Arrest of adult development in debrained pupae of the silkworm, Bombyx mori

The occurrence of developmental arrest after brain removal in pupae of Bombyx mori was examined using a racial hybrid Chinese No. 115 × Japanese No. 122. The results are as follows: (1) Adult development was blocked for a long period in most insects debrained shortly after larval-pupal ecdysis; (2) the earlier the brain removal, the more arrested pupae were obtained; (3) the critical period of brain hormone secretion for adult development was earlier in the female than in the male; (4) the developmental arrest which had been induced in female pupae tended to terminate earlier than that in males; (5) the developmental arrest which was induced by extremely early brain removal terminated earlier than that obtained by later brain removal.     

Differential effects of anoxia on heart rate in developmental stages of the annual killifish Austrofundulus limnaeus that differ in their tolerance of anoxia

Embryos of the annual killifish Austrofundulus limnaeus can experience oxygen deprivation as part of their normal developmental environment. We exposed embryos to anoxia and monitored heart activity for 48 hr, and subsequent aerobic recovery from anoxia for 40 hr. Embryos were tested at four different developmental stages that differ in their tolerance of anoxia. Our results indicate that high tolerance of anoxia is associated with an arrest of heart contractility during the first 24 hr of anoxia. These embryos recover to normoxic levels of heart rate within 16 hr of aerobic recovery. In contrast, embryos from later developmental stages that have a highly reduced ability to survive long-term anoxia experience a severe bradycardia but not an arrest of heart rate. These data illustrate a new and potentially powerful model for investigating the effects of anoxia on the developing cardiovascular system in vertebrates.     

Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone.

Tartrate-resistant acid phosphatase (TRAP) is a characteristic constituent of osteoclasts and some mononuclear preosteoclasts and, therefore, used as a histochemical and biochemical marker for osteoclasts and bone resorption. We now report the isolation of a 1397-base pair (bp) full-length TRAP/tartrate-resistant acid ATPase (TrATPase) cDNA clone from a neonatal rat calvaria lambda gt11 cDNA library. The cDNA clone consists of a 92-bp untranslated 5'-flank, an open reading frame of 981 bp and a 324-bp untranslated 3'-poly(A)-containing region. The deduced protein sequence of 327 amino acids contains a putative cleavable signal sequence of 21 amino acids. The mature polypeptide of 306 amino acids has a calculated Mr of 34,350 Da and a pI of 9.18, and it contains two potential N-glycosylation sites and the lysosomal targeting sequence DKRFQ. At the protein level, the sequence displays 89-94% homology to TRAP enzymes from human placenta, beef spleen, and uteroferrin and identity to the N terminus of purified rat bone TRAP/TrATPase. An N-terminal amino acid segment is strikingly homologous to the corresponding region in lysosomal and prostatic acid phosphatases. The cDNA recognized a 1.5-kilobase mRNA in long bones and calvaria, and in vitro translation using, as template, mRNA transcribed from the full-length insert yielded an immunoprecipitated product of 34 kDa. In neonatal rats, TRAP/TrATPase mRNA was highly expressed in skeletal tissues, with much lower (less than 10%) levels detected in spleen, thymus, liver, skin, brain, kidney, brain, lung, and heart. In situ hybridization demonstrated specific labeling of osteoclasts at endostal surfaces and bone trabeculae of long bones. Thus, despite the apparent similarity of this osteoclastic TRAP/TrATPase with type 5, tartrate-resistant and purple, acid phosphatases expressed in other mammalian tissues, this gene appears to be preferentially expressed at skeletal sites.     

Hochachka's "Hypoxia Defense Strategies" and the development of the pathway for oxygen

Hochachka's "Hypoxia Defense Strategies" identify oxygen signalling, metabolic arrest, channel arrest and coordinated suppression of ATP turnover rates as key factors that determine the ability of organisms to survive exposure to chronic hypoxia. In this review, I assess the developmental role played by these phenomena in the morphogenesis of the gas exchange tissues that define the pathway for oxygen transport to cytochrome c oxidase. Key areas of regulation lie in: (I) the suppression of fetal mitochondrial oxidative function in hand with mitochondrial biogenesis (metabolic arrest), (II) the role of hypoxia-driven oxygen signalling pathways in directing the scope of non-differentiated stem cell proliferation in placenta and lung development and (III) the regulation of epithelial fluid secretion/absorption in the lung through the oxygen-dependent modulation of Na+ conductance pathways. The identification of developmental roles for Hochachka's "Hypoxia Defense Strategies" in directing the morphogenesis of gas exchange structures bears with it the implication that these strategies are fundamental to establishing the scope for aerobic metabolic performance throughout life.     

