Base Sequence

Proteins that belong to the AAA (ATPases associated with various cellular activities) superfamily of mechanochemical enzymes are versatile and control a wide array of cellular functions. Many AAA proteins share the common property of self-association into oligomeric structures and use nucleotide binding and hydrolysis to regulate their biological output. The Escherichia coli transcription activator PspF (phage shock protein F) is a member of the σ 54 -dependent transcriptional activators that belong to the AAA protein family. Nucleotide interactions condition the functional state of PspF, enabling it to self-associate and interact with its target, the σ 54 -RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of σ 54dependent activators remain poorly characterized. In the present study, we have used a fragment of the AAA domain of PspF as a probe to study the nucleotide-conditioned self-association of PspF. Results show that the PspF fragment acts in trans to inhibit specifically self-association of PspF. The PspF fragment prevented efficient binding of nucleotides to PspF, consistent with the observation that the site for nucleotide interactions within an oligomer of AAA proteins is created between two protomers. Using proximity-based footprinting and cross-linking techniques, we demonstrate that the sequences represented in this fragment are close to one protomer-protomer interface within a PspF oligomer. As the sequences represented in this PspF fragment also contain a highly conserved motif that interacts with the σ 54 -RNAP closed complex, we suggest that PspF may be organized to link nucleotide interactions and self-association to σ 54 -RNAP binding and transcription activation.

The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in sigma(54) and core RNA polymerase. The organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using sigma(54) single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early- and late-melted promoters. The results show that sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation. sigma(54) and sigma(54)-RNAP promoter complexes recon...

Enhancer-dependent activator proteins, which act upon the bacterial RNA polymerase containing the σ54 promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-σ54 contact is required for the σ54-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in σ54-RNA polymerase and promoter DNA that lead to DNA opening is poorly understood. Here, band shift and footprinting assays were used to investigate the DNA binding activities of σ54 and σ54-RNA polymerase in the presence of the activator protein PspF bound to poorly hydrolysable analogues of ATP and the ATP hydrolysis transition-state analogue ADP·AlFx. Results show that different nucleotide-bound forms of PspF can change the interactions between σ54, σ54-RNA polymerase, and a DNA fork junction structure present within closed promoter complexes. This provides evidence that in the activation transduction pathway, several functional states of the activator,...

The 54 promoter specificity factor is distinct from other bac- terial RNA polymerase (RNAP) factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotidedependent isomerization of 54 , mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in 54 and core RNA polymerase. The organization of 54 -promoter and 54 -RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using 54 single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early-and late-melted promoters. The results show that 54 undergoes spatial rearrangements of func- tionally important domains upon closed complex formation. 54 and 54 -RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues. Nucleotidedependent alterations in fluorescence resonance energy transfer efficiencies identify different functional states of the activator-54 -RNAP-promoter complex that exist throughout the mechano-chemical transduction pathway of transcriptional activation, i.e. from closed to open promoter complexes. The results suggest that open complex formation only occurs efficiently on replacement of a repressive fork junction with downstream melted DNA.

The abbreviations used are: RNAP, RNA polymerase; AAA, ATPase associated with various cellular activities; PspF, phage shock protein F; OP-Cu, ortho-copper phenanthroline; ATP␥S, adenosine 5Ј-(␥-thio)triphosphate; AMP-PNP, adenosine 5Ј-(␤,␥-imido)triphosphate; GTP␥S, guanosine 5Ј-(␥-thio)triphosphate; ADP A1F x , aluminum fluoride.

Proteins that belong to the AAA (ATPases associated with various cellular activities) superfamily of mechanochemical enzymes are versatile and control a wide array of cellular functions. Many AAA proteins share the common property of self-association into oligomeric structures and use nucleotide binding and hydrolysis to regulate their biological output. The Escherichia coli transcription activator PspF (phage shock protein F) is a member of the σ54-dependent transcriptional activators that belong to the AAA protein family. Nucleotide interactions condition the functional state of PspF, enabling it to self-associate and interact with its target, the σ54–RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of σ54-dependent activators remain poorly characterized. In the present study, we have used a fragment of the AAA domain of PspF as a probe to study the nucleotide-conditioned self-association of PspF. Results show that the PspF fragment act...

Here, we investigated the gene that encodes the main protein component of the byssus in Perna perna (Ppfp) using a consensus fluorescent probe for the Mytilus group and by polymerase chain reaction amplification using specific and random primers. A 524-bp fragment resulting from polymerase chain reaction amplification was found to be homologous to the fp-1 gene of Mytilus coruscus. This fragment was identified as the 3' end of the Ppfp-1 gene, which included the heptapeptide coding sequence for Lys-Pro-Ser-Tyr-Pro-Pro-Ter (probably the incomplete last tandem repeat unit of the giant exon), the stop codon and the polyadenylation signal.

This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly Bacteria; Eucarya and Archaea were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% ␤-and ␥-Proteobacteria (B؉G), 31 to 35% ␣-Proteobacteria (ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G؉C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B؉G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly Bacteria (83 to 101%) and lacked detectable Archaea but differed in that a small fraction (2 to 8%) of Eucarya was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B؉G to ALF, while in the FC microcosms 55 to 65% of the Bacteria community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems.

