CircRNA ZFR stimulates the proliferation of hepatocellular carcinoma through upregulating MAP2K1.

OBJECTIVE: The aim of the study was to illustrate the function of circRNA ZFR in aggravating the development of hepatocellular carcinoma (HCC) by upregulating MAP2K1. PATIENTS AND METHODS: CircRNA ZFR levels in 62 paired HCC and paracancerous species were detected. The influence of circRNA ZFR on clinical data of HCC patients was analyzed. After the overexpression or knockdown of circRNA ZFR, changes in viability and clonality of Bel-7402 and Hep3B cells were assessed, respectively. The involvement of circRNA ZFR/MAP2K1 axis in the development of HCC was explored through Luciferase assay and rescue experiments. RESULTS: CircRNA ZFR was highly expressed in HCC species than the paracancerous ones. Higher level of circRNA ZFR predicted more advanced tumor grading of HCC. The knockdown of circRNA ZFR attenuated the proliferative ability of HCC cells, while the overexpression of circRNA ZFR obtained opposite results. MAP2K1 level was positively correlated to that of circRNA ZFR. Luciferase assay uncovered that circRNA ZFR can be targeted by MAP2K1 through specific binding sites. In addition, the overexpression of MAP2K1 could reverse the influence of silenced circRNA ZFR on proliferative ability of HCC cells. CONCLUSIONS: CircRNA ZFR is upregulated in HCC and closely linked to tumor grading. It promotes proliferative ability in HCC by upregulating MAP2K1.

An ultrastructural investigation of the 'microsclerotia' formed in single cells of root cortical cells associated with corky root rot of tomato.

White and Scott (1973) described two forms of microsclerotia associated with corky root rot of tomato, one being formed in single cells of the outer root cortical cells and the other produced in culture from isolation of diseased roots and in degenerated root debris. By assuming that both forms were of fungal origin, they conducted an ultrastructural study using the microsclerotia produced in culture but not those formed in single cells. They demonstrated that the microsclerotia from culture consisted of hardened, interwoven hyphae of the fungus, Pyrenochaeta lycopersici. Since there have been doubts about the nature of the microsclerotia formed in single cells, an ultrastructural study was conducted. The results demonstrated clearly that the 'microsclerotia' formed in single cells of the root cortical cells were not composed of fungal mycelia, indeed these blackened cells or 'microsclerotia' were filled with numerous crystallines. There was no evidence of the presence of fungal hyphae in or around these cells. Both diseased and healthy, and young and old root cortex cells of many cultivars of tomato contained cells filled with crystallines. Therefore, it is clear that the 'microsclerotia' formed in single cells of the outer cortex described by White and Scott are not microsclerotia of the fungus, P. lycopersici.

Hydroponic chrysanthemum production: cultural and pathological issues.

Hydroponic culture has not replaced soil culture in greenhouse production of chrysanthemum (commonly known as 'mum'). This study examines cultural or pathological conditions that might have affected the conversion from soil to hydroponic production. Cultural factors investigated included hydroponic container size and shape and oxygenation of the nutrient solution. Disorders encountered during the studies included salt wicking during rooting of the cuttings and severe Pythium infection in the third and successive crops in a hydroponic system. Mums did not appear to respond to various shapes and sizes of containers and troughs in which they were grown. Also, increased oxygenation had little effect on pythium root rot and plant growth. Rooting of cuttings in a polyethylene covered hydroponic system reduced wilting, whereas salt wicking was severe without a plastic covering. Pythium disease was severe in the third and successive crops in the same hydroponic system. The disease could be overcome by raising the cuttings in a peat-based growing medium in perforated plastic cells and then transplanting the seedlings along with the peat moss cubes into a hydroponic system.

Effect of soil moisture on seed germination, plant growth and root rot severity of navy bean in Fusarium solani infested soil.

