A large number of new bacteriocins in lactic acid bacteria (LAB) has been characterized in recent years. Most of the new bacteriocins belong to the class II bacteriocins which are small (30-100 amino acids) heat- stable and commonly not post-translationally modified. While most bacteriocin producers synthesize only one bacteriocin, it has been shown that several LAB produce multiple bacteriocins (2-3 bacteriocins). Based on common features, some of the class II bacteriocins can be divided into separate groups such as the pediocin-like and strong anti-listeria bacteriocins, the two-peptide bacteriocins, and bacteriocins with a sec-dependent signal sequence. With the exception of the very few bacteriocins containing a sec-dependent signal sequence, class II bacteriocins are synthesized in a preform containing an N-terminal double-glycine leader. The double-glycine leader-containing bacteriocins are processed concomitant with externalization by a dedicated ABC-transporter which has been shown to possess an N-terminal proteolytic domain. The production of some class II bacteriocins (plantaricins of Lactobacillus plantarum C11 and sakacin P of Lactobacillus sake) have been shown to be transcriptionally regulated through a signal transduction system which consists of three components: an induction factor (IF), histidine protein kinase (HK) and a response regulator (RR). An identical regulatory system is probably regulating the transcription of the sakacin A and carnobacteriocin B2 operons. The regulation of bacteriocin production is unique, since the IF is a bacteriocin-like peptide with a double-glycine leader processed and externalized most probably by the dedicated ABC-transporter associated with the bacteriocin. However, IF is not constituting the bacteriocin activity of the bacterium, IF is only activating the transcription of the regulated class II bacteriocin gene(s). The present review discusses recent findings concerning biosynthesis, genetics, and regulation of class II bacteriocins.

Soybean (Glycine max L.) is a major crop providing an important source of protein and oil, which can also be converted into biodiesel. A major milestone in soybean research was the recent sequencing of its genome. The sequence predicts 69,145 putative soybean genes, with 46,430 predicted with high confidence. In order to examine the expression of these genes, we utilized the Illumina Solexa platform to sequence cDNA derived from 14 conditions (tissues). The result is a searchable soybean gene expression atlas accessible through a browser (http://digbio.missouri.edu/soybean_atlas). The data provide experimental support for the transcription of 55,616 annotated genes and also demonstrate that 13,529 annotated soybean genes are putative pseudogenes, and 1736 currently unannotated sequences are transcribed. An analysis of this atlas reveals strong differences in gene expression patterns between different tissues, especially between root and aerial organs, but also reveals similarities between gene expression in other tissues, such as flower and leaf organs. In order to demonstrate the full utility of the atlas, we investigated the expression patterns of genes implicated in nodulation, and also transcription factors, using both the Solexa sequence data and large-scale qRT-PCR. The availability of the soybean gene expression atlas allowed a comparison with gene expression documented in the two model legume species, Medicago truncatula and Lotus japonicus, as well as data available for Arabidopsis thaliana, facilitating both basic and applied aspects of soybean research.

Peptoids, oligomers of N-substituted glycines, are described as a motif for the generation of chemically diverse libraries of novel molecules. Ramachandran-type plots were calculated and indicate a greater diversity of conformational states available for peptoids than for peptides. The monomers incorporate t-butyl-based side-chain and 9-fluorenylmethoxy-carbonyl alpha-amine protection. The controlled oligomerization of the peptoid monomers was performed manually and robotically with in situ activation by either benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate or bromotris(pyrrolidino)phosphonium hexaflurophosphate. Other steps were identical to peptide synthesis using alpha-(9-fluorenylmethoxycarbonyl)amino acids. A total of 15 monomers and 10 oligomers (peptoids) are described. Preliminary data are presented on the stability of a representative oligopeptoid to enzymatic hydrolysis. Peptoid versions of peptide ligands of three biological systems (bovine pancreatic alpha-amylase, hepatitis A virus 3C proteinase, and human immunodeficiency virus transactivator-responsive element RNA) were found with affinities comparable to those of the corresponding peptides. The potential use of libraries of these compounds in receptor- or enzyme-based assays is discussed.

OBJECTIVE

To assess multiple psychometric characteristics of a new stroke outcome measure, the Stroke Impact Scale (SIS), using Rasch analysis, and to identify and remove misfitting items from the 8 domains that comprise the SIS.

METHODS

Secondary analysis of 3-month outcomes for the Glycine Antagonist in Neuroprotection (GAIN) Americas randomized stroke trial.

METHODS

A multicenter randomized trial performed in 132 centers in the United States and Canada.

METHODS

A total of 696 individuals with stroke who were community-dwelling and independent prior to acute stroke.

METHODS

Not applicable.

