Molecular cloning of a centrin homolog from Marsilea vestita and evidence for its translational control during spermiogenesis

1999 ◽  
Vol 77 (2) ◽  
pp. 101-108 ◽  
Author(s):  
Peter E Hart ◽  
Stephen M Wolniak
Keyword(s):  
Translational Control ◽  
De Novo ◽  
Basal Bodies ◽  
Motile Cells ◽  
Marsilea Vestita ◽  
Stored Mrna ◽  
Eukaryotic Organisms ◽  
Alpha Amanitin ◽  
Made In

Spermiogenesis in the water fern Marsilea vestita is a process that reaches completion 11 h after dry microspores are immersed in an aqueous medium at 20°C. Each microspore produces 32 spermatozoids and each spermatozoid has a coiled cell body and approximately 140 cilia. The spermatids make basal bodies de novo, from a structure known as a blepharoplast. From the onset of development, the spores contain a large quantity of protein and stored mRNA. We have found previously that centrin, a protein involved in the function of microtubule organizing centers and present in association with basal bodies in motile cells, is made in large quantity approximately 4 h after the microspores are placed into liquid medium. In this paper, we show that a centrin cDNA (MvCen1) we isolated from M. vestita closely resembles centrin cDNAs from other eukaryotic organisms. MvCen1, synthesized in Escherichia coli as a GST-fusion protein, reacted with anti-centrin monoclonal antibodies on immunoblots. Northern blot analysis demonstrates that centrin mRNA is present in the dry microspore at the time of imbibition, at levels that remain constant over 10 h of development and are unaffected by treatment of spores with alpha-amanitin. The centrin transcripts, stored in dry microspores, cannot be translated in vitro for at least 30 min after imbibition.Key words: Marsilea vestita, spermatozoid, spermiogenesis, centrin, MTOC.

scholarly journals Centrin Is Necessary for the Formation of the Motile Apparatus in Spermatids of Marsilea

2001 ◽  
Vol 12 (3) ◽  
pp. 761-776 ◽  
Author(s):  
Vincent P. Klink ◽  
Stephen M. Wolniak
Keyword(s):  
Essential Role ◽  
Normal Development ◽  
De Novo ◽  
Basal Bodies ◽  
Double Stranded Rna ◽  
Rna Probes ◽  
Marsilea Vestita ◽  
Stored Mrna ◽  
Novel Method ◽  
Β Tubulin

During spermiogenesis in the water fern, Marsilea vestita, basal bodies are synthesized de novo in cells that lack preexisting centrioles, in a particle known as a blepharoplast. We have focused on basal body assembly in this organism, asking what components are required for blepharoplast formation. Spermiogenesis is a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and stored mRNA, and inhibitors reveal that certain proteins are translated from stored transcripts at specific times during development. Centrin translation accompanies blepharoplast appearance, while β-tubulin translation occurs later, during axonemal formation. In asking whether centrin is an essential component of the blepharoplast, we used antisense, sense, and double-stranded RNA probes made from theMarsilea centrin cDNA, MvCen1, to block centrin translation. We employed a novel method to introduce these RNAs directly into the cells. Antisense and sense both arrest spermiogenesis when blepharoplasts should appear, and dsRNA made from the same cDNA is an effective inhibitor at concentrations at least 10 times lower than either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsileaβ-tubulin permitted normal development until axonemes form. In controls, antisense, sense, and dsRNA, made from a segment of HIV, had no effect on spermiogenesis. Immunoblots suggest that translational blocks induced by centrin-based RNA are gene specific and concentration dependent, since neither β-tubulin- nor HIV-derived RNAs affects centrin translation. The disruption of centrin translation affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These results show that centrin plays an essential role in the formation of a motile apparatus during spermiogenesis of M. vestita.

