Construction of an infectious full-length and eGFP-tagged cDNA clone of a chilli ringspot virus isolate from Yunnan province, China.

Chilli ringspot virus (ChiRSV; genus Potyvirus) was one of several viruses previously detected in pepper samples with severe yellowing and curling symptoms growing in Wenshan, Yunan province, China. We now report the full-length sequence of ChiRSV-YN/Wenshan (MZ269480), which has 88.5-98.9% nucleotide sequence identity to other published ChiRSV isolates. A full-length cDNA infectious clone was constructed. This cDNA and an eGFP-tagged clone were infectious, leading to systemic symptoms in both Nicotiana benthamiana and Capsicum spp. Recombinant clones containing the P1 protein coding region of other ChiRSV isolates differed in their pathogenicity. Single infection by ChiRSV caused mild mosaic or leaf crinkling in Capsicum frutescens L. and Capsicum annuum L.

Pod pepper vein yellows virus, a new recombinant polerovirus infecting Capsicum frutescens in Yunnan province, China.

Pepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5' half of its genome (encoding P0 to P4), PoPeVYV is most similar (93.1% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3'-part encoding P5, where it is most similar (91.7% nt identity) to tobacco vein distorting virus (TVDV, EF529624) suggesting a recombinant origin. Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt, with PeVYV-3 as the major parent but with the region 4126-5192 nt derived from TVDV as the minor parent. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.

α1G T-type calcium channel determines the angiogenic potential of pulmonary microvascular endothelial cells.

Pulmonary microvascular endothelial cells (PMVECs) display a rapid angioproliferative phenotype, essential for maintaining homeostasis in steady-state and promoting vascular repair after injury. Although it has long been established that endothelial cytosolic Ca2+ ([Ca2+]i) transients are required for proliferation and angiogenesis, mechanisms underlying such regulation and the transmembrane channels mediating the relevant [Ca2+]i transients remain incompletely understood. In the present study, the functional role of the microvascular endothelial site-specific α1G T-type Ca2+ channel in angiogenesis was examined. PMVECs intrinsically possess an in vitro angiogenic "network formation" capacity. Depleting extracellular Ca2+ abolishes network formation, whereas blockade of vascular endothelial growth factor receptor or nitric oxide synthase has little or no effect, suggesting that the network formation is a [Ca2+]i-dependent process. Blockade of the T-type Ca2+ channel or silencing of α1G, the only voltage-gated Ca2+ channel subtype expressed in PMVECs, disrupts network formation. In contrast, blockade of canonical transient receptor potential (TRP) isoform 4 or TRP vanilloid 4, two other Ca2+ permeable channels expressed in PMVECs, has no effect on network formation. T-type Ca2+ channel blockade also reduces proliferation, cell-matrix adhesion, and migration, three major components of angiogenesis in PMVECs. An in vivo study demonstrated that the mice lacking α1G exhibited a profoundly impaired postinjury cell proliferation in the lungs following lipopolysaccharide challenge. Mechanistically, T-type Ca2+ channel blockade reduces Akt phosphorylation in a dose-dependent manner. Blockade of Akt or its upstream activator, phosphatidylinositol-3-kinase (PI3K), also impairs network formation. Altogether, these findings suggest a novel functional role for the α1G T-type Ca2+ channel to promote the cell's angiogenic potential via a PI3K-Akt signaling pathway.

Complete genome sequence of yam chlorotic necrosis virus, a novel macluravirus infecting yam.

The complete genome sequence of a novel member of the genus Macluravirus was determined from yam plants with chlorotic and necrotic symptoms in China. The genomic RNA consists of 8,261 nucleotides (nt) excluding the 3'-terminal poly(A) tail, containing one long open reading frame (ORF) encoding a large putative polyprotein of 2,627 amino acids. Its genomic structure is typical of macluraviruses, which lack the P1 protein, N-terminal HC-Pro, and D-A-G motif for aphid transmission that are found in potyviruses. The virus shares 56.3-63.8% sequence identity at the genome sequence level and 49.7-63.9% at the polyprotein sequence level with other members of the genus Macluravirus. Phylogenetic analysis based on the complete polyprotein sequence of representative members of the family Potyviridae clearly places the virus within the genus Macluravirus. These results suggest that the virus, tentatively named "yam chlorotic necrosis virus" (YCNV), should be considered a member of a novel species in the genus Macluravirus.

