The redox state of pyridine nucleotides controls permeability of uncoupled mitochondria to K+

Heart mitochondria respiring in the presence of P_i release endogenous K⁺ to a sucrose medium when an uncoupler is added. The uncoupled mitochondria retain K⁺, however, if the oxidation of NAD(P)H is prevented by the addition of rotenone or antimycin. Addition of rotenone, once the uncoupler-dependent K⁺-efflux has been initiated, results in a rapid reduction of NAD(P) and a simultaneous decrease in permeability to K⁺. These changes are independent of respiration. The results suggest that a latent pathway for K⁺-permeability is present in the membrane, that it can be opened and closed reversibly, and that it reflects, either directly or indirectly, the redox status of mitochondrial pyridine nucleotides. The possible relationship of this putative pathway to those available for Ca²⁺ uptake and release is considered.     

Reduction in postprandial energy expenditure during pregnancy.

Energy expenditure was measured during pregnancy in seven primigravid women at 12-15, 25-28, and 34-36 weeks and after the cessation of lactation. On each occasion the resting metabolic rate and the increase in metabolic rate after ingestion of a liquid test meal were measured by indirect calorimetry. In absolute terms the resting metabolic rate increased steadily during pregnancy but when expressed per unit of body weight no change was found. The energetic response to a mixed constituent meal was significantly reduced by 28% in the middle trimester of pregnancy. These findings suggest a possible maternal adaptation to increase energetic efficiency at a time when the energy demands of the fetus are high.     

Structural basis of human erythrocyte glucose transporter function in reconstituted system. Hydrogen exchange

Hydrogen exchange kinetic behavior of human erythrocyte glucose transporter protein in vesicles was studied in the absence and in the presence of D-glucose or a well known inhibitor, cytochalasin B. This is to detect a proposed channel of water penetrating into the protein through which the sugar molecule passes and to monitor any conformational changes induced by the substrate or inhibitor. Analyses of the kinetic data revealed several classes of hydrogens which exchange with readily distinguishable rates. Of 660 hydrogens detected per transporter, approximately 30% exchanged with rates generally characterized as those of free amide hydrogens indicating they are interfaced to solvent water. Since the transporter is known to be embedded deep in the hydrophobic area of the membrane with minimum exposure to the outside of the membrane lipid bilayer, a significant portion of these free amide hydrogens must be at the purported channel rather than outside of the membrane. D-Glucose and cytochalasin B affected the exchange kinetics of these presumably channel-associated free amide hydrogens rather differently. D-Glucose reduced the apparent rate constants, but not the total number. Cytochalasin B on the other hand reduced the total number to one-half without significantly changing the apparent rate constants. The remaining 70% of the labeled hydrogens exchanged with much slower rates which vary 10-10,000-fold, indicating that they are internally structured peptide amide and side chain hydrogens. Both D-glucose and cytochalasin B further reduced the rates of these hydrogens, indicating a global stabilization of the protein structure.     

Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex.

The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.     

Free fatty acids activate a vigorous Ca(2+):2H(+) antiport activity in yeast mitochondria.

The accumulation and retention of Ca(2+) by yeast mitochondria (Saccharomyces cerevisiae) mediated by ionophore ETH 129 occurs with a variable efficiency in different preparations. Ineffective Ca(2+) transport and a depressed membrane potential occur in parallel, are exacerbated in parallel by exogenous free fatty acids, and are corrected in parallel by the addition of bovine serum albumin. Bovine serum albumin is not required to develop a high membrane potential when either Ca(2+) or ETH 129 are absent, and when both are present membrane potential is restored by the addition of EGTA in a concentration-dependent manner. Respiration and swelling data indicate that the permeability transition pore does not open in yeast mitochondria that are treated with Ca(2+) and ETH 129, whereas fatty acid concentration studies and the inaction of carboxyatractyloside indicate that fatty acid-derived uncoupling does not underlie the other observations. It is concluded that yeast mitochondria contain a previously unrecognized Ca(2+):2H(+) antiporter that is highly active in the presence of free fatty acids and leads to a futile cycle of Ca(2+) accumulation and release when exogenous Ca(2+) and ETH 129 are available. It is also shown that isolated yeast mitochondria degrade their phospholipids at a relatively rapid rate. The activity responsible is also previously unrecognized. It is Ca(2+)-independent, little affected by the presence or absence of a respiratory substrate, and leads to the hydrolysis of ester linkages at both the sn-1 and sn-2 positions of the glycerophospholipids. The products of this activity, through their actions on the antiporter, explain the variable behavior of yeast mitochondria treated with Ca(2+) plus ETH 129.     

Irreversible inhibition of GABA-T by halogenated analogues of beta-alanine

beta-Difluoromethyl-beta-alanine (3-amino-4,4-difluorobutanoic acid) is a potent in vitro and in vivo inhibitor of GABA-T. The rate of inhibition of GABA-T is concentration- and time-dependent. The inactivation is active-site directed. No reactive species escapes from the active site before reacting with the enzyme. The inhibition is irreversible and stereospecific. The use of beta-2H-beta-difluoromethyl-beta-alanine results in a marked primary isotope effect in vitro and in vivo. The use of differently substituted dihalogeno derivatives of beta-alanine suggests that the rate of inhibition is dependent on the nature and position of the leaving group. The mechanism of inhibition is discussed on the basis of spectral changes.     

Nrf2 Negatively Regulates Melanogenesis by Modulating PI3K/Akt Signaling

Nrf2 plays a role in protection of cells against oxidative stress and xenobiotic damage by regulating cytoprotective genes. In this study, we investigated the effect of Nrf2 on melanogenesis in normal human melanocytes (NHMCs). When NHMCs were transduced with a recombinant adenovirus expressing Nrf2, melanin synthesis was significantly decreased. Consistent with this result, overexpression of Nrf2 decreased the expression of tyrosinase and tyrosinase-related protein 1. The inhibitory effect of Nrf2 was reversed by overexpression of Keap1, an intracellular regulator of Nrf2. Interestingly, Nrf2 overexpression resulted in marked activation of PI3K/Akt signaling. Conversely, inhibition of PI3K activity by treatment with wortmannin reversed the depigmentary effects of Nrf2. Taken together, these results strongly suggest that Nrf2 negatively regulates melanogenesis by modulating the PI3K/Akt signaling pathway.     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998