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ATP/ADP translocases transport ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate intracellular bacteria. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen 'Candidatus Liberibacter asiaticus,' the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The 'Ca. Liberibacter asiaticus' gene (nttA) encoding the translocase was subsequently expressed in Escherichia coli and shown to enable E. coli to import ATP directly into the ceil. Competition assays with the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in 'Ca. Liberibacter solanacearum,' the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from 'Ca. Liberibacter asiaticus,' indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment. doi: 10.1128/JB.01279-09

Increased oxidative stress and intracellular calcium levels and mitochondrial overloading of calcium during ischemic renal injury (IRI) favor mitochondrial membrane permeability transition pore (MPTP) opening and subsequent necrotic cell death. Cyclophilin D (CypD) is an essential component of MPTP, and recent findings implicate its role in necrotic, but not apoptotic, cell death. To evaluate the role of CypD following IRI, we tested the hypothesis that CypD gene ablation protects mice from IRI. Renal function as assessed by plasma levels of both creatinine and blood urea nitrogen was significantly reduced in CypD knockout ([CypD.sup.-/-]) mice compared with wild-type mice during the 5-day post-ischemia period. Erythrocyte trapping, tubular cell necrosis, tubular dilatation, and neutrophil infiltration were significantly decreased in [CypD.sup.-/-] mice. To define the mechanisms by which CypD deficiency protect the kidneys, an in vitro model of IRI was employed. Inhibition of CypD using Cyclosporin A in oxidant-injured cultured proximal tubular cells (PTC) prevented mitochondrial membrane depolarization, reduced LDH release, ATP depletion and necrotic cell death. Similarly, oxidant-injured [CypD.sup.-/-] PTC primary cultures were protected from cytotoxicity and necrosis. To conclude, CypD gene ablation offers both functional and morphological protection in mice following IRI by decreasing necrotic cell death possibly via inhibition of MPTP and ATP depletion. acute renal failure; mitochondria; ATP depletion

Phosphofructokinase is a key enzyme of glycolysis that exists as homoand heterotetramers of three subunit isoforms: muscle, liver, and C type. Mice with a disrupting tag inserted near the distal promoter of the phosphofructokinase-M gene showed tissue-dependent differences in loss of that isoform: 99% in brain and 95-98% in islets, but only 50-75% in skeletal muscle and little if any loss in heart. This correlated with the continued presence of proximal transcripts specifically in muscle tissues. These data strongly support the proposed two-promoter system of the gene, with ubiquitous use of the distal promoter and additional use of the proximal promoter selectively in muscle. Interestingly, the mice were glucose intolerant and had somewhat elevated fasting and fed blood glucose levels; however, they did not have an abnormal insulin tolerance test, consistent with the less pronounced loss of phosphofructokinase-M in muscle. Isolated pefifused islets showed about 50% decreased glucosestimulated insulin secretion and reduced amplitude and regularity of secretory oscillations. Oscillations in cytoplasmic free [Ca.sup.2+] and the rise in the ATP/ADP ratio appeared normal. Secretory oscillations still occurred in the presence of diazoxide and high KC1, indicating an oscillation mechanism not requiring dynamic [Ca.sup.2+] changes. The results suggest the importance of phosphofructokinase-M for insulin secretion, although glucoldnase is the overall rate-limiting glucose sensor. Whether the [Ca.sup.2+] oscillations and residual insulin oscillations in this mouse model are due to the residual 2-5% phosphofructoldnase-M or to other phosphofunctokinase isoforms present in islets or involve another metabolic oscillator remains to be determined. OmniBank; calcium; pancreatic islets; adenosine 5'-triphosphate/ adenosine 5'-diphosphate ratio