With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) genes in the channel catfish virus (CCV) genome was identified. IE transcription in CCV-infected cells appears to be restricted to the terminal repeat region, suggesting that CCV is most closely related to the alpha subfamily of herpesviruses. CCV DNA fragments from this region encoding IE transcripts were cloned. Northern analysis with one of these cloned fragments, a 3,927-bp EcoRI-XbaI fragment, indicates that it encodes two IE transcripts. Both transcripts (ie1 and ie2) were characterized by S1 nuclease analysis, primer extension analysis, and analysis of cDNAs. The ie2 transcript is a 1.3-kb bicistronic mRNA containing open reading frame (ORF) 8a and ORF 9. ORF 8a is a 5'-truncated version of ORF 8 which, along with ORF 9, was previously identified (A. J. Davison, Virology 186:9-14, 1992). The ie1 transcript is 0.6 kb in size, contains only ORF 9, and is expressed at a level appro...

Clustered regularly interspaced short palindromic repeats (CRISPRs) comprise a family of short DNA repeat sequences that are separated by non repetitive spacer sequences and, in combination with a suite of Cas proteins, are thought to function as an adaptive immune system against invading DNA. The number of CRISPR arrays in a bacterial chromosome is variable, and the content of each array can differ in both repeat number and in the presence or absence of specific spacers. We utilized a comparative sequence analysis of CRISPR arrays of the plant pathogen Erwinia amylovora to uncover previously unknown genetic diversity in this species. A total of 85 E. amylovora strains varying in geographic isolation (North America, Europe, New Zealand, and the Middle East), host range, plasmid content, and streptomycin sensitivity/resistance were evaluated for CRISPR array number and spacer variability. From these strains, 588 unique spacers were identified in the three CRISPR arrays present in E. amylovora, and these arrays could be categorized into 20, 17, and 2 patterns types, respectively. Analysis of the relatedness of spacer content differentiated most apple and pear strains isolated in the eastern U.S. from western U.S. strains. In addition, we identified North American strains that shared CRISPR genotypes with strains isolated on other continents. E. amylovora strains from Rubus and Indian hawthorn contained mostly unique spacers compared to apple and pear strains, while strains from loquat shared 79% of spacers with apple and pear strains. Approximately 23% of the spacers matched known sequences, with 16% targeting plasmids and 5% targeting bacteriophage. The plasmid pEU30, isolated in E. amylovora strains from the western U.S., was targeted by 55 spacers. Lastly, we used spacer patterns and content to determine that streptomycin-resistant strains of E. amylovora from Michigan were low in diversity and matched corresponding streptomycin-sensitive strains from the background population.

Multidrug resistance protein 1 (ABCC1) is a member of the 'C' class of ATP-binding cassette transporters, which can give rise to resistance to chemotherapy via drug export from cells. It also acts as a leukotriene C4 transporter, and hence has a role in adaptive immune response. Most Cclass members have an additional NH 2 -terminal transmembrane domain versus other ATP-binding cassette transporters, but little is known about the structure and role of this domain. Using electron cryomicroscopy of 2D crystals, data at 1/6 per Å -1 resolution was generated for the full-length ABCC1 protein in the absence of ATP. Analysis using homologous structures from bacteria and mammals allowed the core transmembrane domains to be localised in the map. These display an inward-facing conformation and there is a noteworthy separation of the cytoplasmic nucleotidebinding domains. Examination of non-core features in the map suggests that the additional NH 2terminal domain has extensive contacts on one side of both core domains, and mirrors their inward-facing configuration in the absence of nucleotide.

Multidrug resistance protein 1 (ABCC1) is a member of the 'C' class of ATP-binding cassette transporters, which can give rise to resistance to chemotherapy via drug export from cells. It also acts as a leukotriene C4 transporter, and hence has a role in adaptive immune response. Most Cclass members have an additional NH 2 -terminal transmembrane domain versus other ATP-binding cassette transporters, but little is known about the structure and role of this domain. Using electron cryomicroscopy of 2D crystals, data at 1/6 per Å -1 resolution was generated for the full-length ABCC1 protein in the absence of ATP. Analysis using homologous structures from bacteria and mammals allowed the core transmembrane domains to be localised in the map. These display an inward-facing conformation and there is a noteworthy separation of the cytoplasmic nucleotidebinding domains. Examination of non-core features in the map suggests that the additional NH 2terminal domain has extensive contacts on one side of both core domains, and mirrors their inward-facing configuration in the absence of nucleotide.

