An attempt was made to optimize conditions for introduction of macromolecules into Dictyostelium cells by electroporation. The amount of fluorescein-labeled bovine serum albumin (FITC-BSA) introduced into cells was measured by fluorometry after extraction of FITC-BSA from cells with detergent. The amount increased as the applied voltage and capacitance of the discharger were increased. However, the survival of cells decreased at higher voltages and elevated capacitance. FITC-BSA was introduced into 80-90% of treated cells. FITC-BSA at 0.25mg/ml was introduced into cells under optimum conditions when the concentration of the extracellular protein was 2.5mg/ml. Several discharges in sequence improved the extent of introduction of FITC-BSA although viability decreased. There was a linear correlation between final intracellular concentration and the initial extracellular concentration of FITC-BSA, suggesting the possible quantitative introduction of the protein into cells. The membrane pores that opened during electroporation closed within 2.5 sec after the discharge. FITC-labeled dextran with molecular weights of less than 5×10⁵ were able to pass through these pores. Our results show that electroporation provides a quantitative and reproducible method for introduction of macromolecules into living Dictyostelium cells.

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Triple tandemly repeated sequences and the corresponding complementary sequence are known to exist in the 5'-terminal regulatory region of the human gene for thymidylate synthase (TS). To examine the function of these sequences, a set of deletion mutants was prepared and used in a transient expression assay. The results showed that at least one repeated sequence and its complementary sequence were necessary for the efficient expression of the gene. As another approach to understanding the function of this unique structure, DNA polymorphism in the same region was analyzed. In addition to the TS gene with the triple tandem repeat, the TS gene with a double tandem repeat was found in genomes of normal human subjects at an estimated frequency of 19% when genomes of 21 unrelated Japanese were analyzed. The expression activity of a reporter gene linked to the promoter region of the human TS genes with the two types of repeated sequence was examined and the result showed that the expression activity of the gene with the double repeat was lower than that of the gene with the triple repeat in the transient expression assay. Thus, it appears that the unique repeated sequences in the 5'-terminal region of the human TS gene are polymorphic and contribute to the efficiency of expression of the gene.

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Newly prepared phosphatidylethanolamine (PE) conjugates of glycosaminoglycans (GAGs) enabled us to immobilize GAGs to solid phase through hydrophobic interaction. Using these compounds, called GAG-PEs, we studied the effects of GAGs immobilized to culture plates coated with various extracellular matrix (ECM) proteins in terms of cell-substrate adhesion, DNA synthesis, tyrosine aminotransferase (TAT) induction, and intercellular communication in primary rat hepatocytes. Treatment with chondroitin sulfate (CS)-PE at 10 μg/ml made laminin, fibronectin, vitronectin, and collagens type I-V less adhesive as substrates for cell attachment and inhibited cell spreading on these substrates. The effect on cell attachment was lost after long incubations over 2 h. Dermatan sulfate (DS)-PE was also inhibitory, but less effective. The conjugates of heparan sulfate (HS), heparin, and hyaluronan were much less effective. DNA synthesis initiated by EGF in the culture on laminin substrates was inhibited most strongly by CS-PE, as well as by DS-PE and hyaluronan-PE, but not by either HS-PE or heparin-PE. With type I collagen substrates, GAG-Pes had similar effects but to lesser extent. TAT induction in the culture on laminin substrates but not on type I collagen substrates was significantly enhanced by CS-PE. In terms of DNA synthesis and TAT induction, the culture on laminin substrates treated with CS-PE was comparable to that at higher cell density on the non-treated laminin substrates. Intercellular communication assessed by dye coupling was maintained longer on the substrates treated with CS-PE. Taken together, our results demonstrate that CS immobilized especially onto laminin substrates inhibits the growth of hepatocytes and enhances their differentiated functions by modulating cell-ECM protein interactions.

