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PubMed1977

Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Mitochondrial Respiration and Several Dehydrogenases during Imbibition and Germination.

The influence of low temperature on soybean (Glycine max [L.] Merr. cv. Wells) energy transduction via mitochondrial respiration and dehydrogenases was investigated in this study during imbibition and germination. Mitochondria were isolated from embryonic axes of seeds treated at 10 and 23 C (control) by submergence in H(2)O for 6 hours and maintenance for an additional 42 hours in a moist environment. Arrhenius plots of initial respiration rates revealed that those from cold-treated axes had respiratory control (RC) ratios of near 1.0 above an inflection in the plot at 8 C. Arrhenius plots of control axes mitochondrial respiration showed RC ratios of 2.8 above and 5.0 below an inflection temperature of 12.5 C. Energies of activation for mitochondrial respiration between 20 and 30 C for the cold and control treatments were 7.8 and 15.6 kcal/mole, respectively. These data indicate possible differences in mitochondrial membranes, degree of mitochondrial integrity, and mitochondrial enzyme complement between the two treatments.Glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6P-DH), and NADP-isocitrate dehydrogenase (NADP-ICDH) were assayed from whole seeds and axes (after germination) during the 48 hours of temperature treatments. Activity of these dehydrogenases decreased during the first 6 hours with the exception of MDH. After germination at 23 C (48 hours) all five dehydrogenases increased in activity. Arrhenius plots of cotyledon dehydrogenase activities indicated that one inflection temperature between 6 and 18 C was present for each enzyme assayed. Differences were seen in Arrhenius plots of axes dehydrogenase activities with the two temperature treatments in the cases of GDH and MDH from mitochondrial pellets and with differences in enzyme extraction media. These data suggest that the temperature treatments yield differences in mitochondrial enzyme complement. There were no detectable inflection temperatures for the activities of G6P-DH and ADH extracted from axes. Arrhenius plots of NADP-ICDH activity indicated extreme cold sensitivity. The slopes of the plots for axes NADP-ICDH were very similar to those for mitochondrial respiration (23 C treatment) suggesting that this enzyme may limit mitochondrial respiration at low temperature in soybean tissues grown at moderate temperatures.

Journal Article↗
PubMed1977

Respiration and the energy requirement for nitrogen fixation in nodulated pea roots.

Pisum sativum L. cv. Trapper plants were inoculated and grown in a controlled environment on N-free nutrient solution. After 4 weeks N was supplied to treatment plants as NH(4)NO(3), KNO(3), or NH(4)Cl and rates of C(2)H(2) reduction, root + nodule respiration, and leaf photosynthesis were determined 1 week later. The increase in respiration per unit of C(2)H(2) reduction was not affected by either the form of N added or the light conditions during growth, although the basal respiration rate with no C(2)H(2) reduction increased with irradiance level. The mean regression coefficient from plots of respiration versus C(2)H(2) reduction was 0.23 + 0.04 (P [unk] .01) mg of CO(2) (mumol of C(2)H(2) reduced)(-1) which was very similar to the value for the coefficient of respiration associated with nitrogenase activity estimated by subtracting growth and maintenance respiration. Since the rate of N accumulation in N-free nutrient conditions was proportional to the rate of C(2)H(2) reduction, it appears that the method gives a true estimate of the energy requirements for N fixation which for these conditions was equivalent to 17 grams of carbohydrate consumed per gram of N fixed.

Journal Article↗
PubMed1983

Relation between Respiration and Senescence in Oat Leaves.

The respiration of excised oat (Avena sativa cv Victory) leaves and their sensitivity to inhibitors was followed during senescence under varied conditions. The respiration rate, which in controls reaches its peak on the third day in darkness, is lowered at the time of fastest loss of chlorophyll (as reported earlier) by seven unrelated reagents that all delay dark senescence. When senescence is delayed by white light or by cytokinins, the respiratory rise is correspondingly delayed. Kinetin and l-serine, which act as antagonists on senescence, also act as antagonists on the respiratory rate. However, an exception to this close correspondence between senescence and the respiratory rise is offered by the lower aliphatic alcohols, which delay dark senescence and yet accelerate the onset of the respiratory rise.The respiration of freshly cut leaves is insensitive to KCN up to 8 millimolar, but sensitive to benzhydroxamate (BAM), 1 to 2 millimolar BAM causing 25% promotion and higher concentrations inhibiting. At the respiratory peak, however, part of the respiration becomes KCN-sensitive. Low concentrations of alcohols in darkness, or 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, in light, also render part of the respiration KCN-sensitive, but this sensitivity soon disappears again. Some 10 to 15% of the respiration is insensitive to both inhibitors. Thus, cyanide sensitivity comes and goes, while BAM sensitivity is always present. The current concept of the cyanide-resistant pathway as an overflow, therefore, does not fit well with behavior of these leaves. The respiratory rise in leaf senescence is similar to, but not identical with, the climacteric in ripening fruits and the aging phenomenon in tuber slices.

