A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP

Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.     

Immunotherapeutic potential of whole tumour cells

Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.     

Crystal structure of a tetradecameric assembly of the association domain of Ca2+/calmodulin-dependent kinase II

We report the crystal structure of the 143 residue association domain of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The association domain forms a hub-like assembly, composed of two rings of seven protomers each, which are stacked head to head and held together by extensive interfaces. The tetradecameric organization of the assembly was confirmed by analytical ultracentrifugation and multiangle light scattering. Individual protomers form wedge-shaped structures from which N-terminal helical segments that connect to the kinase domain extend toward the equatorial plane of the assembly, consistent with the arrangement of the kinase domains in a second outer ring. A deep and highly conserved pocket present within the association domain may serve as a docking site for proteins that interact with CaMKII.     

Glucose kinetics and exercise performance during phases of the menstrual cycle: effect of glucose ingestion

To study the effect of menstrual cycle phase and carbohydrate ingestion on glucose kinetics and exercise performance, eight healthy, moderately trained, eumenorrheic women cycled at 70% of peak O(2) consumption for 2 h and then performed a 4 kJ/kg body wt time trial. A control (C) and a glucose ingestion (G) trial were completed during the follicular (F) and luteal (L) phases of the menstrual cycle. Plasma substrate concentrations were similar before the commencement of exercise. Glucose rates of appearance and disappearance were higher (P < 0.05) during the 2nd h of exercise in FC than in LC. The percent contribution of carbohydrate to total energy expenditure was greater in FC than in LC, and subjects performed better (13%, P < 0.05) in FC. Performance improved (19% and 26% in FG and LG compared with FC and LC, respectively, P < 0.05) with the ingestion of glucose throughout exercise. These data demonstrate that substrate metabolism and exercise performance are influenced by the menstrual cycle phase, but ingestion of glucose minimizes these effects.     

Gene Splicing of an Invertebrate Beta Subunit (LCavβ) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels

The accessory beta subunit (Ca_vβ) of calcium channels first appear in the same genome as Ca_v1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Ca_vβ subunits (β₁, β₂, β₃, β₄) which associate with four Ca_v1 channel isoforms (Ca_v1.1 to Ca_v1.4) and three Ca_v2 channel isoforms (Ca_v2.1 to Ca_v2.3). Here we assess the fundamentally-shared features of the Ca_vβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCa_v1, LCa_v2, and LCa_vβ). Invertebrate Ca_vβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Ca_v2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCa_vβ subunit. LCa_vβ will also slow the inactivation kinetics of LCa_v3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Ca_vβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa_vβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Ca_v2.2 and Ca_vβ₃ subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCa_v2 nor LCa_vβ) appears to be compatible with this observed property.     

Assessment of plants from the Brassicaceae family as genetic models for the study of nickel and zinc hyperaccumulation

We report on the second phase of a programme to select a relative of Arabidopsis thaliana for use in large-scale molecular genetic studies of nickel (Ni) and zinc (Zn) hyperaccumulation. We also report on the relatedness among Thlaspi caerulescens accessions and the utility of using O-acetyl-L-serine as a marker for Ni and Zn hyperaccumulation potential. Twenty-seven new accessions of metal-accumulating species collected in the Czech Republic, France, Greece, Italy, Slovenia and the USA during Spring-Summer 2002 were evaluated. The criteria established for selection were hyperaccumulation of metals (Ni and Zn); compact growth habit; reasonable time to flowering; production of > or = 1000 seeds per plant; self-fertility; compact diploid genome; high sequence similarity to A. thaliana; > or = 0.1% transformation efficiency with easy selection. We conclude that the best candidate identified in the first phase was the best candidate overall: T. caerulescens accession St Félix de Pallières.     

Spite and the scale of competition in Pseudomonas aeruginosa

Scale of competition has been shown to be an important factor in shaping the evolution of social interactions. Although many theoretical and experimental studies have examined its effect on altruistic cooperation, relatively little research effort has been focused on spiteful behaviors--actions that harm both the actor and the recipient. In this study, we expand on existing theory by investigating the importance of the global frequency of spiteful alleles, and we determine experimentally how the scale of competition affects selection for spite in the bacterial pathogen Pseudomonas aeruginosa under high and intermediate spatial relatedness. Consistent with our theoretical results, we found in our experiments that spiteful genotypes are more favored under local (rather than global) competition and intermediate (rather than high) spatial relatedness, conditions that have been shown to select against indiscriminate altruism.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.