A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins.

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Mini-TrpRS is essential for IFNγ-induced monocyte-derived giant cell formation.

Truncated tryptophanyl-tRNA synthetase (mini-TrpRS), like any other aminoacyl-tRNA synthetases, canonically functions as a protein synthesis enzyme. Here we provide evidence for an additional signaling role of mini-TrpRS in the formation of monocyte-derived multinuclear giant cells (MGCs). Interferon-gamma (IFNγ) readily induced monocyte aggregation leading to MGC formation with paralleled marked upregulation of mini-TrpRS. Small interfering (si)RNA, targeting mini-TrpRS in the presence of IFNγ prevented monocyte aggregation. Moreover, blockade of mini-TrpRS, either by siRNA, or the cognate amino acid and decoy substrate D-Tryptophan to prevent mini-TrpRS signaling, resulted in a marked reduction in expression of the purinergic receptor P2X 7 (P2RX7) in monocytes activated by IFNγ. Our findings identify mini-TrpRS as a critical signaling molecule in a mechanism by which IFNγ initiates monocyte-derived giant cell formation.

PRMT5 Is Required for Bovine Leukemia Virus Infection In Vivo and Regulates BLV Gene Expression, Syncytium Formation, and Glycosylation In Vitro.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle and is closely related to human T-cell leukemia viruses. We investigated the role of a new host protein, PRMT5, in BLV infection. We found that PRMT5 is overexpressed only in BLV-infected cattle with a high proviral load, but not in those with a low proviral load. Furthermore, this upregulation continued to the lymphoma stage. PRMT5 expression was upregulated in response to experimental BLV infection; moreover, PRMT5 upregulation began in an early stage of BLV infection rather than after a long period of proviral latency. Second, siRNA-mediated PRMT5 knockdown enhanced BLV gene expression at the transcript and protein levels. Additionally, a selective small-molecule inhibitor of PRMT5 (CMP5) enhanced BLV gene expression. Interestingly, CMP5 treatment, but not siRNA knockdown, altered the gp51 glycosylation pattern and increased the molecular weight of gp51, thereby decreasing BLV-induced syncytium formation. This was supported by the observation that CMP5 treatment enhanced the formation of the complex type of N-glycan more than the high mannose type. In conclusion, PRMT5 overexpression is related to the development of BLV infection with a high proviral load and lymphoma stage and PRMT5 inhibition enhances BLV gene expression. This is the first study to investigate the role of PRMT5 in BLV infection in vivo and in vitro and to reveal a novel function for a small-molecule compound in BLV-gp51 glycosylation processing.

Essential Function of the Serine Hydroxymethyl Transferase (SHMT) Gene During Rapid Syncytial Cell Cycles in Drosophila.

Many metabolic enzymes are evolutionarily highly conserved and serve a central function in the catabolism and anabolism of cells. The serine hydroxymethyl transferase (SHMT) catalyzing the conversion of serine and glycine and vice versa feeds into tetrahydrofolate (THF)-mediated C1 metabolism. We identified a Drosophila mutation in SHMT (CG3011) in a screen for blastoderm mutants. Embryos from SHMT mutant germline clones specifically arrest the cell cycle in interphase 13 at the time of the midblastula transition (MBT) and prior to cellularization. The phenotype is due to a loss of enzymatic activity as it cannot be rescued by an allele with a point mutation in the catalytic center but by an allele based on the SHMT coding sequence from Escherichia coli The onset of zygotic gene expression and degradation of maternal RNAs in SHMT mutant embryos are largely similar to that in wild-type embryos. The specific timing of the defects in SHMT mutants indicates that at least one of the SHMT-dependent metabolites becomes limiting in interphase 13, if it is not produced by the embryo. Our data suggest that mutant eggs contain maternally-provided and SHMT-dependent metabolites in amounts that suffice for early development until interphase 13.

Detection of the Epstein-Barr virus and DNA-topoisomerase II- α in recurrent and nonrecurrent giant cell lesion of the jawbones.

