Mutual recognition between polymerized liposomes. IV. Polysaccharide-lectin system.

As a model of cell-cell recognition processes, the association processes of a polysaccharide (mannan)-carrying liposome with a lectin (Concanavalin A, Con A)-carrying polymerized liposome were followed by turbidimetry. The association process was strongly inhibited by the addition of a low molecular weight sugar, methyl-alpha-D-mannopyranoside, which shows that the association between the liposomes is due to the specific interaction between Con A and mannan. The association rate constant obtained was much smaller than the theoretical value for a diffusion-controlled binary association process. This implies that the association rate of liposomes is limited by the recognition between complementary ligands bound on the liposome surfaces. Another reason for the smaller association rate constant in the liposome-liposome system is the repulsive hydration effect. The effect of the surface density of the lectin immobilized on the liposome on the recognition was also examined.     

Ion Efflux during a Single Action Potential of Nitella axilliformis in Medium Lacking Ca2-

Ion efflux during excitation of Nitella axilliformis was measuredconductometrically. In medium lacking Ca²⁺ but with 0.1 mM MgCl₂,the duration of the action potential and the total efflux weremuch larger than those in APW, while the efflux rate, givenas the total efflux divided by the duration, was about halfof that in APW. (Received September 4, 1986; Accepted November 25, 1986)     

Ion Effluxes during Excitation of Characeae

Ion effluxes during excitation of Chara and Nitellopsis measuredby conductometry method were compared with results obtainedby two other analytical methods, Ag-AgCl method for Cl^–and ion chromatography method for K⁺. In both species, the averageefflux measured by the conductometry method was very close tothose of K⁺ and Cl^–. (Received May 12, 1986; Accepted June 25, 1986)     

Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa.

The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

Cytoplasmic Hydration Triggers a Transient Increase in Cytoplasmic Ca2+ Concentration in Nitella flexilis

Cells of Nitella flexilis were made inexcitable by treatmentwith 10 mM KCl for more than 24 h. A Ca²⁺-sensitive photoproteinaequorin was injected into the cytoplasm of such cells. Forvacuolar per fusion, the central part of an aequorin-loadedcell was immersed in silicone oil, and both cell ends bathedin the perfusion medium were cut off. A large light emissionfrom aequorin was observed when the vacuole was perfused witha hypotonic medium whose osmotic pressure was adjusted to halfof the osmotic pressure of the cell sap. This shows that hydrationof the cytoplasm triggers release of Ca²⁺ from internal stores,since influx of Ca²⁺ from silicone oil is excluded. Hydration of cells was induced in another way. Cells were firstdehydrated by transferring them from 10 mM KCl solution to thatwith 250 mM sorbitol added. This procedure did not affect thecytoplasmic streaming. When cells were rehydrated by transferringthem to 10 mM KCl solution, cytoplasmic streaming was eitherstopped or slowed down in a few seconds. A quick light emissionfrom aequorin was observed in the rehydration, evidence thatcytoplasmic streaming was inhibited by an increase in the cytoplasmicCa²⁺ concentration. Both streaming cessation and aequorin lightemission were observed even in KCl-treated cells which werefurther treated with 5 mM EGTA. Thus, the increase in Ca²⁺ isconcluded to be caused by the release of Ca²⁺ from internalstores. These results support our previous hypothesis [Tazawa et al.(1994) Plant Cell Physiol. 35:63] that, in Nitella flexilis,the increase in the concentration of Ca²⁺ in the cytoplasm whichoccurs on the endoosmotic side of the cell during transcellularosmosis is caused by hydration of the cytoplasm. (Received June 6, 1994; Accepted December 26, 1994)     

Demonstration of light-induced potential change in Chara cells lacking tonoplast

Chara cells without tonoplasts, prepared by replacing the cellsap with EGTA-containing media, showed essentially the samepattern of light-induced changes in membrane potential and membraneresistance as normal cells although the concentrations of ionsand ATP in the cytoplasm decreased considerably (1/3–1/10)after loss of the tonoplast. Removal of the tonoplast reducedthe rate of photosynthetic O₂ evolution to about 50% of thatof normal cells but did not affect the magnitude of light-inducedpotential change. Not a full but a certain level of electronflow seems necessary to activate the putative electrogenic H⁺-pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Japan. ² Present address: Niigata College of Pharmacy, Niigata 950-21,Japan. (Received September 4, 1978; )     

A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain.

Using cDNA of the human poliovirus receptor (PVR) as a probe, two types of cDNA clones of the monkey homologs were isolated from a cDNA library prepared from an African green monkey kidney cell line. Either type of cDNA clone rendered mouse L cells permissive for poliovirus infection. Homologies of the amino acid sequences deduced from these cDNA sequences with that of human PVR were 90.2 and 86.4%, respectively. These two monkey PVRs were found to be encoded in two different loci of the genome. Evolutionary analysis suggested that duplication of the PVR gene in the monkey genome had occurred after the species differentiation between humans and monkeys. The NH2-terminal immunoglobulin-like domain, domain 1, of the second monkey PVR, which lacks a putative N-glycosylation site, mediated poliovirus infection. In addition, a human PVR mutant without N-glycosylation sites in domain 1 also promoted viral infection. These results suggest that domain 1 of the monkey receptor also harbors the binding site for poliovirus and that sugar moieties possibly attached to this domain of human PVR are dispensable for the virus-receptor interaction.     

Kinetic study of a membrane enzyme reactor

A flat-membrane dialyzer was used as enzyme reactor by introducing enzyme solution into one of the membrane-separated chambers. The apparent Michaelis constant K_m(app) of urease was always larger (ten times at [urease] = 1 mg/ml) than that of free enzyme because the permeation of substrate through the membrane was rate determining. K_m(app) for urease decreased from 125 to 20mM with increasing flow rate of the substrate solution because of the turbulent flow near the membrane. In the case of glucose oxidase or creatine kinase, the reaction rate was limited by the permeation of less permeable substrates such as oxygen or ATP. Therefore, K_m(app) of more permeable substrates such as glucose or creatine became smaller than that of free enzyme. The reaction amount calculated from the permeation data agreed well with experimental results. By designing spacers for the reactor to give turbulence to the solution, the effectiveness of the reactor was improved fivefold.