Functional consequences of an in vivo mutation in exon 10 of the human GLUT1 gene

The functional consequences of an in vivo heterozygous insertion mutation in the human facilitated glucose transporter isoform 1 (GLUT1) gene were investigated. The resulting frameshift in exon 10 changed the primary structure of the C-terminus from 42 in native GLUT1 to 61 amino acid residues in the mutant. Kinetic studies on a patient's erythrocytes were substantiated by expressing the mutant cDNA in Xenopus laevis oocytes. K(m) and V(max) values were clearly decreased explaining pathogenicity. Targeting to the plasma membrane was comparable between mutant and wild-type GLUT1. Transport inhibition by cytochalasin B was more effective in the mutant than in the wild-type transporter. The substrate specificity of GLUT1 remained unchanged.     

Differential responses of circadian activity onset and offset following GABA-ergic and opioid receptor activation

The circadian pacemaker in the mammalian suprachiasmatic nuclei is responsive to photic and nonphotic stimuli. In the present study, the authors have investigated the response of activity onset and offset to application of nonphotic stimuli: the benzodiazepine midazolam and the opioid receptor agonist fentanyl. In correspondence with previous studies, both stimuli induced phase advances of the activity onset when given in the mid- to late subjective day. In contrast, activity offset did not phase advance following these injections. Injections during the early subjective day induced small phase delays of the activity onset, while large phase delays occurred in activity offset. Phase shifts, induced at both circadian time zones, were paralleled by an increase in the length of daily activity (alpha). The increase in a remained present during several days after the injection. The different kinetics in phase shifting of the activity onset and offset indicate complexity in phase-shifting behavior of the circadian pacemaker in response to nonphotic stimuli. Moreover, the data show responsiveness of the circadian system to GABA-ergic and opioid receptor activation, not only during the mid- to late subjective day but also during the early subjective day. The data implicate that the early subjective day is an interesting phase for analysis of molecular and biochemical processes involved in nonphotic phase shifting.     

Up-regulation of drug resistance-related vaults during dendritic cell development

P-glycoprotein (Pgp) and vaults are associated with multidrug resistance in tumor cells, but their physiological functions are not yet clear. Pgp, the prototypical transmembrane transporter molecule, may also facilitate the migration of skin dendritic cells (DC). Vaults--ribonucleoprotein cell organelles, frequently overexpressed in Pgp-negative drug-resistant tumor cells--have also been associated with intracellular transport processes. Given the pivotal role of DC in dealing with exposure to potentially harmful substances, the present study was set out to examine the expression of Pgp and vaults during differentiation and maturation of DC. DC were obtained from different sources, including blood-derived monocytes, CD34(+) mononuclear cells, and chronic myeloid leukemia cells. Whereas flow cytometric and immunocytochemical analyses showed slightly augmented levels of Pgp, up-regulation of vault expression during DC culturing was strong, readily confirmed by Western blotting, and independent of the source of DC. In further exploring the functional significance of vault expression, it was found that supplementing DC cultures with polyclonal or mAbs against the major vault protein led to lower viabilities of LPS- or TNF-alpha-matured monocytes-DC. Moreover, expression of critical differentiation, maturation, and costimulatory molecules, including CD1a and CD83, was reduced and their capacity to induce Ag-specific T cell proliferative and IFN-gamma release responses was impaired. These data point to a role for vaults in both DC survival and functioning as APC.     

Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity

A major problem in the development of vaccines against Gram-negative bacteria is the endotoxic -activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the O-linked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-alpha. Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O-linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.     

Effects of certain herbicides and their combinations on nitrate and nitrite reduction

A study was made concerning the effect of various herbicides, when used alone or in combination, on nitrite accumulation in excised leaves of wheat (Triticum aestivum L., var. `Centurk'). Treatment of leaves with photosynthetic inhibitor herbicides, known to interfere with the transfer of light energy, caused accumulation of nitrite under illuminated, aerobic conditions. When certain other herbicides, which do not interfere with the photosynthetic process, were applied to leaves and incubated under dark, aerobic conditions, nitrite accumulations were enhanced over those treated with photosynthetic inhibitors or the controls. The combination of photosynthetic inhibitor herbicides and certain other “nonphotosynthetic inhibitor” herbicides caused relatively large amounts of nitrite to accumulate in light or in darkness. Nitrite accumulation occurs when nitrate and nitrite reduction are not in balance. The proposed actions of the herbicides used in this study are discussed. This discussion provides a rationale for the accumulation of nitrite by the herbicide-treated leaves.     

Respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate CD4 T cells

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in children, the elderly, and immune-compromised individuals. CD4 and CD8 T cells play a crucial role in the elimination of RSV from the infected lung, but T cell memory is not sufficient to completely prevent reinfections. The nature of the adaptive immune response depends on innate immune reactions initiated after interaction of invading pathogens with host APCs. For respiratory pathogens myeloid dendritic cell (DC) precursors that are located underneath the epithelial cell layer lining the airways may play a crucial role in primary activation of T cells and regulating their functional potential. In this study, we investigated the role of human monocyte-derived DC in RSV infection. We showed that monocyte-derived DC can be productively infected, which results in maturation of the DC judged by the up-regulation of CD80, CD83, CD86, and HLA class II molecules. However, RSV infection of DC caused impaired CD4 T cell activation characterized by a lower T cell proliferation and ablation of cytokine production in activated T cells. The suppressive effect was caused by an as yet unidentified soluble factor produced by RSV-infected DC.     

The LIM-Only Protein FHL2 Reduces Vascular Lesion Formation Involving Inhibition of Proliferation and Migration of Smooth Muscle Cells

The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.     

Subject-specific geometry affects acetabular contact pressure during gait more than subject-specific loading patterns

Abstract

Finite element modeling (FEM) can predict hip cartilage contact mechanics. This study investigated how subject-specific boundary conditions and joint geometry affect acetabular cartilage contact mechanics using a multi-scale workflow. For two healthy subjects, musculoskeletal models calculated subject-specific hip kinematics and loading, which were used as boundary conditions for FEM. Cartilage contact mechanics were predicted using either generic or subject-specific FEM and boundary conditions. A subject-specific mesh resulted in a more lateral contact. Effects of subject-specific boundary conditions varied between both subjects. Results highlight the complex interplay between loading and kinematics and their effect on cartilage contact mechanics.