Cytokine Stimulation Promotes Increased Glucose Uptake Via Translocation at the Plasma Membrane of GLUT1 in HEK293 Cells

Interleukin‐3 (IL‐3) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) are two of the best‐characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL‐3 and GM‐CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL‐3 and GM‐CSF are present in several non‐hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL‐3 and GM‐CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL‐3 and GM‐CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3‐kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non‐insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non‐hematopoietic cells. J. Cell. Biochem. 110: 1471–1480, 2010. © 2010 Wiley‐Liss, Inc.     

A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.A novel coronavirus (CoV), severe acute respiratory syndrome-CoV (SARS-CoV), is the causal agent of severe acute respiratory syndrome, which afflicted thousands of people worldwide in 2002 and 2003 (10, 39). SARS-CoV is an enveloped, single- and positive-strand RNA virus that encodes four major structural proteins: S, spike glycoprotein (GP); E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein (46, 55). Similar to other coronaviruses, the S glycoprotein of the virus mediates the initial attachment of the virus to host cell receptors, angiotensin-converting enzyme 2 (ACE2) (44) and/or DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-related molecule; also CD209L or L-SIGN[liver/lymph node-SIGN]) (32) and subsequent fusion of the viral and cellular membranes to allow viral entry into susceptible target cells. The S glycoprotein of SARS-CoV (SARS-S) is a 1,255-amino-acid (aa) type I membrane glycoprotein (46) with 23 potential N-linked glycosylation sites (55). The S glycoproteins of some coronaviruses are translated as a large polypeptide that is subsequently proteolytically cleaved into two functional subunits, S1 (harboring the receptor-binding domain [RBD]) and S2 (containing the membrane fusion domains) (1, 31, 51), during biogenesis, but others are not. The S glycoprotein on mature SARS-CoV virions does not appear to be cleaved (50, 61), but sequence alignments with other coronavirus S glycoproteins allow definition of S1 and S2 regions (46, 55). More recently, studies showed the proteolysis of the S glycoprotein of SARS-CoV on mature virions by cathepsin L (CTSL) (28, 59), as well as trypsin (43, 61) and factor Xa (11), suggesting that a critical cleavage event may occur during cell entry rather than during virion biogenesis.Mannose-binding lectin (MBL; also known as mannose-binding or mannan-binding protein [MBP]) is a Ca²⁺-dependent (C-type) serum lectin that plays an important role in innate immunity by binding to carbohydrates on the surface of a wide range of pathogens (including bacteria, viruses, fungi, and protozoa) (8, 14, 18), where it activates the complement system or acts directly as an opsonin (30, 40, 52). In order to activate the complement system, MBL must be in complex with a group of MBL-associated serine proteases (MASPs), MASP-1, -2, and -3. Currently, only the role of MASP-2 in complement activation has been clearly defined (65). The MBL-MASP-2 complex cleaves C4 and C2 to form C3 convertase (C4bC2a), which, in turn, activates the downstream complement cascade. MBL is a pattern recognition molecule (9), and surface recognition is mediated through its C-terminal carbohydrate recognition domains (CRDs), which are linked to collagenous stems by a short coiled-coil of alpha-helices. MBL is a mixture of oligomers assembled from subunits that are formed from three identical polypeptide chains (9) and usually has two to six clusters of CRDs. Within each of the clusters, the carbohydrate-binding sites have a fixed orientation, which allows selective recognition of patterns of carbohydrate residues on the surfaces of a wide range of microorganisms (8, 14, 18). The concentration of MBL in the serum varies greatly and is affected by mutations of the promoter and coding regions of the human MBL gene (45). MBL deficiency is associated with susceptibility to various infections, as well as autoimmune, metabolic, and cardiovascular diseases, although MBL-deficient individuals are generally healthy (13, 37, 67).There are conflicting results with regard to the role of MBL in SARS-CoV infection (29, 42, 72, 73). While the association of MBL gene polymorphisms with susceptibility to SARS-CoV infection was reported in some studies (29, 73), Yuan et al. demonstrated that there were no significant differences in MBL genotypes and allele frequencies among SARS patients and controls (72). Ip et al. observed binding to, and inhibition of, SARS-CoV by MBL (29). However, in other studies, no binding of MBL to purified SARS-CoV S glycoprotein was detected (42).In this study, retroviral particles pseudotyped with SARS-S and in vitro assays were used to characterize the role of MBL in SARS-CoV infection. The data indicated that MBL selectively bound to SARS-S and mediated inhibition of viral infection in susceptible cell lines. Moreover, we identified a single N-linked glycosylation site, N330, on SARS-S that is critical for the specific interactions with MBL.     

Chromatographic analysis of allosteric effects between ibuprofen and benzodiazepines on human serum albumin

The effects of (R)- and (S)-ibuprofen on the binding of benzodiazepines to human serum albumin (HSA) were examined by biointeraction chromatography. The displacement of benzodiazepines from HSA by (R)- and (S)-ibuprofen was found to involve negative allosteric interactions (or possible direct competition) for most (R)-benzodiazepines. However, (S)-benzodiazepines gave positive or negative allosteric effects and direct competition when displaced by (R)- or (S)-ibuprofen. Association equilibrium constants and coupling constants measured for these effects indicated that they involved two classes of ibuprofen binding regions (i.e., low- and high-affinity sites). Based on these results, a model was proposed to explain the binding of benzodiazepines to HSA and their interactions with ibuprofen. This model gave good agreement with previous reports examining the binding of benzodiazepines to HSA.     

