Family in Dictyostelium discoideum, Associates Transiently with Cell Contact Areas, Macropinosomes, and Phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP–RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60c-src or p59fyn results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60c-src or p59fyn to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60c-src is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60c-src from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60c-src to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

An ancestral MADS-box gene duplication occurred before the divergence of plants and animals

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

Regional and strain-specific gene expression mapping in the adult mouse brain

To determine the genetic causes and molecular mechanisms responsible for neurobehavioral differences in mice, we used highly parallel gene expression profiling to detect genes that are differentially expressed between the 129SvEv and C57BL/6 mouse strains at baseline and in response to seizure. In addition, we identified genes that are differentially expressed in specific brain regions. We found that approximately 1% of expressed genes are differentially expressed between strains in at least one region of the brain and that the gene expression response to seizure is significantly different between the two inbred strains. The results lead to the identification of differences in gene expression that may account for distinct phenotypes in inbred strains and the unique functions of specific brain regions.

Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase

Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3′-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2–3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions.

Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice

To elucidate the role of neuropeptide Y (NPY)-Y1 receptor (Y1-R) in food intake, energy expenditure, and other possible functions, we have generated Y1-R-deficient mice (Y1-R−/−) by gene targeting. Contrary to our hypothesis that the lack of NPY signaling via Y1-R would result in impaired feeding and weight loss, Y1-R−/− mice showed a moderate obesity and mild hyperinsulinemia without hyperphagia. Although there was some variation between males and females, typical characteristics of Y1-R−/− mice include: greater body weight (females more than males), an increase in the weight of white adipose tissue (WAT) (approximately 4-fold in females), an elevated basal level of plasma insulin (approximately 2-fold), impaired insulin secretion in response to glucose administration, and a significant changes in mitochondrial uncoupling protein (UCP) gene expression (up-regulation of UCP1 in brown adipose tissue and down-regulation of UCP2 in WAT). These results suggest either that the Y1-R in the hypothalamus is not a key molecule in the leptin/NPY pathway, which controls feeding behavior, or that its deficiency is compensated by other receptors, such as NPY-Y5 receptor. We believe that the mild obesity found in Y1-R−/− mice (especially females) was caused by the impaired control of insulin secretion and/or low energy expenditure, including the lowered expression of UCP2 in WAT. This model will be useful for studying the mechanism of mild obesity and abnormal insulin metabolism in noninsulin-dependent diabetes mellitus.

The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin

The grail of protein science is the connection between structure and function. For myoglobin (Mb) this goal is close. Described as only a passive dioxygen storage protein in texts, we argue here that Mb is actually an allosteric enzyme that can catalyze reactions among small molecules. Studies of the structural, spectroscopic, and kinetic properties of Mb lead to a model that relates structure, energy landscape, dynamics, and function. Mb functions as a miniature chemical reactor, concentrating and orienting diatomic molecules such as NO, CO, O2, and H2O2 in highly conserved internal cavities. Reactions can be controlled because Mb exists in distinct taxonomic substates with different catalytic properties and connectivities of internal cavities.

The role of genetic and genomic attributes in the success of polyploids

In 1950, G. Ledyard Stebbins devoted two chapters of his book Variation and Evolution in Plants (Columbia Univ. Press, New York) to polyploidy, one on occurrence and nature and one on distribution and significance. Fifty years later, many of the questions Stebbins posed have not been answered, and many new questions have arisen. In this paper, we review some of the genetic attributes of polyploids that have been suggested to account for the tremendous success of polyploid plants. Based on a limited number of studies, we conclude: (i) Polyploids, both individuals and populations, generally maintain higher levels of heterozygosity than do their diploid progenitors. (ii) Polyploids exhibit less inbreeding depression than do their diploid parents and can therefore tolerate higher levels of selfing; polyploid ferns indeed have higher levels of selfing than do their diploid parents, but polyploid angiosperms do not differ in outcrossing rates from their diploid parents. (iii) Most polyploid species are polyphyletic, having formed recurrently from genetically different diploid parents. This mode of formation incorporates genetic diversity from multiple progenitor populations into the polyploid “species”; thus, genetic diversity in polyploid species is much higher than expected by models of polyploid formation involving a single origin. (iv) Genome rearrangement may be a common attribute of polyploids, based on evidence from genome in situ hybridization (GISH), restriction fragment length polymorphism (RFLP) analysis, and chromosome mapping. (v) Several groups of plants may be ancient polyploids, with large regions of homologous DNA. These duplicated genes and genomes can undergo divergent evolution and evolve new functions. These genetic and genomic attributes of polyploids may have both biochemical and ecological benefits that contribute to the success of polyploids in nature.

