Automated systems for protein crystallization

Growth of high quality crystals is often the most difficult step in the determination of protein structures by X-ray diffraction. Automation can improve the success of this process both by reducing the amount of protein required for each screen and by relieving the tedium of setting up crystallization experiments by hand. We have been using an automated system for the design and execution of hanging drop crystallization experiments for the last two years. The system includes robots for the preparation of solutions, setup of hanging drops, and automated imaging, as well as a new software package (RoCKS) for managing all phases of the crystallization process. Here, we review the fundamentals of automated protein crystallization and present results from our comparisons of various approaches to screening.     

IFN-alpha induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3

The anti-inflammatory cytokine IL-10 can be induced by type I IFNs, but the molecular mechanisms involved have remained elusive. With in silico analysis of the human IL-10 promoter we identified a module consisting of an IFN regulatory factor 1 (IRF-1) site and a Stat3 site. We demonstrate that IFN-alpha will induce the binding of IRF-1 and Stat3 to the respective motifs. Mutational analysis revealed that inactivation of the IRF-1 motif substantially reduces trans-activation from 5- to 2-fold and that inactivation of the Stat3 motif completely ablates trans-activation by IFN-alpha. The dominant role of Stat3 in this module was confirmed with the blockade of trans-activation by a dominant negative Stat3. By contrast, Stat1 contributes a minor proportion to the DNA binding to the Stat site, and overexpression will counteract Stat3-mediated trans-activation. The data show that IFN-alpha induces the IL-10 gene via a module consisting of interdependent IRF-1 and Stat3 motifs. Of note, LPS-induced trans-activation does not target this module, since it is independent of the IRF-1 motif but completely depends on Stat3.     

Critical role of the alpha 4 integrin/VCAM-1 pathway in cerebral leukocyte trafficking in lupus-prone MRL/fas(lpr) mice

MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in leukocyte recruitment to a wide range of vascular beds, including the cerebral microvasculature. The mechanisms responsible for the leukocyte trafficking to the brain in these animals are not known. Therefore, the aim of this study was to directly examine the cerebral microvasculature in MRL/fas(lpr) mice and determine the molecular mechanisms responsible for this leukocyte recruitment. Intravital microscopy was used to assess leukocyte-endothelial cell interactions (rolling, adhesion) in the pial microcirculation of MRL(+/+) (control) and MRL/fas(lpr) mice at 8, 12, and 16 wk of age. Leukocyte rolling and adhesion were rarely observed in MRL(+/+) mice of any age. MRL/fas(lpr) mice displayed similar results at 8 and 12 wk. However, at 16 wk, significant increases in leukocyte rolling and adhesion were observed in these mice. Histological analysis revealed that the interacting cells were exclusively mononuclear. Leukocyte rolling was reduced, but not eliminated in P-selectin(-/-)-MRL/fas(lpr) mice. However, leukocyte adhesion was not reduced in these mice, indicating that P-selectin-dependent rolling was not required for leukocyte recruitment to the cerebral vasculature in this model of systemic inflammation. E-selectin blockade also had no effect on leukocyte rolling. In contrast, blockade of either the alpha4 integrin or VCAM-1 eliminated P-selectin-independent leukocyte rolling. alpha4 Integrin blockade also significantly inhibited leukocyte adhesion. These studies demonstrate that the systemic inflammatory response that affects MRL/fas(lpr) mice results in leukocyte rolling and adhesion in the cerebral microcirculation, and that the alpha4 integrin/VCAM-1 pathway plays a central role in mediating these interactions.     

Transgenic mice expressing lipoprotein lipase in adipose tissue. Absence of the proximal 3'-untranslated region causes translational upregulation

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and adipocyte metabolism. Defects in LPL can lead to hypertriglyceridemia and the subsequent development of atherosclerosis. The mechanisms of regulation of this enzyme are complex and may occur at multiple levels of gene expression. Because the 3'-untranslated region (UTR) is involved in LPL translational regulation, transgenic mice were generated with adipose tissue expression of an LPL construct either with or without the proximal 3'-UTR and driven by the aP2 promoter. Both transgenic mouse colonies were viable and expressed the transgene, resulting in a 2-fold increase in LPL activity in white adipose tissue. Neither mouse colony exhibited any obvious phenotype in terms of body weight, plasma lipids, glucose, and non-esterified fatty acid levels. In the mice expressing hLPL with an intact 3'-UTR, hLPL mRNA expression approximately paralleled hLPL activity. However in the mice without the proximal 3'-UTR, hLPL mRNA was low in the setting of large amounts of hLPL protein and LPL activity. In previous studies, the 3'-UTR of LPL was critical for the inhibitory effects of constitutively expressed hormones, such as thyroid hormone and catecholamines. Therefore, these data suggest that the absence of the 3'-UTR results in a translationally unrepressed LPL, resulting in a moderate overexpression of adipose LPL activity.     

Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146

Various drugs that elevate cGMP levels and activate cGMP-dependent protein kinase (cGK) inhibit agonist-induced platelet activation. In the present study we identified the LIM and SH3 domain protein (LASP) that was recently cloned from human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C., Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK in human platelets. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed phosphorylation of LASP in vivo. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo. LASP is an actin-binding protein, and the phospho-LASP-mimicking mutant S146D showed reduced binding affinity for F-actin in cosedimentation experiments. Immunofluorescence of transfected PtK2 cells demonstrated the localization of LASP in the tips of cell membrane extensions and at cell-cell contacts. Expression of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the cells. Taken together, these data suggest that phosphorylation of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.     

Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.     

Patterns and dynamics of sex-biased dispersal in a nocturnal primate, the grey mouse lemur, Microcebus murinus

We examined predictions on the proportion of dispersing natal males and females, dispersal distances, the age at dispersal and the potential for inbreeding over a 6-year period in a free-living population of grey mouse lemurs. We used monthly mark-recapture procedures to determine individual locations and interindividual distances. The analysis of seven polymorphic microsatellite markers for 213 (130 males, 83 females) individuals allowed us to estimate relatedness coefficients and kinship relationships. Closely related males ranged further from each other than closely related females and natal males were found further from their potential mothers than were females. Natal males were more likely to disperse from their birth sites than females, although male dispersal was not universal. Male breeding dispersal was detected in half of the long-term observations. Males therefore seem to be the predominant vectors for gene flow between populations and social units. Females usually stayed within one to two home range diameters of their potential mother, facilitating the evolution of cooperative behaviour by kin selection among females. Most dispersal took place before the mating season, indicating an age of less than 7 months for natal dispersal. The analysis of spatiotemporal coexistence revealed the potential for inbreeding in only 3.8% of the potential mother-son dyads, but in 21.9% of the potential father-daughter dyads and in 41.7% of other closely related male-female dyads. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour