ISOLATION AND REGENERATION OF HAPLOID PROTOPLASTS FROM BANGIA ATROPURPUREA (RHODOPHYTA) WITH MARINE BACTERIAL ENZYMES1

Three kinds of enzymes, agarase, β-1,4-mannanase, and β-1,3-xylanase, required for isolation of protoplasts from the red alga Bangia atropurpurea (Roth) C. Ag. were prepared from bacterial culture fluids of Vibrio sp. PO-303, Vibrio sp. MA-138, and Alcaligenes sp. XY-234, respectively, isolated from the sea environment. The optimal pH of all enzymes was around 7.5. Suitable conditions for protoplast isolation from B. atropurpurea were examined. The pretreatment of the fronds with pa-pain solution (20 mM Mes buffer, pH 7.5, containing 2% papain and 0.5 M mannitol) contributed to successful protoplast isolation. When razor-cut fragments of the fronds (about 200 mg in fresh weight) immersed in 20 mM Mes buffer, 7.5, containing 0.5 M mannitol and one unit each of agarase, β-1,4-mannanase, and β-1,3-xylanase were incubated at 22°C for 90 min with gentle agitation, 5.7 × 10⁶ protoplasts were released from them. Many protoplasts regenerated into fronds of regular or irregular shape.     

Sexual process in eight-spored ascus-forming yeast, Schizosaccharomyces japonicus II. Dependency of the sexual process on the carbon source

The carbon-source dependency of the sexual process in Schizosaccharomycesjaponicus was studied. Schiz. japonicus grew well in vegetativemedia containing glucose, sucrose, fructose or raffinose, anddid poorly in one containing mannose. On the other hand, itssexual process proceeded well in sporulation media containingglucose, sucrose or mannose, and was markedly delayed in thosecontaining fructose or raffinose. Neither vegetative growthnor sexual process occurred when non-fermentable carbon sources,such as glycerol, were used. The amount of glucose in the sporulationmedium sufficient for completion of the sexual process varieddepending on the cell-population density. Glucose was requiredfor both zygote and ascus formation but not for spore liberation.Cells were committed to sporulation shortly after the stageof zygote formation. (Received August 3, 1978; )     

Characterization of the edible red alga Meristotheca papulosa (Solieriaceae, Gigartinales) from Japan

The vegetative and reproductive morphology of the edible red alga Meristotheca papulosa (Montagne) J. Agardh (Solieriaceae) was reexamined based on material collected from various localities in Japan. Although the habit of the blades is variable according to the length and width of the axes, the frequency of branching and the abundance of proliferations, rbcL sequence analyses indicate their conspecificity. M. papulosa displays four distinctive reproductive features (presence of an auxiliary cell complex, occurrence of cystocarps on marginal proliferations and the blade surface (although very rare) in addition to the margins of axes, frequent production of spinose outgrowths on the pericarp and tetrasporangial initials typically basally attached to their parental cells) that have not been reported for M. papulosa from other areas. Although these features might warrant recognition of the Japanese entity as a separate species, a better understanding of their possible taxonomic value requires comparisons with M. papulosa from other geographic regions, including the type locality.     

TPA-induced multinucleation of a mesenchymal stem cell-like clone is mediated primarily by karyokinesis without cytokinesis, although cell-cell fusion also occurs

The 5F9A cell, which is a mesenchymal stem cell-like clone established from rat bone marrow substrate adherent cells, can differentiate into adipocytes and osteoblasts in vitro under the appropriate conditions. Multinucleated cells could be also induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in 5F9A cells. This effect was mediated by protein kinase C. Possible mechanisms of multinucleation by TPA were hypothesized to be either karyokinesis without cytokinesis or cell-cell fusion. By observation using time-lapse phase-contrast microscopy, we determined that the multinucleated cells were generated mainly by karyokinesis without cytokinesis. Cell fusion was studied using time-lapse photography, and confocal laser scanning microscopy using two differentially labeled cells. These techniques demonstrated that multinucleated 5F9A cells could be produced by cell fusion, albeit at a low frequency. We conclude that multinucleated 5F9A cells are formed primarily by karyokinesis without cytokinesis, although some cells are also formed by cell-cell fusion.     

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.     

Possible self-fertilization in the brooding coral Acropora (Isopora) brueggemanni

We examined gametogenesis and the periodicity of planula release in the brooding coral Acropora (Isopora) brueggemanni (Brook, 1893) on Akajima Island, Okinawa, Japan. We captured the moment when A. brueggemanni would be in the process of self-fertilization. Whole colonies of this species were cultured separately or together with other colonies in plastic containers. We observed no apparent periodicity of planula release in the collected colonies. A few planulae were released intermittently during the observation period, regardless of whether the colonies were cultured individually or with other colonies. Serial paraffin sections of A. brueggemanni showed follicle-like cells surrounding the oocyte during vitellogenesis. In September and October, some spermaries looked half-broken and some ova were surrounded by sperm or spermaries instead of follicle-like cells. Such ova were heart-shaped and may have been cells at the first cleavage stage. These observations suggest that the migration of spermaries and/or oocytes resulted in the close proximity of oocytes and sperm, which would allow self-fertilization. This possibility, together with the production of planula larvae by isolated colonies, suggests that this brooding coral engages in self-fertilization.     

Cytological analysis of the mother cell death process during sporulation in Bacillus subtilis

We have identified the following events during the late stage in the mother cell in Bacillus subtilis: spore detachment from the polar site of the mother cell, membrane rupture, cell wall collapse, and release of the free spore. The membrane rupture was followed by mother cell lysis. Moreover, we found that NucB, an extracellular nuclease, is involved in DNA degradation after mother cell lysis.     

Anti-predator defence and the complexity-stability relationship of food webs

The mechanism for maintaining complex food webs has been a central issue in ecology because theory often predicts that complexity (higher the species richness, more the interactions) destabilizes food webs. Although it has been proposed that prey anti-predator defence may affect the stability of prey-predator dynamics, such studies assumed a limited and relatively simpler variation in the food-web structure. Here, using mathematical models, I report that food-web flexibility arising from prey anti-predator defence enhances community-level stability (community persistence and robustness) in more complex systems and even changes the complexity-stability relationship. The model analysis shows that adaptive predator-specific defence enhances community-level stability under a wide range of food-web complexity levels and topologies, while generalized defence does not. Furthermore, while increasing food-web complexity has minor or negative effects on community-level stability in the absence of defence adaptation, or in the presence of generalized defence, in the presence of predator-specific defence, the connectance-stability relationship may become unimodal. Increasing species richness, in contrast, always lowers community-level stability. The emergence of a positive connectance-stability relationship however necessitates food-web compartmentalization, high defence efficiency and low defence cost, suggesting that it only occurs under a restricted condition.