白血病抑制因子mRNA的表达受雌激素的调控

运用RT—PCR方法检测了体外培养的大鼠成骨样细胞Ros17／2．8在17β雌二醇(E2)刺激前后细胞中一些细胞因子的mRNA水平．发现在E2刺激后,细胞中白血病抑制因子（LIF）的MRNA水平明显上升,且呈现E2浓度依赖的特点．该结果提示,LIF可能参与替代性治疗过程中雌二醇对于骨质疏松症的缓解作用．这为进一步研究细胞因子与骨质疏松症的相关性,阐明细胞因子在骨代谢中的作用打下了基础,并将有利于发展治疗骨代谢疾病的药物．     

Novel benzenediamine derivative FC99 ameliorates zymosan-induced arthritis by inhibiting RORγt expression and Thl7 cell differentiation

Increased IL-17-producing helper T （Thl7） cells have been observed in patients with rheumatoid arthritis （RA）. The retinoic-acid-related orphan nuclear receptor （RORγt） is the master regulator of Thl7 cells. Our previous research showed that FC99 possesses anti-inflammation activity. However, to date the effects of FC99 on RORγt expression in Thl7 cell differentiation have not been investigated yet. In the present study, we found that FC99 significantly attenu- ated arthritis-like symptoms, i.e., suppressing the develop- ment of paw edema in zymosan-induced arthritis （ZIA） mice. H＆E staining showed that the infdtration of inflamma- tory cells in ankle synovial tissues was significantly suppressed. FC99 also reduced the mRNA levels of pro-in- flammatory cytoklnes in ankle synovial tissues as shown by Q-PCR analysis. The protein levels of the pro-inflammatory cytoklnes in sera were also suppressed after FC99 treatment. Moreover, FC99 decreased the RORγt mRNA level in spleen tissues. Thl7 cell percentage was significantly decreased in spleens and draining lymph nodes （dLNs）. The mRNA and protein levels of IL-17A and IL-23 were reduced after FC99 treatment in ZIA mice. Furthermore, in vitro experiments showed that FC99 inhibited the expression of IL-6 in LPS- induced RAW264.7 cells and BMDCs. Moreover, FC99 sig- nificantly inhibited the RORγt expression in PMA-induced CD4＋ T cells and LPS-induced RAW264.7 cells. These data indicate that FC99 improves arthritis-like pathological symp- toms in vivo and in vitro, which might be related to the inhib- ition of RORγt expression in Thl7 cells. Our findings suggest that FC99 may be a potential therapeutic candidate for the treatment of RA and other inflammatory disorders.     

Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences

To investigate whether a 12 nucleotide DNA-based miniEGSs can silence the expression of human cytomegalovirus （HCMV） UL49 gene efficiently, A HeLa cell line stably expressing UL49 gene was constructed and the putative miniEGSs （UL49-miniEGSs） were assayed in the stable cell line. Quantitative RT-PCR and western blot results showed a reduction of 67% in UL49 expression level in HeLa cells that were transfected with UL49-miniEGSs. It was significantly different from that of mock and control miniEGSs （TK-miniEGSs） which were 1 and 7%, respectively. To further confirm the gene silence directed by UL49-miniEGSs with human RNase P, a mutant of UL49-miniEGSs was constructed and a modified 51 RACE was carried out. Data showed that the inhibition of UL49 gene expression directed by UL49-miniEGSs was RNase P-dependent and the cleavage of UL49 mRNA by RNase P was site specific. As a result, the length of DNA-based miniEGSs that could silence gene expression efficiently was only 12 nt. That is significantly less than any other oligonucleotide-based method of gene inactivation known so far. MiniEGSs may represent novel gene-targeting agents for the inhibition of viral genes and other human disease related gene expression.     

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence, dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3‘ overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNAduplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0-450 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5‘ end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.     

Molecular characterization and functional analyses of a diapause hormone receptor-like gene in parthenogenetic Artemia

In arthropods, mature females under certain conditions produce and release encysted gastrula embryos that enter diapause, a state of obligate dormancy. The process is presumably regulated by diapause hormone (DH) and diapause hormone receptor (DHR) that were identified in the silkworm, Bombyx mori and other insects. However, the molecular structure and function of DHR in crustaceans remains unknown. Here, a DHR-like gene from parthenogenetic Artemia (Ar-DHR) was isolated and sequenced. The cDNA sequence consists of 1410 bp with a 1260-bp open reading frame encoding a protein consisting of 420 amino acid residues. The results of real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and protein of Ar-DHR were mainly expressed at the diapause stage. Furthermore, we found that Ar-DHR was located on the cell membrane of the pre-diapause cyst but in the cytoplasm of the diapause cyst by analysis of immunofluorescence. In vivo knockdown of Ar-DHR by RNA interference (RNAi) and antiserum neutralization consistently inhibited diapause cysts formation. The results indicated that Ar-DHR plays an important role in the induction and maintenance of embryonic diapause in Artemia. Thus, our findings provide an insight into the regulation of diapause formation in Artemia and the function of Ar-DHR.     

Development of neuropeptide analogs capable of traversing the integument: A case study using diapause hormone analogs in Helicoverpa zea

Diapause hormone and its analogs terminate pupal diapause in Helicoverpa zea when injected, but if such agents are to be used as effective diapause disruptors it will be essential to develop simple techniques for administering active compounds that can exert their effect by penetrating the insect epidermis. In the current study, we used two molecules previously shown to have high diapause-terminating activity as lead molecules to rationally design and synthesize new amphiphilic compounds with modified hydrophobic components. An assay for diapause termination identified 13 active compounds with EC₅₀'s ranging from 0.9 to 46.0 pmol per pupa. Three compounds, Decyl-1963, Dodecyl-1967, and Heptyl-1965, selected from the 13 compounds most active in breaking diapause following injection, also successfully prevented newly-formed pupae from entering diapause when applied topically. These compounds feature straight-chain, aliphatic hydrocarbons from 7 to 12 carbons in length; DH analogs with either a short-chain length of 4 or an aromatic phenethyl group failed to act topically. Compared to a high diapause incidence of 80–90% in controls, diapause incidence in pupae receiving a 10 nmole topical application of Decyl-1963, Dodecyl-1967, or Heptyl-1965 dropped to 30–45%. Decyl-1963 and Dodecyl-1967 also remained effective when topically applied at the 1 nmole level. These results suggest the feasibility of developing DH agonists that can be applied topically and suggest the identity of new lead molecules for development of additional topically-active DH analogs. The ability to penetrate the insect epidermis and/or midgut lining is critical if such agents are to be considered for future use as pest management tools.     

家蚕滞育的分子机理2.蛹期滞育激素基因的表达与滞育决定

滞育激素是由食道下神经节分泌的重要昆虫神经肽,诱导昆虫的滞育。选择具有ＲＮＡ聚合酶能够识别的启动子的质粒载体,将滞育激素ｃＤＮＡ克隆进去,在体外大量合成单一的滞育激素ｃＲＮＡ为参照,测定食道下神经节分泌滞育激素ｍＲＮＡ量来确定滞育激素的分泌量。结果证明食道下神经节分泌的滞育激素的数量决定昆虫的滞育。     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

The plurennial life cycles of the European Tettigoniidae (Insecta: Orthoptera)

Summary The effect of drought on embryonic development and on hatching was studied in 13 European Tettigoniidae species. Drought can affect development in three different ways: (1) Embryonic development proceeds slower than if the eggs are in contact with water; (2) it stops (for final diapause) in an earlier embryonic stage; (3) it affects maintenance and termination of the initial embryonic diapause.In many Tettigoniidae species, the initial diapause is prolonged, and may last several years. Without draught stress, between 1 and 7 cold treatments in the laboratory, and with eggs of the Tettigonia-species between 1 and 6 winters in the field were necessary to enable all eggs to complete initial diapause. In Central European species, the number of eggs maintaining initial diapause significantly increased when the eggs had no contact with water at the time when they should recover from diapause. In contrast, termination of initial diapause in Tettigonia caudata from Greece, when the environment became favorable for growth again, was highest in that group of eggs that had lost most water in a preceding period of drought. The importance of the prolonged initial diapause for the survival of unpredictable adverse conditions is discussed.     

