Effects of chloramphenicol on RNA synthesis in Spirodela chlorplasts

RNA from chloroplasts isolated from Spirodela oligorrhiza includedrelatively rapidly-labeled fractions with apparent molecularweights of 2.7; 1.2; 0.7; and 0.5x10⁶. With longer labeling,radioactivity appeared in the mature rRNAs (1.1 and 0.56x10⁶MW). Chloramphenicol inhibited the appearance of labeled maturerRNA, but increased the net labeling and caused the accumulationof the pulse-labeled RNAs, effects similar to those reportedfor bacteria. ¹ 1 Permanent address: Dept. of Biological Sciences, S.U.N.Y.,Binghamton, N.Y. 13901, U.S.A. Supported by a SUNY/ResearchFoundation Faculty Research Fellowship. (Received November 12, 1974; )     

Epidermal growth factor receptor kinase translocation and activation in vivo

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.     

Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures

A protocol employing discontinuous sucrose gradient centrifugation was developed to prepare light mitochondrial (L) and Golgi fraction endosomes from simultaneously prepared parent L and microsomal fractions. As judged by the concentration of labeled hormone postinjection, L intermediate and heavy endosome subfractions were 40- to 175-fold purified and Golgi intermediate and heavy endosome subfractions were 30- to 45-fold purified. On electron microscopy, L endosomal fractions contained a predominance of lipoprotein-filled vesicles and were less heterogeneous than corresponding Golgi endosomal fractions. All endosomal fractions were enriched in receptors for insulin and prolactin but binding sites for the former were more broadly distributed in other subfractions than those for the latter. On Percoll gradient centrifugation, L endosomal fractions yielded one peak (rho 1.057) corresponding to the heavier of two peaks seen in Golgi endosomal fractions. The protein composition of high density L and Golgi endosomes, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar. The bulk of marker enzymes assayed did not migrate with the endosomal components. Combined acid phosphatase cytochemistry and electron microscope radioautography established that about 80% of the L endosomes contained no acid phosphatase. By affinity labeling and immunological titration with insulin receptor antibody, insulin receptors were identical in L and Golgi endosomes. Insulin-stimulable receptor kinase was demonstrable in both L and Golgi endosome fractions. Following in vivo insulin administration, the insulin receptor kinase in both L and Golgi endosomes was significantly activated. This activated state was not inhibited by a large excess of antiserum to insulin and thus not due to insulin contaminating the partially purified receptor preparation. These observations are compatible with the maintenance and/or initiation of hormone-dependent phosphorylations intracellularly.     

Inhibitory Effect of Carbohydrate on Flowering in Lemna perpusilla. I. Interaction of Sucrose With Calcium and Phosphate Ions

Flowering in Lemna perpusilla 6746 grown on tenth-strength Hutner's medium under short days was inhibited by 30 mM sucrose, glucose or fructose, but not by mannitol. The inhibition by sucrose does not appear to be due to sucrose-induced acidification of the medium during growth, or to trace metal contaminants of the sugar. Inhibition was partially prevented by raising either Ca²⁺ or phosphate to levels used in half-strength medium. Possible mechanisms for these effects are discussed.     

Synthesis and processing of RNA in Lemna: Characterization by gel electrophoresis

The synthesis and processing of rapidly labelled RNAs from Lemnaperpusilla 6746 was followed by polyacrylamide electrophoresis,as the first step in a study of the changes which occur withenvironmental changes. Two RNAs with high apparent molecularweights of 2.8 M and 2.3 M were detected. Time-course studyresults were consistent with the idea that the 2.8 M was cleavedto form 2.3 M RNA and "excess" RNA with a molecular weight of0.5 M. The latter had a far lower turnover than the 2.3 M RNA.Another rapidly labelled RNA which has not been described beforein plants, had an apparent weight of 1.2 M. This was followedby labelling the 1.4 M component, then the light rRNA (0.7 M),and finally the heavy rRNA (1.3 M). A relatively large amountof high molecular weight, polydisperse RNA was synthesized understeadystate conditions. Incubation of plants on distilled water reduced synthesis of1.2 M RNA, while other components were less affected. The processingof rRNA precursors was also slowed. (Received December 11, 1972; )     

Muscle metabolism in track athletes, using 31P magnetic resonance spectroscopy.

We tested whether preferred running event in track athletes would correlate with the initial rate of phosphocreatine (PCr) resynthesis following submaximal exercise. PCr recovery was measured in the calf muscles of 16 male track athletes and 7 male control subjects following 5 min of repeated plantar flexion against resistance. Pi, PCr, and pH were measured using phosphorus magnetic resonance spectroscopy (31P MRS) with an 8-cm surface coil in a 1.8-T magnet. During exercise, work levels were gradually increased to deplete PCr to 50-60% of the initial value. No drop in pH was seen in any of the subjects during this exercise. The areas of the PCr peaks following exercise were fit to monoexponential curves. Two or three tests were performed on each subject and the results averaged. Athletes were divided into three groups based on their primary event: sprinters running 400 m or less, middle-distance athletes running 400-1500 m, and long-distance athletes running farther than 1500 m. The maximal rates of PCr resynthesis (mmol.min-1.kg-1 muscle weight) were 64.8 +/- 8.6, for long-distance runners; 41.4 +/- 11, for middle-distance runners; 32.0 +/- 7.0, for sprinters; and 38.6 +/- 10, for controls (mean +/- SE). The faster PCr recovery rates seen in long-distance runners compared with sprinters indicate greater oxidative capacity, which is consistent with the known differences between athletes in these events.     

Rat atrial muscle responses with caffeine: dose-response, force frequency, and postrest contractions.

Caffeine has been reported to have a positive and (or) a negative inotropic effect on cardiac muscle. In this study, the force-frequency and postrest characteristics of rat atrium were studied in the presence of caffeine (1.0-10 mM) to see if the interval between beats affected the response of cardiac muscle to caffeine. When stimulation frequency was 0.5 or 2.0 Hz, there was a positive followed by a negative inotropic response with 1, 5, or 10 mM caffeine. Incomplete relaxation occurred under these circumstances, giving rise to contracture. At low frequency of stimulation (0.1 Hz) caffeine had only a negative inotropic effect, and this effect was greater with 1 mM caffeine than with 5 mM caffeine. In the absence of caffeine, when stimulation at 0.5 or 3 Hz was interrupted, a pause of 2-20 s resulted in potentiation. When caffeine was present (2.0 mM), postrest potentiation was severely attenuated, but the steady-state contraction amplitude within the range 0.5-3.0 Hz was not different. These results are consistent with the hypothesis that caffeine induces a leak of Ca2+ from the sarcoplasmic reticulum, and this Ca2+ is extruded from the cell, possibly by Na+/Ca2+ exchange. Sarcoplasmic reticular uptake of Ca2+ and the translocation to release sites appear not to be affected by caffeine within 1-5 mM concentrations.     

Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody.

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention.