A receptor for formaldehyde-treated serum albumin on human placental brush-border membrane.

Formaldehyde-treated serum albumin (f-Alb) is known to be taken up and degraded by sinusoidal liver cells via receptor-mediated endocytosis. We report that 125I-labeled f-Alb (125I-f-Alb) binding to human placental brush-border membranes also occurs. This binding reached equilibrium within 40 min at 37 degrees C. Kinetic studies demonstrated the presence of saturable binding with an apparent Kd of 2.1 micrograms of f-Alb/ml and a maximal binding of 2.3 micrograms/mg of membrane protein at pH 7.5. Maximal binding was observed at between pH 7.5 and 8.0. 125I-f-Alb binding to the membranes was little inhibited by a 1000-fold molar excess of ovalbumin, human apo-transferrin and native bovine serum albumin. No binding was observed with membranes which had been pretreated with proteinase or trypsin. This f-Alb receptor was extremely heat-stable, since the binding was not abolished even by pretreatment of the membranes at 78 degrees C for 30 min. EDTA, Ca2+ and Mg/4 had no effect on 125I-f-Alb binding, so the binding was independent of divalent cations. These data suggest that a receptor specific for f-Alb exists on human placental brush-border membranes of syncytial trophoblasts.

Maleylated human serum albumin inhibits HIV-1 infection in vitro.

Maleylated-human serum albumin (Mal-HSA) inhibited human immunodeficiency virus type-1 (HIV-1) infection of MT-4 cells in vitro. It was also found to inhibit the fusion between uninfected CD4+ cells (Molt-4 clone 8 cells) and HIV-1 infected cells (Molt-4/HIV-1) to form syncytia. To investigate the mechanism of the inhibition, a study was designed to determine whether Mal-HSA could bind to CD4+ cells. Mal-HSA could bind to both MT-4 cells and Molt-4 clone 8 cells with high affinity, Kd = 2.0 nM and Kd = 5.8 nM, respectively. However, Mal-HSA could neither inhibit anti CD4 antibody Leu 3a binding to Molt-4 clone 8 cells nor modulate the expression of CD4 molecules on the surface of the cells. Mal-HSA binding to Molt-4 clone 8 cells was completely inhibited by sulfated polysaccharides bearing anti-HIV activity, such as dextran sulfate, fucoidan and carrageenan. Other HIV-1 susceptible human T-cell lines, such as Molt-4, CEM-5, H-9 and HuT-78 cells, also have Mal-HSA binding sites showing a high affinity, Kd = 0.9 +/- 0.4 nM. Mal-HSA binding proteins of Molt-4 clone 8 cells were identified by ligand blotting as 155 and 220 kDa proteins. Unlike dextran sulfate, Mal-HSA could not inhibit reverse transcriptase activity of HIV-1. These results indicate that Mal-HSA inhibits HIV-1 infection and syncytia formation, and suggest that 155 and/or 220 kDa proteins of target cells are involved in HIV-1 adsorption and/or the membrane fusion between HIV-1 and target cells.

Human placental ferritin receptor.

Brush-border membranes from human placenta were prepared and their purity was clarified by biochemical and morphological methods. Ferritin binding to these prepared membranes was examined using horse spleen 125I-apoferritin, and was found to be completed within 10 min at 37 degrees C and pH 7.5. The amount of ferritin bound to the membranes was found to be proportional to the amount of membrane added and saturable for a given amount of the membrane in the presence of excess ligand. The membranes exhibited specific ferritin binding with a Ka of 2.3 X 10(7) M-1 at pH 7.5. A competitive binding assay indicated that horse spleen 125I-apoferritin binding was inhibited by a 10-fold molar excess of horse spleen ferric ferritin and a 500-fold molar excess of human transferrin. These results suggest that human placental brush-border membranes have specific receptors for horse spleen apoferritin molecules.

Novel nucleolar protein, midnolin, is expressed in the mesencephalon during mouse development.

