bluuebunny/crossref_metadata_2025 huggingface dataset:
Hierarchy and feedback in the evolution of the
Escherichia coli
transcription network
Authors: Cosentino Lagomarsino M. , Jona P. , Bassetti B. , Isambert H.
Published: March 2007

The
Escherichia coli
transcription network has an essentially feedforward structure, with abundant feedback at the level of self-regulations. Here, we investigate how these properties emerged during evolution. An assessment of the role of gene duplication based on protein domain architecture shows that (
i
) transcriptional autoregulators have mostly arisen through duplication, whereas (
ii
) the expected feedback loops stemming from their initial cross-regulation are strongly selected against. This requires a divergent coevolution of the transcription factor DNA-binding sites and their respective DNA
cis
-regulatory regions. Moreover, we find that the network tends to grow by expansion of the existing hierarchical layers of computation, rather than by addition of new layers. We also argue that rewiring of regulatory links due to mutation/selection of novel transcription factor/DNA binding interactions appears not to significantly affect the network global hierarchy, and that horizontally transferred genes are mainly added at the bottom, as new target nodes. These findings highlight the important evolutionary roles of both duplication and selective deletion of cross-talks between autoregulators in the emergence of the hierarchical transcription network of
E. coli
.
Charged polycyclic aromatic hydrocarbon clusters and the galactic extended red emission
Authors: Rhee Young Min , Lee Timothy J. , Gudipati Murthy S. , Allamandola Louis J. , Head-Gordon Martin
Published: March 2007

The species responsible for the broad extended red emission (ERE), discovered in 1975 and now known to be widespread throughout the Galaxy, still is unidentified. Spanning the range from ≈540 to 900 nm, the ERE is a photoluminescent process associated with a wide variety of different interstellar environments. Over the years, a number of plausible candidates have been suggested, but subsequent observations ruled them out. The objects that present the ERE also emit the infrared features attributed to free polycyclic aromatic hydrocarbon (PAH) molecules, suggesting that closely related materials are plausible ERE carriers. Here, we show that the peculiar spectra and unique properties of closed-shell cationic PAH dimers satisfy the existing observational constraints and suggest that emission from mixtures of charged PAH clusters accounts for much of the ERE. This work provides a view into the structures, stabilities, abundances, and ionization balance of PAH-related species in the emission zones, which, in turn, reflects physical conditions in the emission zones and sheds fundamental light on the nanoscale processes involved in carbon-particle nucleation and growth and carbonaceous dust evolution in the interstellar medium.
Electron transfer in Me-blocked heterodimeric α,γ-peptide nanotubular donor–acceptor hybrids
Authors: Brea Roberto J. , Castedo Luis , Granja Juan R. , Herranz M. Ángeles , Sánchez Luis , Martín Nazario , Seitz Wolfgang , Guldi Dirk M.
Published: March 2007

Bio-inspired cyclopeptidic heterodimers built on β-sheet-like hydrogen-bonding networks and bearing photoactive and electroactive chromophores on the outer surface have been prepared. Different cross-strand pairwise relationships between the side chains of the cyclic α,γ-peptides afford the heterodimers as three nonequivalent dimeric species. Steady-state and time-resolved spectroscopies clearly show an electron transfer process from π-extended tetrathiafulvalene, covalently attached to one of the cyclopeptides, to photoexcited [60]fullerene, located on the complementary cyclopeptide. The charge-separated state was stabilized for up to 1 μs before recombining and repopulating the ground state. Our current example shows that cyclopeptidic templates can be successfully used to form light-harvesting/light-converting hybrid ensembles with a distinctive organization of donor and acceptor units able to act as efficient artificial photosystems.
Transgenic malaria-resistant mosquitoes have a fitness advantage when feeding onPlasmodium-infected blood
Authors: Marrelli Mauro T. , Li Chaoyang , Rasgon Jason L. , Jacobs-Lorena Marcelo
Published: March 2007