Change in gene functions (gene cooption) is one of the key mechanisms of molecular evolution. Genes can acquire new functions via alteration in properties of encoded proteins and/or via changes in temporal or spatial regulation of expression. Here we demonstrate radical changes in the functions of orthologous ATP1B4 genes during evolution of vertebrates. Expression of ATP1B4 genes is brain-specific in teleost fishes, whereas it is predominantly muscle-specific in tetrapods. The encoded [beta]m-proteins in fish, amphibian, and avian species are [beta]-subunits of Na,K-ATPase located in the plasma membrane. In placental mammals [beta]m-proteins lost their ancestral functions, accumulate in nuclear membrane of perinatal myocytes, and associate with transcriptional coregulator Ski-interacting protein (SKIP). Through interaction with SKIP, eutherian [beta]m acquired new functions as exemplified by regulation of TGF-[beta]-responsive reporters and by augmentation of mRNA levels of Smad7, an inhibitor of TGF-[beta] signaling. Thus, orthologous vertebrate ATP1B4 genes represent an instance of gene cooption that created fundamental changes in the functional properties of the encoded proteins. ATP1B4 | gene cooption | skeletal muscle development | TGF-[beta] | Smad7

The transport of carbohydrates by Streptococcus mutans is accomplished by the phosphoenolpyruvatephosphotransferase system (PTS) and ATP-binding cassette (ABC) transporters. To undertake a global transcriptional analysis of all S. mutans sugar transporters simultaneously, we used a whole-genome expression microarray. Global transcription profiles of S. mutans UAI59 were determined for several monosaccharides (glucose, fructose, galactose, and mannose), disaccharides (sucrose, lactose, maltose, and trehalose), a [beta]-glucoside (cellobiose), oligosaccharides (raffinose, stachyose, and maltotriose), and a sugar alcohol (mannitol). The results revealed that PTSs were responsible for transport of monosaccharides, disaccharides, [beta]-glucosides, and sugar alcohol. Six PTSs were transcribed only if a specific sugar was present in the growth medium; thus, they were regulated at the transcriptional level. These included transporters for fructose, lactose, cellobiose, and trehalose and two transporters for mannitol. Three PTSs were repressed under all conditions tested. Interestingly, five PTSs were always highly expressed regardless of the sugar source used, presumably suggesting their availability for immediate uptake of most common dietary sugars (glucose, fructose, maltose, and sucrose). The ABC transporters were found to be specific for oligosaccharides, raffinose, stachyose, and isomaitosaccharides. Compared to the PTSs, the ABC transporters showed higher transcription under several tested conditions, suggesting that they might be transporting multiple substrates.

Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival. A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by the accumulation of PPIX in the skin. We have previously shown that mice with a deficiency in the ABC transporter Bcrp1/Abcg2 display a novel type of protoporphyria. This protoporphyria is mild compared with ferrochelatase-dependent EPP, and in itself not sufficient to cause phototoxicity, but it might exacerbate the consequences of other porphyrias. In this study, we identified the mouse harderian gland as a novel expression site of Bcrp1. Because of its pronounced role in porphyrin secretion, the harderian gland presents a useful tool to study the mechanism of Bcrp1-related protoporphyria and transport of porphyrins. Bcrp[1.sup.-/-] harderian gland displayed a highly increased accumulation of PPIX glycoconjugates, and a similar shift was seen in Bcrp[1.sup.-/-] liver. Tear- and hepatobiliary excretion data suggest that Bcrp1 controls intracellular levels of PPIX by mediating high affinity transport of its glycoconjugates and possibly low-affinity transport of unconjugated PPIX. This mechanism may allow cells to prevent or reduce cytotoxicity of PPIX under excess conditions, without spillage under physiological conditions where PPIX is needed. ATP binding cassette transporter; knockout mice; protoporphyria doi:10.1152/ajpcell.00359.2006

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.

Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. [K.sub.ATP] channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of [K.sub.ATP] channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female [K.sub.ATP] channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in [K.sub.ATP] channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus [K.sub.ATP] channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand. ATP-sensitive [K.sup.+] channel; Kir6.2; magnetic resonance imaging; myocardium; sex

ATP-binding cassette transporter A1 (ABCA1), a pivotal regulator of cholesterol efflux from cells to apolipoproteins, plays an important role in cholesterol homeostasis. As an inflammatory factor, IL-1[beta] has been shown to downregulate ABCA1 in macrophages and facilitates foam cell formation. However, the molecular mechanism underlining the downregulated ABCA 1 by IL-1[beta] is still elusive. In the present study, we demonstrated that IL-1[beta] downregulated ABCA1 but not ABCG1 at mRNA and protein levels in a time- and dose-dependent manner in THP-1 and A549 cells. IL-1[beta] attenuated ABCA1 promoter activity through an LXR (liver X receptor)-independent pathway, since IL-1[beta] did not alter the expression and activities of LXR[alpha]/[beta] and deletion of the LXR responsive element from the ABCA1 promoter failed to reverse the IL-1[beta] effect. In contrast, NF-[kappa]B inhibition by pyrrolidine dithiocarbamate and MG132 prevented the suppression of ABCA1 by IL-1[beta]. Cotransfection with ABCA1 luciferase reporter and the expression plasmids of Rel A decreased ABCA1 promoter activities. An adenovirus expressing NF-[kappa]B inhibitor subunit-[alpha] inhibited NF-[kappa]B activities and also reversed the IL-1[beta] effect at the promoter activity and protein levels of ABCA1. In addition, IL-1[beta] could induce the production of reactive oxygen species (ROS), and N-acetyl-L-cysteine, a scavenger of ROS, reversed the decreased level of ABCA1 induced by IL-1[beta]. [H.sub.2][O.sub.2] decreased ABCA1 at the mRNA and protein levels and the promoter activity. Thus our data provide strong evidence that ROS and NF-[kappa]B, but not LXR, mediate the IL-1[beta]-induced downregulation of ABCA1 via a novel transcriptional mechanism, which might play an important role of proinflammation in the alteration of lipid metabolism. interleukin-1[beta]; nuclear factor-[kappa]B; reactive oxygen species

The genetic factors contributing to the complex disorder of myocardial calcification are largely unknown. Using a mouse model, we fine-mapped the major locus (Dyscalc1) contributing to the dystrophic cardiac calcification (DCC) to an 840-kb interval containing 38 genes. We then identified the causal gene by using an approach integrating genetic segregation and expression array analyses to identify, on a global scale, cis-acting DNA variations that perturb gene expression. By studying two intercrosses, in which the DCC trait segregates, a single candidate gene (encoding the ATP-binding cassette transporter ABCC6) was identified. Transgenic complementation confirmed Abcc6 as the underlying causal gene for Dyscalc1. We demonstrate that in the cross, the expression of Abcc6 is highly correlated with the local mineralization regulatory system and the BMP2-Wnt signaling pathway known to be involved in the systemic regulation of calcification, suggesting potential pathways for the action of Abcc6 in DCC. Our results demonstrate the power of the integrative genomics in discovering causal genes and pathways underlying complex traits. expression quantitative trait locus | transgenic | positional cloning osteopontin

ABC transporters constitute one of the largest gene families in all species. They are mostly involved in transport of substrates across membranes. We have previously demonstrated that the Caenorhabditis elegans ABC family shows poor one-to-one gene orthology with other distant model organisms. To address the evolution dynamics of this gene family among closely related species, we carried out a comparative analysis of the ABC family among the three nematode species C. elegans, C. briggsae, and C. remanei. In contrast to the previous observations, the majority of ABC genes in the three species were found in orthologous trios, including many tandemly duplicated ABC genes, indicating that the gene duplication took place before speciation. Species-specific expansions of ABC members are rare and mostly observed in subfamilies A and B. C. briggsae and C. remanei orthologous ABC genes tend to cluster on trees, with those of C elegans as an outgroup, consistent with their proposed species phylogeny. Comparison of intron/exon structures of the highly conserved ABCE subfamily members also indicates a closer relationship between C. briggsae and C. remanei than between either of these species and C. elegans. A comparison between insect and mammalian species indicates lineage-specific duplications or deletions of ABC genes, while the family size remains relatively constant. Sites undergoing positive selection within subfamily D, which are implicated in very-long-chain fatty acid transport, were identified. The evolution of these sites might be driven by the changes in food source with time.

BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only onethird of the widespread bio Y genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the [alpha]-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coil cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters. biotin transporter | functional genomics | transport systems

Studies have found that cystic fibrosis (CF) lung disease reflects the failure of airways defense against chronic bacterial infection. It is revealed that studies of CF cultures, transgenic mice, and CF patients indicate that the initiating event in the CF airways disease pathogenesis is reduced airway surface liquid (ASL) volume that is dehydration.

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to [approximately equal to] 47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families. ATP-binding cassette | expression | high throughput | transport | tripartite ATP-independent periplasmic

We recently reported the isolation and initial characterization of a transposon-generated mutation that resulted in defects in both morphogenesis and antibiotic production in Streptomyces coelicolor. The insertion identified the SCO7168 open reading frame whose predicted product is a GntR family transcriptional regulator. Here, we show that this gene acts to repress transcription of itself as well as a series of genes immediately adjacent to it on the S. coelicolor chromosome that likely encode an ATP-binding cassette (ABC)-type transporter for carbohydrate uptake. Transcription of this transporter is strongly induced by growth on relatively poor carbon sources such as trehalose and melibiose and weakly induced by lactose and glycerol but not glucose, and induction is not repressed by the presence of glucose. Constructed deletions of the ABC transporter itself resulted in the suppression of the original transposon mutation, suggesting that inappropriate expression of the ABC transporter is responsible, at least in part, for the mutant phenotype. Because this transporter responds to the presence of [alpha]-glucosides and has similarity to two other carbohydrate transporters of this class, we have named the genes of the transporter agl3E, agl3F, and agl3G and the GntR-like protein that regulates transcription of the transporter agl3R in accordance with established nomenclature. We suggest that agl3R is one of a number of homologous proteins in Streptomyces (there are 57 putative GntR family regulators in the S. coelicolor genome) that respond to nutritional and/or environmental signals to control genes that affect morphogenesis and antibiotic production.

The genomes of mammals and birds can be partitioned into megabase-long regions, termed isochores, with consistently high, or low, average C + G content. Isochores with high CG contain a mixture of CG-rich and AT-rich genes, while high-AT isochores contain predominantly AT-rich genes. The two gene populations in the high-CG isochores are functionally distinguishable by statistical analysis of their gene ontology categories. However, the aggregate of the two populations in CG isochores is not statistically distinct from AT-rich genes in AT isochores. Genes tend to be located at local extrema of composition within the isochores, indicating that the CG-enriching mechanism acted differently when near to genes. On the other hand, maximum-likelihood reconstruction of molecular phylogenetic trees shows that branch lengths (evolutionary distances) for third codon positions in CG-rich genes are not substantially larger than those for AT-rich genes. In the context of neutral mutation theory this argues against any strong positive selection. Disparate features of isochores might be explained by a model in which about half of all genes functionally require AT richness, while, in warm-blooded organisms, about half the genome (in large coherent blocks) acquired a strong bias for mutations to CG. Using mutations in CG-rich genes as convenient indicators, we show that [approximately equal to] 20% of amino acids in proteins are broadly substitutable, without regard to chemical similarity.

The regulatory factors, like calcineurin, which promote activation of developmental myosin heavy chain gene expressions during myogenesis, in mouse embryos, are analyzed.

The stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs 1, when replication forks stall. It is shown that replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.