Cutting edge: induction of B7-H4 on APCs through IL-10: novel suppressive mode for regulatory T cells

Multiple modes of suppressive mechanisms including IL-10 are thought to be implicated in CD4+CD25+ regulatory T (Treg) cell-mediated suppression. However, the cellular source, role, and molecular mechanism of IL-10 in Treg cell biology remain controversial. We now studied the interaction between Treg cells and APCs. We demonstrate that Treg cells, but not conventional T cells, trigger high levels of IL-10 production by APCs, stimulate APC B7-H4 expression, and render APCs immunosuppressive. Initial blockade of B7-H4 reduces the suppressive activity mediated by Treg cell-conditioned APCs. Further, APC-derived, rather than Treg cell-derived, IL-10 is responsible for APC B7-H4 induction. Therefore, Treg cells convey suppressive activity to APCs by stimulating B7-H4 expression through IL-10. Altogether, our data provide a novel cellular and molecular mechanism for Treg cell-mediated immunosuppression at the level of APCs, and suggest a plausible mechanism for the suppressive effect of IL-10 in Treg cell-mediated suppression.     

Selective binding of imatinib to the genetic variants of human α1-acid glycoprotein

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to α₁-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of K_a = 1.7(± 0.2) × 10⁶ M^− 1 and n = 0.94 could be determined for the binding on the F1-S variant at 37°. Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nK_a ≈ 3 × 10⁴ M^− 1) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.     

The role of ABC-transporter gene polymorphisms in chemotherapy induced immunosuppression, a retrospective study in childhood acute lymphoblastic leukaemia

We examined the association of functional ABCB1 (MDR1) and ABCG2 (BCRP) polymorphisms with acute side effects of chemotherapy. Analyses were performed on clinical data from 138 patients treated with the ALL-BFM-95 protocol implying several substrates of these transporters. ABCB1 3435T>C, 2677G>T/A 1236C>T and ABCG2 421C>A genotypes were determined. A higher proportion of ABCB1 3435TT patients suffered excessive infectious complications than those harbouring at least one C allele (OR=2.5, p=0.03) during the whole half-year-long intensive phase of chemotherapy. Weaker associations were calculated when ABCB1 1236T-2677T-3435T haplotype homozygotes were tested against the remaining part of the population (OR=2.3, p=0.09). During the reinduction phase of therapy, the occurrence of severe leukocytopenia was similar among ABCB1 genotype groups. The frequency of any toxicities were not shown to differ according to the ABCG2 421C>A genotype. Our data suggest that the ABCB1 3435T>C genotype is associated with the infectious complications of the applied chemotherapy regimen.     

Epidemiology of Helicobacter pylori infection in the Czech Republic

BACKGROUND: Prevalence of Helicobacter pylori infection has been estimated to range from 60 to 95% in the former communist countries of Central and Eastern Europe. The aim of this study was to evaluate H. pylori infection prevalence in a representative sample of the Czech population. The second objective was to describe difference of H. pylori prevalence between different social groups of children and adults. MATERIALS AND METHODS: A total of 2509 persons aged 5-100 years, randomly selected out of 30,012 persons of the general population, took part in the study. H. pylori infection was investigated by means of 13C-urea breath test. Breath samples were analyzed by isotope ratio mass spectrometry. Social and demographic characteristics were based on data from self-completed questionnaires. RESULTS: Using the total Czech population as a standard, we estimated the age-standardized prevalence of H. pylori in males aged 5+ years at 41.9% (95% CI 39.0%, 44.8%) and in females aged 5+ years at 41.4% (95% CI 38.6%, 44.3%) in 2001. Prevalence of H. pylori increased with age but was not related to gender. Children of mothers with basic or lower education, living in crowded accommodations, without access to running warm water, and residing in smaller towns appear to be at the highest risk. Low education and heavy smoking are most strongly associated with prevalence of H. pylori positivity in adults and adolescents. CONCLUSIONS: This is a unique study based on a representative sample of the general population in a Central European country. The overall prevalence of H. pylori is lower than previously assumed and could partly reflect a substantial recent decrease in H. pylori prevalence in the Czech Republic. Consistent with earlier studies, H. pylori infection is strongly influenced by socioeconomic conditions and childhood poverty.     

Conclusions on motor control depend on the type of model used to represent the periphery

Within the field of motor control, there is no consensus on which kinematic and kinetic aspects of movements are planned or controlled. Perturbing goal-directed movements is a frequently used tool to answer this question. To be able to draw conclusions about motor control from kinematic responses to perturbations, a model of the periphery (i.e., the skeleton, muscle–tendon complexes, and spinal reflex circuitry) is required. The purpose of the present study was to determine to what extent such conclusions depend on the level of simplification with which the dynamical properties of the periphery are modeled. For this purpose, we simulated fast goal-directed single-joint movement with four existing types of models. We tested how three types of perturbations affected movement trajectory if motor commands remained unchanged. We found that the four types of models of the periphery showed different robustness to the perturbations, leading to different predictions on how accurate motor commands need to be, i.e., how accurate the knowledge of external conditions needs to be. This means that when interpreting kinematic responses obtained in perturbation experiments the level of error correction attributed to adaptation of motor commands depends on the type of model used to describe the periphery.