Normal T-cell development and immune functions in TRIM-deficient mice

The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4(+) T cells. Previous studies suggested that TRIM is an integral component of the T-cell receptor (TCR)/CD3 complex and might be involved in regulating TCR cycling. To elucidate the in vivo function of TRIM, we generated TRIM-deficient mice by homologous recombination. TRIM(-/-) mice develop normally and are healthy and fertile. However, the animals show a mild reduction in body weight that appears to be due to a decrease in the size and/or cellularity of many organs. The morphology and anatomy of nonlymphoid as well as primary and secondary lymphoid organs is normal. The frequency of thymocyte and peripheral T-cell subsets does not differ from control littermates. In addition, a detailed analysis of lymphocyte development revealed that TRIM is not required for either positive or negative selection. Although TRIM(-/-) CD4(+) T cells showed an augmented phosphorylation of the serine/threonine kinase Akt, the in vitro characterization of peripheral T cells indicated that proliferation, survival, activation-induced cell death, migration, adhesion, TCR internalization and recycling, TCR-mediated calcium fluxes, tyrosine phosphorylation, and mitogen-activated protein family kinase activation are not affected in the absence of TRIM. Similarly, the in vivo immune response to T-dependent and T-independent antigens as well as the clinical course of experimental autoimmune encephalomyelitis, a complex Th1-mediated autoimmune model, is comparable to that of wild-type animals. Collectively, these results demonstrate that TRIM is dispensable for T-cell development and peripheral immune functions. The lack of an evident phenotype could indicate that TRIM shares redundant functions with other transmembrane adaptors involved in regulating the immune response.     

The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration

The model bryophyte Physcomitrella patens exhibits high frequencies of gene targeting when transformed with DNA constructs containing sequences homologous with genomic loci. ‘Targeted gene replacement’ (TGR) resulting from homologous recombination (HR) between each end of a targeting construct and the targeted locus occurs when either single or multiple targeting vectors are delivered. In the latter instance simultaneous, multiple, independent integration of different transgenes occurs at the targeted loci. In both single gene and ‘batch’ transformations, DNA can also be found to undergo ‘targeted insertion’ (TI), integrating at one end of the targeted locus by HR with one flanking sequence of the vector accompanied by an apparent non-homologous end-joining (NHEJ) event at the other. Untargeted integration at nonhomologous sites also occurs, but at a lower frequency. Molecular analysis of TI at a single locus shows that this occurs as a consequence of concatenation of the transforming DNA, in planta, prior to integration, followed by HR between a single site in the genomic target and two of its repeated homologues in the concatenated vector. This reinforces the view that HR is the major pathway by which transforming DNA is integrated in Physcomitrella.     

A 1H STD NMR spectroscopic investigation of sialylnucleoside mimetics as probes of CMP-Kdn synthetase

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and CTP to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP _i . Saturation Transfer Difference (STD) NMR spectroscopy has been employed to investigate the sub-structural requirements of the enzyme’s binding domain. Sialylnucleoside mimetics, where the sialic acid moiety has been replaced by a carboxyl group and a hydrophobic moiety, have been used in NMR experiments, to probe the tolerance of the CMP-Kdn synthetase to such replacements. From our data it would appear that unlike another sialylnucleotide-recognising protein, the CMP-Neu5Ac transport protein, either a phosphate group or other functional groups on the sialic acid framework may play important roles in recognition by the synthetase. Dedicated to the memory of Professor Dr Yasuo Inoue     

Structural analysis of a designed inhibitor complexed with the hemagglutinin-neuraminidase of Newcastle disease virus

Viruses of the Paramyxoviridae family are the leading cause of respiratory disease in children. The human parainfluenza viruses (hPIV) are members of the Paramyxovirinae subfamily, which also includes mumps virus, Newcastle disease virus (NDV), Sendai virus (SV) and simian type 5 virus (SV5). On the surface of these viruses is the glycoprotein hemagglutinin-neuraminidase (HN), which is responsible for cell attachment, promotion of fusion and release of progeny virions. This multifunctional nature of HN makes it an attractive target for the development of inhibitors as a treatment for childhood respiratory diseases. Here we report the crystal structure of NDV HN in complex with a derivative of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, Neu5Ac2en, that has a functional group designed to occupy a large conserved binding pocket around the active site. The purpose of this study was to examine the effect of a bulky hydrophobic group at the O4 position of Neu5Ac2en, given the hydrophobic nature of the binding pocket. This derivative, with a benzyl group added to the O4 position of Neu5Ac2en, has an IC₅₀ of ∼10 μM in a neuraminidase assay against hPIV3 HN. The IC₅₀ value of the parent compound, Neu5Ac2en, in the same assay is ∼25 μM. These results highlight the striking difference between the influenza neuraminidase and paramyxovirus HN active sites, and provide a platform for the development of improved HN inhibitors.     