Altered accumulation of osa-miR171b contributes to rice stripe virus infection by regulating disease symptoms.

Viral infection affects the pattern of plant miRNA expression. It has been presumed that reduction of miR171 and several other miRNAs influences viral symptoms in plants. We here experimentally demonstrate the association of osa-miR171b with rice stripe virus (RSV) symptoms in rice. Inhibition of osa-miR171b caused stunting with reduced chlorophyll content in leaves similar to viral symptoms. Overexpression of osa-miR171b by an artificial miRNA extended vegetative growth and enhanced chlorophyll accumulation in leaves. Tillers were thicker, and panicles were longer with more spikelets in plants overexpressing osa-miR171b than in controls, but there were no differences in tiller numbers. Targets of osa-miR171b, OsSCL6-IIa, OsSCL6-IIb, and OsSCL6-IIc, were respectively up- and down-regulated in plants where osa-miR171b was inhibited or overexpressed. In plants overexpressing osa-miR171b, five positive regulators for heading development, Ehd1, Ehd2, Ehd3, Ehd4, and Hd3a were up-regulated, while the negative regulator Ghd7 was down-regulated. Plants overexpressing osa-miR171b were less susceptible to RSV and virus symptoms were attenuated. Taken together, the results reveal that a reduction of osa-miR171b in RSV-infected rice contributes to RSV symptoms, and provide more insight into the roles of osa-miR171b in rice.

Anti-TMV activity and functional mechanisms of two sesquiterpenoids isolated from Tithonia diversifolia.

Unlike chemical pesticides, antiviral plants are biodegradable, replenishable and safe. In this study, 14 sesquiterpene compounds from Tithonia diversifolia were tested for their activities against Tobacco mosaic virus (TMV) using the half-leaf method. Tagitinin C (Ses-2) and 1ß-methoxydiversifolin-3-0-methyl ether (Ses-5) were found to have in vivo curative activities of 62.86% and 60.27% respectively, at concentrations of 100µg/mL, respectively. In contrast, the in vivo curative inhibition rate of control agent ningnanmycin was 52.48%. Indirect enzyme-linked immunosorbent assay (ID-ELISA) also verified Ses-2 and Ses-5 had higher inhibition activities than the control agent ningnanmycin. Additionally, qRT-PCR showed that both Ses-2 and Ses-5 can partly inhibit the expression of CP and RdRp, two genes that play key roles in TMV infection. When TMV started to systemically spread, Ses-2 inhibited CP expression while Ses-5 inhibited RdRp expression. These results suggest that the two bio-agents have anti-TMV activities and may be used as bio-pesticides to control the plant virus.

Transcriptional regulation of α1H T-type calcium channel under hypoxia.

The low-voltage-activated T-type Ca(2+) channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5'-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site -1,173cacgc-1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region.

Orai1 determines calcium selectivity of an endogenous TRPC heterotetramer channel.

