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PubMed2001

Energetics and litter size variation in domestic dog Canis familiaris breeds of two sizes.

We measured resting metabolic rate (RMR), daily energy expenditure (DEE) and metabolisable energy intake (MEI) in two breeds of dog during peak lactation to test whether litter size differences were a likely consequence of allometric variation in energetics. RMR of Labrador retrievers (30 kg, n=12) and miniature Schnauzers (6 kg, n=4) averaged 3437 and 1062 kJ/day, respectively. DEE of Labradors (n=6) and Schnauzers (n=4) averaged 9808 and 2619 kJ/day, respectively. MEI of Labradors (n=12) was 22448 kJ/day and of Schnauzers (n=7) was 5382 kJ/day. DEE of Labrador pups (2.13 kg, n=19) was 974 kJ/day and Schnauzers (0.89 kg, n=7) were 490 kJ/day. Although Labradors had higher MEIs than Schnauzers during peak lactation, there was no difference in mass-specific energy expenditure between the two breeds. Hence, it is unlikely that litter size variation is a likely consequence of differences in maternal energy expenditure. Individual offspring were relatively more costly for mothers of the smaller breed to produce. Therefore, litter size variations were consistent with the expectation that smaller offspring should be more costly for mothers, but not that smaller mothers should per se invest more resources in reproduction.

Animals↗

PubMed2003

Field energetics of free-living, lactating and non-lactating echidnas (Tachyglossus aculeatus).

We measured daily energy expenditure (DEE) and water turnover rates in lactating and non-lactating short beaked echidnas (Tachyglossus aculeatus) using the doubly labelled water technique during the lactation period in spring. Reproductively inactive echidnas were on average significantly heavier (median: 3354 g; range: 2929-3780 g; N=4) than lactating females (median: 2695 g; range: 2690-2715 g; N=3) during the equivalent time period. The median water flux rate of lactating echidnas (152 ml day(-1); range: 120-198 ml day(-1)) did not differ significantly from that of non-lactating females (170 ml day(-1); range: 128-227 ml day(-1)). The median DEE of echidnas that were lactating was 645 kJ day(-1) (range: 581-850 kJ day(-1)), which was not different from the median DEE of non-reproductive control females (763 kJ day(-1); range: 720-766 kJ day(-1)). Lactating females somehow compensate for the energy costs of milk production, resulting in a daily energy budget that is not different from that of non-reproductive females. At least part of their energy minimising strategy could involve the use of moderate heterothermy, allowing a greater proportion of daily energy expenditure to diverted to milk production.

Animals↗

PubMed1998

Mutagenic and cytotoxic effects of exhaust particulate matter of biodiesel compared to fossil diesel fuel.

The mutagenic and cytotoxic effects of diesel engine exhaust (DEE) from a modern passenger car using rapeseed oil methyl esters (RME, biodiesel) as fuel were directly compared to DEE of diesel fuel (DF) derived from petroleum. Combustion particulate matter was collected on glass fiber filters coated with polytetrafluoroethylene (PTFE) from an exhaust dilution tunnel using three different engine test cycles on a chassis dynamometer. Filters were extracted with dichloromethane in a soxhlet apparatus for 12 h. The mutagenicity of the extracts was tested in the Salmonella typhimurium/mammalian microsome plate-incorporation assay using strains TA97a, TA98, TA100, and TA102. The toxicity to the established cell line L929 (mouse lung fibroblasts) was investigated in the neutral red assay. In the tester strains TA98 and TA100 a significant increase of mutations resulted for the particle extracts of both fuels, but for DF the revertants were significantly higher compared to RME. The highest levels of revertants were observed in tests including a cold start phase. This was probably due to incomplete combustion in the cold engine and a lower conversion rate of the cold catalytic converter. Testing with activated liver S9 fraction induced a slightly lower increase of revertants in most experiments. TA97a and TA102 showed no significant enhancement of spontaneous mutations. In the FTP-75 test cycle RME extracts showed slightly higher toxic effects to the L929 cells than DF, whereas in the other tests no significant differences were observable. These results indicate a higher mutagenic potency of DEE of DF compared to RME. This is probably due to the lower content of polycyclic aromatic compounds (PAC) in RME exhaust, although the emitted masses of RME were higher in most test procedures applied in this study.

Animals↗

PubMed2000

Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes.