The development of root rot and its severity were affected by numerous factors which through their interaction could distort results. In this study, a single factor, soil moisture was considered. Using Fusarium solani infested root rot soil we investigated the effect of three available soil moisture (ASM) levels (i.e. 100%, 70% and 25%) on seed rot, germination, plant growth and root rot severity of navy bean in the greenhouse. Percent germination differed strikingly averaging of 58.5, 89.5 and 87.0% respectively to ASM levels. Apparently, high soil moisture promoted seed rot and impeded seed germination. Root rot incidence and severity were examined two months later. Percent plant with root rot averaged 89.5, 89.0 and 69.8% while root rot severity was 3.01, 2.3 and 1.1 (on a 0-9 scale), respectively to ASM levels. Plant growth was moderate, good and poor with respect to ASM levels. It was apparent that the final plant growth was a sum of seed rot, root rot incidence, disease severity and soil moisture adequacy. However, seed rot appeared to be the largest contributing factor to final plant yield.

First Report of Bean pod mottle virus in Soybean in Canada.

In 2001, soybean fields were surveyed to determine the incidence of viruses because soybean aphids (Aphis glycines Matsamura), known to transmit Soybean mosaic virus (SMV) (2), were found in Ontario. In addition, bean leaf beetle (Cerotoma trifurcata Forster) was found during 2000 to be contaminated with Bean pod mottle virus (BPMV), although soybean plants, on which the beetles were feeding, tested negative (3). In the current survey, young soybean leaves were selected at random in July and August from 20 plants per site at growth stages R4 to R5 (1) from 415 sites representing the entire soybean-producing area in Ontario. Samples were maintained under cool conditions until received at the laboratory, where they were promptly processed. A combined sub-sample was obtained from the 20 plants per site. The 415 sub-samples were tested for SMV, BPMV, Tobacco ringspot virus (TRSV), and Tobacco streak virus (TSV) using polyclonal antibody kits for double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Agdia Inc., Elkart, IN). The ELISA plates were read with a plate reader (MRX, Dynex Technologies Inc., Chantilly, VA), and results were analyzed using ELISA software (Leading Edge Research, Merrickville, Ontario) and compared positive and negative controls (Agdia). TRSV was detected in one sample from Essex County and another sample from Middlesex County. SMV, BPMV, and TSV were not found in commercial soybean fields. However, SMV and BPMV were found in samples originating from two soybean breeding nurseries, one in Essex County and one in Kent County. Seedlings of soybean cv. Williams 82 were inoculated in the greenhouse with sap from leaf samples that tested positive for BPMV. Leaves of plants that developed mosaic symptoms were retested using ELISA and confirmed to be positive for BPMV. SMV and TRSV have been found previously in commercial soybean fields in Ontario (4). To our knowledge, this is the first report of BPMV on soybean plants in Canada. References: (1) W. R. Fehr et al. Merr. Crop. Sci. 11:929, 1971. (2) J. H. Hill et al. Plant Dis. 85:561, 2001. (3) A. U. Tenuta. Crop Pest. 5 (11):8, 2000. (4) J. C. Tu. Can. J. Plant Sci. 66:491, 1986.

An integrated control of Pythium root rot of greenhouse tomato.

Pythium root rot caused by Pythium aphanidermatum is one of the most important diseases of greenhouse tomatoes. Hydroponic culture exacerbates the problem. Both nutrient film technique (NFT) and recirculating growing systems pose a challenge in the control of this disease, because the pathogen, especially the zoospores, can spread easily in the recirculating solution to the whole growing system. Fortunately, hydroponically grown plants are easier to manipulate than soil grown plants, proper manipulation of root environments can lead to excellent disease control. This paper reports the development of an effective integrated control measure for pythium root rot of tomato by integrating pH, bioagent, and ultra-violet irradiation in a specific manner. This integrated control consists of three operations: a) before transplanting, the UV system is connected to sterilize the recirculating solution using 100 mJcm-2; b) after transplanting, the nutrient solution is delivered at pH 5.0 regime for five weeks followed by adjusting pH to 5.8 to 6.2 regime for one week; and c) bacterial bioagent, such as Pseudomonas is introduced into the root zone at 100 mL per plant at 10(8) bacteria mL-1 or added to the nutrient solution to arrive at 10(6) bacteria mL-1 in the solution. This report also discusses the advantages and limitations of this measure in the control of pythium root rot.

Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer.

