The structure of beta-lactoglobulin and its similarity to plasma retinol-binding protein

Since its first isolation, bovine beta-lactoglobulin (BLG) has been an enigma: although it is abundant in the whey fraction of milk, its function is still not clear. The results of the many physicochemical studies on the protein need a structural interpretation. We report here the structure of the orthorhombic crystal form of cow BLG at pH 7.6, at a resolution of 2.8 A. It has an unusual protein fold, composed of two slabs of antiparallel beta-sheet, which shows a remarkable similarity to plasma retinol-binding protein. A possible binding site for retinol in BLG has been identified by model-building. This suggests a role for BLG in vitamin A transport and we have discovered specific receptors for the BLG-retinol complex in the intestine of neonate calves.     

Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T1/Leu-1

The T1/Leu-1/CD5 molecule, a human T-cell surface glycoprotein of relative molecular mass (Mr) 67,000, has been implicated in the proliferative response of activated T cells and in T-cell helper function. A similar involvement in T-cell proliferation has been reported for Ly-1, the murine homologue of T1. Here we report the complete amino-acid sequence of the T1 precursor molecule deduced from complementary DNA clones. The protein contains a classical signal peptide; a 347-amino-acid extracellular segment; a transmembrane region; and a 93-amino-acid intracellular segment. The extracellular segment contains many cysteine residues and is composed of two related structural domains separated by a proline/threonine-rich region. The T1 molecule has structural features characteristic of other receptor molecules.     

A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria

Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA 'exoprotein' onto the cell surface. An additional small immunity protein (CdiI) protects CDI(+) cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E.?coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI(+) cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.     

Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III

Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.     

Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice

Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X?chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X?chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X?chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X?chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.     

Reactive oxygen species produced by NADPH oxidase regulate plant cell growth

Cell expansion is a central process in plant morphogenesis, and the elongation of roots and root hairs is essential for uptake of minerals and water from the soil. Ca2+ influx from the extracellular store is required for (and sets the rates of) cell elongation in roots. Arabidopsis thaliana rhd2 mutants are defective in Ca2+ uptake and consequently cell expansion is compromised--rhd2 mutants have short root hairs and stunted roots. To determine the regulation of Ca2+ acquisition in growing root cells we show here that RHD2 is an NADPH oxidase, a protein that transfers electrons from NADPH to an electron acceptor leading to the formation of reactive oxygen species (ROS). We show that ROS accumulate in growing wild-type (WT) root hairs but their levels are markedly decreased in rhd2 mutants. Blocking the activity of the NADPH oxidase with diphenylene iodonium (DPI) inhibits ROS formation and phenocopies Rhd2-. Treatment of rhd2 roots with ROS partly suppresses the mutant phenotype and stimulates the activity of plasma membrane hyperpolarization-activated Ca2+ channels, the predominant root Ca2+ acquisition system. This indicates that NADPH oxidases control development by making ROS that regulate plant cell expansion through the activation of Ca2+ channels.     

Revised rates for the stellar triple-alpha process from measurement of 12C nuclear resonances

In the centres of stars where the temperature is high enough, three alpha-particles (helium nuclei) are able to combine to form 12C because of a resonant reaction leading to a nuclear excited state. (Stars with masses greater than approximately 0.5 times that of the Sun will at some point in their lives have a central temperature high enough for this reaction to proceed.) Although the reaction rate is of critical significance for determining elemental abundances in the Universe, and for determining the size of the iron core of a star just before it goes supernova, it has hitherto been insufficiently determined. Here we report a measurement of the inverse process, where a 12C nucleus decays to three alpha-particles. We find a dominant resonance at an energy of approximately 11 MeV, but do not confirm the presence of a resonance at 9.1 MeV (ref. 3). We show that interference between two resonances has important effects on our measured spectrum. Using these data, we calculate the triple-alpha rate for temperatures from 10(7) K to 10(10) K and find significant deviations from the standard rates. Our rate below approximately 5 x 10(7) K is higher than the previous standard, implying that the critical amounts of carbon that catalysed hydrogen burning in the first stars are produced twice as fast as previously believed. At temperatures above 10(9) K, our rate is much less, which modifies predicted nucleosynthesis in supernovae.     

A diverse range of gene products are effectors of the type I interferon antiviral response

The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.     

p53 mutant mice that display early ageing-associated phenotypes.

The p53 tumour suppressor is activated by numerous stressors to induce apoptosis, cell cycle arrest, or senescence. To study the biological effects of altered p53 function, we generated mice with a deletion mutation in the first six exons of the p53 gene that express a truncated RNA capable of encoding a carboxy-terminal p53 fragment. This mutation confers phenotypes consistent with activated p53 rather than inactivated p53. Mutant (p53+/m) mice exhibit enhanced resistance to spontaneous tumours compared with wild-type (p53+/+) littermates. As p53+/m mice age, they display an early onset of phenotypes associated with ageing. These include reduced longevity, osteoporosis, generalized organ atrophy and a diminished stress tolerance. A second line of transgenic mice containing a temperature-sensitive mutant allele of p53 also exhibits early ageing phenotypes. These data suggest that p53 has a role in regulating organismal ageing.