Ultrastructural changes induced by juvenile hormone analogue in oöcyte membranes of apterous4Drosophila melanogaster

Egg chambers from apterous⁴ (ap⁴), a female sterile mutant of Drosophila melanogaster, show none of the microvilli or pinocytotic vesicles which are a prominent feature of the membrane of the wild-type vitellogenic oöcyte. The studies reported here show that a juvenile hormone analogue (ZR515) stimulates formation of microvilli and pinocytotic vesicles in oöcytes of ap⁴ flies. Within 12 hr after topical application of ZR515 to homozygous ap⁴ females the oöcyte membranes exhibit extensive microvilli and pinocytotic activity. The follicle-cell surface adjoining the oöcyte also shows some changes. In vitro studies in which ap⁴ ovaries were incubated in Schneider's Drosophila tissue-culture medium in the presence of ZR515 with or without female haemolymph, or in the absence of ZR515, showed that the analogue acts alone directly on the ovary to cause formation of microvilli and pinocytotic vesicles on the oöcyte membrane.     

Effects of a synthetic juvenile hormone and some analogues on Ephestia spp. (Lepidoptera: Phycitidae)

Depending on their stage of development, treatment of mature larvae of Ephestia kühniella with a synthetic juvenile hormone resulted in the production of super larvae (which invariably prolonged larval life) and larval-pupal intermediates. When migrating last-instar larvae were treated with the juvenile hormone analogues (JHA) ethyl-3,7, n-trimethyldodeca-2,4-dienoate (ZR512) and isopropyl ii-methoxy-3,7, ii-trimethyldodeca-2,4-dienoate (ZR515), larval-pupal intermediates and pupal mortality were induced. However, when applied topically, ZR515 appeared more effective than ZR512. Both analogues prevented adult emergence when topically applied to the migrating larvae at doses between 28–52 ng. One-day-old pupae were most susceptible while older individuals became less sensitive with age. When larvae pupated in corrugated cardboard rolls were treated with ZR512 those of both E. cautella and E. kiihniella failed to emerge. At an estimated dose of 179 ng cm^-2, ZR515 prevented 77^-6%E. cautella and 100%E. kühniella larvae from emerging as adults. The control of Ephestia by JHA treatment of the pupation sites is discussed.     

In vivo stimulation of vitellogenesis in Aedes aegypti with juvenile hormone,juvenile hormone analogue (ZR 515) and 20-hydroxyecdysone

Decapitated blood-fed Aedes aegypti mosquitoes do not undergo normal oöcyte maturation. Topical application of 1.25 ng JH analogue (ZR 515) or 250 ng JH-I restored ovarian development in 70–80% of the treated females. The rate of vitellogenin synthesis in these animals was 80% of normal blood-fed controls.When ligated abdomens were treated, 125 pg ZR 515 or 12.5 ng JH-I were sufficient to restore ovarian development in 80% of the animals. The rate of vitellogenin synthesis in these animals was 70% of normal blood-fed controls. On the other hand, injection of 1.25 μg 20-hydroxyecdysone was needed to restore ovarian development and vitellogenin synthesis in decapitated and abdominally ligated females.These experiments indicate that JH concentrations closer to the physiological norm than 20-hydroxyecdysone, can restore ovarian development and vitellogenin synthesis in vivo.     

The role of juvenile hormone in the larval diapause of the European corn borer,Ostrinia nubilalis

The possible role of juvenile hormone (JH) in the induction and termination of larval diapause in the European corn borer, Ostrinia nubilalis, was investigated using topical applications of both JH I and a JH mimic as well as by monitoring JH titers with the Galleria bioassay. Neither JH nor the JH mimic ZR515 was capable of influencing diapause termination when administered topically. The Galleria bioassay revealed little or no JH in the hemolymph of mid diapause (>30 days) insects, indicating no demonstrable role for JH in diapause maintenance. When ZR515 was administered to nondiapause, newly ecdysed fifth instar larvae the pupal molting cycle was delayed. By use of photoperiodic regimes we were able to show that the molting delay was not equivalent to diapause induction. The Galleria bioassay showed differences in JH titer profiles between diapause and nondiapause animals during the final larval stadium. The nondiapause insects showed titers that decline rapidly to trace amounts following the molt to fifth instar then rose prior to pupation. The diapause insects had generally higher titers and exhibited a more gradual decline after the molt. No evidence was obtained to support the hypothesis that JH plays a key role in the induction, maintenance, or termination of larval diapause.     