In members of one of the subfamilies of the bacterial ATP binding cassette (ABC) transporters, the two nucleotide binding domains are fused as a single peptide and the proteins have no membrane-spanning domain partners. Most of the ABC efflux transporters of this subfamily have been characterized in actinomycetes, producing macrolide, lincosamide, and streptogramin antibiotics. Among 40 ABC efflux transporters of Bacillus subtilis, five proteins belong to this subfamily. None of these proteins has been functionally characterized. We examined macrolide, lincosamide, and streptogramin antibiotic resistance in insertional disruptants of the genes that encode these proteins. It was found that only a disruptant of vmlR (formerly named expZ) showed hypersensitivity to virginiamycin M and lincomycin. Expression of the vmlR gene was induced by the addition of these antibiotics in growth medium. Primer extension analysis revealed that transcription of the vmlR gene initiates at an adenosine residue located 225 bp upstream of the initiation codon. From the analysis of the vmlR and lacZ fusion genes, a 52-bp deletion from +159 to +211 resulted in constitutive expression of the vmlR gene. In this region, a typical [rho]-independent transcriptional terminator was found. It was suggested that the majority of transcription ends at this termination signal in the absence of antibiotics, whereas under induced conditions, RNA polymerase reads through the terminator, and transcription continues to the downstream vmlR coding region, resulting in an increase in vmlR expression. No stabilization of vmlR mRNA occurred under the induced conditions.

Glutathione protects cells and organisms from oxygen species and peroxides and is indispensable for aerobically living organisms. Moreover, it acts against xenobiotics and drugs by the formation and excretion of glutathione S conjugates. In this study, we show that the yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a glutathione transporter with the ATP-binding cassette. The transporter imports extracellular glutathione into the cytoplasm in an ATP-dependent manner. This transporter, along with [gamma]-glutamyltranspeptidase, has an important role in E. coli growth with glutathione as a sole sulfur source.

The oligomeric state of ATP binding cassette (ABC)G2 is characterized using various approaches in both drug-selected and transiently transfected cell lines. Chimeric ABCG2 transporters were generated to study ABCG2 as a covalent dimer.

ATP hydrolysis triggers rapid dissociation of the 19S regulatory particles from immunopurified 26S complexes in a manner coincident with release of the bulk of proteasome-interacting proteins. This mechanism leads to quantitative disassembly of the 19S into subcomplexes and free Rpn10, the polyubiquitin binding subunit.

The expression of protein homologues (AlgQ1 and AlgQ2) in strain A1, their interaction with the alginate ATP-binding cassette (ABC) importer, and their characteristics in binding to macromolecules and oligosaccharides, is clarified. The results provide new insights into the bacterial molecular mechanism underlying macromolecule import in periplasm.

The ATP-dependent amide bond forming activity of Streptomyces antibioticus simocyclinone ligase SimL was expressed and purified from Escherichia coli with a variety of polyenoic acids including retinoic acid and fumagillin. The last three enzymes from the novobiocin pathway, namely, NovM, NovP, and NovN, are used to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetranoate moiety.

Site-directed mutagenesis was employed to generate various mutants in order to identify the ATP-binding domain of Epstein-Barr virus thymidine kinase (EBV TK) and to characterize biochemical properties of important residues in this region. It was demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymic activity but are involved in different aspects of its action.

In Streptococcus pneumoniae, an HI03Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an $84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its Pare mutant. The Pare mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration HI03Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.

The effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1) is examined. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis.

The demonstration of FH1-FH2 domain acceleration hydrolysis of ATP coupled to profilin-actin polymerization is presented that uses the derived free energy for processive polymerization increasing 15-fold the rate constant for profillin-actin association to barbed ends. Profillin is required for and takes part in the processive function.

The ATP-binding cassette transporter A3 (ABCA3) is extremely important for the proper formation of lamellar bodies, surfactant function and also may be required for lung function in various pulmonary diseases. Infact, the mutation of the ABCA3 gene would lead to surfactant deficiency among newborns that may turn to be fatal.