Using the gene trap method and the selection of embryonic stem cells in vitro, we have identified several novel genes involved in mouse development. The detailed analysis of one of these, named midnolin (midbrain nucleolar protein), is reported here. Expression of the midnolin gene is developmentally regulated: it is strongly expressed at the mesencephalon (midbrain) of the embryo in day 12.5 (E12.5) mice. The midnolin encodes a protein of 508 amino acids (aa), which contains a Ubiquitin-like domain. The intracellular distribution of the midnolin was studied by using midnolin-green fluorescent protein (GFP) fusion proteins. Midnolin was found to be localized in the nucleus and nucleolus, but not in the cytoplasm. The nucleolar localization signal was determined to be a 28aa peptide (440-QQKRLRRKARRDARGPYHWTPSRKAGRS-467) located at the C-terminal region of the midnolin. Our results suggest that midnolin is involved in regulation of genes related to neurogenesis in the nucleolus.

Immunohistochemical studies on hemoglobin-haptoglobin and hemoglobin catabolism sites.

In order to clarify the catabolism sites of Hb-Hp and free Hb, the organ distributions of [125I]-Hb-Hp and [125I]-Hb were studied, and the cell types in each organ incorporating them were determined by immunohistochemical methods. After administration of [125I]-Hb-Hp in very small amounts to rats, 84.5% was incorporated into the liver, but the renal uptake was only 0.6%. [125I]-Hb was incorporated into the kidneys rather than into the liver when a fivefold greater amount of [125I]-Hb than the binding capacity of plasma Hp was administered. Parenchymal cells, but not Kupffer cells, in the liver were stained with anti-Hb or anti-Hp IgG after administration of Hb in an amount corresponding to the Hb binding capacity of Hp. The proximal tubule cells, but not the distal tubule cells, in the kidney were stained with anti-Hb IgG after administration of a fivefold greater amount of Hb than the binding capacity of Hp. On the basis of these results, we suggest that Hb-Hp was incorporated mainly into liver parenchymal cells and did not traverse glomeruli in the kidney. In contrast to Hb-Hp, free Hb could pass through the glomeruli easily and was incorporated into the proximal tubule cells.

Polymerized albumin receptor on rat liver cells.

The polymerized albumin hypothesis was proposed for the mechanism of a hepatitis B virus (HBV) infection of human liver parenchymal cells on the basis that a receptor for polymerized albumin treated with glutaraldehyde was detected on isolated human liver parenchymal cells. However, some controversy exists regarding this hypothesis, because a receptor for formaldehyde-treated bovine serum albumin (f-BSA) has been found on liver non-parenchymal cells. Therefore, we characterized the uptake of polymerized rat serum albumin (p-RSA) and f-BSA by rat liver in vivo, and their bindings to liver cells in vitro. Most p-RSA and f-BSA was taken up by the liver after intravenous administration, and the uptake of p-RSA was inhibited by a 1,000-fold excess of f-BSA. In addition, more than 80% of p-RSA taken up by the liver was found in the non-parenchymal cells, and the remainder was found in the parenchymal cells. P-RSA as well as f-BSA could bind to isolated rat liver parenchymal and non-parenchymal cells. Furthermore, p-RSA and f-BSA could bind to isolated rat liver cell plasma membranes, and these bindings were completely inhibited by 1,000-fold excess of either f-BSA or p-RSA. These results indicate that there is a receptor, which can recognize both p-RSA and f-BSA, on not only rat liver non-parenchymal cells but also the parenchymal cells. It is also indicated that the receptor on the parenchymal cells as well as the non-parenchymal cells is involved in the in vivo uptake of p-RSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete amino acid sequence of mouse liver metallothionein-II.

The complete amino acid sequence of thionein-II, one of the two major mouse liver thionein components, was determined. The main fragmentation of thionein-II, which consists of 61 amino acid residues, was accomplished by digesting the S-[14C]-carboxymethylated protein and the cyanogen bromide-treated oxidized protein with trypsin. The peptides obtained by papain digestion of S-[14C]carboxymethylated thionein-II were used to align the major tryptic peptides. The sequence was determined by a combination of automated and manual Edman degradation techniques. Remarkable structural homology is observed in mouse thionein-I, mouse thionein-II, and thioneins from man and horse.

