Synaptotagmins (Syt) play important roles in Ca2؉ -induced neuroexocytosis. Insulin secretion of the pancreatic -cell is dependent on an increase in intracellular Ca 2؉ ; however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting TC3 cell. Syt III and VII proteins were identified in rat islets and TC3 and RINm5F -cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F -cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca 2؉ sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde ؉ carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca 2؉ -induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F -cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca 2؉ -induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulincontaining secretory granules, and we suggest that Syt III/VII may be the Ca 2؉ sensor or one of the Ca 2؉ sensors for insulin exocytosis of the -cell.Insulin exocytosis from the -cell of the islets of Langerhans is stimulated by various physiological secretagogues that include glucose, amino acids, and receptor-mediated agonists such as acetylcholine, cholecystokinin, and glucagon like-peptide 1 (1-7). A common mechanism of action for these secretagogues is to cause an increase in cytosolic Ca 2ϩ . Elevation of intracellular Ca 2ϩ is due to an influx of extracellular Ca 2ϩ through voltage-dependent L-type Ca 2ϩ channel and/or mobilization of intracellular Ca 2ϩ from the endoplasmic reticulum (8 -17). However, the mechanisms by which Ca 2ϩ induces insulin granule fusion with the plasma membrane of -cell remain unclear (1,16,18,20,21).1 is a family of membrane proteins initially found to be expressed in brain. At the present, 11 members of Syt have been identified (22,23). The Syt molecule has a single transmembrane domain and two Ca 2ϩ regulatory C 2 domains. The C 2 domains mediate Ca 2ϩ -dependent and Ca 2ϩ -independent interactions with target molecules that may regulate membrane fusion and membrane budding reactions (24,25). Literature concerning the expression and functions of Syt in pancreatic -cell is very limited and contradictory. In an earlier study (26), Syt was found in the non--cell of the islet mantle, but not in the -cell, using a non-isoform-specific antibody, and the mRNAs of Syt A and B were absent in mouse pancreatic -cell and RINm5F cells as demonstrated by in situ hybridization...
Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.
The insulin receptor signaling pathway is present in beta-cells and is believed to be important in beta-cell function. We show here that insulin directly regulates beta-cell function in isolated rodent islets. Long-term insulin treatment caused a sustained increase in [Ca(2+)](i) and enhanced glucose-stimulated insulin secretion in rat islets, but failed to increase insulin content. Chronic activation of insulin receptor signaling by IRS-1 overexpression in the beta-cell inhibited gene expression of SERCA3, an endoplasmic reticulum Ca(2+)-ATPase. Insulin gene transcription was stimulated by insulin receptor signaling and insulin mimetic compound (L-783 281) in a glucose- and Grb2-dependent manner. Thus, beta-cell SERCA3 is a target for insulin regulation, which implies that beta-cell Ca(2+) homeostasis is regulated in an autocrine feedback loop by insulin. This study identifies a novel regulatory pathway of insulin secretion at the molecular level with two main components: (1) regulation of intracellular Ca(2+) homeostasis via SERCA3 and (2) regulation of insulin gene expression.
Escherichia coli O157:H7 remains a continuous public health threat, appearing in meats, water, fruit juices, milk, cheese, and vegetables, where its ingestion at concentrations of perhaps as low as 10 to 100 organisms can result in potent toxin exposure and severe damage to the lining of the intestine. Abdominal pain and diarrhea develop, which in the very young or elderly can progress towards hemolytic uremic syndrome and kidney failure. To assist in the detection of E. coli O157:H7, a recombinant bacteriophage reporter was developed that uses quorum sensing (luxI/luxR) signaling and luxCDABE-based bioluminescent bioreporter sensing to specifically and autonomously respond to O157:H7 serotype E. coli. The bacteriophage reporter, derived from phage PP01, was tested in artificially contaminated foodstuffs including apple juice, tap water, ground beef, and spinach leaf rinsates. In apple juice, detection of E. coli O157:H7 at original inoculums of 1 CFU mL(-1) occurred within approximately 16 h after a 6-h pre-incubation, detection of 1 CFU mL(-1) in tap water occurred within approximately 6.5 h after a 6-h pre-incubation, and detection in spinach leaf rinsates using a real-time Xenogen IVIS imaging system resulted in detection of 1 CFU mL(-1) within approximately 4 h after a 2-h pre-incubation. Detection in ground beef was not successful, however, presumably due to the natural occurrence of quorum sensing autoinducer (N-3-(oxohexanoyl)-L: -homoserine lactone; OHHL), which generated false-positive bioreporter signals in the ground beef samples.
The rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this study, the lux reporter genes were used in a new bioassay that allows pathogen monitoring without multiple sample manipulations or the addition of exogenous substrate. A recombinant phage specific for Escherichia coli O157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli O157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP01-luxI to detect several strains of E. coli O157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PP01-luxI and E. coli O157:H7, and luminescence was monitored. Reporter phages induced light in bioreporter cells within 1 h when exposed to 104 CFU/ml of E. coli O157:H7 and were able to detect 10 CFU/ml in pure culture with a preincubation step (total detection time, 4 h). The detection method was also applied to contaminated apple juice and was able to detect 104 CFU/ml of E. coli O157:H7 in 2 h after a 6-h preincubation.
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