Involvement of the activation loop of ERK in the detachment from cytosolic anchoring

The Extracellular signal-regulated kinases (ERKs) are translocated into the nucleus in response to mitogenic stimulation. The mechanism of translocation and the residues in ERKs that govern this process are not clear as yet. Here we studied the involvement of residues in the activation loop of ERK2 in determining its subcellular localization. Substitution of residues in the activation loop to alanines indicated that residues 173-181 do not play a significant role in the phosphorylation and activation of ERK2. However, residues 176-181 are responsible for the detachment of ERK2 from MEK1 upon mitogenic stimulation. This dissociation can be mimicked by substitution of residues 176-178 to alanines and is prevented by deletion of these residues or by substitution of residues 179-181 to alanines. On the other hand, residues 176-181, as well as residues essential for ERK2 dimerization, do not play a role in the shuttle of ERK2 through nuclear pores. Thus, phosphorylation-induced conformational rearrangement of residues in the activation loop of ERK2 plays a major role in the control of subcellular localization of this protein.     

Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

The hydrodynamic characterization of waxy maize amylopectin in 90% dimethyl sulfoxide–water by analytical ultracentrifugation, dynamic, and static light scattering

Static light scattering of high amylopectin waxy maize starch gently dispersed in 90% dimethyl sulfoxide–water yielded a weight average molecular weight M_w and radius of gyration R_g of 560×10⁶ g/mol and 342 nm, respectively. To obtain an independent hydrodynamic characterization of these solutions, we measured the sedimentation coefficient for the main component in an analytical ultracentrifuge. The value of s⁰, the infinite dilution sedimentation coefficient, was 199 S. The translational diffusion coefficient D⁰ in very dilute solutions was measured by dynamic light scattering at 90° and found to be 2.33×10⁻⁹ cm²/s. An effective hydrodynamic radius R_h was calculated from this diffusion constant using the Stokes–Einstein equation and found to be 348 nm. The structure-related parameter ρ=R_g/R_h was calculated to be 0.98. The weight average molecular weight calculated from the Svedberg equation using the values measured for s⁰ and D⁰ was 593×10⁶ g/mol. This result is in reasonable agreement with the light scattering results. As light scattering results are subject to experimental errors due to the possibility of dust contamination, the presence of microgel or aggregates, and the questionable applicability of light scattering theory to interpret results for macromolecular sizes approaching the wave length of light used as a source for scattering, it is advisable to have corroborating hydrodynamic data when possible to further validate light scattering results in this very high molecular weight range.     

Genomic mapping of chromosomal region 2p15-p21 (D2S378-D2S391): integration of Genemap'98 within a framework of yeast and bacterial artificial chromosomes

The region of chromosome 2 encompassed by the polymorphic markers D2S378 (centromeric) and D2S391 (telomeric) spans an approximately 10-cM distance in cytogenetic bands 2p15-p21. This area is frequently involved in cytogenetic alterations in human cancers. It also harbors the genes for several genetic disorders, including Type I hereditary nonpolyposis colorectal cancer (HNPCC), familial male precocious puberty (FMPP), Carney complex (CNC), Doyne's honeycomb retinal dystrophy (DHRD), and one form of familial dyslexia (DYX-3). Only a handful of known genes have been mapped to 2p16. These include MSH2, which is responsible for HNPCC, FSHR, the gene responsible for FMPP, EFEMP-1, the gene mutated in DHRD, GTBP, a DNA repair gene, and SPTBN1, nonerythryocytic beta-spectrin. The genes for CNC and DYX-3 remain unknown, due to lack of a contig of this region and its underrepresentation in the existing maps. This report presents a yeast- and bacterial-artificial chromosome (YAC and BAC, respectively) resource for the construction of a sequence-ready map of 2p15-p21 between the markers D2S378 and D2S391 at the centromeric and telomeric ends, respectively. The recently published Genemap'98 lists 146 expressed sequence tags (ESTs) in this region; we have used our YAC-BAC map to place each of these ESTs within a framework of 40 known and 3 newly cloned polymorphic markers and 37 new sequence-tagged sites. This map provides an integration of genetic, radiation hybrid, and physical mapping information for the region corresponding to cytogenetic bands 2p15-p21 and is expected to facilitate the identification of disease genes from the area.     