Journal Article↗
PubMed1984

Respiration during Postharvest Development of Soursop Fruit, Annona muricata L.

Fruit of soursop, Annona muricata L., showed increased CO(2) production 2 days after harvest, preceding the respiratory increase that coincided with autocatalytic ethylene evolution and other ripening phenomena. Experiments to alter gas exchange patterns of postharvest fruit parts and tissue cylinders had little success.The respiratory quotient of tissue discs was near unity throughout development. 2,4-Dinitrophenol uncoupled respiration more effectively than carbonylcyanide m-chlorophenylhydrazone; 0.4 millimolar KCN stimulated, 4 millimolar salicylhydroxamic acid slightly inhibited, and their combination strongly inhibited respiration, as did 10 millimolar NaN(3). Tricarboxylic acid cycle members and ascorbate were more effective substrates than sugars, but acetate and glutarate strongly inhibited.Disc respiration showed the same early peak as whole fruit respiration; this peak is thus an inherent characteristic of postharvest development and cannot be ascribed to differences between ovaries of the aggregatetype fruit. The capacity of the respiratory apparatus did not change during this preclimacteric peak, but the contents of rate-limiting malate and citrate increased after harvest.It is concluded that the preclimacteric rise in CO(2) evolution reflects increased mitochondrial respiration because of enhanced supply of carboxylates as a substrate, probably induced by detachment from the tree. The second rise corresponds with the respiration during ripening of other climacteric fruits.

Journal Article↗
PubMed1985

Differential control of ethylene-induced gene expression and respiration in carrot roots.

Ethylene treatment of carrot roots elicits a developmental program encompassing an increase in respiration rate and changes in gene expression. Both phenomena are potentiated when ethylene is administered in O(2). Our previous studies showed that both respiration and a number of ethylene specific mRNAs increase together in response to ethylene through some 21 hours, whereas thereafter respiration continues to rise, while the level of induced mRNAs drops. Herein we ask whether an experimentally effected drop in the respiration rate within the first 21 hours caused by the withdrawal of ethylene, or substitution of air for O(2) in the continued presence of ethylene, is linked to a drop in the level of ethylene-induced mRNA. Quantitative estimation of two ethylene evoked mRNAs by dot blot hybridization with appropriate cDNA clones has shown that under the specified treatment the induced mRNA levels remain constant while the respiration rate drops, suggesting that gene expression, as reflected in induced mRNA levels, and respiration rate are separately regulated facets of the ethylene response.

Journal Article↗
PubMed2006

Real-time monitoring of respiration rhythm and pulse rate during sleep.

A noninvasive and unconstrained real-time method to detect the respiration rhythm and pulse rate during sleep is presented. By employing the a trous algorithm of the wavelet transformation (WT), the respiration rhythm and pulse rate can be monitored in real-time from a pressure signal acquired with a pressure sensor placed under a pillow. The waveform for respiration rhythm detection is derived from the 26 scale approximation, while that for pulse rate detection is synthesized by combining the 2(4) and 2(5) scale details. To minimize the latency in data processing and realize the highest real-time performance, the respiration rhythm and pulse rate are estimated by using waveforms directly derived from the WT approximation and detail components without the reconstruction procedure. This method is evaluated with data collected from 13 healthy subjects. By comparing with detections from finger photoelectric plethysmograms used for pulse rate detection, the sensitivity and positive predictivity were 99.17% and 98.53%, respectively. Similarly, for respiration rhythm, compared with detections from nasal thermistor signals, results were 95.63% and 95.42%, respectively. This study suggests that the proposed method is promising to be used in a respiration rhythm and pulse rate monitor for real-time monitoring of sleep-related diseases during sleep.

Adult↗
PubMed1988

Altered relationship between protonmotive force and respiration rate in non-phosphorylating liver mitochondria isolated from rats of different thyroid hormone status.