Previous studies have demonstrated that tandem binding sites for the inducible transcription factor NF-B are necessary but not sufficient for full cytokine-mediated transcriptional activation. Herein, we demonstrate that full cytokine-induced accumulation of VCAM1 transcript requires protein synthesis. We report the definition of a functional regulatory element in the VCAM1 promoter interacting with the transcriptional activator interferon regulatory factor 1 (IRF-1). DNA-protein binding studies with endothelial nuclear extracts revealed that IRF-1 is cytokine inducible and binds specifically to a consensus sequence motif located 3 of the TATA element. We have identified heterodimeric p65 and p50 as the NF-B species binding to the VCAM1 promoter in TNF-␣-activated endothelial cells. Experiments with recombinant proteins showed that p50/p65 and highmobility-group I(Y) protein cooperatively facilitated the binding of IRF-1 to the VCAM1 IRF binding site and that IRF-1 physically interacted with p50 and with high-mobility-group I(Y) protein. Transient transfection assays in endothelial cells showed that overexpressed IRF-1 resulted in superinduction of TNF-␣-stimulated transcription. Site-directed mutations in the IRF binding element decreased TNF-␣-induced activity and totally abolished superinduction. Cotransfection assays in P19 embryonal carcinoma cells revealed that IRF-1 synergized with p50/p65 NF-B to activate the VCAM1 promoter or heterologous promoter constructs bearing isolated VCAM1 NF-B and IRF binding motifs. Cytokine inducibility of VCAM1 in endothelial cells utilizes the interaction of heterodimeric p50/p65 proteins with IRF-1.

The function of the 5'-flanking region of the mouse major histocompatibility complex gene E4 has been studied by deletion analysis with the chloramphenicol acetyltransferase gene as a transient expression marker in various cell lines. This analysis reveals the presence of several control regions on the 5' side of the gene. Sequences between base pair (bp) -873 and bp -353 have a negative function in human and mouse fibroblasts but not in the mouse macrophage line WEHI-3. Additional positive and negative elements have been mapped between bp -353 and bp -38. A V-interferon response region has been also identified within that sequence. The 5' and 3' boundaries of the -interferon response region have been located between bp -164 and bp -43. Inducible human cell lines showed the same y-interferon response region endpoints with the mouse cell line WEHI-3. A DNA fragment spanning the equivalent region of the mouse E' gene confers yinterferon inducibility to the simian virus 40 and a-globin promoters in an orientation-independent manner. We further provide evidence that the conserved sequence motifs on the 5' side of all major histocompatibility complex class II genes are indispensable for -interferon induction.

Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca2+-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca2+and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca2+dependent pathway is diminished by protein kinase C stim- ulation in Jurkat T cells. This phenomenon is due to mutually Proc. Natl. Acad. Sci. USA 92 (1995)

We have carried out experiments to determine which members of the rel family of transcription factors are involved in virus induction of the beta interferon (IFN-β) gene. First, we examined the inducibility of artificial DNA binding sites that preferentially interact with different homo- or heterodimeric combinations of rel proteins in vitro. We found that only those sites capable of binding the p50/p65 heterodimer are virus inducible. Second, we analyzed a series of mutant rel DNA-binding sites in the context of the intact IFN-β promoter. We found a correlation between (i) sites capable of binding both the p50/p65 heterodimer and the high-mobility-group protein HMG I(Y) and (ii) virus inducibility. Third, cotransfection of the IFN-β gene enhancer/promoter with plasmids capable of expressing several different rel proteins revealed that only the combination of p50 and p65 efficiently activated transcription. Finally, we have used antibodies directed against different rel proteins to ...

To investigate whether or not alternative splicing might be a mechanism by which in Drosophila melanogaster diversity is generated in endoproteases of the novel eukaryotic family of subtilisin-like proprotein processing enzymes, we determined structural and functional characteristics of the Dfurl gene. Northern blot analysis revealed Dfurl transcripts of 7.6, 6.5, 4.5 and 4.0 kb. By comparative nucleotide sequence analysis of Dfurl genomic and cDNA clones, 10 coding exons were identified and, together with Northern blot analysis using exon-specific probes, evidence was obtained that the four transcripts are generated by alternative splicing and polyadenylation. The apparently complete open reading frames of three Dfurl cDNAs revealed that these coded for three furin-like proteins, Dfurinl (892 residues), Dfurinl-CRR (1101 residues) and Dfurinl-X (1269 residues), which possessed common but also unique structural domains. These various isoforms of furin in Drosophila were characterized in gene transfer studies using immunoprecipitation analysis. Differential expression of Dfurl transcripts was found in Northern blot analysis of RNA from various developmental stages of Drosophila. RNA in situ hybridization experiments revealed that the Dfurinl-X and Dfurinl-CRR isoforms are expressed in non-overlapping sets of tissues during Drosophila embryogenesis. In gene transfer experinents in which the Dfurinl, Dfurinl-CRR and Dfurinl-X proteins were expressed at high levels together with the precursor of the O3A-chain of activin-A, a member of the transforming growth factor 3 (TGFO) superfamily, or the precursor of von Willebrand factor, all three proteins appeared capable of processing these substrates. Our studies indicate that the Dfurl gene encodes structurally different subtilisin-like proprotein processing enzymes with distinct physiological functions in Drosophila.

