机械分离的果蝇幼虫中枢神经元全细胞钾电流的特性

培养的果蝇胚胎及幼虫中枢神经元已被广泛用于细胞膜离子通道,突触传递和胞内信使调节等电生理学研究,在本实验中,利用机械震荡分离方法获得了大量的果蝇幼虫中枢神经元,其中大部分为Ⅱ型神经元,运用膜片钳技术,鉴定了Ⅱ型神经元上五种具有不同动力学特性的全细胞钾电流,其中E型电流表型表现出与其它四种电流完全不同的“钟形”激活特性,进一步的研究还表明该类型电流具有明显的钙依赖性,而且它具有与其它四种电流不同的衰减特性。     

Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells

The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.     

Derepression in Saccharomyces cerevisiae can be dissociated from cellular proliferation and deoxyribonucleic acid synthesis.

A method has been developed that permits precise control of release from catabolite repression in Saccharomyces cerevisiae. It consists of transferring cells growing exponentially on 5% glucose to derepression medium at high cell density. Derepression then proceeds with reproducible kinetics and is complete within 6 to 7.5 h for various intra- and extramitochondrial markers, in the absence of any substantial increase in cellular dry weight or protein. Nuclear (and mitochondrial) deoxyribonucleic acid synthesis can be interrupted in certain thermosensitive (cdc) mutants at the nonpermissive temperature; a shift to this temperature before the onset of derepression has no effect on its outcome.     

Receptors and neurons for fly odors in Drosophila

Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila[1]. We systematically examined the trichoid sensilla, one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, by electrophysiological analysis. We find that none respond strongly to food odors but that all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone transferred from males to females during mating [2-4]. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin-female extract as well. Thus, males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. We then systematically tested all members of the Odor receptor (Or) gene family [5-7] that are expressed in trichoid sensilla [8] by using an in vivo expression system [9]. Four receptors respond to fly odors in this system: Two respond to extracts of both males and virgin females, and two respond to cVA. We propose a model describing how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic.     

Mammalian cells contain a DNA-recombining activity that can be dissociated from topoisomerase activity

The role of the topoisomerase enzyme in DNA recombination was investigated by extracting chromosomal deoxyribonucleoproteins from a variety of cultured mammalian ceils and assaying for the formation of recombinant DNA structures. Although each of the crude deoxyribonucleoprotein preparations contained topoisomerase activity, they did not all contain DNA-recombining activity. A distinct, perhaps novel, enzyme may therefore promote DNA recombination in these cell-free systems.     

Innate defence functions of macrophages can be biased by nano-sized ceramic and metallic particles

Nano-sized particles of ceramic and metallic materials are generated by high-tech industrial activities, and can be generated from worn-out replacement and prosthetic implants. The interaction with the human body of such nanoparticles has been investigated, with a particular emphasis on innate defence mechanisms. Human macrophages (PMA-differentiated myelomonocytic U-937 cells) were exposed in vitro to non-toxic concentrations of TiO(2), SiO(2), ZrO(2), or Co nanoparticles, and their inflammatory response (expression of TLR receptors and co-receptors, and cytokine production) was examined. Expression of TLR receptors was generally unaffected by exposure to the different nanoparticles, except for some notable cases. Exposure to nanoparticles of ZrO(2) (and to a lesser extent TiO(2)), upregulated expression of viral TLR receptors TLR3 and TLR7. Expression of TLR10 was also increased by TiO(2) and ZrO(2) nanoparticles. On the other hand, TLR9 expression was decreased by SiO(2) nano-particles, and expression of the co-receptor CD14 was inhibited by Co nanoparticles. Basal and LPS-induced production of cytokines IL-1beta, TNF-alpha, and IL-1Ra was examined in macrophages exposed to nanoparticles. SiO(2) nanoparticles strongly biased naive macrophages towards inflammation (M1 polarisation), by selectively inducing production of inflammatory cytokines IL-1beta and TNF-alpha. SiO(2) nanoparticles also significantly amplified the inflammatory phenotype of LPS-polarised M1 macrophages. Other ceramic nanoparticles had little influence on cytokine production, either in resting macrophages, or in LPS-activated cells. Generally, Co nanoparticles had an overall pro-inflammatory effect on naive macrophages, by reducing anti-inflammatory IL-1Ra and inducing inflammatory TNF-alpha. However, Co nanoparticles reduced production of IL-1beta and IL-1Ra, but not TNF-alpha, in LPS-polarised M1 macrophages. Thus, exposure to different nanoparticles can modulate, in different ways, the defence/inflammatory capacities of macrophages. A thorough analysis of these biasing effects may shed light on the mechanisms of pathogenesis of several diseases based on dysregulation of the immune response (allergies, autoimmunity, tumours).     