The aims of this study were to determine whether the expression of Topo II-α correlates with presence of EBV in giant cell lesion of the jawbones and whether it is predictive of clinical biologic behavior of these lesions. Paraffin-embedded tissues from 8 recurrent and 7 nonrecurrent cases of bony GCLs and 9 peripheral giant cell lesions (PGCLs) as a control group were assessed for the expression of EBV and Topo II-α using immunohistochemistry. The results showed positive staining for Topo II-α in mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs). Student t-test showed that mean Topo II-α labelling index (LI) in recurrent cases was significantly higher than that in non-recurrent cases (P = 0.0001). Moreover, Spearman's correlation coefficients method showed a significant correlation between DNA Topo II-α LI and both of gender and site in these lesions. Moderate EBV expression in relation to the highest Topo II-α LI was observed in two cases of GCT. It was concluded that high Topo II-α LIs could be identified as reliable predicators for the clinical behavior of GCLs. Moreover, EBV has no etiological role in the benign CGCLs in contrast to its role in the pathogenesis of GCTs.

Expression of cathepsin K and tartrate-resistant acid phosphatase is not confined to osteoclasts but is a general feature of multinucleated giant cells: systematic analysis.

OBJECTIVE: Cathepsin K and tartrate-resistant acid phosphatase (TRAP) are two proteins expressed in osteoclastic giant cells. Recently we showed that lesional multinucleated giant cells (MNGs) in pulmonary granulomatosis with polyangiitis expressed these proteins. We aimed to clarify whether the expression of these two proteins has any specificity or is a general feature of MNGs associated with multiple types of granulomatous inflammation. METHODS: In total, 7 Crohn's disease (CD), 5 GCA, 5 giant cell myocarditis (GCM), 11 sarcoidosis and 6 tuberculosis cases were examined for expression of cathepsin K and TRAP using immunohistochemistry (IHC). Protein expression was semi-quantitatively classified as none, weak, moderate or strong. In addition, tissue TRAP activity was examined using an enzymatic reaction. RESULTS: The expression of cathepsin K was robust in >95% of MNGs of all examined disease groups, whereas TRAP expression varied; CD, GCA and tuberculosis showed strong TRAP expression. TRAP expression in sarcoidosis and GCM was weaker (CD vs GCM, P = 0.04; CD vs sarcoidosis, P = 0.06). Compared with IHC, TRAP detection using an enzymatic colour reaction had limited sensitivity. CONCLUSION: Expression of TRAP and cathepsin K is a general feature of MNGs and their expression might be related to histopathological pattern.

Cell wall ingrowths in nematode induced syncytia require UGD2 and UGD3.

The cyst nematode Heterodera schachtii infects roots of Arabidopsis plants and establishes feeding sites called syncytia, which are the only nutrient source for nematodes. Development of syncytia is accompanied by changes in cell wall structures including the development of cell wall ingrowths. UDP-glucuronic acid is a precursor of several cell wall polysaccharides and can be produced by UDP-glucose dehydrogenase through oxidation of UDP-glucose. Four genes in Arabidopsis encode this enzyme. Promoter::GUS analysis revealed that UGD2 and UGD3 were expressed in syncytia as early as 1 dpi while expression of UGD1 and UGD4 could only be detected starting at 2 dpi. Infection assays showed no differences between Δugd1 and Δugd4 single mutants and wild type plants concerning numbers of males and females and the size of syncytia and cysts. On single mutants of Δugd2 and Δugd3, however, less and smaller females, and smaller syncytia formed compared to wild type plants. The double mutant ΔΔugd23 had a stronger effect than the single mutants. These data indicate that UGD2 and UGD3 but not UGD1 and UGD4 are important for syncytium development. We therefore studied the ultrastructure of syncytia in the ΔΔugd23 double mutant. Syncytia contained an electron translucent cytoplasm with degenerated cellular organelles and numerous small vacuoles instead of the dense cytoplasm as in syncytia developing in wild type roots. Typical cell wall ingrowths were missing in the ΔΔugd23 double mutant. Therefore we conclude that UGD2 and UGD3 are needed for the production of cell wall ingrowths in syncytia and that their lack leads to a reduced host suitability for H. schachtii resulting in smaller syncytia, lower number of developing nematodes, and smaller females.