G-protein-coupled receptors (GPCRs) transduce signals from extracellular transmitters to the inside of the cell by activating G proteins. Mutation and overexpression of these receptors have revealed that they can reach their active state even in the absence of agonist, as a result of a natural shift in the equilibrium between their inactive and active conformations. Such agonist-independent (constitutive) activity has been observed for the glutamate GPCRs (the metabotropic glutamate receptors mGluR1a and mGluR5) when they are overexpressed in heterologous cells. Here we show that in neurons, the constitutive activity of these receptors is controlled by Homer proteins, which bind directly to the receptors' carboxy-terminal intracellular domains. Disruption of this interaction by mutagenesis or antisense strategies, or expression of endogenous Homer1a (H1a), induces constitutive activity in mGluR1a or mGluR5. Our results show that these glutamate GPCRs can be directly activated by intracellular proteins as well as by agonists.

Homer proteins regulate coupling of group I metabotropic glutamate receptors to N-type calcium and M-type potassium channels.

Group I metabotropic glutamate receptors (mGluR1 and 5) couple to intracellular calcium pools by a family of proteins, termed Homer, that cross-link the receptor to inositol trisphosphate receptors. mGluRs also couple to membrane ion channels via G-proteins. The role of Homer proteins in channel modulation was investigated by expressing mGluRs and various forms of Homer in rat superior cervical ganglion (SCG) sympathetic neurons by intranuclear cDNA injection. Expression of cross-linking-capable forms of Homer (Homer 1b, 1c, 2, and 3, termed long forms) occluded group I mGluR-mediated N-type calcium and M-type potassium current modulation. This effect was specific for group I mGluRs. mGluR2 (group II)-mediated inhibition of N-channels was unaltered. Long forms of Homer decreased modulation of N- and M-type currents but did not selectively block distinct G-protein pathways. Short forms of Homer, which cannot self-multimerize (Homer 1a and a Homer 2 C-terminal deletion), did not alter mGluR-ion channel coupling. When coexpressed with long forms of Homer, short forms restored the mGluR1a-mediated calcium current modulation in an apparent dose-dependent manner. Homer 2b induced cell surface clusters of mGluR5 in SCG neurons. Conversely, a uniform distribution was observed when mGluR5 was expressed alone or with Homer short forms. These studies indicate that long and short forms of Homer compete for binding to mGluRs and regulate their coupling to ion channels. In vivo, the immediate early Homer 1a is anticipated to enhance ion channel modulation and to disrupt coupling to releasable intracellular calcium pools. Thus, Homer may regulate the magnitude and predominate signaling output of group I mGluRs.

Homer: a link between neural activity and glutamate receptor function.

The proteins of the Homer family bind to proline-rich sequences in group I metabotropic glutamate receptors, inositol trisphosphate receptors, ryanodine receptors, and Shank family proteins. Homer proteins also self associate and function as adaptors to couple interacting proteins. Recent observations indicate a role for Homer complexes in signal transduction, synaptogenesis and receptor trafficking.

Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition.

Homer EVH1 (Ena/VASP Homology 1) domains interact with proline-rich motifs in the cytoplasmic regions of group 1 metabotropic glutamate receptors (mGluRs), inositol-1,4,5-trisphosphate receptors (IP3Rs), and Shank proteins. We have determined the crystal structure of the Homer EVH1 domain complexed with a peptide from mGluR (TPPSPF). In contrast to other EVH1 domains, the bound mGluR ligand assumes an unusual conformation in which the side chains of the Ser-Pro tandem are oriented away from the Homer surface, and the Phe forms a unique contact. This unusual binding mode rationalizes conserved features of both Homer and Homer ligands that are not shared by other EVH1 domains. Site-directed mutagenesis confirms the importance of specific Homer residues for ligand binding. These results establish a molecular basis for understanding the biological properties of Homer-ligand complexes.

Dendritic and axonal targeting of type 5 metabotropic glutamate receptor is regulated by homer1 proteins and neuronal excitation.