Fluxomics with Ratiometric Metabolite Dyes

Today''s major excitement in biology centers on signaling: How can a cell or organism measure the myriad of environmental cues, integrate it, and acclimate to the new conditions? Hormonal signals and second messengers are in the focus of most of these studies, e.g., regulation of glucose transporter GLUT4 cycling by insulin, or regulation of plant growth by auxin or brassinosteroids.^1–3 In comparison, we generally assume that we know almost everything about basic metabolism since it has been studied for many decades; for example we know since the early 80s that allosteric regulation by fructose-2,6-bisphophate plays an important role in regulating glycolysis in plants and animals.⁴ This may be the reason why studies of metabolism appear to be a bit out of fashion. But if we look to other organisms such as E. coli or yeast, we rapidly realize that metabolism is controlled by complex interconnected signaling networks, and that we understand little of these signaling networks in humans and plants.^5,6 As it turns out, the cell registers many metabolites, and flux through the pathways is regulated using complex signaling networks that involve calcium as well as hormones.Key Words: flux, fluxome, glucose, glutamate, phosphate, sucrose, fluorescence resonance energy transfer, biosensorOne of the reasons for the fable for hormones lies in the simple fact that it is easier to observe macroscopic changes, such as changes in the architecture of a plant than to determine metabolite levels, but also here new tools are urgently needed that allow quantification of these small molecules. Visualization of starch levels provided a significant advance, and in combination with mutant screens allowed to identify fundamental components of starch metabolism.^7–9 The biggest advance for the signaling field was the development of advanced chemical and genetically encoded calcium dyes.^10–12 No such dyes are available for hormones or metabolites, as soon as we try to determine levels of metabolites (or signaling molecules), we run into the issues of compartmentation and cellular differences in tissues. Today, the same enzymatic assays used decades ago are still widely used to determine metabolite levels. Although significant advances in chromatography and mass spectrometry based metabolite analysis have moved the study of metabolism to ‘omics’ era, compartmentalization of metabolism still presents a major challenge. Especially the large vacuoles of plant cells are a major obstacle, since even fractionation studies suffer from contamination. Moreover, with the current set of tools it is not possible to determine the dynamic changes in metabolite levels in different subcellular compartments in real time in vivo. Radiotracers have helped a lot to identify and quantify intermediates and to assemble pathways, originally using pulse labeling followed by paper chromatography. Today ¹³C-labeling is used together with mass spectrometry to obtain insights into metabolic flux control.¹³ This tool set for the first time enabled the comparison of mutants and study regulatory networks involved in sugar signaling. While significant, advances in radiotracer experiments do not provide cellular or subcellular information and only limited temporal resolution, they do provide efficient means for studying metabolite fluxes through complex and/or not well-defined pathways. Thus there is a clear need for metabolite specific dyes that can be targeted to subcellular compartments and that would enable flux measurements in response to environmental cues helping to push metabolic research back into the focus of signaling-related biology.In 2002, we developed the first prototype “metabolic dye” FRET sensor for maltose.^14,15 A similar glucose sensor was recently employed for measuring tracer-independent transport of glucose across the ER membrane of liver cells.¹⁶ After resolving some issues such as low signal-to-noise and gene silencing in plants, we are now able to compare glucose levels between cells in an intact root in real time.¹⁷ The parallel development of sucrose and phosphate sensors complements the set of tools, in future experiments providing a comparison of sucrose, phosphate and glucose fluxes in intact tissues with both temporal (below seconds) and spatial resolution (cellular and subcellular).^18,19The first experiments already led to a big surprise: glucose supplied to the root is rapidly taken up and is rapidly metabolized.