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM-N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM-N of cycloheximide or a chelator, 2,2'-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM-N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM-N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM-N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca2+-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.

Algal Proteins↗

PubMed1988

Radioprotection by glutathione esters and cysteamine in normal and glutathione-depleted mammalian cells.

Monoethyl (MEE) and diethyl (DEE) esters of glutathione (GSH) had the capacity to provide some protection of normal and buthionine sulfoximine (BSO) pretreated cells against X-irradiation. Both compounds appeared to be transported through the cell membrane into the cells. MEE was intracellularly partly hydrolysed to GSH and caused a limited rise of intracellular GSH. DEE was intracellularly mainly converted into MEE and partly into GSH. DEE caused a larger rise of the intracellular GSH content than MEE; it also provided a better radioprotection. Radioprotection by the GSH esters may be explained by an increase of intracellular GSH as well as by the presence of the esters themselves. Cysteamine caused no rise of the intracellular GSH content, thus its radioprotection could not be mediated by an increase of intracellular GSH. When the radiosensitivity of GSH-depleted cells protected by cysteamine was compared with the radiosensitivity of non-GSH-depleted cells similarly protected by cysteamine, it appeared that the GSH-depleted cells remained more sensitive to irradiation. Thus, it seems that in this respect cysteamine cannot fully substitute for endogenous GSH.

Animals↗

PubMed2003

Assessment of energy expenditure associated with physical activities in free-living obese and nonobese adolescents.

BACKGROUND: Information on activity patterns and the energy cost of activities is critically missing. OBJECTIVE: We measured the energy cost of and time devoted to various activities in obese and nonobese adolescents. DESIGN: Daily energy expenditure (DEE) and its main components were determined in 27 obese and 50 nonobese adolescents aged 12-16 y by using whole-body calorimetry with the same activity program and the heart rate-recording method in free-living conditions. RESULTS: In whole-body calorimetry, energy expenditures (EEs) during sleep and sedentary activities were 18.9% and 21.5%, respectively, higher in obese subjects than in nonobese subjects (P < 0.001), but not significantly different after adjustment for fat-free mass (FFM). EEs during walking and DEEs were significantly higher in obese than in nonobese subjects, both absolutely (71% and 33%, respectively) and after adjustment for body weight or FFM (16% and 11%, respectively). In free-living conditions, EEs associated with physical activities did not differ significantly between obese and nonobese subjects, but they were 51% lower in obese subjects after adjustment for body weight (P < 0.001). The obese adolescents spent more time in light physical activities but much less time in moderate activities and sports than did the nonobese subjects. The activity-related time equivalent corrected for sedentary EE (ARTE EE(2)) averaged 69 and 122 min/d in obese and nonobese subjects, respectively (P < 0.01). CONCLUSION: Physical activity is low in obese subjects and can be assessed satisfactorily in both obese and nonobese adolescents by using ARTE EE(2) when DEE and the basal metabolic rate are known.

Adolescent↗

PubMed2004

Early enteral nutrition does not decrease hypermetabolism associated with burn injury.

BACKGROUND: A prospective, randomized study was performed to compare the effects of early versus late enteral feeding on postburn metabolism. METHODS: Burn patients were randomized to receive enteral feedings either within 24 hours (early) or 7 days (late) of injury. Basal energy expenditure (BEE) was calculated from Harris-Benedict equations and resting energy expenditure (REE) was obtained from indirect calorimetry. The average daily energy expenditure (DEE) was expressed as REE/BEE. RESULTS: Average age, burn size, infections, and length of stay were similar between groups. Mortality between groups was similar (early, 28%; late, 38%) and not significantly influenced by inhalation injury. When controlled for percentage of total body surface area burn, inhalation injury, and age, the early group had an increased rather than decreased DEE, with a mean DEE calorie 0.17 more than the late group (p = 0.07). CONCLUSION: Early enteral feeding does not decrease the average energy expenditure associated with burn injury.

Adult↗

PubMed2004

Birds sacrifice oxidative protection for reproduction.