The introduction of genes that impairPlasmodiumdevelopment into mosquito populations is a strategy being considered for malaria control. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of this approach. We have previously shown that anopheline mosquitoes expressing the SM1 peptide in the midgut lumen are impaired for transmission ofPlasmodium berghei. Moreover, the transgenic mosquitoes had no noticeable fitness load compared with nontransgenic mosquitoes when fed on noninfected mice. Here we show that when fed on mice infected withP. berghei, these transgenic mosquitoes are more fit (higher fecundity and lower mortality) than sibling nontransgenic mosquitoes. In cage experiments, transgenic mosquitoes gradually replaced nontransgenics when mosquitoes were maintained on mice infected with gametocyte-producing parasites (strain ANKA 2.34) but not when maintained on mice infected with gametocyte-deficient parasites (strain ANKA 2.33). These findings suggest that when feeding onPlasmodium-infected blood, transgenic malaria-resistant mosquitoes have a selective advantage over nontransgenic mosquitoes. This fitness advantage has important implications for devising malaria control strategies by means of genetic modification of mosquitoes.
Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure
Authors: Watkins Adam J. , Platt Duncan , Papenbrock Tom , Wilkins Adrian , Eckert Judith J. , Kwong Wing Yee , Osmond Clive , Hanson Mark , Fleming Tom P.
Published: March 2007

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of
in vitro
culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing
in vivo
. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing
in vivo
. Embryo culture and ET had minimal effects on postnatal growth when compared with
in vivo
development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with
in vivo
development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phospho
enol
pyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth.
Environmental change preceded Caribbean extinction by 2 million years
Authors: O'Dea Aaron , Jackson Jeremy B. C. , Fortunato Helena , Smith J. Travis , D'Croz Luis , Johnson Kenneth G. , Todd Jonathan A.
Published: March 2007

Paleontologists typically treat major episodes of extinction as single and distinct events in which a major environmental perturbation results in a synchronous evolutionary response. Alternatively, the causes of biotic change may be multifaceted and extinction may lag behind the changes ultimately responsible because of nonlinear ecological dynamics. We examined these alternatives for the major episode of Caribbean extinction 2 million years ago (Ma). Isolation of the Caribbean from the Eastern Pacific by uplift of the Panamanian Isthmus was associated with synchronous changes in Caribbean near shore environments and community composition between 4.25 and 3.45 Ma. Seasonal fluctuations in Caribbean seawater temperature decreased 3-fold, carbonate deposition increased, and there was a striking, albeit patchy, shift in dominance of benthic ecosystems from heterotrophic mollusks to mixotrophic reef corals and calcareous algae. All of these changes correspond well with a simple model of decreased upwelling and collapse in planktonic productivity associated with the final stages of the closure of the isthmian barrier. However, extinction rates of mollusks and corals did not increase until 3–2 Ma and sharply peaked between 2 and 1 Ma, even though extinction overwhelmingly affected taxa commonly associated with high productivity. This time lag suggests that something other than environmental change
per se
was involved in extinction that does not occur as a single event. Understanding cause and effect will require more taxonomically refined analysis of the changing abundance and distribution patterns of different ecological guilds in the 2 million years leading up to the relatively sudden peak in extinction.
Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency
Authors: Porubsky Stefan , Speak Anneliese O. , Luckow Bruno , Cerundolo Vincenzo , Platt Frances M. , Gröne Hermann-Josef
Published: March 2007

CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Vα14-Jα18 gene segments in mice and Vα24-Jα18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4+/CD8+cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as α1–3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only byiGb3S, lack of iGb3 in the dorsal root ganglia ofiGb3S-deficient mice (iGb3S−/−), as compared withiGb3S+/−mice, was confirmed.iGb3S−/−mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vβ chains identical to controls. Upon administration of α-galactosylceramide, activation of iNKT and dendritic cells was similar iniGb3S−/−andiGb3S+/−mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-γ in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-γ, TNF-α, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.
Consolidation of learning strategies during spatial working memory task requires protein synthesis in the prefrontal cortex
Authors: Touzani K. , Puthanveettil S. V. , Kandel E. R.
Published: March 2007

Working memory is a temporary memory store where information is held briefly until the appropriate behavior is produced. However, the improvement in the performance of working memory tasks with practice over days points to the existence of a long-lasting component associated with learning strategies that lead to optimal performance. Here we show that the improvement in the performance of mice in a radial maze working memory task required the integrity of the medial prefrontal cortex (mPFC). We further demonstrate that this improvement of working memory performance requires the synthesis ofde novoproteins in the mPFC. We suggest that in addition to storing memory briefly the mPFC is also involved in the consolidation and storage of the long-term learning strategies used in working memory.
Tracking melanosomes inside a cell to study molecular motors and their interaction
Authors: Kural Comert , Serpinskaya Anna S. , Chou Ying-Hao , Goldman Robert D. , Gelfand Vladimir I. , Selvin Paul R.
Published: March 2007

Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured
Xenopus
melanophores is mediated by myosin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial (≈2 nm) and temporal (≈1 msec) localization accuracy. This method enabled us to observe (
i
) stepwise movement of kinesin-2 with an average step size of 8 nm; (
ii
) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (
iii
) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retrograde movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. We observed 35-nm myosin V steps in melanophores containing no IFs. We find that myosin V steps occur faster in the absence of IFs, indicating that the IF network physically hinders organelle transport.
Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription
Authors: Liu Hsiao-Wei , Zeng Yining , Landes Christy F. , Kim Yoen Joo , Zhu Yongjin , Ma Xiaojing , Vo My-Nuong , Musier-Forsyth Karin , Barbara Paul F.
Published: March 2007

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are “chaperoned” by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes
in vitro
has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a “Y” shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the “Y” conformation.
Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a Met antagonist
Authors: Kirchhofer Daniel , Lipari Michael T. , Santell Lydia , Billeci Karen L. , Maun Henry R. , Sandoval Wendy N. , Moran Paul , Ridgway John , Eigenbrot Charles , Lazarus Robert A.
Published: March 2007

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase Met, is secreted as single chain pro-HGF that lacks signaling activity. Pro-HGF acquires functional competence upon cleavage between R494 and V495, generating a disulfide-linked α/β-heterodimer, where the β-chain of HGF (HGF β) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the β-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF β crystal structure shows that V495 inserts into the “activation pocket” near the Met binding site where the positively charged N terminus forms a salt bridge with the negatively charged D672, and the V495 side chain has hydrophobic interactions with main- and side-chain residues. Full-length two-chain HGF mutants designed to interrupt these interactions (D672N, V495G, V495A, G498I, and G498V) displayed <10% activity in Met receptor phosphorylation, cell migration, and proliferation assays. Impaired signaling of full-length mutants correlated with >50-fold decreases in Met binding of the low-affinity HGF β domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF α-chain, full-length mutants maintained normal Met binding and efficiently inhibited HGF-mediated Met activation. Conversion of HGF from agonist to antagonist was achieved by as little as removal of two methyl groups (V495A) or a single charge (D672N). Thus, although serine proteases and HGF have quite distinct functions in proteolysis and Met signal transduction, respectively, they share a similar activation mechanism.
Synthesis of the next-generation therapeutic antibodies that combine cell targeting and antibody-catalyzed prodrug activation
Authors: Abraham Sunny , Guo Fang , Li Lian-Sheng , Rader Christoph , Liu Cheng , Barbas Carlos F. , Lerner Richard A. , Sinha Subhash C.
Published: March 2007

An obstacle in the utilization of catalytic Abs for selective prodrug activation in cancer therapy has been systemic tumor targeting. Here we report the generation of catalytic Abs that effectively target tumor cells with undiminished prodrug activation capability. Ab conjugates were prepared by covalent conjugation of an integrin α
v
β
3
-targeting antagonist to catalytic Ab 38C2 through either sulfide groups of cysteine residues generated by reduction of the disulfide bridges in the hinge region or surface lysine residues not involved in the catalytic activity. Using flow cytometry, the Ab conjugates were shown to bind efficiently to integrin α
v
β
3
-expressing human breast cancer cells. The Ab conjugates also retained the
retro
-aldol activity of their parental catalytic Ab 38C2, as measured by methodol and doxorubicin (dox) prodrug activation. Complementing these Ab conjugates, an evolved set of dox prodrugs was designed and synthesized. Dox prodrugs that showed higher stability and lower toxicity were evaluated both in the presence and absence of the integrin α
v
β
3
-targeting 38C2 conjugates for cell-killing efficacy by using human breast cancer cells. Our study reveals that cell targeting and prodrug activation capabilities can be efficiently combined for selective chemotherapy with novel dox prodrugs.
Endocytosis provides a major alternative pathway for lysosomal biogenesis in kidney proximal tubular cells
Authors: Nielsen Rikke , Courtoy Pierre J. , Jacobsen Christian , Dom Geneviève , Lima Wânia Rezende , Jadot Michel , Willnow Thomas E. , Devuyst Olivier , Christensen Erik I.
Published: March 2007

