Purification and characterization of ferritin fromCampylobacter jejuni

We purified an iron-containing protein from Campylobacter jejuni using ultracentrifugation and ion-exchange chromatography. Electron microscopy of this protein revealed circular particles with a diameter of 11.5 nm and a central core with a diameter of 5.5 nm. The protein was composed of a single peptide of 21 kDa and did not serologically cross-react with horse spleen ferritin. The UV-visible spectrum of the protein showed no absorption peaks in the visible region, indicating that little or no heme is bound. The ratio of Fe:phosphate of C. jejuni ferritin was 1.5:1. From these morphological and chemical examinations, we concluded that the C. jejuni purified protein is a ferritin of the same class as that of Helicobacter pylori and Bacteroides fragilis and differs from the heme-containing bacterioferritin of Escherichia coli. The 30 N-terminal amino acids were sequenced and were found to resemble the sequences of other ferritins strongly (H. pylori ferritin, 73% identity; B. fragilis ferritin, 50% identity; E. coli gene-165 product, 50% identity), and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 26% identity; Azotobacter vinelandii, 26% identity; horse spleen ferritin 30% identity). Proteins that cross-reacted with antiserum against the ferritin of C. jejuni were found in other Campylobacter species and in H. pylori, but not in Vibrio, E. coli, or Pseudomonas aeruginosa. Received: 6 September 1994 / Accepted: 6 February 1995     

Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping

In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.     

Immune cell types involved in early uptake and transport of recombinant mouse prion protein in Peyer’s patches of calves

We have previously reported the early uptake and transport of foreign particles into Peyer’s patches (PPs) of newborn and 2-month-old calves and shown that the peak uptake of particles occurs 6 h after inoculation, in addition to site- and size-related effects on particle uptake. We now report the distribution of immune cells within PPs of the distal ileum in newborn and 2-month-old calves inoculated with carbon black. The types of immune cells involved in the early uptake and transport of recombinant mouse prion protein (rMPrP) within PPs of newborn calf were investigated by using monoclonal antibodies CD11c, CD14, CD68, CD172a, and CD21. CD11c⁺, CD14⁺, CD68⁺, CD172a⁺, and CD21⁺ immune cells were widely distributed in four tissue compartments (villi, dome, interfollicular region, and follicles) of PPs in the distal ileum of newborn and 2-month-old calves, whereas CD11c⁺, CD14⁺, CD172a⁺, and CD21⁺ immune cells were more prominently distributed in the dome areas of newborn calves than in 2-month-old calves. Moreover, CD11c⁺ and CD14⁺ dendritic cells, CD172a⁺ and CD68⁺ macrophages, and CD21⁺ follicular dendritic cells containing rMPrP were primarily observed in the dome and inner follicular regions. The deposition of rMPrP within CD11c⁺, CD14⁺, CD172a⁺, and CD68⁺ cells, but not CD21⁺ cells, was detected in villous regions. rMPrP-positive immune cells within the interfollicular regions included only CD11c⁺ and CD172⁺ cells. Although the particles used in this investigation do not include the infectious prion protein, PrP^Sc, our experimental setup provides a useful model for studying immune cells involved in the early uptake and transport of PrP^Sc.     

Study on immunocapture-chemiluminescence assay of lipase activity in a biological sample.

A new approach for the determination of lipase (triacylglycerol lipase, EC.3.1.1.3) activity in a biological sample was investigated by combining an immunocapture technique with a chemiluminescence (CL) assay method in order to eliminate interference with CL detection. The proposed method consists of an immunocapture step to trap lipase and a subsequent step for CL detection of the activity of the captured lipase. The CL detection is based on the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) reaction and utilizes a proenhancer substrate [a lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI)] which liberates an active enhancer, HDI, by enzymatic hydrolysis. A polyclonal antibody prepared with porcine pancreas lipase was used for the immunocapture. The proposed immunocapture-CL method effectively eliminated the interference with the CL reaction from biological components and enabled the determination of spiked porcine pancreas lipase activity in serum samples in the range 0.41-1.1 U(HDI) (1 U(HDI) corresponds to the amount which liberates 1 pmol HDI/min at 37 degrees C from the substrate). The method was further applied to the assay of the activity for human pancreas lipase in serum and the results showed good correlation (r = 0.871) with those by the conventional colorimetric method.     

Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.     

Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity

Dendritic cell (DC)-based vaccines are being developed for treatment of patients with cancer, in part because DC are potent inducers of CD8(+) CTL. DC MHC class I:antigenic peptide complexes that are required for CTL elicitation are most often generated by incubating DC with peptides or by transfecting (or transducing) DC with cDNAs (or viral vectors) that encode protein Ags. The former approach is feasible when MHC class I Ags and relevant peptides are known. The latter approach may be hampered by inefficient DC transfection (transduction) and/or difficulties associated with preparation and use of viral vectors. Herein we demonstrate that a bacterial recombinant model tumor-associated Ag (OVA) that contains the HIV TAT protein transduction domain (PTD) was readily engineered and purified, efficiently transduced murine lymphocytes and DC, and was processed by proteasomes for MHC class I-restricted presentation to CTL. In addition, PTD-containing rOVA was processed and presented by DC to CD4 T cells as efficiently as native OVA or rOVA lacking the PTD. PTD-OVA-transduced DC induced CTL in vivo in a Th cell-independent fashion and vaccinated against OVA-expressing tumors. In contrast, rOVA lacking the PTD did not enter the DC MHC class I presentation pathway and DC treated with this protein did not prime OVA-specific CTL in vivo. Treatment of mice harboring clinically apparent OVA-expressing tumors with PTD-OVA-transduced DC resulted in tumor regression in some animals. This straightforward vaccination strategy may translate into DC-based treatments for patients with cancer and other serious diseases.     

Molecular basis and functional consequences of the dominant effects of the mutant band 3 on the structure of normal band 3 in Southeast Asian ovalocytosis

Southeast Asian ovalocytosis (SAO) human red cell membranes contain similar proportions of normal band 3 and a mutant band 3 with a nine amino acid deletion (band 3 SAO). We employed specific chemical modification and proteolytic cleavage to probe the structures of band 3 in normal and SAO membranes. When the membranes were modified specifically at lysine residues with N-hydroxysulfosuccinimide-SS-biotin, band 3 Lys-851 was not modified in normal membranes but quantitatively modified in SAO membranes. Normal and SAO membranes showed different patterns of band 3 proteolytic cleavage. Notably, many sites cleaved in normal membranes were not cleaved in SAO membranes, despite the presence of normal band 3 in these membranes. The mutant band 3 changes the structure of essentially all the normal band 3 present in the SAO membranes, and these changes extend throughout the normal band 3 molecules. The results also imply that band 3 in SAO membranes is present as hetero-tetramers or higher hetero-oligomers. The dominant structural effects of band 3 SAO on the other band 3 allele have important consequences on the functional and hematological properties of human red cells heterozygous for band 3 SAO. Analysis of the altered profile of biotinylation and protease cleavage sites suggests the location of exposed surfaces in the band 3 membrane domain and identifies likely interacting regions within the molecule. Our approach provides a sensitive method for studying structural changes in polytopic membrane proteins.