Ali A. Al-Robai

Lichen acids as uncouplers of oxidative phosphorylation of mouse-liver mitochondria

Three lichen acids-namely, (+)usnic acid, vulpinic acid, and atranorin-were isolated from three lichen species (Usnea articulata, Letharia vulpina, and Parmelia tinctorum, respectively). The effects of these lichen products on mice-liver mitochondrial oxidative functions in various respiratory states and on oxidative phosphorylation were studied polarographically in vitro. The lichen acids exhibited characteristics of the 2,4-dinitrophenol (DNP), a classical uncoupler of oxidative phosphorylation. Thus, they released respiratory control and oligomycin inhibited respiration, hindered ATP synthesis, and enhanced Mg(+2)-ATPase activity. (+)Usnic acid at a concentration of 0.75 microM inhibited ADP/O ratio by 50%, caused maximal stimulation of both state-4 respiration (100%) and ATPase activity (300%). Atranorin was the only lichen acid with no significant effect on ATPase. The uncoupling effect was dose-dependent in all cases. The minimal concentrations required to cause complete uncoupling of oxidative phosphorylation were as follows: (+)usnic acid (1 microM), vulpinic acid, atranorin (5 microM) and DNP (50 microM). It was postulated that the three lichen acids induce uncoupling by acting on the inner mitochondrial membrane through their lipophilic properties and protonophoric activities.

Different ouabain sensitivities of Na+/K(+)-ATPase from Poekilocerus bufonius tissues and a possible physiological cost.

1. The properties of Na+/K(+)-transporting ATPase in microsomal preparation from mid-gut of the grasshopper, Poekilocerus bufonius, were investigated and compared with the same enzyme from brain and excretory system. 2. Two components of ATPases activity are present in the three tissues studied. 3. The physiochemical properties of Na+/K(+)-transporting ATPase from mid-gut, brain and excretory system (hind-gut plus Malpighian tubules) are essentially the same. 4. The calculated values of PI50 were 2 (I50 = 1 x 10(-2) M), 3.7 (I50 = 2 x 10(-4) M) and 6.4 (I50 = 3.98 x 10(-7)) for Na+/K(+)-ATPase from mid-gut, excretory system and brain, respectively. The mid-gut contains the most ouabain-resistant Na+/K(+)-ATPase. 5. The results suggest that P. bufonius have developed some tolerance to toxic cardiac glycosides (CGS), but there is a possibility of autotoxicity as indicated by the presence of ouabain-sensitive ATPase from brain tissue. 6. It was concluded that the dissimilarities of Na+/K(+)-ATPases from different tissues of P. bufonius are probably due to tissue-dependent differences in ouabain sensitivity (or isoenzymes pattern) available in the same insect. 7. The atrophy of female flight muscle of P. bufonius suggests the possibility of physiological cost inflicted on insects consuming poisonous plants.

Properties of ouabain-resistant Na+K+-transporting ATPase from the excretory system of Poekilocerus bufonius

Abstract The properties of Na + K + - transporting ATPase in microsomal fractions from the excretory system (Malpighian tubules plus hind-gut) of Poekilocerus bufonius were studied. The optimal conditions required for optimal activity of Na + K + - transporting ATPase were as follows: 1. (1) Maximal activation of Na + K + - transporting ATPase occurred at an ATP:Mg 2+ ratio of 1:1.5. The apparent K m value was 0.84 mM and the V max was 166 nmol Pi lib. mg protein −1 min −1 . 2. (2) Maximal activation of Na + K + - transporting ATPase occurred at 80 mM Na + and 20 mM K + . The apparent K m values were 7.4 mM Na + and 3.6 mM K + . 3. (3) The optimal pH of Na + K + - transporting ATPase was 7.5. 4. (4) The enzyme is rather insensitive to the cardiac glycoside ouabain (I 50 = 2 × 10 −4 M). The sensitivity of Na + K + - transporting ATPase of P. bufonius to Na + , K + and ouabain is lower than that of S. gregaria . However, the low sensitivity of the enzyme to ouabain inhibition observed in this insect would possibly indicate that the presence of this enzyme is an important factor in the tolerance of plant cardiac glycosides in P. bufonius .

Regulatory properties of 6-phosphofructo-1-kinase of the mid-gut of the grasshopper, Poekilocerus bufonius

The fine structure of the mid-gut of Poekilocerus bufonius has been examined and three types of epithelial cells were identified; normal epithelial cells with their apical part possessing well developed microvilli, goblet-like cells containing myelin-like figures and the small basal cells with small and round nuclei, nidi. The regulation of 6-phosphofructo-1-kinase (PFK-1) prepared from the mid-gut of the grasshopper, Poekilocerus bufonius, was studied. Mid-gut PFK-1 displayed cooperativity with respect to fructose-6-phosphate at pH 7.0, and the enzyme was inhibited by high concentrations of ATP. The affinity of the enzyme for fructose-6-phosphate was increased by fru-2,6-P2 whereas the inhibition of the enzyme by high concentrations of ATP was relieved by fru-2,6-P2. The activity of mid-gut PFK-1 was highly stimulated in a simultaneous presence of low concentrations of fru-2,6-P2 and AMP. ADP, AMP and c-AMP were all shown to be activators of the mid-gut PFK-1 with AMP being the greatest effector. The enzyme was not inhibited by citrate either in the presence of low or high concentrations of ATP. These results suggest that the PFK-1 of the mid-gut of the grasshopper is highly regulated with positive stimulators, specially fru-2,6-P2, whereas the enzyme is not regulated by citrate or glucose-1,6-bisphosphate.

