Effect of calmodulin on aldosterone synthesis by a cytochrome P-45011 beta-reconstituted system from bovine adrenocortical mitochondria

Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta.     

Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.     

Pharmacokinetic study of exogenously administered ß-endorphin using a rapid radioreceptor assay in rats

A rapid radioreceptor assay for measuring ß-endorphin (ß-EP) in unextracted serum has been developed. The method is based upon the inhibition by ß-EP of ³H-naloxone binding to the specific receptors on rat brain membranes, prepared in a stable form of pellets. Effect of serum on the assay was minimized by adding pooled serum to the equal dilution of total serum in the assay mixture. Pharmacokinetic analysis of pharmacologically active ß-EP equivalents (ß-EP eq.) in rats was performed using this method. The serum disappearance of ß-EP eq. after iv administration followed a biexponential decline and pharmacokinetic parameters were calculated by a two-compartment open model. The half-lives of α-phase and ß-phase were 2.6 ± 0.5 min and 6.2 ± 1.6 hr (mean ± SE; n=6), respectively. The volume of the central compartment (V₁) and that of steady-state (Vd_ss) were 67 ± 16 and 480 ± 75 ml/kg (mean ± SE; n=6), respectively. The total body serum clearance (CL_tot) was 2.1 ± 0.9 ml/min/kg (mean ± SE; n=6). The serum disappearance curve of ß-EP eq. obtained in the present study was similar to that previously reported by Houghten et al. (Proc. Natl. Acad. Sci. U.S.A. $\text{77}$, 4588–4591 (1980)), in which the disapperance of total radioactivity of tritiated ß-EP in rats was examined.     

Cloning and sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway from maize.

A recombinant clone, pM52, containing cDNA for maize phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) was isolated from a maize leaf cDNA library constructed using an expression vector in Escherichia coli. The screening of the clone was conveniently performed through its ability to complement the phenotype (glutamate requirement) of PEPCase-negative mutant of E. coli. The enzyme encoded by this clone was identical with the major PEPCase in maize, a key enzyme in the C4-pathway, as judged from its allosteric properties and immunological reactivity. The cloned cDNA (3093 nucleotides in length) contained an open reading frame of 2805 nucleotides, the 3'-untranslated region of 222 nucleotides and the poly(dA) tract of 64 nucleotides. The deduced amino acid sequence (935 residues) of the enzyme showed higher homology with that of an enterobacterium, E. coli (43%) than that of a cyanobacterium (blue-green alga), Anacystis nidulans (33%).     

Histamine release from rat mast cells by Vibrio vulnificus protease

Abstract Vibrio vulnificus protease (VVP) stimulated histamine release from isolated mast cells in a dose- and temperature-dependent manner within a range of 0.2–4.0 μ g/0.5 ml. Histamine release was accompanied by degranulation, and no leakage of lactate dehydrogenase from cells was observed, indicating that the histamine release was not due to cytolysis but to exocytosis. This release, completed within 30 s at 37°C, suggested that the mechanism of action of VVP on mast cells is different from that of other proteases, such as trypsin or α-chymotrypsin, which release histamine from the cells slowly.     

Isolation of virus causing hemorrhagic fever with renal syndrome (HFRS) through a cell culture system

Twenty-three rat lung specimens collected in outbreaks of hemorrhagic fever with renal syndrome (HFRS) in three medical institutions were inoculated onto the VERO-E6 cell monolayers. After several blind passages, an agent growing serially in the cell cultures and reacting specifically with known HFRS-positive sera was isolated from two of these specimens. The two isolates were antigenically identical each other. The agent, named strain SR-11, was identified as the causative virus of HFRS by its antigenic identity with E6 cell-adapted HFRS virus, Hantaan 76-118 strain, and the specific reactions with sera from various HFRS cases.     

Surveillance for typhoid fever in Matsuyama city during 1974-1981 and detection of Salmonella typhi in sewage and river waters

Eighty-two cases of typhoid fever were found in Matsuyama city in the period from 1974 to 1981. Seventy-six cases were found to be infected with Salmonella typhi other three with Salmonella paratyphi A, and the remaining three were diagnosed only clinically. The strains of S. typhi isolated from these patients showed such a variety of Vi-phage types as D1, D2, E1, M1, 53 and degraded Vi-positive strain (DVS). The concurrent survey of the city sewage and river waters for typhoid bacilli was conducted with total 578 samples taken therefrom. S. typhi was isolated from 120 of those samples. The Vi-phage types of the isolates were closely related with those of the isolates from the patients. The periodical examinations of the city sewage and the draining river may serve as a useful means for the controlling typhoid fever epidemics.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

The D-JH complex is an intermediate to the complete immunoglobulin heavy-chain V-region gene

We have examined the organization of the immunoglobulin JH segments in three clones derived from a single Abelson murine leukemia virus-transformed cell. Cloning and nucleotide sequence analyses of the JH-containing fragments have revealed the rearrangement from the preformed D-JH complex to the complete VH-D-JH gene, which was accompanied by the expression of the intra-cytoplasmic mu chain. In one case a JH segment downstream to the preformed D-JH was used to create a new VH-D-JH gene. Upon the D-JH and VH-D-JH rearrangements the intervening D segments were deleted from the chromosome. One of the expressed VH genes suffered from a large deletion of the 3' portion (including the 95th cysteine residue) of the VH segment. We discuss the possible mechanism of the allelic exclusion.