Retardation of Regeneration and Division of Blepharisma by Ultraviolet Radiation and Its Photoreversal

Regeneration of Blepharisma undulans variety japonicus from which the hypostome has been removed is retarded by dosages of 3000 to 4600 ergs/mm.² at wavelength 2654A most strongly when the fragment is exposed soon after cutting. Dosages greater than 4600 ergs/mm.² prevent regeneration. Regeneration is also retarded strongly when the Blepharisma are cut soon after irradiation. Starvation retards regeneration and potentiates the effect of ultraviolet radiations. Division after regeneration of Blepharisma is also retarded by ultraviolet radiations about equally, regardless of when the Blepharisma are cut indicating a more lasting effect of the radiations upon the cells. Blepharisma cut after irradiation usually recover from the effects of the radiations sooner than uncut individuals given the same dosage. Retardation of division by ultraviolet radiation is subject to photoreversal by visible light, especially in a nitrogen atmosphere, provided the ultraviolet dose is not excessive. Visible light alone if prolonged, retards regeneration or may even kill the cut fragments of Blepharisma.     

ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE IN CHLAMYDOMONAS REINHARDTII : Influence on Flagellar Function and Regeneration

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.     

Isolation, chemical structure, acute toxicity, and some physicochemical properties of territrem B'' from Aspergillus terreus.

We investigated the production and regeneration of Thiobacillus ferrooxidans spheroplasts. Regeneration frequencies of up to 90% reversion were obtained. Attempts to transform T. ferrooxidans spheroplasts with a recombinant T. ferrooxidans plasmid were unsuccessful.     

Rigenerazione e totipotenza

Abstract

Regeneration and totipotency.—Regeneration and totipotency of cells and nuclei in plants and animals are discussed. The paper consists of the following sections: regeneration in coelenterates and planarians; limb regeneration in amphibians; regeneration of mammalian liver; Wolffian lens regeneration in urodeles; nuclear transplantation in animal eggs; regeneration of plants from single cells (in vivo; in vitro; from somatic cell hybrids); general remarks.     

Three genes control the timing, the site and the size of blastema formation in Drosophila

Regeneration is a vital process to maintain and repair tissues. Despite the importance of regeneration, the genes responsible for regenerative growth remain largely unknown. In Drosophila, imaginal disc regeneration can be induced either by fragmentation and in vivo culture or in situ by ubiquitous expression of wingless (wg/wnt1). Imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing and proliferation at the wound site, forming a regeneration blastema. Most blastema cells maintain their disc-specific identity during regeneration; a few cells however, exhibit stem-cell like properties and switch to a different fate, in a phenomenon known as transdetermination. We identified three genes, regeneration (rgn), augmenter of liver regeneration (alr) and Matrix metalloproteinase-1 (Mmp1) expressed specifically in blastema cells during disc regeneration. Mutations in these genes affect both fragmentation- and wg-induced regeneration by either delaying, reducing or positioning the regeneration blastema. In addition to the modifications of blastema homeostasis, mutations in the three genes alter the rate of regeneration-induced transdetermination. We propose that these genes function in regenerative proliferation, growth and regulate cellular plasticity.     

Regeneration of radiation-damaged digestive tissues in juvenile Pacific oysters (Crassostrea gigas)

Juvenile Pacific oysters, Crassostrea gigas, were irradiated with 16 and 40 krad and their tissues examined histologically. Degenerative syndromes and tissue regeneration processes were determined for the stomach, gut, collecting ducts, and digestive tubules. Following degeneration, tissue regeneration was observed in the digestive tissues of most oysters exposed to 16 krad and in a limited number exposed to 40 krad. Regeneration was first observed in the digestive tubules and subsequently in the stomach, gut, and collecting ducts. Cellular repopulation of the digestive tubules involved epithelialization with large, undifferentiated crypt cells which then differentiated into functional secretory and absorptive cells. Regeneration in the stomach, gut, and collecting ducts was initiated by proliferative islands of small basophilic cells. Mitotic division of those cells and their subsequent differentiation into functional epithelial cells resulted in the rapid restoration and apparent recovery of the affected tissues. The results of these studies indicate that radioresistance of juvenile C. gigas may in part be due to the remarkably efficient regenerative mechanisms involved in replacing injured or lost digestive tissues.     

Suppression of ethylene levels promotes morphogenesis in pepper (Capsicum annuum L.)

