The ability to grow lymphoid and myeloid cells in serum-free culture medium allows researchers to analyze the factors and mechanisms required for hemopoietic cell growth and differentiation without the interference of undefined serum components. Therefore, we used a serum-free medium, RITC 55-9 that consisted of modified Dulbecco's MEM supplemented with bovine serum albumin (BSA), transferrin (Tf) and insulin (Ins) to culture human T lymphoid (Mo), murine myelomonocytoid (WEHI-3B) and murine interleukin (IL)-3-dependent (32Dcl/H4) cell lines. Mo was maintained in RITC for more than 8 months and had a mean viability of 59 % and the same doubling times as in serum-containing medium (SCM). Under these conditions, Mo cells produced hemopoietic colony-stimulating activity that included production of a basophil/eosinophil differentiation factor of similar content to that produced in SCM. WEHI-3B cells grown for more than 12 months in RITC, or for more than 3 months in RITC without Tf and Ins, had a doubling time of 20 h, whereas cells maintained in protein-free RITC showed a 2-fold increase in doubling time then died within 3 months. The IL-3 production by WEHI-3B cells cultured in RITC was higher than the production by cells grown in SCM. When IL-3 was assayed in 32Dcl/H4 cells that had been maintained in RITC for more than 4 months, a lower response to IL-3 was found, an indication that components other than the BSA, Tf and Ins in fetal calf serum are required for optimal cell growth and differentiation.
Concanavalin A at a concentration of 1.0 mg/ml completely inhibited the conjugation of haploid cells of Physarum polycephalum. This inhibitory effect was counteracted by α-methyl-D-mannoside. α-Mannosidase also inhibited conjugation. Neither wheat germ agglutinin nor lentil lectin had any apparent inhibitory effects. When Physarum cells were cultured in the presence of tunicamycin at a concentration of 1 μ/ml, a similar inhibitory effect on conjugation was observed. The plasma membrane from the amoebae partially inhibited conjugation. On the basis of these results, the mannosyl glycoprotein(s) located on the cell surface is considered to function in conju-gation process. Inhibitory compounds could be extracted from the cell surface with the detergent, Triton CF-54, and could be separated by concanavalin A-Sepharose column chromatography.
Migratory properties of normal peripheral blood lymphocyte subsets were studied with a 3-D collagen gel model. Both the T and B subsets entered the gel at the same rate, but once inside the T subsets migrated signifi-cantly faster than the B subsets. In the T subsets, both the T-helper and T-suppressor cells entered the gel at the same rate, but the former migrated significantly faster once inside the gel. The biological significance of their migrations is discussed.
ABSTRACT We compared intracellular K+ and Na+ ion concentrations during cell growth and differentiation of a mouse myeloid leukemia M1 cell line. Cells undergoing mitosis had higher K+ concentrations than quiescent cells. Treatment with a K+ channel blocker and furosemide enhanced cell growth and produced a slight increase in the intracellular K+ concentration. Treatment with reagents that reduced the intracellular K+ concentation stopped cell growth. Induction of differentiation in this cell line produced a decrease in the K+ concentration, which always was accompanied by an increase in the Na+ concentration. Treatment with ouabain, which decreased the intracellular K+ concentration, did not, however, induce differentiation in the Ml cell line. The data suggest that cell growth and differentiation in the M1 cells are accompanied by changes in the intracellular K+ and Na+ concen-trations but that the changes in the contents of these monovalent cations do not necessarily induce differentiation in this cell line.
E-cadherin is a Ca2+-dependent cell-cell adhesion molecule identified as a glycoprotein with a molecular weight (MW) of 124, 000. To study the role of the sugar moieties of this adhesion molecule, we tested the effect of tunicamycin on aggregation mediated by E-cadherin of teratocarci-noma cells. Immunoblot analysis using a monoclonal antibody to E-cadherin showed that in cells treated with tunicamycin this adhesion molecule is converted into two forms with MW of 118, 000 and 131, 000. The smaller one was exposed on the cell surface and showed a trypsin sensitivity characteristic to E-cadherin, suggesting that this is the peptide moiety of E-cadherin whose glycosylation with N-linked oligosaccharides was blocked by tunicamycin. The larger one was not removed by trypsin treatment of cells, suggesting an intracellular location. These tunicamycin-treated cells aggregated in a Ca2+-dependent manner, and the aggregation was inhibited by a monoclonal antibody to E-cadherin. These results suggested that N-linked oligosaccharides are not involved in the functional sites of this adhesion molecule.
Microtubule associated protein-1 of brain and its intracellular 350kd analogues were highly sensitive to purified Ca2+-dependent cysteine proteinase (calpain). After 15 second digestion, we detected intermediate degradation products of MAP-1 by immunoblotting using anti-MAP-1 antibody as 290, 260, 220, 170, 140, 112, 80, 68, and 32kd polypeptides. These values corresponded to the molecular weights of the immunoreactive polypeptides of microtubule-enriched cytoskeletons isolated from HeLa and SV-3Y1 cells, suggesting the action of endogenous calpain on intracellular MAP-1 analogues in vivo or during the course of preparation.
