Membrane Potentials of Heterotrophically Cultured Tobacco Cells

The electrophysiological properties of the membrane of Nicotianatabacum var. Sarnsun cultured cells were determined using amicroelectrode technique in standard medium containing 1 mMKC1, 1 mM NaCl and 1 mu CaCl₂ at pH 7. Tobacco callus was derivedfrom the pith (E_m=–104.4%16.2 mV). The membrane potentialsof the callus cells did not show a symmetrical Gaussian distributionbut were scattered over a wide range. The percentage of highmembrane potential cells increased as the subculture was continueduntil about 11 months and then decreased. The response of themembrane potential to electric stimulus, ionic composition,metabolic inhibitors, sugars and amino acids was characteristicof high (E_m=–{small tilde}–160 220 mV; H-cells)and low (E_m=–80{small tilde}–90 mV; L-cells) membranepotential cells. The membrane potential of H-cells was largelydepolarized by addition of CN^–, carbonium cyanide m-chlorophenylhydrazone,decyclohexylcarbodiimide, and triphenyltin chloride and transientlydepolarized by addition of glucose, galactose, mannose or sucrose,and D-alanine, L-alanine or Llysine, but the membrane potentialof L-cells was not. (Received December 3, 1982; Accepted March 16, 1983)     

Proliferative kinetics and differentiation of murine blast cell colonies in culture: Evidence for variable G0 periods and constant doubling rates of early pluripotent hemopoietic progenitors

Several investigators have described hemopoietic colonies expressing multilineage differentiation in culture. We recently identified a class of murine hemopoletic progenitors which form blast cell colonies with very high replating efficiencies. In order to clarify further the relationship between progenitors for blast cell colonies and progenitors for the multilineage hemopoietic colonies in culture, we carried out analyses of kinetic and differentiation properties of murine blast cell colonies. Serial observations of the development of blast cell colonies into multilineage (and single lineage) colonies in cultures of spleen cells obtained from 5-fluorouracil (5-FU)-treated mice confirmed the transitional nature of the murine blast cell colonies. The data also suggested that the early pluripotent progenitors are in G₀ for variable periods, and that when triggered into cell cycle, they proliferate at relatively constant doubling rates during the early stages of differentiation. The notion that some of the pluripotent progenitors are in G₀ was also supported by long-term thymidine suicide studies in which spleen cells were exposed to ³H-thymidine with high specific activity for 5 days in culture, washed, and assayed for surviving progenitors. Comparison of replating abilities of day-7 and day-16 blast cell colonies from normal as well as 5-FU-treated mice indicated that some of the day-7 blast cell colonies are derived from maturer populations of progenitors which are sensitive to 5-FU. In contrast, progenitors for the day-16 blast cell colonies are dormant in cell cycle and were not affected by 5-FU treatment. Previously we reported that progenitors for day-16 blast cell colonies have a significant capacity for self-renewal. These observations suggest the hypothesis that the capability for self-renewal is accompanied by long periods of G₀, and that once commitment to differentiation takes place, then active cell division occurs.     

Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Extracellular matrix during laminar pattern formation of neocortex in normal and reeler mutant mice

The spatial and temporal distribution of extracellular matrix, which occupied the large extracellular spaces in the developing cerebral cortex, was studied during pre- and perinatal ontogenesis of normal and reeler mutant mice. Colloidal iron-staining material was localized principally in the marginal zone and subplate of normal mice, whereas in reeler mutants, most of the material was found in the outer layers of the cortex. Patterns of extracellular matrix localization in both genotypes followed the laminar pattern formation of cerebral cortex architecture. Histochemical ultrastructural visualization of this extracellular matrix and its susceptibility to enzymatic treatment suggested that the major components are glycosaminoglycans. Their possible role in relation to afferent axon targeting is discussed.     

Molybdic and tungstic heteropolyanions for "ionic fixation" of acetylcholine in cholinergic motor nerve terminals

The treatment of neuromuscular junctions with phosphomolybdic acid (PMA) and silicotungstic acid (STA) heteropolyanions permits the visualization of electron dense precipitates in the synaptic vesicles of the cholinergic motor nerve terminals. At the light microscopic level, the uncolored molybdenum salt is visualized after reduction to molybdenum blue. The blue coloration is confined to the nerve terminals. Since PMA and STA are known as strong precipitating agents of quaternary ammonium compounds (cations) it is supposed that they have insolubilized in situ the acetylcholine (Ach) of the synaptic vesicles by means of a rapid ionic interaction. Furthermore, in spite of the strong acidity of PMA and STA solutions, the ultrastructure of the treated tissue is not significantly altered but on the contrary seems to be well preserved. The ionic insolubilization of Ach, added to the good preservation of the ultrastructure prompted us to use the term "ionic fixation".