We have determined the relationship between rate of respiration and protonmotive force in oligomycin-inhibited liver mitochondria isolated from euthyroid, hypothyroid and hyperthyroid rats. Respiration rate was titrated with the respiratory-chain inhibitor malonate. At any given respiration rate mitochondria isolated from hypothyroid rats had a protonmotive force greater than mitochondria isolated from euthyroid controls, and mitochondria isolated from hyperthyroid rats had a protonmotive force less than mitochondria isolated from euthyroid controls. In the absence of malonate mitochondrial respiration rate increased in the order hypothyroid less than euthyroid less than hyperthyroid, while protonmotive force increased in the order hyperthyroid less than euthyroid less than hypothyroid. These findings are consistent with a thyroid-hormone-induced increase in the proton conductance of the inner mitochondrial membrane or a decrease in the H+/O ratio of the respiratory chain at any given protonmotive force. Thus the altered proton conductance or H+/O ratio of mitochondria isolated from rats of different thyroid hormone status controls the respiration rate required to balance the backflow of protons across the inner mitochondrial membrane. We discuss the possible relevance of these findings to the control of state 3 and state 4 respiration by thyroid hormone.

Animals↗
PubMed1990

Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the 'top-down' approach of metabolic control theory.

The rate of respiration of isolated mitochondria was set at different values by addition of either oligomycin or an ADP-regenerating system (glucose and different amounts of hexokinase). We measured the relationship between respiration rate and membrane potential as respiration was titrated by the addition of malonate under each condition. We used the flux control summation and connectivity theorems and the branching theorem of metabolic control theory to calculate the control over respiration rate exerted by the respiratory chain (and associated reactions), phosphorylating system (and associated reactions) and proton leak at each respiration rate. The analysis also yielded the flux control coefficients of these three reactions over phosphorylation rate and proton leak rate and their concentration control coefficients over protonmotive force. We found that respiration rate was controlled largely by the proton leak under non-phosphorylating conditions, by the phosphorylating system at intermediate rates and by both the phosphorylating system and the respiratory chain in state 3. The rate of phosphorylation was controlled largely by the phosphorylating system itself in state 4 and at intermediate rates, while state 3 control was shared between the phosphorylating system and the respiratory chain; the proton leak had insignificant control. In all states the phosphorylating system had large negative control over the proton leak; the chain and the proton leak both had large positive control coefficients. The protonmotive force was controlled by the chain and by the phosphorylating system; the proton leak had little control.

Adenosine Diphosphate↗
PubMed1997

Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications.

In Escherichia coli the expression of the nuo genes encoding the proton pumping NADH dehydrogenase I is stimulated by the presence of fumarate during anaerobic respiration. The regulatory sites required for the induction by fumarate, nitrate and O2 are located at positions around -309, -277, and downstream of -231 bp, respectively, relative to the transcriptional-start site. The fumarate regulator has to be different from the O2 and nitrate regulators ArcA and NarL. For growth by fumarate respiration, the presence of NADH dehydrogenase I was essential, in contrast to aerobic or nitrate respiration which used preferentially NADH dehydrogenase II. The electron transport from NADH to fumarate strongly decreased in a mutant lacking NADH dehydrogenase I. The mutant used acetyl-CoA instead of fumarate to an increased extent as an electron acceptor for NADH, and excreted ethanol. Therefore, NADH dehydrogenase I is essential for NADH-->fumarate respiration, and is able to use menaquinone as an electron acceptor. NADH-->dimethylsulfoxide respiration is also dependent on NADH dehydrogenase I. The consequences for energy conservation by anaerobic respiration with NADH as a donor are discussed.

Acetates↗
PubMed1997

Chemosensory and cholinergic stimulation of fictive respiration in isolated CNS of neonatal opossum.