Byline: I. I. Titov (1), H.-K. Schroeder (2) Keywords: metabolic network; inorganic phosphate; bacteria; transport; diffusion; ATP Abstract: One of the main problems of metabolic engineering is to determine the genetically controlled limiting links of a metabolic network. We have built a model of the primary transport of inorganic phosphates (P.sub.i), analyzed the [P.sub.i]metabolic network in Gram-negative bacteria, and determined the factors controlling the phosphate exchange. The model explains why the [P.sub.i]primary transport is not observed at the release stage. The nonlinearity of primary transport and the differences in its parameters in the membrane and within the cell give rise to transport asymmetry, i.e., the [P.sub.i]release rate is low as compared with the uptake rate, and is small at the background of secondary transport. Discussed is a general scheme of coordination between primary and secondary transport, which are interconnected through the substrate--product relation. Author Affiliation: (1) Siberian Division, Russian Academy of Sciences, Institute of Cytology and Genetics, Novosibirsk, 630090, Russia (2) Institut fur Physiologische-Chemie, Mainz, Germany Article History: Registration Date: 10/10/2004

The Na+ channel and Na+-K+-ATPase are essential for the perinatal removal of alveolar solute and fluid. It was hypothesized that maternal glucocorticoids (GCs) increased Na+-K+-ATPase gene expression in a fetal lung epithelial cell line because Na+-pump mRNA and activity increase before birth. The results showed that GCs increased Na+-K+-ATPase transcription in a fetal lung epithelial cell line. The findings suggest important therapeutic implications, one of which is optimizing treatment of premature infants by maternal GCs.

We have analyzed the ability of A165V, V169I/D170N, and P536L mutations to suppress pma1 dominant lethal alleles and found that the P536L mutation is able to suppress the dominant lethality of the pma1-R271T, -D378N, -D378E, and -K474R mutant alleles. Genetic and biochemical analyses of site-directed mutants at Pro-536 suggest that this amino acid may not be essential for function but is important for biogenesis of the ATPase. Proteins encoded by dominant lethal pma1 alleles are retained in the endoplasmic reticulum, thus interfering with transport of wild-type Pma1. Immunofluorescence studies of yeast conditionally expressing revertant alleles show that the mutant enzymes are correctly located at the plasma membrane and do not disturb targeting of the wild-type enzyme. We propose that changes in Pro-536 may influence the folding of the protein encoded by a dominant negative allele so that it is no longer recognized and retained as a misfolded protein by the endoplasmic reticulum.

Madin-Darby canine kidney (MDCK) cells have enhanced production of the sodium-potassium antiporter mRNA upon exposure to hyperoxic regimes. The mechanism of gene regulation in MDCK is investigated using promoter transfection techniques and deletion mutants of the promoter sequence. Results reveal that a 61-base pair segment located between nucleotide residues 102 and 41 is responsible for the induction of Na-K-ATPase during hyperoxia.

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the [Beta]-subunit of the mitochondrial [F.sub.1]-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the 'gain-of-function' phenotype found in mgi1 point mutants. The location of Arg435 in the [Beta]-subunit, as deduced from the three-dimensional structure of the bovine [F.sub.1]-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the [Beta]- and [Alpha]- (MGI2) subunits with the [Gamma]-subunit (MGI5) is likely to be affected by the mutations.

When Saccharomyces cerevisiae cells are exposed to temperatures higher than their optimal growing temperature the ATPase activity of the plasma membrane and activity of the gene PMA2 increases, but the activity of the gene PMA1 and the amount of ATPase protein in the membrane decreases. The genes PMA1 and PMA2 are responsible for the plasma membrane's H+-ATPase activity and normally PMA2 is less active than PMA1. The increased ATPase activity is due to either a change in the PMA1 ATPase after it is formed or a change in the fluidity of the plasma membrane.

The ATPase activity of the transcriptional activator FHLA protein is stimulated by the presence of formate and double stranded DNA. DNA which contains specific FHLA binding sites stimulates FHLA more than the DNA which does not contain FHLA binding sites. FHLA belongs to the NTRC family of regulators but its N terminus is different. The binding of formate to FHLA performs the same function as phosphorylation of the NTRC regulators and in the presence of formate, FHLA changes its conformation and undergoes stable interactions with its binding sites present on DNA.

