An improved cellular enucleation method with extracellular matrix and colchicine facilitates the study of nucleocytoplasmic interaction

Enucleated mammalian cells (cytoplasts) have been widely used for studying differential roles of the cytoplasm and nucleus in various cellular processes. Here, we reported an improved enucleation protocol, in which cells were seeded in extracellular matrix (ECM)-coated 24-wells and spun at 4600 g and 35 °C for 60 min in the presence of cytochalasin B and colchicine. When glass-bottom wells were used, cellular structures and organelles in cytoplasts could be examined directly by confocal microscopy. Nuclear envelope rupture did not occur probably due to mild centrifugation conditions used in this study. Addition of paclitaxel or doxorubicin completely blocked proliferation of residual nucleated cells; however, to our surprise, paclitaxel dramatically prolonged the survival of cytoplasts. Results from Annexin V and Propidium Iodide staining showed that cytoplasts died predominantly by apoptosis, which was partially inhibited by ECM and further by paclitaxel. Mitochondria were mostly rod-shaped and formed a connected network in paclitaxel-treated cytoplasts, indicating lack of fusion and fission dynamics. Moreover, paclitaxel increased mitochondrial membrane potential, suggesting that perturbation of mitochondria might be critical to the survival of cytoplasts. In conclusion, we had established an efficient and fast procedure for enucleation of adherent animal cells, which could facilitate the investigation of nucleocytoplasmic interaction.     

Isolation,origin and properties of enucleate plant microplasts

Summary Auxin-induced highly vacuolated thin-walled callus cells of several plant species, when ruptured, released large numbers of subcellular units most of which were enucleate. These enucleate microplasts are surrounded by an inner membrane of the cell, most probably derived from the tonoplast. When microplasts isolated fromSaintpaulia callus were cultured in a medium supplemented with growth substances they formed a thin wall and underwent budding. Microplasts could be useful for various studies in plant cell biology and for genetic manipulations.     

Effects of Enucleation on High-Affinity Binding Sites in Chick Optic Tecta

Abstract: The effect of unilateral eye extirpation on the development of the chick optic tectum has been studied in both the embryo and the newly hatched chick. Although the prevention of normal afferentation of the embryonic tectum retarded its growth, there appeared to be a significant increase of muscarinic acetylcholine binding site in the noninnervated tectum. This phenomenon was repeated also in the posthatch denervated system wherein the functioning optic nerve is severed. A significant increase in the number of binding sites as well as reduced dissociation constant of the interactions of this receptor with [³H]quinuclindinyl benzilate was found in the deafferented optic tectum. This may suggest the presence of a denervation-supersensitivity-like modulation. Similar increases were not detected with other binding sites studied in either the noninnervated embryonic or deafferented posthatch optic lobes. The possibility that acetylcholine is a primary neurotransmitter of the optic system is discussed.     

腹腔镜下子宫血管阻断术联合子宫肌瘤剜除术治疗子宫肌瘤的回顾性研究

摘要 目的：回顾性分析子宫肌瘤剜除术联合腹腔镜下子宫血管阻断术治疗子宫肌瘤的临床疗效。方法：分析2017年6月~2019年7月期间我院收治的子宫肌瘤患者的临床资料（n=150）。根据手术方法的不同将患者分为A组（腹腔镜下子宫肌瘤剜除术治疗,73例）和B组（腹腔镜下子宫血管阻断术联合子宫肌瘤剜除术治疗,77例）,对比两组术中、内分泌激素、术后恢复指标、妊娠情况、并发症发生率及复发率。结果：两组患者手术时间对比无统计学差异（P>0.05）,B组的术中出血量少于A组,术后排气时间、住院天数、下床活动时间短于A组（P<0.05）。A组术前、术后3个月、术后6个月黄体生成素(LH) 、卵泡刺激素(FSH) 、雌二醇(E₂) 水平组内对比无统计学差异（P>0.05）。B组术前、术后3个月、术后6个月E₂水平呈降低后升高趋势,LH、FSH水平呈升高后降低趋势（P<0.05）。B组术后3个月LH、FSH水平高于A组,E2水平低于A组（P<0.05）。与A组相比,B组的并发症发生率明显下降,组间对比有差异（P<0.05）。B组的妊娠率高于A组,子宫肌瘤复发率低于A组（P<0.05）。两组流产率组间对比无统计学差异（P>0.05）。结论：与单纯子宫肌瘤剜除术相比,结合腹腔镜下子宫血管阻断术治疗子宫肌瘤患者可减少术中出血量,促进患者术后恢复,降低并发症发生率及复发率,虽然其对卵巢功能有轻微、短暂性影响,但可逐步恢复,且有利于提高妊娠率。     

Down-regulation of Myc is essential for terminal erythroid maturation

Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes followed by chromatin and nuclear condensation and enucleation. The protein levels of c-Myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G(1) phase and enucleation, suggesting possible roles for c-Myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown but specifically blocked erythroid nuclear condensation and enucleation. Continued Myc expression prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. The histone acetyltransferase Gcn5 was up-regulated by Myc, and ectopic Gcn5 expression partially blocked enucleation and inhibited the late stage erythroid nuclear condensation and histone deacetylation. When overexpressed at levels higher than the physiological range, Myc blocked erythroid differentiation, and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. Gene expression analysis demonstrated the dysregulation of erythropoietin signaling pathway and the up-regulation of several positive regulators of G(1)-S cell cycle checkpoint by supraphysiological levels of Myc. These results reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells.     

