Synthesis of a callosic substance during rhizoid differentiation in Spirogyra

Spirogyra living in running water is anchored to the substratum by rhizoids that form at the ends of the filaments. A new terminal cell differentiates into a rhizoid cell if the filament is injured. The mode of growth changes from diffuse to tip growth when rhizoid differentiation begins. In this study, we found that a callosic substance was synthesized during rhizoid differentiation. Decreasing the cell turgor, lowering extracellular Ca2+ or adding Gd3+, all inhibited the commencement of rhizoid differentiation as well as synthesis of the callose-like substance at the tip of the terminal cell. A callosic substance was also synthesized during formation of the conjugation tube.     

Involvement of microtubules in rhizoid differentiation of Spirogyra species

Summary.  Some species of Spirogyra form rosette-shaped or rod-shaped rhizoids in the terminal cell of the filaments. In the present study, we analyzed an involvement of microtubules (MTs) in rhizoid differentiation. Before rhizoid differentiation, cortical MTs were arranged transversely to the long axis of cylindrical cells, reflecting the diffuse growth. At the beginning of rhizoid differentiation, MTs were absent from the extreme tip of the terminal cell. In the other area of the cell, however, MTs were arranged transversely to the long axis of the cell. In the fully differentiated rosette-shaped rhizoid, MTs were randomly organized. However, at a younger stage of rosette-shaped rhizoids, MTs were sometimes arranged almost transversely in the lobes of the rosette. In the rod-shaped rhizoid, MTs were arranged almost transversely. MT-destabilizing drugs (oryzalin and propyzamide) induced swelling of rhizoids, and neither rosette-shaped nor rod-shaped rhizoids were formed. The role of MTs in rhizoid differentiation was discussed. Received June 17, 2002; accepted November 11, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-1297, Japan.     

Inducible molecular switches for the study of long-term potentiation

This article reviews technical and conceptual advances in unravelling the molecular bases of long-term potentiation (LTP), learning and memory using genetic approaches. We focus on studies aimed at testing a model suggesting that protein kinases and protein phosphatases balance each other to control synaptic strength and plasticity. We describe how gene 'knock-out' technology was initially exploited to disrupt the Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) gene and how refined knock-in techniques later allowed an analysis of the role of distinct phosphorylation sites in CaMKII. Further to gene recombination, regulated gene expression using the tetracycline-controlled transactivator and reverse tetracycline-controlled transactivator systems, a powerful new means for modulating the activity of specific molecules, has been applied to CaMKIIalpha and the opposing protein phosphatase calcineurin. Together with electro-physiological and behavioural evaluation of the engineered mutant animals, these genetic methodologies have helped gain insight into the molecular mechanisms of plasticity and memory. Further technical developments are, however, awaited for an even higher level of finesse.     

Rhizoid differentiation in Spirogyra I. Basic features of rhizoid formation

Several types of rhizoids occurring in the process of differentiationin Spirogyra sp. are described and their interrelation was elucidated.There are two differentiation sequences: P_pPRh_ros or PpPRh_rodRh_ros(for explanation of abbreviations see p. 533), although undersome conditions the sequences ceased halfway through. The initiationtime for rhizoid formation had no relation to the cell cyclestage. The difference in growth patterns between the rhizoidand ordinary filament cells was demonstrated with Calcofluor-stainingand centrifugation. The optimum temperature and pH of the culture medium for rhizoiddifferentiation were 20?C and pH 7, respectively. A contactstimulus was not necessary for induction. Of the several environmental factors examined, light was themost important, for rhizoid formation, since a rhizoid was inducedonly when light was given after cutting the filament. (Received December 14, 1972; )     

Sex differentiation in mammals and tempo of growth: probabilities vs. switches

In the conventional model of sex differentiation in placental mammals, a switch is envisaged to steer the indifferent gonad into the path of either testicular or ovarian development. The immediate cause of the switch is thought to be the presence or absence of Sertoli cells, which in turn is controlled by the presence or absence of the testis-determining factor on the Y chromosome (TDF in humans, Tdy in mice). Quantitative investigations indicate, however, that the rate of growth of XY gonads is faster than that of XX gonads before the formation of Sertoli cells, and furthermore, that XY embryos develop faster than XX embryos long before the formation of gonadal ridges. Since the genetic constitution of the sex chromosomes appears to manifest itself from the earliest embryonic stages onwards, the concept of indifferent gonads being switched into alternate pathways becomes inappropriate. A model is proposed in which gonadal differentiation depends on developmental thresholds: the formation of Sertoli cells needs to occur by a particular stage in time in a sufficiently developed gonad, failing which the gonad will enter the ovarian pathway. While TDF is the principal factor enhancing the rate of gonadal growth, other factors which influence development rates can modulate the probability of a gonad becoming either a testis or an ovary.     