Characterization of a group of pma1 mutants that are temperature sensitive in Saccharomyces cerevisiae helps target (H+)ATPase to the plasma membrane of the yeast. The mutants fail to grow in the absence of ATPase protein activity and targeting, and under restrictive temperature conditions resulting in the delivery and degradation of mutant ATPase to and in the vacuoles, respectively. The mutants help identify a novel AST1 gene, whose protein localizes to the plasma membrane, that redirects the mutant ATPase from the vacuoles to the cell surface and enables growth at restrictive temperatures.

Experimental studies demonstrate presence of a fourth putative human Na+, K+-ATPase alpha-subunit gene, ATP1AL2, in the testis. The polypeptide sequence of the gene encodes 1028 amino acids and is 76-78% homologous to the rat Na+, K+-ATPase alpha1, alpha2 and alpha3 isoforms, with ATP1AL2 as the fourth isoform. Both rat and human testis have abundant levels of a 3.9-kb mRNA expression.

Enzyme-linked immunosorbent assay and Northern blot analysis of the cultured rat vascular smooth muscle cells show that angiotensin II induces and enhances the alpha1- and beta1-mRNA levels of Na-K-adenosintriphosphatase (Na-K-ATPase) gene, modulating its expression. The three- and four-fold increases in the alpha1- and beta1-mRNA levels occur after 6h of angiotensin II treatment and are dose-dependent. The complete elimination of protein kinase C(PKC) activity has no effect on the angiotensin II- induced gene regulation, indicating that alpha1- and beta1-mRNA isoform expression induction is independent of PKC activation.

The par-2 gene in Caenorhabditis elegans is isolated and sequenced to analyze the asymmetric generation and cytoplasmic localization at the molecular level. Molecular analysis of par genes is performed to analyze the role of these genes in partitioning cytoplasms. Observation of two different motifs in the par-2 cDNA protein reveals the par-2 gene as a member of putative zinc-binding proteins.

A novel method for studying glucose metabolism in Escherichia coli resting cells was used to determine the effects high fructose-1,6-biphosphate aldolase levels on glucose flux, fructose-1,6-P2 concentration and the equilibration of both its halves. Results revealed that aldolase only slightly influenced these three variables. A fit was demonstrated between the results obtained with resting cells and with the extra enzyme being active. No special assumptions were required to explain the fit, except that the measured Vmax values should underestimate actual values.

The N-terminal 401 amino acid motor domain (K401) was obtained by truncating the Drosophila kinesin heavy-chain gene and was expressed in Escherichia coli. Biochemical and kinetic chararacterization studies revealed that the purified protein was an active and homogenous preparation and that its steady-state properties were very similar to those exhibited by native kinesin. Results also revealed that the K401 has full catalytic activity with microtubules.

The effects on membrane proton permeability by expressions of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase was examined. The results of the cloned gene expressions suggested that synthesis and assembly of F0 occured in a relatively proton-impermeable form which is transformed to its proton-conducting form upon interaction with F1 subunits. It also indicated that the unassembled subunits generated by overexpression of F0 genes inhibited cell growth without any change in the proton permeability of the membrane.

The Sug1 gene of Saccharomyces cerevisiae is an integral component of the yeast 26S proteasome. Sug1 co-precipitates with proteasome in conventional and nickel-chelate affinity chromatography. The 26S proteasome catalyzes the ATP-dependent degradation of ubiquitin-protein conjugate. Defective proteolysis regulates the effect of Sug1 mutation on transcription.

Study conducted show that hUAP56 has multiple and diverse roles in splicing complex assembly. It is confirmed that hUAP56 is required for prespliceosome assembly at it is also required for the conversion of the prespliceosome to the mature spliceosome.