Alpha-fetoprotein-producing gastric carcinoma: Biological properties of a cultured cell line

We describe a gastric carcinoma cell line that has been maintained in vitro for more than 10 years and retains the capacity to produce a large amount of alpha-fetoprotein. This cell line was isolated from a metastatic lymph node of a 63-year-old male patient with advanced gastric carcinoma (T2N3P0H0M0) who showed high serum levels of alpha-fetoprotein. The primary tumor was moderately differentiated tubular adenocarcinoma and the lymph node was poorly differentiated adenocarcinoma without any particular pattern. The cultured cells grew as densely packed islet-like colonies with small polygonal cells. Electron microscopy revealed cells abundant in cytoplasmic organelles, with some cellular attachments being tight with junctional complexes and some being loose across intercellular spaces. The free cell surface had microvilli. The population doubling-time was 152 h at passage 58. Chromosomal analysis revealed the modal number to be 77, with numerous karyotype abnormalities. The tumorigenicity of the cultured cells in athymic nude mice was positive only when they were subcutaneously transplanted beneath a plastic plate, but when the cells were transplanted subcutaneously or administered by intrasplenic injection in intact or weakly irradiated nude mice, no tumorigenicty was shown. The cell line produced tumor-associated antigens, such as alpha-fetoprotein, carcinoembryonic antigen, and tissue polypeptide antigen. This cell line may be useful for comparative studies of different types of gastric carcinoma and alpha-fetoproteins of different origins.     

Phenotypic analysis in peripheral blood lymphocytes of patients with pulmonary tuberculosis]

In order to clarify the immunological aspects of tuberculosis, phenotype of peripheral blood lymphocytes was analyzed in 96 patients with pulmonary tuberculosis and 43 normal controls. In tuberculosis, CD8 positive T cells were significantly decreased in comparison with normal controls and regarding expression of CD3, CD4, CD19, CD16 and gamma delta T cell receptor (TCR), there was no significant difference between tuberculosis and normal controls, though in tuberculosis CD4 positive T cells tended to be high as compared to normal controls. With respect to previous therapeutic condition, the group with previous chemotherapy had significantly high CD4 positive cells, low CD8 positive cells and low gamma delta TCR positive cells as compared to the group without previous chemotherapy. Between groups divided by Type or Expansion of pulmonary tuberculosis without previous chemotherapy, there was no significant difference regarding any surface markers. Regarding pulmonary tuberculosis with previous chemotherapy, the group of Type II had significantly high CD4 positive cells and low CD8 positive cells as compared to Type III, and in the group of Expansion 3, CD19 positive cells were significantly high as compared to Expansion 2. Characteristic of phenotype in peripheral blood lymphocytes of patients with pulmonary tuberculosis is predominant CD4 positive cells in a corresponding decrease in the proportion of CD8 positive cells and is no elevation of gamma delta TCR positive T cells.     

Aortitis syndrome with fatal acute aortic regurgitation due to aortic dilatation and aortic valve perforation--a case report.

This is a case report of a fifteen-year-old female with aortitis syndrome complicated by acute fatal aortic regurgitation due to the aortic valve perforation, as well as to aortic dilatation, which was confirmed by the autopsy. Aortic valve perforation was first recognized as the important cause of acute aortic regurgitation in the aortitis syndrome in this report.     

Association of insulin resistance with salt sensitivity and nocturnal fall of blood pressure

Insulin resistance was demonstrated in hypertensive patients and in salt-sensitive subjects. It was recently reported that the salt-sensitive state was related to a reduced fall in blood pressure during the night in essential hypertension. In the present study, the relationship among insulin sensitivity, blood pressure response to salt intake, and nocturnal fall in blood pressure was examined in 20 subjects with nondiabetic and nonobese essential hypertension during a low-salt and a high-salt diet. The subjects were maintained on a low-salt diet (50 mmol/d) and a high-salt diet (255 mmol/d) for 1 week each, in random order. On the sixth day of each diet, blood pressure was measured every hour for 24 hours with an automatic device. Insulin sensitivity was measured according to the steady-state plasma glucose (SSPG) method on the seventh day of each diet. Salt-induced increase in blood pressure, which we defined as the change in 24-hour mean arterial pressure between the low and the high dietary salt intakes, was significantly correlated with SSPG (r=0.60, P<0.01) during the high-salt period. There was a significant negative correlation (r=-0.61, P<0.01) between SSPG and a nocturnal fall in mean arterial pressure during the high-salt period. Salt-induced increase in blood pressure was inversely correlated with a nocturnal fall in mean arterial pressure (r=-0.52, P<0.02) with the high-salt diet. These results suggest that insulin resistance, salt sensitivity, and failed nocturnal fall in blood pressure are associated with each other in subjects with essential hypertension.     