High-dimensional switches and the modelling of cellular differentiation

Many genes have been identified as driving cellular differentiation, but because of their complex interactions, the understanding of their collective behaviour requires mathematical modelling. Intriguingly, it has been observed in numerous developmental contexts, and particularly haematopoiesis, that genes regulating differentiation are initially co-expressed in progenitors despite their antagonism, before one is upregulated and others downregulated. We characterise conditions under which three classes of generic "master regulatory networks", modelled at the molecular level after experimentally observed interactions (including bHLH protein dimerisation), and including an arbitrary number of antagonistic components, can behave as a "multi-switch", directing differentiation in an all-or-none fashion to a specific cell-type chosen among more than two possible outcomes. bHLH dimerisation networks can readily display coexistence of many antagonistic factors when competition is low (a simple characterisation is derived). Decision-making can be forced by a transient increase in competition, which could correspond to some unexplained experimental observations related to Id proteins; the speed of response varies with the initial conditions the network is subjected to, which could explain some aspects of cell behaviour upon reprogramming. The coexistence of antagonistic factors at low levels, early in the differentiation process or in pluripotent stem cells, could be an intrinsic property of the interaction between those factors, not requiring a specific regulatory system.     

Hepatocyte growth factor switches orientation of polarity and mode of movement during morphogenesis of multicellular epithelial structures

Epithelial cells form monolayers of polarized cells with apical and basolateral surfaces. Madin-Darby canine kidney epithelial cells transiently lose their apico-basolateral polarity and become motile by treatment with hepatocyte growth factor (HGF), which causes the monolayer to remodel into tubules. HGF induces cells to produce basolateral extensions. Cells then migrate out of the monolayer to produce chains of cells, which go on to form tubules. Herein, we have analyzed the molecular mechanisms underlying the production of extensions and chains. We find that cells switch from an apico-basolateral polarization in the extension stage to a migratory cell polarization when in chains. Extension formation requires phosphatidyl-inositol 3-kinase activity, whereas Rho kinase controls their number and length. Microtubule dynamics and cell division are required for the formation of chains, but not for extension formation. Cells in the monolayer divide with their spindle axis parallel to the monolayer. HGF causes the spindle axis to undergo a variable "seesaw" motion, so that a daughter cells can apparently leave the monolayer to initiate a chain. Our results demonstrate the power of direct observation in investigating how individual cell behaviors, such as polarization, movement, and division are coordinated in the very complex process of producing multicellular structures.     

Rhizoid differentiation in Spirogyra II. Photoreversibility of rhizoid induction by red and far-red light

The optimum light conditions for rhizoid formation in Spirogyrawere determined. Red light was significantly effective on rhizoidformation while green, blue and violet lights had less effect.The dose-effect curve of red light was investigated and theminimum energy needed to saturate the effect was 8.1 Kergs.cm_–2.The effect of red light was completely reversed by subsequentirradiation with far-red light. The doseeffect curve of far-redlight was also obtained. The repeatedly reversible photoresponseswith red and far-red light strongly suggest that the photoreceptorof the rhizoid formation system is phytochrome. The existenceof phytochrome in the Spirogyra cell was also demonstrated spectrophotometrically.The half time for the escape reaction from the reversal effectof far-red light was 2hr. There may be no pigment other thanphytochrome mediating the photoreaction. (Received December 14, 1972; )     

The effective mode of growth and differentiation factor-5 in promoting the chondrogenic differentiation of adipose-derived stromal cells