Formation of a Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10−/− mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10−/− mice suggests that there is a considerable redundancy in the keratin gene family.

Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex.

The primate temporal cortex has been demonstrated to play an important role in visual memory and pattern recognition. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here we address this issue by examining the induction of synaptic plasticity in surgically resected human inferior and middle temporal cortex. The results show that synaptic strength in the human temporal cortex could undergo bidirectional modifications, depending on the pattern of conditioning stimulation. High frequency stimulation (100 or 40 Hz) in layer IV induced long-term potentiation (LTP) of both intracellular excitatory postsynaptic potentials and evoked field potentials in layers II/III. The LTP induced by 100 Hz tetanus was blocked by 50-100 microM DL-2-amino-5-phosphonovaleric acid, suggesting that N-methyl-D-aspartate receptors were responsible for its induction. Long-term depression (LTD) was elicited by prolonged low frequency stimulation (1 Hz, 15 min). It was reduced, but not completely blocked, by DL-2-amino-5-phosphonovaleric acid, implying that some other mechanisms in addition to N-methyl-DL-aspartate receptors were involved in LTD induction. LTD was input-specific, i.e., low frequency stimulation of one pathway produced LTD of synaptic transmission in that pathway only. Finally, the LTP and LTD could reverse each other, suggesting that they can act cooperatively to modify the functional state of cortical network. These results suggest that LTP and LTD are possible mechanisms for the visual memory and pattern recognition functions performed in the human temporal cortex.

Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.

Variant antigens and endothelial receptor adhesion in Plasmodium falciparum.

Parasite-derived proteins expressed on the surface of erythrocytes infected with Plasmodium falciparum are important virulence factors, since they mediate binding of infected cells to diverse receptors on vascular endothelium and are targets of a protective immune response. They are difficult to study because they undergo rapid clonal antigenic variation in vitro, which precludes the derivation of phenotypically homogeneous cultures. Here we have utilized sequence-specific proteases to dissect the role of defined antigenic variants in binding to particular receptors. By selection of protease-resistant subpopulations of parasites on defined receptors we (i) confirm the high rate of antigenic variation in vitro; (ii) demonstrate that a single infected erythrocyte can bind to intercellular adhesion molecule 1, CD36, and thrombospondin; (iii) show that binding to intercellular adhesion molecule 1 and CD36 are functions of the variant antigen; and (iv) suggest that binding to thrombospondin may be mediated by other components of the infected erythrocyte surface.

Evolution of chlorophyll and bacteriochlorophyll: the problem of invariant sites in sequence analysis.

Competing hypotheses seek to explain the evolution of oxygenic and anoxygenic processes of photosynthesis. Since chlorophyll is less reduced and precedes bacteriochlorophyll on the modern biosynthetic pathway, it has been proposed that chlorophyll preceded bacteriochlorophyll in its evolution. However, recent analyses of nucleotide sequences that encode chlorophyll and bacteriochlorophyll biosynthetic enzymes appear to provide support for an alternative hypothesis. This is that the evolution of bacteriochlorophyll occurred earlier than the evolution of chlorophyll. Here we demonstrate that the presence of invariant sites in sequence datasets leads to inconsistency in tree building (including maximum-likelihood methods). Homologous sequences with different biological functions often share invariant sites at the same nucleotide positions. However, different constraints can also result in additional invariant sites unique to the genes, which have specific and different biological functions. Consequently, the distribution of these sites can be uneven between the different types of homologous genes. The presence of invariant sites, shared by related biosynthetic genes as well as those unique to only some of these genes, has misled the recent evolutionary analysis of oxygenic and anoxygenic photosynthetic pigments. We evaluate an alternative scheme for the evolution of chlorophyll and bacteriochlorophyll.