Outcome of the Sauvé–Kapandji procedure for distal radioulnar joint disorder with rheumatoid arthritis or osteoarthritis: Results of one-year follow-up

Objectives: We performed the Sauvé–Kapandji procedure for treating disorders of the distal radioulnar joint (DRUJ) in patients with rheumatoid arthritis (RA) or osteoarthritis (OA). This study aimed to compare and clarify the results of the SK procedure between RA and OA patients. We report the one-year follow-up results of patients who underwent the SK procedure to correct the DRUJ disorder caused by RA or OA.

Methods: The study included 22 wrists of 19 patients with RA and 10 wrists of nine patients with OA. Pain, grip strength and range of motion of the wrist were examined clinically. For the evaluation of the stability of the carpus, ulnar stump and bone union, parameters were measured using radiographs. Shortened disabilities of the arm, shoulder and hand questionnaire (QuickDASH) was used for functional evaluation.

Results: Wrist pain reduced in all cases, and bone union was achieved in all wrists. The QuickDASH score significantly improved in both patients with RA and OA. In patients with RA, the range of motion increased significantly with regard to supination but decreased significantly with regard to palmar flexion. Carpal alignment and ulnar stump stability were maintained well at one-year follow-up.

Conclusion: The Sauvé–Kapandji procedure for treating disorders of the distal radioulnar joint DRUJ showed good results clinically and radiographically, irrespective of RA or OA.     

Blood supply of the sinus node artery in beagle dogs

Summary Postmortem coronary angiography was performed in 20 beagles and 15 mongrels, and the origin and distribution of the sinus node arteries (SNAs) were subsequently investigated using soft X-ray radiography, the tissue clearing method, and histological examination. In 19 of 20 beagles, the SNAs consisted of a single atrial branch originating from the right coronary artery, and the distribution pattern of the atrial branch seemed to be uniform. In contrast, the following three different patterns were recognized in the atrial branches forming the SNAs of mongrels: (1) distribution by two atrial branches, i.e., one originating from the right coronary artery and the other from the left circumflex artery, (2) dual distribution by two atrial branches originating from the right coronary artery, and (3) distribution by a single atrial branch originating from the right coronary artery or from the left circumflex artery. In 26 of the 30 dogs which were histologically examined, the SNAs ran outside of the sinus node and were not centrally located. In the beagles, the proximal atrial branch from the right coronary artery reached the atrioventricuolar node area and supplied blood to the atrioventricular node together with the arterial branches derived from the anterior septal artery and posterior septal artery. In addition, the arterial branch of the SNAs reached the atrioventricular junction area. These findings should contribute to clinical, pharmacological, and pathological studies of the cardiovascular system, including studies on cardiac arrhythmias in beagles.     