Ceramide biosynthesis and metabolism in trophoblast syncytialization.

Sphingolipid mediators such as ceramide are pleiotropic regulators of cellular growth, differentiation and apoptosis. We investigated the role of ceramide biosynthesis, metabolism and actions in term human cytotrophoblasts syncytialized over 7 days in culture. Intracellular C16 ceramide levels increased modestly after 3 days in culture, then declined. Ceramidase was present at particularly high levels in syncytialized trophoblasts; inhibition of ceramidase reduced the degree of cell fusion. Exposure to short chain C8 ceramide or aSMase enhanced secretion of the differentiation marker hCG without affecting fusion or cell viability. In contrast, pharmacological inhibition of ceramidase reduced the extent of fusion. Inhibition of the ceramide-responsive JNK and PP2A pathways did not abolish the effects of ceramide, and JNK phosphorylation was unresponsive to ceramide; however, ceramide significantly inhibited phosphorylation of Akt. This study suggests that changes in ceramide biosynthesis and metabolism play a differential role in the biochemical and morphological features of trophoblast differentiation.

Increased expression of matrix metalloproteinase 9 in cortical lesions from patients with focal cortical dysplasia type IIb and tuberous sclerosis complex.

The malformative cortical lesions in the cerebral cortex that are characteristic of focal cortical dysplasia type IIb (FCDIIb) and tuberous sclerosis complex (TSC) are well-recognized causes of chronic intractable epilepsy in children. Increasing evidence suggests that extracellular matrix molecules play important roles in epileptogenesis. Matrix metalloproteinase 9 (MMP9), a typical extracellular matrix proteolytic protease, has been shown to participate in the occurrence of seizures in experimental models. In the present study, we used immunoblotting to analyze the levels of MMP9 protein in FCDIIb lesions, TSC tubers and control samples, which included epileptic neocortices from temporal lobe epilepsy and non-epileptic normal cortices (CTX). The cellular distribution of MMP9 was further investigated by immunohistochemical methods. Our findings demonstrated the elevated levels of the inactive and active forms of MMP9 protein in FCDIIb and TSC lesions compared with CTX. Furthermore, the immunohistochemical results showed that MMP9 was characteristically expressed in the following misshapen cells: hypertrophic neurons, dysmorphic neurons, balloon cells and giant cells. Additionally, double immunofluorescent staining revealed that the reactive astrocytes, but not the microglia, expressed high levels of MMP9. Taken together, our findings suggest that the overexpression and spatial distribution patterns of MMP9 may be linked with the intractable epilepsy caused by FCDIIb and TSC.

Caspase-mediated apoptosis of trophoblasts in term human placental villi is restricted to cytotrophoblasts and absent from the multinucleated syncytiotrophoblast.