Cell cycle arrest allows centrin translation but not basal body formation during spermiogenesis inMarsilea

2001 ◽  
Vol 114 (23) ◽  
pp. 4265-4272 ◽  
Author(s):  
Chiawei W. Tsai ◽  
Stephen M. Wolniak
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
De Novo ◽  
Cyclin B ◽  
Basal Bodies ◽  
Cycle Arrest ◽  
Essentially Normal ◽  
Marsilea Vestita ◽  
Stored Mrna ◽  
Cytoplasmic Particle

Spermiogenesis in the water fern Marsilea vestita is a rapid process that requires the de novo formation of basal bodies in a cytoplasmic particle known as a blepharoplast. Spermiogenesis is activated by placing dry spores into water and is dependent upon the translation of new proteins from stored mRNAs with little, if any, new transcription. We looked at the necessity of cell division cycles in the gametophyte as a prerequisite for the activation of centrin translation and for the consequent formation of blepharoplasts. Cell cycle arrest was induced by treatments of gametophytes with hydroxyurea, with olomoucine, or after RNAi, employing dsRNA derived from Marsilea cyclin A or cyclin B. In all cases, centrin is translated from stored mRNA at the normal time, approximately 4 hours after imbibition, and it accumulates to maximal levels ∼6 hours after imbibition. In spite of the fact that centrin is translated at essentially normal times and accumulates to nearly normal levels, no blepharoplasts form in the gametophytes where division cycles have been disrupted. These results provide a clear demonstration that the new translation of centrin, by itself, is insufficient for blepharoplast formation, the de novo formation of basal bodies, and the assembly of a motile apparatus.

scholarly journals Analysis of lines of mice selected for fat content. 2. Correlated responses in the activities of enzymes involved in lipogenesis

1990 ◽  
Vol 55 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Ian M. Hastings ◽  
William G. Hill
Keyword(s):  
De Novo ◽  
Fatty Acid Synthetase ◽  
Fat Content ◽  
Acetyl Coa Carboxylase ◽  
Correlated Responses ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Obesity In Mice ◽  
Made In

SummaryEstimates of the activities (Vmax) of six enzymes involved in de novo fat synthesis were made in replicated lines of mice differing in fat content. These lines had been selected high and low for 20 generations with three replicates each of Fat, Control and Lean lines and for a further eight generations high and low as an unreplicated line. The activities of ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), fatty acid synthetase (FAS), cytoplasmic malate dehydrogenase (MDH), malic enzyme (ME) and pyruvate kinase (PK) were determined in vitro in both liver and gonadal fatpad tissues taken at ages five and ten weeks. The activities of ACL, ACC, FAS and ME were significantly higher in the Fat than the Lean lines, and the differences were more pronounced at the earlier age and in the gonadal fatpad where activities in the Fat lines were higher by factors of 3·5, 2·4, 2·5 and 3·5 respectively. The activity of PK was unchanged in each tissue. MDH activity was significantly lower in adipose tissue in the Fat lines than the Lean lines at age ten weeks but not at age five weeks or in liver tissue. Results from replicates indicated that random genetic drift affected enzyme activities but nevertheless significant changes in activity were associated with the direction of selection. The changes in enzyme activity reported here are similar to those known to be associated with major mutations causing obesity in mice.

scholarly journals Phorbol esters induce synthesis of thromboplastin activity in human monocytes

1981 ◽  
Vol 194 (3) ◽  
pp. 699-706 ◽  
Author(s):  
T Lyberg ◽  
H Prydz
Keyword(s):  
Phorbol Esters ◽  
De Novo ◽  
Mouse Skin ◽  
Inhibitory Effect ◽  
Human Monocytes ◽  
Cytotoxic Effects ◽  
Tumour Promotion ◽  
Alpha Amanitin

12-O-Tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-diacetate and phorbol 12,13-didecanoate were all potent inducers of thromboplastin activity in human monocytes in vitro, whereas 4 alpha-phorbol 12,13-didecanoate and 4 alpha-phorbol had no such effect. A concomitant increase in titrable apoprotein III antigen was found (apoprotein III is the protein component of thromboplastin). The increase was inhibited by cycloheximide and actinomycin D and partly by alpha-amanitin. The increase of thromboplastin activity was therefore most likely due to synthesis de novo of apoprotein III. The response was approximately halved in the absence of serum or Ca2+. Retinol had a weak inhibitory effect, and retinoic acid was inhibitory only at concentrations that also induced signs of cytotoxicity. TPA caused an initial rise in monocyte cyclic AMP concentration of about 90-120 min duration. No increase in 45Ca2+ influx was induced over 2 h. Good correlation exists between induction of apoprotein III synthesis in monocytes in vitro and mouse skin-tumour promotion in vivo by the various phorbol derivatives. Substances inactive in tumour promotion do not induce the synthesis of apoprotein III. General activating and cytotoxic effects of TPA were monitored by determining release of lysozyme, beta-glucuronidase and lactate dehydrogenase.

