Improvement of the salt aggregation test to study bacterial cell-surface hydrophobicity

Abstract An improved salt aggregation test (improved SAT) was developed to sensitize the determination of bacterial cell-surface hydrophobicity. One drop of a fresh bacterial suspension standardized to an A ^1cm₅₄₀ of 20 (equivalent to 5 × 10⁹ cfu/ml), and one drop each of ammonium sulphate solutions stained with methylene blue, were mixed on a white hydrophobic paper card using toothpicks. The bacterial suspensions, methylene blue stock solutions and the ammonium sulphate solutions (0.01–4.0 M) were made in 0.02 M sodium phosphate buffer, pH 6.8. Bacterial aggregations were read immediately after mixing the salt/bacterial suspensions while the card was gently rocked. Readings were also confirmed the next day on dried preparations. The results proved independent of reading time and mixture conditions (wet or dry preparations). The improved SAT technique is very rapid and sensitive, the reaction is easily read with the naked eye, and the paper cards can be stored for documentation of aggregation patterns after drying. In the improved SAT, the Staphylococcus cells of different species aggregated in 5 ways: tiny, medial, flaky granular, particulated and macrofilamentous forms; Salmonella strains aggregated in flaky granular, particulated and macrofilamentous forms.     

Mutational analysis of the active site of human insulin-regulated aminopeptidase.

Insulin-regulated aminopeptidase (IRAP) is a type II integral membrane protein belonging to the gluzincin family of metallopeptidases identified by the characteristic Zn(2+)-coordination sequence element, HEXXH-(18-64X)-E. A second conserved sequence element, the GXMEN motif, positioned 22-32 amino acids N-terminal to the Zn(2+)-coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH-(18X)-E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full-length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild-type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino-acid substitutions in the GAMEN motif (G428A, A429G, M430K, M430E, M430I, E431D and E431A) and in the Zn(2+)-binding motif (H464Y, E465D, E465Q, H468Y, E487D and E487Q) resulted in decreased or abolished aminopeptidase activity towards the leucine-para-nitroanilide substrate. The results show that conservation of residues within the GAMEN and Zn(2+)-binding motifs is important for IRAP enzyme activity.     

Synthesis of glycoproteins in the Golgi complex of the mouse gallbladder epithelium during fasting,refeeding, and gallstone formation

Summary The mouse gallbladder epithelium was studied with light microscopic autoradiography and quantitative electron microscopy during fasting, refeeding and experimental gallstone formation. To determine the intracellular pathway of glycoproteins, H³-galactose was injected at different time intervals into the mice. At 10, 25 and 40 min after an intraperitoneal injection the gallbladders were fixed and prepared for light microscopy. As early as 10 min after injection, label was observed in supranuclear cytoplasmic regions and at 25 min, an increased radioactivity was present throughout the apical cytoplasm. At 40 min, silver grains were mainly present at the cell surface. Autoradiographs processed 25 min after an intraperitoneal H³-galactose injection after fasting for 48 h showed decreased supranuclear and apical radioactivity. After refeeding (12 h) there was an enhanced activity in both these regions. Animals fed a lithogenic diet for one month showed a marked increase of radioactive label mainly in cells of crypts and invaginations of the mouse gallbladder mucosa.Morphometric measurements of the Golgi apparatus revealed that deprivation of food significantly diminished the volume density of the Golgi apparatus. Refeeding the animals restored the volume density values to normal levels. In the course of gallstone formation there was a further significant increase in the volume density of the Golgi complexes as compared to controls.     

Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin

Abstract Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori . Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with α-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a k _d 2.88 × 10⁻⁶ M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or α-chymotrypsin after isolation from iron-restricted and iron-containing media.     

IgG antibodies to Lewis type 2 antigens in serum of H. pylori-infected and noninfected blood donors of different Lewis(a,b) blood-group phenotype

Individuals of the Le(b+)/secretor phenotype revealed a stronger natural immune response to Le(x) and Le(y) epitopes irrespective of Helicobacter pylori serologic status. In contrast, H. pylori-infected Le(b-) type individuals showed a significantly higher proportion of strong responders to Le(x) antigen compared with the H. pylori-uninfected subgroup. The data suggest that the immune response to Lewis type 2 determinants is related to both the H. pylori serologic status and the Le(a,b) phenotype of the host.     

Reversible structural changes in a hydrophobic protein,elastin, as indicated by fluorescence probe analysis

Fluorescent probe analysis of purified elastin using 1-anilinonaphthalene-8-sulfonate has been used to investigate reversible structural changes that accompany stretching of this rubberlike protein. There is a specific binding of 1-anilinonaphthalene-8-sulfonate to elastin with a single dye molecule attached per 74,000 molecular-weight protein subunit. When labeled elastin is stretched, the intensity of the 1-anilinonaphthalene-8-sulfonate fluorescence decreases reversibly, and this decrease appears to be linked to an increase in the environmental polarity in the immediate vicinity of the bound dye molecule. The results of experiments carried out in H₂O and D₂O indicate that this polarity change is due to an increase in the exposure of the 1-anilinonaphthalene-8-sulfonate to water as the hydrophobic interior of the protein subunit is unfolded during stretching. The data are consistent with the proposal that the elastin network is a two-phase system of hydrophobic protein globules surrounded by free solvent spaces.     

Production of Bacterial Substrate by Marine Copepods: Effect of Phytoplankton Biomass and Cell Size

Laboratory experiments were performed to evaluate the importanceof grazing activity of the marine copepod Acartia tonsa forproduction of substrate for bacteria. Acartia tonsa were feda range of concentrations of the nanoflagellate Rhodomonas baltica,the diatom Ditylum brightwelli and the dinoflagellate Ceratiumlineatum. Regardless of the concentration of R. baltica, nodetectable response in bacterial biomass was observed due tograzing. However, when A. tonsa grazed the larger phytoplanktoncells of D. brightwelli and C. lineatum, responses in bacterioplanktonwere observed. It was estimated that approximately 54–69%of the grazed carbon was lost to the surroundings when A. tonsawas feeding on these large phytoplankton species. The laboratoryresults were applied to a dataset from a coastal temperate ecosystem.This analysis showed that the copepod contribution to the DOCpool was as important as the leakage from the primary producers.It is concluded, that the DOM contribution from copepods willbe largest when grazing plankton communities are composed oflarge species.