Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis

The enterocytes of the small intestine are occasionally exposed to pathogenic bacteria, such as Salmonella enteritidis 857, an etiologic agent of intestinal infections in humans. The expression of the heat shock response by enterocytes may be part of a protective mechanism developed against pathogenic bacteria in the intestinal lumen. We aimed at investigating whether S. enteritidis 857 is able to induce a heat shock response in crypt- and villus-like Caco-2 cells and at establishing the extent of the induction. To establish whether S. enteritidis 857 interfered with the integrity of the cell monolayer, the transepithelial electrical resistance (TEER) of filter-grown, differentiated (villus-like) Caco-2 cells was measured. We clearly observed damage to the integrity of the cell monolayer by measuring the TEER. The stress response was screened in both crypt- and villus-like Caco-2 cells exposed to heat (40-43 degrees C) or to graded numbers (10(1)-10(8)) of bacteria and in villus-like cells exposed to S. enteritidis 857 endotoxin. Expression of the heat shock proteins Hsp70 and Hsp90 was analyzed by polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies. Exposure to heat or Salmonella resulted in increased levels of Hsp70 and Hsp90 in a temperature-effect or Salmonella-dose relationship, respectively. Incubation of Caco-2 cells with S. enteritidis 857 endotoxin did not induce heat shock gene expression. We conclude that S. enteritidis 857 significantly increases the levels of stress proteins in enterocyte-like Caco-2 cells. However, our data on TEER clearly indicate that this increase is insufficient to protect the cells.     

Chromosomal breakpoint mapping by arrayCGH using flow-sorted chromosomes

Despite the recent completion of the human genome project, the mapping of disease-related chromosomal translocation breakpoints and genes has remained laborious. Here, we describe a novel and rapid procedure to map such translocation breakpoints using flow-sorted chromosomes in combination with array-based comparative genomic hybridization (arrayCGH). To test the feasibility of this approach, we used a t(12;15)(q13;q25)-positive cell line with known breakpoint positions as a model. The derivative 12 chromosomes were flow-sorted, labeled, and hybridized to a genome-wide array containing 3648 well-characterized human genomic clones. The exact locations of the breakpoints on both chromosome 12 and 15 could be determined in a single hybridization experiment. In addition, we have tested the minimal amount of material necessary to perform these experiments and show that it is possible to obtain highly reliable profiles using as little as 10,000 flow-sorted chromosomes.     

Phloem metabolism and function have to cope with low internal oxygen

We have investigated the consequences of endogenous limitations in oxygen delivery for phloem transport in Ricinus communis. In situ oxygen profiles were measured directly across stems of plants growing in air (21% [v/v] oxygen), using a microsensor with a tip diameter of approximately 30 microm. Oxygen levels decreased from 21% (v/v) at the surface to 7% (v/v) in the vascular region and increased again to 15% (v/v) toward the hollow center of the stem. Phloem sap exuding from small incisions in the bark of the stem was hypoxic, and the ATP to ADP ratio (4.1) and energy charge (0.78) were also low. When 5-cm stem segments of intact plants were exposed to zero external oxygen for 90 min, oxygen levels within the phloem decreased to approximately 2% (v/v), and ATP to ADP ratio and adenylate energy charge dropped further to 1.92 and 0.68, respectively. This was accompanied by a marked decrease in the phloem sucrose (Suc) concentration and Suc transport rate, which is likely to be explained by the inhibition of retrieval processes in the phloem. Germinating seedlings were used to analyze the effect of a stepwise decrease in oxygen tension on phloem transport and energy metabolism in more detail. Within the endosperm embedding the cotyledons-next to the phloem loading sites-oxygen decreased from approximately 14% (v/v) in 6-d-old seedlings down to approximately 6% (v/v) in 10-d-old seedlings. This was paralleled by a similar decrease of oxygen inside the hypocotyl. When the endosperm was removed and cotyledons incubated in a 100 mM Suc solution with 21%, 6%, 3%, or 0.5% (v/v) oxygen for 3 h before phloem sap was analyzed, decreasing oxygen tensions led to a progressive decrease in phloem energy state, indicating a partial inhibition of respiration. The estimated ratio of NADH to NAD(+) in the phloem exudate remained low (approximately 0.0014) when oxygen was decreased to 6% and 3% (v/v) but increased markedly (to approximately 0.008) at 0.5% (v/v) oxygen, paralleled by an increase in lactate and ethanol. Suc concentration and translocation decreased when oxygen was decreased to 3% and 0.5% (v/v). Falling oxygen led to a progressive increase in amino acids, especially of alanine, gamma-aminobutyrat, methionine, and isoleucine, a progressive decrease in the C to N ratio, and an increase in the succinate to malate ratio in the phloem. These results show that oxygen concentration is low inside the transport phloem in planta and that this results in adaptive changes in phloem metabolism and function.     

Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2

Serum mannose-binding proteins (MBPs) are C-type lectins that recognize cell surface carbohydrate structures on pathogens, and trigger killing of these targets by activating the complement pathway. MBPs circulate as a complex with MBP-associated serine proteases (MASPs), which become activated upon engagement of a target cell surface. The minimal functional unit for complement activation is a MASP homodimer bound to two MBP trimeric subunits. MASPs have a modular structure consisting of an N-terminal CUB domain, a Ca(2+)-binding EGF-like domain, a second CUB domain, two complement control protein modules and a C-terminal serine protease domain. The CUB1-EGF-CUB2 region mediates homodimerization and binding to MBP. The crystal structure of the MASP-2 CUB1-EGF-CUB2 dimer reveals an elongated structure with a prominent concave surface that is proposed to be the MBP-binding site. A model of the full six-domain structure and its interaction with MBPs suggests mechanisms by which binding to a target cell transmits conformational changes from MBP to MASP that allow activation of its protease activity.     

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.     

Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABPPM

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP_PM) may be a component of this system. To test the hypothesis that FABP_PM is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP_PM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated V_max values of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and K_m values of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The V_max values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP_PM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP_PM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP_PM is a component of this process in muscle.     

Activation of the Ca2+-sensing receptor stimulates the activity of the epithelial Ca2+ channel TRPV5

The extracellular Ca²⁺-sensing receptor (CaR) is a key-player in plasma Ca²⁺ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca²⁺ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca²⁺-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca²⁺ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca²⁺ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR^R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca²⁺ influx via a PMA-insensitive PKC isoform pathway.     

What Makes a Protein Sequence a Prion?

Typical amyloid diseases such as Alzheimer''s and Parkinson''s were thought to exclusively result from de novo aggregation, but recently it was shown that amyloids formed in one cell can cross-seed aggregation in other cells, following a prion-like mechanism. Despite the large experimental effort devoted to understanding the phenomenon of prion transmissibility, it is still poorly understood how this property is encoded in the primary sequence. In many cases, prion structural conversion is driven by the presence of relatively large glutamine/asparagine (Q/N) enriched segments. Several studies suggest that it is the amino acid composition of these regions rather than their specific sequence that accounts for their priogenicity. However, our analysis indicates that it is instead the presence and potency of specific short amyloid-prone sequences that occur within intrinsically disordered Q/N-rich regions that determine their prion behaviour, modulated by the structural and compositional context. This provides a basis for the accurate identification and evaluation of prion candidate sequences in proteomes in the context of a unified framework for amyloid formation and prion propagation.