Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of cytidine monophosphate on the regeneration of sialoproteins on the surface of cultured lymphoma cells

A new approach was used to evaluate the role of surface sialyl transferase activity in the regeneration of surface sialic acid (SSA) on cultured lymphoma cells (Raji). Cells which were made deficient in SSA by neuraminidase treatment were incubated for 18 hours in medium containing CMP, a potent inhibitor of surface sialyl transferase activity. In these cultures, the amount of regenerated SSA was not significantly less than for the controls, even though the surface sialyl transferase activity on these cells was inhibited by an average of 95%. Conversely, emetine, an inhibitor of protein synthesis, effectively inhibited SSA regeneration. Thus, these results support the concept that surface sialo-proteins are largely, if not entirely, synthesized intracellularly instead of being assembled on the cell surface by the surface located transferase system.     

SPATIAL POPULATION STRUCTURE IN THE WHIRLIGIG BEETLE DINEUTUS ASSIMILIS: EVOLUTIONARY INFERENCES BASED ON MITOCHONDRIAL DNA AND FIELD DATA

The spatial population structure of the pond-living water beetle Dineutus assimilis (Coleoptera: Gyrinidae) was investigated through a field study of population dynamics and dispersal, with a concurrent assessment of the spatial distribution of mitochondrial DNA (mtDNA) restriction-fragment-length polymorphism (RFLP). A comprehensive 2-yr survey within a 60-km² study area revealed pronounced fluctuations in local abundances, including extinctions and colonizations. The recapture of marked individuals showed that dispersal among ponds is frequent in both males and females and connects populations on a large geographic scale (maximum observed flight distance: 20 km). The population structure of D. assimilis is thus characterized by both pronounced genetic drift and frequent gene flow. Together, these two forces generate a pattern of very local and transient genetic differentiation. Mitochondrial DNA samples collected within a few kilometers indicate highly significant spatial structure, if newly founded demes or those that experienced recent bottlenecks are included. These results based on four demes within the study area were placed into a regional context by further samples collected at distances of 100 km and 200 km. F_st estimates computed on increasing spatial scales were variable but showed no increasing trend. Thus, gene flow exerts a strong homogenizing force over a wide geographic range but is counteracted locally by genetic drift. These findings highlight the need to supplement estimates of F_st with additional data to arrive at valid interpretations of the genetic information. More generally, this study raises questions about how to capture the relevant features of dynamic, subdivided populations to understand their evolutionary dynamics.     

Leading and lagging strand synthesis at the replication fork of bacteriophage T7. Distinct properties of T7 gene 4 protein as a helicase and primase

Reactions at the replication fork of bacteriophage T7 have been reconstituted in vitro on a preformed replication fork. A minimum of three proteins is required to catalyze leading and lagging strand synthesis. The T7 gene 4 protein, which exists in two forms of molecular weight 56,000 and 63,000, provides helicase and primase activities. A tight complex of the T7 gene 5 protein and Escherichia coli thioredoxin provides DNA polymerase activity. Gene 4 protein and DNA polymerase catalyze processive leading strand synthesis. Gene 4 protein molecules serving as helicase remain bound to the template as leading strand synthesis proceeds greater than 40 kilobases. Primer synthesis for lagging strand synthesis is catalyzed by additional gene 4 protein molecules that undergo multiple association/dissociation steps to catalyze multiple rounds of primer synthesis. The smaller molecular weight form of gene 4 protein has been purified from an equimolar mixture of both forms. Removal of the large form results in the loss of primase activity but not of helicase activity. Submolar amounts of the large form present in a mixture of both forms are sufficient to restore high specific activity of primase characteristic of an equimolar mixture of both forms. These results suggest that the gene 4 primase is an oligomer which is composed of both molecular weight forms. The large form may be the distributive component of the primase which dissociates from the template after each round of primer synthesis.     

Localization of the glucose phosphotransferase to a cytoplasmically accessible site on intracellular membranes

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in rat liver is a glycoprotein of 62 kDa. This acceptor was labeled in liver homogenates through incubation with the 35S-labeled phosphorothioate analogue of UDP-Glc, and its distribution following differential centrifugation was compared to that of the glycoproteins labeled by CMP-[3H]N-acetylneuraminic acid. Whereas 94% of the 3H-labeled macromolecules fractionated to the microsomal pellet, 85% of the 35S-labeled 62-kDa glycoprotein was found in the high-speed supernatant. The distribution of the Glc-phosphotransferase was also examined following differential centrifugation, and the bulk of the activity was found in the 100,000 x g pellet. In contrast to results obtained with the lumenal microsomal markers 4 beta-galactosyltransferase and mannose-6-phosphatase, however, optimal activity of the Glc-phosphotransferase was not dependent on the disruption of microsomal vesicles by detergent. In addition, Glc-phosphotransferase was degraded by exogenous proteases in the absence of detergent, whereas the lumenal markers were not. We conclude, therefore, that the 62-kDa acceptor glycoprotein is cytoplasmic and is glycosylated by the Glc-phosphotransferase at a site accessible to the cytoplasm. This may prove to be a model for the topography of glycosylation of other cytoplasmic glycoproteins as well.     

Ultrastructural injury to human spermatozoa after freezing and thawing

The ultrastructure of human spermatozoa at various stages of the freezing and thawing process was studied. In addition to conventional fixations, a freeze-substitution method was used to examine spermatozoa before they were thawed. Dilution in a glycerol-egg yolk-citrate medium caused slight swelling of the acrosome. During slow freezing, when large ice crystals grow in the diluent, the sperm plasmalemma became tighter, the mitochondria had more angular profiles and there was a reduction in electron density of the acrosomal contents. After thawing, the apical segment of the acrosome usually became swollen and the mitochondria appeared rounded. We deduce that these ultrastructural changes occur either during or after the thawing procedure.