The proprotein processing activity of mutants of the subtilisin-like enzyme furin was studied in transfected mammalian cells. Our studies indicate that the three residues of the catalytic triad of furin, Asp46, His87, and Ser261, are critical not only for substrate processing but also for maturation of furin. Furthermore, evidence is provided that maturation of furin occurs through an intramolecular autocatalytic process. Substitution of the asparagine residue (Asn188) of the oxyanion hole by an alanine residue appears to block substrate processing but not furin maturation. Analysis of carboxyl-terminal deletion mutants revealed that the segment encompassing residues Glu449 to Glu469 of the "middle" domain, which is more than 100 residues downstream of the predicted catalytic domain, contains residues that seem to be critical for processing activity but that the more carboxyl-terminal cysteine-rich region, the transmembrane region, and the cytosolic tail are dispensable. F...

Production of a variety of regulatory eukaryotic proteins, such as growth factors and polypeptide hormones, often involves endoproteolytic processing of proproteins at cleavage sites consisting of paired basic residues. The first known mammalian proprotein processing enzyme with such specificity is the human fur gene product furin. Structurally and functionally, furin is related to the subtilisin-like serine endoprotease kexin (EC 3.4.21.61) of yeast Saccharomyces cerevisiae; unlike kexin, it contains a cysteine-rich region with an unknown function. Here, we describe cloning and sequencing of a 5.8-kbp cDNA of the Dfur2 gene, a fur-like gene of Drosophila melanogaster, which we found expressed during various stages of development. This Dfur2 cDNA has an open reading frame for a 1680-residue protein, called Dfurin2. Dfurin2 contains similar protein domains as mammalian furin, however, it has an extended amino-terminal region and its cysteine-rich region is much larger than that of ma...

Gaining the full activity of the insulin receptor (IR) requires the proteolytic cleavage of its proform by intra-Golgi furin-like activity. In mammalian cells, IR is expressed as two isoforms (IRB and IRA) that are responsible for insulin action. However, only IRA transmits the growth-promoting and mitogenic effects of insulin-like growth factor 2. Here we demonstrate that the two IR isoforms are similarly cleaved by furin, but when this furin-dependent maturation is inefficient, IR proforms move to the cell surface where the proprotein convertase PACE4 selectively supports IRB maturation. Therefore, in situations of impaired furin activity, the proteolytic maturation of IRB is greater than that of IRA, and accordingly, the amount of phosphorylated IRB is also greater than that of IRA. We highlight the ability of a particular proprotein convertase inhibitor to effectively reduce the maturation of IRA and its associated mitogenic signaling without altering the signals emanating from ...

Northern blot analysis of human lung tumors indicated that the gene, which encodes the subtilisin‐like proprotein processing enzyme PC1/PC3, was highly expressed in almost all carcinoid tumors tested. In small cell lung carcinomas (SCLCs), expression varied. In non‐SCLCs and normal lung, no expression was found. Analysis of SCLC cell lines revealed that expression was restricted preferentially to cell lines of the classical type, to lung tumor cells expressing the PC1/PC3 gene, transcripts of 3 kb and 5 kb were detected, the 5 kb mRNA always being the most abundant species. We isolated a cDNA corresponding to the 5 kb human PC1/PC3 transcript, determined the nucleotide sequence of it and deduced the amino acid sequence of the corresponding protein. Furthermore, we conclude that the two PC1/PC3 transcripts have 3′ non‐coding regions of different size and encode the same protein.

Background: Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease. Results: We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age. Conclusions: Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

A total of 358 Shigella dysenteriae strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrheal Disease Research, Bangladesh, between the years 1999 and 2002 were included in this study. S. dysenteriae type 1, the dominant serotype in 1999 (76.4%), declined to 6.5% in 2002. On the other hand, S. dysenteriae types 2 to 12 were isolated with increasing frequencies of 19, 67, 73.5, and 87% in 1999, 2000, 2001, and 2002, respectively. Of these, types 2 and 4 were the most dominant serotypes, accounting for more than 18.7 and 28.5% of the total isolates, respectively. There was no isolation of serotypes 5, 7, 8, and 13 during this period. Twenty-eight (7.8%) of the isolates were atypical and agglutinated only with the polyvalent antiserum of S. dysenteriae . More than 98% of type 1 strains isolated between 1999 and 2001 were resistant to ampicillin, sulfamethoxazole-trimethoprim, and nalidixic acid. Among other serotypes of S. dysenteriae...

The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9-(PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1-and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (؉)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24 -48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.

Purpose: The small leucine-rich repeat proteins (SLRPs) are involved in organizing the collagen fibrils of the sclera and vitreous. The shape of the eyeball is determined by the sclera and vitreous, so defects in SLRP family members may contribute to myopia. The purpose of this study was to test whether mutations in the two members of the class III SLRPs, opticin (OPTC) and dermatan sulfate proteoglycan 3 (EPYC), are responsible for high myopia. Methods: DNA was prepared from venous leukocytes of 93 patients with high myopia (refraction of spherical equivalent <or=-6.00D) and 96 controls (refraction of spherical equivalent between -0.50D and +1.00D). The coding regions and adjacent intronic sequences of OPTC and EPYC were amplified by the polymerase chain reaction (PCR), and the products were then analyzed by cycle sequencing. The detected variations were further evaluated in normal controls and available family members by a heteroduplex-single strand conformation polymorphism (heteroduplex-SSCP) analysis or sequencing. Results: Two substitutions in OPTC, including c.491G>T and c.803T>C, were identified. The c.491G>T mutation (p.Arg164Leu), a novel heterozygous variation, was detected in one of the 93 patients but in none of the 96 controls. The c.803T>C mutation (p.Pro267Leu), a known polymorphism, was detected in 22 of the 93 patients and in 15 of 48 controls. No variation was observed in EPYC. Conclusions: Only one novel variation in OPTC was detected in a Chinese patient with high myopia. Our results imply that OPTC and EPYC are unlikely to play a major role in high myopia.