Oxidative metabolism has reactive oxygen species (ROS) as unavoidable by-products, and the damage ROS inflicts on DNA, proteins and lipids is considered to be a major agent of senescence. Increasing reproductive effort accelerates senescence, but whether reproductive effort is increased at the expense of protection against oxidative damage has not yet been tested. We manipulated reproductive effort in zebra finches through brood size manipulation and measured the activity of two major antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) in the pectoral muscle after 19-20 days of brood rearing. Oxidative stress is reflected by the balance between oxidative protection and ROS exposure, and we therefore scaled SOD and GPx activity to daily energy expenditure (DEE) as an index of ROS production. SOD and GPx activity decreased with increasing brood size by 28% and 24%, respectively. This effect was identical in the two sexes, but arose in different ways: males did not change their DEE, but had lower absolute enzyme activity, and females increased their DEE, but did not change absolute enzyme activity. This result suggests that senescence acceleration by increased reproductive effort is at least in part mediated by oxidative stress.

Aging↗

PubMed2000

Metabolic compensation during high energy output in fasting, lactating grey seals (Halichoerus grypus): metabolic ceilings revisited.

Lactation is the most energetically expensive period for female mammals and is associated with some of the highest sustained metabolic rates (SusMR) in vertebrates (reported as total energy throughput). Females typically deal with this energy demand by increasing food intake and the structure of the alimentary tract may act as the central constraint to ceilings on SusMR at about seven times resting or standard metabolic rate (SMR). However, demands of lactation may also be met by using a form of metabolic compensation such as reducing locomotor activities or entering torpor. In some phocid seals, cetaceans and bears, females fast throughout lactation and thus cannot offset the high energetic costs of lactation through increased food intake. We demonstrate that fasting grey seal females sustain, for several weeks, one of the highest total daily energy expenditures (DEE; 7.4 x SMR) reported in mammals, while progressively reducing maintenance metabolic expenditures during lactation through means not explained by reduction in lean body mass or behavioural changes. Simultaneously, the energy-exported in milk is progressively increased, associated with increased lipoprotein lipase activity in the mammary gland, resulting in greater offspring growth. Our results suggest that females use compensatory mechanisms to help meet the extraordinary energetic costs of lactation. Additionally, although the concepts of SusMR and ceilings on total DEE may be somewhat different in fasting lactating species, our data on phocid seals demonstrate that metabolic ceilings on milk energy output, in general, are not constrained by the same kind of peripheral limitations as are other energy-consuming tissues. In phocid seals, the high ceilings on DEE during lactation, coupled with metabolic compensation, are undoubtedly important factors enabling shortened lactation.

Animals↗

PubMed2006

The energy costs of sexual dimorphism in mole-rats are morphological not behavioural.

Different reproductive strategies of males and females may lead to the evolution of differences in their energetic costs of reproduction, overall energetic requirements and physiological performances. Sexual dimorphism is often associated with costly behaviours (e.g. large males might have a competitive advantage in fighting, which is energetically expensive). However, few studies of mammals have directly compared the energy costs of reproductive activities between sexes. We compared the daily energy expenditure (DEE) and resting metabolic rate (RMR) of males and females of two species of mole-rat, Bathyergus janetta and Georychus capensis (the former is sexually dimorphic in body size and the latter is not) during a period of intense digging when males seek females. We hypothesized that large body size might be indicative of greater digging or fighting capabilities, and hence greater mass-independent DEE values in males of the sexually dimorphic species. In contrast to this prediction, although absolute values of DEE were greater in B. janetta males, mass-independent values were not. No differences were apparent between sexes in G. capensis. By comparison, although RMR values were greater in B. janetta than G. capensis, no differences were apparent between the sexes for either species. The energy cost of dimorphism is most likely to be the cost of maintenance of a large body size, and not the cost of behaviours performed when an individual is large.

Animals↗

PubMedDate not supplied

Exposure to diesel exhaust emissions on board locomotives.

Measurements of diesel exhaust emissions (DEEs) were taken in the cabs of leading and trailing locomotives on 48 runs, under winter and summer conditions, on 9 different routes. The cab windows were kept open during the summer runs and closed during the winter runs. The average measurement duration was 9.5 hours. There was virtually no exposure to DEEs in the lead locomotives during winter or summer and very little in the trailing locomotives during winter. The average elemental carbon (EC) concentration in the trailing units of the summer trials was greater than or equal to the proposed American Conference of Governmental Industrial Hygienists' threshold limit value/time-weighted average of 20 microg/m(3) on 26% of the runs, and was greater than or equal to 10 microg/m(3) on 63%. The concentrations of the gaseous components (nitric oxide, nitrogen dioxide, and carbon monoxide) were from 10 to 20 times below their respective threshold limit values. Mean EC concentration was 2.9 microg/m(3) (detection limit 2 microg/m(3)) during the winter runs and 17.1 microg/m(3) during summer. DEEs appeared to be fairly uniformly distributed in the trailing cabs. Configuration of the locomotives had a major impact on EC concentration, with the mean concentration being nearly three times higher in the forward-backward mode than in the forward-forward mode. Descriptive statistics such as means, medians, standard deviations, and so forth, are provided. Various types of statistical comparisons are reported. Recommendations for controlling exposure are made.