The physiological actions of neurotransmitter receptors are intimately linked to their proper neuronal compartment localization. Here we studied the effect of the metabotropic glutamate receptor (mGluR)-interacting proteins, Homer1a, b, and c, in the targeting of mGluR5 in neurons. We found that mGluR5 was exclusively localized in cell bodies when transfected alone in cultured cerebellar granule cells. In contrast, mGluR5 was found also in dendrites when coexpressed with Homer1b or Homer1c, and in both dendrites and axons when cotransfected with Homer1a. In dendrites, cotransfected mGluR5 and Homer1b/c formed clusters that colocalized with the synaptic marker synaptophysin. Interestingly when transfected alone, the Homer proteins were also translocated to neurites but did not form such clusters. Depolarization of the neurons with a mixture of ionotropic glutamate receptor agonists, NMDA and kainate, or potassium channel blockers, tetraethylammonium and 4-aminopyridine, induced transient expression of endogenous Homer1a and persistent neuritic localization of transfected mGluR5 even long after degradation of Homer1a. These results suggest that Homer1a/b/c proteins are involved in the targeting of mGluR5 to dendritic synaptic sites and/or axons and that this effect can be regulated by neuronal activity. Because the activity-dependent effect of endogenous Homer1a was also long-lasting, the axonal targeting of mGluR5 by this protein is likely to play an important role in synaptic plasticity.

Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.

Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.

Homer 1b regulates the trafficking of group I metabotropic glutamate receptors.

The molecular basis for glutamate receptor trafficking to the plasma membrane is not understood. In the present study, we demonstrate that Homer 1b (H1b), a constitutively expressed splice form of the immediate early gene product Homer (now termed Homer 1a) regulates the trafficking and surface expression of group I metabotropic glutamate receptors. H1b inhibits surface expression of the metabotropic glutamate receptor mGluR5 in heterologous cells, causing mGluR5 to be retained in the endoplasmic reticulum (ER). In contrast, mGluR5 alone or mGluR5 coexpressed with Homer 1a successfully travels through the secretory pathway to the plasma membrane. In addition, point mutations that disrupt mGluR5 binding to H1b eliminate ER retention of mGluR5, demonstrating that H1b affects metabotropic receptor localization via a direct protein-protein interaction. Electron microscopic analysis reveals that the group I metabotropic receptor mGluR1alpha is significantly enriched in the ER of Purkinje cells, suggesting that a similar mechanism may exist in vivo. Because H1b is found in dendritic spines of neurons, local retention of metabotropic receptors within dendritic ER provides a potential mechanism for regulating synapse-specific expression of group I metabotropic glutamate receptors.

Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins.

Homer is a neuronal immediate early gene (IEG) that is enriched at excitatory synapses and binds group 1 metabotropic glutamate receptors (mGluRs). Here, we characterize a family of Homer-related proteins derived from three distinct genes. Like Homer IEG (now termed Homer 1a), all new members bind group 1 mGluRs. In contrast to Homer 1a, new members are constitutively expressed and encode a C-terminal coiled-coil (CC) domain that mediates self-multimerization. CC-Homers form natural complexes that cross-link mGluRs and are enriched at the postsynaptic density. Homer 1a does not multimerize and blocks the association of mGluRs with CC-Homer complexes. These observations support a model in which the dynamic expression of Homer 1a competes with constitutively expressed CC-Homers to modify synaptic mGluR properties.

Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors.

Group I metabotropic glutamate receptors (mGluRs) activate PI turnover and thereby trigger intracellular calcium release. Previously, we demonstrated that mGluRs form natural complexes with members of a family of Homer-related synaptic proteins. Here, we present evidence that Homer proteins form a physical tether linking mGluRs with the inositol trisphosphate receptors (IP3R). A novel proline-rich "Homer ligand" (PPXXFr) is identified in group 1 mGluRs and IP3R, and these receptors coimmunoprecipitate as a complex with Homer from brain. Expression of the IEG form of Homer, which lacks the ability to cross-link, modulates mGluR-induced intracellular calcium release. These studies identify a novel mechanism in calcium signaling and provide evidence that an IEG, whose expression is driven by synaptic activity, can directly modify a specific synaptic function.

Mycoflora and mycotoxins in adzuki and mung beans produced in Ontario, Canada.