1. The aim of the present experiments was to characterize the central chemical drive of fictive respiration in the isolated CNS of the newborn opossum, Monodelphis domestica. This opossum preparation, in contrast to those of neonatal rats and mice, produces respiratory rhythm of high frequency in vitro. 2. Fictive respiration was recorded from C3-C5 ventral roots of the isolated CNS of 4- to 14-day-old opossums using suction electrodes. At room temperature (21-23 degrees C) the frequency of respiration was 43 +/- 5.3 min-1 (mean +/- S.E.M., n = 50) in basal medium Eagle's medium (BMEM) equilibrated with 5% CO2-95% O2, pH 7.37-7.40. Respiratory discharges remained regular throughout 8 h experiments and continued for more than 20 h in culture. 3. Superfusion of the brainstem confirmed that solutions of pH 6.3-7.2 increased both the amplitude and frequency of respiration. High pH solutions (7.5-7.7) had the opposite effect and abolished the rhythm at pH 7.7. Addition of ACh (50-100 microM) or carbachol (0.01-10 microM) to the brainstem superfusion also increased the amplitude and frequency of respiratory activity, as did physostigmine (50-100 microM) or neostigmine (20-50 microM). Conversely, scopolamine (50-100 microM) reduced the amplitude and frequency of the basal respiratory rhythm by about 30%. 4. H(+)- and cholinergic-sensitive areas on the surface of the isolated CNS were explored with a small micropipette (outer tip diameter, 100 microns) filled with BMEM (pH 6.5) or 1 microM carbachol. Carbachol applied to H(+)- and cholinergic-sensitive areas in the ventral medulla mimicked the changes of respiratory pattern produced by low pH application. Responses to altered pH and carbachol were abolished by scopolamine (50 microM). Histochemistry demonstrated several medullary groups of neurons stained for acetylcholinesterase. The superficial location of one of these groups coincided with a functional and anatomically well-defined pH- and carbachol-sensitive area placed medial to the hypoglossal roots. 5. Exploration of chemosensitive areas revealed that application of drugs or solutions of different pH to a single well-defined spot could have selective and distinctive effects upon amplitude and frequency of respiratory activity. 6. These results show that fictive respiration in the isolated CNS of the newborn opossum is tonically driven by chemical- and cholinergic-sensitive areas located on the ventral medulla, the activity of which regulates frequency and amplitude of respiration. They suggest that a cholinergic relay, although not essential for rhythm generation, is involved in the central pH chemosensory mechanism, or that cholinergic and chemical inputs converge upon the same input pathway to the respiratory pattern generator.

Acetylcholinesterase↗
PubMed2001

The role of phosphorylcreatine and creatine in the regulation of mitochondrial respiration in human skeletal muscle.

1. The role of phosphorylcreatine (PCr) and creatine (Cr) in the regulation of mitochondrial respiration was investigated in permeabilised fibre bundles prepared from human vastus lateralis muscle. 2. Fibre respiration was measured in the absence of ADP (V(0)) and after sequential additions of submaximal ADP (0.1 mM ADP, V(submax)), PCr (or Cr) and saturating [ADP] (V(max)). 3. V(submax) increased by 55 % after addition of saturating creatine (P < 0.01; n = 8) and half the maximal effect was obtained at 5 mM [Cr]. In contrast, V(submax) decreased by 54 % after addition of saturating phosphorylcreatine (P < 0.01; n = 8) and half the maximal effect was obtained at 1 mM [PCr]. V(max) was not affected by Cr or PCr. 4. V(submax) was similar when PCr and Cr were added simultaneously at concentrations similar to those in muscle at rest (PCr/Cr = 2) and at low-intensity exercise (PCr/Cr = 0.5). At conditions mimicking high-intensity exercise (PCr/Cr = 0.1), V(submax) increased to 60 % of V(max) (P < 0.01 vs. rest and low-intensity exercise). 5. Eight of the subjects participated in a 16 day Cr supplementation programme. Following Cr supplementation, V(0) decreased by 17 % (P < 0.01 vs. prior to Cr supplementation), whereas ADP-stimulated respiration (with and without Cr or PCr) was unchanged. 6. For the first time evidence is given that PCr is an important regulator of mitochondrial ADP-stimulated respiration. Phosphorylcreatine decreases the sensitivity of mitochondrial respiration to ADP whereas Cr has the opposite effect. During transition from rest to high-intensity exercise, decreases in the PCr/Cr ratio will effectively increase the sensitivity of mitochondrial respiration to ADP. The decrease in V(0) after Cr supplementation indicates that intrinsic changes in membrane proton conductance occur.

Adenosine Diphosphate↗
PubMed1986

Hyperglycemia preserves brain mitochondrial respiration during anoxia.