Carbon↗

PubMed2005

Relationship between daily physical activity and ANS activity in patients with CHF.

PURPOSE: Heart rate variability (HRV) indices are powerful independent prognostic factors of cardiovascular events and all cause mortality in patients with chronic heart failure (CHF). This study evaluates the influence of lifestyle on HRV in CHF patients. METHODS: Thirty-nine CHF patients (33 men, ischemic/dilated cardiomyopathy (18/21), 52.4 +/- 11.2 yr, NYHA I to III, LVEF 33.4 +/- 5.1%) filled out a physical activity questionnaire providing an individual complete qualitative and quantitative picture of their physical activity and daily energy expenditure (PAEE/DEE) corrected for age, weight, severity of the condition and autonomy. Frequency and time domain indices of HRV were calculated from ECG Holter recordings on a typical weekday. Nighttime indices were calculated in order to avoid the confounding factor of physical activity that might alter 24-h frequency analysis of HRV. RESULTS: DEE was significantly different between classes I and II and classes I and III (P = 0.01 both) patients. Time spent in activities above 3 METs decreased significantly with the severity of the condition. Global and parasympathetic indices of 24-h HRV analysis were correlated to DEE and PAEE (Ptot24h = 78.80*PAEE (J x min x kg) -1061.80, R = 0.72, P < 0.0001). Multiple regression analysis revealed that PAEE was the sole independent factor on established HRV prognostic indices (P < 0.05) and especially within PAEE dimensions, only activities above 3 METs were correlated with established prognostic HRV indices (P < 0.05). CONCLUSION: These results indicate that rather than total DEE, moderate to intensive physical activity may counteract the decline in HRV with chronic heart disease. This may be linked to longer time spent in higher intensity activities, and not to total activity time.

Adult↗

PubMed2004

Effects of low sulfur fuel and a catalyzed particle trap on the composition and toxicity of diesel emissions.

In this study we compared a "baseline" condition of uncontrolled diesel engine exhaust (DEE) emissions generated with current (circa 2003) certification fuel to an emissions-reduction (ER) case with low sulfur fuel and a catalyzed particle trap. Lung toxicity assessments (resistance to respiratory viral infection, lung inflammation, and oxidative stress) were performed on mice (C57Bl/6) exposed by inhalation (6 hr/day for 7 days). The engine was operated identically (same engine load) in both cases, and the inhalation exposures were conducted at the same exhaust dilution rate. For baseline DEE, this dilution resulted in a particle mass (PM) concentration of approximately 200 microg/m3 PM, whereas the ER reduced the PM and almost every other measured constituent [except nitrogen oxides (NOx)] to near background levels in the exposure atmospheres. These measurements included PM, PM size distribution, PM composition (carbon, ions, elements), NOx, carbon monoxide, speciated/total volatile hydrocarbons, and several classes of semivolatile organic compounds. After exposure concluded, one group of mice was immediately sacrificed and assessed for inflammation and oxidative stress in lung homogenate. Another group of mice were intratracheally instilled with respiratory syncytial virus (RSV), and RSV lung clearance and inflammation was assessed 4 days later. Baseline DEE produced statistically significant biological effects for all measured parameters. The use of low sulfur fuel and a catalyzed trap either completely or nearly eliminated the effects.

Animals↗

PubMed1999

Urinary continence after staged bladder reconstruction for cloacal exstrophy: the effect of coexisting neurological abnormalities on urinary continence.

