两株棉铃虫胚胎新细胞系的建立及其对杆状病毒侵染的反应

昆虫细胞系的建立在病毒学和昆虫学等领域的研究和应用中发挥着重要的作用。本研究由棉铃虫Helicoverpa armigera胚胎组织建立了两株细胞系, 分别命名为QB-Ha-E-1和QB-Ha-E-5, 在含10％胎牛血清的TNM-FH培养基中已传代60余代。两株细胞系均以圆形和短梭形细胞为主。DAF鉴定结果表明, 两株细胞系均来源于棉铃虫胚胎, 其扩增谱带与其他几种昆虫细胞系明显不同; QB-Ha-E-1和QB-Ha-E-5的第30代细胞群体倍增时间分别为63.7 h和66.9 h。两株细胞系均能被棉铃虫核型多角体病毒（HaSNPV）感染, 4 d的感染率分别为86.6％和56.5％, 对甘蓝夜蛾Mamestra brassicae核型多角体病毒（MbNPV）7 d的感染率均为15％左右, 但对苜蓿银纹夜蛾Autographa californica核型多角体病毒（AcMNPV）侵染的反应不同。DAPI染色和基因组DNA电泳结果表明, AcMNPV可诱导QB-Ha-E-5细胞发生凋亡, 极少数细胞内可形成多角体, 但不能诱导QB-Ha-E-1细胞发生凋亡, 其感染率为55.3％; 两株细胞系均可被1.25 μg/mL的放线菌素D诱导发生凋亡。两株细胞系具有相同的遗传背景, 但对AcMNPV侵染的反应不同, 可作为昆虫病毒和细胞之间相互关系以及细胞凋亡机制研究的理想材料。     

Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress,BAX, and caspase 2

Ischemia-reperfusion (IR) of the testis results in germ-cell-specific apoptosis (GCA) and a reduction in daily sperm production. This has been correlated with and is dependent upon neutrophil recruitment to the testis. In a rat model of testicular IR, this has also been correlated with an increase in reactive oxygen species (ROS). We have investigated ROS in the mouse testis after IR and determined whether the observed GCA is mediated via a mitochondrial caspase-9-dependent pathway involving the upstream mediators caspase 2 and BAX. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. An accumulation of 8-isoprostane, a marker of oxidative stress, occurred 4 h after reperfusion. Activation of a mitochondrial dependent pathway to GCA after testicular IR was determined based on the observations that both BAX and caspase 2 translocated to the mitochondria, and that an increase occurred in cytoplasmic cytochrome c. Moreover, microinfusion of a specific caspase 9 inhibitor significantly reduced active caspase 3 after testicular IR and the number of apoptotic germ cells. These results suggest that oxidative stress products accumulate in the testis following IR and demonstrate that the observed GCA is stimulated through a mitochondrial caspase-9-dependent pathway. The identification of the germ-cell apoptotic pathway induced after testicular IR, including the key players in the pathway subsequent to ROS (BAX, caspase 9, and caspase 2), aids our understanding of IR injury in the testis and provides a wider background for the development of therapeutic interventions to rescue testis function. The work was supported by grants P50 DK45179 and DK53072.     

Hyperthermia inhibits cell proliferation and induces apoptosis: relative signaling status of P53, S100A4, and Notch in heat sensitive and resistant cell lines

The effects of hyperthermia on the expression of p53, the apoptosis-associated genes Bax and Bcl-2, Notch and S100A4 have been studied in the HepG2 cell line and the HUT cell line derived from HepG2, adapted for growth in hyperthermic conditions. Hyperthermia inhibits cell proliferation and induces apoptosis. HepG2 and HUT cells differed in respect of anchorage to growth surface, degree of proliferation and apoptosis and expression of p53, Bax, Bcl-2, Notch, and S100A4 genes. The induction of apoptosis and the inhibition of cell proliferation occurred independently of p53, and independently also of involvement of the apoptosis family genes Bax and Bcl-2. We demonstrate novel and marked differences between transient heat shock and heat adaptation in respect of pathways of signaling and generation of phenotypic effects in vitro. Different signaling patterns have been identified here. Pathways of signaling by S100A4, by its interaction with and sequestration of p53, and by Notch also seem differentially operational in the induction of apoptosis, and both appear to be activated as alternative pathways in the context of hyperthermia signaling independently of p53.     