RATIONALE: Canonical transient receptor potential 4 (TRPC4) contributes to the molecular composition of a channel encoding for a calcium selective store-operated current, I(SOC), whereas Orai1 critically comprises a channel encoding for the highly selective calcium release activated calcium current, I(CRAC). However, Orai1 may interact with TRPC proteins and influence their activation and permeation characteristics. Endothelium expresses both TRPC4 and Orai1, and it remains unclear as to whether Orai1 interacts with TRPC4 and contributes to calcium permeation through the TPRC4 channel. OBJECTIVE: We tested the hypothesis that Orai1 interacts with TRPC4 and contributes to the channel's selective calcium permeation important for endothelial barrier function. METHODS AND RESULTS: A novel method to purify the endogenous TRPC4 channel and probe for functional interactions was developed, using TRPC4 binding to protein 4.1 as bait. Isolated channel complexes were conjugated to anti-TRPC protein antibodies labeled with cy3-cy5 pairs. Förster Resonance Energy Transfer among labeled subunits revealed the endogenous protein alignment. One TRPC1 and at least 2 TRPC4 subunits constituted the endogenous channel (TRPC1/4). Orai1 interacted with TRPC4. Conditional Orai1 knockdown reduced the probability for TRPC1/4 channel activation and converted it from a calcium-selective to a nonselective channel, an effect that was rescued on Orai1 reexpression. Loss of Orai1 improved endothelial cell barrier function. CONCLUSION: Orai1 interacts with TRPC4 in the endogenous channel complex, where it controls TRPC1/4 activation and channel permeation characteristics, including calcium selectivity, important for control of endothelial cell barrier function.

Different Response Mechanisms of Rhizosphere Microbial Communities in Two Species of Amorphophallus to Pectobacterium carotovorum subsp. carotovorum Infection.

Soft rot is a widespread, catastrophic disease caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) that severely damages the production of Amorphophallus spp. This study evaluated the rhizosphere bacterial and fungal communities in Pcc-infected and uninfected plants of two species of Amorphophallus, A. muelleri and A. konjac. Principal component analysis showed that the samples formed different clusters according to the Pcc infection status, indicating that Pcc infection can cause a large number of changes in the bacterial and fungal communities in the Amorphophallus spp. rhizosphere soil. However, the response mechanisms of A. muelleri and A. konjac are different. There was little difference in the overall microbial species composition among the four treatments, but the relative abundances of core microbiome members were significantly different. The relative abundances of Actinobacteria, Chloroflexi, Acidobacteria, Firmicutes, Bacillus, and Lysobacter were lower in infected A. konjac plants than in healthy plants; in contrast, those of infected A. muelleri plants were higher than those in healthy plants. For fungi, the relative abundances of Ascomycota and Fusarium in the rhizosphere of infected A. konjac plants were significantly higher than those of healthy plants, but those of infected A. muelleri plants were lower than those of healthy plants. The relative abundance of beneficial Penicillium fungi was lower in infected A. konjac plants than in healthy plants, and that of infected A. muelleri plants was higher than that of healthy plants. These findings can provide theoretical references for further functional research and utilization of Amorphophallus spp. rhizosphere microbial communities in the future.

The Additional 15 nt of 5' UTR in a Novel Recombinant Isolate of Chilli Veinal Mottle Virus in Solanum nigrum L. Is Crucial for Infection.

An isolate of chilli veinal mottle virus (ChiVMV; genus Potyvirus) of Solanum nigrum L. from southwest China (ChiVMV-YunN/Yuxi) was identified and sequenced (GenBank: OP404087). Comparison with other ChiVMV isolates and recombination analyses suggested a recombinant origin. The most significant recombination event among all 21 complete ChiVMV isolates was an ending breakpoint at 1408-1488 for ChiVMV-YunN/Yuxi with ChiVMV-TaiW and ChiVMV-YunN/Ca operating as the respective major and minor parents. Interestingly, the 5' UTR of ChiVMV-YunN/Yuxi is 15 nucleotides ('AAAAATAAAACAACC') longer than other reported isolates. A full-length clone of ChiVMV-YunN/Yuxi was constructed and was shown to be infectious in Nicotiana benthamiana. The additional 15 nt of 5' UTR in ChiVMV-YunN/Yuxi was stable when transmitted through three generations. Experiments with modified clones showed that the additional 15 nt are essential for infection by this isolate.

Intron retention and 3'-UTR analysis of Arabidopsis Dicer-like 2 transcripts.