Ethylene and polyamines (PAs) are two phytohormones that play important roles during in vitro morphogenesis of several plant species. The interaction between ethylene and PAs has been of interest because both have S-adenosylmethionine as a precursor. To study the influence of ethylene and PAs on in vitro morphogenesis of an ornamental pepper, we added an ethylene scavenger, PAs, a PA inhibitor, and compounds that affect ethylene biosynthesis and activity to the regeneration medium. Regeneration frequencies increased in response to treatment with ethylene inhibitors (aminoethoxyvinylglycine and silver thiosulfate) and an ethylene scavenger (mercury perchlorate). Treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid reduced the regeneration frequency, increased callus formation, and increased ethylene levels; similar results were obtained in response to treatment with the PA inhibitor methylglyoxal-bis(guanylhydrazone). By contrast, treatment with PAs (particularly spermidine and spermine) decreased ethylene levels, increased the regeneration frequency, and increased shoot bud formation. These results suggest a coordinated regulation of ethylene and polyamines because the suppression of ethylene levels using ethylene inhibitors, polyamines, or mercury perchlorate increased the in vitro regeneration frequency and morphogenic responses of Capsicum annuum L.     

Requirement for Wnt and FGF signaling in Xenopus tadpole tail regeneration

We have investigated the requirement for the FGF and Wnt/beta-catenin pathways for Xenopus tadpole tail regeneration. Pathways were modified either by treatment with small molecules or by induction of transgene expression with heat shocks. Regeneration is inhibited by treatment with the FGF inhibitor SU5402, or by activation of a dominant negative FGF receptor, or by activation of expression of the Wnt inhibitor Dkk1. Agents promoting Wnt activity: the small molecule BIO, or a constitutively active form of beta-catenin, led to an increased growth rate. Combination of a Wnt activator with FGF inhibitor suppressed regeneration, while combination of a Wnt inhibitor with a FGF activator allowed regeneration. This suggests that the Wnt activity lies upstream of the FGF activity.Expression of both Wnt and FGF components was inhibited by activation of noggin, suggesting that BMP signalling lies upstream of both Wnt and FGF.The results show that the molecular mechanism of Xenopus tadpole tail regeneration is surprisingly similar to that of the Xenopus limb bud and the zebrafish caudal fin, despite the difference of anatomy.     

Subtractive screen of potential limb regeneration related genes from Pachytriton brevipes

Regeneration capacity varies greatly among different animal species. In vertebrate, amphibian especially the Urodela, has been used as a powerful model system to study the mechanism of tissue regeneration because of the strong ability to regenerate their damaged or lost appendages. Pachytriton brevipes, a species of newt, which is widely distributed in south of China, can completely restore their damaged limbs within several months. In this study, we use modified suppression subtractive hybridization assay and dot-blot screening to identify candidate genes involved in tissue regeneration in P. brevipes. We successfully isolated 81 ESTs from a forward regeneration subtraction library. And we further verified the differential expression of four candidate genes, Rpl11, Cirbp, Ag2 and Trimx, between regenerating blastema and non-regeneration tissues by in situ hybridization. These genes were also be further characterized by phylogenetic and bioinformatic analysis. In general, we provided a comparative experimental approach to study the mechanisms of vertebrate regeneration.     

Morphological,cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2’-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.     

Homeobox Genes Expressed During Echinoderm Arm Regeneration

Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems.     

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.     

Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses

Regeneration of periodontal tissues requires a concerted effort to obtain consistent and predictable results in vivo. The aim of the present study was to test a new family of bioactive polymeric membranes in combination with stem cell therapy for periodontal regeneration. In particular, the novel polyester poly(isosorbide succinate-co-L-lactide) (PisPLLA) was compared with poly(L-lactide) (PLLA). Both polymers were combined with collagen (COL), hydroxyapatite (HA) and the growth factor bone morphogenetic protein-7 (BMP7), and their osteoinductive capacity was evaluated via in vitro and in vivo experiments. Membranes composed of PLLA/COL/HA or PisPLLA/COL/HA were able to promote periodontal regeneration and new bone formation in fenestration defects in rat jaws. According to quantitative real-time polymerase chain reaction (qRT-PCR) and Alizarin Red assays, better osteoconductive capacity and increased extracellular mineralization were observed for PLLA/COL/HA, whereas better osteoinductive properties were associated with PisPLLA/COL/HA. We concluded that membranes composed of either PisPLLA/COL/HA or PLLA/COL/HA present promising results in vitro as well as in vivo and that these materials could be potentially applied in periodontal regeneration.     

