Selective inhibition of hepatic peroxisomal fatty acid beta-oxidation by enoximone.

Although beta-oxidation of fatty acids occurs in both peroxisomes and mitochondria, beta-oxidizing enzymes in these organelles have distinct differences in their specifity and sensitivity to inhibitors. In this study, the effects of the phosphodiesterase inhibitor enoximone on hepatic peroxisomal and mitochondrial beta-oxidation were investigated. In liver homogenates from control rats, cyanide-insensitive peroxisomal beta-oxidation of palmitoyl-CoA was inhibited progressively by increasing concentrations of enoximone. Similar results were obtained in liver homogenates from rats pretreated with the known peroxisomal proliferator diethylhexylphthalate. In contrast, mitochondrial beta-oxidation of palmitoyl-CoA was not inhibited by enoximone. These data show that enoximone selectively inhibits basal as well as induced peroxisomal, but not mitochondrial, beta-oxidation of the CoA thioester of long-chain fatty acids. The availability of specific inhibitors of peroxisomal beta-oxidation should prove useful in elucidating regulatory mechanisms operative in this pathway in normal as well as in proliferated peroxisomes.     

Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.     

Daily variations in colchicine-induced apoptosis in duodenal crypts

Apoptotic cell death can be induced by several agents, among them colchicine, a microtubule disrupting-drug that affects continuously renewing cell populations, such as the intestinal crypt enterocytes. The objectives of this investigation were (1) to confirm in vivo colchicines-inductive effect and (2) to determine the existence of 24 h variations in the crypt enterocytes apoptotic indices. The study was done on C3H/S male adult mice housed under standardized conditions. Starting at midnight until the end of a circadian period, subgroups of mice were sacrificed after having been injected with colchicine or saline i.p. 4h beforehand. Duodenal samples were processed for hematoxylin-eosin staining and TUNEL technique. In order to score the number of apoptosis, the longitudinal sections of the crypts were divided into three regions comprised, respectively, of tiers 1-4, 5-12, and 13-20, proceeding from the bottom to the top of the crypt. Values of each lot were expressed as mean +/- SEM. A highly significant statistical difference in apoptotic indices was found for colchicine-treated animals. The 24 h curve for colchicine-induced apoptosis displayed qualitative and quantitative differences compared to other inducer agents. Highest apoptotic indices were found in the deepest crypt regions. Daily variations were observed in all the crypt sectors of the colchicine-treated animals and in tiers 5-12 of the saline controls. The present work demonstrates that the colchicine cytotoxicity due to its apoptotic-inducing effect depends on the dosing time during the 24 h in this mouse strain.     

Bioconversion of cheese whey permeate into fungal oil by <Emphasis Type="Italic">Mucor circinelloides</Emphasis>

Background

Oleaginous fungi are efficient tools to convert agricultural waste streams into valuable components. The filamentous fungus Mucor circinelloides was cultivated in whey permeate, a byproduct from cheese production, to produce an oil-rich fungal biomass. Response surface methodology was used to optimize the fermentation conditions such as pH and temperature for increased biomass yield and lipid accumulation. Quantification and characterization of the fungal biomass oil was conducted.

Results

Upstream lactose hydrolysis of the whey permeate increased the biomass yield from 2.4 to 7.8 (g dry biomass/L) compared to that of non-hydrolyzed whey permeate. The combination of low pH (4.5) and pasteurization minimized microbial competition, thus favoring fungal growth. A central composite rotatable design was used to evaluate the effects of temperature (22.4–33.6 °C) and a lower pH range (3.6–4.7) on biomass yield and composition. The highest biomass yield and oil content was observed at high temperature (33.6 °C), while the pH range evaluated had a less pronounced effect. The predictive model was validated at the optimal conditions of 33.6 °C and pH 4.5. The fungal biomass yield plateaued at 9 g dry cell weight per liter, while the oil content and lipid yield reached a maximum of 24% dry biomass and 2.20 g/L, respectively, at 168 h. Triacylglycerides were the major lipid class (92%), which contained predominantly oleic (41%), palmitic (23%), linoleic (11%), and γ-linolenic acid (9%).

Conclusions

This study provided an alternative way of valorization of cheese whey permeate by using it as a substrate for the production of value-added compounds by fungal fermentation. The fatty acid profile indicates the suitability of M. circinelloides oil as a potential feedstock for biofuel production and nutraceutical applications.

     

Long-term facilitation in obstructive sleep apnea patients during NREM sleep.

Repetitive hypoxia followed by persistently increased ventilatory motor output is referred to as long-term facilitation (LTF). LTF is activated during sleep after repetitive hypoxia in snorers. We hypothesized that LTF is activated in obstructive sleep apnea (OSA) patients. Eleven subjects with OSA (apnea/hypopnea index = 43.6 +/- 18.7/h) were included. Every subject had a baseline polysomnographic study on the appropriate continuous positive airway pressure (CPAP). CPAP was retitrated to eliminate apnea/hypopnea but to maintain inspiratory flow limitation (sham night). Each subject was studied on 2 separate nights. These two studies are separated by 1 mo of optimal nasal CPAP treatment for a minimum of 4-6 h/night. The device was capable of covert pressure monitoring. During night 1 (N1), study subjects used nasal CPAP at suboptimal pressure to have significant air flow limitation (>60% breaths) without apneas/hypopneas. After stable sleep was reached, we induced brief isocapnic hypoxia [inspired O(2) fraction (FI(O(2))) = 8%] (3 min) followed by 5 min of room air. This sequence was repeated 10 times. Measurements were obtained during control, hypoxia, and at 5, 20, and 40 min of recovery for ventilation, timing (n = 11), and supraglottic pressure (n = 6). Upper airway resistance (Rua) was calculated at peak inspiratory flow. During the recovery period, there was no change in minute ventilation (99 +/- 8% of control), despite decreased Rua to 58 +/- 24% of control (P < 0.05). There was a reduction in the ratio of inspiratory time to total time for a breath (duty cycle) (0.5 to 0.45, P < 0.05) but no effect on inspiratory time. During night 2 (N2), the protocol of N1 was repeated. N2 revealed no changes compared with N1 during the recovery period. In conclusion, 1) reduced Rua in the recovery period indicates LTF of upper airway dilators; 2) lack of hyperpnea in the recovery period suggests that thoracic pump muscles do not demonstrate LTF; 3) we speculate that LTF may temporarily stabilize respiration in OSA patients after repeated apneas/hypopneas; and 4) nasal CPAP did not alter the ability of OSA patients to elicit LTF at the thoracic pump muscle.     