Innate preference in Drosophila melanogaster

Innate preference behaviors are fundamental for animal survival. They actually form the basis for many animal complex behaviors. Recent years have seen significant progresses in disclosing the molecular and neural mechanism underlying animal innate preferences, especially in Drosophila. In this review, I will review these studies according to the sensory modalities adopted for preference assaying, such as vision, olfaction, thermal sensation. The behavioral strategies and the theoretic models for the formation of innate preferences are also reviewed and discussed.     

Disease association mapping in Drosophila can be replicated in the wild

Association and linkage mapping have become important tools in understanding the genetics of complex traits, including diseases in humans. As the success of association mapping is reduced by small effect sizes and limited power, linkage studies in laboratory-based model systems are still heavily used. But whether the results of these studies can be replicated in natural populations has been questioned. Here, we show that a polymorphism in the gene ref(2)P, which had previously been linked to sigma virus resistance in Drosophila melanogaster under laboratory conditions, also provides resistance against the virus in female flies in a wild population in the field. This genetic association is thus upheld in spite of a known genotype-by-genotype interaction and environmental variation.     

Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila

Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/beta-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Our mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. We examined the role of a Dsh-binding PCP molecule, Diego (Dgo), in both Wnt-Fz/beta-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/beta-catenin signaling. Our data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell.     

Broken chromosomal ends can be elongated by conversion in Drosophila melanogaster

The fate of the termini of X chromosomes broken in the regulatory region of the yellow gene was followed in heterozygotes with X chromosomes carrying a point mutation inactivating the yellow gene. Each generation had a loss of about 70 terminal base pairs from the broken chromosome. However, gene conversion restoring the correct sequence at the chromosomal terminus took place with a frequency of about 1×10^–2 per generation. The average length of the conversion track was 2.7 kb. No recombination events occurred. In addition, we found that the normal functioning of the yellow body and wing enhancers located at the tip of the chromosome required about 4 kb of additional upstream sequence. Received: 8 October 1998; in revised form: 18 January 1999 / Accepted: 18 January 1999     

Drosophila cells can be grown to high cell densities in a bioreactor

Summary Cell cultures of the fruitfly, Drosophila melanogaster, were grown in a bioreactor to high cell densities: 5.7×10⁷ cells/ml, in batch, semi-batch and continuous flow modes. Using a semi-batch culture mode 500g wet weight of cell mass was produced in 16 days, using 5 l of cheap, commercially available, serum-free medium.     

The similarity between odors and their binary mixtures in Drosophila

How are odor mixtures perceived? We take a behavioral approach toward this question, using associative odor-recognition experiments in Drosophila. We test how strongly flies avoid a binary mixture after punishment training with one of its constituent elements and how much, in turn, flies avoid an odor element if it had been a component of a previously punished binary mixture. A distinguishing feature of our approach is that we first adjust odors for task-relevant behavioral potency, that is, for equal learnability. Doing so, we find that 1) generalization between mixture and elements is symmetrical and partial, 2) elements are equally similar to all mixtures containing it and that 3) mixtures are equally similar to both their constituent elements. As boundary conditions for the applicability of these rules, we note that first, although variations in learnability are small and remain below statistical cut-off, these variations nevertheless correlate with the elements' perceptual "weight" in the mixture; thus, even small differences in learnability between the elements have the potential to feign mixture asymmetries. Second, the more distant the elements of a mixture are to each other in terms of their physicochemical properties, the more distant the flies regard the elements from the mixture. Thus, titrating for task-relevant behavioral potency and taking into account physicochemical relatedness of odors reveals rules of mixture perception that, maybe surprisingly, appear to be fairly simple.     

