Structure-function studies of adenine nucleotide transport in mitochondria. II. Biochemical analysis of distinct AAC1 and AAC2 proteins in yeast

AAC1 and AAC2 genes in yeast each encode functional ADP/ATP carrier (AAC) proteins of the mitochondrial inner membrane. In the present study, mitochondria harboring distinct AAC proteins and the pet9 Arg96 to HIS mutant (Lawson, J., Gawaz, M., Klingenberg, M., and Douglas, M. G. (1990) J. Biol. Chem. 265, 14195-14201) protein have been characterized. In addition, properties of the different AAC proteins have been defined following reconstitution into proteoliposomes. Deletion of AAC2 but not AAC1 causes a major reduction in the mitochondrial cytochrome content and respiration, and this level remains low even when the level of AAC1 protein is increased to 20% that of the AAC2 gene product. In reconstitution studies, the rate of nucleotide transport by isolated AAC1 protein is approximately 40% that of the AAC2 protein. Thus, the lack of mitochondrial-dependent growth supported by the AAC1 gene product alone may be due to the combination of low abundance and reduced activity. Surprisingly, analysis of the Arg96 to His mutant protein revealed binding and transport activities similar to the functional AAC1 and AAC2 gene products. These observations are discussed in relation to a molecular analysis of this highly conserved small transporter and its function in conjunction with other proteins in the mitochondrial membrane.     

A syngeneic monoclonal anti-idiotypic antibody with internal image properties restricted to a single aldosterone-binding system

Immunization with a murine anti-aldosterone mAb (AAC) resulted in the isolation of a syngeneic monoclonal anti-idiotypic antibody, LH9G4. LH9G4 bound to Fab fragments of AAC and was affinity-purified on an AAC column. LH9G4 inhibited the binding of aldosterone to AAC in a dose-dependent manner with an apparent dissociation constant of 0.5 nM as determined by competitive inhibition assays in ELISA and RIA. LH9G4 and aldosterone have similar relative affinities for AAC. Kinetic studies and Scatchard plot analysis support a reversible and reciprocal competitive inhibition mechanism between LH9G4 and aldosterone for the paratope of AAC. The possibility of a steric hindrance mechanism was eliminated. No cross-reactivity was seen with six other murine anti-aldosterone mAb, with a rabbit polyclonal antibody or with aldosterone receptor. The anti-idiotypic antibody, defined as a "restricted" internal image of aldosterone, is apparently directed at a private idiotope present in the paratope of AAC but not in binding sites of other aldosterone-binding proteins. Biophysical considerations involving characteristics of nonbonded attractive forces can explain these findings. An advantage of the one-step auto-anti-idiotypic procedure for the generation of Ab2-beta or internal image antibodies is discussed.     

ADP/ATP translocator is essential only for anaerobic growth of yeast Saccharomyces cerevisiae

All three genes (AAC1, AAC2 and AAC3) encoding the mitochondrial ADP/ATP translocator, were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator-protein nor carrier-mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of Saccharomyces cerevisiae but not for growth under derepressed conditions. Under anaerobic conditions a non-detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.     

Cardiolipin and mitochondrial carriers

Members of the mitochondrial carrier family interact with cardiolipin (CL) as evident from a variety of functional and structural effects. CL stabilises carrier proteins on isolation with detergents, with the P_i carrier as the prime example. CL is required for transport in reconstituted vesicles, prime examples are the P_i- and ADP/ATP carrier (AAC). CL binds to the AAC in a graded manner; 6 CL/AAC dimer bind tightly as measured on the ³¹P NMR time scale. 2 additional CL/dimer bind reversibly and a fast exchanging envelope of phospholipids includes CL as measured on the ESR time scale. In the crystal structure of the CAT-AAC complex 3 CL bind to the periphery of the AAC in a three-fold pseudo-symmetry. The binding of CL is implicated to contribute lowering the high transition energy barriers in the AAC. Para-functions of the AAC, as in the mitochondrial pore transition (MPT) and in cell death are linked to the CL binding of the AAC. Ca⁺⁺ or oxidants can sequester or destroy AAC bound CL, rendering AAC labile, allowing pore formation and degradation. Thus AAC, by being vital for energy transfer, constitutes an Achilles heel in the eukaryotic cell. AAC together with CL is also engaged in respiratory supercomplexes. Different from AAC the similarly structured uncoupling protein (UCP1) has no tightly bound CL, but CL addition lowers affinity of the inhibitory nucleotide binding that may contribute to the physiological regulation of the uncoupling activity by ATP.     