Arabidopsis thaliana Dicer-like protein 2 (AtDCL2) plays an essential role in the RNA interference pathway. The function of AtDCL2 and other DCLs has been much studied but little has been done to characterize the DCLs transcripts before they are translated into proteins. Here, we investigated AtDCL2 transcripts and showed that all 21 introns of AtDCL2 except intron 9, 18, 20 and 21 could be retained although spliced sequences usually predominated. Intron 10 was more frequently retained and transient expression assays in Nicotiana benthamiana leaves showed that when AG/C at the 3' splicing site of the intron was changed to AG/G, the intron was more frequently spliced out. Conversely, a high retention of intron 18 was obtained if the AG/G at the 3' splicing site was changed to AG/C. These results suggest that the sequence at the 3' splicing site affects the efficiency of intron splicing. The 3'-UTRs of AtDCL2 had lengths between 54 and 154 nts, and the different 3'-UTRs differentially affected the transcriptional levels of fused GFP expressed transiently in N. benthamiana. Further comparisons and mutation experiments suggested that a putative SBF-1 binding site and an AU-rich element in the 3'-UTR both down-regulated expression of the upstream GFP fused to the 3'-UTR. Conversely, a second poly(A) consensus signal sequence in one 3'-UTR up-regulated gene expression. Our results provide insight into the character of AtDCL2 transcripts and demonstrate the potential complexity of factors that affect the frequency and patterns of alternative splicing.

Synergism Among the Four Tobacco Bushy Top Disease Casual Agents in Symptom Induction and Aphid Transmission.

Tobacco bushy top disease (TBTD), caused by multiple pathogens including tobacco bushy top virus (TBTV), tobacco vein distorting virus (TVDV), TBTV satellite RNA (TBTVsatRNA), and TVDV-associated RNA (TVDVaRNA), is a destructive disease in tobacco fields. To date, how these causal agents are co-transmitted by aphid vectors in field and their roles in disease symptom induction remain largely unknown, due mainly to the lack of purified causal agents. In this study, we have constructed four full-length infectious clones, representing the Yunnan Kunming isolates of TVDV, TBTV, TBTVsatRNA, and TVDVaRNA (TVDV-YK, TBTV-YK, TBTVsatRNA-YK, and TVDVaRNA-YK), respectively. Co-inoculation of these four causal agents to tobacco K326 plants caused typical TBTD symptoms, including smaller leaves, necrosis, and plant stunting. In addition, inoculation of tobacco K326 plants with TBTV alone caused necrosis in systemic leaves by 7 dpi. Tobacco K326 and Nicotiana benthamiana plants infected by single virus or multiple viruses showed very different disease symptoms at various dpi. RT-PCR results indicated that co-infection of TVDVaRNA-YK could increase TVDV-YK or TBTV-YK accumulation in N. benthamiana plants, suggesting that TVDVaRNA-YK can facilitate TVDV-YK and TBTV-YK replication and/or movement in the infected plants. Aphid transmission assays showed that the successful transmission of TBTV-YK, TBTVsatRNA-YK, and TVDVaRNA-YK by Myzus persicae depended on the presence of TVDV-YK, while the presence of TBTVsatRNA-YK increased the aphid transmission efficiency of TBTV and TVDV. We consider that these four new infectious clones will allow us to further dissect the roles of these four causal agents in TBTD induction as well as aphid transmission.

Case Report: Fascioliasis Hepatica Precisely Diagnosed by Metagenomic Next-Generation Sequencing and Treated With Albendazole.

The clinical manifestations of fascioliasis hepatica in humans are unspecific. Traditional diagnosis relies on evidence of live parasites or eggs in the bile or feces. However, due to similar imaging manifestations, they are often misdiagnosed as malignant tumors. Here, we report a case of a 43-year-old woman with fever and space-occupying liver disease. Liver biopsy, parasite-specific antibody screening, and stool testing did not find any pathogens. Therefore, metagenomic next-generation sequencing (mNGS) and routine microbiological examinations were performed. Finally, Fasciola hepatica was only identified by mNGS. The body temperature of the patient and the eosinophil count remained normal, and the space-occupying liver lesions were significantly absorbed after more than 7 months of treatment with albendazole. The details of this case highlight the timely use of mNGS to identify parasites and judge therapeutic effects after treatment, providing important help for clinical decision-making.