Our study aimed to find out the most effective mode for chondrogenic differentiation based on time, dose and culture method. ADSCs were cultured and identified by CD44, CD49d, and CD106 immumohistochemical staining method, and their differentiation potential to chondrocyte were detected by Alizarin red staining. ADSCs induced by different concentrations of GDF-5 for chondrogenic differentiation were detected by blue and toluidine blue staining and collagen type II and X immumohistochemical staining. The expression of collagen I, II, X and aggrecan gene in GDF-induced ADSCs cultured in 2- and 3-dimension was identified by real-time PCR. Cell microstructure and proliferation in three-dimensional scaffolds at day 7, 14, 21 and 28 were analyzed by scanning electron microscopy and MTS assay. The ADSCs were successfully identified by CD44 and CD49d, and their differentiation potential was detected by Alizarin red staining. Real-time PCR showed that collagen and aggrecan were expressed at high levels in 100 or 200 ng/mL GDF-5 treated cells. The collagen types (I, II) and aggrecan genes were higher expressed in GDF-5 induced scaffold group than that in monolayer group. MTS showed that the cell counts were not significantly different among different treated time. Both collagen type II and aggrecan gene were highly expressed at day 14, while collagen types I and X gene expressions peaked at day 21 and 28. The 100 ng/mL GDF-5 is effective and cost-effective for chondrogenic differentiation when cultured at day 14 in vitro under three-dimensional culture conditions.     

Phytochrome-dependent modulation and re-induction of growth of the first rhizoid in Dryopteris paleacea Sw

Phytochrome-dependent growth in Dryopteris paleacea Sw. was investigated in young, developing gametophytes with respect to formation and differentiation of rhizoids. Under continuous red light (Rc), the first rhizoids grew synchronously by tip elongation at a constant rate of 240 μm · d⁻¹ until formation and outgrowth of the second rhizoid. Cessation of growth of the first rhizoids and outgrowth of the second rhizoids showed a correlation in time assumed to be mediated by intercellular signaling. The first rhizoids showed two modes of response to actinic irradiations: (i) modulation of rhizoid growth, and (ii) re-induction of growth in non-growing rhizoids. In the former, the promotory effect of actinic irradiations on rhizoids pre-cultured under Rc determined both the time for which rhizoids continued to grow after transfer into darkness and the final rhizoid length. In the latter, re-induced growth was studied using non-growing rhizoids which were obtained after irradiation with a far-red light (FR) pulse at the end of the pre-culture in Rc and transfer into darkness for 3 d to stop growth. Re-induction of growth occurred with a lag phase of 36 to 48 h after formation of the FR-absorbing form of phytochrome (Pfr) by a red light (R) pulse. From the incomplete R/FR reversibility it is evident that, here, coupling of Pfr to signal transduction is possible within minutes. Re-induction of growth possesses the advantage that the effect of actinic irradiations can be studied as an all-or-none response at the level of single gametophytes in future experiments. The present results clearly indicate that the developmental stage of the whole gametophyte, i.e. temporal and spatial patterns undergone during development, affects the regulation of rhizoid growth by the external factor light. Received: 8 June 1998 / Accepted: 22 December 1998     

Proof of the subapical differential growth of the flanks in the Chara rhizoid during graviresponse

The displacement of resin spheres attached to the tip of the gravitropically bending Chara rhizoid was measured by means of time-lapse photography. The relative growth of the flanks which at the beginning of stimulation were opposite to each other, was calculated during the earliest response time. The physically upper subapical flank section continues growing after stimulation at a decreasing rate. The growth of the opposite lower flank section is inhibited immediately after stimulation by the position of the statoliths. As soon as the statoliths are displaced in a basal direction, the growth of this section is transiently promoted. The gravitropic downward bending of the Chara rhizoid is by bowing, not by bulging.     

Entamoeba invadens: influence of 60 Hz magnetic fields on growth and differentiation

Exposure to extremely low-frequency (ELF) electromagnetic fields appears to result in a number of important biological changes. In the present study, we evaluated the effects of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5 and 2.0 mT on growth and differentiation of the protozoan Entamoeba invadens. We demonstrated an inhibitory growth effect when trophozoite cultures were exposed to 1.5 and 2.0 mT. Furthermore, we found that there was not a synergistic effect in cultures co-exposed to MF and Metronidazole, a cytotoxic drug against amoebic cells. In addition, MF exposure inhibited the encystation process of E. invadens.     

