We have analyzed the existence of obligatory steps in the folding reaction of the alpha-spectrin SH3 domain by mutating Asp 48 (D48G), which is at position i+3 of an isolated two-residue type II' beta-turn. Calorimetry and X-ray analysis show an entropic stabilizing effect resulting from local changes at the dihedral angles of the beta-turn. Kinetic analysis of D48G shows that this beta-turn is fully formed in the transition state, while there is no evidence of its formation in an isolated fragment. Introduction of several mutations in the D48G protein reveals that the local stabilization has not significantly altered the transition state ensemble. All these results, together with previous analysis of other alpha-spectrin and src SH3 mutants, indicate that: (i) in the folding reaction there could be obligatory steps which are not necessarily part of the folding nucleus; (ii) transition state ensembles in beta-sheet proteins could be quite defined and conformationally restricted ('mechanic folding nucleus'); and (iii) transition state ensembles in some proteins could be evolutionarily conserved.

Human H3N2 influenza A viruses were known to preferentially bind to sialic acid (SA) in alpha2,6Gal linkage on red blood cells (RBC). However, H3N2 viruses isolated in MDCK cells after 1992 did not agglutinate chicken RBC (CRBC). Experiments with point-mutated hemagglutinin (HA) of A/Aichi/51/92, one of these viruses, revealed that an amino acid change from Glu to Asp at position 190 (E190D) was responsible for the loss of ability to bind to CRBC. A/Aichi/51/92 did not agglutinate CRBC treated with alpha2, 3-sialidase, suggesting that SAalpha2,3Gal on CRBC might not inhibit the binding of the virus to SAalpha2,6Gal on CRBC. However, the virus agglutinated derivatized CRBC resialylated with SAalpha2, 6Galbeta1,4GlcNAc. These findings suggested that the E190D change might have rendered the HA able to distinguish sialyloligosaccharides on the derivatized CRBC containing the SAalpha2,6Galbeta1,4GlcNAc sequence from those on the native CRBC.

It is now recognized that the white adipose tIssue (WAT) produces a variety of bioactive peptIdes, collectively termed "adipokines". Alteration of WAT mass in obesity or lipoatrophy, affects the production of most adipose secreted factors. Since both conditions are associated with multiple metabolic disorders and increased risk of cardiovascular diseases, the Idea has emerged that WAT could be instrumental in these complications, by virtue of its secreted factors. Several adipokines are increased in the obese state and have been implicated in hypertension (angiotensinogen), impaired fibrinolysis (PAI-1) and insulin resistance (ASP, TNFalpha, IL-6, resistin). Conversely, leptin and adiponectin both exert an insulin-sensitizing effect, at least in part, by favoring tIssue fatty-acId oxIdation through activation of AMP-activated kinase. In obesity, insulin resistance has been linked to leptin resistance and decreased plasma adiponectin. In lipoatrophic mice, where leptin and adiponectin circulating levels are low, administration of the two adipokines synergistically reverses insulin resistance. Leptin and adiponectin also have distinct properties: leptin, as a long-term integrative signal of energy store and adiponectin, as a potent anti-atherogenic agent. The thiazolIdinedione anti-diabetic drugs increase endogenous adiponectin production in rodents and humans, supporting the Idea that the development of new drugs targeting adipokines might represent a promising therapeutic approach to protect obese patients from insulin resistance and atherosclerosis.

In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca2+ concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca2+ regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of approximately 90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels ( approximately 18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca2+ that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca2+ to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca2+ deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.

Two genes encoding distinct histidine-rich proteins in a Plasmodium falciparum clone exhibit high levels of homology, suggesting they have originated by duplication and divergence from a common ancestral sequence. Both genes have a similar interrupted structure and an exon that encodes closely related tandem repeats of very high histidine and alanine content. The most common repeat encoded by one gene, the hexapeptide Ala-His-His-Ala-Ala-Asp, differs in the sixth position from the most common repeat encoded by the other gene, the hexapeptide Ala-His-His-Ala-Ala-Asn. The divergence of the repeat domains is greater than that of the flanking regions, which exhibit 85-90% homology, including untranslated sequences. This suggests the tandem repeats are relatively labile elements within the genome that may provide the parasite with a means of rapid evolutionary change.

Striated muscle contraction is regulated by the binding of Ca(2+) to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, beta-adrenergic stimulation induces protein kinase A phosphorylation of cardiac troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the troponin complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca(2+)-dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca(2+) in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca(2+) sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca(2+)-sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca(2+)-binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca(2+)-binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through beta-adrenergic signaling as well as the development of cardiomyopathy.

