TIP,a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.     

The gel-forming behaviour of dextran in the presence of KCl: a quantitative 13C and pulsed field gradient (PFG) NMR study

Although the gel forming ability of certain polysaccharides in the presence of ions is a well-known phenomenon, detailed physicochemical mechanisms of such processes are still unknown. In this investigation high resolution 13C NMR as well as 1H pulsed field gradient (PFG) NMR were used to investigate the mobility of dextran in the sol and in the gel state. Gel-formation of dextran can be easily induced by the addition of large amounts of potassium chloride. No major differences in the T(1) relaxation times of dextran in the sol and in the gel state could be observed. Accordingly, the analysis of the 13C NMR spectroscopic data did not provide any indication of an observable line-broadening upon gel-formation. However, a KCl concentration dependent decrease of signal intensity in comparison to an internal standard was detected. On the other hand, the PFG NMR studies clearly indicated a gradual diminution of the self-diffusion coefficient of the dextran with increasing molecular weight as well as in the presence of potassium chloride. These measurements revealed in agreement with spectroscopic data that at least one potassium ion per monomer subunit (i.e. one glycopyranose residue) is necessary for gel formation.     

Assessing tillage disturbance on assemblages of ground beetles (Coleoptera: Carabidae) by using a range of ecological indices

Many ecological studies have used diversity indices to assess the impact of environmental disturbance. In particular, ground beetles have been advocated as a good group for assessing disturbance. Most studies on various organisms have used only one or two indices. For our study of the impact of tillage disturbance on carabid beetles in farm fields in southern Ontario, Canada, we used seven different diversity indices (richness, Shannon–Wiener, Berger–Parker, Q-statistic, Margalef, and evenness). Few studies have used deviation from diversity abundance models as a measure of disturbance; however, we use three that are applicable to our data (geometric, log-normal and log-series). The indices and models were used to test the null hypothesis that there is no change in diversity with increasing tillage disturbance, and that there is no difference in diversity with different crops or years. We were not able to reject the null hypothesis that there is any diversity difference among farms. We also found that there was no single diversity index or model that was better than any other at detecting disturbance. These results are supplemented by a meta-analysis of 45 published data sets for the same taxon but in different habitats. The meta-analysis supports the conclusions from our field research that diversity indices and models are not useful for detecting the possible effect of disturbance on assemblages of carabid beetles.     

Bcl-2 is a monomeric protein: prevention of homodimerization by structural constraints

The pro-apoptotic activity of the Bcl-2 family member Bax has been shown to be facilitated by homodimerization. However, it is unknown whether Bcl-2 or Bcl-x(L) have to homodimerize to protect cells from apoptosis. Here we show by co-immunoprecipitation and FPLC analyses that while Bax multimerizes and forms heterodimers with Bcl-2, there is no evidence for Bcl-2 homodimerization, even in conditions under which Bcl-2 protects cells from apoptosis. Immunofluorescence studies confirmed that Bax can attract active, soluble Bcl-2 to mitochondrial membranes, but that nuclear/ER membrane-bound Bcl-2 was incapable of dislocating soluble Bcl-2. The failure of Bcl-2 to homodimerize is due to structural constraints as versions of Bcl-2 deleted or mutated in the BH1 and BH2 domains effectively dimerized with wild-type Bcl-2 and were dislocated by Bcl-2 inside cells. These data indicate that naturally occurring Bcl-2 does not expose protein domains that mediate homodimerization and therefore most likely acts as a monomer to protect cells from apoptosis.     

Purification and characterization of perlucin and perlustrin, two new proteins from the shell of the mollusc Haliotis laevigata

Two new proteins, named perlucin and perlustrin, with M(r) 17,000 and 13,000, respectively, were isolated from the shell of the mollusc Halotis laevigata (abalone) by ion-exchange chromatography and reversed-phase HPLC after demineralization of the shell in 10% acetic acid. The sequence of the first 32 amino acids of perlucin indicated that this protein belonged to a heterogeneous group of proteins consisting of a single C-type lectin domain. Perlucin increased the precipitation of CaCO(3) from a saturated solution, indicating that it may promote the nucleation and/or the growth of CaCO(3) crystals. With pancreatic stone protein (lithostathine) and the eggshell protein ovocleidin 17, this is the third C-type lectin domain protein isolated from CaCO(3) biominerals. This indicates that this type of protein performs an important but at present unrecognized function in biomineralization. Perlustrin was a minor component of the protein mixture and the sequence of the first 33 amino acids indicated a certain similarity to part of the much larger nacre protein lustrin A.     

Cutting edge: contribution of NK cells to the homing of thymic CD4+NKT cells to the liver

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.     

Helicobacter pylori infection in wild-type and cytokine-deficient C57BL/6 and BALB/c mouse mutants

Helicobacter pylori causes gastroduodenal ulcer disease in humans. T lymphocytes and their cytokines are thought to play a substantial role in the control of H. pylori infection. To determine the importance of T helper (Th) cytokines and background genes we investigated the natural course of H. pylori infection in BALB/c and C57BL/6 wild-type or mutant mice deficient for either interleukin (IL)-4 or interferon (IFN)-gamma. H. pylori SPM 326 persisted for at least six months in C57BL/6 but was cleared by BALB/c wild-type mice nine weeks postinfection. H. pylori was recovered more frequently from IFN-gamma(-/-) BALB/c and IFN-gamma( -/-) C57BL/6 mice than from the respective wild-type animals. In contrast, IL-4 deficiency had no detectable effect on H. pylori recovery rates from either strain of mice. Our data suggest a protective role of IFN-gamma by mediating inflammation in murine H. pylori infection. In addition, our data emphasize that background genes which differ between BALB/c and C57BL/6 mice regulate the clearance of H. pylori.     

Different signaling pathways are involved in CCK(B) receptor-mediated MAP kinase activation in COS-7 cells

Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCK(B)) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCK(B) receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCK(B) receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kgamma did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species alpha, beta or/and delta in signal transduction via CCK(B) receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCK(B)-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.     

The initiator element of the Drosophila beta2 tubulin gene core promoter contributes to gene expression in vivo but is not required for male germ-cell specific expression

The tissue-specific expression of the Drosophila β2 tubulin gene (B2t) is accomplished by the action of a 14-bp activator element (β2UE1) in combination with certain regulatory elements of the TATA-less, Inr-containing B2t core promoter. We performed an in vivo analysis of the Inr element function in the B2t core promoter using a transgenic approach. Our experiments demonstrate that the Inr element acts as a functional cis-regulatory element in vivo and quantitatively regulates tissue-specific reporter expression in transgenic animals. However, our mutational analysis of the Inr element demonstrates no essential role of the Inr in mediating tissue specificity of the B2t promoter. In addition, a downstream element seems to affect promoter activity in combination with the Inr. In summary, our data show for the first time the functionality of the Inr element in an in vivo background situation in Drosophila.