Effects of interferon-beta in combination with 5-fluorouracil on the growth of esophageal cancer cells in vitro.

The possible antiproliferative potency of human recombinant interferon-beta (hIFN-beta) towards ten human esophageal cancer cell lines was examined in comparison with the activity of the factor towards human malignant melanoma cell lines. The cell growth of esophageal cancer cell lines was inhibited by hIFN-beta in a dose- and time- dependent manner. The 50% inhibitory concentrations (IC50) of hIFN-beta on nine cell lines out of ten ranged between 23 to 332 IU/ml of culture medium. The remaining cell line, T.Tn, was less sensitive to the interferon (IC50, 611 IU/ml). Under the same culture conditions, the melanoma cell lines tested differed markedly in their sensitivity to hIFN-beta. When the esophageal cancer cells were treated with 5-fluorouracil (5-FU) in the presence of a low concentration of hIFN-beta, the effectiveness of 5-FU was markedly enhanced. In particular, the rate of growth inhibition of T.Tn cells was more than the added potencies of 5-FU and hIFN-beta indicating that the interferon is an effective biomodulator of 5-FU. All these data suggest that combination therapy with hIFN-beta and the anticancer drug 5-FU would be beneficial for the treatment of carcinoma of the esophagus.

Acute cadmium intoxication in inbred mice: a study on strain differences.

Male C3H, BALB/c and DBA/2 mice were injected subcutaneously with a single dose of 30 mumol CdCl2/kg. Most of the C3H mice died within 15 h, while all of DBA/2 survived for at least 48 h. At 6 h after Cd-injection, histology revealed severe hemorrhage, fatty change, zonal hepatocyte necrosis and congestion of C3H livers, while these findings were rare in the other strains even at 48 h post-injection. The rate of metallothionein (MT) induction at 6 h was significantly lower in C3H mice than in the other two strains.

[Contralateral pneumothorax after pneumonectomy for bronchogenic carcinoma; report of a case].

A case of contralateral pneumothorax after pneumonectomy was reported. Intrathoracic drainage was performed and pneumothorax was healed. Recurrent pneumothorax was occurred in this patient and intrathoracic drainage was performed again and pneumothorax was healed. We suspected that bulla was the cause of pneumothorax and thought that contralateral pneumothorax after pneumonectomy must be carefully follow-up.

[Multiple lung cancer with cavity; report of a case].

A case of multiple lung cancer with cavity was reported. Chest X-ray and chest computed tomography (CT) showed two abnormal shadows with consolidation in the right S1 and S2b. The shadow in S2b had a cavity. Right upper lobectomy and right middle lobe partial resection was performed and the histopathological examination revealed adenocarcinoma. This case deserves attention of difficulty in differentially diagnosis on the chest X-ray and chest CT from pulmonary tuberculosis.

[Analysis of ovalbumin-specific T cell lines established from patients with hen egg allergy].

Ten ovalbumin (OVA)-specific T cell lines (TCLs) were established from peripheral blood mononuclear cells of 6 patients with hen egg allergy, and the antigen recognition of these TCLs was characterized. Two OVA epitopes were determined by use of 3 synthetic OVA peptides which have been known as murine T cell epitopes. Blocking of antigen-specific T cell proliferation by anti-HLA class II monoclonal antibodies suggest that all 3 HLA class II molecules could act as restriction elements for T cell recognition of OVA. This is the first demonstration of OVA epitopes recognized by T cells in patients with hen egg allergy, as far as we know.

[Antigenic determinants on ovalbumin and ovomucoid: comparison of the specificity of IgG and IgE antibodies].