High-throughput trapping of secretory pathway genes in mouse embryonic stem cells

High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.     

Influence of the facial ruff on the sound-receiving characteristics of the barn owl’s ears

The barn owl, a nocturnal predator, derives its German name (“Schleiereule”, direct English translation “veil owl”) from the conspicuous ruff that covers the ear openings and gives the head a face-like appearance. The ruff is a specialization for the perception of sound. The densely-ramified reflector feathers forming the border of the ruff direct sound to the ear-openings. We studied the influence of the ruff on the behaviorally relevant sound-localization parameters interaural time difference (ITD) and interaural level difference (ILD). The directionality of the ear was much greater when the ruff was intact than when the reflector feathers were removed. With ruff intact, the distribution of ILDs was oblique and the maximum ITD occurred around 110° of azimuth. When all head feathers were removed, the steepest ILD gradient was much closer to the horizontal axis and ITD was maximal at 90°. Many effects were frequency specific. Thus, the ruff reflects some properties of the human pinna. However, by shifting the point where ITD becomes maximal beyond 90°, the ruff also introduces a break of the front–back symmetry of ITD.     

Comparison of Bacteriocins Produced by Lactic-Acid Bacteria Isolated from Boza, a Cereal-Based Fermented Beverage from the Balkan Peninsula

Boza is a low-pH and low-alcohol cereal-based beverage produced in the Balkan Peninsula. From a total population of 9 × 10⁶ colony-forming units ml⁻¹, four isolates (JW3BZ, JW6BZ, JW11BZ, and JW15BZ) produced bacteriocins active against a broad spectrum of Gram-positive bacteria. Bacteriocin JW15BZ inhibited the growth of Klebsiella pneumoniae. The producer strains were identified as Lactobacillus plantarum (strains JW3BZ and JW6BZ) and L. fermentum (strains JW11BZ and JW15BZ). The spectrum of antimicrobial activity, characteristics, and mode of action of these bacteriocins were compared with bacteriocins previously described for lactic-acid bacteria isolated from boza.     

Comparison of four methods for measuring osmotic potential of tree leaves

Osmotic potential (π) of tree leaves is usually determined by pressure-volume (PV) analysis, which is too time consuming for large-scale assessments such as ecological surveys and genetic screening. We investigated methods for rapid measurement of π in sclerophyllous species. The osmotic potential of expressed sap (ES), freeze-thaw leaf discs and hot water extracts – measured using a variety of techniques – were compared with PV analysis using sclerophyllous species; Eucalyptus globulus Labill. , E . camaldulensis Dehnh. , E . cladocalyx F. Muell. and non-sclerophyllous species; Populus nigra L and Alnus jorullensis H.B.K. All methods were sensitive to changes in π and produced results that were unequal but well correlated (r² 0.82–0.90). The proportion of leaf water extracted in ES was reduced by low water content and sclerophylly without affecting method accuracy. PV analysis provided a poor correction for apoplastic dilution of ES. Results of ES osmometry and PV analysis were comparable, and osmometry of ES is proposed as a suitable method for large-scale investigations of leaf π.     

Positional relationships between photoperiod response QTL and photoreceptor and vernalization genes in barley

Winterhardiness has three primary components: photoperiod (day length) sensitivity, vernalization response, and low temperature tolerance. Photoperiod and vernalization regulate the vegetative to reproductive phase transition, and photoperiod regulates expression of key vernalization genes. Using two barley mapping populations, we mapped six individual photoperiod response QTL and determined their positional relationship to the phytochrome and cryptochrome photoreceptor gene families and the vernalization regulatory genes HvBM5A, ZCCT-H, and HvVRT-2. Of the six photoreceptors mapped in the current study (HvPhyA and HvPhyB to 4HS, HvPhyC to 5HL, HvCry1a and HvCry2 to 6HS, and HvCry1b to 2HL), only HvPhyC coincided with a photoperiod response QTL. We recently mapped the candidate genes for the 5HL VRN-H1 (HvBM5A) and 4HL VRN-H2 (ZCCT-H) loci, and in this study, we mapped HvVRT-2, the barley TaVRT-2 ortholog (a wheat flowering repressor regulated by vernalization and photoperiod) to 7HS. Each of these three vernalization genes is located in chromosome regions determining small photoperiod response QTL effects. HvBM5A and HvPhyC are closely linked on 5HL and therefore are currently both positional candidates for the same photoperiod effect. The coincidence of photoperiod-responsive vernalization genes with photoperiod QTL suggests vernalization genes should also be considered candidates for photoperiod effects.