Susan B. Curtis

Expression of the calcium-sensing receptor on human antral gastrin cells in culture.

The presence of the extracellular calcium-sensing receptor on human antral gastrin cells was investigated. Reverse transcription PCR using mRNA isolated from gastrin cell- enriched cell cultures identified a product with a sequence identical to part of the human parathyroid-secreting cell calcium-sensing receptor. Immunocytochemistry with an antibody to the extracellular region of the receptor immunostained all gastrin cells (but not mucin or somatostatin cells), and detected appropriate-sized bands in Western blots of whole cell lysates. Increasing extracellular calcium levels from 0.5 to 9 mM stimulated gastrin release in a concentration-dependent manner, with maximal release obtained at 7.2 mM. A known agonist of the calcium receptor, spermine also stimulated gastrin release. Microfluorimetry of identified gastrin cells demonstrated that increasing extracellular calcium resulted in an initial rapid rise in intracellular calcium followed by a plateau level that returned to basal levels immediately after removal of the elevated calcium. The traces were consistent with activation of a receptor-mediated mechanism rather than a concentration-dependent influx of calcium. In conclusion, these data indicate that G cells express the calcium-sensing receptor, and that activation of the receptor may explain the acid rebound phenomenon associated with calcium-containing antacid preparations.

Helicobacter pylori Infection Targets Adherens Junction Regulatory Proteins and Results in Increased Rates of Migration in Human Gastric Epithelial Cells

ABSTRACT The human gastric pathogen Helicobacter pylori attaches to antral epithelial cells in vivo. Cultured human antral epithelial cells, AGS and NCI-N87 cell lines, were grown in the absence or presence of H. pylori and compared with respect to gene transcript levels, protein expression, organization of the actin cytoskeleton, and the regulation of cell migration. The Clontech Neurobiology array detected differentially expressed transcripts, while Western blots were used to investigate related changes in protein levels. Infection with H. pylori consistently upregulated annexin II, S100 A7, Rho-GTP, and IQGAP-1, whereas SSTR-1 was downregulated upon H. pylori infection. In the adherens junction, E-cadherin and IQGAP-1 were translocated from the plasma membrane to intracellular vesicles. The primary and NCI-N87 cells were similar with respect to cell-cell and cell-matrix adhesion and cell migratory behavior; in contrast the AGS cells were significantly different from the primary gastric epithelial cell preparations, and thus caution must be used when using this cell line for studies of gastric disease. These studies demonstrate a correlation between H. pylori infection and alterations to epithelial cell adhesion molecules, including increased levels of Rho-GTP and cell migration. These data indicate that destabilizing epithelial cell adherence is one of the factors increasing the risk of H. pylori-infected individuals developing gastric cancer.

Human hephaestin expression is not limited to enterocytes of the gastrointestinal tract but is also found in the antrum, the enteric nervous system, and pancreatic β-cells

Hephaestin (Hp) is a membrane protein with ferroxidase activity that converts Fe(II) to Fe(III) during the absorption of nutritional iron in the gut. Using anti-peptide antibodies to predicted immunogenic regions of rodent Hp, previous immunocytochemical studies in rat, mouse, and human gut tissues localized Hp to the basolateral membranes of the duodenal enterocytes where the Hp was predicted to aid in the transfer of Fe(III) to transferrin in the blood. We used a recombinant soluble form of human Hp to obtain a high-titer polyclonal antibody to Hp. This antibody was used to identify the intracellular location of Hp in human gut tissue. Our immunocytochemical studies confirmed the previous localization of Hp in human enterocytes. However, we also localized Hp to the entire length of the gastrointestinal tract, the antral portion of the stomach, and to the enteric nervous system (both the myenteric and submucous plexi). Hp was also localized to human pancreatic beta-cells. In addition to its expression in the same cells as Hp, ferroportin was also localized to the ductal cells of the exocrine pancreas. The localization of the ferroxidase Hp to the neuronal plexi and the pancreatic beta cells suggests a role for the enzymatic function of Hp in the protection of these specialized cell types from oxidative damage.