Recruitment of acid hydrolases to lysosomes generally occurs by intracellular sorting based on recognition of a common mannose 6-phosphate signal in the transGolgi network and selective transport to late endosomes/lysosomes. Here we provide evidence for an alternative, efficient secretion-recapture pathway mediated by megalin and exemplified by cathepsin B in kidney proximal convoluted tubules (PCT). We found that in mouse kidneys with defective megalin expression [megalin knockout (KO)] or apical PCT trafficking (ClC-5 KO), the (pro)cathepsin B mRNA level was essentially preserved, but the protein content was greatly decreased and the enzyme was excreted in the urine as mannose 6-phosphate-devoid species. In polarized PCT-derived cells, purified cathepsin B was avidly and selectively taken up at the apical membrane, and uptake was abolished by the megalin competitor, receptor-associated protein. Direct interaction of cathepsin B with megalin was demonstrated by surface plasmon resonance. Procathepsin B was detected in normal mouse serum. Purified cathepsin B injected into mice was efficiently taken up by kidneys (≈10% of injection) and targeted to lysosomes where it remained active, as shown by autoradiography and subcellular fractionation. A single cathepsin B injection into cathepsin B KO mice could reconstitute full lysosomal enzyme activity in the kidneys. These findings demonstrate a pathway whereby circulating lysosomal enzymes are continuously filtered in glomeruli, reabsorbed by megalin-mediated endocytosis, and transferred into lysosomes to exert their function, providing a major source of enzymes to PCT. These results also extend the significance of megalin in PCT and have several physiopathological and clinical implications.
Stimulation of proliferation of MCF-7 breast cancer cells by a transfected splice variant of growth hormone-releasing hormone receptor
Authors: Barabutis Nektarios , Tsellou Erasmia , Schally Andrew V. , Kouloheri Stavroula , Kalofoutis Anastasios , Kiaris Hippokratis
Published: March 2007

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as an autocrine/paracrine growth factor for various human cancers. A splice variant (SV) of the full-length receptor for GHRH (GHRHR) is widely expressed in various primary human cancers and established cancer cell lines and appears to mediate the proliferative effects of GHRH. To investigate in greater detail the role of SV1 in tumorigenesis, we have expressed the full-length GHRHR and its SV1 in MCF-7 human breast cancer cells that do not possess either GHRHR or SV1. In accordance with previous findings, the expression of both GHRHR and SV1 restored the sensitivity to GHRH-induced stimulation of cell proliferation, with SV1 being more potent than the GHRHR. Furthermore, MCF-7 cells transfected with SV1 proliferated more quickly than the controls, even in the absence of exogenously added GHRH, suggesting the existence of intrinsic, ligand-independent activity of SV1 after its transfection. In agreement with the stimulation of cell proliferation, the levels of proliferation markers cyclin D1, cyclin E, and proliferating cell nuclear antigen were elevated in MCF-7 cells treated with GHRH, cultured in both serum-free and serum-containing media. In addition, SV1 caused a considerable stimulation of the ability of MCF-7 cells to grow in semisolid medium, an assay considered diagnostic for cell transformation. Collectively, our findings show that the expression of SV1 confers oncogenic activity and provide further evidence that GHRH operates as a growth factor in breast cancer and probably other cancers as well.
Additional material of the enigmatic Early Miocene mammal
Kelba
and its relationship to the order Ptolemaiida
Authors: Cote Susanne , Werdelin Lars , Seiffert Erik R. , Barry John C.
Published: March 2007

Kelba quadeemae
, a fossil mammal from the Early Miocene of East Africa, was originally named on the basis of three isolated upper molars.
Kelba
has previously been interpreted as a creodont, a pantolestid, an insectivoran, and a hemigaline viverrid. The true affinities of this taxon have remained unclear because of the limited material and its unique morphology relative to other Miocene African mammals. New material of
Kelba
from several East African Miocene localities, most notably a skull from the Early Miocene locality of Songhor in Western Kenya, permits analysis of the affinities of
Kelba
and documents the lower dentition of this taxon. Morphological comparison of this new material clearly demonstrates that
Kelba
is a member of the order Ptolemaiida, a poorly understood group whose fossil record was previously restricted to the Oligocene Fayum deposits of northern Egypt. Phylogenetic analysis supports the monophyly of the Ptolemaiida, including
Kelba
, and recovers two monophyletic clades within the order. We provide new family names for these groups and an emended diagnosis for the order. The discovery of ptolemaiidans from the Miocene of East Africa is significant because it extends the known temporal range of the order by >10 million years and the geographic range by >3,200 km. Although the higher-level affinities of the Ptolemaiida remain obscure, their unique morphology and distribution through a larger area of Africa (and exclusively Africa) lend support to the idea that Ptolemaiida may have an ancient African origin.
Saposin B is the dominant saposin that facilitates lipid binding to human CD1d molecules
Authors: Yuan Weiming , Qi Xiaoyang , Tsang Pansy , Kang Suk-Jo , Illarionov Petr A. , Besra Gurdyal S. , Gumperz Jenny , Cresswell Peter
Published: March 2007