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X₄ receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X₄ purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s⁻¹), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.In chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channels gated by extracellular ATP (1, 7–9) and are involved in many physiological and pathophysiological processes (10–12). Seven subtypes of the P2X receptors have been identified in mammals (13), and they share ∼40% sequence identity. The P2X₄ receptor is involved in the pathogenesis of chronic neuropathic, inflammatory pain and the endothelial cell-mediated control of vascular tone (11, 14, 15). Compared with ATP, α,β-methylene ATP (α,β-meATP), in which the oxygen atom linking the α- and β-phosphorous atoms of ATP is replaced by a methylene group (Fig. S1A), reportedly induces a lower maximum current in cells expressing the mouse, rat, and human P2X₄ receptors and other P2X receptors (16, 17).Open in a separate windowFig. S1.Characterization of the P2X₄ receptor. (A) Chemical structures of ATP and α,β-meATP. (B and C) TEVC recordings of ATP- and α,β-meATP-evoked currents from rat P2X₄ receptor expressed in Xenopus oocytes, respectively. In B, the currents were evoked twice by ATP (30 μM, 1 min, black bar). In C, the currents were firstly evoked by ATP (30 μM, 1 min, black bar) and subsequently by α,β-meATP (300 μM, 1 min, black bar). (D) TEVC recording of the ATP-evoked current (30 μM, 30 s, black bar) from the N-terminally EGFP-tagged ΔzfP2X₄–A′ construct expressed in Xenopus oocytes. (E) Size exclusion chromatogram of purified EGFP-tagged ΔzfP2X₄–A′ in rHDLs. Elution volumes corresponding to 17.0, 12.2, 10.4, and 7.1 nm Stokes diameters were determined by thyroglobulin, ferritin, catalase, and BSA, respectively. V₀ and 1CV are void volume and single column volume, respectively. (F) SDS/PAGE analyses of purified ΔzfP2X₄–A′ embedded in rHDLs. The samples were analyzed by 12% SDS/PAGE with Coomassie Brilliant Blue staining. (G) Measurement of [³H]ATP saturation binding to the purified ΔzfP2X₄–A′ in rHDLs. (H and I) Estimation of the effects of deuteration based on the crystal structures of zfP2X₄ (PDB ID code 4DW1) and the deuteration incorporation rates. The plots on the Left (without deuteration) and the Right (with deuteration) are the sums of the inverse sixth power of the distances between pseudoatoms centered on the methyl hydrogens of M108, M249, M268, or M325 and each hydrogen atom in the crystal structure of zfP2X₄ (sums of the r⁻⁶) and the sums of the r⁻⁶ multiplied by [1 − (deuterium incorporation rates)] of each hydrogen atom, respectively. The graphs in H and I were calculated from the crystal structure in the apo state (PDB ID code 4DW0) and that in the ATP-bound state (PDB ID code 4DW1), respectively. Sums of the r⁻⁶ of each methionine methyl group and Hαβγ of the intraresidue methionine (green), Hαβγ of the interresidue methionine (light green), Hαβ of tyrosine (light violet), Hδεζη of tryptophan (orange), Hαβδεζ of phenylalanine (pink), Hαβγ of valine (blue), Hαβγδ of leucine (light blue), Hαβγδ of isoleucine (cyan), Hαβγ of threonine (light cyan), Hαβ of alanine (red), Hαβγδ of arginine (dark blue), Hα of glycine (dark green), and Hαβ of serine (magenta) residues, and the other hydrogens connected to carbon atoms (other unexchangeable hydrogens, light gray) are shown with colors. Hydrogen atoms connected to nitrogen, oxygen, or sulfur atoms were not considered in these calculations because these hydrogens should be exchanged with deuterium in D₂O. The deuterium incorporation rates of the hydrogen atoms within each methionine residue (intraresidue) and the deuterium incorporation rates of other methionine residues (interresidue) were set to 98% and 85%, respectively, because the methionine residues would be derived from 85% of [α-, β-, γ-98% ²H-, methyl-¹³C]-methionine and 15% of nonlabeled methionine in the medium.The crystal structures of zebrafish P2X₄ receptor (zfP2X₄) (18, 19), together with mutational analyses (20–26), provided the structural basis for the channel opening of P2X receptors upon ATP binding. In the crystal structures, zfP2X₄ forms a homotrimer (27, 28), in which the transmembrane region of each subunit is composed of two helices (19). In the crystal structure of zfP2X₄ in the ATP-bound state, three ATP molecules are bound to the intersubunit nucleotide binding pockets. In addition, the region that connects the ATP-binding site and the transmembrane region, which is referred to as the “lower body” (Fig. 1 A and B), is expanded by ∼10 Å in the ATP-bound state, and a pore is formed in the transmembrane region, which is proposed to expand by the iris-like movement of the transmembrane helices (18). However, the mechanism underlying the partial activation of P2X receptors is unknown because the structures of the P2X receptors have not been examined in the partial agonist-bound states.Open in a separate windowFig. 1.NMR resonances from the endogenous methionine residues of zfP2X₄ in rHDL. (A and B) Distribution of the methionine residues in the ΔzfP2X₄–A′. One subunit from the crystal structure of zfP2X₄ in the apo form (A) (PDB ID code 4DW0) and one from the ATP-bound form (B) (PDB ID code 4DW1) are shown in ribbons. The lower body and the right flipper are yellow. The A330 residues, the methionine residues, and the residues in which methionine mutations were introduced, L339 and L351, are depicted by green sticks. ATP is depicted by red sticks. Dummy atoms generated by Orientations of Proteins in Membranes (OPM), which represent membrane boundary planes, are gray. (C) Overlaid ¹H-¹³C HMQC spectra of [²H-11AA, α, β-²H, methyl-¹³C-Met]ΔzfP2X₄-A′, embedded in rHDLs, in the apo state (black) and the ATP-bound state (red). The regions with resonances from methionine residues are shown, and the assigned resonances are indicated. The centers of the resonances are indicated with dots. Cross-sections at lines through the centers of each resonance in the ATP-bound state and the cross-sections of the spectra using [α, β-²H, methyl-¹³C-Met]ΔzfP2X₄-A′ are shown on the top of the overlaid spectra. The intensities of the cross-sections were normalized by the concentration of ΔzfP2X₄-A′ and the conditions of the NMR measurements.The P2X₄ receptor used in the previous crystallographic studies was solubilized by detergents, which are widely used for structural investigations of membrane proteins, but the P2X₄ receptor is embedded in lipid bilayers under physiological conditions. It was recently reported that reconstituted high-density lipoproteins (rHDLs), which are also known as nanodiscs (29), can accommodate membrane proteins within a 10-nm-diameter disk-shaped lipid bilayer (30). The rHDLs reportedly provide a lipid environment with more native-like properties, compared with liposomes, in terms of the lateral pressure and curvature profiles because detergent micelles have strong curvature and different lateral pressure profiles from lipid membranes (31). Our NMR analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (32–34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium.Here, we used NMR to observe the conformational equilibrium of the alanine and methionine residues of zfP2X₄ bound to α,β-meATP in rHDLs. Based on the conformational equilibrium, we discuss the mechanism underlying the partial activation of P2X receptors.