Release of somatostatin immunoreactivity from human antral D cells in culture

A primary culture of human antral somatostatin cells has been developed and used in release studies. The phorbol ester, phorbol 12 myristate 13-acetate, caused a concentration-dependent increase in immunoreactive somatostatin secretion with a 1-mumol/L concentration resulting in a 40-fold stimulation (basal 0.28% +/- 0.7% total cell content vs. 13.8% +/- 2.2% TCC, P less than 0.005). The calcium ionophore, A23187, resulted in a significant stimulation only at 1 mumol/L (basal 0.28% +/- 0.7% TCC vs. 2.2% +/- 0.5% total cell content, P less than 0.05). However, addition of the ionophore at 1 mumol/L with the phorbol ester resulted in a potentiation of the response at all concentrations tested. Removal of extracellular calcium by chelation with EGTA reduced the response to that seen with the phorbol ester alone. Forskolin at 0.1 mmol/L resulted in a five-fold increase (basal 0.6% +/- 0.2% total cell content vs. 2.8% +/- 0.9% total cell content, P less than 0.02) and was 1000-fold less potent than the phorbol ester. The peptides bombesin and gastrin at concentrations up to 1 mumol/L had no effect on basal secretion. Cholecystokinin-8 significantly stimulated somatostatin secretion with a maximal effect at 0.1 mumol/L resulting in an eightfold increase (basal 0.2% +/- 0.04% total cell content vs. 1.5% +/- 0.4% total cell content, P less than 0.02). These results indicate that human antral D cells are more responsive to agents acting through the c-kinase pathway (phorbol 12 myristate 13-acetate, A23187, and cholecystokinin) than adenylate cyclase (forskolin).

Biomining with bacteriophage: Selectivity of displayed peptides for naturally occurring sphalerite and chalcopyrite

During mineral processing, concentrates of sulfide minerals of economic interest are formed by froth flotation of fine ore particles. The method works well but recovery and selectivity can be poor for ores with complex mineralogy. There is considerable interest in methods that improve the selectivity of this process while avoiding the high costs of using flotation chemicals. Here we show the first application of phage biotechnology to the processing of economically important minerals in ore slurries. A random heptapeptide library was screened for peptide sequences that bind selectively to the minerals sphalerite (ZnS) and chalcopyrite (CuFeS2). After several rounds of enrichment, cloned phage containing the surface peptide loops KPLLMGS and QPKGPKQ bound specifically to sphalerite. Phage containing the peptide loop TPTTYKV bound to both sphalerite and chalcopyrite. By using an enzyme-linked immunosorbant assay (ELISA), the phage was characterized as strong binders compared to wild-type phage. Specificity of binding was confirmed by immunochemical visualization of phage bound to mineral particles but not to silica (a waste mineral) or pyrite. The current study focused primarily on the isolation of ZnS-specific phage that could be utilized in the separation of sphalerite from silica. At mining sites where sphalerite and chalcopyrite are not found together in natural ores, the separation of sphalerite from silica would be an appropriate enrichment step. At mining sites where sphalerite and chalcopyrite do occur together, more specific phage would be required. This bacteriophage has the potential to be used in a more selective method of mineral separation and to be the basis for advanced methods of mineral processing.

Effects of bacteriophage on the surface properties of chalcopyrite (CuFeS2), and phage‐induced flocculation of chalcopyrite, glacial till, and oil sands tailings

The binding of mineral‐specific phage to the surface of chalcopyrite (CuFeS2) was investigated by using X‐ray photoelectron spectroscopy and scanning Auger microscopy. These studies confirmed the elemental composition of the minerals and confirmed that bacteriophage were bound to the mineral surface. These techniques also revealed that the phage were not forming a continuous film over the entire surface of the CuFeS2 particles, but selectively bound to the slimes coating the particles. In addition, the effect of mineral‐specific phage binding to the surface of CuFeS2 was investigated using induction time and zeta potential measurements. Bacteriophage (1012/mL) increased the induction time (contact time resulting in 50% particle attachment to a bubble) from ∼7.5 to ∼17 ms and reversed the zeta potential from negative to positive. In the course of performing the zeta potential measurements on particles <45 µm in diameter, phage‐induced aggregation was observed. The mechanism of aggregation was explored using a range of pH (3–11) and cation concentrations. Aggregation was observed across the tested pH range and with all cations. Phage also mediated aggregation of glacial till and oil sands tailings in a dose‐dependent and particle size‐dependent manner. We conclude that binding of bacteriophage to the surface of CuFeS2 does alter its surface properties. Biotechnol. Bioeng. 2011; 108:1579–1590.

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost‐efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO4:Ce3+,Tb3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 109 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptides affinity for the fluorescent phosphor components CAT (CeMgAl11O19:Tb3+) and BAM (BaMgAl10O17:Eu2+). No affinity was found for other fluorescent phosphor components such as YOX (Y2O3:Eu3+). The binding specificity of the RCQYPLCS peptide loop was improved 3–51‐fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016–1024.