Product Environmental Life-Cycle Assessment Using Input-Output Techniques

Life-cycle assessment (LCA) facilitates a systems view in environmental evaluation of products, materials, and processes. Life-cycle assessment attempts to quantify environmental burdens over the entire life-cycle of a product from raw material extraction, manufacturing, and use to ultimate disposal. However, current methods for LCA suffer from problems of subjective boundary definition, inflexibility, high cost, data confidentiality, and aggregation.
This paper proposes alternative models to conduct quick, cost effective, and yet comprehensive life-cycle assessments. The core of the analytical model consists of the 498 sector economic input-output tables for the U.S. economy augmented with various sector-level environmental impact vectors. The environmental impacts covered include global warming, acidification, energy use, non-renewable ores consumption, eutrophication, conventional pollutant emissions and toxic releases to the environment. Alternative models are proposed for environmental assessment of individual products, processes, and life-cycle stages by selective disaggregation of aggregate input-output data or by creation of hypothetical new commodity sectors. To demonstrate the method, a case study comparing the life-cycle environmental performance of steel and plastic automobile fuel tank systems is presented.     

Soil flushing: a review of the origin of efficiency variability

Soil flushing using aqueous solutions is employed to solubilise contaminants. As water solubility is the controlling mechanism of dissolution, additives (surfactants, cosolvents, etc.) are used to enhance efficiencies and reduce the treatment time compared to the use of water alone. The use of surfactant alone gives efficiencies of about 80–85 % in laboratory experiments, but the amounts of product to be injected are very important, which does not seem to be economically sustainable. Studies indicate that when soil flushing is applied in the field, efficiency is very variable; it can vary from almost 0 % to almost 100 %. This illustrates the importance of knowledge of the field (soil heterogeneities, type of contamination, etc.). Using only one product (surfactant, cosolvent, cyclodextrin) often gives moderate efficiencies and needs very large amounts of products, with a product:pollutant ratio higher than 100:1. On the other hand, the use of more complex methods involving micro emulsions or several products with polymer injection lead to high efficiencies at first and a product:pollutant ratio that can be lower than 5. The importance of the initial saturation of the non-aqueous phase liquid is highlighted: the higher the initial saturation, the higher the efficiency. For initial saturations lower than 1 %, soil flushing may not be a very efficient technique. This paper provides an overview of recent studies in the area of soil and groundwater remediation, from laboratory columns scale to pilot and real sites. The research has focused on chlorinated solvents as they are extremely difficult to treat.     

Competing uses for China's straw: the economic and carbon abatement potential of biochar

China is under pressure to improve its agricultural productivity to keep up with the demands of a growing population with increasingly resource‐intensive diets. This productivity improvement must occur against a backdrop of carbon intensity reduction targets, and a highly fragmented, nutrient‐inefficient farming system. Moreover, the Chinese government increasingly recognizes the need to rationalize the management of the 800 million tonnes of agricultural crop straw that China produces each year, up to 40% of which is burned in‐field as a waste. Biochar produced from these residues and applied to land could contribute to China's agricultural productivity, resource use efficiency and carbon reduction goals. However competing uses for China's straw residues are rapidly emerging, particularly from bioenergy generation. Therefore it is important to understand the relative economic viability and carbon abatement potential of directing agricultural residues to biochar rather than bioenergy. Using cost‐benefit analysis (CBA) and life‐cycle analysis (LCA), this paper therefore compares the economic viability and carbon abatement potential of biochar production via pyrolysis, with that of bioenergy production via briquetting and gasification. Straw reincorporation and in‐field straw burning are used as baseline scenarios. We find that briquetting straw for heat energy is the most cost‐effective carbon abatement technology, requiring a subsidy of $7 MgCO₂e^?1 abated. However China's current bioelectricity subsidy scheme makes gasification (NPV $12.6 million) more financially attractive for investors than both briquetting (NPV $7.34 million), and pyrolysis ($?1.84 million). The direct carbon abatement potential of pyrolysis (1.06 MgCO₂e per odt straw) is also lower than that of briquetting (1.35 MgCO₂e per odt straw) and gasification (1.16 MgCO₂e per odt straw). However indirect carbon abatement processes arising from biochar application could significantly improve the carbon abatement potential of the pyrolysis scenario. Likewise, increasing the agronomic value of biochar is essential for the pyrolysis scenario to compete as an economically viable, cost‐effective mitigation technology.     