Optimization of Protoplast Formation and Regeneration in Leuconostoc mesenteroides

Conditions are reported for efficient protoplast formation and regeneration in four strains of Leuconostoc mesenteroides. Protoplasts were produced from each strain at frequencies greater than 99%, although the rate of their production was variable from strain to strain. Bovine serum albumin and Mg²⁺ were required for maximal regeneration, while the presence of Ca²⁺ was inhibitory. Regeneration frequencies of 16% could be obtained with strain ATCC 10830. This frequency was four- to eightfold higher than the frequencies of the other strains examined.     

A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta

Regeneration, the ability to replace lost tissues and body parts following traumatic injury, occurs widely throughout the animal tree of life. Regeneration occurs either by remodeling of pre-existing tissues, through addition of new cells by cell division, or a combination of both. We describe a staging system for posterior regeneration in the annelid, Capitella teleta, and use the C. teleta Hox gene code as markers of regional identity for regenerating tissue along the anterior-posterior axis. Following amputation of different posterior regions of the animal, a blastema forms and by two days, proliferating cells are detected by EdU incorporation, demonstrating that epimorphosis occurs during posterior regeneration of C. teleta. Neurites rapidly extend into the blastema, and gradually become organized into discrete nerves before new ganglia appear approximately seven days after amputation. In situ hybridization shows that seven of the ten Hox genes examined are expressed in the blastema, suggesting roles in patterning the newly forming tissue, although neither spatial nor temporal co-linearity was detected. We hypothesized that following amputation, Hox gene expression in pre-existing segments would be re-organized to scale, and the remaining fragment would express the complete suite of Hox genes. Surprisingly, most Hox genes display stable expression patterns in the ganglia of pre-existing tissue following amputation at multiple axial positions, indicating general stability of segmental identity. However, the three Hox genes, CapI-lox4, CapI-lox2 and CapI-Post2, each shift its anterior expression boundary by one segment, and each shift includes a subset of cells in the ganglia. This expression shift depends upon the axial position of the amputation. In C. teleta, thoracic segments exhibit stable positional identity with limited morphallaxis, in contrast with the extensive body remodeling that occurs during regeneration of some other annelids, planarians and acoel flatworms.     

Anteriore und caudale Regeneration bei dem proterandrischen PolychaetenOphryotrocha puerilis Clap. Mecz

Zusammenfassung Vollständige anteriore Regeneration findet nur statt, wenn ein Rest des Prostomiums in situ verbleibt.Die caudale Regeneration verläuft bei und mit und ohne Prostomium völlig gleich. Die Zahl der regenerierten Segmente ist um so größer, je mehr Segmente amputiert wurden.Die Auslösung der caudalen Regeneration durch Amputation hinterer Segmente oder die Regeneration selbst können das Geschlecht der regenerierenden Tiere kurzzeitig beeinflussen.

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Anterior and caudal regeneration in the proterandric polychaeteOphryotrocha puerilis Clap. Mecz

Summary Only when a part of the prostomium remains in situ does complete anterior regeneration take place.The course of caudal regeneration is the same both in males and females whether they have their prostomia in situ or not. The more segments are amputated the more will be regenerated.The amputation of posterior segments which releases the regeneration, or regeneration itself, may influence the sex of regenerating worms for a short time.

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Meinem Lehrer, Herrn Prof. Dr. C. Hauenschild, danke ich sehr für die Förderung dieser Arbeit.     

Oscarella lobularis (Homoscleromorpha,Porifera) Regeneration: Epithelial Morphogenesis and Metaplasia

Sponges are known to possess remarkable reconstitutive and regenerative abilities ranging from common wounding or body part regeneration to more impressive re-building of a functional body from dissociated cells. Among the four sponge classes, Homoscleromorpha is notably the only sponge group to possess morphologically distinct basement membrane and specialized cell-junctions, and is therefore considered to possess true epithelia. The consequence of this peculiar organization is the predominance of epithelial morphogenesis during ontogenesis of these sponges. In this work we reveal the underlying cellular mechanisms used during morphogenesis accompanying ectosome regeneration in the homoscleromorph sponge model: Oscarella lobularis. We identified three main sources of novel exopinacoderm during the processes of its regeneration and the restoration of functional peripheral parts of the aquiferous system in O. lobularis: (1) intact exopinacoderm surrounding the wound surface, (2) the endopinacoderm from peripheral exhalant and inhalant canals, and (3) the intact choanoderm found on the wound surface. The basic morphogenetic processes during regeneration are the spreading and fusion of epithelial sheets that merge into one continuous epithelium. Transdifferentiation of choanocytes into exopinacocytes is also present. Epithelial-mesenchymal transition is absent during regeneration. Moreover, we cannot reveal any other morphologically distinct pluripotent cells. In Oscarella, neither blastema formation nor local dedifferentiation and proliferation have been detected, which is probably due to the high morphogenetic plasticity of the tissue. Regeneration in O. lobularis goes through cell transdifferentiation and through the processes, when lost body parts are replaced by the remodeling of the remaining tissue. Morphogenesis during ectosome regeneration in O. lobularis is correlated with its true epithelial organization. Knowledge of the morphological basis of morphogenesis during Oscarella regeneration could have important implications for our understanding of the diversity and evolution of regeneration mechanisms in metazoans, and is a strong basis for future investigations with molecular-biological approaches.     

Effect of phyllotactic position and cultural treatments toward successful direct shoot organogenesis in dwarf ‘Pixie’ grapevine (Vitis vinifera L.)

In Vitis spp. where somatic embryogenesis-based regeneration predominates, an efficient, reproducible and robust method of direct shoot organogenesis from leaf explant material has been established in the dwarf wine grape ‘Pixie’ (Vitis vinifera). This regeneration system was achieved by testing the response of leaf material in two stages of development, and pre-conditioning the explant material in dark conditions and/or in liquid media prior to excising from the plant and placing it on solidified media. The pre-excision treatments included (1) a dark period of 24 h, with no regeneration medium; (2) soaking in regeneration medium followed by a dark period of 24 h; (3) a dark period of 24 h followed by soaking in liquid VRM (Vitis Regeneration Medium); (4) vacuum infiltration in liquid VRM followed by a dark period of 24 h; and (5) a control of no pre-conditioning treatment. Excised leaves from pre-treated intact plants in vitro significantly increased the frequency of shoot organogenesis. The most responsive explant material consisted of young semi-translucent apical leaves varying in size from 3 to 8 mm in length. The most successful combinations of factors contributing to shoot organogenesis involved the solely dark-exposed apical leaves or the soaking in VRM followed by a dark period. These results are expected to facilitate Vitis-related research in genetics, functional genomics, physiology, and other fields.     

Modelling regeneration effects on protein A affinity chromatography

The effect of in-place regeneration of protein A adsorbents on protein adsorption characteristics is investigated. Regeneration with sodium hydroxide and time of exposure determined the protein capacity of the adsorbent, but no effect was observed on the adsorbent protein affinity. Fixed-bed adsorption of human immunoglobulin G was studied. Breakthrough curves were measured for protein adsorption on fixed-bed columns. These data were analyzed by a simple kinetic model to determine the rate constants for the adsorption process. It was found that forward adsorption rate constant remained constant along the chemical treatment exposure time. Protein A adsorbent selectivity was determined using mouse serum immunoglobulins G ₁ and G ₃ . Column linear gradient elution showed that adsorbent selectivity decreased with the exposure time chemical treatment. The implications of these results on the design and optimization of protein A chromatographic process are discussed.     

Ploidy level stability of adventitious shoots of sour cherry ‘?a?anski Rubin’ and Gisela 5 cherry rootstock

The capacity of regeneration of adventitious shoots from leaf explants was studied in sour cherry ???a?anski Rubin?? (Prunus cerasus L.) and cherry rootstock Gisela 5 (P. cerasus?×?P. canescens). Regeneration assay included thirty different combinations of plant growth regulators. 6-benzyladenine (BA) and thidiazuron (TDZ) were applied either individually or each combined with different concentrations of indole-3-butyric acid, ??-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). ???a?anski Rubin?? showed higher regeneration capacity in comparison with Gisela 5 regarding the total number of treatments inducing regeneration as well as the highest frequency of regeneration achieved. In both genotypes, 8.9???M BA was more effective than both 4.5 and 9.0???M TDZ in inducing adventitious regeneration, but only when combined with auxins. The highest frequency of regeneration (20.8?%) in ???a?anski Rubin?? was achieved on medium supplemented with 8.9???M BA combined with 5.4???M NAA, while in Gisela 5 the highest value (8.3?%) was obtained when BA was combined with 4.5???M 2,4-D. Flow cytometry combined with 4??-6-diamidino-2-phenylindole staining was employed to estimate DNA ploidy levels and relative nuclear DNA content in adventitious regeneration-derived shoots, in vitro shoots of axillary origin and in vivo control plants from open field. No significant differences in nuclear DNA content were detected among plants of different origin. Chromosome counting in root tip meristems also showed normal tetraploid chromosome number (2n?=?4x?=?32) in ???a?anski Rubin?? shoots and normal triploid chromosome number (2n?=?3x?=?24) in Gisela 5 shoots regenerated in vitro. The results obtained suggest that no major genetic instability occurred during adventitious regeneration under the described experimental conditions.