METHODS

Rasch analysis was performed using WINSTEPS, version 3.31, to evaluate 4 psychometric characteristics of the SIS: (1) unidimensionality or fit (the extent to which items measure a single construct), (2) targeting (the extent to which the items are of appropriate difficulty for the sample), (3) item difficulty (the ordering of items from least to most difficult to perform), and (4) separation (the extent to which the items distinguish distinct levels of functioning within the sample).

RESULTS

(1) Within each domain, most of the items measured a single construct. Only 3 items misfit the constructs and were deleted ("add and subtract numbers," "get up from a chair," "feel emotionally connected") and 2 items ("handle money," "manage money") misfit the combined physical domain. These items were deleted to create SIS, version 3.0. (2) Overall, the items are well targeted to the sample. The physical and participation domains have a wide range of items that capture difficulties that most individuals with stroke experience in physical and role functions, while the memory, emotion, and communication domains include items that capture limitations in the most impaired patients. (3) The order of items from less to more difficult was clinically meaningful. (4) The individual physical domains differentiated at least 3 (high, average, low) levels of functioning and the composite physical domain differentiated more than 4 levels of functioning. However, because difficulties with communication, memory, and emotion were not as frequently reported and difficulties with hand function were more frequently reported, these domains only differentiated 2 (high, low) to 3 (high, average, low) strata of patients. Time from stroke onset to administration of the SIS had little effect on item functioning.

CONCLUSIONS

Rasch analysis further established the validity of the SIS. The domains are unidimensional, the items have an excellent range of difficulty, and the domain scores differentiated patients into multiple strata. The activities of daily living/instrumental activities of daily living, mobility, strength, composite physical, and participation domains have the most robust psychometric characteristics. The composite physical domain is most able to discriminate difficulty in function in individuals after stroke, while the communication, memory, and emotion domain items only capture limitations in function in the more impaired groups of patients.

Caspase-1 cleaves the inactive IL-1beta and IL-18 precursors into active inflammatory cytokines. In Salmonella-infected macrophages, caspase-1 also mediates a pathway of proinflammatory programmed cell death termed "pyroptosis." We demonstrate active caspase-1 diffusely distributed in the cytoplasm and localized in discrete foci within macrophages responding to either Salmonella infection or intoxication by Bacillus anthracis lethal toxin (LT). Both stimuli triggered caspase-1-dependent lysis in macrophages and dendritic cells. Activation of caspase-1 by LT required binding, uptake, and endosome acidification to mediate translocation of lethal factor (LF) into the host cell cytosol. Catalytically active LF cleaved cytosolic substrates and activated caspase-1 by a mechanism involving proteasome activity and potassium efflux. LT activation of caspase-1 is known to require the inflammasome adapter Nalp1. In contrast, Salmonella infection activated caspase-1 through an independent pathway requiring the inflammasome adapter Ipaf. These distinct mechanisms of caspase-1 activation converged on a common pathway of caspase-1-dependent cell death featuring DNA cleavage, cytokine activation, and, ultimately, cell lysis resulting from the formation of membrane pores between 1.1 and 2.4 nm in diameter and pathological ion fluxes that can be blocked by glycine. These findings demonstrate that distinct activation pathways elicit the conserved cell death effector mechanism of caspase-1-mediated pyroptosis and support the notion that this pathway of proinflammatory programmed cell death is broadly relevant to cell death and inflammation invoked by diverse stimuli.

Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.

The search for tumour-specific markers is one of the chief goals in cancer biology. We show that the translocation t(12;16)(q13:p11) in malignant myxoid liposarcoma can be a fusion of the CHOP dominant negative transcription factor gene with a novel gene, FUS, which can result in fusion of the FUS glycine-rich protein with the whole CHOP coding region. The data support the concept that protein fusion may commonly occur in solid tumours resulting in tumour-specific markers of potential clinical importance. The data also indicate the importance of transcription disruption in the pathogenesis of solid tumours.

Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin. Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp. Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide. These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin. In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin. Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin. These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities.

YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. It activates or represses many genes during cell growth and differentiation and is also required for the normal development of mammalian embryos. Previous studies have established that YY1 interacts with histone acetyltransferases p300 and CREB-binding protein (CBP) and histone deacetylase 1 (HDAC1), HDAC2, and HDAC3. Here, we present evidence that the activity of YY1 is regulated through acetylation by p300 and PCAF and through deacetylation by HDACs. YY1 was acetylated in two regions: both p300 and PCAF acetylated the central glycine-lysine-rich domain of residues 170 to 200, and PCAF also acetylated YY1 at the C-terminal DNA-binding zinc finger domain. Acetylation of the central region was required for the full transcriptional repressor activity of YY1 and targeted YY1 for active deacetylation by HDACs. However, the C-terminal region of YY1 could not be deacetylated. Rather, the acetylated C-terminal region interacted with HDACs, which resulted in stable HDAC activity associated with the YY1 protein. Finally, acetylation of the C-terminal zinc finger domain decreased the DNA-binding activity of YY1. Our findings suggest that in the natural context, YY1 activity is regulated through intricate mechanisms involving negative feedback loops, histone deacetylation, and recognition of the cognate DNA sequence affected by acetylation and deacetylation of the YY1 protein.

Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca(2+) ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

Urethane is widely used as an anesthetic for animal studies because of its minimal effects on cardiovascular and respiratory systems and maintenance of spinal reflexes. Despite its usefulness in animal research, there are no reports concerning its molecular actions. We designed this study to determine whether urethane affects neurotransmitter-gated ion channels. We examined the effects of urethane on recombinant gamma-aminobutyric acid(A), glycine, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Urethane potentiated the functions of neuronal nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors, and it inhibited N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors in a concentration-dependent manner. At concentrations close to anesthetic 50% effective concentration, urethane had modest effects on all channels tested, suggesting the lack of a single predominant target for its action. This may account for its usefulness as a veterinary anesthetic. However, a large concentration of urethane exerts marked effects on all channels. These findings not only give insight into the molecular mechanism of anesthetics but also caution that neurophysiologic measurements from animals anesthetized with urethane may be complicated by the effects of urethane on multiple neurotransmitter systems. Our results also suggest that small changes in multiple receptor systems can produce anesthesia.

CONCLUSIONS

Urethane modestly affects multiple neurotransmitter systems at an anesthetic concentration. Our findings suggest that these degenerate effects of urethane can produce anesthesia and that urethane has a potential to influence neuronal measurements made in in vivo preparations.

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.

We have cloned and sequenced cDNAs of the strychnine-binding subunit of the rat glycine receptor, a neurotransmitter-gated chloride channel protein of the CNS. The deduced polypeptide shows significant structural and amino-acid sequence homology with nicotinic acetylcholine receptor proteins, indicating that there is a family of genes encoding neurotransmitter-gated ion channels.

Glutamate excitotoxicity, oxidative stress, and acidosis are primary mediators of neuronal death during ischemia and reperfusion. Astrocytes influence these processes in several ways. Glutamate uptake by astrocytes normally prevents excitotoxic glutamate elevations in brain extracellular space, and this process appears to be a critical determinant of neuronal survival in the ischemic penumbra. Conversely, glutamate efflux from astrocytes by reversal of glutamate uptake, volume sensitive organic ion channels, and other routes may contribute to extracellular glutamate elevations. Glutamate activation of neuronal N-methyl-D-aspartate (NMDA) receptors is modulated by glycine and D-serine: both of these neuromodulators are transported by astrocytes, and D-serine production is localized exclusively to astrocytes. Astrocytes influence neuronal antioxidant status through release of ascorbate and uptake of its oxidized form, dehydroascorbate, and by indirectly supporting neuronal glutathione metabolism. In addition, glutathione in astrocytes can serve as a sink for nitric oxide and thereby reduce neuronal oxidant stress during ischemia. Astrocytes probably also influence neuronal survival in the post-ischemic period. Reactive astrocytes secrete nitric oxide, TNFalpha, matrix metalloproteinases, and other factors that can contribute to delayed neuronal death, and facilitate brain edema via aquaporin-4 channels localized to the astrocyte endfoot-endothelial interface. On the other hand erythropoietin, a paracrine messenger in brain, is produced by astrocytes and upregulated after ischemia. Erythropoietin stimulates the Janus kinase-2 (JAK-2) and nuclear factor-kappaB (NF-kB) signaling pathways in neurons to prevent programmed cell death after ischemic or excitotoxic stress. Astrocytes also secrete several angiogenic and neurotrophic factors that are important for vascular and neuronal regeneration after stroke.

Human synovial sarcomas contain a recurrent and specific chromosomal translocation t(X;18)(p11.2;q11.2). By screening a synovial sarcoma cDNA library with a yeast artificial chromosome spanning the X chromosome breakpoint, we have identified a hybrid transcript that contains 5' sequences (designated SYT) mapping to chromosome 18 and 3' sequences (designated SSX) mapping to chromosome X. An SYT probe detected genomic rearrangements in 10/13 synovial sarcomas. Sequencing of cDNA clones shows that the normal SYT gene encodes a protein rich in glutamine, proline and glycine, and indicates that in synovial sarcoma rearrangement of the SYT gene results in the formation of an SYT-SSX fusion protein. Both SYT and SSX failed to exhibit significant homology to known gene sequences.

BACKGROUND

The issue of whether regular use of an inhaled beta2-adrenergic agonist worsens airflow and clinical outcomes in asthma is controversial. Retrospective studies have suggested that adverse effects occur in patients with a genetic polymorphism that results in homozygosity for arginine (Arg/Arg), rather than glycine (Gly/Gly), at aminoacid residue 16 of the beta2-adrenergic receptor. However, the existence of any genotype-dependent difference has not been tested in a prospective clinical trial.

