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1.
J Infect Dis ; 218(12): 1983-1994, 2018 11 05.
Article in English | MEDLINE | ID: mdl-30016475

ABSTRACT

Background: Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity. Methods: P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 Å); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo. Results: Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target. Conclusions: Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.


Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Conserved Sequence , Pseudomonas aeruginosa/metabolism , Antibodies, Bispecific , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Epitopes , Humans , Models, Molecular , Operon , Protein Conformation , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/immunology , Whole Genome Sequencing
2.
J Biol Chem ; 274(29): 20603-10, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10400691

ABSTRACT

We previously identified stearoyl-CoA desaturase 2 (SCD2) as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear sterol regulatory element binding proteins (SREBPs) or adipocyte determination and differentiation factor 1 (ADD1). A novel sterol regulatory element (SRE) (5'-AGCAGATTGTG-3') identified in the proximal promoter of the mouse SCD2 gene is required for induction of SCD2 promoter-reporter genes in response to cellular sterol depletion (Tabor, D. E., Kim, J. B., Spiegelman, B. M., and Edwards, P. A. (1998) J. Biol. Chem. 273, 22052-22058). In this report, we demonstrate that this novel SRE is both present in the promoter of the SCD1 gene and is critical for the sterol-dependent transcription of SCD1 promoter-reporter genes. Two conserved cis elements (5'-CCAAT-3') lie 5 and 48 base pairs 3' of the novel SREs in the promoters of both the SCD1 and SCD2 murine genes. Mutation of either of these putative NF-Y binding sites attenuates the transcriptional activation of SCD1 or SCD2 promoter-reporter genes in response to cellular sterol deprivation. Induction of both reporter genes is also attenuated when cells are cotransfected with dominant-negative forms of either NF-Y or SREBP. In addition, we demonstrate that the induction of SCD1 and SCD2 mRNAs that occurs during the differentiation of 3T3-L1 preadipocytes to adipocytes is paralleled by an increase in the levels of ADD1/SREBP-1c and that the SCD1 and SCD2 mRNAs are induced to even higher levels in response to ectopic expression of ADD1/SREBP-1c. We conclude that transcription of both SCD1 and SCD2 genes is responsive to cellular sterol levels and to the levels of nuclear SREBP/ADD1 and that transcriptional induction requires three spatially conserved cis elements, that bind SREBP and NF-Y. Additional studies demonstrate that maximal transcriptional repression of SCD2 reporter genes in response to an exogenous polyunsaturated fatty acid is dependent upon the SRE and the adjacent CCAAT motif.


Subject(s)
Gene Expression Regulation, Enzymologic , Isoenzymes/metabolism , Stearoyl-CoA Desaturase/metabolism , Transcription Factors/metabolism , 3T3 Cells , Animals , Base Sequence , CCAAT-Enhancer-Binding Proteins , Cell Line , DNA-Binding Proteins/genetics , Electrophoresis/methods , Genes, Reporter , Humans , Isoenzymes/chemistry , Mice , Nuclear Proteins/genetics , Promoter Regions, Genetic , RNA, Messenger/genetics , Stearoyl-CoA Desaturase/chemistry , Stearoyl-CoA Desaturase/genetics , Sterol Regulatory Element Binding Protein 1
3.
J Biol Chem ; 273(34): 22052-8, 1998 Aug 21.
Article in English | MEDLINE | ID: mdl-9705348

ABSTRACT

To identify genes that are transcriptionally activated by sterol regulatory element-binding proteins (SREBPs), we utilized mRNA differential display and mutant cells that express either high or low levels of transcriptionally active SREBP. This approach identified stearoyl-CoA desaturase 2 (SCD2) as a new SREBP-regulated gene. Cells were transiently transfected with reporter genes under the control of different fragments of the mouse SCD2 promoter. Constructs containing >199 base pairs of the SCD2 proximal promoter were activated following incubation of cells in sterol-depleted medium or as a result of co-expression of SREBP-1a, SREBP-2, or rat adipocyte determination and differentiation factor 1 (ADD1). Electromobility shift assays and DNase I footprint analysis demonstrated that recombinant SREBP-1a bound to a novel cis element (5'-AGCAGATTGTG-3') in the proximal promoter of the SCD2 gene. The finding that the endogenous SCD2 mRNA levels were induced when wild-type Chinese hamster ovary fibroblasts were incubated in sterol-deficient medium is consistent with a role for SREBP in regulating transcription of the gene. These studies identify SCD2 as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear SREBP/ADD1. In addition, the sterol regulatory element in the SCD2 promoter is distinct from all previously characterized motifs that confer SREBP- and ADD1-dependent transcriptional activation.


Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Enzymologic , Helix-Loop-Helix Motifs , Isoenzymes/genetics , Nuclear Proteins/metabolism , Stearoyl-CoA Desaturase/genetics , Transcription Factors/metabolism , Transcriptional Activation , Animals , Cricetinae , DNA Footprinting , Mice , RNA, Messenger/metabolism , Rats , Sterol Regulatory Element Binding Protein 1 , Sterol Regulatory Element Binding Protein 2
5.
Biochem Mol Med ; 59(1): 44-51, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8902193

ABSTRACT

We have utilized SSCP analysis to identify disease-causing mutations in a cohort with arginase deficiency. Each of the patient's mutations was reconstructed in vitro by site-directed mutagenesis to determine the effect of the mutations on enzyme activity. In addition we identified six areas of cross-species homology in the arginase protein, four containing conserved histidine residues thought to be important to Mn(2+)-dependent enzyme function. Mapping patient mutations in relationship to the conserved regions indicates that substitution mutations within the conserved regions and randomly occurring microdeletions and nonsense mutations have a significant effect on enzymatic function. In vitro mutagenesis was utilized to create nonpatient substitution mutations in the conserved histidine residues to verify their importance to arginase activity. As expected, replacement of histidine residues with other amino acids dramatically reduces arginase activity levels in our bacterial expression system.