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A thymic epithelial cell line Mm2T was cultured in a medium containing a high concentration (100μg/ml) of methylated vitamin B₁₂ (CH₃-B₁₂). After 19 days, cells were found to have a flat phenotype, to have lost the floating cells which were observed in the control cells at the con fluent stage, and to have acquired a resistance to trypsin. However, treatment of the CH₃-B₁₂-treated cells with EDTA resulted in a dissociation of cell-to-cell contact and reaggregation was achieved by addition of Ca²⁺, indicating the involvement of Ca²⁺ ion in cell-to-cell contact. Electron microscopic analyses revealed that the CH₃-B₁₂-treated cells were nearly square in their vertical section, which was in contrast to the dome-shaped feature of the control cells, and their cell-to-cell contact area was significantly widespread, as compared to those of the control, indicating that Mm2T cells acquires an adhesive property by treatment with CH₃-B₁₂. Biochemical analyses of both cells indicated that the concentration of glucosylceramide in the CH₃-B₁₂-treated cells was higher than that of the control. Free glucose characteristically inhibited the reattachment of cells dissociated with EDTA, suggesting the involvement of glucose in the cell-to-cell adhesion of CH₃-B₁₂-treated cells. In addition, WGA-binding glycoconjugates were intensely observed in the boundary region of CH₃-B₁₂-treated cells by immunohistochemical staining, but not in that of the control cells. It is suggested that CH₃-B₁₂ may affect the morphological alteration of Mm2T by enhancing cell adhesion through elevated expression of the C-type lectin.

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We have investigated the factor(s) present in serum that stimulate cumulus expansion (accumulation of hyaluronic acid in the intercellular space of cumulus cells) and male pronucleus (mPN) formation in porcine oocytes matured and fertilized in vitro. After 24 hr of culture, oocyte-cumulus complexes that matured in the serum of pig, cow, rabbit and rat showed an expansion of the surrounding cumulus cell matrices. The cumulus expansion stimulating effect is not species-specific. Porcine serum was fractionated by ultracentrifugation at 220, 000×g at 10°C for 48 hr, resulting in four fractions. The oocyte-cumulus complexes that matured in the 2nd fraction exhibited a marked expansion of the cumulus cell layers, including the corona radiata. Complexes cultured in the remaining fractions, however, showed very little or no expansion. There was no expansion of the oocytectomized complexes matured in the 2nd fraction. Serum was not effective in the formation of the male pronucleus. The active factor(s) was heat stable at 100°C for 30 min and showed a relatively strong activity at a molecular weight of >1.0 and <6.5 kDa. These results indicate that serum contains a low molecular weight, heat stable factor(s) that acts on the oocyte to stimulate cumulus expansion.

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To investigate the importance of SH-groups in the energy metabolism of liver cells, isolated rat hepatocytes were exposed to various SH-blocking compounds. After 1.0hr exposure, the cells were analyzed for the content of glycogen, lactate, pyruvate, ATP and the rate of oxygen consumption.
Without affecting the cell viability, PCMB, PCMBS, mersalyl, NEM, DTP and DSF were found to decrease glycogen levels, whereas the disulphide reagent CDPS did not affect this endogenous energy reserve. Lactate and pyruvate levels were decreased by the organic mercury compounds, whereas NEM, DTP and DSF stimulated the formation of lactate, without affecting the levels of pyruvate.
In both situations the oxygen consumption was slightly decreased. The FCCP uncoupled oxygen consumption was not affected. Up to the point of loss of cell viability, as measured by trypan blue exclusion and LDH leakage, the liver cells maintained their ATP levels, during exposure to the various SH-reagents.
In conclusion, the results with organic mercury compounds suggest a reaction of these agents with SH-groups in the outer membrane of cells, having an inhibiting effect on the glucose uptake. The most prominent effect of DTP, DSF and NEM was an increased lactate formation, implying an intracellular effect, most probably in the TCA-cycle.

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The vegetative role of the germinal micronucleus of Tetrahymena thermophila was studied. Amicronucleate cells that had spontaneously arisen in star strains (very aged clones with defective micronuclei) were observed in B^* as well as in A^* and in C^* and were found to lack the oral apparatus and to have disordered ciliary rows. To reduce uncertainty given the very small micronucleus and possible effects of aging factors, we induced amicronucleate cells in a young clone by treatment with the antitubulin drug, nocodazole, and observed their cortical structure and nuclei. Amicronucleate cells gradually lost their oral apparatus and then their ciliary rows became disordered, even without cell division, and they became crinkled cells. It can be concluded that the vegetative micronucleus appears to play a critical role in the maintenance of the cortical structure, especially of the oral apparatus.

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