Human placental villi are surfaced by a multinucleated and terminally differentiated epithelium, the syncytiotrophoblast, with a subjacent layer of mononucleated cytotrophoblasts that can divide and fuse to replenish the syncytiotrophoblast. The objectives of this study were i) to develop an approach to definitively identify and distinguish cytotrophoblasts from the syncytiotrophoblast, ii) to unambiguously determine the relative susceptibility of villous cytotrophoblasts and syncytiotrophoblast to constitutive and stress-induced apoptosis mediated by caspases, and iii) to understand the progression of apoptosis in villous trophoblasts. Confocal microscopy with co-staining for E-cadherin and DNA allowed us to clearly distinguish the syncytiotrophoblast from cytotrophoblasts and identified that many cytotrophoblasts are deeply interdigitated into the syncytiotrophoblast. Staining for specific markers of caspase-mediated apoptosis indicate that apoptosis occurs readily in cytotrophoblasts but is remarkably inhibited in the syncytiotrophoblast. To determine if an apoptotic cell or cell fragment was from a cytotrophoblast or syncytiotrophoblast, we found co-staining with E-cadherin along with a marker for apoptosis was essential: in the absence of E-cadherin staining, apoptotic cytotrophoblasts would easily be mistaken as representing localized regions of apoptosis in the syncytiotrophoblast. Regions with perivillous fibrin-containing fibrinoid contain the remnants of trophoblast apoptosis, and we propose this apoptosis occurs only after physical isolation of a region of the syncytium from the main body of the syncytium. We propose models for the progression of apoptosis in villous cytotrophoblasts and for why caspase-mediated apoptosis does not occur within the syncytium of placental villi.

Cyclooxygenase-2 expression in central giant cell lesion of the jaws: an immunohistochemical study.

Central Giant Cell Lesion (CGCL) is an uncommon benign jaw lesion, with uncertain etiology, and a variable clinical behavior. Studies of molecular markers of CGCL, may help understanding better the nature and behavior of this lesion, and eventually may represent a definitive target to pharmacological approach in the treatment of CGCL. Chronic inflammation has been found to mediate a wide variety of diseases including neoplasms. Among the gene products involved in the induction of the inflammatory process, Cyclooxygenase 2 (COX-2) has been shown to have a close relationship with tumorigenesis, however COX-2 expression has never been evaluated in CGCL. The aim of the study was to investigate the expression of COX-2 in CGCL. Immunohistochemical assessment for COX-2 expression was performed in 18 patients previously diagnosed with CGCL. Multinucleated giant cells (MGC) and mononucleated stromal cells (MSC) were used in the slide analysis. Among the patients studied, 10 were male and 8 were female, with a median age of 15.4 years. Lesions in the mandible were observed in 11 cases and 7 were found in the maxilla. There were 9 aggressive and 9 non-aggressive CGCLs. COX-2 immunopositivity was present in only 3 cases stained in both MGC and MSC. All 3 cases presented with ulcerations in the mucosa lesion, suggesting that the COX-2 expression is due to the presence of inflammation. This study does not support the involvement of COX-2 in the etiophatogenesis of CGCL.

Reduced bone formation in alcohol-induced osteopenia is associated with elevated p21 expression in bone marrow cells in aldehyde dehydrogenase 2-disrupted mice.

INTRODUCTION: High consumption of alcohol is one of the risk factors for osteoporosis. Approximately 45% of Chinese and Japanese individuals have the inactive aldehyde dehydrogenase 2 (Aldh2) phenotype. The absence of the ALDH2*2 allele is found to adversely influence the risk of osteoporosis. The aim of this study is to clarify the effects of alcohol consumption on osteoblast differentiation in bone marrow and trabecular bone formation in Aldh2-disrupted mice. MATERIALS AND METHODS: Seven-week-old male Aldh2 knockout mice (Aldh2(-/-)) and wild-type (Aldh2(+/+)) mice were fed with water (groups Aldh2(-/-)/Wa and Aldh2(+/+)/Wa) or with 5% ethanol (groups Aldh2(-/-)/Al and Aldh2(+/+)/Al) for 4 weeks. At the age of 12 weeks, bone histomorphometry was performed at the secondary spongiosa of the tibias. Bone marrow cells from the bilateral femurs and tibias were used for mRNA expression analysis. RESULTS: Histomorphometrical study revealed that trabecular bone was significantly reduced in the Aldh2(-/-)/Al group compared with that in the Aldh2(-/-)/Wa and Aldh2(+/+)/Wa groups. Bone formation rate was significantly decreased in Aldh2(-/-)/Al compared with the other three groups. Quantitative RT-PCR revealed a significant decrease in type I collagen, osterix, osteopontin, and osteocalcin mRNA expressions in Aldh2(-/-)/Al compared with Aldh2(-/-)/Wa. In bone marrow cell cultures, mineralized nodule formation in Aldh2(-/-)/Al was significantly decreased compared with that in Aldh2(+/+)/Wa and Aldh2(-/-)/Wa, while PAK18, a p21-activated kinase inhibitor, recovered the decreased mineralized nodule formation in Aldh2(-/-)/Al. CONCLUSION: Alcohol consumption suppressed the differentiation and mineralization of osteoblasts and then reduced trabecular bone formation and bone volume in association with the elevated p21 expression in bone marrow cells, especially in aldehyde dehydrogenase 2-disrupted mice.