Subject(s)
Arginase/genetics , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , Humans , Manganese/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Neurospora , Pedigree , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Rats , Sequence Alignment , Xenopus
6.
Somat Cell Mol Genet ; 20(4): 313-25, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7974006

ABSTRACT

Functional and DNA binding analyses were used to investigate transcriptional regulation of liver arginase, a mammalian urea cycle enzyme with marked tissue specificity. Reporter constructs containing the proximal 111 bp of the gene from man and Macaca fascicularis showed over sixfold background activity in HepG2 hepatoma cells, which express significant levels of liver arginase, and 12-fold background activity in minimally expressing HEK cells. Longer constructs, active in both cell lines, showed greater activity in the liver cell line. The constructs showed no activity in arginase-negative NIH 3T3 fibroblasts. A 54-bp dyad insert present in the human sequence and absent in M. fascicularis did not affect function. DNA binding analyses localized multiple liver-specific complexes as well as complexes shared among cell types. Little binding was evident in fibroblast extracts. Despite liver-specific binding, there was no evidence of a strong liver-specific enhancer. HEK and NIH 3T3 nuclear extracts showed strikingly different patterns of DNA binding. These studies demonstrate that molecular regulation of liver arginase transcription is complex and that control mechanisms differ among tissue types.


Subject(s)
Arginase/genetics , Liver/enzymology , Macaca fascicularis/genetics , Promoter Regions, Genetic , 3T3 Cells , Animals , Biological Evolution , Cell Line , DNA-Binding Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Mice , Organ Specificity , Plasmids , Transcription, Genetic , Tumor Cells, Cultured
7.
Laryngoscope ; 104(2): 127-34, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8302113

ABSTRACT

Cloning techniques allow the engineering and production of highly purified DNA. Further advances in molecular biology have provided the means to identify DNA sequences in a rapid fashion. Sequencing methods can identify mutations, deletions, polymorphisms, or confirm a known genetic sequence. The use of these techniques in clinical medicine has made it possible to accurately diagnose infectious diseases and determine the molecular etiology of many genetic disorders and malignancies. In this study, DNA extracted from archival, celloidin-embedded temporal bone sections has been cloned and sequenced using these techniques. We amplified, cloned, and sequenced varicella-zoster viral DNA extracted from archival temporal bone sections from patients who had herpes zoster oticus. The application of cloning and sequencing techniques to DNA extracted from archival temporal bones provides the methodology to study temporal bone pathology at the molecular level.


Subject(s)
DNA, Viral/genetics , Genome, Viral , Herpes Zoster Oticus/diagnosis , Herpesvirus 3, Human/genetics , Temporal Bone/pathology , Base Sequence , Cloning, Molecular , Herpesvirus 3, Human/isolation & purification , Humans , Molecular Sequence Data , Polymerase Chain Reaction
9.
Somat Cell Mol Genet ; 20(1): 61-5, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8197478

ABSTRACT

Because of a subtle anomaly we encountered upon an analytical gel while characterizing a point mutation in an exon of a patient, we decided to perform expensive and time-consuming procedures to characterize the anomaly. Although initial and subsequent Southern blots and PCR analyses of this patient's mutation suggested that his mutation lay directly within a TaqI recognition site, further characterization revealed that the mutation actually lay in a base immediately outside the recognition site. Had we included an appropriate double-stranded DNA control in the restriction enzyme digestion of this patient's PCR-amplified exon, we could have arrived at the correct conclusion as to the location of the mutation without incurring high costs and time loss. This brief report depicts the use of DNA controls of appropriate length and base composition as a means of avoiding erroneous conclusions and expense in routine mutational analyses in the clinical setting.


Subject(s)
DNA Mutational Analysis/methods , Point Mutation , Polymerase Chain Reaction/methods , Alleles , Base Composition , DNA , DNA Restriction Enzymes , Humans , Templates, Genetic
11.
Somat Cell Mol Genet ; 17(4): 369-75, 1991 Jul.
Article in English | MEDLINE | ID: mdl-1887333

ABSTRACT

While routinely mapping point mutations within the arginase locus of a collection of hyperargininemic patients, we discovered that a base immediately outside a restriction endonuclease recognition site (TaqI) can eliminate cleavage of this site by this enzyme. The genetic lesion lay in a base immediately flanking a TaqI recognition site within exon 8 of the arginase locus and abolished cutting by approximately 80%. We wish to emphasize the necessity of heeding subtle cues frequently encountered while generating restriction enzyme data, because neither Southern blot maps nor endonuclease digestion of polymerase chain reaction amplified products of exon 8 accurately predicted where the point mutation lay. To our knowledge, this is the first instance of inhibition of cleavage by flanking bases occurring on natural (nonsynthetic) DNA substrates, i.e., within the clinical setting of characterization of a human genetic disorder.


Subject(s)
Deoxyribonucleases, Type II Site-Specific , Mutation , Restriction Mapping , Base Sequence , Blotting, Southern , Densitometry , Humans , Hydrolysis , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/genetics , Molecular Sequence Data , Polymerase Chain Reaction
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