Transfer of reducing equivalents into mitochondria by the interconversions of proline and Δ1-pyrroline-5-carboxylate

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers ³H from [1-³H]glucose to NADP⁺ to Δ¹-pyrroline-5-carboxylate and yields [³H]proline. The formation of [³H]proline depends on the presence of NADP, Δ¹-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and Δ¹-pyrroline-5-carboxylate reductase. The production of [³H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to Δ¹-pyrroline-5-carboxylate by mitochondrial proline oxidase. Δ¹-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by Δ¹-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of Δ¹-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.     

Generation of nitric oxide by human neutrophils

Human neutrophils were evaluated for their ability to generate nitric oxide. Neutrophils incubated with superoxide dismutase at 37 degrees C produce nitrite anion at a rate of 1.8 nmols/2 x 10(6) cells/30 min, providing indirect evidence of nitric oxide production. Incubation of the neutrophils with concentrations of serum-opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine, or phorbol myristate acetate sufficient to stimulate the respiratory burst and lysosomal enzyme release caused no additional nitrite anion production. Glass wool-adherent neutrophils exhibited a similar dissociation of nitrite anion production from the respiratory burst and lysosomal enzyme release. Direct evidence for nitric oxide production was also obtained using nitric oxide-specific chemiluminescence. These results demonstrate that human neutrophils are capable of generating nitric oxide.     

The phagovar variability of Listeria strains under the influence of virulent and temperate bacteriophages

Selected strains of Listeria spp. were challenged against a variety of bacteriophages usually employed for phage typing. The resistant mutants derived were characterized by the loss of sensitivity to defined groups of phages. When different phages were used in succession multiple mutants could be obtained. They eventually became insensitive to all phages employed. Our results indicate the possibility of shifting phagovars among Listeria strains grown in mixed culture, due to the potential action of free bacteriophages.     

Lipid composition and food quality of some freshwater phytoplankton for cladoceran zooplankters

The nutritional value of several planktonic algae was testedby means of feeding trials with three cladoceran zooplankters.The algae were monocultures and included two blue-greens, fourgreens and four flagellates with a size range of 5–48µm. The specific growth rates of the zooplankters werechosen as the measure of the nutritional value of the algae.The three cladocerans showed large differences in growth ratein the different algae, but the two cryptomonads were withoutdoubt best suited as food for all. The fatty acid compositionfor the cryptomonads were different from the other algae. Theycontained high percentages of the polyunsaturated fatty acids20:5æ3 (EPA) and 22:6æ3 (DHA), which also are commonin fish. It is suggested that the lipid composition is a probablefactor determining the nutritional quality of the algae.     

BRAIN TISSUE AND PLASMA ASSAY OF L-DOPA AND α-METHYLDOPA METABOLITES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

Using reverse phase high-performance liquid chromatography and electrochemical detection with mobile phases composed of simple acids, we have developed an assay technique to measure multiple catecholamines and their catechol metabolites in plasma or brain tissue with sensitivity to the picomole level. Ion-pairing chromatography with nitric or trichloroacetic acid as the mobile phase permits separation and quantitation of norepinephrine, α-methylnorepinephrine, epinephrine, dopamine, α-methyldopamine, l -DOPA, α-methyldopa, carbidopa, and DOPAC. Alumina extraction selectively isolates catechols which are then separated on a reverse-phase column and measured by a commercially available electrochemical detector. This method has been applied to measurement of L-DOPA metabolites in patients with Parkinson's disease treated with L-DOPA and carbidopa and to measurement of catecholamines in rat hypothalamus in the course of studies on L-DOPA and α-methyldopa metabolism. Dihydroxybenzylamine is added as an internal standard and standard curves are linear over two orders of magnitude in concentration with coefficients of variation averaging 3.1%. Quantitation is routinely done to 20 pmol with absolute sensitivity possible to 0.5 pmol.     