Characterization of prolactin-releasing peptide: binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor

The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K_d in the 10⁻⁹ M range and a B_max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with ¹²⁵I-PrRP31 with a similar K_i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.     

Infectivity of Anticarsia gemmatalis nucleopolyhedrovirus to different insect cell lines: Morphology, viral production, and protein synthesis

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is currently used as an efficient biological pesticide for the control of the velvetbean caterpillar (A. gemmatalis), an important pest of soybean in Brazil. Until now, production of the virus has been achieved mainly by infection of larvae on local soybean farms. Studies for the development of in vitro systems and the optimization of mass production in insects reared on artificial diets is now important to help to meet the actual demand for the bioinsecticide. We therefore, investigated the infectivity of AgMNPV in cell culture, which might contribute to the selection of suitable cell lines that may be used for in vitro production of this virus. The cytopathic effects induced by the virus, the production of viral particles and the synthesis of viral polypeptides were examined and compared in the cell lines from A. gemmatalis (UFL-AG-286), Trichoplusia ni (BTI-Tn-5B1-4 and TN-368), Spodoptera frugiperda (IPLB-SF-21AE and Sf9), Lymantria dispar (IPLB-LD-652Y), and Bombyx mori (BM-5). Whereas, Tn-5B1-4 and AG-286 cells produced large numbers of occlusion bodies, no polyhedra were visualized in either Ld-652Y or BM-5 cells, although extensive cell lysis was observed in BM-5. Analysis of the kinetics of viral protein synthesis by SDS–PAGE after pulse labeling with [³⁵S]methionine, showed similar protein patterns in most of the cell lines tested. Exceptions were the LD-652Y and BM-5 cells, in which viral polypeptides, including polyhedrin, were not synthesized. In parallel, measurement of viral titers (budded virus) by the endpoint dilution method showed that Tn-5B1-4, AG-286, and SF-21AE cells were highly productive. Their TCID₅₀ values, at 48 h p.i., were about 10⁷ IU/ml. In addition to the lower formation of polyhedra, the viral titers determined in Sf9 and TN-368 cells were about 5 to10-fold lower. As expected, the viral titers obtained in LD-652Y and BM-5 cells were similar to basal levels.     

G1P3, an interferon inducible gene 6-16, is expressed in gastric cancers and inhibits mitochondrial-mediated apoptosis in gastric cancer cell line TMK-1 cell

Expression of an interferon inducible gene 6-16, G1P3, increases not only in type I interferon-treated cells but also in human senescent fibroblasts. However, the function of 6-16 protein is unknown. Here we report that 6-16 is 34 kDa glycosylated protein and localized at mitochondria. Interestingly, 6-16 is expressed at high levels in gastric cancer cell lines and tissues. One of exceptional gastric cancer cell line, TMK-1, which do not express detectable 6-16, is sensitive to apoptosis induced by cycloheximide (CHX), 5-fluorouracil (5-FU) and serum-deprivation. Ectopic expression of 6-16 gene restored the induction of apoptosis and inhibited caspase-3 activity in TMK-1 cells. Thus 6-16 protein has anti-apoptotic function through inhibiting caspas-3. This anti-apoptotic function is expressed through inhibition of the depolarization of mitochondrial membrane potential and release of cytochrome c. By two-hybrid screening, we found that 6-16 protein interacts with calcium and integrin binding protein, CIB/KIP/Calmyrin (CIB), which interacts with presenilin 2, a protein involved in Alzheimers disease. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyses are need. These results overall indicate that 6-16 protein may have function as a cell survival protein by inhibiting mitochondrial-mediated apoptosis.     