Structure-function studies of adenine nucleotide transport in mitochondria. I. Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria

The gene encoding the major ADP/ATP carrier in yeast AAC2 (pet9; Lawson, J., and Douglas, M. (1988) J. Biol. Chem. 263, 14812-14818) has been disrupted (delta AAC2) by itself and in combination with a disruption of a second translocator gene AAC1 (delta AAC1). Disruption of AAC2 like the pet9 mutation renders yeast unable to grow on a nonfermentable carbon source. The AAC1 AAC2 double disruption exhibits a phenotype identical to the AAC2. This provides the host strain for the analysis of point mutations in the AAC protein. We have initiated this structure-function analysis by characterizing and confirming that the pet9 mutation is a G to A transition resulting in an arginine to histidine change at position 96. Site-directed replacements at Arg96 confirm its essential function for growth on a nonfermentable carbon source. These data also suggest that in the absence of functional AAC1 and AAC2 gene products, adenine nucleotide transport across the mitochondrial inner membrane must occur by an as yet unidentified translocator or translocation mechanism or that within these cells separate intra- and extramitochondrial adenine nucleotide pools can exist to support growth.     

Cardiolipin,a critical determinant of mitochondrial carrier protein assembly and function

The ability of phospholipids to act as determinants of membrane protein structure and function is probably best exemplified by cardiolipin (CL), the signature phospholipid of mitochondria. Early efforts to reconstitute individual respiratory complexes and members of the mitochondrial carrier family, most notably the ADP/ATP carrier (AAC), often demonstrated the importance of CL. Over the past decade, the significance of CL in the organization of components of the electron transport chain into higher order assemblies, termed respiratory supercomplexes, has been established. Another protein required for oxidative phosphorylation, AAC, has received comparatively little attention likely stemming from the fact that AACs were thought to function in isolation as either homodimers or monomers. Recently however, AACs have been demonstrated to interact with the respiratory supercomplex, other members of the mitochondrial carrier family, and the TIM23 translocon. Interestingly, many if not all of these interactions depend on CL. As the paradigm for the mitochondrial carrier family, these discoveries with AAC suggest that other members of this large group of important proteins may be more gregarious than anticipated. Moreover, it is proposed that AAC and perhaps additional members of the mitochondrial carrier family might represent downstream targets of pathological states involving alterations in CL.     

A third ADP/ATP translocator gene in yeast

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.     

An integrated environmental and cost assessment method based on LCA and LCC for automobile interior and exterior trim design scheme optimization

Purpose

This paper provided an integrated method to evaluate environmental impact and life cycle cost (LCC) of various alternative design schemes in the early design and development stages of complex mechanical product; an optimization method of product design schemes based on life cycle assessment (LCA) and LCC is proposed as a supporting design tool to achieve optimal integration of environmental impact and cost of the design.

Methods

The applied research methods include product level deconstruction model, LCA/LCC integrated analysis model, and the product design scheme optimization method. In the life cycle environmental assessment, GaBi software and CML2001 evaluation method are used to evaluate product environmental impact. In terms of product design configuration scheme optimization, the TOPSIS method is used to optimize the design schemes generated. Taking the internal and external trim of automobile as an example, the specific implementation process of the method is illustrated.