The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-alpha/beta); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in approximately 15- to 50-fold more efficient establishment of persistent replication in hamster (BHK21) and human (HEK293 and HEp-2) cell lines. Transfection with a reporter plasmid carrying the luciferase gene under the control of the IFN-beta promoter resulted in approximately 6- to 7-fold-higher luciferase expression in HEp-2 cells stably expressing KUN replicon RNA with an Ala30-to-Pro mutation in the NS2A gene compared to that observed in HEp-2 cells stably expressing KUN replicon RNA with the wt NS2A gene. Moreover, cotransfection of plasmids expressing individual wt or Ala30-to-Pro-mutated NS2A genes with the IFN-beta promoter reporter plasmid, followed by infection with Semliki Forest virus to activate IFN-beta promoter-driven transcription, showed approximately 7-fold inhibition of luciferase expression by the wt but not by the Ala30-to-Pro-mutated NS2A protein. The results show for the first time a role for the flavivirus nonstructural protein NS2A in inhibition of IFN-beta promoter-driven transcription and identify a single-amino-acid mutation in NS2A that dramatically reduces this inhibitory activity. The findings determine a new function for NS2A in virus-host interactions, extend the range of KUN replicon vectors for noncytopathic gene expression, and identify NS2A as a new target for attenuation in the development of live flavivirus vaccines.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.

Specific chromosomal translocations encoding chimeric transcription factors are considered to play crucial oncogenic roles in a variety of human cancers but the fusion proteins themselves seldom represent suitable therapeutic targets. Oncogenic TFE3 fusion proteins define a subset of pediatric renal adenocarcinomas and one fusion (ASPL-TFE3) is also characteristic of alveolar soft part sarcoma (ASPS). By expression profiling, we identified the MET receptor tyrosine kinase gene as significantly overexpressed in ASPS relative to four other types of primitive sarcomas. We therefore examined MET as a direct transcriptional target of ASPL-TFE3. ASPL-TFE3 binds to the MET promoter and strongly activates it. Likewise, PSF-TFE3 and NONO-TFE3 also bind this promoter. Induction of MET by ASPL-TFE3 results in strong MET autophosphorylation and activation of downstream signaling in the presence of hepatocyte growth factor (HGF). In cancer cell lines containing endogenous TFE3 fusion proteins, inhibiting MET by RNA interference or by the inhibitor PHA665752 abolishes HGF-dependent MET activation, causing decreased cell growth and loss of HGF-dependent phenotypes. MET is thus a potential therapeutic target in these cancers. Aberrant transcriptional up-regulation of MET by oncogenic TFE3 fusion proteins represents another mechanism by which certain cancers become dependent on MET signaling. The identification of kinase signaling pathways transcriptionally up-regulated by oncogenic fusion proteins may reveal more accessible therapeutic targets in this class of human cancers.

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.

Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.

The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5.

Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the gamma2 subunit of GABA(A) receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-beta (SERZ-beta) are the main members of the DHHC family of enzymes that are able to palmitoylate the gamma2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-beta show indistinguishable palmitoylation-dependent interaction with the gamma2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZ(C157S)) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABA(A) receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABA(A) receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.

The active migration of tumor cells through extracellular matrices has been proposed to play a role in certain aspects of metastasis. Metastatic tumor cells migrate in vitro in response to substratum-bound adhesive glycoproteins such as fibronectin. The present studies use affinity-purified proteolytic fragments of fibronectin to determine the nature of adhesion- and/or motility-promoting domains within the protein. Two distinct fragments were identified with cell adhesion-promoting activities. By a number of criteria, the adhesive activity promoted by these two fragments was distinct. One fragment, a 75-kD tryptic fragment purified by monoclonal antibody chromatography, promoted the adhesion, spreading, and haptotactic motility of melanoma cells. Experiments using a synthetic cell attachment peptide in solution indicated that at least part of the attachment activity exhibited by the 75-kD fragment is mediated by the sequence arg-gly-asp-ser. It was not possible to demonstrate migration-stimulating activity using a small (11.5 kD) peptic fragment containing this sequence (Pierschbacher, M.D., E. G. Hayman, and E. Ruoslahti, 1981, Cell, 26:259-267) suggesting that another cell-binding activity within the 75 kD fragment distinct from arg-gly-asp-ser might be required for motility. The second fragment that stimulated melanoma adhesion was a 33-kD tryptic/catheptic carboxyl-terminal heparin-binding fragment, which is localized to the A chain of fibronectin. This fragment promotes adhesion and spreading but not the motility of these cells. Melanoma adhesion to this heparin-binding fragment was sensitive to the effects of cycloheximide, which contrasted adhesion to the haptotaxis-promoting fragment. Importantly, these studies illustrate that haptotaxis in response to fibronectin is not due to simple adhesion gradients of this protein. The results are discussed in light of a model for multiple distinct cell surface constituents mediating cell adhesion and motility on fibronectin.