Drug-Induced Changes in Blood Pressure Lead to Changes in Extracellular Concentrations of Epinephrine, Dihydroxyphenylacetic Acid, and 5-Hydroxyindoleacetic Acid in the Rostral Ventrolateral Medulla of the Rat

Neurochemical changes in the extracellular fluid of the rostral ventrolateral medulla (RVLM) were produced by changes in arterial blood pressure. Blood pressure was raised or lowered with systemic infusions of phenylephrine or nitroprusside and neurochemicals were recovered from RVLM by in vivo microdialysis. A dialysis probe 300 microns in diameter and 500 microns in length was stereotaxically implanted in the RVLM of the urethane-anesthetized rat. Sterile physiological Ringer's solution was perfused at a rate of 1.5 microliter/min. The perfusate was collected under ice-cold conditions every 15 min for the assay of epinephrine, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), ascorbic acid, and uric acid. After stable baseline neurochemical concentrations were achieved, animals were infused with phenylephrine or nitroprusside intravenously to raise or lower the blood pressure. Increasing blood pressure 50 mm Hg above the baseline value by phenylephrine led to a significant reduction in heart rate and a reduction in extracellular epinephrine and DOPAC concentrations. The 5-HIAA concentration was increased during the hypertensive drug infusion. There were no changes in the concentrations of ascorbic acid or uric acid. Hypotension produced by nitroprusside (-20 mm Hg) led to neurochemical changes which were the reciprocal of those seen during hypertension. During hypotension, heart rate increased as did the extracellular fluid epinephrine concentration. The 5-HIAA concentration fell with hypotension and remained depressed following the nitroprusside infusion. Ascorbic acid and uric acid concentrations did not change during hypotension but ascorbic acid did increase after the nitroprusside infusion stopped. These data provide direct evidence that epinephrine release in RVLM is linked to changes in systemic blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

One- and two-dimensional proton NMR studies of cys-102 S-methylated yeast isozyme-1 ferricytochrome c.

The effect of S-methylating cysteine-102 (cys-102) (SH----SSCH3) of yeast isozyme-1 (iso-1) ferricytochrome c has been studied using proton NMR spectroscopy. COSY, NOESY, and one-dimensional nuclear Overhauser effect (NOE) difference spectroscopies have all been used. The NMR spectrum of this derivative is very similar to that of native yeast iso-1 ferricytochrome c. The advantage of using the cys-102 S-methylated derivative is that it is unable to spontaneously dimerize in solution, like native iso-1 monomer does. This makes the derivative a simple, ideal protein for long NMR experiments. This work yields many proton resonance assignments for S-methylated yeast iso-1 monomer and confirms all of the assignments for iso-1 monomer that were previously made using only the one-dimensional NOE method.     

Carolina Chickadee (Aves, Paridae, Poecile carolinensis) Vocalization Rates: Effects of Body Mass and Food Availability under Aviary Conditions

We evaluated the effect of body mass and several environmental factors on vocalization rates of Carolina chickadees ( Poecile carolinensis ) housed in an aviary. Two different nonsong vocalizations (tseet and chick-a-dee) and song (fee-bee fee-bay) were recorded. Food was delivered from a feeder and three different levels of food access were presented to each bird: 10, 40 and 55 min/d. Two scales of body mass were measured: 'dawn mass' and 'focal mass' (mass during a focal observation divided by dawn mass). Across all birds, there was a significant negative correlation between both nonsong vocalization rates and body mass (both dawn and focal mass) and the effect of mass on call rate was greatest for days when food was relatively abundant. Nonsong vocalizations were also given at higher rates when food was limited (10 min/d) than when food was more abundant (40 and 55 min/d). No changes in call rates with time of day were observed. Overall, song rates were substantially lower than nonsong rates. Unlike nonsong rates, song rates were highest in birds that had relatively high dawn mass. No significant correlation between song rates and focal mass was observed, and no significant correlation between song rates and time of day was observed. Finally, vocalizations from nonfocal birds had little effect on vocalization rates of focal birds. Our results suggest that nonsong communicative signals are more important for birds facing energetic stress, while song is more important for birds that are not energetically stressed.