PURPOSE: We determined whether there is a difference in the incidence of urinary continence in cloacal and classic bladder exstrophy after staged bladder neck reconstruction using the Young-Dees-Leadbetter technique. MATERIALS AND METHODS: We reviewed the records of patients with cloacal and classic bladder exstrophy who underwent staged bladder neck reconstruction from 1971 to 1997. RESULTS: The Young-Dees-Leadbetter bladder neck reconstruction was completed in 23 patients with cloacal exstrophy, of whom 5 (22%) became continent and 18 (78%) have persistent incontinence. A clinically apparent neurological abnormality significantly hindered the achievement of continence. Specifically 1 of the 13 children (7%) with versus 4 of the 10 (40%) without a neurological abnormality became continent (p <0.05). In contrast, staged reconstruction of classic bladder exstrophy resulted in urinary continence in 67 of the 82 patients (82%). Of the 67 continent patients 23 (34%) cannot void and require intermittent catheterization. None of the patients with classic exstrophy had a neurological deficit. CONCLUSIONS: The ability of the Young-Dees-Leadbetter bladder neck reconstruction to result in urinary continence significantly differs in the cloacal and classic bladder exstrophy populations (22 versus 82%, p <0.001). Our findings also suggest that a coexisting neurological abnormality significantly hinders the ability to reconstruct a functional bladder in patients with cloacal exstrophy.

Bladder Exstrophy↗

PubMed1999

Strategies for reconstruction after unsuccessful or unsatisfactory primary treatment of patients with bladder exstrophy or incontinent epispadias.

PURPOSE: Following unsuccessful or unsatisfactory primary treatment in patients with the epispadias/exstrophy complex, the options for a surgical solution to preserve the upper urinary tract, to achieve complete continence, and to reconstruct the external and female internal genitalia are limited. We reviewed the records of the patients treated at our institution to determine a surgical compromise between ingenious operative constructions and patient desires, both of which are secondary to stabilization of renal function. MATERIALS AND METHODS: From 1967 to December 1997, 128 patients with bladder exstrophy/epispadias complex were treated, of 80 whom had received previous unsuccessful or unsatisfactory treatment. Of these patients 72 were followed for an average of 22.4 years after the first surgical intervention. There were 40 patients referred to our institution after primary bladder closure and bladder neck reconstruction, 10 after rectal bladder, 7 after ureterosigmoidostomy and 5 after incontinent diversion. RESULTS: At the latest followup 19 patients had a rectal reservoir, 38 an ileocecal pouch, 12 a conduit diversion and 3 an augmented Young-Dees procedure. The upper urinary tract remained stable in 95% of the renal units with rectal reservoir, 95% with ileocecal pouch, 96% with a colonic conduit and 100% with an augmented Young-Dees procedure. Day and night continence was achieved in 95% of the patients with a rectal reservoir and 97% of those with an ileocecal pouch were continent, whereas only 2 of the 3 patients with an augmented Young-Dees procedure were continent. Of the women 16 were satisfied with the cosmetic results and 6 delivered 8 children by cesarean section. Only 1 man was dissatisfied with the final cosmetic result. All adults but 1 engaged in sexual intercourse. CONCLUSIONS: The first operative intervention in patients with bladder exstrophy/epispadias complex determines their fate. After failure of primary treatment, the upper urinary tract must be stabilized. In patients with severely impaired renal function the colonic conduit is our method of choice while in those with a normal or slightly dilated upper urinary tract and intact anal sphincter we performed a rectal reservoir. In the remaining patients an ileocecal pouch guarantees continence day and night. The results of genital reconstruction are satisfactory.

Adult↗

PubMed1995

Urinary diversion in bladder exstrophy and incontinent epispadias: 25 years of experience.

PURPOSE: To determine the optimal surgical approach in achieving complete urinary continence with preservation of the upper urinary tract in the exstrophy-epispadias complex we reviewed the records of patients treated at our institution. MATERIALS AND METHODS: From 1968 to July 1994, 115 patients with bladder exstrophy/incontinent epispadias were treated of whom followup was available for 104 and 2 died of causes unrelated to urinary diversion. Mean followup is 16.7 years. In 43 of the 102 patients surgery was primarily performed at our institution (urinary diversion in 39, a modified Young-Dees procedure in 1 and sling plasty in 3). In another 59 patients urinary diversion was done secondarily after therapy elsewhere (bladder closure/bladder neck reconstruction in 34 and failed urinary diversion with incontinence in 22). Urinary diversion was performed in 49 patients, a Young-Dees procedure in 7 and genital reconstruction alone in 3. RESULTS: The present continence rates are 96% for the rectal reservoir, 97% for the Mainz pouch I and 67% for the modified Young-Dees augmentation. Upper tracts have remained stable and no bowel neoplasms have developed. CONCLUSIONS: Rectal reservoirs represent our urinary diversion of choice. After failed reconstruction/insufficient anal sphincter a Mainz pouch I is constructed and when the upper tract has deteriorated a colon conduit is created.

