Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.     

Identification of a Receptor for Neuropeptide VGF and Its Role in Neuropathic Pain

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca²⁺ levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca²⁺ levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.     

Action of powerful ultrashort laser impulses on the permeability of the outer membrane of HeLa cells

It was shown that in HeLa cells, at the stationary growth phase, the permeability of the outer membrane for thymidine increased 1 h after irradiation with two ultrashort impulses (Nd3+: YAG laser, lambda = 266, 532 or 1064 nm, the impulse duration of 3 X 10(-11) s, I = 2 or 20 MW/cm2). The effect was dependent on the time interval between the impulses and virtually independent of the radiation wave-length. The maximum increase in the membrane permeability (by approximately 3 times) was noted after irradiation at 4-6 s intervals between the impulses.     

Cells that express MyoD mRNA in the epiblast are stably committed to the skeletal muscle lineage

The epiblast of the chick embryo contains cells that express MyoD mRNA but not MyoD protein. We investigated whether MyoD-positive (MyoDpos) epiblast cells are stably committed to the skeletal muscle lineage or whether their fate can be altered in different environments. A small number of MyoDpos epiblast cells were tracked into the heart and nervous system. In these locations, they expressed MyoD mRNA and some synthesized MyoD protein. No MyoDpos epiblast cells differentiated into cardiac muscle or neurons. Similar results were obtained when MyoDpos cells were isolated from the epiblast and microinjected into the precardiac mesoderm or neural plate. In contrast, epiblast cells lacking MyoD differentiated according to their environment. These results demonstrate that the epiblast contains both multipotent cells and a subpopulation of cells that are stably committed to the skeletal muscle lineage before the onset of gastrulation. Stable programming in the epiblast may ensure that MyoDpos cells express similar signaling molecules in a variety of environments.     

The complexity of mitochondrial outer membrane permeability and VDAC regulation by associated proteins

Previous studies have shown that class II β-tubulin plays a key role in the regulation of oxidative phosphorylation (OXPHOS) in some highly differentiated cells, but its role in malignant cells has remained unclear. To clarify these aspects, we compared the bioenergetic properties of HL-1 murine sarcoma cells, murine neuroblastoma cells (uN2a) and retinoic acid - differentiated N2a cells (dN2a). We examined the expression and possible co-localization of mitochondrial voltage dependent anion channel (VDAC) with hexokinase-2 (HK-2) and βII-tubulin, the role of depolymerized βII-tubuline and the effect of both proteins in the regulation of mitochondrial outer membrane (MOM) permeability. Our data demonstrate that neuroblastoma and sarcoma cells are prone to aerobic glycolysis, which is partially mediated by the presence of VDAC bound HK-2. Microtubule destabilizing (colchicine) and stabilizing (taxol) agents do not affect the MOM permeability for ADP in N2a and HL-1 cells. The obtained results show that βII-tubulin does not regulate the MOM permeability for adenine nucleotides in these cells. HL-1 and NB cells display comparable rates of ADP-activated respiration. It was also found that differentiation enhances the involvement of OXPHOS in N2a cells due to the rise in their mitochondrial reserve capacity. Our data support the view that the alteration of mitochondrial affinity for ADNs is one of the characteristic features of cancer cells. It can be concluded that the binding sites for tubulin and hexokinase within the large intermembrane protein supercomplex Mitochondrial Interactosome, could be different between muscle and cancer cells.     

Wild and Rare Self-Incompatibility Allele S17 Found in 24 Sweet Cherry (Prunus avium L.) Cultivars

The pollination of self-incompatible diploid sweet cherry is determined by the S-locus alleles. We resolved the S-alleles of 50 sweet cherry cultivars grown in Estonia and determined their incompatibility groups, which were previously unknown for most of the tested cultivars. We used consensus primers SI-19/20, SI-31/32, PaConsI, and PaConsII followed by allele-specific primers and sequencing to identify sweet cherry S-genotypes. Surprisingly, 48% (24/50) of the tested cultivars, including 17 Estonian cultivars, carry the rare S-allele S₁₇, which had initially been described in wild sweet cherries in Belgium and Germany. The S₁₇-allele in Estonian cultivars could originate from ‘Leningradskaya tchernaya’ (S₆|S₁₇), which has been extensively used in Estonian sweet cherry breeding. Four studied cultivars carrying S₁₇ are partly self-compatible, whereas the other 20 cultivars with S₁₇ have not been reported to be self-compatible. The recommended pollinator of seven self-incompatible sweet cherries is of the same S-genotype, including four with S₁₇-allele, suggesting heritable reduced effectiveness of self-infertility. We classified the newly genotyped sweet cherry cultivars into 15 known incompatibility groups, and we proposed four new incompatibility groups, 64–67, for S-locus genotypes S₃|S₁₇, S₄|S₁₇, S₅|S₁₇, and S₆|S₁₇, respectively, which makes them excellent pollinators all across Europe. Alternatively, the frequency of S₁₇ might be underestimated in Eastern European populations and some currently unidentified sweet cherry S-alleles might potentially be S₁₇.

     

Importance of polyamines in cell cycle kinetics as studied in a transgenic system

Polyamines are organic cations, which are considered essential for normal cell cycle progression. This view is based on results from numerous studies using a variety of enzyme inhibitors or polyamine analogues interfering with either the metabolism or the physiological functions of the polyamines. However, the presence of non-specific effects may be hard to rule out in such studies. In the present study, we have for the first time used a transgenic cell system to analyze the importance of polyamines in cell growth. We have earlier shown that expression of trypanosomal ODC in an ODC-deficient variant of CHO cells (C55.7) supported growth of these otherwise polyamine auxotrophic cells. However, one of the transgenic cell lines grew much slower than the others. As shown in the present study, the level of ODC activity was much lower in these cells, and that was reflected in a reduction of cellular polyamine levels. Analysis of cell cycle kinetics revealed that reduction of growth was correlated to prolongation of the G₁, S, and G₂ + M phases in the cells. Providing exogenous putrescine to the cells resulted in a normalization of polyamine levels as well as cell cycle kinetics indicating a causal relationship.     

The effect of irradiation by a He-Ne laser and phytohemagglutinin on lymphocyte mitochondria

Electron-microscopic morphometry has been applied to study mitochondria on ultrathin sections of lymphocytes from human peripheral blood. It has been shown that the stimulation of lymphocytes by the mitogen phytohemagglutinin (PHA) 1 h causes increases in the quantity of mitochondria per cellular section (17%) as well as in the total area of mitochondria per cell section (35%), i.e. an increase in mitochondrial mass. Taking into account known facts about growth and division of mitochondria in late phases of cellular cycle, one can suppose that described above changes in mitochondria during G0----G1 transition under action of PHA belong to an early phase of biogenesis of mitochondria. In the contrary, irradiation of lymphocytes with He-Ne-laser (lambda = 632.8 nm) in dose 56 J/m2 which does not cause the G0----C1 transition, results in the increase in the number of mitochondria per cellular section (20%) but not increase in the total area of mitochondria per cell section. The last finding indicates to some modification of space configuration of the mitochondria without any changes in their mass. The increase in the quantity of mitochondria per cellular section after the irradiation could be related with the increase in electrochemical proton gradient and in phosphorylating activity of mitochondria. He-Ne-laser radiation as well as mitogen PHA cause some deaggregation of mitochondria (this is more pronounced in case of PHA) which may be related to their functional activation.