Endophthalmitis of probable endogenous origin caused by Scedosporium boydii: A case report

BackgroundMycotic ocular infections caused by the Scedosporium apiospermum species complex are challenging to treat because of the delayed diagnoses and poor responses to antifungal drugs and surgical treatment.Case reportA case of a 69-year-old male patient with a history of diabetes mellitus type 2 and prior surgery on the right femur is described. In the 10 days prior to the ophthalmic consultation he started with ocular pain, adding to a previous and progressive loss of visual acuity in his right eye. The diagnosis of endophthalmitis of probable endogenous origin was established. Despite medical treatment, the patient's condition worsened and, due to the imminent risks, an enucleation was performed. Smears of the enucleation tissue revealed fungal cells, and the cultures yielded a fungus belonging to the S. apiospermum species complex, which was identified as Scedosporium boydii by morphological characteristics and sequencing of a PCR amplicon.ConclusionsA diagnosis of endophthalmitis of probable endogenous origin in the right eye was based on a previous right femur surgery. Potential risk to the patient led to enucleation.     

An analysis of morphogenesis of the reproductive whorl of Acetabularia acetabulum

Acetabularia acetabulum (Linn.) P.C. Silva, is a useful system for studying changes in shape because it is large, morphologically complex unicell. The middle, or gametophore lobe of the cap grows radially from the stalk axis as a disc and the fully grown cap can be one of several shapes: flat, concave, convex, and saddle. The shape of the cap normally changes during the first three and a half weeks of reproductive development: individual caps within a population change shape in a stereotypical progression, with the majority proceeding from concave to flat to saddle. Marking the existing surface of caps with carbon grains revealed that the majority of growth occurs near the center, not at the perimeter, of caps. The shape of the mature cap appeared to be independent of algal height, number of gametophores per cap, and final cap diameter. Removing the rhizoid, which contains the nucleus, suggested that the contribution of the nucleus may be important for changes in shape during early cap growth. Based on these data, we present a simple model of cap shape development that suggests both differential growth and biophysical factors may contribute to the final shape of caps of A. acetabulum. Received: 7 January 1998 / Accepted: 7 March 1998     

A Comparative Study on the Efficiency of Two Enucleation Methods in Pig Somatic Cell Nuclear Transfer: Effects of the Squeezing and the Aspiration Methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72–78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

Effects of illumination and enucleation on substance-P-immunoreactive structures in subcortical visual centers of golden hamster and Wistar rat

The undecapeptide substance P is found in different entities of the visual system that control eye movement and synchronize endogenous rhythms with the light cycle (i.e., superior colliculus, suprachiasmatic nucleus, intergeniculate leaflet). Immunocytochemical methods were used to compare the reactivity to substance P in the brain of five groups of golden hamsters and two groups of Wistar rats: (1) untreated hamsters kept under 14L:10D and sacrificed at noon; (2) identically maintained animals sacrificed at midnight; (3) enucleated animals kept under control conditions; (4) hamsters kept under constant darkness; (5) hamsters kept under the same conditions as the controls, but intraventricularly injected with colchicine. The results obtained in golden hamsters of groups (1) and (3) were compared with findings in Wistar rats treated accordingly [groups (6) and (7)]. Substance P-immunoreactive perikarya were found in the suprachiasmatic nucleus and superior colliculus of hamsters and Wistar rats. Substance P-immunoreactive nerve fibers were abundant in the hypothalamic area ventral to the paraventricular nucleus, in the intergeniculate leaflet, in some thalamic nuclei, and in the superior colliculus. Immunoreactivity to substance P in the suprachiasmatic nucleus and intergeniculate leaflet did not vary among the experimental groups. However, a conspicuous decrease in reactivity to substance P was observed in the superficial layers of the superior colliculus of enucleated hamsters and rats, compared with all other groups. These results indicate that substance P immunoreactivity in the superior colliculus, but not that in the suprachiasmatic nucleus or intergeniculate leaflet, depends on the integrity of the retinal projection.     

Birth of Beagle dogs by somatic cell nuclear transfer

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 μs electrical stimulation, chemically activated after 1 h of fusion using 10 μM calcium ionophore for 4 min and cultured 4 h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4–10, 11–25 and 26–40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P > 0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4–10 embryos (n = 7) and 26–40 embryos (n = 12) while pregnancy was detected in 23.53% recipients received a group of 11–25 embryos (n = 17). Among them, five (1.76%) live puppies were born (P < 0.05). These data show an increase in the overall efficiency of SCNT in canine species.