Fine-root morphology and uptake of 32P and 35S in a Norway spruce (Picea abies (L.) Karst.) stand subjected to various nutrient and water supplies

An investigation of fine (< 1 mm in diameter) and small (1–2 mm in diameter) roots in the organic soil layer was carried out in a Norway spruce forest stand with different treatments of water and nutrients, including control (C); ammonium sulphate application (NS); nitrogen-free fertilization (V); irrigation with liquid fertilization (a complete nutrient solution) (IF); NS followed by artificial drought (ND); V followed by artificial drought (VD). In order to evaluate the vitality and function of the fine roots, the following approaches were used: i) classification of fine roots, based on morphological characteristics; ii) nutrient uptake bioassay, using ³²P-phosphate and ³⁵S-sulphate; iii) nutrient concentration in fine roots and its relation to nutrient uptake. The NS treatment showed effects on the fine and small roots, with a decrease in amount of living roots, and a decrease in the total amount of fine and small roots. The VD treatment resulted in increased amounts of living small roots, while the ND treatment showed the opposite, as compared with the V and NS treatments, respectively. The uptake of P was negatively related to the P supply, with a higher P uptake for C and NS fine roots than for IF and V fine roots. The specific root length (SRL, m g^-1 DW) decreased for NS fine roots and increased for IF fine roots, indicating a further increase in uptake for NS roots and a decreased uptake for IF roots if calculated on a root length basis. So far, the NS and IF treatments maintain a considerable increase in above-ground biomass with a significantly reduced root biomass and standing crop.     

Arene-Ru(II)-chloroquine complexes interact with DNA, induce apoptosis on human lymphoid cell lines and display low toxicity to normal mammalian cells

The complexes [Ru(η⁶-p-cymene)(CQ)Cl₂] (1), [Ru(η⁶-benzene)(CQ)Cl₂] (2), [Ru(η⁶-p-cymene)(CQ)(H₂O)₂][BF₄]₂ (3), [Ru(η⁶-p-cymene)(en)(CQ)][PF₆]₂ (4), [Ru(η⁶-p-cymene)(η⁶-CQDP)][BF₄]₂ (5) (CQ = chloroquine base; CQDP = chloroquine diphosphate; en = ethylenediamine) interact with DNA to a comparable extent to that of CQ and in analogous intercalative manner with no evidence for any direct contribution of the metal, as shown by spectrophotometric and fluorimetric titrations, thermal denaturation measurements, circular dichroism spectroscopy and electrophoresis mobility shift assays. Complexes 1-5 induced cytotoxicity in Jurkat and SUP-T1 cancer cells primarily via apoptosis. Despite the similarities in the DNA binding behavior of complexes 1-5 with those of CQ the antitumor properties of the metal drugs do not correlate with those of CQ, indicating that DNA is not the principal target in the mechanism of cytotoxicity of these compounds. Importantly, the Ru-CQ complexes are generally less toxic toward normal mouse splenocytes and human foreskin fibroblast cells than the standard antimalarial drug CQDP and therefore this type of compound shows promise for drug development.     

Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: Tolerance to NaCl and freezing stress

Sixty-seven hydroxyproline-resistant (hyp^r) cell lines were selected from cell suspensions of a diploid potato ( Solanum tuberosum L., clone H²578) after plating on 5 and 10 m M hydroxyproline (hyp). Resistant colonies were obtained with a spontaneous frequency of 2.9×10⁻⁶. No clear influence could be shown from treatment with N-ethyl-N-nitrosourea (10 or 50 μ M ). Ninety % of the variant lines contained more proline than the wild type when cells were grown away from hyp for 1 month. Total free amino acid content was increased 2.2 to 6.8 times. When the lines were grown for another 2–5 months on non-selective medium, the content of proline and other amino acids and hyp resistance decreased. After this period the values were, however, still substantially higher than in the wild type. When tested for growth on media with other amino acid analogues (azetidine-2-carboxylic acid and dehydroproline, analogues of proline; aminoethyl-cysteine, analogue of lysine and 3-fluorotyrosine, analogue of tyrosine) and on media with inhibitory concentrations of lysine + threonine. lines H4a and H4b4 were cross resistant to these compounds. When tested on media with inhibitory NaCl concentrations, variant lines H2a, H4a, and H6 showed better tolerance than the wild type. One variant cell line (H4a) was successfully regenerated into plants. Preliminary results showed an increased frost tolerance in the leaves of these plants (−4.5°C compared to −3°C for the wild type), accompanied by a higher leaf proline content. Callus initiated from leaves of the regenerated clones was more resistant to hyp than wild type callus, indicating that the variant trait might be due to a mutation.     