Biological and Genetic Characterization of Pod Pepper Vein Yellows Virus-Associated RNA From Capsicum frutescens in Wenshan, China.

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with some viruses of the genus Polerovirus. Pod pepper vein yellows virus (PoPeVYV) was recently reported as a new recombinant polerovirus causing interveinal yellowing, stunting, and leaf rolling in Capsicum frutescens plants at Wenshan city, Yunnan province, China. The complete genome sequence of its associated RNA has now been determined by next-generation sequencing and reverse transcription (RT) polymerase chain reaction (PCR). PoPeVYV-associated RNA (PoPeVYVaRNA) (GenBank Accession No. MW323470) has 2970 nucleotides and is closely related to other group II tlaRNAs, particularly tobacco bushy top disease-associated RNA (TBTDaRNA, GenBank Accession No. EF529625). In infection experiments on Nicotiana benthamiana and C. frutescens plants, synergism between PoPeVYVaRNA and PoPeVYV was demonstrated, leading to severe interveinal yellowing of leaves and stunting of plants. The results provide further information on the genetic and biological properties of the various agents associated with pepper vein yellows disease (PeVYD).

Identification of two novel poleroviruses and the occurrence of Tobacco bushy top disease causal agents in natural plants.

Tobacco bushy top disease (TBTD) is a devastating tobacco disease in the southwestern region of China. TBTD in the Yunnan Province is often caused by co-infections of several plant viruses: tobacco bushy top virus (TBTV), tobacco vein distorting virus (TVDV), tobacco bushy top virus satellite RNA (TBTVsatRNA) and tobacco vein distorting virus-associated RNA (TVDVaRNA). Through this study, two new poleroviruses were identified in two TBTD symptomatic tobacco plants and these two novel viruses are tentatively named as tobacco polerovirus 1 (TPV1) and tobacco polerovirus 2 (TPV2), respectively. Analyses of 244 tobacco samples collected from tobacco fields in the Yunnan Province through RT-PCR showed that a total of 80 samples were infected with TPV1 and/or TPV2, and the infection rates of TPV1 and TPV2 were 8.61% and 29.51%, respectively. Thirty-three TPV1 and/or TPV2-infected tobacco samples were selected for further test for TBTV, TVDV, TBTVsatRNA and TVDVaRNA infections. The results showed that many TPV1 and/or TPV2-infected plants were also infected with two or more other assayed viruses. In this study, we also surveyed TBTV, TVDV, TBTVsatRNA and TVDVaRNA infections in a total of 1713 leaf samples collected from field plants belonging to 29 plant species in 13 plant families and from 11 provinces/autonomous regions in China. TVDV had the highest infection rates of 37.5%, while TVDVaRNA, TBTV and TBTVsatRNA were found to be at 23.0%, 12.4% and 8.1%, respectively. In addition, TVDV, TBTV, TBTVsatRNA and TVDVaRNA were firstly detected of co-infection on 10 plants such as broad bean, pea, oilseed rape, pumpkin, tomato, crofton weed etc., and 1 to 4 of the TBTD causal agents were present in the samples collected from Guizhou, Hainan, Henan, Liaoning, Inner mongolia and Tibet autonomous regions. The results indicated that TBTD causal agents are expanding its host range and posing a risk to other crop in the field.

Effect of hypernatremia on injury caused by energy deficiency: role of T-type Ca2+ channel.

