Changes in cytoplasmic Ca 2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca 2+ entry involves receptor-mediated depletion of intracellular Ca 2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca 2+ sensor responsible for activation of Ca 2+ releaseactivated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca 2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca 2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex. IntroductionThe regulation of intracellular Ca 2+ ([Ca 2+ ] i ) is essentially involved in signaling processes in virtually all cells. In nonexcitable cells, including hematopoietic cells, Ca 2+ is released from the ER via inositol 1,4,5-triphosphate-mediated (IP 3 -mediated) receptor activation triggered by ligand-activated plasma membrane receptors. If the limited Ca 2+ reservoir of the ER becomes exhausted, extracellular Ca 2+ enters the cytoplasm by a mechanism known as store-operated Ca 2+ entry (SOCE) (1, 2). Although electrophysiologically well defined for more than a decade, the molecular identity of the pivotal proteins undoubtedly involved in SOCE has been discovered only recently. Stromal interaction molecule 1 (Stim1) is an ER resident protein necessary for the detection of ER Ca 2+ depletion (3-6). The 4-transmembrane domain protein Orai1, or CRACM, was reported recently to confer SOC activity (4,(7)(8)(9)(10)(11)(12). In T cells, Orai1 appears to be the predominant SOC (9), despite the fact that the C-terminal region of Stim1 has been shown to also interact with other SOC candidates such as transient receptor potential channels (TRPCs) 1,

Bordetella pertussis is readily killed after uptake by professional phagocytes, whereas its close relative Bordetella bronchiseptica is not and can persist intracellularly for days. Phagocytosis of members of either species by a mouse macrophage cell line results in transport of the bacteria to a phagosomal compartment positive for the lysosome-associated membrane protein 1, the protease cathepsin D, and the late endosomal vacuolar proton-pumping ATPase but negative for the early endosome antigen 1 and the early endosomal transferrin receptor. In addition, we demonstrate that Bordetella-containing phagosomes rapidly acidify to pH 4.5 to 5.0. Taken together, these data demonstrate that Bordetella-containing phagosomes rapidly mature to an acidic late endosomal/lysosomal compartment. Following up on this observation, we determined that B. pertussis does not survive in bacterial growth media adjusted to a pH of 4.5, whereas this pH has only minor effects on the growth of B. bronchiseptica. Raising the intracellular pH in infected macrophages by the addition of bafilomycin A 1 , ammonium chloride, or monensin increases the survival of acid-sensitive B. pertussis but, surprisingly, decreases that of acid-tolerant B. bronchiseptica. In summary, we hypothesize that the differential survival of B. pertussis and B. bronchiseptica in macrophages is, at least in part, due to the differences in their acid tolerance.Bordetella pertussis is the causative agent of whooping cough (27). Its close relative Bordetella bronchiseptica causes infections of the respiratory tract in a variety of mammals and occasionally in humans (18, 63). Although they were previously considered to be extracellular pathogens, several recent reports have indicated significant cell invasive properties of these bacteria, e.g., for various typically nonphagocytic epithelial cell types (11,32,54,55). However, the bacterial factors involved in the uptake of either species appear to be different, because invasion by B. pertussis depends on the presence of factors transcriptionally activated by the BvgAS two-component system (11,32), the master regulator of virulence in these bacteria (5), whereas invasion by B. bronchiseptica was shown to occur independently of these factors (54,55).While the invasion of epithelial cells requires dedicated bacterial features, these are not necessary for the uptake by professional phagocytes, such as macrophages. When macrophages ingest bacteria, they wrap them with their plasma membrane and incorporate the newly formed so-called phagosomes. Phagosomes are not static organelles, but structures which undergo several maturation steps that transform the newly formed phagosomes into phagolysosomes. In detail, phagosome maturation is characterized by the sequential acquisition and loss of early endosomal, late endosomal, and lysosomal structural and compositional features (9, 10). The transition of an "early phagosome" into a phagolysosome is also accompanied by the exposure of the ingested bacteria to a number of potentially bact...

The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the char- (29): (i) the dehydrogenase variant in which the intermediate tetrahydrodipicolinate (THDP) common to all three pathways is converted in a single step to DAP, (ii) the succinylase variant involving two succinylated intermediates, and (iii) the acetylase variant using the acetyl residue instead of succinyl as the blocking group (Fig. 1).In the succinylase pathway, THDP is converted by the succinyltransferase (DapD) to N-succinyl-2-amino-6-ketopimelate, which is the substrate of the aminotransferase DapC. Its product, N-succinyl-L,L-DAP, is converted by DapE, a desuccinylase, to the common product of both the succinylase and acetylase pathways, L,L-DAP (27). The acetylase and/or dehydrogenase pathways are found among members of the genus Bacillus (38), while the succinylase pathway is present in Escherichia coli (18). In Corynebacterium glutamicum, both the succinylase and dehydrogenase pathways can operate in D,L-DAP and L-lysine biosynthesis (30). This high variability and flexibility of DAP pathways might ensure the availability of a sufficient amount of meso-DAP for cell wall synthesis under different environmental conditions (37). In addition to the vital role of DAP in the cross-linking of the glycan backbones in the bacterial cell wall and in providing lysine for protein biosynthesis, DAP is a central constituent in the Bordetella pertussis tracheal cytotoxin, which is an important virulence factor that causes several pathological effects in epithelial cells (7,21).Since DAP is neither produced nor required by humans, many efforts have been made to study DAP biosynthetic enzymes (8,24,28), and DAP analogs are evaluated for their potential to inhibit bacterial growth. Furthermore, the use of DAP auxotrophic mutants of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Salmonella subspecies, or Helicobacter pylori as attenuated vaccine strains or for the maintenance of cloning vectors expressing foreign antigens in such attenuated strains has been proposed (9, 17, 23). Although the biochemistry of the DAP-lysine pathway is very well understood, the genes encoding enzymes involved in this pathway have not been completely characterized. Indeed, only three out of the four genes required for the succinyl pathway of E. coli, dapD, dapE, and dapF, encoding THDP succinylase, and ...

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.

Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl-N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.