Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+-depleted conditions and its recovery upon addition of insulin and LDL

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [³H]thymidine. The progression was arrested in a chemically defined medium in which K⁺ was replaced by Rb⁺ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K^?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.     

Examination of the method for measuring soil respiration in cultivated land: Effect of carbon dioxide concentration on soil respiration

An acceleration of soil respiration with decreasing CO₂ concentration was suggested in the field measurements. The result supporrs that obtained in laboratory experiments in our previous study. The CO₂ concentrations in a chamber of the alkali absorption method (the AA-method) were about 150–250 parts/10⁶ lower than that in the atmosphere (about 350 parts/10⁶), while those observed in the open-flow IRGA method (the OF-method) were nearly equal to the soil surface CO₂ levels. The AA-method at such low CO₂ levels in the chamber appears to overestimate the soil respiration. Our results showed that the rates obtained by the AA-method were about twice as large as those by the OF-method in field and laboratory measurements. This finding has important consequences with respect to the validity of the existing data obtained by the AA-method and the estimation of changes in the terrestrial carbon flow with elevated CO₂     

Field measurements of nitrogen-fixing activity of intact saplings ofAlnus maximowiczii in the subalpine zone of Mt Fuji

Nitrogen-fixing (acetylene-reducing) activity of intact saplings ofAlnus maximowiczii was measured under natural conditions in the subalpine zone of Mt Fuji. The nitrogen-fixing activity was detected from the middle of June when expansion of leaves had just begun to the end of October when the shedding of leaves was almost completed. Diurnal changes in the activity were almost parallel with those of ground temperature. The measured nitrogen-fixing activity was related to ground temperature and total leaf area. Using this relation, annual nitrogen fixation was estimated from the data of ground temperature and leaf area measured in the field. The amount of annual nitrogen fixation was almost the same as that of nitrogen used for annual growth. It was concluded that nitrogen fixation by nodules made a considerable contribution to the nitrogen economy in the saplings ofA. maximowiczii.     

Chemical composition of cell walls as a taxonomical marker

Matrix sugar composition ofChlorella is species-specifically different. The rigid wall consists of either glucosamine or glucose and mannose. Ruthenium red stainability and anisotropy of cell wall are either plus or minus species-specifically. The cell wall is specifically degraded by the lytic enzyme of the cell itself.     

Efficient and gentle elution of antibodies from an immobilized polypeptide antigen BT saturated magnesium chloride

Summary A model antigen, rabbit immunoglobulin G, was immobilized onto polyester cloth by adsorption. The antigen cloth was reacted with sheep anti-rabbit IgG antibody. Antibody bound to the antigen cloth was nearly quantitatively eluted by saturated MgCl₂, whereas a commercial antibody eluent slowly eluted only about 70 % of the antibody. Exposure of antibody to saturated MgCl₂ for 30 min resulted in no loss of immunoactivity. Saturated MgCl₂, therefore, is an ideal eluent in immunoaffinity purification of antibodies.     

Glycoblotting enables seamless and straightforward workflow for MALDI-TOF/MS-based sulphoglycomics of N- and O-glycans

Sulfated N- and O-glycans exist in trace levels which are challenging to detect, especially when abundant neutral and sialylated glycans are present. Current matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based sulfoglycomics approaches effectively utilize permethylation to discriminate sulfated glycans from sialyl-glycans. And a charge-based separation to isolate the sulfated glycans from the rest of the permethylated neutral and sialyl-glycans. However, these approaches suffer from concomitant sample losses during cleanup steps. Herein, we describe Glycoblotting as a straightforward complementary method with seamless glycan purification, enrichment, methylation, and labeling on a single platform to address sulfated glycan enrichment, sialic acid methylation, and sample loss. Glycoblottings’ on-bead chemoselective ligation of reducing sugars with hydrazide showed excellent recovery of sulfated glycans, allowing the detection of more sulfated glycan species. On-bead methyl esterification of sialic acid using 3-methyl-1-p-tolyltriazene (MTT) effectively discriminates sulfated glycans from sialyl-glycans. Furthermore, we have shown that using MTT as a methylating agent allowed us to simultaneously detect and differentiate sulfate from phosphate groups in isobaric N-glycan species. We believe that Glycoblotting will contribute significantly to the MALDI-TOF MS-based Sulphoglycomics workflow.     