青海云杉花芽分化期内源激素含量的变化特征

采用酶联免疫法测定并分析了中国青藏高原东北边缘特有树种青海云杉花芽分化过程中内源激素的变化,以期为调控青海云杉花期调控提供理论依据。结果表明:(1)内源激素吲哚乙酸(IAA)、赤霉素(GAs)、玉米素核苷(ZR)和脱落酸(ABA)含量的变化存在一定的相似性,它们分别在青海云杉花芽生理分化前期和形态分化前期出现高峰;青海云杉叶片ZR/GAs、ZR/IAA之值在花芽生理分化期达到峰值,而ABA/GAs值在花芽生理分化期总体呈递增趋势。(2)青海云杉花芽生理分化期,其顶芽、侧芽中可溶性糖及蛋白质皆出现高峰;形态分化前期,顶芽及侧芽中蛋白质含量下降,但可溶性糖含量持续上升;而花芽生理分化期间,叶片中核酸含量总体皆呈递增趋势。研究认为,较高的ZR/GAs、ZR/IAA有利于青海云杉花芽生理分化,但对维持花芽形态分化可能不是必须的,而高的ABA/GAs、ABA/IAA可能是花芽形态分化能够顺利完成所不可缺少的;可溶性糖、蛋白质、核酸等结构物质和能量物质的积累有利于青海云杉花芽生理分化的完成。     

Arabidopsis Zinc Ribbon 3 is the ortholog of yeast mitochondrial HSP70 escort protein HEP1 and belongs to an ancient protein family in mitochondria and plastids

ZR proteins belong to a phylogenetically conserved family of small zinc-ribbon proteins in plastids and mitochondria of higher plants. The function of these proteins is so far unclear. The mitochondrial proteins share sequence similarities with mitochondrial Hsp70 escort proteins (HEP) from Saccharomyces cerevisiae (HEP1) and human. Expression of the mitochondrial ZR protein from Arabidopsis, ZR3, rescued a hep1 knockout mutant from yeast. Accordingly, ZR3 was found to physically interact with mitochondrial Hsp70 from Arabidopsis. Our findings support the idea that mitochondrial and plastidic ZR proteins from higher plants are orthologs of HEP proteins.

Structured summary of protein interactions

ZR3physically interacts with mtHSC70-2 by pull down (View interaction)ZR3physically interacts with mtHSC70-1 by pull down (View interaction)     

BH3-only proteins BIM and PUMA in the regulation of survival and neuronal differentiation of newly generated cells in the adult mouse hippocampus

Neurogenesis persists in the adult hippocampus, where several thousand neurons are born every day. Most of the newly generated cells are eliminated by apoptosis, possibly because of their failure to integrate properly into neural networks. The BH3-only proteins Bim and Puma have been shown to mediate trophic factor withdrawal- and anoikis-induced apoptosis in various systems. We therefore determined their impact on proliferation, survival, and differentiation of adult-generated cells in the mouse hippocampus using gene-deficient mice. Wild-type, bim-, and puma-deficient mice showed similar rates of precursor cell proliferation, as evidenced by 5-bromo-2-deoxyuridine (BrdU)-incorporation. Deficiency in either bim or puma significantly increased the survival of adult-born cells in the dentate gyrus (DG) after 7 days. Consistently, we detected increased numbers of doublecortin (DCX)-positive and fewer terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelled-positive cells in the DG of bim- and puma-deficient mice. Bim and puma deficiency did not change early markers of neuronal differentiation, as evidenced by BrdU/DCX double-labelling. However, BrdU/NeuN double-labelling revealed that deficiency of bim, but not puma, accelerated the differentiation of newly generated cells into a neuronal phenotype. Our data show that Bim and Puma are prominently involved in the regulation of neuronal progenitor cell survival in the adult DG, but also suggest that Bim has an additional role in neuronal differentiation of adult-born neural precursor cells.     

Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

High cyclic AMP levels in young chick embryonic hearts.

Acid phosphatase (Acph) activities and protein content were measured in developing ovaries of adult flies. Acph and protein increased approximately logarithmically for the first 2 days of adult life and then plateaued at about 80 and 35 times, respectively, the levels present at eclosion. The specific activity of Acph was constant for the first 15 hr and then increased by a factor of three over the next 2 days. Analysis of staged follicles showed that the specific activity of Acph starts to increase at stage 10. Ovaries from homozygotes for Acph-1ⁿ⁴, a null activity mutant, showed constant low specific activity, indicating that this gene codes for the major ovarian Acph. Ovarian transplantations between Acph-1ⁿ⁴ and wild type showed that Acph is made by the ovary. Ovaries from isolated abdomens failed to increase in Acph activity or protein, but treating isolated abdomens with ZR-515, a juvenile hormone analog, caused nearly normal levels to be attained. Ovaries of the female sterile mutant ap⁴ failed to develop Acph activity unless they were implanted into a normal host or treated with ZR-515. Ovaries from the female sterile mutant fs(3)L3 developed no increase in Acph activity even when treated with ZR-515. The results demonstrate that the activity of a genetically localized enzyme is controlled by a chemically defined hormone in a genetically favorable higher eucaryote.     

A role for the myogenic determination gene Myf5 in adult regenerative myogenesis

The myogenic determination genes Myf5, Myod and Mrf4 direct skeletal muscle cell fate prenatally. In adult myogenesis, Myod has been shown to regulate myoblast differentiation, however, our understanding of satellite cell regulation is incomplete since the roles of Myf5 and Mrf4 had not been clearly defined. Here we examine the function of Myf5 and Mrf4 in the adult using recently generated alleles. Mrf4 is not expressed in normal or Myf5 null satellite cells and myoblasts, therefore excluding a role for this determination gene in adult muscle progenitors. Skeletal muscles of adult Myf5 null mice exhibit a subtle progressive myopathy. Crucially, adult Myf5 null mice exhibit perturbed muscle regeneration with a significant increase in muscle fibre hypertrophy, delayed differentiation, adipocyte accumulation, and fibrosis after freeze-injury. Satellite cell numbers are not significantly altered in Myf5 null animals and they show a modest impaired proliferation under some conditions in vitro. Mice double mutant for Myf5 and Dystrophin were more severely affected than single mutants, with enhanced necrosis and regeneration. Therefore, we show that Myf5 is a regulator of regenerative myogenesis and homeostasis, with functions distinct from those of Myod and Mrf4.     

Complementarity of ATP-induced and light-induced absorbance changes around 515 nm

A comparative study of the light-induced and the ATP-induced changes of P-515 absorbance gave the following results: (1) Following light activation of the latent ATP-hydrolase, ATP can induce a ΔA(515) of about the same size as that observed either in continuous light or by a saturating light flash. The ATP-induced ΔA(515) is stable in the dark as long as ATP is hydrolysed. (2) Any preceding ATP-induced ΔA(515) reduces the size of a consequent light-induced ΔA(515), and vice versa. The total P-515 absorbance change which can be induced by ATP and light is constant; there is strict complementarity of ATP- and light-induced ΔA(515). (3) The suppression of the flash-induced ΔA(515) by a preceding ATP-induced ΔA(515) is accompanied by an about 15-fold acceleration of the overall dark-decay rate, which is not further accelerated by addition of 0.2 μM valinomycin. (4) Adopting the kinetic model of Schapendonk (Doctoral Thesis, Wageningen, 1980) it is concluded that the apparent acceleration of the overall dark-decay rate results from a specific elimination of the slowly decaying ‘Reaction II’ component. ATP hydrolysis is suggested to produce and to maintain the Reaction II-type electrochromic pigment shift in the dark. (5) The data offer an alternative explanation to the prevailing notion that increased proton conductance via the activated ATPase is the main cause for the apparent acceleration of the overall decay rate of the flash-induced ΔA(515) following preillumination or under ‘phosphorylating conditions’. (6) On the basis of the presented data it is argued that the total number of available sites which can produce a Reaction II-type electrochromic pigment shift is strictly limited. Consequently, the notion of a localized ATP- or light-induced field is favored. The properties of this localized field would suggest a close link to energy-dependent changes at the coupling factor complex and to the electrogenic reactions coupled with cyclic photophosphorylation.     

Analysis and characterization of DCMU-resistant Euglena gracilis

Dark-grown, DCMU-adapted Euglena gracilis Z (ZR) are able to undergo light-induced chloroplast development in the presence or absence of DCMU. The differentiated chloroplasts are photosynthetically active and are resistant not only to DCMU, but also to an analog, o-phenanthrolene. When DCMU overdoses are added to ZR cells or to chloroplasts isolated from these cells, photosynthesis is partially inhibited. A brief period of darkness removes this inhibition. This recovery phenomenon is related to DCMU resistance, since it is not exhibited by non-resistant control cells. The chloroplast protein synthesis apparatus is not involved in DCMU resistance. Rather, this phenomenon is apparently related to new characteristics of thylakoids. It is shown that photosynthetic recovery by ZR cells depends on the accessibility and fluid properties of membranes. The analysis of fluorescence induction kinetics shows that changes in the environmental conformation of photosystem II units occur during recovery.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ZR DCMU-adapted Euglena gracilis Z I and II=Calvayrac et al., in press (a, b)     

Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation

Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5^mC) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.     

Urothelial transdifferentiation to prostate epithelia is mediated by paracrine TGF-β signaling

The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF-β signaling directed the prostatic induction. UGM from TGF-β type II receptor stromal conditional knockout mice (Tgfbr2^fspKO) or control mice (Tgfbr2^{floxE2/floxE2}) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2^{floxE2/floxE2} UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2^fspKO UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2^fspKO UGM compared with Tgfbr2^{floxE2/floxE2} UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF-β activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF-β signaling to mediate epithelial Wnt activity.     

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells^1-3.Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines⁴. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors^5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies⁷. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types⁸. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC^2,3.The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)³. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.