(Na+, K+)ATPase was purified from rat renal outer medulla by concanavalin A-and wheat germ agglutinin-lectin Sepharose affinity chromatographies. The antibody, which was raised in rabbits, markedly inhibited ATPase activity. The monospecificity of this antibody was assayed by the Ouchterlony double immunodiffusion and Western blotting tests. The endoplasmic reticulum (ER)-rich, and Golgi-rich subfractions were prepared from the rat kidney microsomal fraction by sucrose density gradient centrifugation. On the immuoblot, the molecular weight of the a subunit in both fractions was 95 kilodalton (Kd); whereas, that of the β subunit was 50 Kd in the ER-rich fraction and 54 Kd in the Golgi-rich fraction. When treated with endoglucosidase H, the 50 Kd component was converted to 38 Kd, but the 54 Kd component was endoglucosidase H resistant. These results suggest that the β subunit (38 Kd) is glycosylated cotranslationally in the ER (50 Kd) then is converted to the mature type subunit (54 Kd) in theGolgi apparatus.
Since the plasticity of the plasma membrane is correlated with changes in its dynamic behavior and biological functions, the content of cholesterol in this membrane is thought to have an important role in the regulation of cell metabolism. The research reported here was done to clarify the role of cholesterol in the interaction of the innersurface of plasma mem-brane with cytoplasmic proteins in relation to the regulatory mechanism of signal transduction. Many cytoplasmic proteins of EATC or rat liver cells were found to associate with DPPC liposomes and they induced a transient increase in membrane permeability at the phase transition temperature of the liposomal lipid. The association and the permeability increase were inhibited by the introduction of cholesterol into DPPC liposomes, and the sensitivity of individual proteins to the action of cholesterol differed. F-actin, but not G-actin, associated selectively with the liposomes. Also, the main endothermic peak of the unilamellar DPPC liposomes was shifted from 37°C to 43°C by this protein association, evidence of transformation from a unito multi-lamellar structure. The introduction of cholesterol into the liposomal membrane caused a reduction in the energy content of the phase transition and in the inhibition of protein-membrane interaction. We concluded that the cholesterol in the plasma membrane contributes to the regulation of cell surface signal trans-duction.
Dietary lectins of gluten origin have been suggested to play an important role in the mechanisms leading to the characteristic morphology of the intestine found in patients with celiac disease. To further explore this issue we have used Wheat Germ Agglutinin (WGA) or Concanavalin A (Con A) to challenge rat small intestine and study the ultrastructural changes of such a treatment. Both lectins affected the enterocytes at the base of the villi more than those at the top. The morphological findings included disarrange-ment of the cytoskeleton, increased endocytosis and shortening of the microvilli. The interrelationship between the observed changes, and their relevance for similar morphological alterations found in patients with celiac disease are discussed. In conclusion, the morphological findings in our rat model resemble early changes in patients with celiac disease, thus supporting the idea that lectins or lectin-like substances are involved in the pathogenesis of this disease.
To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.
An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulinimmunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.
DNA polymerase α combined with the endoplasmic reticulum (ER) was isolated from unfertilized sea urchin eggs. NaCl treatment of this fraction released DNA polymerase α from the ER. The molecular size (the S value) of the ER-free DNA polymerase α changed with the concentration of NaCl used; being 23 S, 11-15 S and 6-8 S in the presence of 0.05-0.12 M, 0.12-0.24 M and more than 0.24 M NaCl. DNA polymerase α activity decreased concomitantly with the reduction in molecular size. The 6-8 S form of DNA polymerase α did not aggregate by itself nor with other cellular components nonspecifically, when the 23 S form was present. These results are evidence of the presence of 6-8 S DNA polymerase α as a high molecular weight form (23 S-form) in sea ruchin eggs.
Modification of rectified Nomarski differential interference contrast optics (Nikon) and the epi-illumination system (Nikon IGS-cube) improved the detection of colloidal gold particles with analog video enhanced microscopy. Immuno-gold labelled microtubules of Haemanthus endosperm are visualized at a level of detection unmatched in conventional light micro-scopy. Single gold, or gold silver enhanced particles in suspension viewed with the modified epi-illumination after pressure injection into cells, are well distinguished from other granular cell components. Immuno-gold has also been detected on the surface of chromosomes and the nuclear envelopes in cells during the rapid experimental disassembly of microtubules. Thus, under certain conditions tubulin in a form other than microtubules may be detected. Practical applications of this "optical stain" for non fading immuno-gold 5-40 nm markers are discussed.