Purpose: There are few genetic studies and clinical descriptions of Asian patients with X-linked ocular albinism (OA1). In the present study, the mutation analysis of G protein-coupled receptor 143 gene (GPR143) and clinical characteristics were assessed in Chinese patients with OA1. Methods: Six families with OA1 were recruited from our pediatric and genetic eye clinic. Genomic DNA was prepared from venous leukocytes. The coding regions of GPR143 were amplified by polymerase chain reaction, and subsequently analyzed by direct sequencing. The variations detected were further evaluated in available family members as well as controls. Results: Mutations in GPR143 were identified in each of the six families: c.849delT (p.Val284SerfsX15); c. 238_240delCTC (p.Leu80del); c.658+1G>A, c.353G>A (p.Gly118Glu); g.1103_7266del6164 (p.Gly84AlafsX65), which resulted in a deletion of exons 2 and 3; and g.25985_26546del562 (p.Gly296ValfsX26), which resulted in a deletion of exon 8. Of these six, c.353G>A is a known mutation, while the other five are novel. All affected patients had nystagmus, poor visual acuity, and foveal hypoplasia. However, hypopigmentation of the iris and fundus was very mild in these patients. Conclusions: Five novel mutations and one known mutation were identified in six Chinese families with OA1. These results expand the mutation spectrum of GPR143, and demonstrate the clinical characteristics of OA1 among the Chinese. X-linked ocular albinism (OA1; OMIM 300500), also called ocular albinism type 1, is the most common form of ocular albinism. Most affected white males exhibit nystagmus, poor visual acuity, iris translucency, foveal hypoplasia, an albinotic fundus, and normally pigmented skin and hair . Other findings may include photophobia, strabismus, and misrouting of the optic tracts, resulting in loss of stereoscopic vision . In some OA1 patients of black people, O'Donnell found hypopigmentation of the iris and retina may be subtle, such that these patients present a nonalbinotic fundus and little iris translucency . However, the characteristics of OA1 have not been well defined in Asians. The misdiagnosis of OA1 as congenital nystagmus is not uncommon, and is usually due to lack of clinical experience or lack of phenotypic characterization for certain ethnic groups . Such misdiagnoses might have gone undetected before the OA1 gene was identified. OA1 is caused by mutations in the G protein-coupled receptor 143 (GPR143) gene (OMIM 300500), originally also known as the OA1 gene, located at Xp22.32 . Various types of mutations in GPR143 have been identified in Caucasians living in the Netherlands,

Purpose: Dominant optic atrophy (DOA) is the most common form of autosomal inherited optic neuropathy, mainly caused by mutations in the optic atrophy 1 (OPA1) gene. The purpose of this study was to detect OPA1 gene mutations and associated phenotypes in Chinese patients with suspected hereditary optic neuropathy. Methods: A cohort of 193 Chinese families with suspected hereditary optic neuropathy was collected, which had been excluded from the three common primary mitochondrial DNA mutations associated with Leber hereditary optic neuropathy in our prior screening. Sanger sequencing was used to analyze variants in the coding and adjacent regions of OPA1. Results: In this study, 11 heterozygous OPA1 mutations, among which eight were novel and three were known, were identified in 12 of the 193 families (6.2%) but in none of the 192 control individuals. These novel mutations consisted of two nonsense mutations (p.E707* and p.K797*), two missense mutations (p.T330S and p.V377I), two deletions (p.S64fs and p.L331fs), one small insertion (p.L17fs), and one splice site mutation (c.2614-2A>G). Of the 12 families, three had a family history of optic neuropathy while nine were sporadic cases. Analysis of the family members in the two sporadic cases demonstrated that one parent in each of the two families had the OPA1 mutation and mild phenotype of optic atrophy. A 4-year-old boy with severe ocular phenotype was found to be compound heterozygous for two OPA1 mutations, a p.S64fs frameshift deletion and a p.V377I missense mutation, possibly implying an additive effect. Conclusions: This study implies that the frequency of DOA is much lower than that of Leber hereditary optic neuropathy in Chinese compared with other ethnic groups. Lack of awareness of the mild phenotype of DOA may contribute to the low frequency of OPA1-related DOA in Chinese. The phenotype associated with compound heterozygous OPA1 mutations may suggest a possible addictive effect.

