A study on the mechanism of energy coupling in the redox chain

The present study demonstrates that the mitochondrial respiratory chain includes not three but four energy coupling sites, the fourth site being localized at the NADPH→NAD⁺ step.

1. The NADPH→NAD⁺-directed transhydrogenase reaction in sonicated beef heart submitochondrial particles energizes the particle membrane as judged by two membrane potential probes, i.e. uptake of a penetrating anion, phenyldicarbaundecaborane (PCB^?), and enhancement of anilinonaphthalene sulfonate (ANS^?) fluorescence.
2. The reverse reaction (NADH→NADP⁺) is accompanied by the oppositely directed anion movement, i.e. PCB^? efflux.
3. Being insensitive to rotenone, antimycin, cyanide, and oligomycin, both the influx and efflux of PCB^? coupled with transhydrogenase reaction can be prevented or reversed by uncouplers.
4. Equalization of concentrations of the transhydrogenase substrates and products also prevents (or reverses) the PCB^? influx coupled with oxidation of NADPH by NAD⁺, as well as the PCB^? efflux coupled with reduction of NADP⁺ by NADH.
5. The transhydrogenase-linked PCB^? uptake depends linearly on the energy yield of the oxidation reaction calculated according to formula $$\Delta G = RTln\frac{{[NADPH] x [NAD^ + ]}}{{[NADP^ + ] x [NADH]^ \cdot }}$$ No threshold value of Δ was found. Measurable PCB^? transport was still observed at Δ≤0.5 kcal/mole NADPH oxidized.
6. Partial uncoupling of transhydrogenase reaction and PCB^? transport, induced by low concentrations ofp-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), dinitrophenol, or by removing coupling factor F₁, results in the decrease of the slope of the straight line showing the PCB^? uptake as a function of Δ. Oligomycin improves the coupling in F₁-deprived particles, the slope being increased. Rutamycin, dicyclohexylcarbodiimide (DCCD) and reconstitution of particles with F₁, also increase the coupling.
7. In phosphorylating particles oxidizing succinate by O₂, both the energy-dependent NADH→NADP⁺ hydrogen transfer and PCB^? influx possess equal sensitivity to FCCP, which is lower than the sensitivity of oxidative phosphorylation. Similarly, the decrease in the succinate oxidation rate induced by malonate arrests first phosphorylation and then under higher malonate concentration, PCB^? influx. The rate of NADPH→NAD⁺ transhydrogenase reaction was found to be lower than the threshold value of rate of succinate oxidation, still coupled with phosphorylation. Respectively, the values of PCB^? uptake under transhydrogenase reaction are lower than those inherent in phosphorylating oxidation of succinate.

The conclusion is made that the NADPH→NAD⁺-directed transhydrogenase reaction generates the membrane potential of the same polarity as respiration and ATP hydrolysis but of a lower magnitude (“plus” inside particles; the forward hydrogen transfer). The NADH→NADP⁺-directed transhydrogenase reaction forms the membrane potential of the opposite polarity (“minus” inside particles; the reverse hydrogen transfer). Under conditions used, the transhydrogenase-produced membrane potential proves to be too low to support ATP synthesis (and, most probably, the synthesis of any other high-energy compound) maintaining, nevertheless, some electrophoretic ion fluxes. A conclusion is made that transhydrogenase forms a membrane potential with no high-energy intermediates involved.     

Subcellular distribution and properties of rabbit liver aminoacyl-tRNA synthetases under myocardial ischemia

Subcellular distribution of aminoacyl-tRNA synthetase activities has been studied in normal rabbit liver and under experimental myocardial ischemia (EMI). An increase in the activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal supernatants from rabbit liver has been determined 12 hr after EMI. Gel chromatography of the postribosomal supernatant on Sepharose 6B shows that aminoacyl-tRNA synthetase activities are distributed among the fractions with M_r 1.82×10⁶, 0.84×10⁶ (high-M_r aminoacyl-tRNA synthetase complexes) and 0.12–0.35×10⁶. In the case of EMI aminoacyl-tRNA synthetase activities are partly redistributed from the 1.82×10⁶ complex into the 0.84×10⁶ complex. The catalytic properties of both free and complex leucyl-tRNA synthetases have been compared. K_M for all the substrates are the values of the same order in norm and under EMI. A decrease in some aminoacyl-tRNA synthetase activities associated with polyribosomes has been observed 12 hr after EMI. The interaction of aminoacyl-tRNA synthetases with polyribosomes stimulates the catalytic activity of some enzymes and protects them from heat inactivationin vitro. It is assumed that the changes in association of aminoacyl-tRNA synthetases with high-M_r complexes and compartmentalization of these enzymes on polyribosomes may be related to the alteration of protein biosynthesis under myocardial ischemia.     

Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts

The genetic manipulation of perennial woody tree species presents a range of additional challenges compared to that of annual weedy crop species. These include long generation times and reproductive cycle, the heterogeneity of plants under investigation and, when investigating wood properties, a number of physical and biochemical limitations to microscopical and molecular experimentation. The use of in vitro wood formation systems for molecular studies and Agrobacterium-mediated introduction of transgenes overcomes many of these obstacles. Using a commercially relevant Eucalyptus species as model organism, we demonstrate here that in vitro wood formation systems can be readily employed to introduce transgenes into growing wood-producing tissue, initially leading to frequent transient gene expression in a range of cell types. Stable transformation events were observed as sectors of transformed tissue derived from primary transformation events in individual cells. The usefulness of such systems for the analysis of gene function during the process of wood formation and wood quality determination, as well as for constructing developmental fate maps of cambial derivatives, is discussed.     

Whole blood DNA aberrant methylation in pancreatic adenocarcinoma shows association with the course of the disease: a pilot study

Pancreatic tumors are usually diagnosed at an advanced stage in the progression of the disease, thus reducing the survival chances of the patients. Non-invasive early detection would greatly enhance therapy and survival rates. Toward this aim, we investigated in a pilot study the power of methylation changes in whole blood as predictive markers for the detection of pancreatic tumors. We investigated methylation levels at selected CpG sites in the CpG rich regions at the promoter regions of p16, RARbeta, TNFRSF10C, APC, ACIN1, DAPK1, 3OST2, BCL2 and CD44 in the blood of 30 pancreatic tumor patients and in the blood of 49 matching controls. In addition, we studied LINE-1 and Alu repeats using degenerate amplification approach as a surrogate marker for genome-wide methylation. The site-specific methylation measurements at selected CpG sites were done by the SIRPH method. Our results show that in the patient's blood, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while repeats were slightly less methylated compared to control blood. This was found to be significantly associated with higher risk for pancreatic ductal adenocarcinoma. Additionally, high methylation levels at TNFRSCF10C were associated with positive perineural spread of tumor cells, while higher methylation levels of TNFRSF10C and ACIN1 were significantly associated with shorter survival. This pilot study shows that methylation changes in blood could provide a promising method for early detection of pancreatic tumors. However, larger studies must be carried out to explore the clinical usefulness of a whole blood methylation based test for non-invasive early detection of pancreatic tumors.     

Ecology and importance of Oligochaeta in the biocenosis of zoobenthos in lakes of the National Park of the Lithuanian SSR

This paper aims to describe the ecology and determine the distribution of Oligochaeta according to sediment type, depth and other environmental factors in 21 lakes of the National Park of the Lithuanian SSR. Diagrams are included showing the distribution of the lakes according to the index of density and biomass of Oligochaeta in the trophic structure of biocenosis of the zoobenthos. Light is thrown on the problem of sediment composition as a significant factor in the distribution and development of aquatic Oligochaeta.