Immunohistochemical expression of mast cell tryptase in giant cell fibroma and inflammatory fibrous hyperplasia of the oral mucosa.

This study analysed the immunohistochemical expression of mast cell tryptase in giant cell fibromas (GCFs). In addition, the possible interaction of mast cells with stellate giant cells, as well as their role in fibrosis and tumour progression, was investigated. For this purpose, the results were compared with cases of inflammatory fibrous hyperplasia (IFH) and normal oral mucosa. Thirty cases of GCF, 30 cases of IFH and 10 normal mucosa specimens used as control were selected. Immunoreactivity of mast cells to the anti-tryptase antibody was analysed quantitatively in the lining epithelium and in connective tissue. In the epithelial component (p=0.250) and connective tissue (p=0.001), the largest mean number of mast cells was observed in IFHs and the smallest mean number in GCFs. In connective tissue, the mean percentage of degranulated mast cells was higher in GCFs than in IFHs and normal mucosa specimens (p<0.001). Analysis of the percentage of degranulated mast cells in areas of fibrosis and at the periphery of blood vessels also showed a larger mean number in GCFs compared to IFHs and normal mucosa specimens (p<0.001). The percent interaction between mast cells and stellate giant cells in GCFs was 59.62%. In conclusion, although mast cells were less numerous in GCFs, the cells exhibited a significant interaction with stellate giant cells present in these tumours. In addition, the results suggest the involvement of mast cells in the induction of fibrosis and modulation of endothelial cell function in GCFs.

Arabidopsis peroxidase AtPRX53 influences cell elongation and susceptibility to Heterodera schachtii.

Cyst nematodes establish and maintain feeding sites (syncytia) in the roots of host plants by altering expression of host genes. Among these genes are members of the large gene family of class III peroxidases, which have reported functions in a variety of biological processes. In this study, we used Arabidopsis-Heterodera schachtii as a model system to functionally characterize peroxidase 53 (AtPRX53). Promoter assays showed that under non-infected conditions AtPRX53 is expressed mainly in the root, the hypocotyl and the base of the pistil. Under infected conditions, the AtPRX53 promoter showed upregulation at the nematode penetration sites and in their migration paths. Interestingly, strong GUS activity was observed in H. schachtii-induced syncytia during the early stage of infection and remained strong in the syncytia of third-stage juveniles. Also, AtPRX53 showed upregulation in response to wounding and jasmonic acid treatments. Manipulation of AtPRX53 expression through overexpression and knockout mutation affected both plant morphology and nematode susceptibility. While AtPRX53 overexpression lines exhibited short hypocotyls, aberrant flower development and reduced nematode susceptibility to H. schachtii, the atprx53 mutant showed long hypocotyls and a 3-carpel silique phenotype as well as a non significant increase of nematode susceptibility. Taken together these data, therefore, indicate diverse roles of AtPRX53 in the wound response, flower development and syncytium formation.

Overexpression of matrix metalloproteinase (MMP)-1 and -9 in central giant cell lesions of the jaws: implications for clinical behavior.