CD1d molecules bind lipid antigens in the endocytic pathway, and access to the pathway is important for the development of CD1d-restricted natural killer T (NKT) cells. Saposins, derived from a common precursor, prosaposin, are small, heat-stable lysosomal glycoproteins required for lysosomal degradation of sphingolipids. Expression of prosaposin is required for efficient lipid binding and recognition of human CD1d molecules by NKT cells. Despite high sequence homology among the four saposins, they have different specificities for lipid substrates and different mechanisms of action. To determine the saposins involved in promoting lipid binding to CD1d, we expressed prosaposin deletion mutants lacking individual saposins in prosaposin-negative, CD1d-positive cells. No individual saposin proved to be absolutely essential, but the absence of saposin B resulted in the lowest recognition of α-galactosylceramide by NKT cells. When recombinant exogenous saposins were added to the prosaposin-negative cells, saposin B was the most efficient in restoring CD1d recognition. Saposin B was also the most efficient in mediating α-galactosylceramide binding to recombinant plate-bound CD1d and facilitating NKT cell activation. Saposin B could also mediate lipid binding to soluble CD1d molecules in a T cell-independent assay. The optimal pH for saposin B-mediated lipid binding to CD1d, pH 6, is higher than that of lysosomes, suggesting that saposin B may facilitate lipid binding to CD1d molecules throughout the endocytic pathway.
Control of human thymocyte migration by Neuropilin-1/Semaphorin-3A-mediated interactions
Authors: Lepelletier Yves , Smaniotto Salete , Hadj-Slimane Réda , Villa-Verde Déa Maria Serra , Nogueira Ana Cristina , Dardenne Mireille , Hermine Olivier , Savino Wilson
Published: March 2007

It is largely established that molecules first discovered in the nervous system are also found in the immune system. Neuropilin-1 (NP-1) was initially identified to mediate semaphorin-induced chemorepulsion during brain development and is also involved in peripheral T cell/dendritic cell interactions. Herein, we studied NP-1 during T cell development in the human thymus. NP-1 is expressed in both cortex and medulla of thymic lobules, being found in distinct CD4/CD8-defined thymocyte subsets. NP-1 is also found in thymic epithelial cells (TEC)
in situ
and
in vitro
, and is recruited at the site of TEC–thymocyte contact. Moreover, NP-1 was rapidly up-regulated during thymocyte stimulation by T cell receptor (TCR) and IL-7 or after adhesion to TEC. Semaphorin-3A (Sema-3A), a natural ligand of NP-1, is also present in human thymus, both in TEC and thymocytes, being up-regulated in thymocytes after TCR engagement. Functionally, Sema-3A decreases the adhesion capacity of NP-1
+
thymocytes and induces their migration by a repulsive effect. In conclusion, we show here that NP-1/Sema-3A-mediated interactions participate in the control of human thymocyte development.
Alteration in the gene expression pattern of primary monocytes after adhesion to endothelial cells
Authors: Thomas-Ecker Sybill , Lindecke Antje , Hatzmann Wolfgang , Kaltschmidt Christian , Zänker Kurt S. , Dittmar Thomas
Published: March 2007

Monocytes originate from precursors made in the bone and remain in the circulation for nearly 24 h. Much effort has been done to identify the molecules regulating transendothelial migration of monocytes during inflammatory conditions. In contrast, considerably less is known about the process of constitutive monocyte emigration although nearly 340 million monocytes leave the circulation each day in healthy individuals. Previous studies indicated that chemokines were up-regulated in monocytes cocultured with endothelial cells that induce the retraction of the latter cell type, thereby increasing vascular permeability. Thus, we hypothesized that the utilities required for efficient constitutive monocyte extravasation are generated by monocytes themselves because of adhesion to naïve endothelial cells. To test this hypothesis, cDNA microarray analysis was performed to determine the changes in the gene expression pattern of primary monocytes that have been attached to endothelial cells compared with monocytes that were held in suspension, and we were able to identify three major groups of genes. The first group includes genes such as matrix metalloproteinase 1, monocyte chemoattractant protein 1, and tissue transglutaminase 2, which are likely required for monocyte extravasation. The second group consists of genes that are expressed in phagocytes such as caveolin-1 and CD74. Finally, the third group comprises genes that are expressed in cells of endothelial tissue and cartilage including E-selectin, fibronectin-1, matrix Gla protein, and aggrecanase-2. In summary, we conclude that adhesion of peripheral blood monocytes to naïve endothelial cells has two effects: mandatory extravasation-specific genes are regulated, and the differentiation program of monocytes is initiated.
Bacterial superantigen facilitates epithelial presentation of allergen to T helper 2 cells
Authors: Ardern-Jones Michael R. , Black Antony P. , Bateman Elizabeth A. , Ogg Graham S.
Published: March 2007