Hypernatremia exerts multiple cellular effects, many of which could influence the outcome of an ischemic event. To further evaluate these effects of hypernatremia, isolated neonatal cardiomyocytes were chronically incubated with medium containing either normal (142 mM) or elevated sodium (167 mM) and then transferred to medium containing deoxyglucose and the electron transport chain inhibitor amobarbital. Chronic hypernatremia diminished the degree of calcium accumulation and reactive oxygen species generation during the period of metabolic inhibition. The improvement in calcium homeostasis was traced in part to the downregulation of the Ca(V)3.1 T-type calcium channel, as deficiency in the Ca(V)3.1 subtype using short hairpin RNA or treatment with an inhibitor of the Ca(V)3.1 variant of the T-type calcium channel (i.e., diphenylhydantoin) attenuated energy deficiency-mediated calcium accumulation and cell death. Although hyperosmotically stressed cells (exposed to 50 mM mannitol) had no effect on T-type calcium channel activity, they were also resistant to death during metabolic inhibition. Both hyperosmotic stress and hypernatremia activated Akt, suggesting that they initiate the phosphatidylinositol 3-kinase/Akt cytoprotective pathway, which protects the cell against calcium overload and oxidative stress. Thus hypernatremia appears to protect the cell against metabolic inhibition by promoting the downregulation of the T-type calcium channel and stimulating cytoprotective protein kinase pathways.

Alpha1G T-type calcium channel selectively regulates P-selectin surface expression in pulmonary capillary endothelium.

Regulated P-selectin surface expression provides a rapid measure for endothelial transition to a proinflammatory phenotype. In general, P-selectin surface expression results from Weibel-Palade body (WPb) exocytosis. Yet, it is unclear whether pulmonary capillary endothelium possesses WPbs or regulated P-selectin surface expression and, if so, how inflammatory stimuli initiate exocytosis. We used immunohistochemistry, immunofluorescence labeling, ultrastructural assessment, and an isolated perfused lung model to demonstrate that capillary endothelium lacks WPbs but possesses P-selectin. Thrombin stimulated P-selectin surface expression in both extra-alveolar vessel and alveolar capillary endothelium. Only in capillaries was the thrombin-stimulated P-selectin surface expression considerably mitigated by pharmacologic blockade of the T-type channel or genetic knockout of the T-type channel alpha(1G)-subunit. Depolarization of endothelial plasma membrane via high K(+) perfusion capable of eliciting cytosolic Ca(2+) transients also provoked P-selectin surface expression in alveolar capillaries that was abolished by T-type channel blockade or alpha(1G) knockout. Our findings reveal an intracellular WPb-independent P-selectin pool in pulmonary capillary endothelium, where the regulated P-selectin surface expression is triggered by Ca(2+) transients evoked through activation of the alpha(1G) T-type channel.

Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer.

BACKGROUND: Activated leukocyte cell adhesion molecule (ALCAM) is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs. RESULTS: A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters. CONCLUSION: Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

A role of functional T-type Ca2+ channel in hepatocellular carcinoma cell proliferation.

The role of T-type Ca2+ channels in hepatocellular carcinoma cell proliferation was investigated in vitro. Eleven hepatocellular carcinoma cell lines and one immortalized liver cell line (LO2) were examined for the status of T-type Ca2+ channels with RT-PCR and voltage-clamp recordings. Except HBxF344, other cell lines tested had one, two or all three of alpha1-subunits (alpha1G, alpha1H and alpha1I) mRNA expression. Obvious T-type current was recorded in SNU449 cells, while others exhibited a minimal or no T-type current. SNU449, PLC/PRF5, Hep3B and LO2 cell lines were subjected to growth assay in the presence of Mibefradil, a T-type Ca2+ channel blocker, only the proliferation of SNU449 cell which had functional T-type Ca2+ channel was reduced by Mibefradil treatment. Furthermore, the persistent increase of phosphorylated ERK1/2 in SNU449 cells was found when treated with Mibefradil. A microarray assay also demonstrated some down-regulated genes were mainly associated with cell cycle and cell proliferation in Mibefradil treated SNU449 cells. In conclusion, this study showed that the functional T-type Ca2+ channels probably participate in modulating the proliferation of some hepatocellular carcinoma cells. The cell proliferation reduction of SNU449 with Mibefradil treatment is possibly associated with the persistent increase of phosphorylated ERK1/2.

Molecular cloning and characterization of the Dicer-like 2 gene from Brassica rapa.

Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3' end of BrDCL2, clones with three different lengths of 3' untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.