Engineering a high-throughput clone for industrial-scale production of long-acting GLP-1 analogue with retained bio-efficacy.

Type 2 diabetes mellitus (T2DM) and obesity are critical global health issues with rising incidence rates. Glucagon-like peptide-1 (GLP-1) analogues have emerged as effective treatments due to their ability to regulate blood glucose levels and gastric emptying through central nervous signals involving hypothalamic receptors, such as leptin. To address the short plasma half-life of native GLP-1, a C-16 fatty acid was conjugated to lysine in the GLP-1 analogue sequence to enhance its longevity.

Recombinant approach for the production of HIV fusion inhibitor Enfuvirtide using Escherichia coli.

The use of antiretroviral drugs is gaining importance in the recent past for the treatment of human immunodeficiency virus infection. Enfuvirtide (T20) is one of the fusion inhibitors, inhibits the fusion between the virus and healthy target CD4 cells. The treatment with T20 involves very high therapeutic dose.

DNA sequence conservation and diversity in transposable element IS605 of Helicobacter pylori.

Background: IS605, a transposable element-like sequence identified in the virulence-associated cag region of Helicobacter pylori reference strain NCTC11638, is unusual in containing two oppositely-oriented open reading frames whose products are homologues of the single transposases of the unrelated elements, IS200 and IS1341.

Methods: One hundred independent H. pylori isolates from different parts of the world were screened by PCR and dot blot hybridization to determine the presence of IS605.

Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kappa B in gastric epithelial cells.

In vivo, gastric infection with Helicobacter pylori leads to substantial production of the inflammatory cytokines IL-1, IL-6, TNF-alpha, and IL-8. H. pylori strains that contain the cag pathogenicity island (cag+) and are associated with ulceration and gastric carcinoma induce greater cytokine production than cag- strains.

Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced.

Role of vacuolating cytotoxin in gastritis due to Helicobacter pylori in gnotobiotic piglets.

To investigate the role of the Helicobacter pylori cytotoxin in the pathogenesis of gastritis, gnotobiotic piglets were colonized with either toxigenic H. pylori or a nontoxigenic isogenic mutant. Only piglets given the toxigenic strain developed toxin-neutralizing antibodies (indicating that toxin is expressed in vivo), but there was no difference in bacterial colonization, epithelial vacuolation, or gastritis between the two groups of piglets.

Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216.

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids.

Effect of growth phase and acid shock on Helicobacter pylori cagA expression.

Helicobacter pylori strains possessing cagA are associated with peptic ulceration. To understand the regulation of expression of cagA, picB, associated with interleukin-8 induction, and ureA, encoding the small urease subunit, we created gene fusions of cagA, ureA, and picB of strain 3401, using a promoterless reporter (xylE). Expression of XylE after growth in broth culture revealed that basal levels of expression of cagA and urea in H.

Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA.

Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage. In this study, we sought to test the hypothesis that two H. pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo.

Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells.

Approximately 60% of Helicobacter pylori strains are cagA+ and this genotype is more frequently associated with duodenal ulcer disease. Although most wild-type cagA+ strains are both cytotoxigenic and induce enhanced Interleukin-8 (IL-8) secretion in gastric epithelial cells, isogenic cagA- mutants retain full activity in these assays; thus, cagA appears to be a marker of enhanced virulence. Delineation of the nucleotide sequence of a 4 kb region upstream of cagA allowed the identification of 966 bp (picA) and 2655 bp (picB) open reading frames encoding 36 kDa and 101 kDa polypeptides, respectively.

Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease.

The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]).

Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration.

Approximately 50% of Helicobacter pylori strains produce a cytotoxin, encoded by vacA, that induces vacuolation of eukaryotic cells. Analysis of a clinically isolated tox- strain (Tx30a) indicated secretion of a 93-kDa product from a 3933-base pair vacA open reading frame. Characterization of 59 different H.

Nucleotide sequence and characterization of peb4A encoding an antigenic protein in Campylobacter jejuni.

The 29-kDa protein PEB4, a major antigen of Campylobacter jejuni, is present in all C. jejuni strains tested and elicits an antibody response in infected patients. By screening a lambda gt11 library of chromosomal DNA fragments of C.

Segmental conservation of sapA sequences in type B Campylobacter fetus cells.

