A Comparison between Quin-2 and Aequorin as Indicators of Cytoplasmic Calcium Levels in Higher Plant Cell Protoplasts.

Assessment of the regulation of plant metabolism by the calcium ion requires a knowledge of its intracellular levels and dynamics. Technical problems have prevented direct measurement of the concentration of intracellular Ca(2+) in plant cells in all but a few cases. In this study we show that electropermeabilized protoplasts of Daucus carota and Hordeum vulgare took up the Ca(2+) indicating fluorescent dye methoxyquinoline(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Quin-2) and the Ca(2+) indicating photoprotein, aequorin. These protoplasts subsequently recovered their plasma membrane integrity. However, up to 10% of intracellularly trapped Quin-2 was associated with a protoplast vacuolar fraction. Also, Quin-2 loading reduced total ATP levels by approximately 60% and inhibited subsequent protoplast division whereas aequorin loading reduced ATP content by only 20% and did not prevent division. Therefore, the basal cytoplasmic Ca(2+) level measured with aequorin (less than 200 nanomolar) may more reliably reflect that found in vivo in the unperturbed protoplast than that measured with Quin-2 (120-360 nanomolar). However, measurements made with aequorin were found to be inaccurate at Ca(2+) levels below 200 nanomolar, Quin-2 proving complementary in indicating these low Ca(2+) concentrations. Cytosolic Ca(2+) was observed to increase on treatment with azide and silver ions.

Application of X-ray microanalysis to cell suspensions of oil palm (Elaeis guineensis Jacq.).

X-ray microanalysis has been used to determine the elemental composition of oil-palm (Elaeis guineesis) cell suspensions without the use of cryoprotectants. Results based on individual cells were gathered over a typical growth cycle of 14 d. During the log phase (5-7 d) there is an increase in the number of cells containing high concentrations of both K (400 mmol kg(-1) dry weight) and P (400 mmol kg(-1) dry weight). Morphologically these cells had thin cell walls and were frequently joined to other cells (two to five cells per clump).

Distance Race Injuries: An Analysis of Runners' Perceptions.

In brief: This report examines the type and frequency of self-reported injuries that occurred at the Chicago Distance Classic, a 20km race held each year in July. Questionnaires were sent to the nearly 5,000 entrants. The runners were asked about their medical history and if they had suffered injuries or illnesses from the race. Most entrants had been running two to five years. More than half stretched regularly before and after running. The knee was most often injured (10%), and the foot was second. Most orthopedic, knee, and foot injuries occurred to beginners, and most entrants were between ages 31 and 40. Many of the entrants said they were affected by the heat; 31 said they suffered severely from the heat.

An investigation of the growth characteristics of oil-palm (Elaeis guineensis) suspension cultures using 31P NMR.

High-resolution 31P nuclear-magnetic-resonance (NMR) spectra are reported for oil-palm (Elaeis guineensis) cells in suspension culture. The spectra are a significant improvement on the results that have appeared for other cultures and they are comparable with the spectra of the meristematic tissue in seedling roots. The NMR technique was used in parallel with other analytical methods to investigate the growth characteristics of the suspension culture, including the effect of 2,4-dichlorophenoxyacetic acid.

Cardiac and renal responses to cross-clamping of the descending thoracic aorta.

The present study was performed to document the relative efficacy of commonly applied techniques used adjunctively during 1 hour of descending thoracic aortic cross-clamping. Renal and cardiac responses were determined by standard laboratory methods. There were four experimental groups: (1) heparin-bonded shunt; (2) partial femoral-femoral bypass; (3) sodium nitroprusside; (4) control. Each of the experimental groups showed abnormal hemodynamic responses during cross-clamping. Elevations in left ventricular end-diastolic pressure (LVEDP) and systolic blood pressure were common events during clamping, and cardiac output often decreased. Nevertheless, left ventricular performance curves after cross-clamping showed similar increases in left ventricular stroke work (LVSW) with increasing preload. In addition, left ventricular biopsy specimens showed preservation of myocardial high-energy phosphate stores and essentially normal ultrastructural integrity. Radioactive microspheres generally showed increased myocardial blood flow during and after cross-clamping, but no evidence of preferential subendocardial ischemia. Examination of renal function showed a marked decrease in urine output, glomerular filtration rate, and renal plasma flow during cross-clamping. Following the release of the cross-clamp, renal function returned to 50% to 85% of baseline status. Since we could find no major advantage of any of the techniques employed under the present experimental conditions, we suggest that all of the techniques should be part of the surgical armamentarium and the particular preoperative and/or intraoperative findings in a specific case should determine which technique is most appropriate for a given patient.

The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.

Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin.

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.

Anti-insulin receptor antibodies mimic the effects of insulin on the activities of pyruvate dehydrogenase and acetylCoA carboxylase and on specific protein phosphorylation in rat epididymal fat cells.

Previous studies have shown that autoantibodies against insulin receptors found in certain patients with severe insulin resistance stimulate glucose transport and metabolism in fat cell and muscle preparations. The present studies show that preincubation of rat epididymal adipose tissue with 1 : 1000 dilution on one such serum results in a two fold increase in the initial activities of pyruvate dehydrogenase and acetylCoA carboxylase. These increases are similar to the maximum effects of insulin. Incubation of isolated fat cells with the serum at the same concentration also resulted in the increased phosphorylation of three intracellular proteins with subunit molecular weights of 130,000, 35,000 and 22,000 to the same extent as observed with insulin. These findings lend further support to the view that the short term effects of insulin do not involve the entry of the insulin molecule (or part thereof) into cells of target tissues.

Adrenaline and the regulation of acetyl-coenzyme A carboxylase in rat epididymal adipose tissue. Inactivation of the enzyme is associated with phosphorylation and can be reversed on dephosphorylation.

1. Exposure of rat epididymal fat-pads or isolated fat-cells to adrenaline results in a decrease in acetyl-CoA carboxylase activity measured both in initial extracts and in extracts incubated with potassium citrate; in addition the concentration of citrate required to give half-maximal activation may also be increased. 2. Incorporation of 32Pi into acetyl-CoA carboxylase within intact fat-cells was investigated and evidence is presented that adrenaline increases the extent of phosphorylation of the enzyme. 3. Dephosphorylation of 32P-labelled acetyl-CoA carboxylase was studied in cell extracts. The rate of release of 32P is increased by 5mM-MgCl2 plus 10--100 microM-Ca2+, whereas it is inhibited by the presence of bivalent metal ion chelators such as EDTA and citrate. 4. The effects of adrenaline on the kinetic properties of acetyl-CoA carboxylase disappear if pad or cell extracts are treated with Mg2+ and Ca2+ under conditions that also lead to dephosphorylation of the enzyme. 5. The results of this study represent convincing evidence that adrenaline inactivates acetyl-CoA carboxylase in adipose-tissue preparations by increasing the degree of phosphorylation of the enzyme.

Demonstration of the phosphorylation of acetyl-coenzyme A carboxylase within intact rat epididymal fat-cells.

Intact rat epididymal fat-cells were incubated with 32Pi and the intracellular proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the phosphorylated proteins has the same RF value as [14C]biotin-labelled acetyl-CoA carboxylase purified from fat-cells and is specifically precipitated after incubation with antiserum raised against acetyl-CoA carboxylase. No significant changes in the extent of phosphorylation of acetyl-CoA carboxylase were detected after exposure of the cells to insulin.