A Tie2‐driven BAC‐TRAP transgenic line for in vivo endothelial gene profiling

Recent technological innovations including bacterial artificial chromosome‐based translating ribosome affinity purification (BAC‐TRAP) have greatly facilitated analysis of cell type‐specific gene expression in vivo, especially in the nervous system. To better study endothelial gene expression in vivo, we have generated a BAC‐TRAP transgenic mouse line where the L10a ribosomal subunit is tagged with EGFP and placed under the control of the endothelium‐specific Tie2 (Tek) promoter. We show that transgene expression in this line is widely, but specifically, detected in endothelial cells in several brain regions throughout pre‐ and postnatal development, as well as in other organs. We also show that this line results in highly significant enrichment of endothelium‐specific mRNAs from brain tissues at different stages. This BAC‐TRAP line therefore provides a useful genetic tool for in vivo endothelial gene profiling under various developmental, physiological, and pathological conditions. genesis 54:136–145, 2016. © 2016 Wiley Periodicals, Inc.     

Bovine immature oocytes acquire developmental competence during meiotic arrest in vitro

To test the hypothesis that oocytes require time to acquire developmental competence during meiotic arrest, we investigated the effects of butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinase, on the developmental competence of bovine oocytes after in vitro fertilization (IVF) following release from meiotic arrest. In the present study, 4 culture conditions were used: addition of BSA or fetal bovine serum (FBS) under 2 oxygen tensions (5% vs. 20%) during meiotic arrest with 100-microM BL I. The developmental competence to the blastocyst stage was higher (P < 0.01) in oocytes that were arrested in FBS-supplemented medium under 5% O2 (37%) than in oocytes that were arrested under other conditions (5%-24%) or that matured directly following follicle aspiration (23%). The time course of nuclear maturation of BL I-treated oocytes was also examined. The results demonstrated that oocytes treated with BL I start germinal vesicle (GV) breakdown and reach the metaphase II stage 5.5-6.0 h earlier than nonarrested oocytes. The developmental rates to the blastocyst stage of BL I-treated oocytes matured for 15.5 and 21 h were higher (P < 0.05) than those of nontreated oocytes matured for 21 and 26.5 h, respectively. These results demonstrate that bovine immature oocytes, which were arrested at the GV stage with BL I in FBS-supplemented medium under low oxygen tension, acquire higher developmental competence during meiotic arrest.     

Allosteric aptamers: targeted reversibly attenuated probes

Aptamers are unique nucleic acids with regulatory potentials that differ markedly from those of proteins. A significant feature of aptamers not possessed by proteins is their ability to participate in at least two different types of three-dimensional structure: a single-stranded folded structure that makes multiple contacts with the aptamer target and a double-helical structure with a complementary nucleic acid sequence. We have made use of this structural flexibility to develop an aptamer-based biosensor (a targeted reversibly attenuated probe, TRAP) in which hybridization of a cis-complementary regulatory nucleic acid (attenuator) controls the ability of the aptamer to bind to its target molecule. The central portion of the TRAP, between the aptamer and the attenuator, is complementary to a target nucleic acid, such as an mRNA, which is referred to as a regulatory nucleic acid (regNA) because it regulates the activity of the aptamer in the TRAP by hybridization with the central (intervening) sequence. The studies reported here of the ATP-DNA TRAP suggest that, as well as inhibiting the aptamer, the attenuator also acts as a structural guide, much like a chaperone, to promote proper folding of the TRAP such that it can be fully activated by the regDNA. We also show that activation of the aptamer in the TRAP by the complementary nucleic acid at physiological temperatures is sensitive to single-base mismatches. Aptamers that can be regulated by a specific nucleic sequence such as in an mRNA have potential for many in vivo applications including regulating a particular enzyme or signal transduction pathway or imaging gene expression in vivo.     

Hypoxia and nitric oxide induce a rapid, reversible cell cycle arrest of the Drosophila syncytial divisions

Cells can respond to reductions in oxygen (hypoxia) by metabolic adaptations, quiescence or cell death. The nuclear division cycles of syncytial stage Drosophila melanogaster embryos reversibly arrest upon hypoxia. We examined this rapid arrest in real time using a fusion of green fluorescent protein and histone 2A. In addition to an interphase arrest, mitosis was specifically blocked in metaphase, much like a checkpoint arrest. Nitric oxide, recently proposed as a hypoxia signal in Drosophila, induced a reversible arrest of the nuclear divisions comparable with that induced by hypoxia. Syncytial stage embryos die during prolonged hypoxia, whereas post-gastrulation embryos (cellularized) survive. We examined ATP levels and morphology of syncytial and cellularized embryos arrested by hypoxia, nitric oxide, or cyanide. Upon oxygen deprivation, the ATP levels declined only slightly in cellularized embryos and more substantially in syncytial embryos. Reversal of hypoxia restored ATP levels and relieved the cell cycle and developmental arrests. However, morphological abnormalities suggested that syncytial embryos suffered irreversible disruption of developmental programs. Our results suggest that nitric oxide plays a role in the response of the syncytial embryo to hypoxia but that it is not the sole mediator of these responses.     

Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane

Although the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the translocon-associated protein (TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property of the signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as a functional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.     

心肺脑复苏后如何进行早期氧疗

心肺脑复苏（Cardiopulmonary—cerebralResuscitation,CPCR）是抢救心跳骤停（CardiacArrest）患者的重要手段,而早期氧疗（AcuteOxygenTherapy）是提高心肺脑复苏成功率的重要辅助措施。以往人们一直认为CPCR后应尽早给予患者高浓度氧疗;然而,近年认为早期氧疗不当非但达不到挽救心跳骤停患者生命、降低致残率之目的,反而会降低复苏成功率。基础研究和临床研究提示,与暴露于正常空气或低浓度氧气组相比,大脑缺氧后早期暴露于高浓度氧气中的动物或患者,脑组织损伤更加严重。其可能机制主要有高浓度氧含量引起的氧化应激和乳酸堆积造成的脑组织损伤。此外,复苏后高浓度氧疗还可造成心肌损伤,其主要机制有大量活性氧簇（ReactiveOxygenSpecies,ROS）造成心脏的继发性损伤、Ca2＋通道激活,引起血管收缩加重心肌缺血、K＋ATP通道关闭,造成心肌受损、血管紧张素Ⅱ释放增多和缩血管物质20-HETE生成增多,加重心肌缺血等。因此,在对复苏后病人进行氧疗过程中,目前主张限制复苏后早期氧疗。     

Gene expression patterns associated with suppression of odontogenesis in mouse and vole diastema regions

Rodents have a toothless diastema region between the incisor and molar teeth which may contain rudimentary tooth germs. We found in upper diastema region of the mouse (Mus musculus) three small tooth germs which developed into early bud stage before their apoptotic removal, while the sibling vole (Microtus rossiaemeridionalis) had only a single but larger tooth germ in this region, and this developed into late bud stage before regressing apoptotically. To analyze the genetic mechanisms of the developmental arrest of the rudimentary tooth germs we compared the expression patterns of several developmental regulatory genes (Bmp2, Bmp4, Fgf4, Fgf8, Lef1, Msx1, Msx2, p21, Pitx2, Pax9 and Shh) between molars and diastema buds of mice and voles. In diastema tooth buds the expression of all the genes differed from that of molars. The gene expression patterns suggest that the odontogenic program consists of partially independent signaling cascades which define the exact location of the tooth germ, initiate epithelial budding, and transfer the odontogenic potential from the epithelium to the underlying mesenchyma. Although the diastema regions of the two species differed, in both species the earliest difference that we found was weaker expression of mesenchymal Pax9 in the diastema region than in molar and incisor regions at the dental lamina stage. However, based on earlier tissue recombination experiments it is conceivable that the developmental arrest is determined by the early oral epithelium. Received: 1 February 1999 / Accepted: 30 March 1999     

Continuity versus split and reconstitution: exploring the molecular developmental corollaries of insect eye primordium evolution

Holometabolous insects like Drosophila proceed through two phases of visual system development. The embryonic phase generates simple eyes of the larva. The postembryonic phase produces the adult specific compound eyes during late larval development and pupation. In primitive insects, by contrast, eye development persists seemingly continuously from embryogenesis through the end of postembryogenesis. Comparative literature suggests that the evolutionary transition from continuous to biphasic eye development occurred via transient developmental arrest. This review investigates how the developmental arrest model relates to the gene networks regulating larval and adult eye development in Drosophila, and embryonic compound eye development in primitive insects. Consistent with the developmental arrest model, the available data suggest that the determination of the anlage of the rudimentary Drosophila larval eye is homologous to the embryonic specification of the juvenile compound eye in directly developing insects while the Drosophila compound eye primordium is evolutionarily related to the yet little studied stem cell based postembryonic eye primordium of primitive insects.     

Effects of acute asphyxia on brain energy metabolism in fetal guinea pigs near term.

In a previous study we suggested that--unlike other forms of asphyxia--acute asphyxia caused by arrest of uterine blood flow is accompanied by a fall in oxygen delivery to the fetal brain (Jensen et al., 1987). This may change cerebral energy metabolism by causing an increase in the glycolytic rate. To test this hypothesis we studied the time course of the changes in the levels of high-energy phosphates and glycolytic intermediates in the cerebral cortex of unanaesthetized fetal guinea pigs near term before and after 2 and 4 min of acute asphyxia. During asphyxia there was a progressive fall of adenosine triphosphate, creatine-phosphate, glucose and fructose-1,6-diphosphate concentrations, whereas adenosine diphosphate, adenosine monophosphate and lactate concentrations increased. Pyruvate concentrations did not change. We conclude that fetal cerebral energy metabolism becomes increasingly anaerobic during acute asphyxia caused by arrest of uterine blood flow, because oxygen delivery to the fetal brain falls.     

