Increased ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn.

Transgenic tomato plants Lycopersicon esculentum (Solanaceae) cv. Heinz 902 expressing the bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, under the transcriptional control of either two tandem 35S cauliflower mosaic virus promoters (constitutive expression), the rolD promoter from Agrobacterium rhizogenes (root specific expression) or the pathogenesis related PRB-1b promoter from tobacco, were compared to non-transgenic tomato plants in their ability to grow in the presence of Cd, Co, Cu, Mg, Ni, Pb, or Zn and to accumulate these metals. Parameters that were examined include metal concentration and ACC deaminase activity in both plant shoots and roots; root and shoot development; and leaf chlorophyll content. In general, transgenic tomato plants expressing ACC deaminase, especially those controlled by the PRB-1b promoter, acquired a greater amount of metal within the plant tissues, and were less subject to the deleterious effects of the metals on plant growth than were non-transgenic plants.

Substrate profile in rat soleus muscle fibers after hindlimb unloading and fatigue.

Limb muscles from rats flown in space and after hindlimb unloading (HU) show an increased fatigability, and spaceflight has been shown to result in a reduced ability to oxidize fatty acids. The purpose of this investigation was to determine the effects of HU on the substrate content in fast- and slow-twitch fibers and to assess the substrate utilization patterns in single slow type I fibers isolated from control and HU animals. A second objective was to assess whether HU altered the ability of the heart or limb muscle to oxidize pyruvate or palmitate. After 2 wk of HU, single fibers were isolated from the freeze-dried soleus and gastrocnemius muscles. HU increased the glycogen content in all fiber types, and it increased lactate, ATP, and phosphocreatine in the slow type I fiber. After HU, the type I fiber substrate profile was shifted toward that observed in fast fibers. For example, fiber glycogen increased from 179 +/- 16 to 285 +/- 25 mmol/kg dry wt, which approached the 308 +/- 23 mmol/kg dry wt content observed in the post-HU type IIa fiber. With contractile activity, the type I fiber from the HU animal showed a greater utilization of glycogen and accumulation of lactate compared with the control type I fiber. HU had no effect on the ability of crude homogenate or mitochondria fractions from the soleus or gastrocnemius to oxidize pyruvate or palmitate. The increased fatigability after HU may have resulted from an elevated glycolysis producing an increased cell lactate and a decreased pH.

Identification of DNA sequences that regulate the expression of the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylic acid deaminase gene.

Analysis of the DNA sequence upstream of the previously isolated Enterobacter cloacae UW4 ACC deaminase gene (Shah et al. 1998) suggests that this segment contains several features that are thought to be involved in the transcriptional regulation of this gene. These features include half of a CRP (cAMP receptor protein) binding site, an FNR (fumarate-nitrate reduction) regulatory protein binding site, an LRP (leucine responsive regulatory protein) binding site, and an LRP-like protein coding region. ACC deaminase activity was measured following growth of either various Escherichia coli strains carrying a plasmid that contained the Enterobacter cloacae UW4 ACC deaminase gene, or of Enterobacter cloacae UW4. Variables that were compared include aerobic versus anaerobic conditions, the presence and absence of ACC in the growth medium, addition of leucine to the medium, and bacterial strains that did or did not contain either lrp or fnr genes. The data reported are consistent with the involvement of most, if not all, of the above mentioned potential regulatory regions in the expression of ACC deaminase.

Substrate and enzyme profile of fast and slow skeletal muscle fibers in rhesus monkeys.

Results from the Russian Cosmos program suggest that the rhesus monkey is an excellent model for studying weightlessness-induced changes in muscle function. Consequently, the purpose of this investigation was to establish the resting levels of selected substrate and enzymes in individual slow- and fast-twitch muscle fibers of the rhesus monkey. A second objective was to determine the effect of an 18-day sit in the Spacelab experiment-support primate facility [Experimental System for the Orbiting Primate (ESOP)]. Muscle biopsies of the soleus and medial gastrocnemius muscles were obtained 1 mo before and immediately after an 18-day ESOP sit. The biopsies were freeze-dried, and individual fibers were isolated and assayed for the substrates glycogen and lactate and for the high-energy phosphates ATP and phosphocreatine. Fiber enzyme activity was also determined for the glycolytic enzymes phosphofructokinase and lactate dehydrogenase (LDH) and for the oxidative markers 3-hydroxyacyl-CoA dehydrogenase (beta-OAC) and citrate synthase. Consistent with other species, the fast type II fibers contained higher glycogen content than did the slow type I fibers. The ESOP sit had no significant effects on the metabolic profile of the slow fibers of either muscle or the fast fibers of the soleus. However, the fast gastrocnemius fibers showed a significant decline in phosphocreatine and an increase in lactate. Also, similar to other species, the fast fibers contained significantly higher LDH activities and lower 3-hydroxyacyl-CoA dehydrogenase activities. For the muscle enzymes, the quantitatively most important effect of the ESOP sit occurred with LDH where activities increased in all fiber types postsit except the slow type I fiber of the medial gastrocnemius.