Purpose: Mutations in the glutamate receptor metabotropic 6 gene (GRM6) have been identified in patients with congenital stationary night blindness (CSNB1B). High myopia is usually observed in CSNB1B patients. This study tested if any mutations in GRM6 were solely responsible for high myopia. Methods: DNA was prepared from the venous leukocytes of 96 Chinese patients with high myopia (refraction of spherical equivalent of at least -6.00 diopters [D]) and 96 controls (refraction of spherical equivalent between -0.50 D and +2.00 D with normal visual acuity). The coding regions and adjacent intronic sequence of GRM6 were amplified by a polymerase chain reaction (PCR) and then analyzed by cycle sequencing. Detected variations were evaluated in normal controls by heteroduplex-single-strand-conformation (SSCP) polymorphism analysis or restriction fragment polymorphism (RFLP). Results: Four novel variations predicted to have potential functional changes were identified: c.67-82delCAGGCGGGCCTGGCGCinsT (p.Gln23_Arg28delinsCys), c.858-5a>g (r.spl?), c.1172G>A (p.Arg391Gln), and c.1537G>A (p.Val513Met). Except for c.1172G>A, the other three were not detected in the 96 controls. In addition, five rare variations-(c.72G>A, c.504+10g>t, c.726-50g>c, c.1359C>T, and c.1383C>T)-and one common variation (c.2437-6g>a) without predicted functional consequences and nine known single nucleotide polymorphisms (SNPs) were also detected. Conclusion: Three novel variations with potential functional consequences were identified in the GRM6 of patients with high myopia, suggesting a potential role in the development of myopia in rare cases.

Purpose: To identify mutations in 6 genes of 9 Chinese families with congenital cataract and microcornea. Methods: Nine unrelated families with congenital cataract and microcornea were collected. Cycle sequencing was used to detect variants in the coding and adjacent regions of the crystallin alpha A (CRYAA), crystallin beta B1 (CRYBB1), crystallin beta A4 (CRYBA4), crystallin gamma C (CRYGC), crystallin gamma D (CRYGD), and gap junction protein alpha 8 (GJA8) genes. Results: Upon complete analysis of the 6 genes, three mutations in 2 genes were detected in 3 families, respectively. These mutations were not present in 96 normal controls. Of the three mutations, two novel heterozygous mutations in GJA8, c.136G>A (p.Gly46Arg) and c.116C>G (p.Thr39Arg), were found in two families with congenital cataract and microcornea. The rest one, a heterozygous c.34C>T (p.Arg12Cys) mutation in CRYAA, was identified in three patients from a family with nuclear cataract, microcornea with axial elongation. No mutation in the 6 genes was detected in the remaining 6 families. Conclusions: Mutations in GJA8 and CRYAA were identified in three families with cataract and microcornea. Elongation of axial length accompanied with myopia was a novel phenotype in the family with the c.34C>T mutation in CRYAA. Our results expand the spectrum of GJA8 mutations as well as their associated phenotypes.

Retinitis pigmentosa (RP, OMIM 268000) is the most common and highly heterogeneous genetic group of hereditary retinal degeneration diseases, affecting one in about 3,500-5,000 individuals worldwide . So far, mutations in over 60 genes have been reported to be responsible for about half of nonsyndromic RP . Phenotypic and molecular genetic overlap has been observed in different forms of retinal degeneration [7,; for example, RP might be the main sign of syndromic RP or other related diseases [9]. Mutations in a few genes have been shown to cause different forms of retinal dystrophy [7], while a few genes originally held responsible for other forms of retinal dystrophy have been found to cause RP as well . Systematic analysis of all genes responsible for other forms of retinal dystrophy in patients with RP is limited, especially in Chinese cohorts . Therefore, systemic evaluation of the frequency of mutations in all genes responsible for other forms of retinal dystrophy, apart from known RP genes, would be valuable. Our previous whole exome sequencing study detected potential pathogenic mutations in 73 known genes in 86 of 157 patients with RP . Due to the highly heterogeneous and genetically and clinically complicated features of RP, mutations in the genes related to more severe or syndromic diseases might be ignored, and mutations in previously analyzed genes might be mistakenly used in molecular diagnosis and clinical evaluation. Therefore, it might be interested to know if mutations in genes associated with other forms of retinal dystrophy may also contribute to RP. In the present study, variants in 129 genes responsible for other forms of retinal dystrophy were analyzed based on the exome data set of the same cohort of 157 patients. This study conformed to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of the Zhongshan Ophthalmic Center. Written informed

Purpose: To identify mutations in 12 genes in Chinese families with congenital cataracts. Methods: Twenty five families with congenital cataracts involved in this study. The coding exons and adjacent intronic regions of 12 genes were analyzed by cycle sequencing, including the alpha A crystallin (CRYAA), alpha B crystallin (CRYAB), beta A1 crystallin (CRYBA1), beta A4 crystallin (CRYBA4), beta B1 crystallin (CRYBB1), beta B2 crystallin (CRYBB2), beta B3 crystallin (CRYBB3), gamma C crystallin (CRYGC), gamma D crystallin (CRYGD), gamma S crystallin (CRYGS), alpha 3 gap junction protein (GJA3), and alpha 8 gap junction protein (GJA8) genes. Novel variants were further evaluated in 96 normal controls. Results: Nine mutations were identified in 10 of the 25 families (40%), including 5 novel (c.350_352delGCT in CRYAA, c.205C>T in CRYAB, c.106G>C in CRYGD, c.77A>G in CRYGS, c.1143_1165del23 in GJA3) and 4 known (c.292G>A in CRYAA; c.215+1G>A and c.272_274delGAG in CRYBA1, and c.176C>T in GJA3). All novel mutations were predicted to be pathogenic and were not present in 96 controls. Conclusions: Mutations in the 12 genes encoding crystallins and connexins were responsible for 40% Chinese families with congenital cataracts. Our results enriched our knowledge on the molecular basis of congenital cataracts in Chinese population.

