Regulation of acid hydrolysis of sucrose in acid lime juice sac cells

The sucrose content of acid lime [ Citrus aurantifolia (Christm.) Swing.] juice tissue was measured at time 0 and at various times following incubation at 15.5, 26.6 and 37.7°C. The decline in sucrose content in fruit stored at 15.5°C paralleled the expected values for a sucrose solution at pH 2.1. At higher temperatures, the in vivo sucrose content decreased at significantly lower rates than the expected values. In fruit stored at 26.6 and 37.7°C, the vacuolar pH increased 0.11 and 0.23 units, respectively. When sucrose hydrolysis was recalculated at the increased vacuolar pH of juice cells stored at 26.6 and 37.7°C, the calculated values were similar to the measured values obtained in vivo. It is concluded that within the limits of the experimental conditions, the rates of sucrose acid hydrolysis are regulated by changes in the vacuolar H⁺ concentration.     

Antibody-nucleic acid interactions. Antibodies to psoralen-modified RNA as probes of RNA structure.

Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells.     

Repair response of human fibroblasts to bleomycin damage

The ability of human fibroblasts to repair the specific types of DNA damage caused by bleomycin (BLM) was examined in whole-cell experiments. The method utilized for analysis was alkaline sucrose-gradient centrifugation of DNA. The results of these studies show that a repair pathway exists for the damage produced in DNA by bleomycin. DNA from BLM-treated cells shows a decrease in molecular weight, caused by chemical or enzymatic incision at sites of drug action. If the drug is removed, the DNA rapidly returns to high molecular weight, demonstrating reformation of damaged DNA. This repair response to BLM-damage was also confirmed in fibroblasts isolated from patients with putative DNA-repair defects. We observed that the response (to BLM) of cells from patients with Fanconi anemia was altered in that the fall in molecular weight of DNA from treated cells was not as great as that observed in other cell strains after drug treatment.     

PRODUCTION AND CHARACTERIZATION OF MULTIPLE-LAYERED POPULATIONS OF ANIMAL CELLS

Dense populations containing 129 x 10⁶ Jensen sarcoma, 134 x 10⁶ DON Chinese hamster, 28.9 x 10⁶ WI-38 human diploid, 61.8 x 10⁶ HEp-2 human carcinoma, and 67.4 x 10⁶ WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 10⁶, in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca ±0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 10⁶ WI-38 and 250 x 10⁶ DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments.     

Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.     

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.     

Sustained effects of biofeedback-assisted relaxation therapy in essential hypertension

The usefulness of biofeedback-assisted relaxation as an adjunct or substitute for pharmacotherapy in essential hypertension can be enhanced if the effects are shown to persist after formal treatment has ended. Patients with essential hypertension successfully treated with biofeedback-assisted relaxation were recalled for follow-up yearly after the termination of treatment. Twenty-six of 40 patients met the BP criterion for success. At one-, two-, and three-year follow-up, 31%, 38%, and 27% of the successful completers continued to meet the criterion for success. The pretreatment-posttreatment decreases in BP were accompanied by decreases in forehead muscle tension and urinary cortisol. Forehead muscle tension, urinary cortisol, and anxiety levels were significantly lower than pretreatment one year after the end of treatment. Self-report data were used to assess continued relaxation practice. No relationship was found between practice and any other dependent measure. It appears that some patients trained in biofeedback-assisted relaxation can maintain lowered blood pressure, muscle tension, anxiety, and cortisol levels over the long term; however, the role of relaxation practice in maintaining these lowered levels remains unclear.     

Effects of N6-endonorbornan-2-yl-9-methyladenine, N0861, on negative chronotropic and vasodilatory actions of adenosine in the canine heart in vivo.

The pharmacology of N6-endonorbornan-2-yl-9-methyladenine (N0861), a new selective antagonist of adenosine at the A1 adenosine receptor subtype (A1-AdoR), was studied in vivo using a canine model. First, the pharmacokinetics of N0861 were determined in anesthetized dogs. The time-dependent decay of plasma levels of N0861 fitted a two-compartment polyexponential model with alpha-phase t1/2 = 3.80 min and beta-phase t1/2 = 80.55 min. Secondly, the effect of N0861 on the negative chronotropic and vasodilatory actions of adenosine in the canine heart were determined. N0861 attenuated the negative chronotropic action of adenosine (1-6 mumol/kg; rapid bolus into the right atrium) on sinus node pacemaker activity in a dose-dependent manner (pA2 = 4.23). For example, the maximal prolongation of sinus cycle length induced by 6 mumol/kg adenosine was 82 +/- 13% under baseline conditions and 57 +/- 10, 34 +/- 5 and 34 +/- 6% during infusion of N0861 at incremental rates leading to plasma levels of 7.75 +/- 1.02, 14.15 +/- 0.87, and 19.71 +/- 1.83 micrograms/mL, respectively. In contrast, N0861 did not inhibit but had a tendency to potentiate the vasodilatory action of adenosine (thought to be mediated by the A2 adenosine receptor subtype (A2-AdoR)) on the left anterior descending and circumflex coronary arteries. These data indicate that two different receptors, similar to the typical A1-AdoR and A2-AdoR, mediate the electrophysiologic and vasodilatory actions of adenosine in the canine heart, respectively, and that N0861 is a selective antagonist of adenosine at A1-AdoR in the canine heart in vivo.     

Isolation and characterization of nuclear genes coding for subunits of the yeast ubiquinol-cytochrome c reductase complex

Nuclear genes coding for the M_r 17000, 14000 and 11000 subunits of the ubiquinol-cytochrome c reductase complex (complex III) in yeast have been isolated from a clone bank of yeast nuclear DNA by use of a mRNA hybridization-competition assay. This is based on our observations that levels of mRNAs for these subunits are much reduced during glucose repression and in cytoplasmic petite mutants and the procedure should be of general application for the isolation of other inducible or repressible genes coding for mRNAs present at low levels in the cell. A first characterization of the clones is presented. The genes are not closely linked in the genome and those coding for M_r 14000 and 11000 subunits are present in unique genomic environments, which suggests that there are only single copies of each in the nuclear genome.