Space Travel Shortens Diapause in Gypsy Moth Eggs

Field-collected and laboratory-reared gypsy moth eggs were exposed to microgravity, cosmic radiation, sub-freezing temperatures, unusual vibrations, and other extraterrestrial phenomena while they were sealed for 6 days, in January, in a Get-Away-Special (GAS) canister in the open bay of a NASA earth-orbiting spacecraft, the Columbia. Insects were not exposed to light after preparation for and during space flight. Under field conditions, out-of-doors, the eggs should have hatched in April, after 3-4 months of chilling temperatures and should not have hatched after the 6 days of chilling to -11 ^oC during flight in the Columbia spacecraft. However by April 1, more than 4000 larvae had hatched from eggs that had travelled in space, as opposed to β350 from a similar number of control, earthbound eggs. These results indicate that the period of a circannual rhythm in field- and lab-reared insects had been shortened, presumably as result of exposure to microgravity, other factors associated with space flight, and/or conditions of outer space. These results suggest that it may be possible to develop methods for rearing the gypsy moth year round, without the necessity of three months chilling interspersed in the development process. This, in turn, would facilitate production of large numbers of insects for sterile male release or for use as a rearing medium for parasites, predators and pathogens of the gypsy moth.     

Effect of photoperiod on winter and summer diapause of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae)

To understand the geographical differences between diapause systems and synchronization of adult occurrence in the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae), we examined the timing of winter diapause termination and intensity of summer diapause using univoltine and potentially bivoltine individuals in Iwate, Japan. In laboratory rearing experiments of mature larvae maintained at constant temperature (20 °C), winter diapause intensity weakened by January without photoperiodic responses. Meanwhile, summer diapause was maintained by the long day length and presumably terminated with the photoperiodic transition from long to short day length. The intensity of summer diapause was stronger for cocoons that transitioned from a 16 h light to 8 h dark (LD 16:8) to a LD 15:9 photoperiod than for those that transitioned from LD 15:9 to LD 14:10. These results suggest that populations distributed in relatively low-latitude areas, with partly or potentially bivoltine individuals, would have a weaker summer diapause or none at all. Moreover, sexual differences in the number of days to emergence were not detected when individuals experienced a photoperiodic transition from long to short day length, suggesting that the summer diapause system may function to synchronize the emergence of males and females in the population examined.     

Why is the number of days required for induction of adult diapause in the linden bug Pyrrhocoris apterus fewer in the larval than in the adult stage?

Adult females of Pyrrhocoris apterus, programmed for diapause by short-day (SD) photoperiod and those programmed for reproduction by long-day (LD) retain photoperiodic information in continuous darkness (DD) until death. However, if the interruption of SD by DD is made in the course of diapause programming in adults, then the incidence of diapause depends on the number of SD cycles received before DD, with no evidence that the photoperiodic clock is free-running at DD to complete diapause induction. These results indicate that the photoperiodic clock is stopped after transfer to DD and the information accumulated before transfer to DD is maintained. Diapause programming in the adult stage requires 9–10 SD cycles to induce diapause in 80% of individuals. However, if the diapause programming starts after ecdysis of LD-larvae to the last instar, only 3 SD cycles before transfer to DD are required for diapause in 80% of individuals. Surprisingly, if the newly ecdysed last instar LD-larvae, sensitive to photoperiod, are transferred to DD (thus they did not experience any SD), diapause occurs in 40% of the individuals. Thus, diapause ‘information’ is present in LD-larvae and is responsible for a lower number of SD required for diapause induction in the larval than in the adult stage.