Purpose: To identify the genetic defect in a large Chinese family with autosomal dominant cerulean cataract. Methods: Genomic DNA and clinical data were collected from the family. Candidate gene sequencing and genome-wide linkage analysis were used to disclose the molecular basis responsible for cerulean cataract in the family. Results: Initially, sequencing analysis of the three genes (beta-B2-crystallin [CRYBB2], gamma-D-crystallin [CRYGD], and V-MAF avian musculoaponeurotic fibrosarcoma oncogene homolog [MAF]) known to cause cerulean cataract failed to find any mutation. Then, genome-wide linkage analysis mapped the disease to chromosome 12q13-q22 between D12S85 and D12S351, with a maximum lod score of 4.10 at θ=0. Sequence analysis of the major intrinsic protein of lens fiber gene (MIP), a gene known to cause other types of cataract in the linkage interval, detected a novel heterozygous initiation codon mutation, c.2T>C (p.Met1?). This mutation was present in all patients with cerulean cataract but was not present in any of the 13 unaffected family members nor in 96 control individuals. Conclusions: Cerulean cataract was found in a large family and is caused by a novel initiation codon mutation in MIP. This study adds a new member in the existing list of genes causing cerulean cataract and expands the mutation spectrum and phenotypic association of MIP mutations.

Purpose: Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector–based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model. Experimental Design: To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector–based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo. Results: Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil ...

Against an unprecedented water crisis in our country, balancing water supply and demand is necessary for a secure and sustainable water supply. This challenge requires systems capable of delivering the necessary quantities while conserving resources. Numerous research initiatives focus on addressing water distribution challenges with the help of smart water systems to optimize network operations and minimize water demand. Based on these advancements, this paper proposes a new smart water distribution system for southwest of Algeria. The system integrates the Internet of Things (IoT), information and communication technologies, and smart technologies to address critical attributes for enhancing efficiency. To achieve the efficient management of two-way flows (both water and data) based on water demand and its availability, two innovative architectures have been proposed, using various measurements of water quantity and quality parameters. Algorithms to automate and optimize water distribution are also proposed. According to obtained results, performance has improved, with an accuracy rate of over 98%. These results establish the suggested system as a strong option for intelligent and sustainable water resource management by demonstrating its efficacy and durability.

The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls,

Este trabalho tem como objetivo demonstrar a necessidade da reconstrução da Responsabilidade Social da Empresa (RSE) por meio da ética do discurso. O primeiro momento será utilizado para analisar o conceito da RSE e como ele é, com frequência, utilizado de uma perspectiva dos ganhos empresariais com a sua adoção, pois, como será abordado, esse entendimento parte da ação estratégica na teoria habermasiana da sociedade, uma ação que busca ter como resultado a
ampliação dos lucros. Assim, no segundo momento, abordaremos a necessidade de uma reconstrução da RSE para inclusão da ética em sua concepção, uma ética em perspectiva universalista, na qual a ação seja orientada para a produção de ganhos sociais e a promoção de valores constitucionais. Por fim, será proposto o modelo da ética do discurso habermasiano como forma de superação do atual paradigma de RSE, em que, por meio da ação comunicativa, auxilie no cumprimento de valores constitucionais. O método utilizado para o estudo é o reconstrutivo, visando produzir um ambiente comunicativo apto para a emancipação.

Recoverin is a member ofthe EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was iden- tified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is ex- pressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified initially by Western blot analysis of the CAR patient's biopsy tissue by using anti- recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse tran- scription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlap- ping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser- Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmunemediated diseases for which the specific self-antigen is known.

Few clades of plants have proven as difficult to classify as cacti. One explanation may be an unusually high level of convergent and parallel evolution (homoplasy). To evaluate support for this phylogenetic hypothesis at the molecular level, we sequenced the genomes of four cacti in the especially problematic tribe Pachycereeae, which contains most of the large columnar cacti of Mexico and adjacent areas, including the iconic saguaro cactus (Carnegiea gigantea) of the Sonoran Desert. We assembled a high-coverage draft genome for saguaro and lower coverage genomes for three other genera of tribe Pachycereeae (Pachycereus, Lophocereus, and Stenocereus) and a more distant outgroup cactus, Pereskia We used these to construct 4,436 orthologous gene alignments. Species tree inference consistently returned the same phylogeny, but gene tree discordance was high: 37% of gene trees having at least 90% bootstrap support conflicted with the species tree. Evidently, discordance is a product of l...