To delineate the antigenic determinants on ovalbumin (OA) and ovomucoid (OVM) recognized by specific IgG and IgE antibodies in sera from patients with allergy to hen's egg, we studied the binding activities of IgG and IgE antibodies to native OA or OVM and seven different OA or OVM preparations, i.e. heated OA or OVM (100 degrees C for 3 min and 80 degrees C for 30 min) or 0.3 M NaOH, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), 6 M urea or HCl treated OA or OVM. Eight patients with IgE anti-OA antibodies and 12 patients with IgE anti-OVM antibodies were used in these studies. The binding activities of IgG and IgE antibodies to physically or chemically denatured OA were partially decreased compared with those to native OA, whereas IgG and IgE antibodies in sera from patients bound well to denatured OVM with similar binding activities to native OVM. These results strongly suggest that antibodies to OA recognize partially conformational antigenic determinants on OA, whereas antibodies to OVM mainly recognize sequential antigenic determinants on OVM, and that antigenic determinants of OVM recognized by antibodies in sera from patients are more stable than those of OA under these denaturation conditions. In addition, the binding activities of IgE antibodies to denatured OA or OVM were significantly different from those of IgG antibodies to these OA or OVM, suggesting that the specificities of IgE antibodies to OA or OVM may be different from those of IgG antibodies to OA or OVM.

[Treatment of solid tumor by a direct electric current].

This is a case report of a 37-year-old Japanese married female with laryngeal sarcoma, treated by direct electric current so as to obtain remission for more than 4 years without signs of recurrence. Due to her hoarseness and laryngeal numb feeling she underwent a laryngeal examination including a biopsy, resulting in a diagnosis of sarcoma. She refused a total laryngectomy and was given Cobalt treatment of 40 Grey. In the following several months, no improvement was observed, objectively or subjectively. In Nagoya University Hospital the patient then received direct electric current therapy of 36 Coulombs through two platinum electrodes inserted into the tumor under a CT guide, pericutaneously. Two months later, as the hoarseness remained in spite of some improvement, she underwent another session of direct electric current therapy of 14.4 Coulombs through the platinum electrodes by bronchoscope-guided direct insertion. Her hoarseness soon disappeared thereafter and there was a regression of the tumor in 6 months. She did well thereafter without any signs of recurrence for 4 years. Clinical treatment of solid tumors with electric treatment with direct electric current has been done in more than 8,000 cases with CR in 25% of all cases and PR in 50%. Its mechanism, however, remains unclear. In our experimental animal studies, apoptosis was observed. It is considered that this electric therapy using direct electric current will be recognized as one method to treat solid tumors.

Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross-allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level.

BACKGROUND: Pollens from species of Cupressaceae family are one of the most important causes of respiratory allergies worldwide. In Japan, many patients with pollinosis have specific IgE to both pollens of Japanese cypress (Chamaecyparis obtusa) and Japanese cedar (Cryptomeria japonica). The sequences between Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar pollens, respectively, are 80% identical. OBJECTIVE: This study was undertaken to identify T cell epitopes in Cha o 1, and to elucidate the mechanism of cross-allergenicity between Cha o 1 and Cry j 1, at the T cell level. METHODS: T cell epitopes in Cha o 1 were identified by the reactivity of T cell lines, generated from 19 patients, to stimulation with overlapping peptides. The subsets of T cell clones specific to rCha o 1 were determined according to their ability to produce IL-4 and IFN-gamma. Peptide specificities of two T cell clones were determined by stimulation with the peptides from Cha o 1 and Cry j 1. RESULTS: Four dominant and six subdominant T cell epitopes were identified in Cha o 1. While four T cell epitopes, p11-30, p211-230, p251-270 and p331-350, were common to Cha o 1 and Cry j 1, 4 T cell epitopes, p61-80, p71-90, p311-330 and p321-340, were considered to be unique to Cha o 1. The subsets of T cell clones were predominantly of T helper2-type. One T cell clone recognized p16-30, which is common to Cha o 1 and Cry j 1, but another recognized p321-330, which is unique to Cha o 1. CONCLUSION: Presence of both T cells reactive to T cell epitopes common to Cha o 1 and Cry j 1 and T cells specific to T cell epitopes unique to Cha o 1 in patients with pollinosis contributes to prolonged symptoms after the cedar pollen season in March and the following cypress pollen season in April.

A ligand-receptor binding assay by receptor immobilization.

