Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy.

Hypertension and pregnancy-related hypertension are major public health problems of largely unknown causes. We describe a mutation in the mineralocorticoid receptor (MR), S810L, that causes early-onset hypertension that is markedly exacerbated in pregnancy. This mutation results in constitutive MR activity and alters receptor specificity, with progesterone and other steroids lacking 21-hydroxyl groups, normally MR antagonists, becoming potent agonists. Structural and biochemical studies indicate that the mutation results in the gain of a van der Waals interaction between helix 5 and helix 3 that substitutes for interaction of the steroid 21-hydroxyl group with helix 3 in the wild-type receptor. This helix 5-helix 3 interaction is highly conserved among diverse nuclear hormone receptors, suggesting its general role in receptor activation.

DNA-binding mechanism of the monomeric orphan nuclear receptor NGFI-B.

The 2.7 A X-ray crystal structure of the DNA-binding domain (DBD) of the orphan nuclear receptor, nerve growth factor-induced-B (NGFI-B), complexed to its high-affinity DNA target, represents the first structure analysis of a nuclear receptor DBD bound as a monomer to DNA. The structure of the core DBD and its interactions with the major groove of the DNA are similar to previously crystallographically solved DBD-DNA complexes in this superfamily; however, residues C-terminal to this core form a separate and unique substructure that interacts extensively and in a sequence-specific way with the minor groove of its DNA target, in particular with the characteristic 3 A-T base-pair identity element that extends 5' to the usual nuclear receptor half-site (AGGTCA).

Antibodies against malondialdehyde (MDA) in MRL/lpr/lpr mice: evidence for an autoimmune mechanism involving lipid peroxidation.

Previous studies have shown that lipid peroxidative processes may play a role in disease pathogenesis in lupus-prone MRL/lpr mice. Studies were thus performed to determine if an immune response against malondialdehyde (MDA), a highly reactive byproduct of lipid peroxidation, was present in these mice. By using MDA-modified mouse serum albumin (MSA) as antigens in ELISA, we found that these mice produce high levels of MDA-specific antibodies in the complement-fixing IgG2a and IgG2b subclasses. Anti-MDA antibodies were also found in MRL/+ mice but in significantly lower levels. The specificity of these antibodies was verified by inhibition ELISA. MDA may contribute to disease pathogenesis in these mice by altering the immunogenicity of self molecules, eliciting an immune response and forming immune complexes that may deposit in tissues.

Malondialdehyde modified proteins and their antibodies in the plasma of control and streptozotocin induced diabetic rats.

One of the possible mechanisms of diabetes-related tissue damage is modification of various proteins via lipid peroxidation byproducts such as malondialdehyde (MDA). To determine the extent of MDA derivatization of plasma proteins, Western blots were carried out using anti-MDA antisera to study plasma proteins in control and streptozotocin (STZ)-induced diabetic rats. Since MDA can modify proteins and may alter or enhance their antigenicity, we screened plasma samples for anti-MDA antibodies using enzyme-linked immunosorbent assay (ELISA), and confirmed antibody specificity by inhibition ELISA. Circulating immune complexes containing MDA were also assayed. This study is the first demonstration of the existence in plasma of MDA-modified proteins with a molecular weight of approximately 100 Kd. Both control and diabetic rats have similar concentrations of plasma anti-MDA antibodies and circulating immune complexes. These results do not support the notion that diabetes alters the immune response to MDA modified proteins. Whether MDA modification of proteins participate in immunological processes that lead to tissue injury remains to be demonstrated.

Immunochemical properties of malondialdehyde-protein adducts.

Malondialdehyde (MDA), a product of lipid peroxidation, can bind to and modify proteins by adduct formation. To determine whether MDA adducts were immunogenic, MDA was added to rabbit serum albumin (RSA) in order to characterize MDA-proteins and to immunize rabbits. Bound MDA was proportional to the concentration of MDA added in the range of 2.5-20 mM as measured by thiobarbituric acid reactivity. MDA adducts of RSA migrated further toward the anode than native serum protein in zone and immunoelectrophoresis indicating increased negative charge. Rabbits immunized with MDA-RSA produced high titers of IgG antibodies to MDA-RSA as measured by enzyme-linked immunosorbent assay (ELISA). Hapten specificity of the antibody was demonstrated by antisera reactivity with MDA-RSA but not with unaltered RSA. Our findings support the possibility that MDA may serve as a hapten to form neoantigens which may represent a pathway by which lipid peroxidation could produce tissue damage via an immunologic mechanism.

Acetaldehyde-albumin adduct formation: possible relevance to an immunologic mechanism in alcoholism.

Acetaldehyde (A), an ethanol metabolite, was incubated with rabbit serum albumin (RSA) and human serum albumin (HSA) to form the corresponding soluble haptenized proteins, A-RSA and A-HSA respectively. Both protein adducts showed conjugation with acetaldehyde evidenced by more rapid anodal migration compared to RSA and HSA. A-RSA immunized rabbits produced titers greater than 1:100,000 by enzyme-linked immunosorbent assay (ELISA). Rabbit serum antibodies to A-RSA diluted 1:10,000 showed high specificity for the hapten when reacted with acetaldehyde conjugates of RSA, HSA, bovine serum albumin, bovine gamma globulin and human gamma globulin. Our findings support the possibility that acetaldehyde may serve as a hapten to form neoantigens relevant to an immunologic mechanism which might be associated with alcoholic liver disease.