scholarly journals Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz

2003 ◽  
Vol 163 (6) ◽  
pp. 1197-1204 ◽  
Author(s):  
James E. Wilhelm ◽  
Meredith Hilton ◽  
Quinlan Amos ◽  
William J. Henzel
Keyword(s):  
Translational Control ◽  
Mrna Localization ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Translational Repressor ◽  
Eukaryotic Initiation Factor 4E ◽  
Microtubule Transport ◽  
Rnp Complex ◽  
Made In

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end–directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation.

scholarly journals Recent advances in our understanding of the mechanisms of lung alveolarization and bronchopulmonary dysplasia

2019 ◽  
Vol 317 (6) ◽  
pp. L832-L887 ◽  
Author(s):  
Ettore Lignelli ◽  
Francesco Palumbo ◽  
Despoina Myti ◽  
Rory E. Morty
Keyword(s):  
Preterm Infants ◽  
Bronchopulmonary Dysplasia ◽  
De Novo ◽  
50Th Anniversary ◽  
Mouse Genetics ◽  
Histopathological Feature ◽  
Chronic Lung Diseases ◽  
Made In

Bronchopulmonary dysplasia (BPD) is the most common cause of morbidity and mortality in preterm infants. A key histopathological feature of BPD is stunted late lung development, where the process of alveolarization—the generation of alveolar gas exchange units—is impeded, through mechanisms that remain largely unclear. As such, there is interest in the clarification both of the pathomechanisms at play in affected lungs, and the mechanisms of de novo alveoli generation in healthy, developing lungs. A better understanding of normal and pathological alveolarization might reveal opportunities for improved medical management of affected infants. Furthermore, disturbances to the alveolar architecture are a key histopathological feature of several adult chronic lung diseases, including emphysema and fibrosis, and it is envisaged that knowledge about the mechanisms of alveologenesis might facilitate regeneration of healthy lung parenchyma in affected patients. To this end, recent efforts have interrogated clinical data, developed new—and refined existing—in vivo and in vitro models of BPD, have applied new microscopic and radiographic approaches, and have developed advanced cell-culture approaches, including organoid generation. Advances have also been made in the development of other methodologies, including single-cell analysis, metabolomics, lipidomics, and proteomics, as well as the generation and use of complex mouse genetics tools. The objective of this review is to present advances made in our understanding of the mechanisms of lung alveolarization and BPD over the period 1 January 2017–30 June 2019, a period that spans the 50th anniversary of the original clinical description of BPD in preterm infants.

Einfluß der Glucose auf die [14C] Thymidin-Einbaurate von Ehrlich-Ascitescarcinomzellen in vitro

1969 ◽  
Vol 08 (02) ◽  
pp. 196-206 ◽  
Author(s):  
Dieter. Kummer
Keyword(s):  
De Novo

ZusammenfassungIn nahezu glucosefreier Suspension von Ehrlich-Ascitescarcinomzellen bewirkt die Zufuhr von Glucose 2,5 × 10–4 bis 10–2 M:1. Hemmung der [14C] Thymidin-Einbaurate in die Zellen.2. Aktivierung des Ribonucleotid-Reductase-Systems und damit Stimulierung der Desoxyribonucleotidsynthese (auch der Thymidintriphosphat-de-novo-Synthese).3. Blockierung der Thymidinkinase über Endprodukthemmung, wodurch die Minderung des [14C] Thymidin-Einbaus in die Zellen erklärbar ist.

