Social factors and the neurobiology of pathogen avoidance

Although the evolutionary causes and consequences of pathogen avoidance have been gaining increasing interest, there has been less attention paid to the proximate neurobiological mechanisms. Animals gauge the infection status of conspecifics and the threat they represent on the basis of various sensory and social cues. Here, we consider the neurobiology of pathogen detection and avoidance from a cognitive, motivational and affective state (disgust) perspective, focusing on the mechanisms associated with activating and directing parasite/pathogen avoidance. Drawing upon studies with laboratory rodents, we briefly discuss aspects of (i) olfactory-mediated recognition and avoidance of infected conspecifics; (ii) relationships between pathogen avoidance and various social factors (e.g. social vigilance, social distancing (approach/avoidance), social salience and social reward); (iii) the roles of various brain regions (in particular the amygdala and insular cortex) and neuromodulators (neurotransmitters, neuropeptides, steroidal hormones and immune components) in the regulation of pathogen avoidance. We propose that understanding the proximate neurobiological mechanisms can provide insights into the ecological and evolutionary consequences of the non-consumptive effects of pathogens and how, when and why females and males engage in pathogen avoidance.     

A regulatory role for CD37 in T cell proliferation

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.     

Mouse models of K-ras-initiated carcinogenesis

Activating mutations of the oncogene K-ras are found in one third of all human cancers. Much of our knowledge on K-ras signal transduction and its influence on tumor initiation and progression comes from in vitro studies with cell lines. However, mouse models of human cancer allow a much more faithful recapitulation of the human disease, and the in vivo perspective is crucial for our understanding of neoplasia. In recent years, several new murine models for K-ras-induced tumorigenesis have been described. They allow new insights into the specific role that oncogenic K-ras proteins play in different solid tumors, and they permit the molecular dissection of the pathways that are initiated by somatic mutations in subsets of cells. Key advances have been made by the use of tissue-specific and inducible control of expression, which is achieved by the Cre/LoxP technology or the tetracycline system. from these sophisticated models, a common picture emerges: The effects of K-ras on tumor initiation depend strongly on the cellular context, and different tissues vary in their susceptibility to K-ras transformation.     

Improved photobiological H2 production in engineered green algal cells

Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e-), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e- to drive hydrogen (H2) production via the chloroplast hydrogenases HydA1 and A2 (H2 ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H2 production in Chlamydomonas, we have developed a new approach to increase H+ and e- supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e- transfer around photosystem I, eliminating possible competition for e- with H2ase. Selected strains were further screened for increased H2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves (i.e. enhanced substrate availability), and a low dissolved O2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H2 production rates of Stm6 were 5-13 times that of the control WT strain over a range of conditions (light intensity, culture time, +/- uncoupler). Typically, approximately 540 ml of H2 liter(-1) culture (up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h(-1) (efficiency = approximately 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H2 production systems.     

Gene disruption of Spred-2 causes dwarfism

The impact of the fibroblast growth factor receptor 3 (FGFR3)-mediated signaling pathway on bone growth has been demonstrated by various genetic approaches. Overexpression of fibroblast growth factors (FGFs), several gain-of-function mutations in the FGFR3, and constitutive activation of mitogen-activated protein kinase (MAPK) kinase (MEK1) in chondrocytes have been shown to cause dwarfism in mice by activation of the MAPK signaling pathway. To investigate the previously reported inhibitory role of Spred in the FGFR3/MAPK pathway, we generated mice with a trapped Spred-2 gene. Here we show that lack of functional Spred-2 protein in mice caused a dwarf phenotype, similar to achondroplasia, the most common form of human dwarfism. Spred-2(-/-) mice showed reduced growth and body weight, they had a shorter tibia length, and showed narrower growth plates as compared with wild-type mice. We detected promoter activity and protein expression of Spred-2 in chondrocytes, suggesting an important function of Spred-2 in chondrocytes and bone development. Stimulation of chondrocytes with different FGF concentrations showed earlier and augmented ERK phosphorylation in Spred-2(-/-) chondrocytes in comparison to Spred-2(+/+) chondrocytes. Our observations suggest a model in which loss of Spred-2 inhibits bone growth by inhibiting chondrocyte differentiation through up-regulation of the MAPK signaling pathway.     

Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the Gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity. Received: 19 November 1999 / Accepted: 21 December 1999     

The 1.1 A resolution crystal structure of the p130cas SH3 domain and ramifications for ligand selectivity

The Crk-associated tyrosine kinase substrate p130cas (CAS) is a docking protein containing an SH3 domain near its N terminus, followed by a short proline-rich segment, a large central substrate domain composed of 15 repeats of the four amino acid sequence YxxP, a serine-rich region and a carboxy-terminal domain, which possesses consensus binding sites for the SH2 and SH3 domains of Src (YDYV and RPLPSPP, respectively). The SH3 domain of CAS mediates its interaction with several proteins involved in signaling pathways such as focal adhesion kinase (FAK), tyrosine phosphatases PTP1B and PTP-PEST, and the guanine nucleotide exchange factor C3G. As a homolog of the corresponding Src docking domain, the CAS SH3 domain binds to proline-rich sequences (PxxP) of its interacting partners that can adopt a polyproline type II helix. We have determined a high-resolution X-ray structure of the recombinant human CAS SH3 domain. The domain, residues 1-69, crystallized in two related space groups, P2(1) and C222(1), that provided diffraction data to 1.1 A and 2.1 A, respectively. The crystal structure shows, in addition to the conserved SH3 domain architecture, the way in which the CAS characteristic amino acids form an atypically charged ligand-binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by the CAS SH3 domain. The structure enables modelling of the docking interactions to its ligands, for example from focal adhesion kinase, and supports structure-based drug design of inhibitors of the CAS-FAK interaction.     

Impaired trafficking of mutants of lysosomal glucocerebrosidase in Gaucher's disease

Gaucher's disease is the most inherited lysosomal storage disorder. Except for a few cases, the broad phenotypic heterogeneity of Gaucher's disease can be neither predicted from defined mutations nor from differences in residual enzyme activity. Here, we analyse the intracellular trafficking of glucocerebrosidase as an underlying mechanism for the expression of the clinical phenotype. Biosynthetic labeling studies combined with immunofluorescence analyses with fibroblasts from patients with the defined mutations N370S, L444P, D409H and G202R unequivocally demonstrate a retarded transport of glucocerebrosidase carrying the mutation N370S and a transport block in the ER of the enzyme with the mutations G202R, L444P and D409H. We asked whether cellular components in the patients' fibroblasts other than glucocerebrosidase are implicated in the onset of the disease. For this, mutant cDNA's corresponding to the phenotypes N370S, G202R and L444P were expressed in the mouse fibroblasts NIH3T3. Essentially similar biochemical and cellular features were revealed as compared to the patients' fibroblasts strongly suggesting that these mutations are exclusively responsible for the characterized phenotypes. Interestingly, the immunoglobulin binding protein (BiP) binds wild type and the mutant N370S but not the G202R and L444P variants suggesting a discriminatory role played by this chaperone associated with the severity of the disease.     

Recent highlights on photolithic oligonucleotide array in situ synthesis

Light directed synthesis of high-density oligonucleotide microarrays is currently performed using either ortho-nitro-benzyl-type [MeNPOC] (Pease, A.C.; Solas, D.; Sullivan, E.J.; Cronin, T.M.; Holmes, C.P.; Fodor, S.P.A. Proc. Natl. Acad Sci U.SA. 1994, 91, 6333.) or ortho-nitrophenylethyl-type [NPPOC] (Hasan, A.; Stengele, K.P.; Giegrich, H.; Cornwell, P.; Isham, K.R.; Sachleben, R.A.; Pfleiderer, W.; Foote, R.S. Tetrahedron 1997, 53, 424Z) protecting groups as the 5'-O-carbonate ester of the phosphoramidite building block. The synthesis cycle uses a combinatorial approach attaching one specific base per cycle, thus as many as 100 cycles need to be run to make an array of 25-mers. Time needed for deprotection/activation of the growing oligo chain determines overall manufacturing time and consequently also cost. In this report we demonstrate the development of photoprotected posphoramidite monomers for light directed array synthesis with increasing sensitivity to the UV light used. If combined with maskless array synthesis, this technology allows for synthesis of arrays with >780,000 different 25-mer oligonucleotides in about one hour and allows for high flexibility in array design and reiterative redesign. The arrays synthesized show high quality and reproducibility in our standard hybridization based assay.