Although clinical and laboratory evidence support roles for both staphylococcal infection and environmental allergens in the pathogenesis of atopic dermatitis, human studies have largely considered these variables independently. We sought to test the hypothesis that staphylococcal superantigen influences the allergen-specific T cell response. We first mapped a Der p 1 epitope and used HLA DRB1*1501 class II tetramer-based cell sorted populations to show that specific CD4+T cells were able to recognize the peptide presented by HLA DR-matched keratinocytes. We observed that staphylococcal enterotoxin B (SEB) enhanced the IL-4 Der p 1-specific T cell response. This response was mediated by two synergistic mechanisms: first, SEB-induced IFN-γ promoted class II and intercellular adhesion molecule-1 expression by presenting keratinocytes; and second, SEB-induced IL-4 directly amplified allergen-specific CD4+T cell production of many cytokines. We propose that handling of staphylococcal infection is a critical step in the amplification of the allergen-specific T cell response, linking two common disease associations and with implications for the prevention and treatment of atopic disease.
Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS
Authors: Van Hoewyk Douglas , Abdel-Ghany Salah E. , Cohu Christopher M. , Herbert Stephen K. , Kugrens Paul , Pilon Marinus , Pilon-Smits Elizabeth A. H.
Published: March 2007

NifS-like proteins provide the sulfur (S) for the formation of iron-sulfur (Fe-S) clusters, an ancient and essential type of cofactor found in all three domains of life. Plants are known to contain two distinct NifS-like proteins, localized in the mitochondria (MtNifS) and the chloroplast (CpNifS). In the chloroplast, five different Fe-S cluster types are required in various proteins. These plastid Fe-S proteins are involved in a variety of biochemical pathways including photosynthetic electron transport and nitrogen and sulfur assimilation.
In vitro
, the chloroplastic cysteine desulfurase CpNifS can release elemental sulfur from cysteine for Fe-S cluster biogenesis in ferredoxin. However, because of the lack of a suitable mutant allele, the role of CpNifS has not been studied thus far
in planta
. To study the role of CpNifS in Fe-S cluster biogenesis
in vivo
, the gene was silenced by using an inducible RNAi (interference) approach. Plants with reduced CpNifS expression exhibited chlorosis, a disorganized chloroplast structure, and stunted growth and eventually became necrotic and died before seed set. Photosynthetic electron transport and carbon dioxide assimilation were severely impaired in the silenced plant lines. The silencing of CpNifS decreased the abundance of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S proteins and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. These findings indicate that CpNifS is necessary for the maturation of all plastidic Fe-S proteins and, thus, essential for plant growth.
The implications of alternative splicing in the ENCODE protein complement
Authors: Tress Michael L. , Martelli Pier Luigi , Frankish Adam , Reeves Gabrielle A. , Wesselink Jan Jaap , Yeats Corin , Ólason Páll ĺsólfur , Albrecht Mario , Hegyi Hedi , Giorgetti Alejandro , Raimondo Domenico , Lagarde Julien , Laskowski Roman A. , López Gonzalo , Sadowski Michael I. , Watson James D. , Fariselli Piero , Rossi Ivan , Nagy Alinda , Kai Wang , Størling Zenia , Orsini Massimiliano , Assenov Yassen , Blankenburg Hagen , Huthmacher Carola , Ramírez Fidel , Schlicker Andreas , Denoeud France , Jones Phil , Kerrien Samuel , Orchard Sandra , Antonarakis Stylianos E. , Reymond Alexandre , Birney Ewan , Brunak Søren , Casadio Rita , Guigo Roderic , Harrow Jennifer , Hermjakob Henning , Jones David T. , Lengauer Thomas , A. Orengo Christine , Patthy László , Thornton Janet M. , Tramontano Anna , Valencia Alfonso
Published: March 2007

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.
Perturbed ATPase activity and not “close confinement” of substrate in the cis cavity affects rates of folding by tail-multiplied GroEL
Authors: Farr George W. , Fenton Wayne A. , Horwich Arthur L.
Published: March 2007