Campylobacter fetus cells may exist as either of two defined serogroups (type A or B) based on their lipopolysaccharide (LPS) composition. Wild-type strains contain surface array proteins (S-layer proteins) that have partial antigenic cross-reactivity but bind exclusively to LPS from homologous (type A or B) cells. Type A cells possess 8 homologs of sapA, which encodes a 97-kDa S-layer protein; the gene products of these homologs have a conserved N terminus of 184 amino acids.

Serologic detection of infection with cagA+ Helicobacter pylori strains.

Approximately 60% of Helicobacter pylori isolates possess the cagA gene and express its 120- to 140-kDa product (CagA). In this study, the cagA gene was detected in H. pylori isolates from 26 (81.

Interleukin-8 response of gastric epithelial cell lines to Helicobacter pylori stimulation in vitro.

Gastric infection with Helicobacter pylori activates a mucosal inflammatory response by mononuclear cells and neutrophils that includes expression of cytokines interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha, and IL-8. In this study, we analyzed the IL-8 response of human gastric cancer cell lines (Kato III, AGS, and MKN28) to H. pylori infection in vitro.

A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the conserved N terminus of a family of silent and divergent homologs.

Campylobacter fetus cells can produce multiple S-layer proteins ranging from 97 to 149 kDa, with a single form predominating in cultured cells. We have cloned, sequenced, and expressed in Escherichia coli a sapA homolog, sapA2, which encodes a full-length 1,109-amino-acid (112-kDa) S-layer protein. Comparison with the two previously cloned sapA homologs has demonstrated two regions of identity, approximately 70 bp before the open reading frame (ORF) and proceeding 550 bp into the ORF and immediately downstream of the ORF.

High-frequency S-layer protein variation in Campylobacter fetus revealed by sapA mutagenesis.

Campylobacter fetus utilizes paracrystalline surface (S-) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full-length S-layer proteins.

Mutation of the cytotoxin-associated cagA gene does not affect the vacuolating cytotoxin activity of Helicobacter pylori.

Helicobacter pylori now is recognized as an etiological agent in chronic superficial gastritis and peptic ulcer disease. Although only about 60% of H. pylori isolates produce an immunodominant 128-kDa antigen (CagA; cytotoxin-associated gene product), virtually all H.

Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains.

Approximately 50% of Helicobacter pylori isolates produce a cytotoxin in vitro that induces vacuolation of eukaryotic cells. Screening a lambda ZapII library of H. pylori 60190 chromosomal fragments permitted the identification of a 3864-base pair (bp) open reading frame (vacA) that encoded the vacuolating cytotoxin, and a > or = 567-bp upstream gene that was homologous to Escherichia coli cysteinyl-tRNA synthetase.

Rearrangement of sapA homologs with conserved and variable regions in Campylobacter fetus.

The Campylobacter fetus surface-layer (S-layer) proteins mediate both complement resistance and antigenic variation in mammalian hosts. Wild-type strain 23D possesses the sapA gene, which encodes a 97-kDa S-layer protein, and several sapA homologs are present in both wild-type and mutant strains. Here we report that a cloned silent gene (sapA1) in C.

Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production.

A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pylori 84-183 by using lambda ZapII.

Characterization of the Campylobacter fetus sapA promoter: evidence that the sapA promoter is deleted in spontaneous mutant strains.

Wild-type Campylobacter fetus cells possess S-layer proteins (S+ phenotype), whereas after laboratory passage, spontaneous stable mutants that do not express these proteins (S- phenotype) arise. To determine the molecular mechanisms by which C. fetus changes to the S- phenotype, we studied wild-type strain 23D, from which the sapA gene encoding the 97-kDa S-layer protein has been cloned, and strain 23B, a spontaneous S- mutant.

The in vitro conversion of chorismate to isochorismate catalyzed by the Escherichia coli entC gene product. Evidence that EntA does not contribute to isochorismate synthase activity.

The entC and entA genes, coding for the enzymes isochorismate synthase and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, respectively, were subcloned behind the T7 promoter in the expression plasmid pGEM3Z. Their protein products were overproduced and partially purified for in vitro analysis of the conversion of chorismate to isochorismate. Whereas previous genetic experiments suggested that the EntA enzyme has a role in this conversion, this study clearly indicates that EntC alone catalyzes the reaction.