OBJECTIVE: The aim of this study was to investigate the relationship between the immunohistochemical expression of MMP-1 and MMP-9 with the clinical behavior of central giant cell lesions (CGCLs) of the jaws. STUDY DESIGN: Paraffin-embedded tissue from 30 aggressive and 12 nonaggressive CGCLs was assessed for the expression of MMP-1 and MMP-9 using immunohistochemistry. RESULTS: Although cellular immunolocalization patterns of MMP-1 and MMP-9 were similar, mean values of expression estimation/SID scores of each protease were significantly higher in aggressive CGCLs in comparison with nonaggressive lesions. Moreover, linear regression analysis showed that there was a reasonably good correlation not only between the expression estimation but also among SID scores of the 2 proteolytic enzymes. CONCLUSION: The findings of this study suggest a role for MMP-1 and MMP-9 in the resorptive activity of different cellular groups in CGCLs and indicate that differences in immunoreactivity of these 2 proteolytic enzymes may underlie the distinct clinical behavior.

Evidence for the role of matrix metalloproteinase-13 in bone resorption by giant cell tumor of bone.

Giant cell tumor of bone (GCT) is an aggressively osteolytic primary bone tumor that is characterized by the presence of abundant multinucleated osteoclast-like giant cells, hematopoietic monocytes, and a distinct mesenchymal stromal cell component. Previous work in our laboratory has shown that matrix metalloproteinase (MMP)-13 is the principal proteinase expressed by the stromal cells of GCT. The release of cytokines, particularly interleukin-1beta, by the giant cells of GCT acts on stromal cells to stimulate a surge in MMP-13 secretion. The purpose of this study was to determine the bone resorption capabilities of the cellular elements of GCT and the significance of the MMP-13 expression involved in GCT bone resorption. We present a 3-dimensional histomorphometric technique developed to analyze resorption pit depth and yield an accurate measurement of bone resorption with a direct physical view of lacunae on bone slices. In this study, we demonstrate that the mesenchymal stromal cells and the multinucleated giant cells of GCT are independently capable of bone resorption. However, coculture of these 2 cell fractions shows a synergistic increase in bone resorption. In addition, inhibition of MMP-13 reduces resorptive activity of the cells indicating that MMP-13 likely plays an important role in this tumor. This cell-cell cooperation involves giant cell-derived cytokine up-regulation of MMP-13 in the stromal cells, which in turn assists the giant cells in bone resorption. Future research will involve elucidation of the role of cell-cell/matrix communication pathways in bone resorption and tumorigenesis in GCT.

The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice.

The dynamin-related guanosine triphosphatase Drp1 mediates the division of mitochondria and peroxisomes. To understand the in vivo function of Drp1, complete and tissue-specific mouse knockouts of Drp1 were generated. Drp1-null mice die by embryonic day 11.5. This embryonic lethality is not likely caused by gross energy deprivation, as Drp1-null cells showed normal intracellular adenosine triphosphate levels. In support of the role of Drp1 in organelle division, mitochondria formed extensive networks, and peroxisomes were elongated in Drp1-null embryonic fibroblasts. Brain-specific Drp1 ablation caused developmental defects of the cerebellum in which Purkinje cells contained few giant mitochondria instead of the many short tubular mitochondria observed in control cells. In addition, Drp1-null embryos failed to undergo developmentally regulated apoptosis during neural tube formation in vivo. However, Drp1-null embryonic fibroblasts have normal responses to apoptotic stimuli in vitro, suggesting that the apoptotic function of Drp1 depends on physiological cues. These findings clearly demonstrate the physiological importance of Drp1-mediated organelle division in mice.

Myo-inositol oxygenase genes are involved in the development of syncytia induced by Heterodera schachtii in Arabidopsis roots.