Light control of porphyrin accumulation in acifluorfen-methyl-treated Lemna pausicostata

In Lemna pausicostata Hegelm. 6746, light is required for sufficient acifluorfenmethyl (AFM) stimulation of protoporphyrin IX (Proto IX) accumulation to cause significant herbicidal action. In darkness, AFM causes Proto IX levels to increase for about 2 h, after which Proto IX content is stable at levels significantly lower than those accumulated in light. In darkness, sucrose cannot increase levels of AFM-induced Proto IX. However, addition of δ-aminolevulinic acid (ALA) increases Proto IX levels in AFM-treated plants in darkness, demonstrating that the herbicide blocks the porphyrin pathway in darkness as it does in the light. Thus, Proto IX accumulation in darkness appears to be limited by ALA availability. This is supported by the finding that dioxoheptanoic acid caused more ALA to accumulate in light than in darkness. Heme is a feedback inhibitor of ALA synthesis, and heme synthesis is inhibited by AFM. However, total extractable heme levels were reduced by AFM by about the same amount in both light and darkness. Exogenously supplied hemin reduced AFM-caused Proto IX accumulation and herbicidal damage in the light and also reduced Proto IX accumulation caused by AFM or AFM plus ALA in darkness. AFM-stimulated Proto IX accumulation was inversely proportional to the log of the photon flux density between 5 and 500 μmol in m⁻² s⁻¹. Reduced effects of higher photon fluxes on AFM-stimulated Proto IX accumulation are probably due to both increased photobleaching of Proto IX and reduced porphyrin synthesis because of herbicidal damage. AFM-stimulated Proto IX accumulation in darkness could not be demonstrated to be under phytochrome control, but it appeared to be under the negative influence of protochlorophyllide levels.     

LIFE HISTORY AND SYSTEMATIC POSITION OF PSEUDOCHORDA GRACILIS SP. NOV. (LAMINARIALES,PHAEOPHYCEAE)1

Pseudochorda gracilis sp. Nov. (Pseudochordacease, Laminariales) is described from the Japan Sea coast of Hokkaido/ the species is subtidal, epilithic and annual, appearing in spring and maturing in winter. Erect thalli grow solitary or in tufts on a small discoid holdfast. They are simple, cord-shaped and hollow, with inner hyphal filaments, cylindrical medullary cells and paraphyses consisting of 3–6 cells. Hair tufts are observed only in young thalli. Unilocular sporangia are sessile and narrowly ovate. In culture, P. gracilis shows a heteromorphic life history with oogamy, characteristic of the order Laminariales. Gametophytes are dioecious and dimorphic. Gametophytes mature under lower temperature conditions (usually below 10°C), and sporophytes mature under low temperature and short-day conditions (5°C, SD). The seasonal growth pattern of the species results from the photoperiod-temperature conditions controlling saprophyte maturation.     

PCR method for genotyping and zygosity-testing of RasH2 transgenic mice.

In short-term carcinogenicity testing using CB6F1-TgrasH2 mice, sibling nonTgrasH2 mice are used as a negative control. However, selection of TgrasH2 and nonTgrasH2 mice has been performed by PCR with only transgene specific primers by the conventional method. Therefore, the conventional method involves the risk of false negative results due to reaction failure, and contamination with TgrasH2 mice in the control mice group. Based on the nucleotide sequence information around the pre-integration site, we developed a genotyping method for distinguishing not only TgrasH2 mice (hemizygous for the Tg allele) but also nonTgrasH2 (homozygous for the nonTg allele) in a positive manner.