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.     

Induction of the Cytoprotective Enzyme Heme Oxygenase-1 by Statins Is Enhanced in Vascular Endothelium Exposed to Laminar Shear Stress and Impaired by Disturbed Flow

In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H₂O₂-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.The efficacy of 3-hydroxy-3-methylglutaryl-coenzyme A reductase antagonists (statins) in reducing low density lipoprotein cholesterol, cardiovascular morbidity, and mortality is widely recognized (1). The observation that beneficial actions of statins on vascular function are detectable prior to any fall in serum cholesterol, extend to normocholesterolemic patients and exceed those of other lipid-lowering drugs despite comparable falls in total cholesterol (2, 3), suggest the existence of low density lipoprotein-cholesterol-independent effects (4, 5). Judging from in vitro studies, these may include immunomodulatory, anti-inflammatory, anti-adhesive, anti-thrombotic, and cytoprotective actions (6). However, the experimental work demonstrating these pleiotropic effects has predominantly used statin concentrations exceeding those achieved by therapeutic dosing, raising questions concerning clinical relevance (4).Heme oxygenase-1 (HO-1)² acts as the rate-limiting factor in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide (CO). Biliverdin is subsequently converted to bilirubin by biliverdin reductase, whereas intracellular iron induces expression of heavy chain-ferritin and the opening of Fe²⁺ export channels (7). The biologic activity of HO-1 represents an important adaptive response in cellular homeostasis, as revealed by widespread inflammation and persistent endothelial injury in human HO-1 deficiency (8).Expression of HO-1 in atherosclerotic lesions, and its ability to inhibit vascular smooth muscle cell proliferation, exert anti-inflammatory, antioxidant, and antithrombotic effects, suggests a protective role during atherogenesis (9, 10). HMOX1 promoter polymorphisms affecting HO-1 expression may influence susceptibility to intimal hyperplasia and coronary artery disease, whereas a low serum bilirubin constitutes a cardiovascular risk factor (11). Moreover, overexpression of HO-1 inhibited atherogenesis, whereas Hmox1^−/− mice bred onto an ApoE^−/− background developed more extensive and complex atherosclerotic plaques (12, 13).Recent interest has focused on the therapeutic potential of HO-1 and its products, with probucol, statins, rapamycin, nitric oxide donors, and aspirin being shown to induce HO-1 (reviewed in Ref. 10). Indeed, induction of HO-1 may represent an important component of the vasculoprotective profile of statins, with simvastatin, atorvastatin, and rosuvastatin variously shown to increase HMOX1 promoter activity and mRNA levels, to induce enzyme activity and increase antioxidant capacity in human endothelial cells (EC) (14–18). However, induction of HO-1 in vascular EC in vivo has not yet been demonstrated.Vascular endothelium exposed to unidirectional, pulsatile laminar shear stress (LSS) >10 dynes/cm² is relatively protected against atherogenesis. LSS increases nitric oxide (NO) biosynthesis, prolongs EC survival, and generates an anticoagulant, anti-adhesive cell surface. In contrast, endothelium exposed to disturbed blood flow, with low shear reversing or oscillatory flow patterns, such as that located at arterial branch points and curvatures, is atheroprone. Thus endothelial cells exposed to disturbed blood flow exhibit reduced levels of endothelial nitric-oxide synthase (eNOS), increased apoptosis, oxidative stress, permeability to low density lipoprotein, and leukocyte adhesion (19).The atheroprotective influence of unidirectional LSS and the overlap between these actions and those of statins led us to hypothesize that LSS increases endothelial responsiveness to statins. We demonstrate for the first time that treatment of mice with atorvastatin induces HO-1 expression in the aortic endothelium and that this occurs preferentially at sites exposed to LSS. In vitro, pre-conditioning human EC with an atheroprotective, but not an atheroprone waveform, significantly reduces the concentration of atorvastatin required to enhance HO-1-mediated cytoprotection against oxidant-induced injury. A synergistic relationship between LSS and statins is revealed, resulting in maximal Akt phosphorylation and dependence upon eNOS, Kruppel-like factor 2 (KLF2), and NF-E2-related factor-2 (Nrf2) activation.     

Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most potent toxins known in nature and are characterized as category A select agents since they are considered potential bioterrorism threats (3). BoNTs can be distinguished immunologically into seven serotypes by using homologous antitoxins, designated A to G. BoNT/A is of particular interest, since it is frequently implicated in cases of botulism and is a significant threat in bioterrorism (1, 10).BoNT is a 150-kDa protein composed of a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulfide bond and noncovalent molecular interactions (24). The heavy chain (H) has two functional domains, a transmembrane domain and a receptor binding domain. The light chain (L) is a zinc-dependent protease which specifically cleaves one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors, resulting in the blockage of evoked acetylcholine release at the skeletal neuromuscular junction (8).Previous studies have found that the bont genes of all strains of C. botulinum and neurotoxigenic strains of Clostridium butyricum and Clostridium baratii have a set of genes located upstream of the bont and ntnh genes that are organized as gene clusters (5, 7, 23). The two known primary types of clusters are (i) a hemagglutinin (ha) cluster and (ii) an orfX cluster with open reading frames (ORFs) of unknown functions. The ha cluster consists of genes encoding HA17, HA33, HA70, BotR, and NTNH. The orfX cluster consists of genes encoding ORFX3, ORFX2, ORFX1, P47, P21, and NTNH. Previous studies indicate that BoNT/A subtypes possess either a ha cluster or an orfX cluster associated with their expressed bont gene, depending on the subtype and strain (5, 11, 13-15, 33).It has been shown that the BoNT complex can form stable toxin complexes (TCs) of various sizes, including LL-TC (∼900 kDa), L-TC (∼500 kDa), and M-TC (∼300 kDa) composed of various combinations of HA proteins, NTNH, and BoNT (19, 21, 23, 29, 31, 34). M-TC contains BoNT and NTNH but has no HA proteins, whereas LL-TC and L-TC contain different ratios of the BoNT, NTNH, and HA proteins (21, 22, 29, 34). The biological and structural roles of the complex proteins are not completely characterized, although it has been proposed that they serve the role of protecting BoNT from harsh conditions, including pH, salt, temperature, and digestive enzymes, and that they assist BoNT translocation across the intestinal epithelial layer (2, 6, 17). A recent report indicated that the nontoxic proteins serve as adjuvants and contribute to the immunogenicity of BoNT/A (25).The production of botulinum TCs is known to vary with different serotypes and strains, medium composition, and culture conditions (21, 24, 31). The LL-TC has only been observed in proteolytic strains (group I). Serotype A to D strains produce M-TC and L-TC in their culture medium, while serotype E and F strains produce only M-TC (17, 18).In 1986, a Japanese group isolated four HA-negative C. botulinum strains from infant botulism cases that produced only M-TC (300 kDa). They assigned the strains to subtype A2 (14, 30). In 2004, our laboratory confirmed on a genomic level that the BoNT/A2 subtype contained the orfX cluster instead of the ha cluster (12). Since then, more arrangements and combinations of neurotoxin gene clusters were characterized along with more BoNT subtypes (13, 20, 33). However, the function of the orfX genes and the role of the presumptive protein products and their role in the TCs are still unknown, including whether ORFX proteins can form a TC with the expressed toxin analogous to the ha cluster proteins.In this study, the BoNT/A2 TC was purified from a native culture to determine if the orfX cluster proteins remain associated with BoNT/A2. To better understand the role of the orfX cluster genes, the orfX cluster proteins of C. botulinum A2 strains (ORFX1, ORFX3, P47, and the middle part of NTNH) was expressed using either an Escherichia coli or a C. botulinum expression system in this study. Antibodies against individual expressed orfX cluster proteins were then raised by immunizing a rabbit and mice. These antibodies were then used as probes to investigate the expression pattern of the orfX cluster genes in the native A2 culture. ORFX2, which could not be expressed, was detected by N-terminal protein sequencing.     

The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.     

Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people

The higher counts or particular groups (Firmicutes/Bacteroidetes) of intestinal microbiota are related to host metabolic reactions, supporting a balance of human ecosystem. We further explored whether intestinal lactobacilli were associated with some principal cellular and metabolic markers of blood in 38 healthy >65-year-old persons. The questionnaire, routine clinical and laboratory data of blood indices as much as the oxidized low-density lipoprotein (ox-LDL) and baseline diene conjugates in low-density lipoprotein (BDC-LDL) of blood sera were explored. The PCR-based intestinal Lactobacillus sp. composition and counts of cultivable lactobacilli (LAB) were tested. The facultative heterofermentative lactobacilli (Lactobacillus casei and Lactobacillus paracasei) were the most frequent (89 and 97%, respectively) species found, while Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri were present in almost half of the elderly persons. The number of species simultaneously colonizing the individuals ranged from 1 to 7 (median 4). In elderly consuming probiotics the LAB counts were significantly higher than in these not consuming (median 7.8, range 4.2–10.8 vs. median 6.3, range 3.3–9.7 log cfu/g; p = 0.005), adjusted (OR = 1.71, CI95 1.04–2.82; p = 0.035) for age and body mass index (BMI). The colonization by L. acidophilus was negatively related (r = ?0.367, p = 0.0275) to L. reuteri, staying significant after adjusting for age, sex and BMI (OR = 0.16, CI95 0.04–0.73; p = 0.018). However, the blood glucose concentration showed a tendency for a negative correlation for colonization with Lactobacillus fermentum (r = ?0.309, p = 0.062) adjusted for BMI (Adj. R² = 0.181; p = 0.013) but not for age and sex. The higher white blood cells (WBC) count was positively related (r = 0.434, p = 0.007) to presence of Lactobacillus reuteri adjusted for age, sex and BMI (Adj. R² = 0.193, p = 0.027). The lower values of ox-LDL were predicted by higher counts of cultivable lactobacilli adjusted by sex, age and BMI (r = ?0.389, p = 0.016; Adj. R² = 0.184 p = 0.029). In conclusion, the pilot study of elderly persons shows that the intestinal lactobacilli are tightly associated with WBC count, blood glucose and content of ox-LDL which all serve as risk markers in pathogenesis of inflammation, metabolic syndrome and cardiovascular disease (CVD).     

Toosendanin: Synthesis of the AB-ring and investigations of its anti-botulinum properties (Part II)

Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. The natural product toosendanin, a limonoid, is a traditional Chinese medicine that has reported anti-botulinum properties in animal models. Toosendanin effectively inhibits the biological activity of BoNT/A in neuronal cells at concentrations of 200 nM, and partial inhibition can be observed with concentrations as low as 8 nM. Mechanistically, toosendanin’s inhibition is due to prevention of transduction of the BoNT LC through the HC channel. Intriguing questions as to the molecular architecture of toosendanin as related to its anti-botulinum properties have focused our attention on a synthesis of toosendanin’s unusual AB-ring, containing a unique bridged hemi-acetal. Within the current work, a synthetic strategy allowing access to the AB-fragment of toosendanin was achieved from a trans-decalin system. In addition, this fragment was examined for its modulation of BoNT/A intoxication in a rat spinal cord cellular assay.     

Effect of polyamine deficiency on proteins involved in Okazaki fragment maturation

Polyamine depletion causes S phase prolongation, and earlier studies indicate that the elongation step of DNA replication is affected. This led us to investigate the effects of polyamine depletion on enzymes crucial for Okazaki fragment maturation in the two breast cancer cell lines MCF-7 and L56Br-C1. In MCF-7 cells, treatment with N(1),N(11)-diethylnorspermine (DENSPM) causes S phase prolongation. In L56Br-C1 cells the prolongation is followed by massive apoptosis. In the present study we show that L56Br-C1 cells have substantially lower basal expressions of two Okazaki fragment maturation key proteins, DNA ligase I and FEN1, than MCF-7 cells. Thus, these two proteins might be promising markers for prediction of polyamine depletion sensitivity, something that can be useful for cancer treatment with polyamine analogues. DENSPM treatment affects the cellular distribution of FEN1 in L56Br-C1 cells, but not in MCF-7 cells, implying that FEN1 is affected by or involved in DENSPM-induced apoptosis.     

Tropomyosin expression and dynamics in developing avian embryonic muscles

The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.     

Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes

Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39-57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1-HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.     

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.