Folding of substrate proteins inside the sequestered and hydrophilic GroEL-GroES cis cavity favors production of the native state. Recent studies of GroEL molecules containing volume-occupying multiplications of the flexible C-terminal tail segments have been interpreted to indicate that close confinement of substrate proteins in the cavity optimizes the rate of folding: the rate of folding of a larger protein, Rubisco (51 kDa), was compromised by multiplication, whereas that of a smaller protein, rhodanese (33 kDa), was increased by tail duplication. Here, we report that this latter effect does not extend to the subunit of malate dehydrogenase (MDH), also 33 kDa. In addition, single-ring versions of tail-duplicated and triplicated molecules, comprising stable cis complexes, did not produce any acceleration of folding of rhodanese or MDH, nor did they show significant retardation of the folding of Rubisco. Tail quadruplication produced major reduction in recovery of native protein with both systems, the result of strongly reduced binding of all three substrates. When steady-state ATPase of the tail-multiplied double-ring GroELs was examined, it scaled directly with the number of tail segments, with more than double the normal ATPase rate upon tail triplication. As previously observed, disturbance of ATPase activity of the cycling double-ring system, and thus of “dwell time” for the folding protein in the cis cavity, produces effects on folding rates. We conclude that, within the limits of the ≈10% decrease of cavity volume produced by tail triplication, there does not appear to be an effect of “close confinement” on folding in the cis cavity.
Genome plasticity of BCG and impact on vaccine efficacy
Authors: Brosch Roland , Gordon Stephen V. , Garnier Thierry , Eiglmeier Karin , Frigui Wafa , Valenti Philippe , Dos Santos Sandrine , Duthoy Stéphanie , Lacroix Céline , Garcia-Pelayo Carmen , Inwald Jacqueline K. , Golby Paul , Garcia Javier Nuñez , Hewinson R. Glyn , Behr Marcel A. , Quail Michael A. , Churcher Carol , Barrell Bart G. , Parkhill Julian , Cole Stewart T.
Published: March 2007

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette–Guérin (BCG) vaccines,
Mycobacterium bovis
BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene,
glpD2
, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding σ-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.
Extreme genomic variation in a natural population
Authors: Small Kerrin S. , Brudno Michael , Hill Matthew M. , Sidow Arend
Published: March 2007

Whole-genome sequence data from samples of natural populations provide fertile grounds for analyses of intraspecific variation and tests of population genetic theory. We show that the urochordate
Ciona savignyi
, one of the species of ocean-dwelling broadcast spawners commonly known as sea squirts, exhibits the highest rates of single-nucleotide and structural polymorphism ever comprehensively quantified in a multicellular organism. We demonstrate that the cause for the extreme heterozygosity is a large effective population size, and, consistent with prediction by the neutral theory, we find evidence of strong purifying selection. These results constitute in-depth insight into the dynamics of highly polymorphic genomes and provide important empirical support of population genetic theory as it pertains to population size, heterozygosity, and natural selection.
Structure of limonene synthase, a simple model for terpenoid cyclase catalysis
Authors: Hyatt David C. , Youn Buhyun , Zhao Yuxin , Santhamma Bindu , Coates Robert M. , Croteau Rodney B. , Kang ChulHee
Published: March 2007

The crystal structure of (4
S
)-limonene synthase from
Mentha spic ata
, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-Å; resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate–metal ion coordination and substrate binding conformation in the course of the multistep reaction.
Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay
Authors: Rauch Tibor , Wang Zunde , Zhang Xinmin , Zhong Xueyan , Wu Xiwei , Lau Sean K. , Kernstine Kemp H. , Riggs Arthur D. , Pfeifer Gerd P.
Published: March 2007

De novo
methylation of CpG islands is a common phenomenon in human cancer, but the mechanisms of cancer-associated DNA methylation are not known. We have used tiling arrays in combination with the methylated CpG island recovery assay to investigate methylation of CpG islands genome-wide and at high resolution. We find that all four
HOX
gene clusters on chromosomes 2, 7, 12, and 17 are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. CpG islands associated with many other homeobox genes, such as
SIX
,
LHX
,
PAX
,
DLX
, and
Engrailed
, were highly methylated as well. Altogether, more than half (104 of 192) of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Analysis of paralogous
HOX
genes showed that not all paralogues undergo cancer-associated methylation simultaneously. The
HOXA
cluster was analyzed in greater detail. Comparison with ENCODE-derived data shows that lack of methylation at CpG-rich sequences correlates with presence of the active chromatin mark, histone H3 lysine-4 methylation in the
HOXA
region. Methylation analysis of
HOXA
genes in primary squamous cell carcinomas of the lung led to the identification of the
HOXA7
- and
HOXA9
-associated CpG islands as frequent methylation targets in stage 1 tumors. Homeobox genes are potentially useful as DNA methylation markers for early diagnosis of the disease. The finding of widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.
Sonic hedgehog acts at multiple stages during pancreatic tumorigenesis
Authors: Morton Jennifer P. , Mongeau Michelle E. , Klimstra David S. , Morris John P. , Lee Yie Chia , Kawaguchi Yoshiya , Wright Christopher V. E. , Hebrok Matthias , Lewis Brian C.
Published: March 2007