Drosophila adult and larval pheromones modulate larval food choice

Insects use chemosensory cues to feed and mate. In Drosophila, the effect of pheromones has been extensively investigated in adults, but rarely in larvae. The colonization of natural food sources by Drosophila buzzatii and Drosophila simulans species may depend on species-specific chemical cues left in the food by larvae and adults. We identified such chemicals in both species and measured their influence on larval food preference and puparation behaviour. We also tested compounds that varied between these species: (i) two larval volatile compounds: hydroxy-3-butanone-2 and phenol (predominant in D. simulans and D. buzzatii, respectively), and (ii) adult cuticular hydrocarbons (CHs). Drosophila buzzatii larvae were rapidly attracted to non-CH adult conspecific cues, whereas D. simulans larvae were strongly repulsed by CHs of the two species and also by phenol. Larval cues from both species generally reduced larval attraction and pupariation on food, which was generally—but not always—low, and rarely reflected larval response. As these larval and adult pheromones specifically influence larval food search and the choice of a pupariation site, they may greatly affect the dispersion and survival of Drosophila species in nature.     

Hid can induce, but is not required for autophagy in polyploid larval Drosophila tissues

The major cell death pathways are apoptosis and autophagy-type cell death in Drosophila. Overexpression of proapoptotic genes in developing imaginal tissues leads to the activation of caspases and apoptosis, but most of them show no effect on the polytenic cells of the fat body during the last larval stage. Surprisingly, overexpression of Hid induces caspase-independent autophagy in the fat body, as well as in most other larval tissues tested. Hid mutation results in inhibition of salivary gland cell death, but the disintegration of the larval midgut is not affected. Electron microscopy shows that autophagy is normally induced in fat body, midgut and salivary gland cells of homozygous mutant larvae, suggesting that Hid is not required for autophagy itself. Constitutive expression of the caspase inhibitor p35 produces identical phenotypes. Our results show that the large, post-mitotic larval cells do not react or activate autophagy in response to the same strong apoptotic stimuli that trigger apoptosis in small, mitotically active imaginal disc cells.     

The recessive phenotype of forked can be uncovered by heat shock in Drosophila

Heat shock uncovers the recessive forked phenotype when heterozygotes between f^36a and wild-type are heated during sensitive periods in pupal development. We call the phenocopy of a mutant in such a heterozygote a heterocopy. The heterocopy in f^36a/+ is virtually identical to the mutant phenotype; however, bristles on different parts of the body are affected during different sensitive periods. We discuss the hypothesis that the heat shock acts by affecting expression of the wild-type gene product corresponding to the mutant gene. The sensitive period for heterocopy induction in a specific tissue is proposed to correspond to the normal time of gene expression for the forked gene product in a particular tissue.     

Neural crest induction by the canonical Wnt pathway can be dissociated from anterior-posterior neural patterning in Xenopus

While Wnt signaling is known to be involved in early steps of neural crest development, the mechanism remains unclear. Because Wnt signaling is able to posteriorize anterior neural tissues, neural crest induction by Wnts has been proposed to be an indirect consequence of posteriorization of neural tissues rather than a direct effect of Wnt signaling. To address the relationship between posteriorization and neural crest induction by Wnt signaling, we have used gain of function and loss of function approaches in Xenopus to modulate the level of Wnt signaling at multiple points in the pathway. We find that modulating the level of Wnt signaling allows separation of neural crest induction from the effects of Wnts on anterior-posterior neural patterning. We also find that activation of Wnt signaling induces ectopic neural crest in the anterior region without posteriorizing anterior neural tissues. In addition, Wnt signaling induces neural crest when its posteriorizing activity is blocked by inhibition of FGF signaling in neuralized explants. Finally, depletion of beta-catenin confirms that the canonical Wnt pathway is required for initial neural crest induction. While these observations do not exclude a role for posteriorizing signals in neural crest induction, our data, together with previous observations, strongly suggest that canonical Wnt signaling plays an essential and direct role in neural crest induction.