Septo-optic dysplasia - novel insights into the aetiology

Septo-optic dysplasia (SOD) is a highly heterogeneous condition comprising a variable phenotype of optic nerve hypoplasia, midline brain abnormalities and pituitary hypoplasia with consequent endocrine deficits. The majority of cases are sporadic and several aetiologies have been suggested to account for the pathogenesis of the condition. However, a number of familial cases have been described and the identification of mutations in key developmental genes including HESX1, SOX2 and SOX3 in patients with SOD and associated phenotypes suggests that a genetic causation is likely in the more common sporadic cases of the condition. The precise aetiology of SOD is most likely multifactorial involving contributions from environmental factors in addition to an important role for crucial developmental genes. The variability of the penetrance and phenotypes within a single SOD pedigree may also suggest a complex interaction between genetics and the environment, and at present, the understanding of these interactions is rudimentary. Further study of these critical factors may shed light on the aetiology of this complex disorder. We have reviewed recent literature selecting relevant references based on the keywords HESX1, SOX2, SOX3, Septo-optic dysplasia, genetics and pituitary development.     

Evaluation of acetylcholinesterase and adenosine deaminase activities in brain and erythrocytes and proinflammatory cytokine levels in rats submitted to neonatal hypoxia-ischemia model

Perinatal hypoxic-ischemic (HI) brain injury is a common problem with severe neurologic sequelae. The definitive brain injury is a consequence of pathophysiological mechanisms that begin at the moment of HI insult and may extend for days or weeks. In this context, the inflammatory response and the formation of reactive oxygen species seem to play a key role during evolution of brain damage after injury. Thus, the aim of this study was to describe the chronological sequence of acetylcholinesterase (AChE) activity and the lipid peroxidation changes in the cerebral cortex using the classic model of neonatal HI. Furthermore, the erythrocyte AChE and adenosine deaminase (ADA) activities as well as the serum levels of proinflammatory cytokines were assessed. We observed that neonatal HI caused an increase of lipid peroxidation immediately after HI insult, which remained for several days afterward. There was a time-related change in the AChE activity in the cerebral cortex and the same was observed in erythrocyte AChE and ADA activities. In addition, immediately after HI, ADA activity showed a strong positive correlation with all proinflammatory cytokines assessed. Together, these findings may help the understanding of some mechanism related to the pathophysiology of neonatal HI, therefore highlighting the putative therapeutic targets to minimize brain injury and enhance recovery.     

Restauration de la fertilité masculine par la spermatogenèsein vitro

Anin-vitro culture technique, previously tested with germ cells from men with complete spermatogenesis, was applied in assisted reproduction treatment for cases of meiotic and postmeiotic maturation arrest. Some primary spermatocytes from men with maturation arrest at the pachytene stage developed up to the late elongated spermatid stages and were capable of fertilizing the spouse’s oocytes and of giving rise to embryos that were transferred into the spouse’s uterus and subsequently developed to term. In other cases, round spermatids blocked in vivo before the process of spermiogenesis developed to elongated spermatidsin vitro; with the use of suchin-vitro formed spermatids, the first term pregnancies in cases of complete spermiogenesis arrest were achieved. These findings show that certain in-vivo developmental blocks in male germ cells from patients with severe testiculopathies can be overcome byin-vitro culture, probably by modifying control mechanisms acting at developmental checkpoints.     

High levels of p66shc and intracellular ROS in permanently arrested early embryos

A high incidence of permanent embryo arrest occurs during the first week of in vitro development. We hypothesize that this developmental arrest event is regulated by the stress adaptor protein p66shc, a genetic determinant of life span in mammals, which regulates ROS metabolism, apoptosis, and cellular senescence. The aim of this study was to assess the relationship between intracellular oxidative stress levels with the incidence of embryo arrest and the expression of senescent-associated genes in embryos produced under different oxygen tensions. Embryos cultured under 20% oxygen conditions showed approximately 10-fold increase in oxidative stress, 2-fold increase in the percentage of 2- to 4-cell arrest, and significantly lower developmental capabilities compared to embryos cultured under a 5% oxygen environment. Quantification by real-time PCR and by semiquantitative immunofluorescence showed significantly higher p66shc mRNA and protein levels, respectively, in embryos cultured in 20% versus those cultured in 5% oxygen atmosphere. No significant changes in p53 mRNA and protein levels were detected among embryos derived from both oxygen tensions. Taken together, these results demonstrate that p66shc, but not p53, is significantly more abundant in an embryo population that exhibits higher frequencies of embryo arrest and quantities of intracellular ROS. These results further substantiate that p66shc and oxidative stress are associated with a p53-independent embryonic arrest event for in vitro-produced embryos.