Особенности морфогенеза редкого вида Allium neriniflorum (Herb.) Backer в условиях in vitro

2019 ◽  
pp. 37-41 ◽  
Author(s):  
Альбина Шамсуновна Ахметова ◽  
Альфия Ануровна Зарипова
Keyword(s):  
De Novo

Показана возможность эффективного применения метода культуры тканей для размножения Allium neriniflorum (Herb.) Backer. Исследуемый вид является декоративным растением, размножение которого затруднено из-за низкой всхожести семян и ослабленной способности к формированию дочерних луковиц. Разработана технология клонального микроразмножения из стерильных луковиц. В качестве исходного материала использовали семена A. neriniflorum. Подобраны условия стерилизации, позволяющие достичь максимального числа (75 %) жизнеспособных эксплантов. Поверхностную стерилизацию проводили в ламинар-боксе с использованием в качестве стерилизующего агента 0,1 % раствор диацида. Семена сначала обрабатывали 70 % этанолом, затем стерилизующим раствором. Экспозиция стерилизующих растворов составляла от 5 до 9 мин. Показано, что способность к индуцированному морфогенезу существенно зависит от состава питательной среды. Максимальное число луковиц образовывалось на среде QL — 9 шт./эксплант. Исследуемые виды обладали высокой способностью к мультипликации и формированию полноценных растений при подобранных условиях культивирования in vitro. Выявленная морфогенетическая активность зачаточного побега, сегментов чешуй и донца стерильной луковицы A. neriniflorum, проявляющаяся в способности регенерировать побеги de novo, что возможно только в культуре in vitro, обеспечивает формирование полноценных луковиц. Луковицы, полученные in vitro, включали в последующие циклы микроразмножения. Культура тканей и органов in vitro позволяет размножать A. neriniflorum с более высоким коэффициентом размножения. От одной стерильной луковицы можно получить до 7000 луковиц в год. При традиционном вегетативном способе размножения материнская луковица формирует 1, редко 2 дочерние луковицы.

Study of Phosphocalcic Glasses from SiO2-CaO-P2O5 System with and Without Silver II. The bioactivity analysis by FTIR, SEM methods and microbiological study of silver-doped glasses

2017 ◽  
Vol 68 (6) ◽  
pp. 1188-1192
Author(s):  
Daniela Avram ◽  
Nicolae Angelescu ◽  
Dan Nicolae Ungureanu ◽  
Ionica Ionita ◽  
Iulian Bancuta ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Body Fluid ◽  
Pathogenic Bacteria ◽  
De Novo ◽  
Microbiological Examination ◽  
Doped Glasses ◽  
P2o5 System ◽  
Scanning Electron

The study in vitro of the glass powders bioactivity was performed by soaking them in simulated body fluid for 3 to 21 days at a temperature of 37�C and pH = 7.20. The synthesis de novo of hydroxyapatite, post soaking was confirmed by Fourier Transform Infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The study of the antimicrobial activity was performed by microbiological examination on two strains of pathogenic bacteria involved in postoperative nosocomial infections.

Potential of Nanoparticles in Combating Candida Infections

2019 ◽  
Vol 16 (5) ◽  
pp. 478-491 ◽  
Author(s):  
Faizan Abul Qais ◽  
Mohd Sajjad Ahmad Khan ◽  
Iqbal Ahmad ◽  
Abdullah Safar Althubiani
Keyword(s):  
Targeted Delivery ◽  
Recent Progress ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Fold Increase ◽  
Antifungal Drugs ◽  
Low Toxicity ◽  
Candida Infections ◽  
Made In

Aims: The aim of this review is to survey the recent progress made in developing the nanoparticles as antifungal agents especially the nano-based formulations being exploited for the management of Candida infections. Discussion: In the last few decades, there has been many-fold increase in fungal infections including candidiasis due to the increased number of immunocompromised patients worldwide. The efficacy of available antifungal drugs is limited due to its associated toxicity and drug resistance in clinical strains. The recent advancements in nanobiotechnology have opened a new hope for the development of novel formulations with enhanced therapeutic efficacy, improved drug delivery and low toxicity. Conclusion: Metal nanoparticles have shown to possess promising in vitro antifungal activities and could be effectively used for enhanced and targeted delivery of conventionally used drugs. The synergistic interaction between nanoparticles and various antifungal agents have also been reported with enhanced antifungal activity.

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