Results and discussion

The case study indicates that, when comprehensively considering the environmental impact and cost, the composite indices of the optimal and worst schemes are 0.8667 and 0.3001, respectively; their costs are ¥164.87 and ¥179.68, respectively; and the eco points of environmental impact are 14.74 and 39.78, respectively. The cost of the two schemes are not much different, but the environmental impact of the optimal scheme is only 37.1% of the worst scheme’s; When cost is the only factor to be considered, the lowest cost design scheme is about 36.7% of the maximum scheme’s cost, and the environmental impact of the lowest cost design scheme is about 1.6 times of the maximum cost scheme’s. When environmental impact is the only factor to be considered, the least environmental impact of design scheme accounts about 31.7% of the largest; the cost of design scheme with the least environmental impact accounts for about 58.1% of the largest one’s. Integrating LCA and LCC, scientific suggestions can be provided from several perspectives.

Conclusions

By considering the environmental impact and LCC, this paper proposes a method of product design scheme optimization as a supporting design tool which could evaluate the design options of the product and identify the preferred option in the early stage of product design. It is helpful to realize the sustainability of the product. In order to improve the applicability of this method, the weighting factors of environmental impact and cost could be adjusted according to the requirements of energy saving and emission reduction of different enterprises.

     

Separate genes encode functionally equivalent ADP/ATP carrier proteins in Saccharomyces cerevisiae. Isolation and analysis of AAC2

Genetic and biochemical analysis of Saccharomyces cerevisiae containing a disruption of the nuclear gene (AAC1) encoding the mitochondrial ADP/ATP carrier has revealed a second gene for this protein. The second gene, designated AAC2, has been isolated by genetic complementation and sequenced. AAC2 contains a 954-base pair open reading frame coding for a protein of 318 amino acids which is highly homologous to the AAC1 gene product except that it is nine amino acids longer at the NH2 terminus. The two yeast genes are highly conserved at the level of DNA and protein and share identity with the ADP/ATP carriers from other organisms. Both genes complement an ADP/ATP carrier defect (op1 or pet9). However, the newly isolated gene AAC2 need be present only in one or two copies while the previously isolated AAC1 gene must be present in multiple copies to support growth dependent on a functional carrier protein. This gene dosage-dependent complementation combined with the high degree of conservation suggest that these two functionally equivalent genes may be differentially expressed.     

Removal of Ni(II) from aqueous solution by adsorption onto hazelnut shell activated carbon: equilibrium studies

Carps are the mainstay of Indian aquaculture, contributing over 90% to the total fish production, which was estimated to be 1.77 million metric tonnes in 1996. Carp culture has a great potential for waste utilization and thus for pollution abatement. Many wastes such as cow, poultry, pig, duck, goat, and sheep excreta, biogas slurry, effluents from different kinds of factories/industries have been efficiently used for enhancing the productivity of natural food of carps and related species. Besides, several organic wastes/byproducts such as plant products, wastes from animal husbandry, and industrial by-products have been used as carp feed ingredients to lower the cost of supplementary feeding. However, to ensure the continued expansion of fish ponds and the pollution control, there must be a market for the fish (carps) produced in these ponds. The carps have, however, a low market value due to the presence of intra-muscular bones, which reduces their consumer acceptability. Thus, a need was felt to develop some boneless convenience products for enhancing the consumer acceptability of the carps. Efforts were made to prepare three value-added fish products, namely fish patty, fish finger and fish salad from carp flesh and were compared with a reference product ('fish pakoura'). Sensory evaluation of these products gave highly encouraging results. The methods of preparation of these products were transferred to some progressive farmers of the region who prepared and sold these products at very attractive prices. Carp processing has a great potential for the establishment of a fish ancillary industry and thus for boosting the production of these species. In Punjab alone, there is a potential of consuming 32,448 metric tonnes per annum of such value-added products (which would require 54,080 metric tonnes of raw fish). The development of value-added products has a significant role in raising the socio-economic status of the people associated with carp culture. The average cost of production of these products was estimated to be INR 80 per kg. With a sale price of INR 110 per kg, and a sale of 50 kg per day of the value-added products (26 days a month), the average monthly income of a carp-processing unit comes to be INR 39,000 (929 USD, approximately).     

