Changes in Osmotic Pressure and Swelling in Horseshoe Crab Embryos During Development

Water influx accompanying the swelling of embryos during normal development of horseshoe crabs, Limulus polyphemus and Tachypleus tridentatus , following a rupture of the chorion, was analyzed. The increase in volume of perivitelline fluid during deveopment was about 90 percent of the increase in total embryo weight. Considerable water discharge was observed on drying the embryos in air and a reversible water influx occurred with a second immersion in sea water, even though the embryos died as a result of this treatment. Since the osmotic pressure of the perivitelline fluids decreased markedly during development until the end of swelling, a close correlation between swelling and osmotic pressure was recognized. These results indicate that certain osmoactive substances may be produced in the perivitelline fluid at the initial stage of swelling.     

PHYSIOLOGICAL ACTIVITIES OF HELMINTHOSPOROL AND HELMINTHOSPORIC ACID: II. EFFECTS ON EXCISED PLANT PARTS

Effects of H-ol and H-acid were observed using excised partsof several plant species. Both H-ol and H-acid were active inelongation of oat coleoptile and mesocotyl, expansion of Raphanusleaf disk, but were inactive in elongation of wheat coleoptileand of green stem of pea. They gave also negative results inthe standard Avena curvature test and in the split pea test.In expansion of lettuce cotyledon, H-ol was inactive while H-acidwas active. In excised plant parts, as in intact plants, theactivity of H-ol and H-acid resembles rather gibberellins thanauxins and cytokinins. (Received August 20, 1966; )     

Keratinocyte Extracellular Matrix-Mediated Regulation of Normal Human Melanocyte Functions

Active roles of cell-cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell-cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation-associated protein (MEL-5) and proliferation-associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell-matrix interactions with those of direct melanocyte-keratinocyte, cell-cell contact in co-culture on melanocyte functions. Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs. Expression of pigmentation-associated protein detected by monoclonal antibody, MEL-5, was not changed on the kECM, while it was increased in melanocytes in co-culture with keratinocytes. Expression of PCNA/cyclin in melanocytes cultured on kECMs was generally downregulated on kECM and in co-culture compared to that in a control culture. We demonstrated that the kECMs play important roles in the melanocyte morphology and proliferation. These observations suggest that environmental (UVB) and physiological (Ca⁺⁺) stimuli can regulate melanocyte functions through the keratinocyte extracellular matrix in vivo.     

The diversity of hemi-epiphytic figs (Ficus ; Moraceae) in a Bornean lowland rain forest

The diversity and niche specificity of hemi-epiphytic figs in a lowland dipterocarp forest in Sarawak were investigated in 1998. Twenty-seven fig species (264 individuals, c. 120 ha) colonized a diversity of host taxa (35 families), but densities were very low and only 1.77% of trees> 30 cm d.b.h. were occupied. There were no significant associations with host taxa or host-bark roughness but among 11 common species (≥9 individuals) the distributions of all other parameters (host-d.b.h., height and position of colonization, crown illumination, soil-texture and slope-angle) were significantly different, and we identified five fig guilds. The guilds corresponded to canopy strata, and appeared to reflect the establishment microsite requirements of different species. A fundamental trade-off within the hemi-epiphytic habit was revealed: Species colonizing larger hosts were rarer, because of lower host densities and more specific microsite requirements, but had better light environments and attained a larger maximum size. The single strangler species appeared to escape many of these constraints, and an important source of mortality caused by host-toppling, indicating the advantages of this strategy. Thus, the hemi-epiphytic figs in this community have come to fill a remarkable diversity of niches, despite low levels of competition, through the exigencies of a complex environment.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 439–455     

INFLUENCE OF INORGANIC NITROGENOUS COMPOUNDS ON TOBACCO SEED GERMINATION INDUCED BY GIBBERELLIN A3, KINETIN AND AMMONIUM SALTS OF ORGANIC ACIDS

1. Investigations were made on the influence of inorganic nitrogenouscompounds upon the the germination of tobacco seeds (Nicotianatabacum L. var. virginica (AGDH.) COM. "Bright Yellow") inducedby GA₃, kinetin and ammonium salts of organic acids. Potassiumnitrate, ammonium chloride, ammonium sulfate and ammonium nitrategreatly increase the germination of the seeds induced by theabove reagents, while these inorganic salts, given alone, arealmost ineffective in causing germination.
2. Kinetin was shownto induce germination of tobacco seeds inthe dark. The discrepancywith the results of previous investigationsin this respectwas discussed.
3. It was inferred that nitrogenous metabolismis involved in theprocess of dark-germination of tobacco seedsas induced by theabove-stated stimulating factors and promotedby inorganic nitrogenoussubstances.