Activation of sonic hedgehog (Shh) signaling occurs in the majority of pancreatic ductal adenocarcinomas. Here we investigate the mechanisms by which Shh contributes to pancreatic tumorigenesis. We find that Shh expression enhances proliferation of pancreatic duct epithelial cells, potentially through the transcriptional regulation of the cell cycle regulators cyclin D1 and p21. We further show that Shh protects pancreatic duct epithelial cells from apoptosis through the activation of phosphatidylinositol 3-kinase signaling and the stabilization of Bcl-2 and Bcl-X
L
. Significantly, Shh also cooperates with activated K-Ras to promote pancreatic tumor development. Finally, Shh signaling enhances K-Ras-induced pancreatic tumorigenesis by reducing the dependence of tumor cells on the sustained activation of the MAPK and phosphatidylinositol 3-kinase/Akt/mTOR signaling pathways. Thus, our data suggest that Shh signaling contributes to tumor initiation in the pancreas through at least two mechanisms and additionally enhances tumor cell resistance to therapeutic intervention. Collectively, our findings demonstrate crucial roles for Shh signaling in multiple stages of pancreatic carcinogenesis.
Loss of the Par-1b/MARK2 polarity kinase leads to increased metabolic rate, decreased adiposity, and insulin hypersensitivity
in vivo
Authors: Hurov Jonathan B. , Huang Mei , White Lynn S. , Lennerz Jochen , Choi Cheol Soo , Cho You-Ree , Kim Hyo-Jeong , Prior Julie L. , Piwnica-Worms David , Cantley Lewis C. , Kim Jason K. , Shulman Gerald I. , Piwnica-Worms Helen
Published: March 2007

Obesity is a major factor central to the development of insulin resistance and type 2 diabetes. The identification and characterization of genes involved in regulation of adiposity, insulin sensitivity, and glucose uptake are key to the design and development of new drug therapies for this disease. In this study, we show that the polarity kinase Par-1b/MARK2 is required for regulating glucose metabolism
in vivo
. Mice null for Par-1b were lean, insulin hypersensitive, resistant to high-fat diet-induced weight gain, and hypermetabolic.
18
F-FDG microPET and hyperinsulinemic–euglycemic clamp analyses demonstrated increased glucose uptake into white and brown adipose tissue, but not into skeletal muscle of Par-1b null mice relative to wild-type controls. Taken together, these data indicate that Par-1b is a regulator of glucose metabolism and adiposity in the whole animal and may be a valuable drug target for the treatment of both type 2 diabetes and obesity.
Loss of blood–brain barrier integrity in the spinal cord is common to experimental allergic encephalomyelitis in knockout mouse models
Authors: Fabis Marzena J. , Scott Gwen S. , Kean Rhonda B. , Koprowski Hilary , Hooper D. Craig
Published: March 2007

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is used to model certain parameters of multiple sclerosis. To establish the relative contributions of T cell reactivity, the loss of blood–brain barrier (BBB) integrity, CNS inflammation, and lesion formation toward the pathogenesis of EAE, we assessed the incidence of EAE and these parameters in mice lacking NF-κB, TNF-α, IFN-αβ receptors, IFN-γ receptors, and inducible nitric oxide synthase. Although increased myelin oligodendrocyte glycoprotein-specific T cell reactivity was generally associated with a more rapid onset or increased disease severity, the loss of BBB integrity and cell accumulation in spinal cord tissues was invariably associated with the development of neurological disease signs. Histological and real-time RT-PCR analyses revealed differences in the nature of immune/inflammatory cell accumulation in the spinal cord tissues of the different mouse strains. On the other hand, disease severity during the acute phase of EAE directly correlated with the extent of BBB permeability. Thus, the loss of BBB integrity seems to be a requisite event in the development of EAE and can occur in the absence of important inflammatory mediators.