The effect of hexokinase and tricarboxylic acid-cycle intermediates on fatty acid oxidation and formation of ketone bodies by rat-liver mitochondria

1. The oxidation of butyrate, hexanoate and octanoate by rat-liver mitochondria suspended in a tris-potassium chloride medium in the presence of malate and serum albumin has been investigated. 2. The oxidation of butyrate to acetoacetate was markedly decreased by the addition of a system competitive for ATP (hexokinase-glucose). 3. Serum albumin or tricarboxylic acid-cycle intermediates prevented the inhibition by hexokinase and in their presence a greater proportion of the oxygen consumption was contributed by the tricarboxylic acid cycle. The results suggest that the energy supply for fatty acid activation is either compartmentalized in a spatial or kinetic sense or there exists a special activating mechanism not involving ATP. 4. Malate and other tricarboxylic acid-cycle intermediates caused substantial reduction (to beta-hydroxybutyrate) of the acetoacetate formed during the oxidation of butyrate, hexanoate and octanoate.     

Intramuscular accumulation of prostaglandins during static contraction of the cat triceps surae.

We previously demonstrated that muscle afferent endings are sensitized by exogenous prostaglandins during static contraction of skeletal muscle. The purpose of this study was to determine whether 30 s of static hindlimb contraction, induced by electrical stimulation of the cat sciatic nerve, increases the concentration of immunoreactive prostaglandin E2 (iPGE2) and 6-ketoprostaglandin F1 alpha (i6-keto-PGF1 alpha, the stable metabolite of prostaglandin I2) in muscle tissue. In addition, the role of ischemia in augmenting prostanoid production was examined. Gastrocnemius muscle was obtained by freeze-clamping tissue, and prostaglandins were extracted from muscle homogenates and measured by radioimmunoassay. Compared with precontraction values, high-intensity (68% of maximal tension) static contraction elevated gastrocnemius iPGE2 and i6-keto-PGF1 alpha by 45 and 53%, respectively (P less than 0.01). Likewise, when blood flow to the gastrocnemius was attenuated by arterial occlusion during and 2 min before low-intensity contraction (29% maximal tension), the intramuscular iPGE2 concentration was increased by 71% (P less than 0.01). Conversely, low-intensity contraction (30% of maximal tension) and arterial occlusion without contraction did not alter the concentration of either prostanoid. Our findings demonstrate that prostaglandins accumulate in muscle during static contraction. We believe that local muscle ischemia may provide a stimulus for this phenomenon. These prostaglandins therefore are available to sensitize afferent endings responsible for reflex adjustments during static muscle contraction.     

The Gibberellin Status of lip1, a Mutant of Pea That Exhibits Light-Independent Photomorphogenesis

Dark-grown seedlings of the lip1 (light independent photomorphogenesis) mutant of Pisum sativum L. display many features of de-etiolated growth and are similar in many respects to wild-type (WT) seedlings grown in the light. The involvement of gibberellins (GAs) with the mutant phenotype was examined by applying GA1 and GA20 to the mutant and WT, and by quantifying endogenous GA1, GA8, GA19, GA20, and GA29 levels in the two genotypes. These experiments were conducted in both the light and the dark. In neither environment could GA application restore elongation in the mutant to that in GA-treated WT plants. Quantification of GAs provided further evidence that the mutant phenotype is not attributable to a deficiency in endogenous GA1. However, dark-grown lip1 seedlings contained lower levels of GA19 and higher levels of GA20 than dark-grown WT plants, whereas in the light, the effect of the mutation on the ratio of GA19 to GA20 was reversed. Thus, there appears to be a complex interaction between the lip1 mutation, the light regime, and the step GA19 to GA20.     

Improved coronary vascular function evoked by high-intensity treadmill training is maintained in arteries exposed to ischemia and reperfusion.

We hypothesized that myocardial contractile function and coronary arterial function are greater after ischemia and reperfusion in high-intensity treadmill-trained vs. sedentary rats. Rats performed 10 x 4-min bouts of treadmill running consisting of 2 min at 13 m/min + 2 min at 45-60 m/min (Etr) or were sedentary (Sed) for 12 wk. Animals then were instrumented to measure left ventricular (LV) contractility in response to three 15-min coronary occlusion (O) and 5-min reperfusion (R) cycles (Isc) or a sham operation (Sham). After the Isc and Sham protocols, hearts were excised and coronary arterial ( approximately 105 microm ID) function was evaluated by using isometric techniques. LV developed pressure, the first derivative of LV pressure at a developed pressure of 40 mmHg, and systolic blood pressure were not different between Etr (n = 14) and Sed (n = 7) rats before or after the Sham protocol. Furthermore, hemodynamic variables were similar in Etr (n = 14) and Sed (n = 13) animals before the Isc protocol and were depressed to the same degree by the three O-R cycles. Therefore, Etr did not alter myocardial contractile function in rats that were (i.e., Isc) or were not (i.e., Sham) exposed to ischemia and reperfusion. Acetylcholine-evoked relaxation (10-8 to 3 x 10-5 M) was greater (P < 0.05) in coronary arteries from Sham-Etr vs. Sham-Sed animals (5 of 8 doses tested) and Isc-Etr vs. Isc-Sed rats (3 of 8 doses tested). Maximal relaxation produced by sodium nitroprusside (10-4 M) was similar among groups. Vasocontractile responses produced by KCl (10-100 mM) and endothelin-1 (10-11-10-4 M) were greater (P < 0.05) in the presence vs. the absence of nitric oxide synthase inhibition (10-6 M NG-monomethyl-l-arginine) in vessels from Sham-Etr but not Sham-Sed rats and from Isc-Etr but not Isc-Sed rats. These findings suggest that Etr-evoked improvements in coronary function are maintained in small arteries even when exposed to ischemia and reperfusion.     

The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

Avocado sunblotch viroid: primary sequence and proposed secondary structure.

The sequence of the 247 nucleotide residues of the single strand circular RNA of avocado sunblotch viroid (ASBV) was determined using partial enzymic cleavage methods on overlapping viroid fragments obtained by partial ribonuclease digestion followed by 32p-labelling in vitro at their 5'-ends. ASBV is much smaller than potato spindle tuber viroid (PSTV; 359 residues) and chrysanthemum stunt viroid (CSV; 356 residues). A secondary structure model for ASBV is proposed and contains 67% of its residues base paired. In contrast to the extensive (69%) sequence homology of CSV with PSTV, only 18% of the ASBV sequence is homologous to PSTV and CSV. There are eight potential polypeptide translation products with chain lengths from 4 to 63 amino acid residues coded for by the plus (infectious) strand and four potential translation products (2 to 60 residues) coded for by the minus strand. An improved method is described for the synthesis of gamma-32p-ATP of high specific activity.     

A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts

Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.     

Protein metabolism. 6. Trichostrongylus colubriformis: skeletal protein catabolism in primary and secondary infections of the guinea pig

The urinary excretion of 3-methylhistidine (3-MH) was used as an index of muscle protein catabolism in primary and secondary infections of the guinea pig with Trichostrongylus colubriformis and in uninfected animals fed quantitatively reduced rations. Catabolism, which was depressed in all three groups, was directly related to a fall in food consumption. Possible explanations for the greater depression of catabolism in the primary infection than in the uninfected guinea pigs and its fall in the secondary infection in spite of little change in consumption are briefly discussed. It was concluded that the faster rate of whole-body protein turnover reported earlier in this series on protein metabolism in intestinal nematode infection was not partly due to a faster rate of muscle protein catabolism. It was shown that the urinary excretion of 3-MH could be validly expressed in terms of unit creatinine.