Effect of Massive Small Bowel Resection on Components of the Peptidergic Innervation of the Rat Small Intestine

The effect of massive small bowel resection on the immunostaining of neuropeptides in the submucous plexus of the retained small intestine was examined. The neuropeptides chosen were somatostatin and vasoactive intestinal polypeptide because these are markers for two of the major populations of neurons in the plexus. Three different methods were used to assess the effect of resection on the enteric nervous system. Firstly immunocytochemical staining of neuropeptide containing neurons and nerve fibers was compared between test and control animals. The results demonstrated a significant increase in the number and size of the vasoactive intestinal polypeptide containing neurons with no change in the number of somatostatin neurons although these were also increased in size. Secondly the possibility that the increase in neuron number might be the result of neuronal division was examined by 3H-thymidine incorporation experiments. The results demonstrated that no neuronal elements were labelled. Finally the possibility that the increase in vasoactive intestinal peptide was the result of an increase in transcription was assessed by Northern blot analysis. The results demonstrated a small but significant increase in mRNA levels. It was concluded that massive small bowel resection directly affects neuropeptide levels in the submucous plexus, resulting in an increase in vasoactive intestinal polypeptide-immunoreactive neurons.

Identification of mineral-binding peptides that discriminate between chalcopyrite and enargite

Innovative approaches to the separation of minerals and subsequent extraction of metals are imperative owing to the increasing mineralogical complexity of ore deposits that are difficult or even impossible to separate into slurries or solutions containing only the minerals or metals of interest. Low recovery of metal is typical for these complex deposits leading to significant losses to tailings. In addition, the minerals often contain impurities, some toxic, which are difficult and costly to control or manage during the processing of a concentrate or other mineral product. One example of this complex situation is the significant economic and environmental costs associated with diluting and processing copper concentrates containing arsenic (in the form of the mineral enargite, Cu3AsS4) in the production of pure copper. To overcome these separation problems, we have utilized phage display to identify peptides that demonstrate selective recognition of enargite and the arsenic‐free copper sulfide, chalcopyrite. Screening of two random peptide phage display libraries resulted in the identification of an enargite‐selective peptide with the sequence MHKPTVHIKGPT and a chalcopyrite‐selective peptide with the sequence RKKKCKGNCCYTPQ. Mineral‐binding selectivity was demonstrated by binding studies, zeta potential determination and immunochemistry. Peptides that have the ability to discriminate between enargite and chalcopyrite provide a greener option for the separation of arsenic containing contaminants from copper concentrates. This represents the first step towards a major advance in the replacement or reduction of toxic collectors as well as reducing the level of arsenic‐bearing minerals in the early stages of mineral processing. Biotechnol. Bioeng. 2017;114: 998–1005.

Bacteriophage-induced aggregation of oil sands tailings.

Very large quantities of tailings are produced as a result of processing oil sands. After the sand particles settle out, a dense stable mixture of clay, silt, water with residual bitumen, salts, and organics called mature fine tailings (MFT) can remain in suspension for decades. Research into developing methods that would allow consolidation and sedimentation of the suspended particles is ongoing. We have studied the ability of a filamentous bacteriophage (called VP12 bearing the peptide DSQKTNPS at the N‐terminus of the major coat protein pVIII) to aggregate MFT. To understand the biophysical basis of the aggregation, phage‐induced aggregation of diluted MFT was measured at room temperature under varying conditions of pH, salt, detergent. Phage at concentrations of 5.0 × 1011/mL to 1012/mL induced rapid settling of the diluted MFT. The addition of sodium chloride (10 mM) lowered the concentration of phage required to induce aggregation. Since the non‐ionic detergents Triton‐X 100 and Tween‐20, and the ionic detergent sodium deoxycholate had little effect, hydrophobic interactions do not appear to be a major contributor to the phage‐induced aggregation of MFT. However, aggregation was prevented at pH values higher than 9.0 suggesting that positively charged amino acid residues are required for MFT aggregation by phage. Genetic engineering of the pVIII peptide sequence indicated that hydrogen bonding also contributes to phage‐induced aggregation. In addition, replacing the basic residue lysine with an alanine in the recombinant peptide of VP12 completely prevented phage‐induced aggregation. Three other phage displaying different amino acid sequences but all containing a lysine in the same position had variable aggregation efficiencies, ranging from no aggregation to rapid aggregation. We conclude that not only are the functional groups of the amino acids important, but the conformation that is adopted by the variable pVIII peptide is also important for phage‐induced MFT aggregation. Biotechnol. Bioeng. 2013; 110: 803–811.