METHODS

Patients with mild asthma, not using a controller medication, were enrolled in pairs matched for forced expiratory volume in 1 s (FEV1) according to whether they had the Arg/Arg (n=37; four of 41 matches withdrew before randomisation) or Gly/Gly (n=41) genotype. Regularly scheduled treatment with albuterol or placebo was given in a masked, cross-over design, for 16-week periods. During the study, as-needed albuterol use was discontinued and ipratropium bromide was used as needed. Morning peak expiratory flow rate (PEFR) was the primary outcome variable. The primary comparisons were between treatment period for each genotype; the secondary outcome was a treatment by genotype effect. Analyses were by intention to treat.

RESULTS

During the run-in period, when albuterol use was kept to a minimum, patients with the Arg/Arg genotype had an increase in morning PEFR of 23 L/min (p=0.0162); the change in patients with the Gly/Gly genotype was not significant (2 L/min; p=0.8399). During randomised treatment, patients with the Gly/Gly genotype had an increase in morning PEFR during treatment with regularly scheduled albuterol compared with placebo (14 L/min [95% CI 3 to 25]; p=0.0175). By contrast, patients with the Arg/Arg genotype had lower morning PEFR during treatment with albuterol than during the placebo period, when albuterol use was limited (-10 L/min [-19 to -2]; p=0.0209). The genotype-attributable treatment difference was therefore -24 L/min (-37 to -12; p=0.0003). There were similar genotype-specific effects in FEV1, symptoms, and use of supplementary reliever medication.

CONCLUSIONS

Genotype at the 16th aminoacid residue of the beta2-adrenergic receptor affects the long-term response to albuterol use. Bronchodilator treatments avoiding albuterol may be appropriate for patients with the Arg/Arg genotype.

Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100-150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.

Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 +/- 3% to 14 +/- 2%. Return of contraction was observed in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H2O2) increased approximately 2.5-fold. As a substitute for hypoxia, the addition of H2O2 (15 micromol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic acid, and the anion channel inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, all of which also abrogated protection. ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor L-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection. Furthermore, mitochondrial anion channels and cytosolic dismutation to H2O2 may be important steps for oxidant induction of hypoxic preconditioning.

Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection-transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.

Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the gamma-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.

The salt tolerance gene SOS3 (for salt overly sensitive3) of Arabidopsis is predicted to encode a calcium binding protein with an N-myristoylation signature sequence. Here, we examine the myristoylation and calcium binding properties of SOS3 and their functional significance in plant tolerance to salt. Treatment of young Arabidopsis seedlings with the myristoylation inhibitor 2-hydroxymyristic acid caused the swelling of root tips, mimicking the phenotype of the salt-hypersensitive mutant sos3-1. In vitro translation assays with reticulocyte showed that the SOS3 protein was myristoylated. Targeted mutagenesis of the N-terminal glycine-2 to alanine prevented the myristoylation of SOS3. The functional significance of SOS3 myristoylation was examined by expressing the wild-type myristoylated SOS3 and the mutated nonmyristoylated SOS3 in the sos3-1 mutant. Expression of the myristoylated but not the nonmyristoylated SOS3 complemented the salt-hypersensitive phenotype of sos3-1 plants. No significant difference in membrane association was observed between the myristoylated and nonmyristoylated SOS3. Gel mobility shift and (45)Ca(2)+ overlay assays demonstrated that SOS3 is a unique calcium binding protein and that the sos3-1 mutation substantially reduced the capacity of SOS3 to bind calcium. The resulting mutant SOS3 protein was not able to interact with the SOS2 protein kinase and was less capable of activating it. Together, these results strongly suggest that both N-myristoylation and calcium binding are required for SOS3 function in plant salt tolerance.

Protein kinases targeted by small-molecule inhibitors develop resistance through mutation of the 'gatekeeper' threonine residue of the active site. Here we show that the gatekeeper mutation in the cellular forms of c-ABL, c-SRC, platelet-derived growth factor receptor-alpha and -beta, and epidermal growth factor receptor activates the kinase and promotes malignant transformation of BaF3 cells. Structural analysis reveals that a network of hydrophobic interactions-the hydrophobic spine-characteristic of the active kinase conformation is stabilized by the gatekeeper substitution. Substitution of glycine for the residues constituting the spine disrupts the hydrophobic connectivity and inactivates the kinase. Furthermore, a small-molecule inhibitor that maximizes complementarity with the dismantled spine (compound 14) inhibits the gatekeeper mutation of BCR-ABL-T315I. These results demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases and provide structural insights to guide the development of next-generation inhibitors.