Biotype status and distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) in Shandong province of China based on mitochondrial DNA markers

Bemisia tabaci has caused significant crop losses in China during the last decade. Recent research has shown that two potentially invasive variants, biotypes B and Q, have been found in several regions of China. Our objective was to determine the biotype status and the distribution of B. tabaci in Shandong province, an important agricultural region of China. Based on mitochondrial DNA markers, both biotypes B and Q were detected, with B being the predominant biotype. The results indicate that the more recently introduced biotype Q has not only been located in China but also has established and spread in some regions.     

Hemopoietic cell expression of the chemokine decoy receptor D6 is dynamic and regulated by GATA1

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.     

Investigation of Sexual Process in Interspecific Crosses Between Gossipium hirsutum and G. arboreum

The present investigation is the first of a series of cytological researches on inter-specific hybridization of cottons. The present report deals with the cytological behavior of sex and related cells revealed in crossing Gossipium hirsutum (female parent) with G. arboreum. Self-pollenated G. hirsutum served as control. The germination of pollen grains on the stigma and the growth of pollen tubes in the style and the entry of pollen tubes into the micropyles in crosses are in a fairly normal' manner when compared with controls. The percentages of pollen tubes entering ovules of the crossed and controls have no significant difference, being 48.2% in crosses and 54.7% in controls. Fertilization proceeds also normal in cross pollinated ovules. About 92.7% of pollinated ovules are found to have completed the fusion of sexual elements. It is thus: shown that the growth of foreign pollen tubes and fusion of female elements with foreign sperms are not the factors which cause failure in hybridization. The development of endosperm in crosses is abnormal. The number of free nuclei of hybrid endosperm is much smaller than that of controls; the formation of hybrid cellular endosperm is 6 or 7 days earlier than that of controls. The hybrid endosperm cells start to abort soon after their formation. About 14 days after pollination they become completely disintegrated. The hybrid embryo which thus far developed normally becomes arrested in its development and aborts also after that time. It may be concluded that the sterility of crosses between G. hirsutum and G. arboreum is primarily attributed to the abortion of hybrid endosperm. The sound development of the hybrids should be sought in the improvement nutrition of the hybrid embryo from resource other than their own endosperm.     

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.     

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.     

Molecular Imaging Reveals a Progressive Pulmonary Inflammation in Lower Airways in Ferrets Infected with 2009 H1N1 Pandemic Influenza Virus

Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [¹⁸F]-2-deoxy-2-fluoro-D-glucose ([¹⁸F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [¹⁸F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7–7.0). By days 2 and 3, consolidation (CT) and inflammation ([¹⁸F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0–7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [¹⁸F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman’s ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time.     

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

Apoptotic effects of over-expressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner

Epidemiologic studies reported that the prevalence of hereditary non-polyposis colon cancer (HNPCC) in male is about 1.5-fold higher than that in female. Decreases in circulatory estradiol (E2) have been reported to downregulate the expression of E2 receptor (ER) and significantly increase the risk of colorectal cancer. Patients that received E2 replacement therapy were found to have a reduction in the incidence of colon adenoma and carcinoma. Furthermore, significant decreases in the expression of ER have been found in colorectal cancer specimens. These data strongly suggest the protective roles of E2 and ER against colorectal cancer. However, the mechanisms remain unexplored. LoVo cells were transient transfected to overexpress ER-beta, DNA fragmentation and caspase activity assay were performed to evaluate apoptotic effects. Western blotting was used to evaluate protein levels, and luciferase activity assay to measure the TNF-alpha promoter activity. Our data clearly demonstrated that E2 and ER-beta alone could upregulate p21 and p27 proteins, which further activate caspase-8 and caspase-9 to induce apoptosis in LoVo cell, and the ER-beta. effects were enhanced by E2. However, overexpressed ER-beta did not influence the expression and promoter activity of TNF-alpha. In addition, E2 and overexpressed ER-beta downregulated the beta-catenin proteins which cause the downregulation of its target genes, cyclin D1 and Rb, to inhibit the cell cycle and cell proliferation. The results indicate that overexpressed ER-beta may induce LoVo cell apoptosis and anti-proliferation by increasing p53 signaling in a ligand-dependent manner, and without hTNF-alpha involvement. Efforts aiming at enhancing ER-beta expression and/or activity may prove to be an attractive alternative therapy against colorectal cancer.     

Ammonia-induced structural changes of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy

Light-induced Fourier transform infrared difference spectroscopy has been applied to studies of ammonia effects on the oxygen-evolving complex (OEC) of photosystem II (PSII). We found that NH(3) induced characteristic spectral changes in the region of the symmetric carboxylate stretching modes (1450-1300 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra of PSII. The S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the controlled samples was very likely upshifted to 1379 cm(-1) in that of NH(3)-treated samples; however, the frequency of the corresponding S(1) carboxylate mode at 1402 cm(-1) in the same spectrum was not significantly affected. These two carboxylate modes have been assigned to a Mn-ligating carboxylate whose coordination mode changes from bridging or chelating to unidentate ligation during the S(1) to S(2) transition [Noguchi, T., Ono, T., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200; Kimura, Y., and Ono, T.-A. (2001) Biochemistry 40, 14061-14068]. Therefore, our results show that NH(3) induced significant structural changes of the OEC in the S(2) state. In addition, our results also indicated that the NH(3)-induced spectral changes of the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the temperature of the FTIR measurement. Among the temperatures we measured, the strongest effect was seen at 250 K, a lesser effect was seen at 225 K, and little or no effect was seen at 200 K. Furthermore, our results also showed that the NH(3) effects on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the concentrations of NH(4)Cl. The NH(3)-induced upshift of the 1365 cm(-1) mode is apparent at 5 mM NH(4)Cl and is completely saturated at 100 mM NH(4)Cl concentration. Finally, we found that CH(3)NH(2) has a small but clear effect on the spectral change of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. The effects of amines on the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (NH(3) > CH(3)NH(2) > AEPD and Tris) are inverse proportional to their size (Tris approximately AEPD > CH(3)NH(2) > NH(3)). Therefore, our results showed that the effects of amines on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are sterically selective for small amines. On the basis of the correlations between the conditions (dependences on the excitation temperature and NH(3) concentration and the steric requirement for the amine effects) that give rise to the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII and the conditions that give rise to the altered S(2) state multiline EPR signal, we propose that the NH(3)-induced upshift of the 1365 cm(-1) mode is caused by the binding of NH(3) to the site on the Mn cluster that gives rise to the altered S(2) state multiline EPR signal. In addition, we found no significant NH(3)-induced change in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum at 200 K. Under this condition, the OEC gives rise to the NH(3)-stabilized g = 4.1 EPR signal and a suppressed g = 2 multiline EPR signal. Our results suggest that the structural difference of the OEC between the normal g = 2 multiline form and the NH(3)-stabilized g = 4.1 form is small.     

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.