Integrating life cycle cost analysis and LCA

The private sector decision making situations which LCA addresses mustalso eventually take theeconomic consequences of alternative products or product designs into account. However, neither the internal nor external economic aspects of the decisions are within the scope of developed LCA methodology, nor are they properly addressed by existing LCA tools. This traditional separation of life cycle environmental assessment from economic analysis has limited the influence and relevance of LCA for decision-making, and left uncharacterized the important relationships and trade-offs between the economic and life cycle environmental performance of alternative product design decision scenarios. Still standard methods of LCA can and have been tightly, logically, and practically integrated with standard methods for cost accounting, life cycle cost analysis, and scenario-based economic risk modeling. The result is an ability to take both economic and environmental performance — and their tradeoff relationships — into account in product/process design decision making.     

The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli

The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.     

Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.     

The Environmental Impacts of Reuse: A Review

The fear that human consumption is causing climate change, biodiversity loss, and mineral scarcity has recently prompted interest in reuse because of the intuitive belief that it reduces new production and waste. The environmental impacts of reuse have, however, received little attention—the benefits typically assumed rather than understood—and consequently the overall effects remain unclear. In this article, we structure the current work on the topic, reviewing the potential benefits and pitfalls described in the literature and providing a framework for future research. Many products’ use‐phase energy requirements are decreasing. The relative importance of the embodied impacts from initial production is therefore growing and the prominence of reuse as an abatement strategy is likely to increase in the future. Many examples are found in the literature of beneficial reuse of standardized, unpowered products and components, and repairing an item is always found to be less energy intensive than new production. However, reusing a product does not guarantee an environmental benefit. Attention must be paid to restoring and upgrading old product efficiencies, minimizing overspecification in the new application, and considering whether more efficient, new products exist that would be more suitable. Cheap, reused goods can allow many consumers access to products they would otherwise have been unable to afford. Though socially valuable, these sales, which may help minimize landfill in the short term, can represent additional consumption rather than a net environmental benefit compared to the status quo.     

Estimating cell and product yields

The balance equations for carbon, reduction potential, and energy during cell growth and product formation are rederived in a general form. Cells are treated simply as a very complex product, and the Y(ATP) concept is extended to products. Limitations on the theoretical yield are discussed for different product types. Simple aerobic products cannot be energy limited unless the maintenance requirement is large, while complex products cannot be reduction limited. A maximum yield is defined for products much more oxidized than their substrate (carbon limited) because the theoretical yield conditions may violate the energy balance. For reduced complex products the yield on available electrons is related to Y(ATP), the P/O ratio, and the product composition. Narrow bounds are established on the actual yields in simple anaerobic fermentations, and the significance of the yields in the linear growth equation is discussed.     

Mechanistic characterization of the bifunctional aminoglycoside-modifying enzyme AAC(3)-Ib/AAC(6')-Ib' from Pseudomonas aeruginosa

A recently discovered bifunctional antibiotic-resistance enzyme named AAC(3)-Ib/AAC(6')-Ib', from Pseudomonas aeruginosa, catalyzes acetylation of aminoglycoside antibiotics. Since both domains are acetyltransferases, each was cloned and purified for mechanistic studies. The AAC(3)-Ib domain appears to be highly specific to fortimicin A and gentamicin as substrates, while the AAC(6')-Ib' domain exhibits a broad substrate spectrum. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism. The use of dead-end and product inhibition and solvent-isotope effect reveals that both domains catalyze their reactions by a steady-state ordered Bi-Bi kinetic mechanism, in which acetyl-CoA is the first substrate that binds to the active site, followed by binding of the aminoglycoside antibiotic. Subsequent to the transfer of the acetyl group, acetylated aminoglycoside is released prior to coenzyme A. The merger of two genes to create a bifunctional enzyme with expanded substrate profile would appear to be a recent trend in evolution of resistance to aminoglycoside antibiotics, of which four examples have been documented in the past few years.