Protective effect of olive and juniper leaves extracts on nephrotoxicity induced by thioacetamide in male mice

This study, for the first time, evaluates the effect of olive and juniper leaves extracts and their combination on thioacetamide (TAA)-induced nephrotoxicity in male mice. The experimental mice were divided into eight groups. Group 1 was served as control. Group 2 was exposed to TAA. Group 3 was treated with TAA and olive leaves extract. Group 4 was subjected to TAA and juniper leaves extract. Group 5 was exposed to TAA and olive and juniper leaves extracts. Groups 6, 7 and 8 were treated with olive, juniper, and olive and juniper leaves extracts respectively. In mice treated with only TAA, significant increases of blood urea nitrogen and uric acid were observed after six weeks. Moreover, levels of serum creatinine, blood urea nitrogen and uric acid were statistically increased in mice administrated with only TAA for twelve weeks. Insignificant alterations in levels of these haematobiochemical parameters were noted in other treated groups after six and twelve weeks. Histopathological evaluations of renal sections from mice treated with only TAA for twelve weeks showed severe damage of the renal corpuscles. Furthermore, the renal sections from mice treated with TAA and olive leaves extract, TAA and juniper leaves extract, TAA and olive and juniper leaves extracts, olive leaves extract, juniper leaves extract, and olive and juniper leaves extracts showed normal structures. In addition, it is conceivable therefore, that these extracts exhibit protective influences against TAA-induced nephrotoxicity, probably mediated through the antioxidative pathway roles.

Effect of Olea oleaster and Juniperus procera leaves extracts on thioacetamide induced hepatic cirrhosis in male albino mice.

The effect of Olea oleaster and Juniperus procera leaves extracts and their combination on thioacetamide (TAA)-induced hepatic cirrhosis were investigated in male albino mice. One hundred sixty mice were used in this study and were randomly distributed into eight groups of 20 each. Mice of group 1 served as controls. Mice of group 2 were treated with TAA. Mice of group 3 were exposed to TAA and supplemented with O. oleaster leaves extracts. Mice of group 4 were treated with TAA and supplemented with J. procera leaves extracts. Mice of group 5 were subjected to TAA and supplemented with O. oleaster and J. procera leaves extracts. Mice of groups 6, 7 and 8 were supplemented with O. oleaster, J. procera, and O. oleaster and J. procera leaves extracts respectively. Administration of TAA for six and twelve weeks resulted in a decline in body weight gain and increased the levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin. Histopathological evaluations of hepatic sections from mice treated with TAA showed severe alterations including increase of fibrogenesis processes with structural damage. Treatment of mice with these extracts showed a pronounced attenuation in TAA induced hepatic cirrhosis associated with physiological and histopathological alterations. Finally, this study suggests that the supplementation of these extracts may act as antioxidant agents and could be an excellent adjuvant support in the therapy of hepatic cirrhosis.

Properties of the V-type ATPase from the excretory system of the usherhopper, Poekilocerus bufonius

The bafilomycin A(1) and N-ethylmaleimide (NEM)-sensitive (V-type) ATPase was partially purified from the apical membrane-rich fractions of excretory system (Malpighian tubules and hind gut) of P. bufonius. Enzymatic activity was inhibited by bafilomycin A(1) (IC(50) = 1.3 nM) and NEM (IC(50) = 10.1 microM). The V-type ATPase activity is confined to the apical membrane fraction, while the activity of Na(+)/K(+) -ATPase forms the major part of the basal membrane fraction. The optimal pH required for maximal activity of V-type ATPase was pH 7.5. The effect of 30 mM of various salts on ATPase activity was investigated. NaCl and KCl caused increases of 175% and 184%, respectively. Other chloride salts also caused an increase in activity in the following ascending order: RbCl, LiCI, choline Cl, NaCI, KCl and tris-HCl. The activity of V-type ATPase was stimulated by a variety of different anions and cations, and HCO(3)(-) was found to be the most potent cationic activator of ATPase activity. The present results show that the properties of V-type ATPase of P. bufonius are similar to those reported for other insect tissues.

6-phosphofructo-1-kinase from the flight muscle of the grasshopper, Poekilocerus bufonius

Abstract 1. 1. The ultrastructure of the flight muscle of the adult male grasshopper, Poekilocerus bufonius, showed a variation in the shape and size of each individual myofibril profile which was in almost parallel register. 2. 2. Moderate number of mitochondria are present as well as high numbers of compact cristae. 3. 3. 6-Phosphofructo-1-kinase (PFK) from the flight muscle of P. bufonius was purified more than 1000-fold to homogeneity with a yield of 77%. 4. 4. The sodium dodecyl sulphate-treated purified enzyme migrated as a single band in 7.5% polyacrylamide gel. 5. 5. The enzyme is a tetramer, with a monomer Mr 82,000 ± 2000. 6. 6. The regulatory properties of the purified enzyme were studied at pH 7.0 and the affinity of the enzyme for fructose 6-phosphate was slightly increased by fructose 2,6-bisphosphate whereas the enzyme inhibition by high concentrations of ATP was slightly relieved by fructose 2,6-bisphosphate. 7. 7. The activity of flight muscle PFK was markedly inhibited by glucose 1,6-bisphosphate and phosphoarginine, and was weakly activated by ADP, AMP and fructose 2,6-bisphosphate. 8. 8. These data indicate that flight muscle PFK is not strongly affected by activators or by synergism between AMP and fructose 2,6-bisphopshate which is consistent with the fact that this insect is incapable of a long flight and the flight muscles of the adult females are very small and reduced to a thread-like structure.