Bladder Exstrophy↗

PubMed1998

Bis(dialkyl)dithiocarbamato cobalt(III) complexes of bidentate nitrogen mustards: synthesis, reduction chemistry and biological evaluation as hypoxia-selective cytotoxins.

Cobalt(III) complexes [Co(R2dtc)2(L)]+ containing two dithiocarbamate ligands (R = Me, Et, pyrrolidine) and a bidentate nitrogen mustard ligand (L) have been prepared as potential hypoxia-selective cytotoxins. The complexes were synthesized by treatment of the binuclear precursor [Co2(R2dtc)5]+ with the diamine mustards N,N'-bis(2-chloroethyl)ethylenediamine (BCE) and N,N-bis(2-chloroethyl)ethylenediamine (DCE), or their non-alkylating analogues [N,N-diethylethylenediamine (DEE) and N,N'-diethylethylenediamine (BEE)]. Cyclic voltammetry of the complexes in MeCN reveals quasi-reversible behaviour for the Co(III)/Co(II) couple, with E1/2 increasing in the order DCE < DEE approximately BCE < BEE. In MeCN/H2O electrochemical reduction is irreversible, indicating rapid substitution of H2O into the coordination sphere of the Co(II) intermediate. This fast ligand loss was confirmed by pulse radiolysis of [Co(Me2dtc)2(DEE)]+, while steady-state radiolysis showed that the initial intermediate disproportionates to [CoII(H2O)6]2+ + 2[CoII(Me2dtc)3]. The latter species reduces additional parent complex to give an overall stoichiometry of 3 mol parent complex/mol reductant. [Co(Me2dtc)2(DCE)]+ decays rapidly by an analogous mechanism in hypoxic culture medium. This reaction is not inhibited by O2, indicating that reoxidation of the Co(II) intermediate by O2 is not rapid enough to compete with ligand dissociation. The resulting free R2dtc-ligands, rather than the released mustards, are primarily responsible for growth inhibition by [Co(R2dtc)2(L)]+ complexes, although DCE release does contribute to clonogenic cell killing. Clonogenic cell killing is not appreciably enhanced under hypoxic conditions for any of the dithiocarbamato complexes. This finding, coupled with their instability in culture medium, suggests that [Co(R2dtc)2(L)]+ complexes are probably not suited for further development as bioreductive anticancer drugs.

Animals↗

PubMed1992

Subunit interactions of GTP-binding proteins.

Fluorescence energy transfer [cf. Förster, T. (1948) Ann. Phys. 6, 55-75] was tested for its suitability to study quantitative interactions of subunits of G0 with each other and these subunits or trimeric G0 with the beta 1-adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., Im, M.-J., Klein, H. W., Hekman, M., Holzhöfer, A, Dees, C., Levitzki, A., Helmreich, E. J. M. & Pfeuffer, T. (1986) EMBO J. 5, 1509-1514]. For this purpose, alpha 0- and beta gamma-subunits and trimeric G0 purified from bovine brain, the beta gamma-subunits from bovine rod outer segment membranes and the beta 1-adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodamine maleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of alpha 0- and beta gamma-interactions, specific high-affinity binding interactions (Kd approximately 10 nM) and nonspecific low-affinity binding interactions (Kd approximately 1 microM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 microM guanosine 5'-O-[gamma-thio]triphosphate and 10 mM MgCl2 where only low-affinity binding interactions remained. Interactions between alpha 0- and beta gamma-subunits from bovine brain or from bovine retinal transducin did not differ much. The beta gamma-subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone-activated and the nonactivated beta 1-receptor reconstituted in lipid vesicles: Kd = 100 +/- 20 and 120 +/- 20 nM, respectively; however, beta gamma-subunits from transducin appeared to bind more weakly to the beta 1-adrenoceptor than beta gamma-subunits from bovine brain. Separated purified homologous alpha 0- and beta gamma-subunits from bovine brain interfered mutually with each other in binding to the beta 1-adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein-protein interactions of G-proteins and G-protein-linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Fröhlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167-176] that beta gamma-subunits can bind to the nonactivated beta 1-adrenoceptor.

Animals↗