Observations on the dynamics of argyrophilic nucleolar material in the nuclei of mice spermatids

Applying a new silver staining technique it could be shown that in very early spermatids strong argyrophilia in nucleoli is confined to their granular and fibrillar components; fibrillar centers are devoid of silver. During subsequent developmental stages remnants of these nucleolar components are present in the form of intensively silver stained clusters of coiled fibers. As chromatin condensation proceeds, these fibrous structures decrease in size and density and are finally completely absent. The nucleus of the mature sperm contains only the space in which they formerly existed, now silver negative, as the so-called 'nuclear vacuole'.     

Observations on the dynamics of argyrophilic nucleolar material in the nuclei of mice spermatids

Summary Applying a new silver staining technique it could be shown that in very early spermatids strong argyrophilia in nucleoli is confined to their granular and fibrillar components; fibrillar centers are devoid of silver. During subsequent developmental stages remnants of these nucleolar components are present in the form of intensively silver stained clusters of coiled fibers. As chromatin condensation proceeds, these fibrous structures decrease in size and density and are finally completely absent. The nucleus of the mature sperm contains only the space in which they formerly existed, now silver negative, as the so-called nuclear vacuole.Acknowledgment. This investigation was supported partly by grant from the Universitätspreis der Wiener Wirtschaft.     

Ultrastructural aspects of nucleolar fibrillar centres in meristematic cells ofAllium cepa

Summary This paper deals with the fine structure of the fibrillar centres of the nucleolus inAllium cepa cells in ultrathin, sections of in vivo fixed roots. The ultrastructural observations have allowed us to consider each nucleolar fibrillar centre as an active zone in the nucleolar chromatin loop, and to propose a possible model for the organization of the different components of the nucleolus within it.Acknowledgments. The author wishes to thank Dr.M. C. Risueño for her valuable comments and discussion. This work has been partially supported by the III Plan de Desarrollo of Spain and by a grant of the Foundation Rodriguez Pascual.     

Effect of actinomycin D on the quail oocyte nucleolus during meiotic prophase I

Summary Actinomycine D alters profoundly the distribution of the nucleolar constituents in the quail oocyte at prophase I of meiosis. As a consequence of nucleolar segregation the normally existing relations between the nucleolus fibrillar centers and the microchromosomes are ruptured. The relations between the fibrillar center and the dense fibrills which surround it remain intact, suggesting that they constitute together a functional unit.With the technical assistance of Mrs A. Calisti.     

Inositol pyrophosphates: structure, enzymology and function

The stereochemistry of the inositol backbone provides a platform on which to generate a vast array of distinct molecular motifs that are used to convey information both in signal transduction and many other critical areas of cell biology. Diphosphoinositol phosphates, or inositol pyrophosphates, are the most recently characterized members of the inositide family. They represent a new frontier with both novel targets within the cell and novel modes of action. This includes the proposed pyrophosphorylation of a unique subset of proteins. We review recent insights into the structures of these molecules and the properties of the enzymes which regulate their concentration. These enzymes also act independently of their catalytic activity via protein–protein interactions. This unique combination of enzymes and products has an important role in diverse cellular processes including vesicle trafficking, endo- and exocytosis, apoptosis, telomere length regulation, chromatin hyperrecombination, the response to osmotic stress, and elements of nucleolar function.     

Nucleolar localization and circadian regulation of Per2S,a novel splicing variant of the Period 2 gene

In this work, we show for the first time that a second splicing variant of the core clock gene Period 2 (Per2), Per2S, is expressed at both the mRNA and protein levels in human keratinocytes and that it localizes in the nucleoli. Moreover, we show that a reversible perturbation of the nucleolar structure acts as a resetting stimulus for the cellular clock. Per2S expression and periodic oscillation upon dexamethasone treatment were assessed by qRT-PCR using specific primers. Western blot (WB) analysis using an antibody against the recombinant human PER2 (abRc) displayed an intense band at a molecular weight of ~55 kDa, close to the predicted size of Per2S, and a weaker band at the expected size of Per2 (~140 kDa). The antibody raised against PER2 pS662 (abS662), an epitope absent in PER2S, detected only the higher band. Immunolocalization studies with abRc revealed a peculiar nucleolar signal colocalizing with the nucleolar marker nucleophosmin, whereas with abS662 the signal was predominantly diffuse all over the nucleus and partially colocalized with abRc in the nucleolus. The analysis of cell fractions by WB confirmed the enrichment of PER2S and the presence of PER2 in the nucleolar compartment. Finally, a pulse (1 h) of actinomycin D (0.01 μg/ml) induced reversible nucleolar disruption, PER2S de-localization and circadian synchronization of clock and Per2S genes. Our work represents the first evidence that the Per2S splicing isoform is a clock component expressed in human cells localizing in the nucleolus. These results suggest a critical role for the nucleolus in the process of circadian synchronization in human keratinocytes.     