Studies of kissing bug-sensitive patients: evidence for the lack of cross-reactivity between Triatoma protracta and Triatoma rubida salivary gland extracts.

In the southern and western sections of the United States, bites from the reduviid bug, commonly known as the kissing bug, genus Triatoma, may induce serious life-threatening allergic reactions. This study was undertaken to identify the allergens responsible for patient sensitization and to determine the extent of cross-reactivity of these allergens. The Triatoma spp. most commonly encountered in California and Arizona, T. protracta and T. rubida, were obtained, maintained in the laboratory, and dissected to prepare extracts for testing. Extracts were prepared from T. protracta and T. rubida for study by RAST, lymphocyte transformation, leukocyte histamine release, and RAST inhibition. Sera and cells were collected from patients who had generalized reactions to Triatoma bites. Our results indicate that T. protracta and T. rubida antigens to which patients are sensitized are present in extracts that contain saliva and that human responses are specific for T. protracta or T. rubida, i.e., allergic cross-reactivity could not be demonstrated.

Immunoprecipitation of the solubilized membrane receptor for IgE of human cultured lymphoblastoid cells.

The putative Fc receptor for IgE (Fc epsilon) of cultured human lymphoblastoid cells was characterized by using a goat anti-receptor antiserum. The antiserum was prepared against NP-40-solubilized cell components of Wil-2WT cells which bound to an IgE-Sepharose-4B immunoadsorbent column. The antiserum specifically inhibited binding of 125I-labelled IgE to Fc epsilon-positive RPMI-8866 lymphoblastoid cells. Absorption of the antiserum with Fc epsilon-negative Raji and Molt-4 cells and with IgE and IgG did not change this inhibitory activity. Antiserum extensively absorbed with Raji and Molt-4 cells precipitated 7% of the radioactivity of lysates of 125I-lactoperoxidase-labelled RPMI-8866 cells but only 0.5-1.8% of that of Raji and Molt-4 cells. Autoradiography of SDS-PAGE analyses demonstrated two major labelled peptides of 86,000 and 47,000 mol. wt. in reduced immunoprecipitates from RPMI-8866 but not from Raji or Molt-4 cell lysates. As determined by Sepharose-6B gel filtration, the approximate molecular weight of the solubilized radiolabelled membrane component that reacted with the antiserum was 250,000 solubilized in NP-40 and 125,000 in NP-40-4 M urea. Both the 250,000 and 125,000 mol. wt. material consisted of the 86,000 and 47,000 peptides. The data demonstrate that the anti-receptor antiserum inhibited binding of 125I-labelled IgE to Fc epsilon-receptor-positive lymphoblastoid cells and suggest that the receptor may consist of two non-covalently linked polypeptides that remain associated in NP-40 and NP-40-4 M urea.

Isolation and characterization of the major capsid protein of bovine papilloma virus type 1.

The major capsid protein of bovine papilloma virus type 1 (BPV-1) was isolated by gel filtration following disruption of purified virus particles with guanidine hydrochloride. The capsid protein, VP1, has a mol. wt. of about 53 500. Amino acid composition studies of VP1 showed that it is a highly acidic protein containing almost twice the average number of acidic residues than basic residues. Relatedness was observed between VP1 and the major capsid proteins of simian virus 40 (SV40) and polyoma virus.

Enrichment of antigen-specific B lymphocytes by the direct removal of B cells not bearing specificity for the antigen.

Antigen-specific B cells (ASC) were purified from other B cells by prior incubation with specific antigen followed by rosetting with erythrocytes conjugated with anti-mouse Ig and sedimenting on Ficoll-Isopaque. This procedure allowed the removal of most of the B cells, while those speicifc for the antigen used in incubation were retained. Relative to the B-cell content, ASC were enriched 64- to 132-fold. The method is highly specific in that B cells primed to two different antigens, turkey gamma globulin and sheep erythrocytes, could be separated from each other. The advantages of this indirect purification procedure over purification procedures which obtain ASC directly are the simplicity of obtaining the ASC and the ability of the ASC of respond to antigen without the addition of other cells.

In Vivo Studies with the Partially Purified Protease (Elastase) from Pseudomonas aeruginosa.

An extracellular protease from Pseudomonas aeruginosa exhibiting elastase activity was characterized in vivo after an 80- to 100-fold purification by chemical and chromatographic procedures. The lethality of different samples for white, female mice was determined by intravenous, intranasal, intraperitoneal, and subcutaneous injections. The purified protease exhibited the following 48-hr LD(50) values: intraperitoneally, 9.0 protease units; intranasally, 31.5 protease units; and intranasally, 0.3 protease unit. In the concentrations tested no lethality was observed when the subcutaneous route was employed. Gross and microscopic studies revealed that purified protease was capable of eliciting a variety of tissue responses in mice depending upon its route of administration. Intraperitoneal injections resulted in gastrointestinal tract serosal hemorrhage and necrosis. Intranasal and intravenous injections produced pulmonary hemorrhage, whereas subcutaneous injections resulted in black, necrotic, ulcerating lesions.