Chloroplast DNA restriction site polymorphism inGenisteae (Leguminosae) suggests a common origin for European and American lupines

Restriction site polymorphism in cpDNA of 35 legumes was studied in order to address natural relationships and geographic distribution within the tribeGenisteae. 386 sites were studied, 277 were polymorphic, 207 were informative. Phylogenetic inferences with distance and parsimony methods suggest that the American and MediterraneanLupinus species belong to a monophyletic group which arose from a single center of diversification. The data furthermore indicate thatLupinus should not be included in the tribeGenisteae since at the level of cpDNA polymorphismAnagyris foetida (tribeThermopsideae) appears more closely related to otherGenisteae thanLupinus does.     

Inhibiting effect of a hepatoma extract on the mitotic rate of regenerating liver

Aqueous tumor extracts were prepared by the homogenization of a fast-growing, undifferentiated, transplantable malignant murine hepatoma in distilled water. After centrifugation, an aliquot of 0.01 ml of the supernatant g body weight was injected intraperitoneally into partially hepatectomized mice. Control animals were injected with saline. Groups of mice were killed at various times in relation to the hepatectomy. Four h before killing the animals were given Colcemid (1 microgram/g body weight). The number of Colcemid-arrested mitoses in the hepatocytes and in the littoral cells, respectively, were counted in 140 microscopic fields. The extract significantly inhibited the mitotic rate in hepatocytes when the injection was given between 22 h before, and up to 26 h after hepatectomy. In the littoral cells, a slight initial stimulation was followed by a slight but significant inhibition which occurred when the injection was given at hepatectomy or until 18 h after hepatectomy. The effect was not modified by exposing the extracts to temperatures of 47 degrees C for 30 min or 22 degrees C for 24 h, but 10 min of boiling destroyed their inhibitory effect. Lyophilization and storing at -18 degrees C for up to 4 weeks did not modify the effect. The mitosis-inhibiting effect was also measurable when the extract was injected subcutaneously. There was an almost linear dose-response curve. The results are discussed in relation to circadian rhythms, the pattern of liver cell proliferation after hepatectomy, and recent similar reports from the literature. The conclusion is drawn that extracts of a hepatoma contain one or more growth-inhibitory factors significantly active on regenerating liver cells, and less significantly on littoral cells.     

Identification of two types of specific endothelin receptors in rat mesangial cell

Two types of receptors specific for endothelin were identified using cross-linking technique in cultured rat mesangial cells. The molecular weights of these receptors were approximately 58,000 and 34,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The binding of radioiodinated-endothelin to its receptors was inhibited by excess of unlabeled endothelin, but not by nifedipine, nicardipine, verapamil, diltiazem, angiotensin II or [Arg8]-vasopressin. The endothelin binding proteins were solubilized with 1% digitonin and fractionated under non-denaturing conditions by gel filtration. Two endothelin binding peaks eluted at the positions corresponding to the molecular weights of 65,000 and 43,000. These observations indicate that there are two types of specific endothelin receptors in rat mesangial cells which are distinct from voltage dependent L-type calcium channel.     

Effect of airway impedance on CO2 retention and respiratory muscle activity during NREM sleep

We hypothesized that a sleep-induced increase in mechanical impedance contributes to CO2 retention and respiratory muscle recruitment during non-rapid-eye-movement (NREM) sleep. The effect NREM sleep on respiratory muscle activity and CO2 retention was measured in healthy subjects who increased maximum total pulmonary resistance (RLmax, 1-81 cmH2O.l-1.s) from awake to NREM sleep. We determined the effects of this sleep-induced increase in airway impedance by steady-state inhalation of a reduced-density gas mixture (79% He-21% O2, He-O2). Both arterialized blood PCO2 (PaCO2) and end-tidal PCO2 (PETCO2) were measured. Inspiratory (EMGinsp) and expiratory (EMGexp) respiratory muscle electromyogram activity was measured. NREM sleep caused 1) RLmax to increase (7 +/- 3 vs. 39 +/- 28 cmH2O.l-1.s), 2) PaCO2 and/or PETCO2 to increase in all subjects (40 +/- 2 vs. 44 +/- 3 Torr), and 3) EMGinsp to increase in 8 of 9 subjects and EMGexp to increase in 9 of 17 subjects. Compared with steady-state air breathing during NREM sleep, steady-state He-O2 breathing 1) reduced RLmax by 38%, 2) decreased PaCO2 and PETCO2 by 2 Torr, and 3) decreased both EMGinsp (-20%) and EMGexp (-54%). We concluded that the sleep-induced increase in upper airway resistance accompanied by the absence of immediate load compensation is an important determinant of CO2 retention, which, in turn, may cause augmentation of inspiratory and expiratory muscle activity above waking levels during NREM sleep.