Using transferrin-transferrin receptor binding as a model of ligand-receptor binding, we have developed a new and simple binding assay for the solubilized receptor. Solubilized membrane proteins containing transferrin receptor were immobilized by covalent binding to beads having chemical reactive epoxide groups, and then 125I-labeled transferrin was added to the beads. Dose-dependent, ligand-specific, and saturable binding of 125I-labeled transferrin to the immobilized membrane proteins were demonstrated and a Scatchard analysis derived affinity of Kd = 1.8 X 10(-9) M was obtained. These results indicate that the immobilization of receptors onto beads may be useful in a simple binding assay of the solubilized receptor.

Characterization of transferrin binding and specificity of the placental transferrin receptor.

This study systematically examined the characteristics of specific binding of adult diferric transferrin to its receptor using a Triton X-100 solubilized preparation from human placentas as the receptor source. The following information was obtained. The ionic strength for maximal binding is in the range of 0.1-0.3 M NaCl. The pH optimum for specific binding extends over the range, from pH 6.0-10.0. Specific binding of diferric transferrin is not affected by 2.5 approximately 50 mM CaCl2 or by 10 mM EDTA. Triton X-100 in the concentration range of 0.02-3.0% does not affect specific binding. Specific binding is saturated within 10 min at 25 or 37 degrees C in the presence of excess amounts of diferric transferrin. The binding is reversible and the dissociation of diferric transferrin from the transferrin receptor is complete within 40 min at 25 degrees C. Apotransferrin, both adult and fetal, showed less binding than the holotransferrin species by competitive binding assay in the presence of 10 mM EDTA independent of up to 20 mM CaCl2. A 1500-fold molar excess of adult and fetal apotransferrin is required to give 40% inhibition for 125I-labeled diferric transferrin binding. Since calcium ion is not a factor, and since apotransferrin has such high binding affinity for iron (Ka = 1 X 10(24], this experiment suggests that the EDTA was necessary to prevent conversion of apotransferrin to holotransferrin from available iron in the reaction system. The specificity of the transferrin receptor for transferrin was examined by competitive binding studies in which 125I-diferric transferrin binding was measured in the presence of a series of other proteins. The proteins tested in the competitive binding studies were classified into three groups; in the first group were human serum albumin and ovalbumin; in the second group were proteins containing iron ions, such as hemoglobin, hemoglobin-haptoglobin complex, heme-hemopexin complex, ferritin, and diferric lactoferrin; in the third group were the metal-binding serum proteins, ceruloplasmin and metallothionein. None of these proteins except ferritin showed inhibition of diferric transferrin binding to the receptor. The effect of ferritin was small since a 700- to 1500-fold molar excess of ferritin is required for 50% inhibition of binding of diferric transferrin to the receptor.

Placental transferrin receptor. Evaluation of the presence of endogenous ligand on specific binding.

The effect of endogenous ligand on transferrin binding to receptor was investigated using a Triton X-100 extract of acetone powder from human placentae as the source of transferrin receptor. This extract contained 2.0 +/- 0.4 micrograms of endogenous transferrin per mg of protein. The studies showed that it is necessary to account for the concentration of endogenous transferrin when calculating the specific binding and the association constant (Ka) of transferrin to the receptor. The attempts to remove endogenous transferrin from the extract by washing with agents such as 2.0 M KSCN and glycine/NaOH, pH 10.0, with 1 M NaCl or by immunoabsorption with anti-transferrin were unsuccessful in that substantial amounts (10%) of endogenous ligand remained. Under the assumption that endogenous and exogenous transferrin have similar affinity of binding to the receptor, the effect of endogenous ligand on specific binding could be accurately determined by measuring the amounts of transferrin in the placental extract by radioimmunoassay and then accounting for it in the total transferrin concentration in the experiments. The Ka value corrected for endogenous transferrin present, 2.39 +/- 0.35 X 10(9) M-1 (n = 6), was approximately 3 times higher than the value obtained without consideration of endogenous transferrin, 0.87 +/- 0.20 X 10(9) M-1 (n = 5). From these studies, it appears that a true Ka value for ligand-receptor binding cannot readily be determined experimentally in the presence of substantial amounts of endogenous ligand but that the endogenous ligand must be quantitatively measured and the amounts present corrected for in the calculation of the Ka value.