Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

Dynamics of plasmid maintenance in a CSTR upon square-wave perturbations in the dilution rate

The effects of forced oscillations in the dilution rate on a population of Escherichia coli K12 harboring the plasmid pBR322 in a chemostat with a nonselective medium were studied. In the constant dilution rate control experiments, the percentage of plasmid-containing cells decreased after a long lag time. Eventually, the culture approached a population consisting of 100% plasmid-free ells. However, under forced perturbations of the dilution rate, the culture maintained a mixed population of plasmid-free and plasmid-carrying cells for a longer period of ime. An unstructured model was developed to describe the above observations. Our results indicate that transient conditions created by dilution-rate perturbations provide a favorable environment for the plasmid-carrying population. In addition, experiments with different cycling frequencies suggest that adaptation by the culture to these transient conditions will reduce or totally eliminate such an advantage.     

Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.     

Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase

Pressure dissociation of yeast glyceraldehydephosphate dehydrogenase (GAPDH) was studied by fluorescence spectroscopy. Observations in the range of -5 to 30 degrees C indicate that monomer association into the tetramer proceeds with an enthalpy change of -14 kcal mol-1 and a large increase in entropy which at 25 degrees C amounts to 18 kcal mol-1. The large conformational drift and the low-temperature stability of the tetramer recovered after decompression facilitated a comparison of its properties with those of the native tetramer. Significant differences in absorption and fluorescence-excitation polarization spectra, yield of tryptophan fluorescence, and binding of anilinonaphthalenesulfonate and NADH were observed. At 0 degree C the standard free energies of association of the monomers into the native and drifted tetramers were respectively -32 and -29 kcal mol-1. The volume change upon association measured from the pressure span of the compression curves was 200-230 mL mol-1 but four times as large when derived from the displacement of the compression curves with total protein concentration. This large discrepancy can be explained by the existence in the native tetramer population of a distribution of free energies of association with a dispersion from the mean of about 6 kcal mol-1. At 0 degree C and 1 bar ATP and ADP decreased the stability of the GAPDH tetramer by changes in free energy of association of +3.7 and +4.1 kcal mol-1, respectively. NAD and c-AMP stabilized it by -2.3 and -1.3 kcal mol-1. The variation in sign and magnitude of the ligand-induced changes in free energy of association observed in this case, and previously in hexokinase [Ruan, K., & Weber, G. (1988) Biochemistry 27, 3295], and the heterogeneity of the free energy of association of GAPDH, revealed as indicated above, lead to the conclusion that oligomeric aggregates exist in a variety of conformations that depend upon the protein concentration, temperature, pressure, and the presence of specific ligands. The multiplicity of species revealed by the energetics raises questions about the significance of the structures of oligomeric proteins determined by X-ray crystallography.     

Inhibition of epidermal growth factor receptor biosynthesis caused by the src oncogene product, pp60v-src.

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.     

The effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold

The purpose of this investigation was to determine the effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold (PWCFT). Ten adult males (mean age 23 years, SD 3) volunteered as subjects for this study. During the first laboratory visit the subjects performed a maximal bicycle ergometer test for the determination of maximum oxygen consumption (VO2max). Between 48 and 72 h later, the subjects pedaled to exhaustion at a power output which corresponded to a mean of 76% of VO2max (range, 72-80%) for the purpose of glycogen depletion. For the next 3 days, the subjects were fed a 10.5 MJ.day-1 low carbohydrate diet which consisted of 7.5% carbohydrates, 22.0% protein and 70.5% fat. The subjects then performed an incremental cycle ergometer test to the onset of fatigue or PWCFT, which was estimated from integrated electromyographic voltages of the vastus lateralis muscle. For the next 3 days the subjects were fed a 10.5 MJ high carbohydrate diet which consisted of 72.2% carbohydrates, 12.4% protein and 15.4% fats for the purpose of glycogen supercompensation. The subjects then performed a second PWCFT test. A paired t-test indicated that there was no significant (p greater than 0.05) difference between the means of the PWCFT values (depletion 246 W, SD 30; supercompensation 265 W, SD 28) and they were highly correlated at r = 0.884. The results of this investigation suggested that the methods commonly used to affect glycogen depletion or supercompensation had no effect on PWCFT.