¹⁷ Roots expressing the highest affinity sensor FLIPglu170n responded to glucose perfusion suggesting that the steady state glucose level in the root is less than 100 nM, the estimated detection limit for this sensor in these first experiments. The first experiments were limited by the mixing kinetics in the bath used for perfusion, while improvement of the chamber now allow for faster for glucose exchange. We estimate that glucose levels fall from a steady state level of approximately 5 mM in the cytosol when perfused with 5 mM glucose to below 100 nM in about three minutes. For the sensor with an affinity of 600 µM the rate of glucose accumulation, which is composed of the various rates that affect the steady state in the cytosol such as metabolism, compartmentation and transport across the plasma membrane, is in the range of 527 ± 77 µM glucose/min and that for glucose removal is 317 ± 37 (Fig. 1; Chaudhuri B, Frommer WB, unpublished). Questions that arise are: Which transport systems drive uptake? How much does the vacuole contribute to the observed flux and steady state levels? Is the capacity of hexokinase at levels below its K_m still sufficient to phosphorylate glucose efficient enough to pull glucose below 100 nM or does hexokinase have different properties in vivo compared to what we know from the purified enzyme? Are there different transporters and enzymes contributing to flux in the low (1–10 mM) and the ultrahigh affinity (low µM) phases? Are there spatial differences in the root? Why do roots take up glucose so efficiently in the first place? The combination of the sensors with information from the expression-LEDs from Birnbaum and Benfey²⁰ and specific knock-out mutants should help answering some of these questions.Open in a separate windowFigure 1Quantitative analysis of glucose flux from an Arabidopsis root expressing FLIPglu-600µΔ13, a FRET sensor for glucose with an affinity of 600 µM. The root of a 10 day-old seedling was placed into a perfusion chamber and perfused with hydroponic medium with or without 5 mM glucose. eCFP was excited and emission was recorded for eCFP and eYFP every 10 seconds (essentially as decsribed in ref. 17). The emission intensities for a region-of-interest were averaged and the emission ratio was determined at the two wavelengths for each image of a time series and plotted on the Y-axis against time on the X-axis. Addition of glucose is indicated.Another big surprise is the dramatic gradient of glucose across the plasma membrane, which has important implications for our understanding of transport processes across the plasma membrane as well as the intracellular membranes.¹⁷ Information about the gradients is relevant in the context of apo- and symplasmic unloading routes in roots²¹ and the contribution of proton-coupled transporters in cellular export.²² It will thus be interesting to follow the extracellular levels using surface-anchored sensors. Now that besides high sensitivity glucose FLIPs¹⁷ we also generated nanosensors for sucrose¹⁹ and phosphate,¹⁸ complementing the similar tool sets for calcium²³ and pH,²⁴ it is possible to compare multiple parameters and to follow flux at different levels and to calibrate against other influences.The improvements of the signal-to-noise ratio of the FRET-based metabolite sensors²⁵ makes the FLIPs a standard tool for every lab interested in measuring ion-, sugar- or amino acid flux in living cells. Since the nanosensors are genetically encoded, they can be used to characterize intracellular fluxes^16,26 in any organism for which transformation protocols have been established. The existing sets of sensors are simple to use, constructs are available through Addgene and Arabidopsis lines from the Arabidopsis Stock Center. Detailed instructions for imaging can be found at: http://carnegiedpb.stanford.edu/research/frommer/research_frommer_protocols.php. These tools will hopefully become a standard system not only for physiological analyses, but in addition provide a new way for high throughput fluxomics studies.     

Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 M induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 M of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (_m) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC₆(3), a cationic lipophilic dye into mitochondria indicating the disruption of _m. This drop in _m was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation excess ROS production GSH depletion oxidative stress disruption of _m release of cytochrome C and other apoptosis related proteins to cytosol apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.     

A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation

Background

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn''s disease.

Methods and Findings

To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.

Conclusion

These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.     

DIROM: an experimental design interactive system for directed mutagenesis and nucleic acids engineering

A computer system DIROM for oligonucleotide-directed mutagenesisand artificial gene design has been designed for better experimentalplanning and control. DIROM permits searching for optimal oligonucleotideswith respect to certain important parameters, namely sufficientenergy of oligonucleotide-target hybridization, the secondarystructure of oligonuc-tide and target DNA, the presence of alternatebinding sites in the target DNA and terminal G/C pairs. It canalso be used to plan polymerase chain reaction experiments,for optimal primer selection, in sequencing, etc. DIROM enablesone to search for both existing and potential restriction sites,to perform vector + target sequence construction. The systemconsists of a set of original algorithms that formalize theempirical knowledge of oligonucleotide action as primers.