We examined brain mitochondrial function in normo- (5 mM) and hyperglycemic (50 mM) cats after 8 min of anoxia. In anoxic normoglycemic cats, mitochondrial state 3 respiration with NAD-linked substrates glutamate or pyruvate (both plus malate) was inhibited 30-50%. The uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) maximally stimulated respiration, indicating that inhibition of phosphorylation, not impairment of electron transport, substrate transport, or oxidation was present. State 3 respiration with succinate (plus rotenone) was unaffected. Mitochondrial respiratory control ratios trended toward reductions whereas ADP/O ratios remained unchanged. In contrast, brain mitochondria from anoxic hyperglycemic cats showed no such inhibition of state 3 respiration and no differences in function from normo- and hyperglycemic control animals except for trends toward loose coupling. Significantly higher brain tissue glucose concentrations were present in hyperglycemic controls as the only metabolite difference compared to normoglycemic controls. At the end of anoxia, hyperglycemic cats exhibited significantly higher cortical lactate and glucose levels but similarly reduced high-energy phosphate concentrations compared to normoglycemic cats. These results demonstrate that increased availability of glucose to gray matter as a consequence of hyperglycemia maintains normal mitochondrial state 3 respiration during exposure to anoxia. Previous survival studies have shown that lower serum glucose concentrations during anoxia are relatively brain protective. This result indicates that the presently described alterations in mitochondrial respiration must be fully reversible.

Animals↗
PubMed1990

Effect of peroxide, sodium, and calcium on brain mitochondrial respiration in vitro: potential role in cerebral ischemia and reperfusion.

Mitochondrial pyruvate-supported respiration was studied in vitro under conditions known to exist following ischemia, i.e., elevated extramitochondrial Ca2+, Na+, and peroxide. Ca2+ alone (7-10 nmol/mg) decreased state 3 and increased state 4 respiration to 81 and 141% of control values, respectively. Sodium (15 mM) and/or tert-butyl hydroperoxide (tBOOH; up to 2,000 nmol/mg protein) alone had no effect on respiration; however, Na+ or tBOOH in combination with Ca2+ dramatically altered respiration. Respiratory inhibition induced by Ca2+ and tBOOH does not involve pyruvate dehydrogenase (PDH) inhibition since PDH flux increased linearly with tBOOH concentration (R = 0.96). Calcium potentiated tBOOH-induced mitochondrial NAD(P)H oxidation and shifted the redox state of cytochrome b from 67 to 47% reduced. Calcium (5.5 nmol/mg) plus Na+ (15 mM) decreased state 3 and increased state 4 respiratory rates to 55 and 202% of control values, respectively. Sodium- as well as tBOOH-induced state 3 inhibition required mitochondrial Ca2+ uptake because ruthenium red addition before Ca2+ addition negated the effect. The increase in state 4 respiration involved Ca2+ cycling since ruthenium red immediately returned state 4 rates back to control values. The mechanisms for the observed Ca2(+)-, Na(+)-, and tBOOH-induced alterations in pyruvate-supported respiration in vitro are discussed and a multifactorial etiology for mitochondrial respiratory dysfunction following cerebral ischemia in vivo is proposed.

Animals↗
PubMed2003

Respiration-correlated spiral CT: a method of measuring respiratory-induced anatomic motion for radiation treatment planning.

We describe a method for generating CT images at multiple respiratory phases with a single spiral CT scan, referred to as respiratory-correlated spiral CT (RCCT). RCCT relies on a respiration wave form supplied by an external patient monitor. During acquisition this wave form is recorded along with the initiation time of the CT scan, so as to "time stamp" each reconstructed slice with the phase of the respiratory cycle. By selecting the appropriate slices, a full CT image set is generated at several phases, typically 7-11 per cycle. The CT parameters are chosen to optimize the temporal resolution while minimizing the spatial gap between slices at successive respiratory cycles. Using a pitch of 0.5, a gantry rotation period of 1.5 s, and a 180 degrees reconstruction algorithm results in approximately 5 mm slice spacing at a given phase for typical respiration periods, and a respiratory motion within each slice that is acceptably small, particularly near end expiration or end inspiration where gated radiotherapy is to occur. We have performed validation measurements on a phantom with a moving sphere designed to simulate respiration-induced tumor motion. RCCT scans of the phantom at respiratory periods of 4, 5, and 6 s show good agreement of the sphere's motion with that observed under fluoroscopic imaging. The positional deviations in the sphere's centroid between RCCT and fluoroscopy are 1.1+/-0.9 mm in the transaxial direction (average over all scans at all phases +/-1 s.d.) and 1.2+/-1.0 mm in the longitudinal direction. Reconstructed volumes match those expected on the basis of stationary-phantom scans to within 5% in all cases. The surface distortions of the reconstructed sphere, as quantified by deviations from a mathematical reference sphere, are similar to those from a stationary phantom scan and are correlated with the speed of the phantom. A RCCT scan of the phantom undergoing irregular motion, demonstrates that successful reconstruction can be achieved even with irregular respiration. Limitations from x-ray tube heating in our current CT unit restrict the length of the scan region to 9 cm for the RCCT settings used, though this will not be a limitation for a multislice scanner. RCCT offers an alternative to the current method of respiration-triggered axial scans. Multiple phases of respiration are imaged with RCCT in approximately the same scanning time required to image a single phase with a triggered axial scan. RCCT scans can be used in connection with respiratory-gated treatment to identify the patient-specific phase of minimum tumor motion, determine residual tumor motion within the gate interval, and compare treatment plans at different phases.