(Received July 17, 1961; )     

Pigmented Human Skin Equivalent: New Method of Reconstitution by Grafting an Epithelial Sheet Onto a Non-Contractile Dermal Equivalent

We have established a new protocol for reconstituting a pigmented human skin equivalent (PSE) and have evaluated its functional responses to environmental stimulus, UVB. The PSE is reconstituted by grafting an epithelial sheet consisting of keratinocytes and melanocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active fibroblasts. i) The PSE has a multilayered, well-differentiated epidermis with cuboidal basal cells and highly organised dermis with newly synthesised extracellular matrix components. ii) Ki67-positive proliferating keratinocytes (18.1 ± 7.4%) were detected on the basal layer of the epidermis. iii) Melanocytes located exclusively within the basal layer were detected by monoclonal antibody against tyrosinase-related protein (TRP-1). iv) After exposure to UVB (100 mJ/cm² per day) for 7 consecutive days, the intensity of TRP-1 staining was increased in the PSE, showing their functional state, whereas the number of melanocytes was not changed. This non-contractile and functioning new PSE is potentially useful as a model for studying the role of melanocyte-keratinocyte-fibroblast interactions in photoprotection of the skin in more complex cutaneous microenvironment than monolayer culture, and for developing in vitro disease models and therapeutic protocols with genetically altered cells both in epidermis and dermis.     

Marked Amplification and Diversification of Products of ras Genes from Rat Brain,Rab GTPases,in the Ciliates Tetrahymena thermophila and Paramecium tetraurelia

ABSTRACT. Small GTPase Rab (products of ras genes from rat brain) is a widely conserved molecular switch among eukaryotes and regulates membrane trafficking pathways. It is generally considered that the number of Rab encoded in the genome correlates with multicellularity; however, we found that unicellular ciliates Tetrahymena thermophila (Tt) and Paramecium tetraurelia (Pt) possess many more Rab genes in their genome than the 64 HsRab genes in the human genome. We succeeded in isolating 86 cDNA clones of 88 TtRab genes in the Tetrahymena genome. By comparing the amino acid sequence of Rab in humans and the budding yeast Saccharomyces cerevisiae, 42 TtRab belonged to subfamilies functionally characterized and designated as conventional Rab, while the remaining 44 TtRab were considered to be species‐specific. To examine the diversity of Rab in ciliates, we searched for Rab genes in the genome database of P. tetraurelia. Overall, 229 PtRab genes were found and categorized as 157 conventional and 72 species‐specific PtRab, respectively. Among them, nine PtRab genes showed high homology to seven TtRab, suggesting the conservation of ciliate‐specific Rab. These data suggested that the range of Rab is markedly amplified and diversified in ciliates, which may support the elaborate cellular structures and vigorous phagocytosis of those organisms.     

Modulation of Normal Human Melanocyte Dendricity by Growth-Promoting Agents

Dendrite formation and extension, which comprise a characteristic morphology of human normal melanocytes in the skin, represent one of the functional activities of melanocytes, the ability to transfer melanosomes into neighboring keratinocytes. However, the morphology of the melanocyte in vitro is usually quite different from that observed in vivo. it is probably due to the hyperproliferative condition of the melanocytes in culture. No studies have ever compared the effects of a single factor on both dendricity and proliferation at the same time. Therefore, we have compared the effects of six growth-promoting agents commonly used for melanocyte cultures on dendrite formation and proliferation. The addition of agents that increase the intracellular levels of cyclic adenosine monophosphate (cAMP)—dibutyryl cyclic adenosine monophosphate (db cAMP; 1 mM) or isobutylmethyl xanthine (IBMX; 0.1 mM)—had a strong effect on dendrite formation and a negative effect on proliferation. This was especially true with db cAMP. In the presence of 2% or 5% of heat-inactivated fetal bovine serum (FBS), dendrite formation was significantly increased as was proliferation. The number of dendrites was decreased in the culture with 12-o-tetradecanoylphorbol-13-acetate (TPA), but cell growth was slightly increased. With human recombinant basic fibroblast growth factor (bFGF) (0.5, 1.0 ng/ml) in the presence of bovine pituitary extract (BPE) (60 μg/ml), cell growth was increased. With 2 ng/ml of bFGF, however, a strong inhibitory effect on proliferation was observed. However, dendrite formation was constant at all concentrations of bFGF tested (0.5, 1.0 or 2.0 ng/ml) with BPE (30 or 60 μg/ml). In this study, we have demonstrated that dendrite formation was suppressed by the reagents that stimulate melanocyte proliferation, and vice versa, with the only exception being heat-inactivated FBS. Both dendrite formation and proliferation were induced by the heat-inactivated FBS. This approach is crucial to the development of an adequate culture system for proliferation and/or dendrite formation of normal human melanocytes. It is necessary to keep these aspects in mind as we further investigate the biology of melanocytes, especially the cell-to-cell interactions between melanocytes and keratinocytes, involved in melanogenesis and melanin pigmentation in vivo. This study also provides practical and important information for a future reconstitutive skin system composed of melanocytes, keratinocytes, and fibroblasts in a single culture medium.