This is the first report on the detection of fumonisin B1 (FB1) in Fusarium-infected adzuki bean (Phaseolus angularis) and mung bean (Phaseolus aureus). The infected beans had either a mouldy appearance or a distinct discoloration. Seed coats of infected adzuki beans changed from dark red to light red and those of mung beans from green to dark or brownish green. Fusarium spp. isolated from mouldy and discoloured beans included F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. moniliforme, F. oxysporum, F. solani, F. sporotrichoides and a few unidentified species. Healthy beans without any apparent discoloration and diseased beans with discoloration and mouldy appearance were analysed for mycotoxins. Diacetoxyscripenol, deoxynivalenol and T-2 toxin (T-2) were not detected in either healthy or discoloured adzuki and mung bean samples by thin layer chromatography (TLC). FB1 was detected by TLC in discoloured adzuki and mung bean samples but not in the healthy samples. TLC results were confirmed by high performance liquid chromatography (HPLC). The quantification of FB1 by HPLC revealed that discoloured adzuki and mung bean samples contained 261 +/- 43.8, and 230 +/- 21.6 micrograms g-1 of FB1, respectively. This investigation emphasizes the need for more detailed research in dealing with possible mycotoxin contamination in various foodstuffs including legumes.

Natural occurrence of mycotoxins in Fusarium infected beans.

This is the first report on the discovery of several mycotoxins in naturally infected mouldy navy beans (Phaseolus vulgaris L.) with distinct whitish grey to pink discoloration. Species of Fusarium isolated from the mouldy beans included F. avenaceum, F. culmorum, F. graminearum, F. moniliforme, F. oxysporum, F. solani and two Fusarium spp. Three types of bean samples were analysed for mycotoxins: (a) health beans without any apparent discoloration, (b) beans with pink discoloration, and (c) a mixture of beans with whitish grey and pink discoloration. The results indicated that diacetoxyscripenol (DAS), deoxynivalenol (DON), T-2 toxin (T-2) and fumonisin B1 (FB1) were present in type B and C samples but not in the healthy type A samples. Quantification of DAS, DON, and T-2 and of FB1 were achieved by gas chromatography and high performance liquid chromatography, respectively. The results showed that DAS, DON, T-2 and FB1 were not evident in the healthy samples, but in type B and type C samples, DAS, DON, T-2 and FB1 in the amounts of 9.2, 6.5, 5.5 and 0.5 microgram/g, and of 3.3, 3.1, 13.5 and 1.1 micrograms/g, respectively, were detected. Further research is under way to determine which species of Fusarium is responsible for the production of specific toxins.

Comparison of the profiles of seedborne fungi and the occurrence of aflatoxins in mould-damaged beans and soybeans.

The average percentage of fungal infection in twenty bean and soybean lots was determined by visual inspection (24.5 and 16.8%, respectively), and by laboratory isolation (58.5 and 33.7%), respectively. The mycoflora were more diverse in bean than soybean. Bean was more susceptible to fungal infection than soybean under similar environmental and cultural conditions. The most frequently isolated fungal genera for bean and soybean were Aspergillus, Penicillium, Rhizopus, Eurotium and Curvularia. Aflatoxins were not detected in the non-infected beans and soybeans, but aflatoxins (B1, B2, G1 and G2) were detected in the infected beans but not in the infected soybeans. The absence of aflatoxin in the fungal infected soybeans is discussed.

Oxalic acid induced cytological alterations differ in beans tolerant or susceptible to white mould.

Leaf tissues of two white bean (Phuscolus vulgaris L.) cultivars, one susceptible and one tolerant to white mould [Sclerotinia sclerotinrum (Lib.) de Bary], were treated for different times with oxalic acid. Tissues were prepared by both thin sectioning and freeze-fraeture techniques for electron microscopy. The plasma membrane of the resistant cultivar appeared more tolerant to damage induced by oxalic acid than that of the susceptible cultivar. At the same concentration of oxalic acid the plasma membrane and chloroplasts of the susceptible cultivar were affected more, and ruptured more quickly, than those of the tolerant one. In thin sections, the ruptured organelles were partially or completely disorganized. In replicas of freeze-fractured preparations of the plasma membrane, increased protrusions, wrinkles, breakages and ruptures were associated with increasing exposure to oxalic acid. Damage was more severe in the plasma membrane of the susceptible cultivar than in that of the tolerant one. Measurements of the conductivity of water bathing leaf discs of the two cultivars showed that the susceptible cultivar led to a higher conductivity than the resistant one, indicating that the injury and rupturing of the plasma membrane were closely related to membrane stability.