Microbial biomass C,N and P in two arctic soils and responses to addition of NPK fertilizer and sugar: implications for plant nutrient uptake

The soil microbial carbon (C), nitrogen (N) and phosphorus (P) pools were quantified in the organic horizon of soils from an arctic/alpine low-altitude heath and a high-altitude fellfield by the fumigation-extraction method before and after factorial addition of sugar, NPK fertilizer and benomyl, a fungicide. In unamended soil, microbial C, N and P made up 3.3–3.6%, 6.1–7.3% and 34.7% of the total soil C, N and P content, respectively. The inorganic extractable N pool was below 0.1% and the inorganic extractable P content slightly less than 1% of the total soil pool sizes. Benomyl addition in spring and summer did not affect microbial C or nutrient content analysed in the autumn. Sugar amendments increased microbial C by 15 and 37% in the two soils, respectively, but did not affect the microbial nutrient content, whereas inorganic N and P either declined significantly or tended to decline. The increased microbial C indicates that the microbial biomass also increased but without a proportional enhancement of N and P uptake. NPK addition did not affect the amount of microbial C but almost doubled the microbial N pool and more than doubled the P pool. A separate study has shown that CO₂ evolution increased by more than 50% after sugar amendment and by about 30% after NPK and NK additions to one of the soils. Hence, the microbial biomass did not increase in response to NPK addition, but the microbes immobilized large amounts of the added nutrients and, judging by the increased CO₂ evolution, their activity increased. We conclude: (1) that microbial biomass production in these soils is stimulated by labile carbon and that the microbial activity is stimulated by both labile C and by nutrients (N); (2) that the microbial biomass is a strong sink for nutrients and that the microbial community probably can withdraw substantial amounts of nutrients from the inorganic, plant-available pool, at least periodically; (3) that temporary declines in microbial populations are likely to release a flush of inorganic nutrients to the soil, particularly P of which the microbial biomass contained more than one third of the total soil pool; and (4) that the mobilization-immobilization cycles of nutrients coupled to the population dynamics of soil organisms can be a significant regulating factor for the nutrient supply to the primary producers, which are usually strongly nutrient-limited in arctic ecosystems.     

Ride to cell wall: Arabidopsis XTH11, XTH29 and XTH33 exhibit different secretion pathways and responses to heat and drought stress

The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re-joining hemicellulose chains in the xyloglucan–cellulose network. Here, in a homologous system, we compare the secretion patterns of XTH11, XTH33 and XTH29, three members of the Arabidopsis thaliana XTH family, selected for the presence (XTH11 and XTH33) or absence (XTH29) of a signal peptide, and the presence of a transmembrane domain (XTH33). We show that XTH11 and XTH33 reached, respectively, the cell wall and plasma membrane through a conventional protein secretion (CPS) pathway, whereas XTH29 moves towards the apoplast following an unconventional protein secretion (UPS) mediated by exocyst-positive organelles (EXPOs). All XTHs share a common C-terminal functional domain (XET-C) that, for XTH29 and a restricted number of other XTHs (27, 28 and 30), continues with an extraterminal region (ETR) of 45 amino acids. We suggest that this region is necessary for the correct cell wall targeting of XTH29, as the ETR-truncated protein never reaches its final destination and is not recruited by EXPOs. Furthermore, quantitative real-time polymerase chain reaction analyses performed on 4-week-old Arabidopsis seedlings exposed to drought and heat stress suggest a different involvement of the three XTHs in cell wall remodeling under abiotic stress, evidencing stress-, organ- and time-dependent variations in the expression levels. Significantly, XTH29, codifying the only XTH that follows a UPS pathway, is highly upregulated with respect to XTH11 and XTH33, which code for CPS-secreted proteins.     

Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: Benefits and drawbacks

The baculovirus expression vector system (BEVS) is a versatile and powerful platform for protein expression in insect cells. With the ability to approach similar post-translational modifications as in mammalian cells, the BEVS offers a number of advantages including high levels of expression as well as an inherent safety during manufacture and of the final product. Many BEVS products include proteins and protein complexes that require expression from more than one gene. This review examines the expression strategies that have been used to this end and focuses on the distinguishing features between those that make use of single polycistronic baculovirus (co-expression) and those that use multiple monocistronic baculoviruses (co-infection). Three major areas in which researchers have been able to take advantage of co-expression/co-infection are addressed, including compound structure-function studies, insect cell functionality augmentation, and VLP production. The core of the review discusses the parameters of interest for co-infection and co-expression with time of infection (TOI) and multiplicity of infection (MOI) highlighted for the former and the choice of promoter for the latter. In addition, an overview of modeling approaches is presented, with a suggested trajectory for future exploration. The review concludes with an examination of the gaps that still remain in co-expression/co-infection knowledge and practice.     

Differentiating responses of lung cancer cell lines to Doxorubicin exposure: in vitro Raman micro spectroscopy,oxidative stress and bcl‐2 protein expression

The potential of Raman micro spectroscopy as an in vitro, non‐invasive tool for clinical applications has been demonstrated in recent years, specifically for cancer research. To further illustrate its potential as a high content and label free technique, it is important to show its capability to elucidate drug mechanisms of action and cellular resistances. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 24 hr exposure of lung cancer cell lines, A549 and Calu‐1, to the commercially available drug, doxorubicin (DOX). Raman spectroscopy, coupled with Confocal Laser Scanning Microscopy and Flow Cytometry, was used to track the DOX mechanism of action, at a subcellular level, and to study the mechanisms of cellular resistance to DOX. Biomarkers related to the drug mechanism of action and cellular resistance to apoptosis, namely reactive oxygen species (ROS) and bcl‐2 protein expression, respectively, were also measured and correlated to Raman spectral profiles. Calu‐1 cells are shown to exhibit spectroscopic signatures of both direct DNA damage due to intercalation in the nucleus and indirect damage due to oxidative stress in the cytoplasm, whereas the A549 cell line only exhibits signatures of the former mechanism of action.

PCA of nucleolar, nuclear and cytoplasmic regions of A549 and Calu‐1 with corresponding loadings of PC1 and PC2     

Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites

Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[a]pyrene (B[a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor alpha-naphthoflavone (alpha-NF), the p53 inhibitor pifithrin-alpha (PFT-alpha), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[a]P as measured by the generation of tetrols and by covalent binding of B[a]P to macromolecules. In contrast, the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, had neither an effect on the cyp1a1 expression nor the B[a]P-metabolism. In order to avoid these unspecific effects, we characterized the mechanisms involved in the apoptotic effects of B[a]P-metabolites. B[a]P and the B[a]P-metabolites B[a]P-7,8-DHD and BPDE-I induced apoptosis, whereas B[a]P-4,5-DHD had no effect. B[a]P, B[a]P-7,8-DHD and BPDE-I induced an accumulation and phosphorylation of p53, while the Bcl-2 proteins Bcl-xl, Bad and Bid were down-regulated. Interestingly, the levels of anti-apoptotic phospho-Bad were up-regulated in response to B[a]P as well as to B[a]P-7,8-DHD and BPDE-I. Both p38 MAPK and JNK were activated, but the p38 MAPK inhibitors were not able to inhibit BPDE-I-induced apoptosis. PFT-alpha reduced the BPDE-I-induced apoptosis, while both the PI-3 kinase inhibitor and the ERK inhibitors increased the apoptosis in combination with BPDE-I. BPDE-I also triggered apoptosis in primary cultures of rat lung cells. In conclusion, often used cell signaling inhibitors both enhanced the expression and the level of cyp1a1 and more directly acted as inhibitors of cyp1a1 metabolism of B[a]P. However, studies with the B[a]P-metabolite BPDE-I supported the previous suggestion that p53 has a role in the pro-apoptotic signaling pathway induced by B[a]P. Furthermore, these studies also show that the reactive metabolites of B[a]P induce the anti-apoptotic signals, Akt and ERK. Neither the induction nor the activity of p38 MAPK and JNK seems to be of major importance for the B[a]P-induced apoptosis.