Ultrastructural identification of the nucleolus organizer by the silver staining technic]

The nucleolus organizer regions can be selectively stained in metaphase chromosome preparations by the Goodpasture and Bloom's technique which was adapted to electron microscopy analysis of cells during interphase. Using this technique, a selective accumulation of silver grains was observed over nucleolus light areas. This selective accumulation allows the identification of the interphase fibrillar centers as the nucleolus organizer regions. Ultrastructural relationships between fibrillar centers and dense fibrillar component are discussed.     

Assessing heterogeneity in oligomeric AAA+ machines

ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.     

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion.

The role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca2+, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca2+ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 mumol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 mumol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca2+. Increasing the Ca2+ concentration to 1.26 mmol/l in TCM 199 during the 22-24 h exposure to glucose (16.7 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca2+ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Targeting mitochondria by α-tocopheryl succinate overcomes hypoxia-mediated tumor cell resistance to treatment

Rapidly proliferating tumor cells easily become hypoxic. This results in acquired stability towards treatment with anticancer drugs. Here, we show that cells grown at 0.1 % oxygen are more resistant towards treatment with the conventionally used anticancer drugs doxorubicin and cisplatin. The stimulation of apoptosis, as assessed by the number of cells in the SubG1 fraction of the cell cycle, release of cytochrome c into the cytosol, activation of caspase-3, and cleavage of PARP, was markedly suppressed under low oxygen content or when hypoxia was mimicked by deferoxamine. Hypoxia or deferoxamine treatment was accompanied by stabilization of the hypoxia-inducible factor (HIF-1). The downregulation of HIF-1 using siRNA technique restored cell sensitivity to treatment under hypoxic conditions to the levels detected under normoxic conditions. In contrast to cisplatin or doxorubicin, α-tocopheryl succinate (α-TOS), a compound that targets mitochondria, stimulated cell death irrespective of the oxygen concentration. Moreover, under hypoxic condition cell death induced by α-TOS was even enhanced. Thus, α-TOS can successfully overcome resistance to treatment caused by hypoxia, which makes α-TOS an attractive candidate for antitumor therapy via mitochondrial targeting.     

Nucleolar structure during the prophase of Physarum polycephalum]

With Sugihara's fixation technique, electron microscopic study of early-prophase nucleolus in the Physarm polycephalum showed the existence of particular fibrillar structures. The characteristic feature of these spherical structures is an electron-lucid center surrounded by a dense fibrillar component. Their relationships between the "fibrillar centers" and between the rate of ribosomal RNA synthesis are studied.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Toxicity of statins on rat skeletal muscle mitochondria

We investigated mitochondrial toxicity of four lipophilic stains (cerivastatin, fluvastatin, atorvastatin, simvastatin) and one hydrophilic statin (pravastatin). In L6 cells (rat skeletal muscle cell line), the four lipophilic statins (100 micromol/l) induced death in 27-49% of the cells. Pravastatin was not toxic up to 1 mmol/l. Cerivastatin, fluvastatin and atorvastatin (100 micromol/l) decreased the mitochondrial membrane potential by 49-65%, whereas simvastatin and pravastatin were less toxic. In isolated rat skeletal muscle mitochondria, all statins, except pravastatin, decreased glutamate-driven state 3 respiration and respiratory control ratio. Beta-oxidation was decreased by 88-96% in the presence of 100 micromol/l of the lipophilic statins, but only at higher concentrations by pravastatin. Mitochondrial swelling, cytochrome c release and DNA fragmentation was induced in L6 cells by the four lipophilic statins, but not by pravastatin. Lipophilic statins impair the function of skeletal muscle mitochondria, whereas the hydrophilic pravastatin is significantly less toxic.     

Ultrastructural localization of catalase and D-amino acid oxidase in ‘normal’ fetal mouse liver

Summary In the hepatocytes of normal fetal mice from mothers which were carriers of muscular dysgenesis, catalase and D-amino acid oxidase (DAAO) positive as well as negative peroxisomes were observed. DAAO reaction product was occasionally localized in patches around cell membranes and DAAO-positive peroxisomes were frequently observed near mitochondria.     

纳米Fe3O4协同微生物对黄褐土中PCB30的降解

研究了纳米Fe3O4协同微生物降解黄褐土中的2,4,6 三氯联苯(PCB30),以PCB30为唯一碳源时降解菌的生长状况,以及微生物接菌量、纳米Fe3O4投加量、PCB30浓度对黄褐土中PCB30降解的影响。PCBs降解菌经1 6S rDNA鉴定为假单胞菌Pseudomonas sp.,与草莓假单胞菌Pseudomonas sp.fragi同源性为75%。环境因素对黄褐土中PCB30降解效果有明显影响。微生物接菌量在0~0.8 mL(1×1 09cfu·mL-1)、纳米Fe3O4投加量在0~1 6.7 g·kg-1、PCB30浓度在0~1 0 mg·kg-1范围内时,PCB30残留率随着微生物接菌量、纳米Fe3O4投加量以及PCB30浓度的增大而降低。当三者都分别达到各自范围的上限时,微生物单一体系、纳米Fe3O4单一体系和纳米Fe3O4/微生物协同体系中PCB30残留率在反应7 d后分别为63.1 8%、43.27%和26.28%;纳米Fe3O4/微生物协同体系的降解效果明显优于微生物和纳米Fe3O4单一体系。