An expression vector for human papillomavirus type 16 (HPV 16) E7-glutathion S transf erase fusion protein was constructed. The E7 gene was confirmed by southern blot analysis with a digoxygenin 11-dUTP labeled and simultaneously amplified E7 DNA probe by polymerase chain reaction (PCR). The fusion protein was expressed in E. coli, recoverd as inclusion bodies, and was confirmed by western blot analysis using a monoclonal antibody against HPV 16-E7 protein. A polyclonal antibody against the HPV 16-E7 protein was raised in the serum of mice hyper-immuned with the fusion protein. This antibody will be available for screening of patients with cervical cancer.

This study describes the molecular mechanism by which treatment with 3‐AB, a potent inhibitor of PARP, allows human osteosarcoma MG‐63 cells to restrict growth and enter differentiation. Our findings show that in MG‐63 cells, aberrant gene expression keeps Rb protein constitutively inactivated through hyperphosphorylation and this promotes uncontrolled proliferation of the cells. After 3‐AB‐treatment, the poly(ADP‐ribosyl)ation of nuclear proteins markedly decreases and this results in an increase in both the hypophosphorylated active form of Rb and pRb/E2F complexes. These effects are accompanied by G1 arrest, downregulation of gene products required for proliferation (cyclin D1, β‐catenin, c‐Jun, c‐Myc and Id2) and upregulation of those implicated in the osteoblastic differentiation (p21/Waf1, osteopontin, osteocalcin, type I collagen, N‐cadherins and alkaline phosphatase). Our study suggests that use of PARP inhibitors may induce a remodeling of chromatin with the reprogramming o...

Isolation and characterization of methane-oxidizing bacteria from a subtropical paddy field of Ishigaki, Japan, were performed. Two strains, namely R16 and R18, of Methylosinus-like organisms were isolated from methane enrichment cultures of rice rhizosphere. The isolates were motile, and stained Gram-negative. Cells of strains R16 and R18 were short rods, while slightly curved cells and coccobacillus-like cells were also observed for strains R16 and R18, respectively. Strain R16 was slightly smaller in size than R18. The organisms had an optimal pH range of 6.3^6.8 and a temperature optimum of 29^30³C. Methane and methanol were used as sole carbon and energy sources. Nitrate and ammonium sulfate could serve as nitrogen sources. The acetylene reduction method showed that the strains were able to fix nitrogen. On the basis of morphological and physiological properties, and 16S rRNA gene sequence analysis, the organisms were identified as genus Methylosinus. Strain R16 shows the highest 16S rRNA gene similarity of 95.5% with Methylosinus trichosporium, whereas R18 shows the highest 16S rRNA gene similarity with Methylosinus sporium (95.6%).

Patients with intestinal-type sinonasal adenocarcinoma (ITAC) have an unfavorable prognosis and new therapeutic approaches are needed to improve clinical management. Genetic analysis of 96 ITACs was performed by microarray comparative genomic hybridization and immunohistochemistry and correlated to previously obtained mutation, methylation, and protein expression data, and with pathological characteristics and clinical outcome. Seven copy number alterations (CNAs) were significantly associated with unfavorable clinical outcome: gains at 1q22-23, 3q28-29, 6p22, and 13q31-33, and losses at 4p15-16, 4q32-35, and 10q24. Unsupervised cluster analysis resulted in 5 subgroups of ITAC with significantly distinct genetic signatures and clinical outcomes, independently of disease stage or histological subtype. These data may guide studies to identify driver genes and signaling pathways involved in ITAC. In addition, the subclassification of genetic subgroups of patients with distinct clinical...

The roles of eukaryotic DNA methylation in the repression of mRNA transcription and in the formation of heterochromatin have been extensively elucidated over the past several years. However, the role of DNA methylation in transcriptional activation remains a mystery. In particular, it is not known whether the transcriptional activation of methylated DNA is promoter-specific, depends directly on sequence-specific DNA-binding proteins, or is facilitated by the methylation. Here we report that the sequence-specific DNA-binding protein, RFX, previously shown to mediate the transition from an inactive to an active chromatin structure, activates a methylated promoter. RFX is capable of mediating enhanceosome formation on a methylated promoter, thereby mediating a transition from a methylation-dependent repression of the promoter to a methylation-dependent activation of the promoter. These results indicate novel roles for DNA methylation and sequence-specific DNA-binding proteins in transcriptional activation. * This work was supported by National Institutes of Health Grants R01 CA81497 (to G. B.), R01 CA089242 (to J. Q. C.), R01 CA63136 (to S. C.), and R01 AI34000 and R01 GM47310 (to J. M. B.

European chickens were introduced into the American continents by the Spanish after their arrival in the 15th century. However, there is ongoing debate as to the presence of pre-Columbian chickens among Amerindians in South America, particularly in relation to Chilean breeds such as the Araucana and Passion Fowl. To understand the origin of these populations, we have generated partial mitochondrial DNA control region sequences from 41 native Chilean specimens and compared them with a previously generated database of ≈1,000 domestic chicken sequences from across the world as well as published Chilean and Polynesian ancient DNA sequences. The modern Chilean sequences cluster closely with haplotypes predominantly distributed among European, Indian subcontinental, and Southeast Asian chickens, consistent with a European genetic origin. A published, apparently pre-Columbian, Chilean specimen and six pre-European Polynesian specimens also cluster with the same European/Indian subcontinent...