* In plants, UDP-glucuronic acid is synthesized by the oxidation of UDP-glucose by UDP-glucose dehydrogenase or the oxygenation of free myo-inositol by myo-inositol oxygenase (MIOX). In Arabidopsis, myo-inositol oxygenase is encoded by four genes. Transcriptome analysis of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots revealed that MIOX genes are among the most strongly upregulated genes. * We have used beta-glucuronidase (GUS) analysis, in situ reverse transcription polymerase chain reaction (RT-PCR), and real-time RT-PCR to study the expression of all four MIOX genes in syncytia induced by H. schachtii in Arabidopsis roots. All these methods showed that MIOX genes are strongly induced in syncytia. GeneChip data were analysed for the expression of genes related to the MIOX pathway (mapman). * Two complementary double mutants were used to study the importance of MIOX genes. Results of the infection assay with double mutants in two combinations (Deltamiox1+2, Deltamiox4+5) showed a significant reduction (P < 0.05) in the number of females per plant when compared with the wild-type. Furthermore, syncytia in double mutants were significantly smaller than in wild-type plants. * Our data demonstrate an important role of the MIOX genes for syncytium development and for the development of female nematodes.

Integrin-linked kinase can facilitate syncytialization and hormonal differentiation of the human trophoblast-derived BeWo cell line.

BACKGROUND: In the fusion pathway of trophoblast differentiation, stem villous cytotrophoblast cells proliferate and daughter cells differentiate and fuse with existing syncytiotrophoblast to maintain the multi-nucleated layer. Integrin-linked kinase (ILK) is highly expressed in 1st and 2nd trimester villous cytotrophoblast cells, yet barely detectable in syncytiotrophoblast, thus we examined the potential role of ILK in aiding trophoblast fusion. METHODS: The temporal/spatial expression and activity of ILK were determined in BeWo cells undergoing syncytialization by immunoblot and immunofluorescence analyses. BeWo cells were also transfected with pEGFP expression vectors containing wildtype or two mutant ILK cDNA constructs. The incidence of cell fusion in transfected cells grown under syncytialization conditions was then scored by the presence or absence of E-cadherin immunostaining. Beta-hCG expression in transfected cells, a marker of syncytiotrophoblast hormonal differentiation, was also similarly assessed. RESULTS: ILK catalytic activity increased and ILK began to increasingly localize to BeWo cell nuclei during syncytialization in correlation with increased pAkt and Snail protein expression. Syncytialization was also significantly elevated (p < 0.05) in BeWo cells expressing constitutively active (ca)-ILK vs cells containing empty vector or dn-ILK. Furthermore, cytoplasmic Beta-hCG expression markedly increased (p < 0.05) in cells expressing wt- and ca-ILK. CONCLUSION: ILK-facilitated syncytialization is dependent, at least in part, on ILK catalytic activity while hormonal differentiation appears dependent on both ILK-associated protein interactions and catalytic activity. This study demonstrates that ILK plays a novel role in BeWo syncytialization and differentiation, perhaps through an ILK-Akt-Snail pathway, and implicates ILK in the same process in villous cytotrophoblasts in vivo.

Role of NADPH oxidase in formation and function of multinucleated giant cells.

Macrophages play essential roles in a wide variety of physiological and pathological processes. One of the unique features of these phagocytic leukocytes is their ability to fuse, forming multinucleated giant cells. Multinucleated giant cells are important mediators of tissue remodeling and repair and are also responsible for removal or sequestration of foreign material, intracellular bacteria and non-phagocytosable pathogens, such as parasites and fungi. Depending on the tissue where fusion occurs and the inflammatory insult, multinucleated giant cells assume distinctly different phenotypes. Nevertheless, the ultimate outcome is the formation of large cells that can resorb bone tissue (osteoclasts) or foreign material and pathogens (giant cells) extracellularly. While progress has been made in recent years, the mechanisms and factors involved in macrophage fusion are still not fully understood. In addition to cytokines and a number of adhesion proteins and receptors, it is becoming increasingly clear that NADPH oxidase-generated reactive oxygen species (ROS) also play an important role in macrophage fusion. In this review, we provide an overview of macrophage multinucleation, with a specific focus on the role of NADPH oxidases and ROS in macrophage fusion and in the function of multinucleated giant cells. In addition, we provide an updated overview of the role of these cells in inflammation and various autoimmune diseases.