Artifacts↗
PubMed2003

Quantifying the predictability of diaphragm motion during respiration with a noninvasive external marker.

The aim of this work was to quantify the ability to predict intrafraction diaphragm motion from an external respiration signal during a course of radiotherapy. The data obtained included diaphragm motion traces from 63 fluoroscopic lung procedures for 5 patients, acquired simultaneously with respiratory motion signals (an infrared camera-based system was used to track abdominal wall motion). During these sessions, the patients were asked to breathe either (i) without instruction, (ii) with audio prompting, or (iii) using visual feedback. A statistical general linear model was formulated to describe the relationship between the respiration signal and diaphragm motion over all sessions and for all breathing training types. The model parameters derived from the first session for each patient were then used to predict the diaphragm motion for subsequent sessions based on the respiration signal. Quantification of the difference between the predicted and actual motion during each session determined our ability to predict diaphragm motion during a course of radiotherapy. This measure of diaphragm motion was also used to estimate clinical target volume (CTV) to planning target volume (PTV) margins for conventional, gated, and proposed four-dimensional (4D) radiotherapy. Results from statistical analysis indicated a strong linear relationship between the respiration signal and diaphragm motion (p<0.001) over all sessions, irrespective of session number (p=0.98) and breathing training type (p=0.19). Using model parameters obtained from the first session, diaphragm motion was predicted in subsequent sessions to within 0.1 cm (1 sigma) for gated and 4D radiotherapy. Assuming a 0.4 cm setup error, superior-inferior CTV-PTV margins of 1.1 cm for conventional radiotherapy could be reduced to 0.8 cm for gated and 4D radiotherapy. The diaphragm motion is strongly correlated with the respiration signal obtained from the abdominal wall. This correlation can be used to predict diaphragm motion, based on the respiration signal, to within 0.1 cm (1 sigma) over a course of radiotherapy.

Artifacts↗
PubMed1978

Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration.

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Respiring bacteria deposit accumulated INT-formazan intracellularly as dark red spots. Corresponding to electron transport system activity, these deposits attain a size and a degree of optical density which allows them to be examined by light microscopy. If polycarbonate filters and epifluorescence microscopy are applied to analyze an INT-treated water sample, it is possible to differentiate between respiring and apparently nonrespiring bacteria. This differentiation, which permits determinations of the total number of bacteria and the proportion thereof involved in respiration, is realized directly within one and the same microscopic image. Initial applications of the present method for hydrobiological purposes showed that the proportion of respiring aquatic bacteria ranged between 6 to 12% (samples taken from coastal areas of the Baltic Sea) and 5 to 36% (samples taken from freshwater lakes and ponds). Cells of 1.6 to 2.4 micrometer (freshwater) and 0.4 micrometer (Baltic Sea) account for the highest proportion of respiring bacteria.

Bacteria↗
PubMed1982

Respiration of tritiated substrates in heterotrophic activity assays.

The respiration percentage of H-labeled glucose by heterotrophic microorganisms in water samples was determined by a method based on equimolar addition of [H]- and [C]glucose to samples. The respiration percentage of [H]glucose exceeded that of [C]glucose by 10% over the assayed range of respiration rates. Respiration percentages diminished with decreasing temperature, the relation between respiration of [H]- and [C]glucose remaining the same. The significance of tritiated water respiration with regard to experimental design is emphasized.

Journal Article↗
PubMed2003

Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA.

Our goal was to develop a field soil biodegradation assay using (13)C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived (13)C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of (13)C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for (13)CO(2) respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of (13)CO(2) emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF(6), that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired (13)CO(2). Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of (13)CO(2) released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of (13)C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with (13)C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.

Bacteria↗