Phytochrome-mediated phototropism inAdiantum cuneatum young leaves

Phototropism of youngAdiantum fern leaves is induced by red light as well as blue light. The red light response is mediated by phytochrome. This is the first evidence of phytochrome action in diploid fern tissue. The blue light response is mainly mediated not by phytochrome, but probably by a blue light-absorbing pigment as in the case of almost all plants and fungi. The red light-induced phototropism becomes detectable within 2 hr after the onset of unilateral light. The highest bending rate is about 10 degrees/hr, which occurs between 3–5 hr after the induction of the tropic response. The bending region is about 6–8 mm from the highest point of the coiled crozier where the growth rate becomes slow.     

Stable isotopic structure of aquatic ecosystems

Isotopic, biogeochemical and ecological structure can provide a new dimension for understanding material flows, and the simultaneous function and structure of an ecosystem. Distributions ofδ ¹³C andδ ¹⁵N for biogenic substances in the Nanakita river estuary involving Gamo lagoon in Japan were investigated to construct isotope biogeochemical and ecological structure for assessing fate and transfer of organic matter, and food web structure. The isotopic framework of the ecosystem was successfully described in aδ ¹⁵N–δ ¹³C map. In this estuary the variations of isotope ratios of biogenic substances were clearly explained by the mixing of land-derived organic matter, and marine-derived organic matter. A trophic-level effect of¹⁵N enrichment was clearly observed. Organisms were classified into three groups depending upon the contribution of land-derived organic matter in a food chain. Almost all biota except mollusca in the lagoon depend on organic matter of marine origin. The contributions of both land and marine organic matter were comparable for mollusca in the lagoon.     

Cell Sorting and Chondrogenic Aggregate Formation in Limb Bud Recombinants and in Culture

The cartilage pattern of the developing chick limb changes along the proximal-distal (PD) axis. It is assumed that these spatial changes are brought about by differences in the cellular properties of distal mesoderm, the progress zone (PZ). To examine whether these differences are actually maintained in the individual cells composing the PZ, we dissociated early (stage 20) and late (stage 25) PZ tissues into single cells, then mixed and recombined them with ectodermal jackets. The recombinants were grafted to limb bud stumps and allowed to develop into limb-like structures. Early PZ cells were distributed within whole cartilage elements along the PD axis of the limb-like structures, while cells from late PZ participated only in the formation of distal cartilage elements.
A difference in distribution pattern between the cells of early and late PZ in mixed culture was also observed. Cells of early PZ aggregated rapidly in patches and formed cartilage nodules, while the cells of late PZ distributed in regions surrounding these cell aggregates and gradually differentiated to cartilage cells. These results suggest that the cellular properties in the PZ concerning the rate of chondrogenic aggregate formation change during limb bud development, and that this change may relate to the cartilage pattern formation along the PD axis.     

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium docecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65°C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20–25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35–40% in the SDS denaturation.     

Specific bindings of [3H](+)PN200-110 and [125I]ω-conotoxin to crude membranes from differentiated NG108-15 cells

The characteristics of the specific bindings of [³H](+)PN200-110 (PN: L-type Ca channel antagonist) and [¹²⁵I]-conotoxin G VI A (-CgTX: neuronal L-or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma x glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt₂cAMP) were examined, because we have already observed that the magnitude and rate of KCL-stimulated⁴⁵Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt₂cAMP (unpublished results). The specific binding of [³H](+)PN to these crude membranes was saturable at various concentrations of 2.5–5.0 nM [³H](+)PN. Scatchard analysis showed that the specific binding of [³H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt₂cAMP, but that the apparent Kd value for the specific binding of [³H](+)PN was not influenced by treatment with Bt₂cAMP. The specific binding of [³H](+)PN to crude membranes from Bt₂cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN>nitrendipine>(–)PNBay K 8644diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [³H(+)PN. On the other hand, [¹²⁵I]-CgTX at concentrations of 0.075–0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions. These results suggest that the increase in magnitude or rate of KCl-stimulated⁴⁵Ca uptake during differentiation of NG108-15 cells is partially due to quantitative alteration of voltage-sensitive Ca channels in the cells, and that there are scarcely any specific binding sites for [¹²⁵I]-CgTX on Bt₂cAMP-treated or untreated NG108-15 cells.     

Characteristics of specific125I-ω-conotoxin GVIA binding in rat whole brain

Characteristics of specific¹²⁵I-omega-conotoxin (-CgTX) binding were systematically investigated in crude membranes from rat whole brain. Kd and Bmax Values for the binding were 49.7 pM and 181.5 fmol/mg of protein, respectively. The effects of various types of Ca channel antagonists on the binding were investigated. Dynorphin A (1–13), in particular, specifically inhibited¹²⁵I--CgTX binding, but not that of [³H](+)PN200-110. Spider venom fromPlectreurys tristes did not specifically inhibit specific binding of¹²⁵I--CgTX, because the venom also inhibited the binding of [³H](+)PN200-110 to a similar degree. The amount of specific binding of¹²⁵I--CgTX was less in the cerebellum than that in any other area of whole brain. The cross-linker disuccinimidyl suberate did not label with¹²⁵I--CgTX and its binding sites in rat whole brain, although it did in chick whole brain, which was used as a positive control. These findings suggested that dynorphine A (1–13) was a selective blocker of -CgTX-sensitive Ca channels in crude membranes from rat whole brain and that -CgTX-sensitive Ca channels were mainly present a rat brain except cerebellum.     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. II. Formation of the preprophase band and cell division in centrifuged and non-centrifuged cells

Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the PPB is discussed.     

The Circular Arrangement of Cortical Microtubules around the Subapex of Tip-Growing Fern Protonemata Is Sensitive to Cytochalasin B

Effects of cytoskeletal inhibitors on circular arrays of microtubules(MTs) and microfilaments (MFs) around the subapex of fern protonematawere examined. Colchicine and amiprophos-methyl disrupted arraysof MTs but not of MFs. By contrast, cytochalasin B disruptedboth MF and MT arrays, suggesting the dependence of MT arrayson MFs. (Received June 28, 1991; Accepted November 14, 1991)     

The localized melting of mini-F origin by the combined action of the mini-F initiator protein (RepE) and HU and DnaA of Escherichia coli

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50^° bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaC complex, much as has been documented for oriC- dependent replication.     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.