Poliovirus switches to an eIF2-independent mode of translation during infection

Inhibition of translation is an integral component of the innate antiviral response and is largely accomplished via interferon-activated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). To successfully infect a host, a virus must overcome this blockage by either controlling eIF2α phosphorylation or by utilizing a noncanonical mode of translation initiation. Here we show that enterovirus RNA is sensitive to translation inhibition resulting from eIF2α phosphorylation, but it becomes resistant as infection progresses. Further, we show that the cleavage of initiation factor eIF5B during enteroviral infection, along with the viral internal ribosome entry site, plays a role in mediating viral translation under conditions that are nonpermissive for host cell translation. Together, these results provide a mechanism by which enteroviruses evade the antiviral response and provide insight into a noncanonical mechanism of translation initiation.     

Multiple switches between vegetative and reproductive growth in annual plants

A model of growth and reproduction in annual plants was developed by Cohen (1971, J. Theor. Biol.33, 299–307) to determine the allocation strategy which maximizes seed yield. The model divides the plant into vegetative and reproductive parts and predicts that yield is maximized by a strategy consisting of a switch from purely vegetative to strictly reproductive growth. We generalize Cohen's model to include vegetative and reproductive loss terms. Both growth and loss rates are allowed to vary with time. Using optimal control theory we find that seed yield is maximized by a strategy consisting of multiple switches between vegetative and reproductive growth, for certain ranges of the model parameters. In natural systems a predictable vegetative loss burst may be necessary to promote multiple switches.     

Photoisomerization of retinoic acid and its influence on regulation of human keratinocyte growth and differentiation

Retinoic acid constantly undergoes structural inter-conversions among the geometrical isomers (all-trans-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid and 9-13-di-cis-retinoic acid) by photoisomerization under natural light. Geometric isomers of retinoic acid thus formed showed different effects on human epidermal keratinocyte growth and differentiation. The ability of the isomers to inhibit the synthesis of cornified envelope (terminal event in the keratinocyte differentiation program) changed rapidly when illuminated by white fluorescent light. The 11-cis-retinoic acid had a 3-fold stronger activity to inhibit the growth of keratinocytes than the other geometric isomers. On the other hand, all-trans-retinoic acid, 9-cis-retinoic acid and 9-13-di-cis-retinoic acid exhibited a 3-fold greater ability to inhibit synthesis of involucrin, transglutaminase and the cornified envelopes. The regulation of keratin expression by the geometric isomers of retinoic acids was extremely complex. Level of keratin-1 (K1) mRNA was increased by 11-cis-retinoic acid and 13-cis-retinoic acid, but suppressed by 9,13-di-cis-retinoic acids while all-trans-retinoic acid and 9-cis-retinoic acid had no effect. Level of keratin-10 (K10) mRNA was strongly inhibited by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid as compared to 13-cis-retinoic acid and 9,13-di-cis-retinoic acids. The mRNA level of keratin-14 (K14) was suppressed by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid but not influenced by 13-cis-retinoic acid and 9,13-di-cis-retinoic acid. Natural light induced structural inter-conversions among the geometric isomers of retinoic acids in tissues-especially the skin, might play a crucial role in the regulation of growth and differentiation of keratinocytes.     

Inducible DNA repair and differentiation in Bacillus subtilis: interactions between global regulons

The SOS response of Escherichia coli has become a paradigm for the study of inducible DNA repair and recombination processes in many different organisms. While these studies have demonstrated that the components of the SOS response appear to be highly conserved among bacterial species, as with most models, there are some significant variations. Perhaps the best example of this comes from an analysis of the SOS-like system of the developmental organism, Bacillus subtilis. Accordingly, the most striking difference is the complex developmental regulation of the SOS system as this organism differentiates into its competent state. In this review we have given an overview of the elements that comprise the SOS system of B. subtilis. Additionally, we have summarized our most recent findings regarding the regulation of this regulon. Using these results along with new findings from other laboratories we have provided provocative molecular models for the regulation of the B. subtilis SOS system in response to DNA damage and during competent cell formation.