Sequence analysis was performed of an ovine hypothalamic 41-residue polypeptide that had been postulated to be a putative corticotropin-releasing factor (CRF) because of its high intrinsic corticotropin releasing activity. The NH2-terminal 39 residues of CRF were determined by Edman degradation of 0.6-3.5 nmol of peptide in a Wittmann-Liebold modified Beckman 890C spinning cup sequencer with reverse-phase high-pressure liquid chromatography for the identification of amino acid phenylthiohydantoins (direct micro-sequence analysis). Evidence for residue 40 (isoleucine) was provided by direct micro-sequence analysis of 2.0 nmol of acetylated CRF selectively cleaved at its arginine residues by trypsin prior to analysis. The thermolytic COOH-terminal fragment isoleucyl-alanineamide was characterized as its dansyl derivative. Based on the analytical data, the following primary structure is proposed for ovine hypothalamic CRF: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp -Ile-Ala-NH2. In agreement with this proposal, the synthetic replicate of CRF is highly potent in stimulating secretion of both corticotropin and beta-endorphin-like immunoactivities.

Thymocytes and class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes express predominantly heterodimeric alpha/beta CD8. By interacting with non-polymorphic regions of MHC class I molecules CD8 can mediate adhesion or by binding the same MHC molecules that interact with the T-cell antigen receptor (TCR) function as coreceptor in TCR-ligand binding and T-cell activation. Using TCR photoaffinity labelling with a soluble, monomeric photoreactive H-2Kd-peptide derivative complex, we report here that the avidity of TCR-ligand interactions on cloned cytotoxic T cells is very greatly strengthened by CD8. This is primarily explained by coordinate binding of ligand molecules by CD8 and TCR, because substitution of Asp 227 of Kd with Lys severely impaired the TCR-ligand binding on CD8+, but not CD8- cells. Kinetic studies on CD8+ and CD8- cells further showed that CD8 imposes distinct dynamics and a remarkable temperature dependence on TCR-ligand interactions. We propose that the ability of CD8 to act as coreceptor can be modulated by CD8-TCR interactions.

Pseudomonas exotoxin (PE), a single-chain polypeptide toxin of 613 amino acids, consists of three functional domains: an amino-terminal receptor-binding domain, a middle translocation domain, and a carboxyl-terminal ADP-ribosylation domain. Deletion of as few as 2 or as many as 11 amino acids from the carboxyl terminus of PE does not affect ADP-ribosylation activity but produces noncytotoxic molecules. Deletions and substitutions between positions 602 and 611 of PE show that the last 5 amino acids of PE are very important for its cytotoxic action. The carboxyl-terminal sequence of PE is Arg-Glu-Asp-Leu-Lys. Mutational analysis indicates that a basic amino acid at 609, acidic amino acids at 610 and 611, and a leucine at 612 are required for full cytotoxic activity. Lysine at 613 can be deleted or replaced with arginine but not with several other amino acids. Mutant toxins are able to bind normally to target Swiss mouse 3T3 cells and are internalized by endocytosis, but apparently they do not penetrate into the cytosol. A PE molecule that ends with Lys-Asp-Glu-Leu, which is a well defined endoplasmic reticulum retention sequence [Munro, S. and Pelham, R. B. (1987) Cell 48, 899-907], is fully cytotoxic, suggesting that a common factor may be involved in intoxication of cells by PE and retention of proteins in the lumen of the endoplasmic reticulum. Sequences similar to those at the carboxyl end of PE are also found at the end of Cholera toxin A chain and Escherichia coli heat-labile toxin A chain.

Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn induces dopaminergic neuronal cell death remains unclear. In the present study, we sought to investigate the key downstream apoptotic cell signaling events that contribute to Mn-induced cell death in mesencephalic dopaminergic neuronal (N27) cells. Mn exposure induced a dose-dependent increase in neuronal cell death in N27 cells. The cell death was accompanied by sequential activation of mitochondrial-dependent proapoptotic events, including cytochrome c release, caspase-3 activation, and DNA fragmentation, but not caspase-8 activation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers Mn-induced apoptosis. Notably, Mn treatment proteolytically activated protein kinase Cdelta (PKCdelta), a member of a novel class of protein kinase C. The caspase-3 specific inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK) significantly blocked PKCdelta cleavage and its kinase activity, indicating that caspase-3 mediates the proteolytic activation. Cotreatment with the PKCdelta inhibitor rottlerin or the caspase-3 inhibitor Z-DEVD-FMK almost completely blocked Mn-induced DNA fragmentation. Additionally, N27 cells expressing a catalytically inactive PKCdelta(K376R) protein (PKCdelta dominant negative mutant) or a caspase cleavage resistant PKCdelta(D327A) protein (PKCdelta cleavage resistant mutant) were found to be resistant to Mn-induced apoptosis. To further establish the proapoptotic role of PKCdelta, RNA interference-mediated gene knockdown was performed. Small interfering RNA suppression of PKCdelta expression protected N27 cells from Mn-induced apoptotic cell death. Collectively, these results suggest that caspase-3-dependent proteolytic activation of PKCdelta plays a key role in Mn-induced apoptotic cell death.

Bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris work in tandem to modulate synthesis of the light-harvesting complex LH4 in response to light. Although RpBphP2 and RpBphP3 share the same domain structure with 52% sequence identity, they demonstrate distinct photoconversion behaviors. RpBphP2 exhibits the "classical" phytochrome behavior of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states, whereas RpBphP3 exhibits novel photoconversion between Pr and a near-red (Pnr) light-absorbing states. We have determined the crystal structure at 2.2-A resolution of the chromophore binding domains of RpBphP3, covalently bound with chromophore biliverdin IXalpha. By combining structural and sequence analyses with site-directed mutagenesis, we identify key residues that directly modulate the photochemical properties of RpBphP3 and RpBphP2. Remarkably, we identify a region spanning residues 207-212 in RpBphP3, in which a single mutation, L207Y, causes this unusual bacteriophytochrome to revert to the classical phenotype that undergoes reversible photoconversion between the Pr and Pfr states. The reverse mutation, Y193L, in the corresponding region in RpBphP2 significantly diminishes the formation of the Pfr state. We propose that residues 207-212 and the spatially adjacent conserved residues, Asp-216 and Tyr-272, interact with the chromophore and form part of the interface between the chromophore binding domains and the PHY domain that modulates photoconversion.

Of 150 clinical isolates of Neisseria gonorrhoeae recovered in 2001, we examined 55 clinical isolates of N. gonorrhoeae for which cefixime MICs were>> or=0.125 microg/ml and randomly selected 15 isolates for which cefixime MICs were < or =0.06 microg/ml for analysis of alterations in the penicillin-binding protein 2 (PBP 2) gene. We found insertion of an extra codon (Asp-345a) in the transpeptidase domain of PBP 2, and this insertion occurred alone or in conjunction with other amino acid substitutions. We also found a mosaic PBP 2 that was composed of fragments of the PBP 2 proteins from Neisseria cinera and Neisseria perflava. This mosaic PBP 2 was significantly associated with decreased susceptibilities to penicillin and cephalosporins, especially oral cephalosporins. For most of the isolates with a mosaic PBP 2, the cefixime MICs were>> or =0.5 microg/ml and the cefdinir MICs were>> or =1 microg/ml. Analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis revealed that most isolates with the mosaic PBP 2 were genetically similar. The recombination events that generated the mosaic PBP 2 would likely have contributed to the decreased sensitivities to cephalosporins. Isolates with the mosaic PBP 2 appear to threaten the efficacy of the currently recommended regimen with cefixime. The emergence of such strains may be the result of the in vivo generation of clones in which interspecies recombination occurred between the penA genes of N. gonorrhoeae and commensal Neisseria species.

We demonstrate that PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, which is required for myosin II assembly. We demonstrate that PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis.

Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family.

A clone that transforms the RKa mutant of Synechocystis PCC6803 defective in inorganic carbon (Ci) transport to the wild-type phenotype was isolated from a cyanobacterial genomic library. The clone contained an 11.8-kilobase-pair DNA insert. Sequencing of the insert DNA in the region of the mutation in RKa revealed an open reading frame (designated as ndhB), which showed extensive amino acid sequence homology to the subunit-2 genes of NADH dehydrogenase (EC 1.6.99.3) (ndhB) of chloroplasts and mitochondria. The homology was much stronger with the chloroplast genes. Sequence analysis of the ndhB gene of RKa mutant revealed a G----A substitution that results in a Gly----Asp substitution in the deduced amino acid. A defined mutant (M55), constructed by inactivating the ndhB gene in wild-type Synechocystis, required high CO2 conditions for growth and was unable to transport CO2 and HCO3- into the intracellular Ci pool. The results indicate that the ndhB gene is required for Ci transport. Dark respiration was also depressed by the inactivation of the ndhB gene. A possible role of the ndhB gene product in the energization of Ci transport is discussed.

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.